CN1195997A - Flow electroporation chamber and method - Google Patents
Flow electroporation chamber and method Download PDFInfo
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- CN1195997A CN1195997A CN96193177A CN96193177A CN1195997A CN 1195997 A CN1195997 A CN 1195997A CN 96193177 A CN96193177 A CN 96193177A CN 96193177 A CN96193177 A CN 96193177A CN 1195997 A CN1195997 A CN 1195997A
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Abstract
The present invention relates to a method and apparatus for the encapsulatio n of biologically-active substances in red blood cells, characterized by an optionally automated, continuous-flow, self-contained electroporation system (5) which allows withdrawal of blood from a patient, separation of red blood cells, and optional recombination of blood plasma and the modified red blood cells thereby producing blood with modified biological characteristics. The present invention is particularly suited for use to encapsulate allosteric effectors of hemoglobin, thereby reducing the affinity of erythrocytes for oxygen and improving the release of oxygen from erythrocytes in tissues. The blood is introduced into the system (5) at inlet (11) and mixed with an anticoagulant (27). The blood and anticoagulant mixture is passed through a filter (18) and to a blood separation and wash bowl (44). Plasma and white blood cells are admitted into the plasma reservoir (89) while the red blood cells are retained in the wash bowl (44). The separated red blood cells are pumped out and admixed at junction (67) withan IHP solution from reservoir (50). This mixture is cooled by cooling coil (68) and then pumped to electroporation chamber (72) for treatment.
Description
Technical field
The present invention relates to be used for bioactive substance is wrapped in the method and apparatus of various cell masses.More particularly, the present invention relates to a kind of being used for the hemoglobin allosteric effector to be wrapped in erythrocyte, thereby realize the method and apparatus of required variation in the treatment of hemoglobin physical characteristic in the born of the same parents by electroporation.
Background of invention
In an adult vascular system, the volume of blood is approximately 5 to 6 liters.Wherein only about half of volume is that cell is occupied, comprises erythrocyte (erythrocyte), leukocyte (leukocyte) and platelet.Erythrocyte has been formed the most cell component of blood.Blood plasma, i.e. the liquid part of blood, water and 10% various solutes for about 90%.These solutes comprise plasma protein, organic metabolite, refuse and inorganic compound.
Erythrocytic major function is to transport oxygen from pulmonary to in-vivo tissue, and transports carbon dioxide so that remove from tissue to pulmonary.Because oxygen only is oligodynamical in aqueous solution, so blood plasma transports very small amount of oxygen.Most of oxygen that blood carries is transported by erythrocytic hemoglobin.Mammiferous erythrocyte does not contain nucleus, mitochondrion or any other born of the same parents' inner cell organ, and they do not consume oxygen in the metabolism of self.Erythrocyte contains the hemoglobin of about 35% weight fraction, and they are in combination and transport in the oxygen and work.
Hemoglobin is that a kind of molecular weight is approximately 64,500 daltonian protein.It comprises that 4 polypeptide chains and 4 and iron atom are with the bonded haemachrome prothetic group of ferrous state.Common globin, promptly the protein portion of haemoglobin molecule comprises two α chains and two β chains.Article four, each bar in the chain all has a distinctive tertiary structure, and chain wherein folds.Four thereby polypeptide chain is arranged to combine with about level Four and is constituted the distinctive quarternary structure of hemoglobin.A hemachrome group is arranged on each bar polypeptide chain, and it can reversible molecule oxygen in conjunction with a part.When hemoglobin and combination with oxygen, form HbO2 Oxyhemoglobin.When discharging oxygen, HbO2 Oxyhemoglobin is reduced into deoxyhemoglobin.
Transport oxygen to tissue and depend on many factors, these factors comprise, the volume flow of blood, erythrocytic quantity, the concentration of hemoglobin in the erythrocyte, hemoglobin is to the affinity of oxygen and formal at some, and corpuscular hemoglobin and mol ratio high and low oxygen affinity in the born of the same parents still are not limited to above-mentioned factor.Hemoglobin also depends on 4 factors to the affinity of oxygen, i.e. the dividing potential drop of (1) oxygen; (2) pH value; (3) play 2 of allosteric effector effect in the hemoglobin, the concentration of 3-diphosphoglyceric acid (DPG); And (4) concentration of carbon dioxide.In pulmonary, when the dividing potential drop of oxygen was 100mmHg, about 98% circulation hemoglobin was saturated by oxygen.This represents total oxygen transporting power of blood.Behind complete oxygenate, 100 milliliters of complete mammiferous blood can carry about 21 milliliters gaseous state oxygen.
The influence in conjunction with the oxygen ability preferably illustrates by the oxygen saturation curve of measuring hemoglobin to hemoglobin for oxygen partial pressure and pH value.The oxygen saturation curve has been drawn when oxygen partial pressures different in haemoglobin molecule solution and the gas phase is in balance, is the percentage ratio in total combination with oxygen site of the occupied haemoglobin molecule of oxygen molecule.
The oxygen saturation curve of hemoglobin is a S shape.Therefore, the oxygen in conjunction with first molecule has increased the affinity of remaining hemoglobin in conjunction with the additional oxygen molecule.Increase with oxygen partial pressure reaches a peak value, locates each haemoglobin molecule all by saturated and contain the upper limit of 4 molecule oxygen at this.
According to reaction equation:
In pulmonary, the oxygen partial pressure of air space is approximately 90 to 100mmHg, and pH value also is higher than the pH value (reaching 7.6) of corresponding normal blood.Therefore, trend towards in pulmonary's hemoglobin almost saturated by institute's oxygen to greatest extent.At this pressure and pH value, hemoglobin nearly 98% is saturated by oxygen.On the other hand, in the blood capillary of organization internal, oxygen partial pressure has only about 25 to 40mmHg and pH value also relatively low (about 7.2 to 7.3) around.Because the myocyte consumes oxygen fast, thereby has reduced the local concentration of oxygen, therefore the bonded oxygen of release portion is favourable in tissue.When blood during by the blood capillary in the muscle, oxygen will be discharged into blood plasma near in the saturated hemoglobin from erythrocyte, enter the myocyte then.When hemoglobin when the muscle blood capillary, it will discharge about 1/3rd in conjunction with oxygen, so when it leaves muscle, it will have only about 64% by saturated.Usually, leave the hemoglobin organized in the circulation venous blood by between between about 65% and 97% circulation oxygen institute is saturated repeatedly in pulmonary and surrounding tissue.Therefore, oxygen partial pressure and pH value influence the release of oxygen jointly by hemoglobin.
The 3rd key factor of regulating hemoglobin Oxygenation degree is allosteric effector 2,3-diphosphoglyceric acid (DPG).DPG is the common physiological effect thing of hemoglobin in the mammalian erythropoietin.DPG regulates the oxygen-binding affinity that is pressed with hemoglobin in the erythrocyte of pass with the pulmonary oxygen edema caused by disorder of QI.Usually, the concentration of DPG is high more in cell, and hemoglobin is low more to the affinity of oxygen.
When reducing the transmission of oxygen in tissue lentamente, the concentration of DPG increases in normal person's the erythrocyte.For example, oxygen partial pressure is obviously lower aloft.Accordingly, the oxygen partial pressure in the tissue is also lower.In several hours after a normal person moves to eminence more, the content of DPG increases in the erythrocyte, and causes more DPG combined, and the oxygen affinity of hemoglobin reduces.The increase of DPG content also can occur among the patient who suffers from anoxia in the erythrocyte.This adjusting makes hemoglobin can easierly discharge the Oxygenation that bonded oxygen descends in pulmonary's hemoglobin with compensation.Opposite variation can take place when people adapt to eminence and drop to lower.
When separating from blood usually, hemoglobin contains considerable DPG.When hemoglobin " was peelled off " DPG, it demonstrated the affinity higher to oxygen.When increasing DPG, the combination with oxygen affinity of hemoglobin reduces.Therefore the normal release of oxygen in tissue is essential to hemoglobin as the physiology allosteric effector of DPG.
Though DPG is the normal physiological effect thing of hemoglobin in the mammal erythrocyte, find that the inositol of phosphorylation plays similar effect in the erythrocyte of some birds and reptile.Although IHP can not pass the cell membrane of mammalian erythropoietin, it can combine with the erythrocytic hemoglobin of mammal at the binding site of DPG to improve the other structure structure of hemoglobin, and the result of its effect reduces the affinity of hemoglobin to oxygen.For example, DPG can be replaced by phytic acid (IHP), is reducing IHP even more effective than DPG aspect the oxygen affinity of hemoglobin.IHP to the affinity of hemoglobin than DPG high 1000 times (R.E.Benesch etc., biochemistry, the 16th volume, 2594-2597 page or leaf (1977)) and at 37 ℃, pH value is 7.4 o'clock P with hemoglobin
50Be increased to 96.4mmHg (journal of biological chemistry, the 250th volume, 7093-7098 page or leaf (1975)).
Can strengthen the erythrocytic oxygen releasability of mammal by introduce certain hemoglobin allosteric effector to erythrocyte, thereby reduce hemoglobin to the affinity of oxygen and improved the economic utilization of oxygen in the blood.This phenomenon shows to exist to be used for the treatment of because pulmonary or circulatory function lack makes tissue experience the patient's of low oxygenated effect multiple medical application.
Because use these improved erythrocyte can realize potential medical science interests, developed multiple technologies in the prior art and the hemoglobin allosteric effector has been wrapped up in erythrocyte so that make.Correspondingly, many equipment have been designed with auxiliary or simplification encapsulation process.Packaging method known in the prior art comprises rebuilding of infiltration pulse (expansions) and cell, controls cracking and joint filling again (resealing), the mixing and electroporation of liposome.Be suitable on the commercial scale of using, existing electroporation method make this technology commercial be unpractiaca.
Following list of references described by the liposome that is loaded with IHP interaction with polyphosphoric acids be incorporated into method in the erythrocyte: Gersonde etc. " by polyphosphoric acids is incorporated into original erythrocyte with the oxygen affinity of hemoglobin in the improvement born of the same parents and strengthen the release of oxygen in the blood capillary system ", Biblthca.Haemat., 46 volumes, 81-92 page or leaf (1980); Gersonde etc. " strengthen human erythrocytic O after mixing phytic acid with the effector fusion that contains lipid vesicle
2Releasability and Bohr effect ", the collaborative combination origin of hemoglobin, (1982); And Weiner, " by the liposome technology Hb-O that in the erythrocyte that can survive, moves to right
2Dissociate " cytobiology, the 47th the volume, (1983).
In addition, the United States Patent (USP) 4,192,869,4,321,259 and 4,473,563 of Nicolau etc. has been described a kind of by fluid-charged lipid vesicle and erythrocyte being merged the method for its inclusions of storage in erythrocyte.In this way, allosteric effector such as phytic acid may be transported in the erythrocyte, in this cell, because higher binding constant, IHP has replaced the DPG on the binding site that is positioned at hemoglobin.
According to liposome technology, IHP is dissolved in the phosphate buffer saturated and the lipid vesicle mixture is suspended in the solution up to solution.Then float is carried out supersound process or injection process, centrifugal subsequently.The upper strata float comprises the small pitch pocket bubble that contains IHP, collects them then.The lipid vesicle that contains IHP and erythrocytic cell membrane merge during in, join in the suspension after the collection erythrocyte and incubation, thereby its inclusions be saved in erythrocytic inside.The erythrocyte of washing improvement and joining in the blood plasma then to generate product.
The shortcoming of liposome technology comprises the low repeatability of the IHP concentration that is incorporated in the erythrocyte and with erythrocytic obvious haemolysis in the post processing.In addition, because this process is tediously long and complicated, its commercialization is unpractiaca.
In order to solve the shortcoming of liposome technology, a kind of cracking and the erythrocytic method of joint filling have again been developed.This method is described in following publication to some extent: Nicolau etc. " mix the hemoglobin allosteric effector in erythrocyte.Physiological effect, " Biblthca.Haemat., the 51st volume, 92-107 page or leaf (1985).The related U.S. patent 4 of Ropars etc., 752,586 and 4,652,449 also described a kind of by control erythrocytic cracking and again joint filling the material of biologically active is wrapped in method in the mankind or the animal erythrocyte, this method has been avoided the interaction of RBC-liposome.
Being characterized as of this technology maximum to be similar to the continuous-flow dialysis system that the infiltration pulse technique works.Specifically, at least one the dialysis element a dividing plate erythrocytic liquid float is provided continuously, it is hypotonic aqueous solution that the secondary dividing plate of the element of dialysing simultaneously contains with respect to the red blood cell suspension thing.Hypisotonic solution causes erythrocyte splitting.Thereby the erythrocyte splitting thing is contacted with bioactive substance is incorporated in the erythrocyte it.In order to make the joint filling again of erythrocyte cell membrane, increase the infiltration and/or the oncotic pressure of erythrocyte splitting thing, reclaim the red blood cell suspension of joint filling again.
In the related U.S. patent 4,874,690 and 5,043 of Gooddrish etc., a kind of correlation technique that relates to the erythrocyte lyophilization and rebuild is disclosed in 261.As a part that rebuilds the erythrocyte process, described in the literary composition and added various polyanions, comprise phytic acid.According to disclosed method, handle erythrocyte and can generate active unaffected cell.Presumably, in rebuilding process, IHP is incorporated into cell, thereby keeps the activity of hemoglobin.
At the United States Patent (USP) 4,478,824 and 4,931 of Franco etc., described in 276 by effective cracking and will comprise that with joint filling again the potent agent of phytic acid is incorporated into second relevant method and apparatus in the mammiferous erythrocyte.This process is known as " infiltration pulse technique ".In implementing the infiltration pulse technique, suspend in the solution of the chemical compound of cell and the erythrocyte raw material of incubation after wrapping up containing to be easy to spread, wherein this compound concentrations should be oozed and is as the criterion to cause that enough it is diffused in the cell so that make the inclusions of cell become height.Next, introduce in the presence of the reagent at least a required waiting, dilute by a kind of isoosmotic basically liquid medium and to contain the solution that height oozes cell and stride the film ion gradient, thereby cause that water is diffused in the cell and causes expanding and the increase of epicyte permeability with formation.Should " infiltration pulse " cause that water is diffused in the cell and has caused the expansion of cell, above-mentioned expansion can increase the permeability of okioplast film to required reagent.The increase of membranous permeation rate can be kept a period of time, only is enough in during this period of time to cell and transports at least a reagent and chemical compound diffuses out cell.
Operable polyanion comprises pyrophosphoric acid, tripolyphosphate, the inositol of phosphorylation in implementing the infiltration pulse, 2,3-diphosphoglyceric acid (DPG), adenosine triphosphate, heparin and water soluble and to the nondestructive polycarboxylic acid of the outer duplicature of erythrocytic lipid.
The infiltration pulse technique has several shortcomings, and these shortcomings comprise that the parcel productive rate is low, incomplete joint filling again, the minimizing of the loss of cell component and corresponding cell survival.This technology is tediously long, and is complicated and be unsuitable for automation mechanized operation.Owing to these reasons, the infiltration pulse technique does not almost have the commerce of success to use.
The another kind of method that is used at the various bioactive substances of erythrocyte parcel is an electroporation, electroporation has been used for wrapping up exogenous molecules at different cell types, comprise as Mouneimne etc. " causing that by using electroporation in erythrocyte, to wrap up phytic acid the stable of Oxygenation dissociation curve moves to right ", FEBS, 275 volumes, 1,2 phase, the IHP erythrocyte described in the 117-120 page or leaf (1990).
The method of electroporation relate to by contain cell or vesicle (vesicles) thus the both sides of liquid cellular suspension apply electric field pulse at cell membrane or form micropore in the vesicle arbitrarily.In perforation procedure, cell suspension experiences an electric field pulse then in liquid medium.This medium can be an electrolyte, non-electrolyte, or the mixture of electrolyte and non-electrolyte.Affacting electric field intensity on the suspension and pulse length (affacting the time of the electric field on the cell suspending liquid) changes according to the type of cell.In order to produce micropore on the adventitia of cell, the electric field that is applied must have time enough length and enough voltage so that produce the voltage of a setting and generate micropore in the both sides of cell membrane in sufficiently long a period of time.
There are four phenomenons in the process of electroporation, to work.First phenomenon is an insulation breakdown.Insulation breakdown refers to high electric field and produce the little micropore or the ability in hole on cell membrane.In case generation micropore, cell just can the load bioactive substances.Second phenomenon is the insulation bunching effect, and it refers to, and the stacking (placement) by vesicle produces mutual self-gravitation in uniform electric field.The 3rd phenomenon is that vesicle merges.Vesicle merge refer to produce micropore by insulation breakdown the film of biological vesicle closely in and its trend that combines in the insulation breakdown site.The 4th phenomenon is the trend that cell is arranged in straight line along their axle in the presence of high-frequency electric field.Therefore, for load with unload the cell vesicle, electroporation relates in vesicle rotation pre-determined bit, the application in the dielectric constant of vesicle perforation and vesicle.
Electroporation has been used for mixing at erythrocyte the allosteric effector of hemoglobin effectively.At Mouneimne, one piece of article of Y etc. is (referring to " causing that by using electroporation to wrap up phytic acid in erythrocyte the stable of Oxygenation dissociation constant moves to right ", FEBS, the 275th volume, 1,2 phases, 11-120 page or leaf (November nineteen ninety)) in, Mouneimne and colleague thereof have reported that can realize in the erythrocyte that mixes IHP in containing of handling that hemoglobin-oxygen is dissociated moves to right.The mensuration of 24 hours and 48 hours shows and has a stable P behind the load IHP
50, this shows that erythrocytic joint filling again is persistent.And the result shows that the erythrocyte of load phytic acid has normal 11 day half-life.But, by Mouneimne and with resulting result shows in 24 hours of blood transfusion nearly 20% the loss cell that re-enters.
Disclosed in the prior art electroporation method is unsuitable for handling a large amount of samples can not use high or multiple charging.And this method is not suitable for successive or " mobile " electroporation chamber.Available electroporation chamber is designed to only use in static state.That is batch process sample.The continuous use of " static state " chamber causes this chamber overheated and increase the cracking of cell.And prior art can not be mixed the oxygen carrying capacity of the IHP of q.s with remarkable change blood in the pending cell of enough percentage ratio.In addition, the method for prior art needs purified equipment and is unsuitable for the erythrocyte of load patient during nursing.Therefore, this process is consuming time and be not suitable for commercial size.
People are needed to be to be used for simple at the erythrocyte wrapping biological active matter, method effectively and fast, and meanwhile, this method can also keep cell integrity and biological function.The potential therapeutic use of bio-modification blood cell show need be in original erythrocyte wrapping biological active matter, comprise more simple, the more effective and complete method of the allosteric effector of hemoglobin.
Many clinical cases will be benefited from this treatment, and the tissue that it can increase the oxygen that is attached to hemoglobin transports.For example, the major causes of death of the U.S. is a cardiovascular disease now.The serious symptoms and the condition of illness of many cardiovascular disease comprise congestive heart failure, myocardial infarction, and apoplexy, intermittent claudication and sicklemia, and these are to be caused by the oxygen supply deficiency in the liquid that soaks tissue.Similarly, hemorrhage, wound or the postoperative oxygen supply that can cause vitals that loses blood seriously reduce.Do not have oxygen, the tissue at the dirty far place of centroid, even heart self all can not produce enough energy to keep its normal function.The result of forfeiture oxygen is that tissue die and organ damage.
Although concentrating on for a long time, the attention of American public alleviates on the required preventive measure of heart disease, as exercise, and the alcohol consumption of suitable dietary habit and appropriateness, death still continues to take place with surprising rapidity.Because dead the forfeiture by oxygen causes that it causes disorganization and/or organ loss of function again conversely.A kind of life-threatening method of cardiovascular disease that alleviates is the Oxygenation that increases tissue under condition of serious stress of soil.Same method also be applicable to lost blood or chronic hypoxia disease imbalance as the people of congestive heart failure.
Benefiting from raising oxygen is anemia to the another kind of situation that tissue transports.The hospital patient of quite a few suffers from the anemia that caused by the quantity not sufficient of erythrocyte in the blood or hemoglobin or low " crit ".This cause they the tissue in insufficient Oxygenation and thereupon the generation complication.General, the doctor can temporarily correct this situation to the erythrocyte unit that patient imports encapsulation.
Strengthening blood oxygenation also can reduce the quantity of allos blood transfusion and can use autotransfusion under more situation.The existing method that is used for the treatment of the loss of anoxia or supplemental blood is the complete mankind's of input a blood.Three to 4,000,000 patients are arranged because operation or needs of medical treatment are accepted blood transfusion every year in the U.S. according to estimates.Having under the situation of more time, said method helps avoiding fully using blood donor or allos blood and replaces the use self-blood.
The amount of the blood that can extract out and store before the operation of being everlasting has limited autohemic use.General, surgical patient does not have time enough to contribute the blood of q.s before operation.An operation needs the available blood of several units (unit).Because each unit needs the time in several weeks and can not be less than before operation in time in two weeks to finish during donating blood, it often is impossible obtaining enough blood supplies.By handling self-blood with IHP, with needs still less blood and may avoid importing allos blood fully.
Because it is to be untreated erythrocytic 2-3 doubly that the erythrocyte that IHP handled transports the amount of oxygen, in many cases, the doctor need import the erythrocyte that still less unitary IHP handled.This make patient still less accept allos blood, reduced from the scope of blood donor's infective virus disease and made because transfusional immunologic function disorder drops to minimum.Importing more effective erythrocytic ability also is being good under the situation of patient blood volume surplus., when losing the oxygen transporting power, improve the Oxygenation ability of tissue of patient rapidly and can save life more under the serious situation at other.
Although it is conspicuous that the enhancing oxygen delivery has potential medical application to the method for organizing, also not can be used for increasing at present the method for the tissue conveying that is attached to the oxygen on the hemoglobin clinically.Use DPG or DPG precursor molecule, storage temporary transient, that improved oxygen in 6 to 12 hours appears in the newspapers in animal experiment.But DPG is synthetic in vivo regulates relative short biological half-life with it naturally and has limited the PO that organizes after the concentration of DPG and the improvement
2Persistent period, thereby limited its medical applications.
People are needed to be a kind of destruction under the erythrocytic situation, is used at the erythrocyte wrapping biological active matter, as the method simply, effectively and fast of IHP.
Summary of the invention
The present invention relates to a kind of method and apparatus that is used at different cell mass wrapping biological active matters.More particularly, the invention provides and a kind ofly can form one automatically, the electroporation chamber of supporting (self-contained) streaming (flow) equipment, it is used in erythrocyte parcel chemical compound or compositions (as phytic acid), thereby reduces hemoglobin is transported oxygen to the affinity and the enhancing of oxygen through ability from erythrocyte to tissue.Parcel is preferably realized by electroporation; But, can expect that other packaging method also can be used to implement the present invention.Method and apparatus of the present invention comprises that described electroporation chamber is equally applicable to the various bioactive substances of parcel in various cell masses.
Equipment of the present invention and method are applicable to mixes various bioactive substances in cell and lipid vesicle.Method of the present invention, equipment and chamber can be used for chemical compound or bioactive substance are incorporated into vesicle, and no matter this vesicle is generation genetic modification or natural.For example, equipment of the present invention, method and chamber can be used for IHP is introduced erythrocyte.
According to the present invention, in erythrocyte, wrap up phytic acid by electroporation and cause hemoglobin that the obvious reduction of the affinity of oxygen is not influenced life-span, ATP content, K
+The normal rheology of content or cell.In addition, except making O
2The binding curve outer Bohr effect that moves to right does not change yet.Reducing erythrocytic oxygen affinity has increased erythrocyte and has dissociated in conjunction with the ability of oxygen, thereby has improved the oxygen supply of tissue.Strengthen erythrocytic oxygen releasability and produce tangible physiological effect, as reducing of heart output, the increase of arteriovenous difference and the improvement of tissue oxygenation.
The improvement erythrocyte with improved oxygen releasability according to the present invention's preparation can be applied under the following situation: 1. under the condition of low oxygen partial pressure, as eminence; 2. when the oxygen exchange surface of pulmonary reduces, as situation about in edema due to disorder of QI, taking place; 3. when having the oxygen diffusional resistance that increases, as situation about in pneumonia or asthma, taking place in pulmonary; 4. when the oxygen transporting power of erythrocyte reduces, as situation about taking place in erythrocytopenia or the anemia maybe when using arteriovenous shunt; 5. treatment blood circulation disorder is as arteriosclerosis, thromboembolism, organ infraction, congestive heart failure, cardiac insufficiency or ischemia; 6. treat the situation of the high oxygen affinity of hemoglobin, as hemoglobin mutant, the chemical modification of-terminal amino acid or the enzyme defect in erythrocyte in the hemoglobin chain; 7. quicken detoxification processes by the supply that improves oxygen; 8. the oxygen affinity of the blood after reducing to store; Or the effectiveness of the various treatments of cancer of 9. improvement.
According to method and apparatus of the present invention, might produce the improvement erythrocyte that the oxygen economic utilization of the improvement of blood is made contributions.The erythrocyte of these improvement is that the electroporation by the erythrocyte cell membrane obtains to mix allosteric effector (as IHP).
(comprise the hemoglobin allosteric effector is wrapping in the erythrocyte) in the cell according to method of the present invention bioactive substance is incorporated into and carry out external by automatic flow electroporation equipment.Briefly, cell suspending liquid is introduced in the separation and washing roller (bowl) chamber of streaming parcel equipment.Cell is separated washing and being suspended in again in the solution of the bioactive substance of waiting to introduce cell from suspension.This suspension is incorporated into electroporation chamber's incubation then.Behind electroporation and the incubation, with cell washing with separate.Carry out impurity detects to confirm that all not wrapping biological active matters are removed not essentially.Then, preparation is used for storing or being reintroduced to the intravital cell of patient.
According to the present invention and with reference to embodiment preferred, in the patient body, extract blood out, separating red corpuscle from the blood of extracting out is by erythrocyte and the blood plasma that mixes allosteric effector improvement erythrocyte and rebuild improvement.In this way, preparation and store contains IHP-to improve erythrocytic blood is possible.
Equipment of the present invention provides a kind of improved method that is used at the cell wrapping biological active matter, thereby this equipment comprise a kind of supporting also be aseptic equipment (it can handle a large amount of cells in the time bar of a weak point), a kind of equipment with improved impurity detection, cooling and incubation element, in case a kind of fully automatically and sample be incorporated into the equipment that does not just need technical staff's ACTIVE CONTROL in the device.
Therefore, an object of the present invention is to provide a kind of automatically, the parcel equipment of continuous-flow.
Another purpose of the present invention provides a kind of automatic, the electroporation device of continuous-flow.
Another purpose of the present invention provides a kind of parcel equipment of continuous-flow, and this equipment can be produced the homogeneous group of load cell or vesicle.
Another object of the present invention provides a kind of electroporation device of continuous-flow, and this equipment can be produced the homogeneous group of load cell or vesicle.
Another object of the present invention provide a kind of in cell the aseptic and athermic method of wrapping biological active matter.
Another object of the present invention provides and a kind ofly causes stable cell or the vesicle method and apparatus of joint filling again behind electroporation, so that the cracking minimum of cell of improveing behind electroporation or vesicle.
Another object of the present invention provides a kind of streaming parcel equipment, and this equipment can be produced the not cell mass of the improvement of the bioactive substance of parcel of therefrom having removed all external sources.
Another object of the present invention provides a kind of electroporation device, and this equipment can be produced the not cell mass of the improvement of the bioactive substance of parcel of therefrom having removed all external sources.
Another object of the present invention provide a kind of make can be in cell or vesicle colony the method and apparatus of wrapping biological active matter continuously.
Another purpose of the present invention provides a kind of purpose of above-mentioned definition, method and apparatus of feature and advantage of can reaching in single circulation.
Another object of the present invention provides a kind of continuous flow type electroporation chamber.
Another object of the present invention provide a kind of improved and than existing methods availalbe more effective in cell the method for encapsulated bioactive substance.
Another purpose of the present invention provides a kind of disease and/or ill compositions that is applicable to that treatment is caused by Oxygenation shortage or reduction.
Read the detailed description of the following preferred embodiment of the invention in conjunction with the drawings with claims, other purpose of the present invention, feature and advantage can be apparent.
Brief Description Of Drawings
Fig. 1 is the sketch map of first embodiment of continuous flow type parcel equipment.
Fig. 2 is the sketch map of second embodiment of continuous flow type parcel equipment.
Fig. 3 is the vertical view of first embodiment that has the flow electroporation chamber of electrode.
Fig. 4 is the vertical view of second embodiment of electroded flow electroporation chamber not.
Fig. 5 is the side view of first embodiment of flow electroporation chamber.
Fig. 6 is the upward view of first embodiment of flow electroporation chamber.
Fig. 7 is the side view of the electrode that first embodiment adopted of flow electroporation chamber.
Fig. 8 is the front view of Fig. 7 electrode.
Fig. 9 is the exploded perspective illustration of second embodiment of flow electroporation chamber.
Figure 10 is the perspective view of the flow electroporation chamber after Fig. 9 assembles.
Figure 11 be under static or flox condition, the comparison diagram of different field intensity effects, it is a benchmark with the percent of the erythrocytic Oxygenation of parcel IHP.
Figure 12 be under static or flox condition, the comparison chart of different field intensity effects, it is with the erythrocytic P of parcel IHP
50Value is benchmark.
Figure 13 is under static and mobile condition, under different field intensity, the comparison chart of the erythrocyte survival rate of experience electroporation.
Figure 14 is the front view according to the supporting member of the 3rd embodiment of electroporation chamber of the present invention.
Figure 15 is the cross-sectional view strength that cuts open Figure 14 supporting member of getting along the straight line 15-15 of Figure 14.
Figure 16 is the enlarged drawing of the circle 16 indications part of Figure 15.
Figure 17 is the exploded perspective illustration according to the 3rd embodiment of electroporation chamber of the present invention and the support column that is installed to thereof.
Figure 18 is the perspective view that shows the electroporation chamber of being installed to the Figure 17 on the support column.
Figure 19 is the anterior elevational view of being installed to the 3rd embodiment of electroporation chamber on the support column.
Figure 20 is the electroporation chamber of Figure 19 and the perspective cut-away schematic view of support column.
Figure 21 is the sketch map of the supporting electroporation device of an electroporation chamber that comprises Figure 14-20.
Figure 22 is the accompanying drawing that shows the resistance of several IHP solution.
Figure 23 is the sketch map of the 3rd embodiment of continuous flow type parcel equipment.
Figure 24 is the cutaway view of a cell washing equipment.
Figure 25 is the side view of cell plates 526, and it shows the floor (ridge) that limits labyrinth (labyrinth) and shows cell suspending liquid circulation pipeline.
Figure 26 is the cutaway view of second embodiment of cell washing equipment.
Figure 27 is the side sectional view of elastic chamber.
Figure 28 is the figure that shows representative electroporation voltage.
Detailed Description Of The Invention
The invention provides a kind of for wrapping up such as the other structure compound of phytic acid or automatic, the supporting streaming equipment of composition such as erythrocytic cell. In a specific embodiment, equipment of the present invention combines a blood plasma and removes the feature of equipment and a flow electroporation equipment, thereby has formed a kind of automatic, supporting flow electroporation equipment. The present invention also comprises a kind of new flow electroporation chamber, and it can be so that except using this electroporation chamber the quiescent conditions under the condition that flows. Can expect that the method and apparatus that comprises electroporation chamber of the present invention can be used at the various bioactivators of various cell colony parcels.
In addition, the invention provides a group improvement cell, these cells have to be needed or benefits from the particularly advantageous physical characteristic of disease that oxygen transports increase in the tissue treatment. According to method of the present invention, can obtain the red blood cell homogeneous group of load IHP, impurity also reduces to some extent simultaneously, and the characteristic of cracking behind the parcel also descends to some extent. The red blood cell of processing demonstrates in blood circulation to have the normal life-span. Adopt the present invention, the patient's of a needs treatment red blood cell can and turn back in patient's the blood circulation by rapid load.
Relevant International Application PCT/the U.S.94/03189 that submits on March 23rd, 1994 is incorporated herein by reference, and this application is the continuity of the U. S. application submitted on March 23rd, 1993 number 035,467.
With reference to the preferred using method of present device, the method for operating of equipment of the present invention is described below, i.e. the method for parcel hemoglobin allosteric effector in red blood cell. Phytic acid is preferred allosteric effector used in the present invention. Also can use other sugar phosphate, such as IP5, inositol tetrakisphosphate, inositoltriphosphoric acid, inositol diphosphate and two phosphatidylinosital biphosphate. Other suitable allosteric effector comprises polyphosphate, such as the nucleotides triphosphoric acid, and nucleotides diphosphonic acid, nucleotide monophosphate and pure phosphate. In the situation of certain sudden change (such as " Zurich " hemoglobin) of hemoglobin, organic anion such as polycarboxylic acid, can be used as allosteric effector. At last, can adopt inorganic anion, such as six cyano group ferrates, phosphate or chloride are made allosteric effector.
According to load of the present invention the red blood cell of phytic acid can be used for the treatment of the disease and morbid state of a very wide kind. Can be with the red blood cell of the load IHP that makes according to the present invention to the patient's administration that is just suffering from a heart complaint, thus improve oxygen to the transmission of ischemic heart tissue and reduce simultaneously heart output. The red blood cell of the load IHP that makes according to the present invention also can be used for the treatment of any one ischemic situation, this situation comprises " hemorrhagic " anaemia, operation complication, apoplexy, diabetes, sickle cell disease, burn, Charcot's syndrome, wind-puff, hypothermia, peripheral vascular disease, congestive heart failure, angina, instantaneous ischaemic disease, scatters intravascular clotting, adult respiratory distress syndrome (ARDS) and cystic fibrosis, but is not limited thereto. Hereinafter provided the detailed description of the medical use of the composition for preparing according to method of the present invention. Continuous flow type parcel equipment
Below with reference to the best using method of equipment of the present invention, the allosteric effector that namely wraps up hemoglobin by electroporation in red blood cell is described the method for operating of present device. Should recognize and to transform this equipment so that it adapts to other cell colony or vesicle and other bioactivator. Can transform this equipment in addition so that utilization other packaging method except electroporation.
In brief, be that a blood sample is incorporated in the continuous flow type parcel equipment according to method of the present invention. If collecting erythrocytic words, blood or can directly extract out in the patient body or can extract out in advance. At the beginning blood is divided into red blood cell, blood plasma and white blood cell and waste product. Waste product is included in diluent and the various blood dissolves thing that retains in after centrifugal in the supernatant. They are stored in the refuse storage tank of this equipment. When red blood cell when material to be wrapped mixes, blood plasma and white blood cell also are retained in the storage tank of this system. Then erythrocytic suspension is carried out electroporation. After the electroporation, can make again this cell of incubation under the condition of joint filling of red blood cell. Process then and wash these cells to remove external source, not wrapped bioactivator. After this cell was processed, containing the red blood cell that is wrapped material can optionally rebuild with blood plasma and white blood cell. The blood that rebuilds directly can be turned back in the patient body or after storing then and re-use. Although this process is described with discontinuous step, be continuous on this process nature.
Described first embodiment of the present invention with reference to Fig. 1, Fig. 1 is with the structure of schematic view illustrating continuous flow type parcel of the present invention equipment.
According to the present invention, whole blood are sent in the electric perforating system 5 at charging aperture 11.Blood sample can be optionally directly extracted out in the patient body and is entered electric perforating system 5 or can extract blood out in the time more early and stored earlier before drawing-in system 5.Opening valve 12 makes this sample enter system 5.Meanwhile, open valve 25 and open pump 22 and come into operation so that from anticoagulant storage tank 27, supply with anticoagulant.Although can adopt other anticoagulant, a kind of suitable anticoagulant is a heparin.Preferred anticoagulant is ACD.Valve 15 and 36 also is opened and opens pump 40.The mixture of anticoagulant and whole blood passes the mobile piezometry system 19 of this equipment by filter 18 and monitoring, and collects this mixture at one when opening in the blood separation that started in the pump 40 and the washing roller 44.When being full of erythrocyte in blood separation and the washing roller 44, pick off can be indicated.When it is filled, stop the supply of blood.Can pass through a plasma removing equipment, such as (the plasma removing equipment of MA) being produced is realized the step relevant with separating of blood constituent for Haemonetices company, Braintree by Haemonetices company.
Just as explained above, when pump 40 rotated in a clockwise direction, the suspension that starts blood separation and washing roller 44 and centrifugal anticoagulant and whole blood was with separated plasma, leukocyte, erythrocyte and refuse.Open valve 87, thereby blood plasma and leukocyte are sent in the blood plasma storage tank 89.
Optionally and depend on that cell mass by this device processes, washing are retained in the cell in blood separation and the washing roller 44. Open valve 33,15 and 36, contain erythrocytic blood separation and wash in the roller 44 thereby will send into from the brine buffer solution of diluent storage tank 30.Pump 40 is still in running.Wash then and centrifugal erythrocyte.Although other physiology isotonic buffer solution also can be as diluent and washing erythrocyte, preferred brine buffer solution is 0.9% sodium chloride solution.In washing process, open valve 54, thereby refuse is sent in the refuse storage tank 57.Refuse is stored in again in the refuse storage tank, and this erythrocyte is retained in blood separation and washs in the roller 44.Words if necessary repeat this washing step.
By the experiment of under the multiple variation of pulse length and field intensity, carrying out, have been found that rectangular pulse can cause people's the effect of erythrocyte parcel IHP bad.It is seemingly inadequate that the macropore that produces on cell membrane enters erythrocyte to IHP outside the born of the same parents.This shows after the hole produces, exist one than IHP to the more complicated process of cellular invasion.Should be understood that electric pulse must realize two tasks.First task is to produce the hole on cell membrane, and second task is that IHP passes above-mentioned hole and enters erythrocytic electrophoresis fast and move.These can be by exponential pulse (1.5 to 1.75 kV/cm in low voltage, 5ms) adopt high-tension rectangular pulse (2.13 kV/cm afterwards immediately, 2 ms) realize, thereby cause the parcel of IHP in erythrocyte to improve 50% than the parcel of common exponential pulse mode.Under the electroporation threshold value, exponential pulse self is suitable.By adopting exponential pulse can realize this two tasks most effectively, promptly the hole forms with electrophoresis and moves.Another specific embodiment be earlier with cellular exposure under a high-voltage rectangular pulse, be exposed to then and a series ofly tend to order about IHP and enter under the erythrocytic action of low-voltage pulse, thereby cause the more effective IHP load that enters cell.In the middle of using, will pass the cellular exposure of electroporation chamber of the present invention under the series of pulses string.Train of pulse is between 80 and 512 pulses, and wherein preferred pulse number is 312 pulses.Change polarity then, and apply second train of pulse to cell.When applying the 3rd cover pulse, another changes polarity.For given cell, when it passes the length of electroporation chamber, will apply three to five trains of pulse, wherein the reverse of polarity between each train of pulse.
Erythrocyte turns to pump 40 through after separating, closes pump 22, valve- off 12,15,33,36,25,87 and 54, and open valve 97 and 64.With pump erythrocyte in the suction cooling worm 68, is being extracted out IHP solution from blood separation and washing roller 44 from IHP storage tank 50.Pipeline mixture of red blood cell and IHP solution at the equipment that is arranged in 67 places, meeting point make it pass through cooling worm 68 with pump then.In an embodiment preferred of the present invention and as hereinafter explain in detail, before IHP solution and erythrocyte are sent into cooling worm 68 also can separate with wash roller 44 in mix them.
The preferred concentration of IHP is between about 10mMol and 100mMol in the solution, and one of them more preferred concentration is approximately 22.5 to 50mMol, and most preferred concentration is 35mMol.The preferred concentration of KCl is between about 10mM and 5mM in the IHP solution.MgCl
2Preferred concentration be between about 2mM and 0.5mM.The preferred concentration of sucrose is between about 67.5mM and 270mM in the IHP solution.Should recognize and also can use other sugar or polymer to come place of sucrose.
Employed in the present invention solution is the resistance enhance fluid.Notice that this IHP solution should have high resistivity and minimum electrolyte is important.The IHP of Aldrich chemical company or Matrea chemical company does not contain the electrolyte of any sodium chloride and minimum other.Therefore above-mentioned IHP does not have significantly to reduce the resistivity of this solution.The milliosmolarity of this solution should be between about 300 and 500.Its resistivity should be between about 87 Ω cm and 185 Ω cm.Its conductivity should be between about 4 to 8nS/cm.Its actual salinity should between about 4 and 9ppt between, and the NaCl equivalent should between about 4.5 and 9.0ppt between.
Preferably between about 30 and 80, wherein most preferred hematocrit is approximately 40 to the hematocrit of suspension.From erythrocyte response after measured in immobilized cell (its spacing is 0.4cm) high pressure should not surpass 800 volts, this is corresponding to 2kV/cm.For flow cell, they have the gap of 0.3cm, so the voltage in the cell both sides will be restricted to 600 volts (+/-300V).After tested many different electroporation fluid compositions.Table A has been listed six samples and characteristic thereof.Solution under the E hurdle is preferred electroporation solution.Pump 40 is used for extracting out erythrocyte and IHP solution and can regulates, thereby can be predetermined the final hematocrit that enters cooling worm 68.
Table A
A 10 mmol KCl, 2 mmol MgCl
2, 270 mmol sucrose, 35 mmol
| A a | B b | C c | D d | E e | CBR f | |
| Conductivity (nS/cm) | 2.78 | 8.92 | 11.2 | 8.67 | 7.07 | 16.8 |
| Resistivity (ohm-cm) | ?361 | ?112 | ?89.1 | ?115 | ?134 | ?59.3 |
| ?mOsm | ?379 | ?472 | ?408 | ?397 | ?314 | ?452 |
| Actual salinity (ppt) | ?1.54 | ?5.43 | 6.91 | 5.24 | ?4.45 | 11 |
| NaCl equivalent (ppt) | ?1.71 | ?5.43 | 6.76 | 5.25 | ?4.59 | 10.2 |
| pH | ?7.39 | ?7.346 | 7.185 | 7.316 | 7.4 | 7.42 |
| Phytic acid (IHP) | Aldric h IHP | Sigma IHP | Aldric h IHP | Aldric h IHP | Matrey a IHP | Sigma IHP |
IHP b except the potassium salt of IHP, all the other and the Dulbecco s 125 mmol Su d Dulbecco s phosphate-buffered saline of all identical c Iscove of A s improvement, 125 mmol sucrose e, 5 mmol KCl, 1 mmol MgCl
2, 135 mmol sucrose f, 33 mmol K
2HPO
4, 7.0 mmol NaH
2PO
4, 30.6 mmol KCl, 6.4
Mmol NaCl, 7.3 mmol sucrose, 5.0 mmol ATP
After mixing, make erythrocyte-IHP suspension by cooling worm 68 with pump.Can adopt water-bath or adopt a cooling system to cool off based on heat-electricity.For example, cooling worm 68 is immersed in the cooling bath in the cooling reservoir 69.When erythrocyte-IHP suspension passes through cooling worm 68, the temperature of this suspension is cooled between about 1 ℃ and 12 ℃, preferably approximately be 4 ℃.The cooling erythrocyte has been guaranteed retaining of the hole that produces on the cell membrane in electroporation process.By improving the surface area that this sample contacts with cooling element, adopt cooling worm to help quickening cooling.This cooling worm can surround with a heat-electric heating pump not essentially.
May before electroporation, heat cell suspending liquid under some occasion.In this case, can replace cooling worm 68 with heating coil.The patient maximum temperature of erythrocyte is approximately 37 ℃.
The electrical heating pump is by extracting heat energy to reduce the temperature of himself in a special area, and (heatsink) work comes in the zone for " heat sink " that then heat energy entered again a higher temperature.In cold meeting point, when the low-lying level of electronics from p-type semiconductor element changes energy level higher on the n-type semiconductor element over to, electronics will absorb energy.Power supply provides mobile electron to pass the energy of this system.In the meeting point of heat, when electronics from a high level element (n-type) when an element than low-lying level (p-type) is mobile, energy is discharged in the heat abstractor.
Thermoelement all is solid-state and does not have mobile machine components or also do not resemble needs working fluid steam-recycle unit.But, thermoelectric heatpump can realize with based on the both vapor compression or the same refrigerating function of Absorption Refrigerator of freon.The thermoelectric heatpump high safety is reliable, and size and volume are little, and cost is low, and is in light weight, and it is more safer and can manage precise dose control in fact than many other cooling devices in fact.
The preferred thermoelectric heatpump of Shi Yonging is that MELCOR material electronics Products Co., Ltd by the Trenton of New Jersey is produced in the present invention.Thermocouple is made by the efficient crystallization semi-conducting material.This semi-conducting material is a Tellurobismuthite., and the quaternary alloy of a kind of bismuth, tellurium, selenium and antimony has mixed impurity in this alloy and the process processing is directed polycrystalline quasiconductor with generating feature.Be connected in series on the electricity and full-boiled process on the galvanic couple that is connected in parallel be integrated into assembly.This component package between gold-plated earthenware slab, thereby best electric insulation and conduction of heat is provided under the high mechanical properties in compression.This assembly can be installed in parallel to improve heat-transfer effect or can pile up to realize high temperature difference by plural serial stage.The transmission electric current passes heat pump and can cause the temperature difference of striding thermocouple, and its highest rated value is 70 ℃ or higher.
After cooling, erythrocyte-IHP suspension enters electroporation chamber 72, in this chamber the electric pulse from pulse generator 75 is applied on erythrocyte-IHP suspension, thereby causes at the inner opening that forms of erythrocytic cell membrane.Optionally, when being full of erythrocyte-IHP suspension in the chamber 72, an automatic checkout system can unbalanced pulse generator 75.When in chamber 72, being full of the cell that is not wrapped, just apply electric pulse to this suspension.When this equipment be operating as static state the time, promptly when the cell of handling single, discontinuous batch, can adopt conventional electroporation chamber.In an embodiment preferred of the present invention, adopted a kind of flow electroporation chamber.In one embodiment, flow electroporation chamber 72 be constitute by pure polrvinyl chloride and contain two opposite electrodes, its space length is at a distance of 7mm.Interelectrode distance is along with flow and field intensity and change.This flow electroporation chamber 72 is preferably easy-to-handle.Electroporation chamber also can be made by polysolfone, and it is preferred for certain sterilization process, such as autoclaving.The structure of this flow electroporation chamber and the detailed description of structure are provided below.
Erythrocyte-IHP suspension passes between two electrodes of electroporation chamber 72.When untreated cell suspending liquid inlet chamber 72, continue 1 to 4 millisecond with producing one 1 field intensity and this field intensity, and be preferably 2 milliseconds for 1.8 milliliters chamber to 3KV/cm.The IHP-erythrocyte of unit volume preferably experiences three high-voltage pulses, and wherein the field intensity of each pulse is approximately 2600 to 3200V/cm.Pulse current passes cell membrane and makes the cell membrane electrical breakdown, produces micropore on film.IHP is diffused in the cell by these micropores then.
After the electroporation, erythrocyte-IHP suspension enters incubation chamber 78, suspension incubation at room temperature in this chamber, and the incubation time, the preferred incubation time was 30 to 60 minutes between about 15 minutes to 120 minutes.Erythrocyte-IHP suspension can optionally be approximately about 5 minutes of 37 ℃ of following incubations in temperature, and at room temperature is at least 15 minutes.The incubation chamber can optionally be surrounded by heater 80.For example, heater can be water-bath or thermoelectric heatpump.
Table B
Again joint filling buffer solution
| I. form | |
| Sodium chloride and potassium chloride sodium phosphate magnesium sulfate dextrose adenine inositol benzyl penicillin chloramphenicol | 150mMol 8mMol 6mMol 2mMol |
| II. additive | |
| BSA calcium chloride | 3.5％ 2mMol |
In the preferred embodiment of the invention, do not use joint filling buffer solution again.
After the incubation, open valve 51, primer pump 40, erythrocyte-IHP suspension turn back to from incubation chamber 78 and separate and washing roller 44.By centrifugal excessive IHP solution is removed from the erythrocyte suspension.Useless IHP solution imports waste water storage tank 57. Opening valve 33,15 and 36 then allows the diluent of 1 volume enter blood separation and washing roller 44.The operable diluent of the present invention is shown in table C.
Table C
Dilution buffer
| I. form | |
| Sodium chloride magnesium chloride calcium chloride magnesium sulfate glucose 0.1% penicillin, (interchangeable) 0.1% streptomycin, (interchangeable) | 0.9％ 2mM 2mM 2mMol 10mMol 0.1％ 0.1％ |
Centrifugal then erythrocyte-IHP suspension enters useless storage tank 57 by valve 54 with supernatant, and erythrocyte is then stayed blood separation and washed in the roller 44.From diluent storage tank 30 saliniferous buffer solution is joined the erythrocyte of improvement.Wash this cell and in centrifugal back abandoning supernatant.Can repeat this washing process if desired.
When removing refuse from separation and washing roller, thereby this refuse can optionally any free IHP confirm that IHP external source, that wrap up does not remove in the waste liquid from the erythrocyte of improveing to detect by an impurity detector 46.The impurity detector system depends on the optical change of washing buffer solution.The erythrocyte of improvement through washing and centrifugal after, supernatant passed through impurity detector 64 earlier before waste water storage tank 57 storage.If IHP external source, that wrap up does not remain in the washing buffer solution, the solution that then discards can be muddy.Turbidity is because the reaction of a component calcium of IHP and washing buffer solution causes.Impurity detector 46 is used the optical detection system.Preferably, light source is LED, and detector is a photodiode.The voltage of photodiode shows the quantity of IHP in the wash solution.Impurity detector 46 is selectable.
After the washing, IHP-erythrocyte product can optionally rebuild with the blood plasma and the leukocyte that are retained in the storage tank 89.The erythrocyte of handling can be collected in re-injection (reinjection) bag or in anticorrosion medium or in patient's self blood plasma.
The erythrocyte of the load IHP that obtains can directly turn back in the patient body and maybe can store for future use.IHP in the normal storage life in the erythrocyte can not discharge.
An embodiment preferred of the present invention is described with reference to Fig. 2, and this figure has schematically illustrated the structure of the electroporation device of continuous-flow of the present invention.And the operational approach of this equipment is described with reference to its preferred using method (that is, wrapping up the method for hemoglobin allosteric effector by electroporation in erythrocyte).Should recognize and to transform cell mass or vesicle and other bioactive substance to this equipment to be applicable to other.Can transform to be suitable for other packaging method this equipment in addition.
According to the present invention, the sample of whole blood is sent in the electric perforating system 10 at inlet 11.Open valve 12 sample is sent into system 10.Simultaneously, open valve 25 and primer pump 22 so that from anticoagulant storage tank 27, introduce anticoagulant.Open valve 15 and 36 and primer pump 40.
The mixture of anticoagulant and whole blood is by filter 18 and piezometry system 19, collects in blood separation and washing roller 44, and wherein this roller also is activated in the time of unlatching pump 40.Pick off can show when being full of erythrocyte in blood separation and the washing roller 44.
When pump 40 rotated along clockwise direction, the suspension that starts blood separation and washing roller 44 and centrifugal anticoagulant and whole blood was with separated plasma, leukocyte, erythrocyte and refuse.Opening valve 87 allows blood plasma and leukocyte enter blood plasma storage tank 89.
Optionally will remain in then separate with washing roller 44 in cell washing and centrifugal. Open valve 33,35, thereby 15 and 36 allow brine buffer solution from diluent storage tank 30 enter to contain in erythrocytic blood separation and the washing roller 44.Valve-off 12, pump 40 is kept rotation.
In washing process, open valve 54 and allow waste water enter waste water tank 57.Waste water is stored in again in the waste water tank 57, and erythrocyte is stayed blood separation and washed in the roller 44.If needed, repeat this washing process.Can optionally be installed in impurity detector between separation and washing roller 44 and the waste water tank 57 with the control washing process.
The erythrocyte after separating with pump 40 counter-rotatings, is closed pump 22, and valve- off 12,15,33,35,36,25,87 and 54 is opened valve 97.If washed cell is closed pump 22 in advance, valve 12 and 25 close.Extraction IHP solution is sent into and is contained erythrocytic separation and wash in the roller 44 from IHP storage tank 50.In this roller, erythrocyte mixes the formation suspension with IHP.
To between the 100mMol, between the 35mMol, most preferred concentration is 35mMol to preferred concentration to the preferred concentration of IHP at about 23mMol at about 10mMol in the solution.Preferred IHP solution contains the following compounds of following concentration:
The neutral IHP of 35mM (six sodium salts) (Matreya chemical company)
5mM?KCl
1.0mM?MgCl
2
135mM sucrose
The IHP of Aldrich chemical company does not contain other electrolyte of any sodium chloride and minimum, does not therefore have significantly to reduce the resistivity of this solution.Should recognize that the component that can use other high-resistance solution and solution in the present invention is not crucial.As long as the Penetration Signature of solution is not destroyed cell, as erythrocyte, and the resistivity height of solution, it just is suitable for using in the present invention.Measured the resistivity of several compositionss, the result as shown in figure 22." CBR fluid " listed in Table A.
Preferably between about 30 to 60, most preferred hematocrit is approximately 40 to the hematocrit of suspension.Pump 40 is designed to extracts erythrocyte and IHP solution out and can adjust so that can pre-determine the final hematocrit that enters in the cooling worm 68.
After erythrocyte and the combination of IHP solution, the pump 40 that reverses once more, valve-off 97 is opened valve 64.Then with in erythrocyte-IHP suspension suction thermoelectric-cooled coil 68.A kind of blood bag from blood warming apparatus (blood bag) is for example by the Baxter health care Fenwal that company produced
The blood bag that provides in the blood warming apparatus can be used as cooling worm 68.When erythrocyte-IHP suspension when being arranged in the cooling worm 68 of cooling reservoir 69, cooling suspension to temperature approximately between 1 ℃ to 12 ℃, preferably approximately be 4 ℃.Can optionally join the equipment place that is between cooling worm 68 and cooling reservoir 69 and the electroporation chamber to a pump, thereby guarantee the volume fluctuation that constant flow velocity and compensation take place when cooling worm 68 is full of.
Optionally can save pre-cooled step and after mixing, erythrocyte-IHP suspension directly be introduced electroporation chamber 72.In this case, can wrap up from continuous flow type and save cooling worm 68 and cooling reservoir 69 equipment 10.Be in 4 ℃ if the temperature of electroporation chamber is enough cold to keep this cell suspending liquid, before electroporation, can cool off.
After the cooling, erythrocyte-IHP suspension enters electroporation chamber 72.The temperature maintenance of chamber 72 is at about 4 ℃.When erythrocyte-IHP suspension when the flow electroporation chamber 72, cause on the suspension on the erythrocytic cell membrane and produce the hole thereby the electric pulse from pulse generator 75 is added to.
Erythrocyte-IHP suspension passes through between two electrodes of electroporation chamber 72.Fig. 3 has described this electroporation chamber to Figure 10.In the preferred embodiment of the invention, when the suspension inlet chamber 72 of untreated cell, the IHP-erythrocyte suspension of unit volume will experience about three high-voltage pulses or train of pulse, and wherein each pulsed field is powerful is about 2600 to 3200V/cm.Confirmed that using pulse train to replace individual pulse to transport IHP to erythrocyte can be more effective for IHP is introduced blood.Umber of pulse best in each train of pulse is between about 10 pulse to 512 pulses, and preferred number is about 312 pulses.The polarity that changes electric field between pulse or train of pulse also is good.Demonstrate two representative trains of pulse among Figure 28.The electric charge that produces in the cell membrane both sides causes cell membrane to puncture, thereby produces the hole on cell membrane.IHP is diffused in the cell by these holes then.In addition, although not necessarily need following hypothesis, people be sure of that IHP is actually and enter cell under effect of electric field.
In electroporation process, produce one 1 to 3KV/cm electric field and keep 1 to 4 millisecond.Preferred pulse length is 3 to 4 milliseconds, and the pulse length of best choosing is 2 milliseconds.Pulse length or burst length are defined as 1/e.Under the flow velocity of about 10.6 ml/min, preferred umber of pulse is 3 to 5, and preferred pulse rate is 0.29Hz.Field intensity is defined as the ratio of two interelectrode voltages and distance.Two distance between electrodes are centimetre being that unit is measured.Preferred electrical quantity is as follows:
Pulse length or burst length=1.5～2.5 millisecond
Field intensity=2.7～3KV/cm
Electroporation chamber optionally can be a pick off, and it can monitor the resistivity of the cell solution that passes electroporation chamber in a sense.When the change in resistance of cell solution, exist the feedback circuit that to adjust the cell pulse to keep best electroporation efficiency.For example, when electroporation blood in IHP solution, different blood samples can have different resistivity.By the resistivity of monitoring of blood, can on basis, apply best pulse strength and burst length to resistivity measurement.In addition, if electroporation chamber introduces bubble, because the variation of resistivity, this feedback circuit can detect the existence of bubble, and closes pulse and leave this chamber up to bubble.
Behind the electroporation, erythrocyte-IHP suspension enters incubation chamber 78, in this chamber under room temperature about 10 minutes to 120 minutes of incubation suspension, the wherein preferred incubation time is 30 minutes.Erythrocyte-IHP suspension can be optionally about 5 minutes of about 37 ℃ of following incubations, incubation 15 minutes at least at room temperature.Incubation chamber 78 can be surrounded by heater 80.When enforcement is of the present invention, can use any heater.Preferred heater 80 is water-bath or thermoelectric heatpump.
Behind the incubation, when opening valve 51 and primer pump 40, erythrocyte-IHP suspension returns blood separation and washs in the roller 44.By centrifugal excessive IHP solution is separated from the erythrocyte suspension.Useless IHP solution imports waste water storage tank 57.Open then valve 33,37,15 and 36 allow from a volume of storage tank 31 in back (post) wash solution enters blood separation and washing roller 44.In the preferred embodiment of the invention, contain 0.9%NaCl solution at after scouring solution, this solution comprises 2.0mM CaCl
2With 2.0mM MgCl
2Can use any normal saline.
After being added in after scouring solution, centrifugal erythrocyte-IHP suspension, and supernatant is entered in the refuse storage tank 57 by valve 54 is stayed erythrocyte in blood separation and the washing roller 44 simultaneously.Repeat this washing process up to removing all not IHP of parcel.
When refuse from separate with washing roller 44 when removing, it can be optionally by an impurity detector 46 detecting any free IHP in this waste liquid, thereby confirm external source, the IHP of parcel does not remove from the erythrocyte of improvement.Impurity detector is selectable.
After the washing, the erythrocyte that contains IHP can rebuild with the blood plasma and the leukocyte that are stored in the storage tank 89.Primer pump 40 and open valve 87,36 and 92.The improvement erythrocyte, blood plasma and leukocyte with being pumped into storage tank 96.A filter can be installed to remove any aggregation or other impurity that comes from the improvement blood that rebuilds between storage tank 96 and valve 92.
The erythrocyte of the load IHP that obtains according to method of the present invention can directly be sent back in the patient body and maybe this erythrocyte be stored for future use.In the normal storage life, the IHP in the erythrocyte can not discharge.
Can expect and to improve to be applicable to other packaging method continuous flow type parcel equipment of the present invention.
And, can expect and can improve so that handle various cell masses this continuous flow type parcel equipment.And this equipment can also be used at artificial vesicle wrapping biological active matter.
Can expect that also continuous flow type parcel equipment of the present invention can be used to wrap up the bioactive substance of wide range.Flow electroporation chamber
In electroporation process, the speed of mixing IHP is linearly dependent on the voltage that is added on the cell.In general, voltage is high more, and the IHP of parcel is many more.But the cracking of cell also can increase and the survival of cell reduces.The efficient of electric perforating system can be judged by the survival of cell behind the electroporation.The amplitude of electric pulse and persistent period cause the electrical breakdown of cell membrane and are being parallel to upward generation micropore of the polar cap of electric field (pole caps).Therefore, the factor that need consider at electric perforating system of design comprises field intensity, pulse length and umber of pulse.
The shape of an ideal electroporation target resembles a ball, so that its direction does not influence the efficient of the electric field that is applied.When target when being spheric, adopt the individual pulse of a field intensity more than threshold values can puncture 100% target.Erythrocyte is discoidal.Because their shape and direction in electroporation chamber, so in the individual pulse process, have only about 40% cell by electrical breakdown.Other 60% in order to puncture, can increase field intensity.This will increase erythrocytic stressed with the direction over against electric field, and cause the survival rate of cell lower.
In order when reducing lysis and dead the generation, to reach more effective parcel, developed a kind of flow electroporation chamber that uses short multistage pulses of persistent period.Under stable through-rate and stable field voltage, can measure a plurality of pulses and will mix the IHP of maximum under the complete cracking minimum 2 hours to 24 hours.The multiple-pulse system can improve the survival rate of cell under the situation that does not increase field intensity.When using the multiple-pulse system, harsh the direction of cell does not resemble when using monopulse system.Lower field intensity is to the erythrocyte milder.Be easier to each cell of electrical breakdown in the multiple-pulse system, this is because the flow velocity of erythrocyte by this chamber and synchronous (timing) between the electroporation pulse, and the direction of cell do not resemble in the individual pulse system key.Compare with the individual pulse method, a plurality of Pulse Electric perforation systems that flow have also increased erythrocytic short-term and secular survival rate.
Figure 11 to 13 has shown that under static or flox condition different field intensity is to the influence of the erythrocyte Oxygenation percent of parcel IHP in certain oxygen pressure scope; Erythrocyte P to parcel IHP
50The influence of value (the IHP solution to two concentration contrasts); And to the influence of erythrocyte survival rate of experience electroporation.All data are acquisition in 24 hours behind electroporation.The result shows that multiple-pulse produces best parcel result under low relatively field intensity.
One in electroporation process preferred refrigerative electroporation chamber under stationary temperature, preserve erythrocyte, thereby increase its survival rate.This can realize by drain the too much heat that is produced by electric pulse in electroporation process.Too much heat can or cool off whole flow electroporation chamber and get rid of by cooling electrode.According to one embodiment of the invention, electrode carries out self cooling.
In electroporation process, with pump blood is sent into electroporation chamber by inlet, and they will experience a series of electric pulses when erythrocyte passes this chamber.Opposite side erythrocyte in this chamber flows out.This chamber can be made by any one insulant, comprises pottery, politef, and lucite, glass, plastics, silicon, rubber or other synthetic material are confined to this but have more than.This chamber preferably is made up of glass or polysulfones.No matter the composition of this chamber how, the inner surface of chamber should be slick to reduce the logical out of date turbulent flow of fluid.The shell of this chamber should be nonconducting and be biologically inert.In commerce is used, can expect that this chamber should be tractable.
In an embodiment preferred of the present invention, forming the electrode of the part of electroporation device can be made by the raw material (stock material) of the hollow of any one conduction or heat conduction, comprise pyrite, rustless steel, gold-plated rustless steel, gold-plated glass, gold-plated plastics or contain the metal of plastics, but be not limited thereto.The surface of electrode is preferably gold-plated.Gold-plated playing eliminated Oxidation and minimizing hemoglobin and other cell particle in the accumulative effect of electrode.The surface of electrode should be slick.
Electrode can be a hollow, thereby can cool off or electrode can be solid-state by liquid or gas, thereby can adopt the conduction of thermoelectric or any other type to cool off.Except cooling electrode, also can realize cooling by cooling electroporation chamber self.
Electroporation chamber is preferably tractable.The detailed description of three embodiments of electroporation chamber of the present invention hereinafter is provided.
In one embodiment, flow electroporation chamber is made of pure polrvinyl chloride, and contains two comparative electrodes that spacing is about 7 millimeters.This electroporation chamber is the improvement of a chamber obtaining from the BTX electronics corporation that adds the inferior Santiago of livre Buddhist nun.But when this electroporation chamber of continuous use, its is understood overheated and reduces in time through the cell branch survival rate that this device processes is crossed.The problems of excessive heat that takes place when using this equipment in order to correct in the continuous-flow mode has designed a kind of continuous flow type electroporation chamber.The detailed description of continuous flow type electroporation chamber structure hereinafter is provided.
Fig. 3 provides an embodiment of flow electroporation chamber 72 of the present invention to Fig. 8.As can be seen from Figure 3, flow electroporation chamber 72 comprises the shell 100 that contains two electrodes 102, and these two electrodes are inserted in the relative both sides of the shell 100 of electroporation chamber 72.Shell 100 comprises an access road 104 that is positioned at an end and the exit passageway 106 that is positioned at the other end.Access road 104 and exit passageway 106 comprise connection tube 108 and 109 respectively, and wherein preferred Luer class is inserted accessory (male Luervariety ).Connection tube 108 and 109 be hollow and form access road 104 and the exit passageway 106 enter electroporation chamber 72 inside.
From Figure 4 and 5 as can be seen, inner room 110 has occupied shell 100 most length and its size is as the criterion so that two electrodes 102 to be installed.Inner room 110 comprises the inclined surface that is used for installing electrodes 102 inner edges.Therefore form inner room 110 by the inner surface of electrode 102 and the inner surface of shell 100.Inner room 110 joins with access road 104 and exit passageway 106.
From Fig. 7 and 8 as can be seen, Fig. 3 is made of flat, elongated hollow shell to the electrode 102 of the electroporation chamber 72 of Fig. 6.Electrode 102 comprises cooling inlet 112 and the coolant outlet 114 that is positioned at end.As mentioned above, the inclined surface 111 of being close to shell 100 among the rear surface of electrode 102 or Fig. 7 towards the surface on a left side is installed.
In order in electroporation process, to keep relative stationary temperature, with pump cooling liquid or refrigerating gas are sent into and flowed out from coolant outlet 114 from cooling inlet 112 and be approximately 4 ℃ so that keep electrode 102.
Fig. 9 and 10 has shown second embodiment of flow electroporation chamber 172.As can be seen, flow electroporation chamber 172 comprises the shell that is rectangle basically 200 of a hollow from Fig. 9 and 10.Two inside that electrode 202 directly inserts shell 200 relative to one another are close to the wall of shell 200 and are installed.Flow electroporation chamber 172 also comprises access road 204 that is positioned at shell 200 1 ends and the exit passageway 206 that is positioned at the other end.Access road 204 and exit passageway 206 comprise connection tube 208 and 209, and this connection tube is connected to cell suspending liquid feeder channel (supply) by pipeline 216, and this feeder channel provides cell suspending liquid to electroporation chamber 172, i.e. IHP-erythrocyte suspension.Connection tube 208 and 209 and enter the mouth 204 and outlet 206 play the effect that cell suspending liquid is imported and derives shell 200.
As can be seen from Figure 10, an end of an end of access road 204 and exit passageway 206 extends to the inside formation inner room 210 of shell 200.Therefore, inner room 210 is to be formed by the inner surface of electrode 202, the inner surface of shell 200 and the inner surface of access road 204 and exit passageway 206.
As can be seen, the electrode 202 of flow electroporation chamber 172 comprises flat, elongated, hollow shell from Fig. 9 and 10.Electrode 202 comprises cooling inlet 212 and the coolant outlet 214 that is positioned at its end, by they can intake-gas or liquid pass electrode 202 so that in electroporation process, keep stationary temperature.By cable 220 electrode 202 is connected on the pulse generator.
Chamber as mentioned above is designed to the electroporation chamber 172 of Fig. 9 and 10 and can allows the suspension of waiting to experience electroporation enter electroporation chamber 172 and extend to by liquid inlet 204 to be full of inner room 210.When erythrocyte suspension flow during, between two electrodes 202, the width both sides of passing inner room 210 apply a pulse or electric charge through inner room 210.In order in electroporation process, to keep relative stationary temperature, cooling liquid or refrigerating gas are sent into the cooling inlet 212 of electrode 202 by connection tube 208 and extract out through connection tube 209 with pump from coolant outlet 214, be approximately 4 ℃ thereby keep electrode 202.Access road 204, exit passageway 206 and connection tube 208 and 209 can be made the glass component of a solidified one, rather than the parts that separate.
Can expect that flow electroporation chamber 172 can be by (drawn) glass or any other press polished material manufacture of drawing.The inner surface of preferred electroporation chamber 172 is smooth as much as possible to reduce the generation of turbulent surface flow.The composition of the glass that draws can meet the requirement of perfect surface finish well.And mentioned component is stablized and blood constitutent is inertia.Mentioned component is also quite cheap, and this is required just to a kind of easy-to-handle electroporation chamber.
Electrode also can be formed and adopted aurosol to electroplate by the glass that draws.Electrode surface also should be press polished, highly conducts, but is inertia on the biology.The metal electroplate is lasting and cheap.Fluidic connection can use common available parts to realize.
Flow electroporation chamber or can be used as the part of whole streaming parcel equipment and construct or also can be used as specific installation and construct.In order to wrap up special cell mass, can be connected to flow electroporation equipment on the plasma removing machine that can obtain by commercial sources.For example, by hardware or software electronics or that move, can be connected to flow electroporation equipment on the plasma removing equipment that can obtain by commercial sources.A pinched valve array (array) and controller by PC driven by program (driven) also can optionally be used to control flow electroporation equipment.Similarly, power supply can be set up running flow electroporation chamber or the required energy level of streaming parcel equipment.
The 3rd embodiment of continuous flow type electroporation chamber described referring now to Figure 14-20.At first with reference to Figure 14-16, supporting member 300 is made of flexible silicone rubber.Supporting structure 300 is diamond basically and comprises a upper end 301 and a lower end 302.The major part of supporting structure 300 has formed latticed " waffle " pattern thereon, this supporting structure by thicker fin 303 (rib section) and between fin 303 thin fin 304.Along the edge of supporting structure 300, many thin slices 305 are provided, a hole 306 of passing this thin slice is all arranged on each thin slice.
A pair of continuous band-shaped electrode 330A, the 330B that is made of the metal tape (tape) or the thin slice (foil) of conduction is positioned on the supporting structure 300.All some is arranged in the passage 308 and the whole length by passage 308 basically for each electrode 330A, 330B.Perhaps preferably can in Figure 16, find out, electrode 330A, 330B are installed on sidewall 310, the 312 formed concave portion opposite 332 by passage 308.Near the top and bottom of passage 308, each continuous band-shaped electrode 330A, 330B leave passage 308 by a close-fitting slit that forms on conduit wall.Continuous band-shaped then electrode 330A, 330B are outwardly-bent, are arranged essentially parallel to the circumferential extension of supporting structure 300 and inwardly separate each other from this.For purpose as described below,, lax (slack) part 334 is set on each continuous band-shaped electrode 330A, 330B along the center line of supporting structure 300 and near its outer rim.
In each side of passage 308 and be directly adjacent to the passage place, form many rectangular holes 340 that are generally.As hereinafter explaining in more detail, it is optionally to hold the Peltier thermoelement for the cooling purpose that hole 340 is set.On each side, circular hole 342 is set near passage 308 top and bottom, as described below, can regulate this hole the reel (capstan) that is used to strain continuous band-shaped electrode 330A, 330B is installed.Just as will be described in more detail, along the center line of supporting structure 300 and near its edge, can regulate pair of holes 344 and install and be used for passing electrically contacting of this hole to electrode 330A, 330B charging.
Referring now to Figure 17, supporting member 300 is installed on the transparent polycarbonate frame 350.Framework 350 comprises a plane antetheca 352.Medial wall 354 extends back from the incline of plane antetheca 352.Between two medial walls 354, form an open channel 356 backward.354 the trailing edge in the medial wall, a pair of rear wall 358 stretches out.Pair of outside wall 360 extends forward from the outer rim of rear wall 358.Between lateral wall 360 and medial wall 354, form an open forward passage 362.The hanging stick 363 of removable installation provides a kind of device easily that is used for hanging fluid reservoir bag in passage on each open forward passage 362.
Supporting structure 300 is installed on the rear surface of antetheca of polycarbonate frame 350.Supporting structure 300 adheres to and is combined on the framework 350 so that the antetheca 352 of framework 350 seals up the open upper end of the passage that forms on the surface of supporting structure 300 308.Sealing like this, passage 308 just defines a fluidic passage or " flow cell " 364.In addition, supporting structure 300 and framework 350 correspondingly portions define electroporation chamber 366.
With further reference to Figure 17, support column 370 has one and is generally rectangular cross section.Form a shape and a consistent hole 372 of the degree of depth with supporting structure 300 at 371 places, front of support column 370.Lay respectively at each side and have the Peltier thermoelement 374 of many Tellurobismuthite .s to be fixedly mounted in this hole along the major axis place in hole 372 and from the hole 372 bottom stretch out forward.Peltier thermoelement 374 has heat to get in touch with a radiator 375 that is installed in support column 370 inside.Be installed in electric fan 376 near support column 370 parts and produce flowing of air by support column so that from radiator 375 heat radiations.
The size of polycarbonate frame 350 is as the criterion with the inside that support column 370 fitly is installed in framework open channel 356 backward.When framework 350 was positioned on the support column 370, the supporting structure 300 that is installed in antetheca 352 back sides of framework 350 was assemblied in the hole 372 that forms on the front 371 of support column 370 just.Shelf 381 is positioned on the front 371 of lucky support column 370 below hole 372, with the lower edge of supporting polycarbonate frame 350.
As shown in figure 19, by framework 350 so is installed on the support column 370, the various elements relevant with hole 372 and support column 370 jointly mesh together with supporting structure 300.More particularly, thermoelectric-cooled sheet 374 stretches out by the hole on supporting structure 300 340 and the wall of contact channels 308.Reel 377 is passed in the hole 342 of the top and bottom of supporting structure 300.Stretch out in the hole 344 that electrode contact 378 is passed on the supporting structure 300.Positioning needle 379 is arranged on the thin slice 305 of supporting structure 300 in the corresponding hole 306.Ventilative and liquid-tight cylinder 380 is passed in the hole 326 in 324 li in the hole 320 in 318 li in hole on the upper end 301 of supporting structure 300 and the hole on the lower end 302.
Referring now to Figure 20, at least one is used to support the reel 377 of each electrode 330A, 330B by 382 tensions are in tension to keep continuous band-shaped electrode as take-up device.As also seeing among Figure 20, each electrode contact 378 has a slit 383 that forms in its surface, and the slack 334 relevant with continuous band-shaped electrode 330A or 330B is by this slit.Running and each electrode contact 378 with the motor 384 of transmission engagement (driving engagement) with the rotation electrode joint, thereby around a part of joint rotation electrode 330A or 330B and tension slack.This spinning movement plays the adjection that increases surface contact between electrode contact 378 and electrode 330A that is correlated with or the 330B, thereby has strengthened electrically contacting with electrode.
Ventilative and the liquid-tight cylinder 380 that is positioned at flow cell 364 top and bottom (Figure 20 has only shown its upper end) and vacuum source are got in touch with fluid by connection tube 388 and pipeline 390.Figure 20 also demonstrates, and radiator 375 disperses the heat of being collected by thermoelectric-cooled sheet 374.
The control fluid is to realize by being installed in the peristaltic-type pump arrangement 392 on the support column 350 and the pinched valve 394 of electromagnetic excitation along suitable runner turnover flow cell 364 mobile.Pump installation 392 and pinched valve 394 can operated under the control in the appropriate algorithm of the computer installation (not demonstrating) that operation place is attached thereto.
Cooled plate 396 is installed in the side of support column 370.Cooling bag 398 in the groove 362 that is clipped in framework 350 is stuck, so that the reprocessed fluid of cooling electroporation with plate 396 tight contacts.Depend on environment and pending biological substance, optionally hot plate 396 is so that be higher than the inclusions of keeping bag 398 under the predetermined temperature of room temperature.
Figure 21 has illustrated a supporting electroporation device 400.Equipment 400 comprises a handbarrow 402 that plays shell and supporting structure effect.Be installed to the support column 370 that has electroporation chamber 366 on this handbarrow and extend upward thus.Handbarrow 402 has a chassis structure 404, and this structure is equipped with wheel 406 and is transported to another position to quicken handbarrow 402 from a position.Be installed on the chassis structure 404 is power capacitor 408.Power-supply radiator 410 carries out heat exchange to disperse the heat that is produced by power capacitor with power capacitor 408.
Circuit board determinator 412 also is installed in the chassis structure 404.Come drive circuit board determinator 412 by the power supply circuit board 414 that is installed in chassis structure 404 inside, close circuit board determinator.The power-supply radiator 416 that carries out heat exchange with power supply circuit board 414 disperses the heat that is produced by power supply circuit board.The cooling fan 418 that is installed in front apron 420 lower ends of chassis structure 404 deflates from chassis structure so that take away heat from radiator 410,416.
Be installed in handbarrow 402 top panel 428 inside be centrifugal roller 430.The centrifugal drive motor 432 that is installed in the chassis structure 404 meshes with transmission with centrifugal roller 430.Centrifugal roller 430 comprises the pipe 434 that is rotatably connected, and by this connection tube blood is imported centrifugal roller.
With reference now to Figure 21, is described in the supporting electroporation device 400 that contains the 3rd concrete electroporation chamber 366 processing to biomone.Blood supply bag 450 is suspended on the hanging stick 363 of groove 362 inside that are positioned at framework 350.Pipeline 452 pumping bloods are to centrifugal roller 430, and wherein blood enters centrifugal roller by the pipe 434 that is rotatably connected.Centrifugal blood is isolated erythrocyte so that from blood plasma in leukocyte and the refuse.Then erythrocyte and material to be wrapped are mixed.Transport this mixture and be introduced into the inlet 328 that is positioned at container 364 lower ends through pipeline 454.This mixture is upwards flowed between electrode 330A and 330B, pass flow cell 364.This electrode is the pulse mode charging in second embodiment as described above.Top and bottom by being arranged in this container ventilative and liquid-tight cylinder 380 can be removed mixture and can cause electrolytic gas.Mixture after the processing flows out from the outlet 324 that is positioned at this container upper end, outlet 456 transports in mixture to a cooling bag 460 after the processing, and this cooling bag is suspended on the hanging stick 363 of another groove 362 inside that are positioned at framework 350 and with cooled plate 396 and contacts.Then this fluid is transported in the cooling and reservoir bag 462 that is suspended near the post processing on the hanging stick 363 of cooling bag 460.
The speed of pump (and flow velocity thus), valve operation, centrifugally operated, the operation of Peltier thermoelement and the pulse charge of electrode all are subjected to the control of circuit board determinator 412.The processing speed of Utopian centrifugal roller 430 should be complementary with the flow velocity of flow cell 364.But,, a storage tank can be set between centrifugal roller 430 and flow cell 364 optionally in order to adapt to any unmatched situation.Therefore, if centrifugal roller 430 processing blood are more treatable when rapider than 364 of flow cell, the mixture that this storage tank will store any surplus can " pick up the pace " until flow cell.Similar therewith, if centrifugal roller 430 processing blood are more treatable when slower than 364 of flow cell, just circuit board determinator 412 can accumulate mixture at the beginning in storage tank.Then when centrifugal roller 430 treated during the blood of enough volumes, can be transported to this mixture the flow cell 364 from storage tank.When the volume of mixture in the storage tank has been used up, this centrifugal roller also will be finished the processing of the blood of aequum.
One of above-mentioned electroporation device 400 is optional is characterized as a succession of Peltier heat that can control separately-electric cooling fin 374, thereby can provide higher or the more cooling of low degree than other locational other cooling fin that is in along flow cell 364 at a locational cooling fin that is in along flow cell 364.Owing to, therefore may need more cooling than close charging aperture place when flow cell 364 moves at the discharging opening place of close flow cell 364 when biomone is being heated.As long as the above-mentioned variation of adaptation is satisfied in the independent control of different heat-electric cooling fins 374.Different heat-electric cooling fin 374 or can be by controlling with the corresponding different heat of the temperature of experiencing-electric cooling fin in the temperature and the control of placing heat sensor along the diverse location place of flow cell, experience to 412 inputs of circuit board determinator.Perhaps, different heat-electric cooling fins 374 can be controlled along predetermined " on average " variations in temperature of flow cell according to biomone.Other method that is used to control different heat-electric cooling fin 374 is that those skilled in the art institute is thinkable.
As what those those of ordinary skill in the art understood, there are several reasons that need not to reuse former electroporation chamber.At first, may be existing the probability that infects composition to be delivered in other patient bodies.And, because the high voltage at electrode surface two ends has improved the potential of arc discharge, so the electrical efficiency of this electrode surface can reduce.In order to prevent these and other problem, of the 3rd disclosed embodiment is characterized as provides a kind of device of guaranteeing that this chamber is no longer reused of being used to.At the end of this technology and before framework 350 is removed from support column 370, be in the motor 384 of electrode contact 378 and driven automatically and excessive-rotation with the transmission engagement, exceeding stretching electrode 330A under the situation of tensile strength, 330B and break electrode.Therefore along with electrode 330A, 330B breaks, and can not reuse this chamber again.
A kind of known danger relevant with electroporation device for the gas that causes by electrolysis be not intended to produce.Known by the unnecessary overcompression meeting that accumulation caused of these gases cause the blast.In order to reduce this probability, the present invention has used a kind of flow cell 364 that is limited in three sides by softish silicone rubber.Under the situation of instantaneous overcompression, the elasticity of supporting structure 300 can adapt to the expansion of flow cell 364, thereby reduces the probability of blast.In addition, flow cell 364 is clipped between support column 370 and the polycarbonate frame 350 tightly, thereby further provides protection in case any possible blast.
When describing particularly with reference to disclosed embodiment when of the present invention, should recognize in the spirit and scope described in the additional claim of the present invention and can realize many changes and improvements.Cell washing equipment
Figure 24 is to use the cross-sectional schematic of the cell washing equipment 500 that filters dialysis, preferred adverse current filtration dialysis.This complete device has end face and the sidewall that has comprised its internal part fully.Another aspect of the present invention is a cell washing equipment, and this equipment has adopted the adverse current dialysis of passing perforated membrane removing IHP solution, and replaces a kind of solution compatible with erythrocyte, and this solution comprises containing normal saline but having more than and is confined to this.As shown in figure 24, cell washing equipment 500 comprises and containing by first storage tank 505 of the cell of electroporation.By in the cell of electroporation, these cells will pass electroporation chamber 72 and be in the solution that contains superfluous IHP in the presence of IHP.In the direction of arrows the cell behind the electroporation is taken out piping 515 by pump 510 then.At entrance 520 places that are positioned on the cell washing device housings 523, cell suspending liquid is introduced cell washing equipment 500.The cell passage that is in equipment 500 inside is limited by cell plates 526, and these cell plates have the floor 525 that limits the labyrinth that this cell suspending liquid flows through.
A preferred labyrinth is presented among Figure 25, the figure illustrates the side view of cell plates 526 and demonstrates the floor that is positioned on the plate that limits the labyrinth.Extruding cell plates 526 make it pasting the first side wall 577 of semipermeable membrane 575, wherein pressure want enough greatly in case cell along by labyrinth that floor 525 limited and stressed.Should recognize that the labyrinth that is limited by floor 525 can be an arbitrary form, as long as cell suspending liquid contacts with semipermeable membrane 575.Therefore, when cell suspending liquid is flowed through cell washing equipment 500, can closely contact between this suspension and the semipermeable membrane 575.
Opposite side at semipermeable membrane 575 is identical saline plate 536, and it has and the identical floor 555 of those floors on the cell plates 526.Promote the saline plate and make its second sidewall 578 that is pasting semipermeable membrane 575, thereby define a labyrinth that becomes mirror image with the labyrinth that is limited by floor 525.By 565 wash solutions compatible of pump (for example saline) with cell biological from contain the fluidic storage tank 540 of bio-compatible, extract out, the pipeline 567 of flowing through, thereby enter cell washing equipment 500 at wash solution 550 places that enter the mouth.
Should recognize that this wash solution can be any one and the compatible solution of cell biological to be washed.It comprises isotonic saline solution, hypertonic saline, hypotonic saline, Krebs-Ringer bicarbonate buffer, Earles balanced salt, Hanks balanced salt, BES, BES-Tris, HEPES, MOPS, TES and Tricine but have more than and be confined to this.Cell culture medium also can be used as a kind of wash solution, and they comprise eagle s culture medium, CMRL-1066, minimum essential medium (MEM) and the RPMI-1640 of culture medium 199, Dulbecco s improvement, are confined to this but have more than.In addition, also can be used as a kind of wash solution as the defined solution of joint filling again in this article.At last, the combination in any of above-mentioned solution also can be used as a kind of wash solution.
By pump 565 suction bio-compatible solution, make it along the labyrinth flow that is limited by floor 555 to flow out cell washing equipment 500 until this bio-compatible solution at outlet 560 places through this equipment.Discard bio-compatible solution by floss hole 570 then.Point out that equipment 500 will be the most effective if according to extracting bio-compatible solution in the opposite direction with the flow of solution that contains this cell, this point is important.But can expect in the present invention also can be according to aspirating this bio-compatible solution with the flow of solution that contains this cell to identical direction.
Employing contains IHP from the cell suspension thing of the cell behind the electroporation as an embodiment, when with two kinds of solution processes of pump suction cell washing equipment 500, the cell suspending liquid that contains IHP will diffuse through semipermeable membrane 575, and meanwhile bio-compatible solution will diffuse through semipermeable membrane 575 with opposite direction.Along with the continuation of diffusion, cell suspending liquid is diluted gradually and replace bio-compatible solution, till the content of IHP reaches an acceptable level.
The profile of cell washing equipment 500 can be any one shape that comprises a bulge, and wherein the inside of this bulge is contained cell suspending liquid and separated by the outside of semipermeable membrane 575 with this bulge.The solution that bulge 500 can rotate lentamente to help to contain cell passes semipermeable membrane 575, thereby removes impurity.
This cell washing equipment can be made by any one material compatible with cell biological to be washed in this equipment.Cell plates 526 and wash plate 536 can be made by flexible silicone rubber.
Figure 26 demonstrates another embodiment of cell washing equipment, and it can be used to replace to wash the centrifuge of the cell behind the electroporation.In second concrete cell washing equipment 600, the principal character of this washing facility be one by elastomeric material, the elastic chamber 605 of making as silicone rubber.Translate into Figure 27 now, elastic chamber 605 is the moulded parts that contain a semipermeable membrane 610 at the center of this chamber.In the both sides of semipermeable membrane 610 is the indenture 615 of level, and this indenture has formed a labyrinth and spread all over the whole length of this elastic chamber.
As shown in figure 26, elastic chamber 605 has inlet 625, the outlet 630 of removing wash solution of introducing wash solution, introduces and contain the inlet 635 of the fluidic cell of electroporation and remove the outlet 640 that contains the fluidic cell of electroporation.Therefore, the wash solution annular of a side of the semipermeable membrane from elastic chamber 605 610 introducing flows through the labyrinth and flows out in outlet 640.The electroporation solution that contains the cell behind the electroporation is introduced the opposite side of semipermeable membrane 610, and this solution annular flows through the labyrinth and flows out at outlet 640 places.
Should recognize that semipermeable membrane 610 has separated both sides fully, and any interchange between these both sides is undertaken by semipermeable membrane 610 all.Semipermeable membrane has can make solution pass through the hole of film 610, but but can not allow particle, passes through semipermeable membrane 610 as cell.This semipermeable membrane can be with cell suspending liquid in any one material of cytocompatibility.The semipermeable membrane that can be used for cell washing equipment of the present invention comprises polypropylene (Travenol laboratory), cellulose diacetate (Asahi Medical), polyvinyl alcohol (Kuraray), polymethyl methacrylate (Toray) and polrvinyl chloride (Cobe laboratory), but is not limited thereto.At erythrocyte, the bore dia in the semipermeable membrane should be greater than 1 micron but can be little many.
Can be placed into this elastic chamber in the framework 655, and sidewall 660 can overturn on hinge (hinge) 665 and 666,, thereby between sidewall 660 and sidewall 665, wedge the elastic chamber tightly so that sidewall 660 blocks the elastic chamber 605 near sidewall 665.Sidewall 660 is one can heat or cool off the thermoelement of elastic chamber 610.Sidewall 665 is a pulsatile machinery that contains roller 670, and wherein this roller moves on 675 and can sequentially push the elastic chamber when moving with 675 and sequentially vertically passing on the pliable and tough hanging stick 677 of sidewall 665 short transverses and exerting pressure when roller.
In operation, the elastic chamber is put into framework 665 and sidewall 660 (thermoelement) is closed in the elastic chamber 605.Certainly, sidewall 660 can be a plate that does not have thermoelement.On the first side wall 615, inlet is connected on the wash solution pipeline that links to each other with a wash solution storage tank (not demonstrating).Outlet 630 is connected to a discharge tube (not demonstrating).Opposite side in the elastic chamber is connected to inlet 635 to contain on cell and the fluidic storage tank of electroporation (not demonstrating).Outlet 640 is connected to one sends cell and electroporation fluid on the pipeline of cell storage tank back to.
In operation, wriggling promotes activator (peristaltic activator) 670 to aspirate lightly in wash solution one side, thereby forces fluid to flow to outlet one side from inlet side.Can optionally pass through two labyrinths to two kinds of solution suctions according to the similar fashion shown in the cell washing equipment 500 by external pump.Because this wriggling promotes activator extruding elastic chamber, so improved the fluidic transportation of passing semipermeable membrane 650 by the quality transfer function.This effect continues to the electroporation fluid always and is washed basically till the fluid replacement.Erythrocytic application through the IHP processing
The invention provides a kind of new method that is used to improve erythrocyte oxygen carrying capacity.According to method of the present invention, IHP and hemoglobin combine and have changed its oxygen releasability with a kind of stable manner.The erythrocyte that contains the IHP-hemoglobin can discharge more oxygen than the erythrocyte per molecule that contains hemoglobin separately, and therefore the circulation blood of per unit is had more oxygen and can be used to diffuse into tissue.Under common environment, IHP is deleterious and can not be tolerated as a kind of common medicine.But, in this new technology, IHP is connected to the toxicity of the IHP that but neutralized on the hemoglobin.Under the situation that lacks serious chronic blood loss, adopt the compositions for preparing according to this method to treat and produced to continue about 90 days beneficial effect.
Erythrocytic another benefit of handling through IHP is that this erythrocyte is not lost glass ear effect when storing.After 24 hours, just can not recover oxygen carrying capacity that it is maximum according to the stored common erythrocyte of conventional method.This is to have left haemoglobin molecule because be arranged in the DGP diffusion of common erythrocyte in storage process, and must be replaced by substance in vivo (body) after blood transfusion.In contrast, the erythrocyte after handling according to the present invention has kept its maximum oxygen carrying capacity in the process that stores, so they can go immediately maximum oxygen being delivered in the tissue after people or animal blood transfusion.
The purposes of RBC after IHP handles is very widely, it comprises the many acute and chronic diseases of treatment, and these diseases include but not limited to the inpatient, operation on vessels of heart, chronic anaemia, anemia after the major operation, crown infraction and relevant problem, chronic lung disease, cardiovascular patient, autotransfusion, the erythrocyte infusion of encapsulation is (hemorrhage, wound or operation) increase, congestive heart failure, myocardial infarction (heart attack), peripheral vascular disease, intermittent claudication, the circulation shock, hemorrhagic shock, anemia and chronic hypoxia disease, breathe the alkali mass formed by blood stasis, metabolic alkalosis, sicklemia, by pneumonia, the lung capacity that operation causes reduces, pneumonia, wound, the breast puncture, gangrene, anaerobism infects, angiopathy, as diabetes, what treated the altitude chamber substituting or replenishing, erythrocytic utilization again in the operation, cardiac insufficiency, anoxia-be only second to chronic disease, organ transplantation, carbon monoxide, nitrogen oxide and cyanide poisoning.
At above-mentioned any or multiple disease, can (blood of handling through IHP according to the present invention's preparation between 1 unit=500ml) be treated the human or animal between about 0.5 to 6 unit by input in human or animal body.Under certain conditions, may exist the blood of handling with IHP to replace the situation of intravital all normal blood of patient basically fully.Erythrocyte after the IHP processing is depended on symptom to be treated to the volume of patient or animals administer.In addition, the erythrocytic volume handled of IHP also depends on the erythrocytic concentration that IHP handled in the erythrocyte suspension.Should recognize that erythrocyte after IHP handled is not strict to the amount of patient's administration and can in very wide scope, change and still effective.
Encapsulation erythrocyte per unit after IHP handles can be to the oxygen of 2 to 3 times of organized delivery, and the encapsulation RBC after IHP handles is similar to common erythrocyte.Therefore the encapsulation erythrocyte after the doctor IHP that can select to use 1 unit handles, rather than select the common erythrocyte of 2 units.Except comprising IHP is wrapped in the treatment step in the cell, can be through the encapsulation erythrocyte that IHP handles according in blood treatment, being prepared with the similar method of this blood processing.
Though described the present invention in detail with reference to disclosed embodiment, should understand, in the spirit and scope described in the additional claim of the present invention, can realize many changes and improvements.
Claims (11)
1. be used for the equipment of biomone perforation, this equipment comprises:
Limit the wall of fluid flowing passage;
Along the electrode that said fluid flowing passage opposing sidewalls is arranged, this electrode comprises and is used to place the device that this electrode of electrical communication is arranged with high-voltage pulse power source, thereby makes the impulse electric field of biomone experience that moves along said fluid flowing passage;
At least one said wall that limits said fluid flowing passage that is characterized as of said equipment is made by yielding elastomeric material;
Instantaneous pressure in said fluid flowing passage increases to a rare part by the said elastomeric material buffering of said wall thus.
2. the equipment of claim 1, wherein said at least one limit said fluid flowing passage and comprise two said walls of making by yielding plastic material by the said wall that yielding elastomeric material is made.
3. the equipment of claim 1, wherein said electrode comprises continuous band-shaped electrode.
4. be used for the equipment of biomone perforation, this equipment comprises:
Limit the wall of fluid flowing passage;
Along the electrode that said fluid flowing passage opposing sidewalls is arranged, this electrode comprises and is used to place the device that this electrode of electrical communication is arranged with high-voltage pulse power source, thereby makes the impulse electric field of biomone experience that moves along said fluid flowing passage;
Have heat to get in touch and the chiller arranged with said fluid flowing passage, this device is used for cooling off this particle at said biomone when said fluid flowing passage moves;
Be used to measure the signal sensor of the temperature of the said biomone that moves along said fluid flowing passage;
The said temperature that measures is produced the control device of response, and this device is used at said biomone controlling the cooling degree of said particle when said fluid flowing passage moves.
5. the equipment of claim 4, wherein said chiller comprises the many heat-electric cooling fin that has heat to get in touch with said fluid flowing passage.
6. the equipment of claim 5, wherein said heat-electric cooling fin contacts with the wall that limits said fluid flowing passage.
7. be used for the equipment of biomone perforation, this equipment comprises:
Limit the wall of fluid flowing passage;
Along the electrode that said fluid flowing passage opposing sidewalls is arranged, this electrode comprises and is used to place the device that this electrode of electrical communication is arranged with high-voltage pulse power source, thereby makes the impulse electric field of biomone experience that moves along said fluid flowing passage; And
Have heat to get in touch and a heat extraction-electric cooling fin of arranging with said fluid flowing passage, this cooling fin is used to cool off the biomone that moves along this fluid flowing passage.
8. the equipment of claim 7 wherein has heat to get in touch with said fluid flowing passage and a said heat extraction-electric cooling fin of arranging comprises a heat extraction-electric cooling fin that contacts with the said wall that limits said fluid flowing passage.
9. be used for the equipment of biomone perforation, this equipment can be installed on the supporting member in activity, and it comprises:
Limit the wall of fluid flowing passage;
Along the electrode that said fluid flowing passage opposing sidewalls is arranged, this electrode comprises and is used to place the device that this electrode of electrical communication is arranged with high-voltage pulse power source, thereby makes the impulse electric field of biomone experience that moves along said fluid flowing passage; And
Be used for before said equipment is removed from said supporting member, that destroys said electrode operates relevant device with this electrode;
Said thus equipment can not be reused.
10. the equipment of claim 9,
Wherein being used to place with power supply has the said device of the said electrode of electrical communication to comprise an axle, and said electrode is around the periphery parcel of a reference axis at least a portion; With
The wherein said device that is used to destroy said electrode comprises a kind of like this device, and it is used to rotate said axle, so that the stretching electrode is to exceeding its limit of stretch, thereby breaks said electrode and makes this electrode can not carry out electrical operation.
11. be used for the equipment of biomone perforation, this equipment comprises:
Limit the wall of fluid flowing passage;
Along the electrode that said fluid flowing passage opposing sidewalls is arranged, this electrode comprises and is used to place the device that this electrode of electrical communication is arranged with high-voltage pulse power source, thereby makes the impulse electric field of biomone experience that moves along said fluid flowing passage;
Be used to pump installation that said biomone is moved along said fluid flowing passage; And
Make said biomone produce the control device of response along the speed that said fluid flowing passage moves to said pump, this device is used to control the pulse spacing of said high voltage electric energy.
When the said biomone that moves along said fluid flowing passage was exposed between the said electrode, this biomone experienced the pulse of predetermined number thus.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/402,145 US5720921A (en) | 1995-03-10 | 1995-03-10 | Flow electroporation chamber and method |
| US08/402,145 | 1995-03-10 | ||
| US490695P | 1995-10-06 | 1995-10-06 | |
| US60/004,906 | 1995-10-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1195997A true CN1195997A (en) | 1998-10-14 |
Family
ID=26673634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96193177A Pending CN1195997A (en) | 1995-03-10 | 1996-03-11 | Flow electroporation chamber and method |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0814855A4 (en) |
| JP (2) | JP3859709B2 (en) |
| KR (1) | KR19980702848A (en) |
| CN (1) | CN1195997A (en) |
| AU (1) | AU5305096A (en) |
| CA (1) | CA2214800C (en) |
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| US6773669B1 (en) | 1995-03-10 | 2004-08-10 | Maxcyte, Inc. | Flow electroporation chamber and method |
| US7029916B2 (en) | 2001-02-21 | 2006-04-18 | Maxcyte, Inc. | Apparatus and method for flow electroporation of biological samples |
| US7141425B2 (en) | 2001-08-22 | 2006-11-28 | Maxcyte, Inc. | Apparatus and method for electroporation of biological samples |
| US7771984B2 (en) | 2004-05-12 | 2010-08-10 | Maxcyte, Inc. | Methods and devices related to a regulated flow electroporation chamber |
| CN101857836A (en) * | 2009-11-10 | 2010-10-13 | 北京大学 | Flow electroporation device and system |
| CN101928666A (en) * | 2010-07-30 | 2010-12-29 | 北京大学 | Flow type electroporation device and system |
| CN102665453A (en) * | 2009-12-23 | 2012-09-12 | 曼海姆/奥克森福特旭德楚克股份公司 | Reactor system for electroporation |
| CN1965079B (en) * | 2004-06-12 | 2012-12-05 | 英杰生命新加坡私人有限公司 | Electroporator having an elongated hollow member |
| CN103060191A (en) * | 2004-06-12 | 2013-04-24 | 英杰生命新加坡私人有限公司 | Electroporator system |
| CN107530486A (en) * | 2015-05-08 | 2018-01-02 | 生物磁溶液有限公司 | The apparatus and method of immunomagnetic cell separation |
| CN107872982A (en) * | 2015-03-31 | 2018-04-03 | 昂科赛克医疗公司 | System and method for the improved electroporation based on tissue sensing |
| CN108795750A (en) * | 2018-05-28 | 2018-11-13 | 深圳先进技术研究院 | A kind of electroporation device with refrigerating function |
| US11071860B2 (en) | 2019-02-06 | 2021-07-27 | Oncosec Medical Incorporated | Systems and methods for detecting fault conditions in electroporation therapy |
| CN113260697A (en) * | 2018-12-20 | 2021-08-13 | 国立大学法人丰桥技术科学大学 | Electroporation device and method for producing foreign substance-introduced cell |
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| AU3752200A (en) * | 1999-03-17 | 2000-10-04 | Transfusion Technologies Corporation | System and method for processing blood |
| US7601268B2 (en) * | 2003-05-27 | 2009-10-13 | Haemonetics Corporation | Continuous blood filtration and method of use |
| KR101084528B1 (en) * | 2008-04-15 | 2011-11-18 | 인비트로겐 싱가포르 피티이. 엘티디. | Pipette tip for electroporation device |
| GB201012521D0 (en) | 2010-07-27 | 2010-09-08 | Univ Strathclyde | Integrated perfusion system |
| US10206735B2 (en) * | 2013-03-13 | 2019-02-19 | Nuvaira, Inc. | Fluid delivery system and method for treatment |
| JP6683598B2 (en) * | 2013-03-15 | 2020-04-22 | マックスサイト インコーポレーティッド | Methods and compositions for producing stable transfected cells |
| KR20170135966A (en) | 2015-04-13 | 2017-12-08 | 맥스시티 인코포레이티드 | Methods and compositions for transforming genomic DNA |
| CA3018505A1 (en) * | 2016-04-05 | 2017-10-12 | General Electric Company | Activated platelet composition with tunable growth factor level |
| JP6985508B2 (en) * | 2017-09-30 | 2021-12-22 | インスクリプタ， インコーポレイテッド | Flow-through electroporation equipment organization |
| SG11202102008XA (en) | 2018-08-31 | 2021-03-30 | Charles Stark Draper Laboratory Inc | Method and apparatus for high throughput high efficiency transfection of cells |
| JP7248721B2 (en) * | 2021-02-17 | 2023-03-29 | ゼネラル・エレクトリック・カンパニイ | Activated platelet composition capable of modulating growth factor levels |
| WO2022224803A1 (en) | 2021-04-22 | 2022-10-27 | 富士フイルム株式会社 | Electric punching device, introduction method of bioactive material, producing method of tissue-derived biomaterial, and tissue-derived biomaterial |
| WO2023223931A1 (en) * | 2022-05-16 | 2023-11-23 | 富士フイルム株式会社 | Electroporation device and electroporation method |
-
1996
- 1996-03-11 EP EP96909619A patent/EP0814855A4/en not_active Withdrawn
- 1996-03-11 KR KR1019970706255A patent/KR19980702848A/en not_active Application Discontinuation
- 1996-03-11 JP JP52774896A patent/JP3859709B2/en not_active Expired - Lifetime
- 1996-03-11 CN CN96193177A patent/CN1195997A/en active Pending
- 1996-03-11 CA CA002214800A patent/CA2214800C/en not_active Expired - Lifetime
- 1996-03-11 AU AU53050/96A patent/AU5305096A/en not_active Abandoned
-
2006
- 2006-07-25 JP JP2006202504A patent/JP4171755B2/en not_active Expired - Lifetime
Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6773669B1 (en) | 1995-03-10 | 2004-08-10 | Maxcyte, Inc. | Flow electroporation chamber and method |
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Also Published As
| Publication number | Publication date |
|---|---|
| JP4171755B2 (en) | 2008-10-29 |
| AU5305096A (en) | 1996-10-02 |
| CA2214800A1 (en) | 1996-09-19 |
| JP3859709B2 (en) | 2006-12-20 |
| JP2007007430A (en) | 2007-01-18 |
| EP0814855A1 (en) | 1998-01-07 |
| JPH11502133A (en) | 1999-02-23 |
| EP0814855A4 (en) | 2002-07-17 |
| CA2214800C (en) | 2002-01-22 |
| KR19980702848A (en) | 1998-08-05 |
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