JP4171755B2 - Flow electroporation chamber and method - Google Patents

Description

  The present invention relates to a method and apparatus for encapsulating biologically active substances in various cell populations. More specifically, the present invention relates to a method for encapsulating an allosteric effector of hemoglobin in erythrocytes by electroporation in order to change the physical characteristics of hemoglobin in the cells to be favorable for treatment. And an apparatus.

BACKGROUND OF THE INVENTION Within the adult vasculature, the volume of blood is about 5-6 liters. About half of this volume is occupied by cells such as red blood cells, white blood cells and platelets. Red blood cells constitute the majority of the cellular components of blood. Plasma, the liquid part of blood, is about 90% water and 10% various solutes. These solutes include plasma proteins, organic intermediates and waste products, and inorganic compounds.

  The primary function of red blood cells is to transport oxygen from the lungs to body tissues and to transport carbon dioxide from tissues to the lungs for removal. Most oxygen is not transported by the plasma because oxygen is only slightly soluble in the aqueous solution. Most of the oxygen carried by the blood is transported by red blood cell hemoglobin. Mammalian erythrocytes do not contain the nucleus, mitochondria or any other intracellular organelle and do not use oxygen in their metabolism. Red blood cells contain about 35% by weight of hemoglobin, which binds and transports oxygen.

  Hemoglobin is a protein having a molecular weight of about 64,500 daltons. Hemoglobin contains four polypeptide chains and four heme prosthetic groups in which iron atoms are bound in a divalent state. Normal globin, which is the protein portion of the hemoglobin molecule, is composed of two α chains and two β chains. Each of the four chains has a characteristic tertiary structure in which the chains are folded. The four polypeptide chains are arranged approximately in a tetrahedron and are combined together to form a characteristic quaternary structure of hemoglobin. One heme group capable of reversibly binding to one molecule of molecular oxygen is bound to each polypeptide chain. When hemoglobin binds to oxygen, oxyhemoglobin is formed. When oxygen is released, oxyhemoglobin is reduced to deoxyhemoglobin.

  Transport of oxygen to tissues includes blood flow volume, red blood cell count, hemoglobin concentration in red blood cells, oxygen affinity of hemoglobin, and in some species, the molar ratio of hemoglobin in red blood cells with high and low oxygen affinity, etc. Depends on several factors, but not limited to these. Furthermore, the oxygen affinity of hemoglobin depends on four factors. That is, (1) oxygen partial pressure, (2) pH, (3) concentration of 2,3-diphosphoglycerate (DPG) having an allosteric effect in hemoglobin, and (4) carbon dioxide concentration. In the lungs, approximately 98% of circulating hemoglobin is saturated with oxygen at an oxygen partial pressure of 100 mmHg. This represents the total oxygen transport capacity of the blood. When fully oxygen bound, 100 ml of mammalian whole blood can carry about 21 ml of gaseous oxygen.

  The effect of oxygen partial pressure and pH on the ability of hemoglobin to bind oxygen is best explained by examining the oxygen saturation curve of hemoglobin. The oxygen saturation curve plots the percentage of total oxygen binding sites of hemoglobin molecules occupied by oxygen molecules when the solution of hemoglobin molecules is in equilibrium with various oxygen partial pressures in the gas phase.

  The oxygen saturation curve of hemoglobin is S-shaped. Thus, the binding of the first oxygen molecule increases the affinity of the remaining hemoglobin for binding to other oxygen molecules. As the oxygen partial pressure increases, each hemoglobin molecule saturates and approaches a plateau containing four oxygen molecules as the upper limit.

  The reversible binding of hemoglobin and oxygen involves the release of protons according to the following formula:

  Thus, increasing the pH shifts the equilibrium to the right, and at a given partial pressure, hemoglobin binds with more oxygen. As the pH decreases, the amount of oxygen bound decreases.

  In the lung, the oxygen partial pressure in the air space is approximately 90-100 mmHg, and the pH is also relatively higher than the normal blood pH (below 7.6). Thus, in the lung, hemoglobin tends to be almost maximally saturated with oxygen. At that pressure and pH, hemoglobin is approximately 98% saturated with oxygen. On the other hand, in the capillary inside the peripheral tissue, the oxygen partial pressure is only about 25 to 40 mmHg, and the pH is also relatively low (about 7.2 to 7.3). Muscle cells consume oxygen at a fast pace and lower the local concentration of oxygen, making it easier for some of the bound oxygen to be released into the tissue. As blood passes through the capillaries in the muscles, oxygen is released from hemoglobin, which is almost saturated in the red blood cells, into the plasma and thus into the muscle cells. As hemoglobin passes through the muscle capillaries, it releases approximately one third of the bound oxygen, so that only about 64% is saturated when the hemoglobin leaves the muscle. In general, hemoglobin in venous blood leaving tissue repeats between about 65% and 97% oxygen saturation in a repetitive circuit between the lung and peripheral tissue. Thus, the oxygen partial pressure and pH work together, causing hemoglobin to release oxygen.

  A third important factor that regulates the extent of hemoglobin oxygenation is the allosteric effector 2,3-diphosphoglycerate (DPG). DPG is the normal physiological effector of hemoglobin in mammalian erythrocytes. DPG, in conjunction with pulmonary oxygen tension, regulates the oxygen binding affinity of hemoglobin in erythrocytes. In general, as the intracellular DPG concentration increases, the affinity of hemoglobin for oxygen decreases.

  When oxygen transport to the tissue is chronically reduced, the concentration of DPG in red blood cells increases in normal individuals. For example, the oxygen partial pressure is quite low at high altitudes. Correspondingly, the oxygen partial pressure in the tissue is low. Within a few hours of a normal subject moving to a high altitude, the DPG value in the red blood cells rises, more DPG binds, and the oxygen affinity of hemoglobin decreases. Increased DPG levels in red blood cells also occur in patients with hypoxia. This regulation allows oxygen bound to hemoglobin to be released more rapidly into the tissue, making up for the decreased oxygenation of hemoglobin in the lung. If a person adapts to a higher altitude and then descends to a lower altitude, the opposite change occurs.

  Hemoglobin contains a significant amount of DPG when normally separated from blood. When DPG is “removed” from hemoglobin, the affinity of hemoglobin for oxygen becomes higher. As DPG increases, the oxygen binding affinity of hemoglobin decreases. Thus, physiological allosteric effectors such as DPG are essential for normal release of oxygen from hemoglobin in tissues.

While DPG is a normal hemoglobin physiological effector in mammalian erythrocytes, phosphorylated inositol has been found to play a similar role in certain avian and reptile erythrocytes. IHP cannot pass through the mammalian erythrocyte membrane, but can bind to the hemoglobin of mammalian erythrocytes at the binding site of DPG and modify the allosteric conformation of hemoglobin, which reduces the oxygen affinity of hemoglobin It is to let you. For example, DPG can be replaced by inositol hexaphosphate (IHP), which reduces the oxygen affinity of hemoglobin more potently than DPG. IHP has an affinity for hemoglobin 1000 times that of DPG [R. E. Benesch et al., Biochemistry, Vol. 16, pp. 2594-2597 (1977)], increasing the P ₅₀ of hemoglobin to 96.4 mmHg at pH 7.4 and 37 ° C. [J. Biol. Chem., Vol. 250, pp. 7093-7098 (1975)].

  The ability of mammalian erythrocytes to release oxygen can be enhanced by introducing some allosteric effectors of hemoglobin into erythrocytes, thereby reducing the oxygen affinity of hemoglobin and using blood oxygen efficiently (oxygen economy) is improved. This phenomenon suggests a variety of medical applications for treating individuals whose tissue oxygen supply is reduced due to inadequate lung or circulatory system function.

  Due to the potential medical benefits achieved by using these modified red blood cells, various techniques have been developed in the prior art to encapsulate the allosteric effector of hemoglobin into the red blood cells. It became possible. Therefore, a number of devices have been designed to assist or simplify the encapsulation operation. Encapsulation methods known in the art include osmotic pulse (swelling) and reconstitution of cells, controlled lysis and resealing, liposome uptake, and electroporation. Current methods of electroporation are not commercially practical to operate on a scale suitable for commercial use.

In the following literature, uptake of polyphosphate into erythrocytes by the interaction of liposomes encapsulating IHP is described. Gersonde, et al., "Modification of the Oxygen Affinity of Intracellular Haemoglobin by Incorporation of Polyphosphates into Intact
Red Blood Cells and Enhanced O2 Release in the Capillary System ", Biblthca. Haemat., No. 46, pp. 81-92 (1980); Gersonde, et al.," Enhancement of the O2 Release Capacity and of the Bohr-Effect of Human Red Blood Cells after Incorporation of Inositol Hexaphosphate by Fusion with Effector-Containing Lipid Vesicles ", Origins of Cooperative Binding of Hemoglobin, (1982); and Weiner," Right Shifting of Hb-O ₂ Dissociation in Viable Red Cells by Liposomal Technique , "Biology of the Cell, Vol. 47, (1983).

  Further, US Pat. Nos. 4,192,869, 4,321,259 and 4,473,563 by Nicolau et al. Fused a fluid-encapsulated lipid vesicle with an erythrocyte cell membrane, and the contents of the vesicle. Is described in a method for placing erythrocytes in red blood cells. In this way, allosteric effectors such as inositol hexaphosphate can be transported into erythrocytes, where IHP replaces DPC at the binding site in hemoglobin because of the higher binding constant of IHP in erythrocytes.

  According to liposome technology, IHP is dissolved in phosphate buffer until saturated, and a mixture of lipid vesicles is suspended in the solution. This suspension is then subjected to a sonication or injection step and then centrifuged. The upper suspension contains small lipid vesicles containing IHP, which are then collected. Red blood cells are added to the collected suspension and incubated, during which time lipid vesicles containing IHP fuse with the red blood cell membrane and the contents of the vesicles enter the red blood cells. The modified red blood cells are then washed and added to plasma to complete the product.

  The disadvantages with liposome technology are the low reproducibility of the concentration of IHP incorporated into erythrocytes and the considerable lysis of erythrocytes after treatment. Furthermore, commercialization is impractical because the operation is cumbersome and complicated.

  In an attempt to solve the drawbacks associated with liposome technology, a method of lysing and resealing red blood cells has been developed. This method is described in the following announcement. Nicolau, et al., "Incorporation of Allosteric Effectors of Hemoglobin in Red Blood Cells. Physiologic Effects," Biblthca. Haemat., No. 51, pp. 92-107, (1985). Related, US Pat. Nos. 4,752,586 and 4,652,449 by Ropars et al. Also describe human or animal by controlled lysis and resealing of red blood cells, avoiding RBC and liposome interactions. Describes a method of encapsulating a biologically active substance in red blood cells

  This technique is best characterized as a continuous flow dialysis system that functions in a manner similar to the osmotic pulse technique. Specifically, the first compartment of at least one dialysis element is continuously fed with an aqueous suspension of red blood cells, while the second compartment of the dialysis element is hypotonic with respect to the red blood cell suspension. Contains an aqueous solution. This hypotonic solution lyses red blood cells. The red blood cell lysate is then contacted with a biologically active substance that is incorporated into the red blood cells. To reseal the erythrocyte membrane, the osmotic pressure and / or colloid osmotic pressure of the erythrocyte lysate is increased and the resealed erythrocyte suspension is recovered.

  Related U.S. Pat. Nos. 4,874,690 and 5,043,261 by Goodrich et al. Disclose related techniques including lyophilization and reconstitution of red blood cells. As part of the method of reconstituting red blood cells, the addition of various polyanions including inositol hexaphosphate has been described. Treatment of red blood cells by the disclosed method results in cells whose activity is not altered. Presumably, IHP is taken up into cells during the reconstitution process, thereby maintaining the activity of hemoglobin.

  U.S. Pat. Nos. 4,478,824 and 4,931,276 by Franco et al. Describe effective drugs, including inositol hexaphosphate, in mammalian erythrocytes by effectively lysing and resealing cells. A second related method and apparatus for introduction into the device is described. This method is described as the “osmotic pulse technique”. In performing the osmotic pulse technique, the encapsulated red blood cell feed is suspended and incubated in a solution containing a compound that diffuses rapidly into and out of the cell, and the concentration of the compound is then introduced into the cell. Sufficient to diffuse, resulting in hypertonic cell contents. A transmembrane ion gradient is then generated by diluting the solution containing hypertonic cells with an essentially isotonic aqueous medium in the presence of at least one desired agent to be introduced, thereby As a result, it causes the diffusion of water into the cell with cell swelling and increased permeability of the outer membrane. This “osmotic pulse” causes the cells to swell, causing water to diffuse into the cells, resulting in increased permeability of the outer cell membrane to the desired drug. The increase in membrane permeability is maintained only for a period of time sufficient to transport at least one drug into the cell and diffuse the compound out of the cell.

  Polyanions that can be used to perform osmotic pulse technology include water-soluble, pyrophosphate, tripolyphosphate, phosphorylated inositol, 2,3-diphosphoglycerin that do not disrupt the outer bilayer membrane of erythrocyte lipids Acid (DPG), adenosine triphosphate, heparin, and polycarboxylic acid are included.

The osmotic pulse technique has several disadvantages including low encapsulation yield, incomplete resealing, loss of cell contents, and correspondingly shorter cell life. is there. This technique is cumbersome and complex and is not suitable for automation. For these reasons, osmotic pulse technology has been almost unsuccessful commercially.

  Another method for encapsulating various biologically active substances in erythrocytes is electroporation. Electroporation has been used to encapsulate foreign molecules into various cell types, including IHP erythrocytes, as described by Mouneimne et al. ("Stable rightward shifts of the oxyhemoglobin dissociation curve induced by encapsulation of inositol hexaphosphate in red blood cells using electroporation, "FEBS, Vol. 275, No. 1, 2, pp. 117-120 (1990)).

  The electroporation process involves the formation of pores in the cell membrane or any vesicle by application of an electric field pulse across the liquid cell suspension containing the cells or vesicles. In this poration process, the cells are suspended in a liquid medium and then subjected to an electric field pulse. This medium may be an electrolyte, a non-electrolyte, or a mixture of an electrolyte and a non-electrolyte. The electric field strength applied to the suspension and the length of the pulse (the time that the electric field is applied to the cell suspension) vary with the cell type. In order to form pores in the outer membrane of the cell, the electric field must be applied for a length and voltage that creates a specific potential across the cell membrane for a length of time sufficient to form the pore. I must.

  Four phenomena appear to play a role in the electroporation process. The first is a phenomenon of dielectric breakdown. Dielectric breakdown means the ability of a high electric field to form small holes or holes in the cell membrane. Once the pores are formed, the biologically active substance can be encapsulated in the cells. The second phenomenon is the dielectric bunching effect, which means mutual self attraction created by placing vesicles in a uniform electric field. The third phenomenon is the fusion of vesicles. Vesicle fusion represents the tendency of a membrane of biological vesicles, where the membrane is perforated by dielectric breakdown and the vesicles join together when the dielectric breakdown sites approach. The fourth phenomenon is a tendency for cells to line up along one direction of the axis in the presence of a high frequency electric field. Thus, electroporation relates to the use of vesicles for rotational prealignment, vesicle bunching and relative permittivity, or for the purpose of encapsulating and non-encapsulating vesicles.

Electroporation has been used effectively for the purpose of incorporating hemoglobin allosteric effectors into red blood cells. Mouneimne, Y et al. ("Stable Rightward
In Shifts of Oxyhemoglobin Disassociation Constant Induced by Encapsulation of Inositol Hexaphosphate in Red Blood Cells Using Electroporation ", FEBS, Vol. 275, No. 1, 2, pages 11-120 (November 1990)), Mouneimne et al. Incorporated IHP. Reported that hemoglobin-oxygen dissociation can be shifted to the right in treated erythrocytes, measured 24 hours and 48 hours after encapsulating IHP, indicating that erythrocyte resealing is permanent. Stable P ₅₀ values were obtained and moreover, the half-life of erythrocytes encapsulating inositol hexaphosphate was shown to be normal 11 days, however, the results obtained by Mouneimne et al. It has been shown that approximately 20% of the cells made were lost within the first 24 hours after injection.

The electroporation methods disclosed in the prior art are not suitable for processing large samples or using high or repetitive charges. Furthermore, these methods are not suitable for use in a continuous or “flow” electroporation chamber. Available electroporation chambers are designed for fixed use only. That is, sample processing in batches. Continuous use of a “static” chamber will overheat the chamber and increase cell lysis. In addition, existing technologies are unable to incorporate a sufficient amount of IHP at a sufficient percentage of treated cells to dramatically change the oxygen carrying capacity of blood. In addition, the prior art methods require sophisticated equipment and are not suitable for encapsulating the patient's red blood cells just before treatment. Thus, this method requires a lot of time and is not suitable for use on a commercial scale.

  What is needed is a simple, efficient and rapid method of encapsulating biologically active substances in erythrocytes while preserving cell integrity and biological function. Biologically altered blood cells may be applied therapeutically, making it easier, more effective and complete to encapsulate biologically active substances, including allosteric effectors of hemoglobin in intact red blood cells There is a need for a new method.

  There are many clinical conditions in which treatment to increase tissue transport of oxygen bound to hemoglobin is beneficial. For example, the major cause of death in the United States today is cardiovascular disease. Acute symptoms and pathologies of various cardiovascular diseases, including congestive heart failure, myocardial infarction, stroke, intermittent claudication, and sickle cell anemia, are inadequate supply of oxygen in the fluid that soaks the tissue Arises from being. Similarly, bleeding, traumatic injury, or acute blood loss after surgery reduces oxygen supply to living organs. Without oxygen, tissues far from the heart and even the heart itself cannot produce enough energy to maintain its normal function. The result of oxygen deprivation is tissue death and organ failure.

  Although the Americans have long focused on preventive measures needed to alleviate heart disease, such as exercise, proper eating habits, and alcohol consumption restrictions, deaths have occurred at a surprising rate. continuing. One approach to reducing the life-threatening consequences of heart disease is to increase tissue oxygenation during acute stress, since death occurs due to oxygen deprivation that causes tissue destruction and / or organ failure. It is. The same approach is also suitable for patients suffering from bleeding and chronic hypoxic diseases such as congestive heart failure.

  Another condition in which increased transport of oxygen to tissues is effective is anemia. A significant proportion of hospitalized patients experience anemia and low “crit” due to insufficient blood red blood cells or hemoglobin. This leads to inadequate oxygen supply to the tissue and complications. Typically, the physician can temporarily correct this condition by transfusing several units of red blood cell preparations to the patient.

  By enhancing blood oxygen supply, the number of heterologous transfusions is reduced in more cases and autologous blood transfusions are possible. The current method used to treat anemia or replace blood loss is transfusion of human whole blood. Each year in the United States, it is estimated that 3-4 million patients receive blood transfusions at the time of surgery or treatment. In more time-consuming situations, it is advantageous to completely avoid the use of donor blood, i.e. foreign blood, and use autologous blood instead.

  The use of autologous blood is often limited by the amount of blood that can be collected and stored prior to surgery. Typically, surgical patients do not have enough time to collect a sufficient amount of blood prior to surgery. The surgeon wants to make several units of blood available. Collecting blood requires several weeks for each unit, and since it is not possible to collect blood within 2 weeks prior to surgery, it is often impossible to collect a sufficient amount of supply blood. By treating autologous blood with IHP, the amount of blood required is reduced and transfusions of different types of blood can be completely avoided.

Because IHP-treated red blood cells carry 2-3 times as much oxygen as untreated red blood cells, in many cases physicians will need less transfusions of IHP-treated red blood cells . This reduces patient contact with dissimilar blood, reduces the degree of contact with blood donor-derived viral diseases, and minimizes immune dysfunction resulting from blood transfusions. The ability to infuse more efficient red blood cells is advantageous even when the patient's blood volume is excessive. In other more severe conditions where oxygen transport is lacking, life is saved by the ability to quickly improve the patient's tissue oxygenation.

There are obvious medical applications for ways to enhance oxygen supply to tissues, but there is currently no clinically available method to increase tissue transport of oxygen bound to hemoglobin . In experimental animals using DPG or a compound that is a precursor of DPG, a transient 6-12 hour increase in oxygen attachment has been reported. However, due to the natural regulation of DPG synthesis in vivo and its relatively short biological half-life, the duration of elevation of DPG concentration and tissue PO ₂ is limited, thus limiting the usefulness of DPG in therapy. Is done.

What is needed is a simple, efficient and rapid method of encapsulating biologically active substances such as IHP within erythrocytes without damaging the erythrocytes.
US Pat. No. 4,192,869 US Pat. No. 4,321,259 US Pat. No. 4,473,563 US Pat. No. 4,752,586 U.S. Pat. No. 4,652,449 U.S. Pat. No. 4,874,690 US Pat. No. 5,043,261 U.S. Pat. No. 4,478,824 US Pat. No. 4,931,276 R. E. Benesch et al., Biochemistry, Vol. 16, pp. 2594-2597 (1977) J. Biol. Chem., Vol. 250, pp. 7093-7098 (1975) Gersonde, et al., "Modification of the Oxygen Affinity of Intracellular Haemoglobin by Incorporation of Polyphosphates into Intact Red Blood Cells and Enhanced O2 Release in the Capillary System", Biblthca. Haemat., No. 46, pp. 81-92 (1980 ) Gersonde, et al., "Enhancement of the O2 Release Capacity and of the Bohr-Effect of Human Red Blood Cells after Incorporation of Inositol Hexaphosphate by Fusion with Effector-Containing Lipid Vesicles", Origins of Cooperative Binding of Hemoglobin, (1982) Weiner, "Right Shifting of Hb-O2 Dissociation in Viable Red Cells by Liposomal Technique," Biology of the Cell, Vol. 47, (1983) Nicolau, et al., "Incorporation of Allosteric Effectors of Hemoglobin in Red Blood Cells. Physiologic Effects," Biblthca. Haemat., No. 51, pp. 92-107, (1985) "Stable rightward shifts of the oxyhemoglobin dissociation curve induced by encapsulation of inositol hexaphosphate in red blood cells using electroporation," FEBS, Vol.275, No. 1,2, pp.117-120 (1990))

The present invention relates to a method and apparatus for encapsulating biologically active substances in various cell populations. More specifically, the present invention provides an electroporation chamber that can form part of an automated, self-contained flow device for encapsulating a compound or composition such as inositol hexaphosphate in red blood cells. Thereby reducing the affinity of hemoglobin for oxygen and enhancing the transport of oxygen to the tissue by red blood cells. Encapsulation is preferably accomplished by electroporation, but it is anticipated that other methods of encapsulation can be used in the practice of the invention. The methods and devices of the present invention involving electroporation chambers are equally suitable for encapsulating various biologically active agents within various cell populations.

  The devices and methods of the present invention are suitable for incorporating various biologically active substances into cells and lipid vesicles. The methods, devices and chambers of the present invention can be used to introduce a compound or biologically active substance into a vesicle that is manufactured or naturally occurring. For example, the devices, methods, and chambers of the present invention can be used to introduce IHP into red blood cells.

According to the present invention, the encapsulation of inositol hexaphosphate into erythrocytes by electroporation does not affect the lifetime, ATP value, K + value, or normal rheological ability of cells, without affecting hemoglobin to oxygen. Affinity is significantly reduced. In addition, the Bohr effect does not change except that the O ₂ bond curve shifts to the right. The reduction in red blood cell oxygen affinity increases the ability of red blood cells to dissociate bound oxygen, thus improving the oxygen supply to the tissue. The enhancement of red blood cell oxygen release results in significant physiological effects such as decreased cardiac output, increased arteriovenous differences, and improved tissue oxygenation.

The modified erythrocytes prepared according to the present invention have improved oxygen release capacity and can be used in situations as shown below.
1. Under low oxygen partial pressure conditions such as high altitudes
2. If the lungs have a reduced oxygen exchange surface, such as the development of emphysema,
3. If resistance to oxygen diffusion is increasing in the lung, such as the development of pneumonia or asthma,
4). If red blood cell oxygen transport capacity is reduced, such as the development of erythropenia or anemia, or if you are using an arteriovenous shunt
5. To treat blood circulation disorders such as arteriosclerosis, thromboembolism, tissue infarction, congestive heart failure, cardiac dysfunction or ischemia,
6). To treat hemoglobin high oxygen affinity conditions, such as hemoglobin mutations, chemical modification of the N-terminal amino acid of the hemoglobin chain, or erythrocyte enzyme deficiency,
7). To accelerate the detoxification process by improving the oxygen supply,
8). 8. to reduce oxygen affinity of stored blood, or To improve the effectiveness of various cancer treatments.

  According to the method and apparatus of the present invention, it is possible to produce modified red blood cells that contribute to improving the efficient use of oxygen in the blood. This modified red blood cell is obtained by incorporating an allosteric effector such as IHP by electroporation of the red blood cell membrane.

  Incorporation of biologically active substances into cells according to the method of the present invention, including encapsulation of hemoglobin allosteric effectors into erythrocytes, is carried out in vitro by an automated flow electroporation device. Briefly, the cell suspension is introduced into the separation washing tank chamber of the flow electroporation apparatus. Cells are separated from the suspension, washed, and resuspended in a solution of biologically active agent that is introduced into the cells. This suspension is introduced into the electroporation chamber and then incubated. After electroporation and incubation, the cells are washed and separated. An optional contamination check is performed to ensure that all unencapsulated biologically active material has been removed. The cells are then prepared for storage or reintroduction into the patient.

  In accordance with the present invention and in preferred embodiments, blood is collected from the patient, red blood cells are separated from the collected blood, the red blood cells are modified by incorporating an allosteric effector, and the modified red blood cells and plasma are reconstituted. In this method, blood containing red blood cells modified with IHP can be prepared and stored.

  The device of the present invention provides an improved method for encapsulating biologically active substances in cells and is self-contained, thus a sterile device, which can process large volumes of cells in a shortened time Includes devices capable of detecting contamination, improved cooling and incubation elements, devices that are fully automated and do not require manual control of the technician once the sample has been introduced into the device.

  Thus, an object of the present invention is to provide an automated continuous flow encapsulation device.

  It is a further object of the present invention to provide an automated continuous flow electroporation apparatus.

  It is a further object of the present invention to provide a continuous flow encapsulation device that produces a uniform population of encapsulated cells or vesicles.

  Another object of the present invention is to provide a continuous flow electroporation device that produces a uniform population of encapsulated cells or vesicles.

  Another object of the present invention is to provide a sterile, non-pyrogenic method for encapsulating biologically active substances in cells.

  Another object of the present invention is a method and apparatus for stably resealing cells or vesicles following electroporation to minimize lysis of the modified cells or vesicles after electroporation. Is to provide.

  Another object of the present invention is to provide a flow-type encapsulation device that produces a modified cell population from which all exogenous unencapsulated biologically active substances have been removed.

  Another object of the present invention is to provide an electroporation apparatus for producing a modified cell population from which all exogenous unencapsulated biologically active substances have been removed.

  Another object of the present invention is to provide a method and apparatus for continuously encapsulating biologically active substances within a population of cells or vesicles.

  It is a further object of the present invention to provide a method and apparatus that achieves the objects, features and advantages defined above in one cycle.

  Another object of the present invention is to provide a continuous flow electroporation chamber.

  Another object of the present invention is to provide an improved method of encapsulating biologically active substances in cells that is more efficient than currently available methods.

  A further object of the present invention is to provide a composition suitable for the treatment of diseases and / or disease states resulting from a lack or reduction of oxygen supply.

  Other objects, features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments of the invention, along with the drawings and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an automated, self-contained flow device that encapsulates an allosteric compound or composition such as inositol hexaphosphate in cells such as red blood cells. In one embodiment, the device of the present invention combines the features of a plasmaphoresis device with the features of a flow electroporation device to form an automated self-contained flow electroporation device. The present invention further includes a novel flow electroporation chamber that can be used under flow conditions rather than static conditions. It is contemplated that the method and apparatus comprising the electroporation chamber of the present invention can be used to encapsulate various biologically active substances within a diverse cell population.

  In addition, the present invention provides modified cell populations with physical properties that make cells particularly useful in the treatment of diseases that require and benefit from increased transport of oxygen to tissues. According to the method of the present invention, it is possible to obtain a uniform population of erythrocytes encapsulating IHP with reduced contamination of impurities and reduced tendency to lyse after encapsulation. Treated red blood cells exhibit a normal life span in the blood circulation. Using the present invention, patient red blood cells in need of treatment can be quickly encapsulated back into the patient's blood circulation.

  The related international application number PCT / US94 / 03189, filed March 23, 1994, which is a continuation-in-part of US Patent Application No. 035,467, filed March 23, 1993, is incorporated by reference. Included in this specification.

  The method of operation of the device of the present invention is described below with respect to the preferred use of the device, ie with respect to the encapsulation of the allosteric effector of hemoglobin in red blood cells. Inositol hexaphosphate is a preferred allosteric effector used in the present invention. Other sugar phosphates such as inositol pentaphosphate, inositol tetraphosphate, inositol triphosphate, inositol diphosphate and diphosphatidylinositol diphosphate can also be used. Other suitable allosteric effectors include polyphosphates such as nucleotide triphosphates, nucleotide diphosphates, nucleotide monophosphates, and alcohol phosphate esters. For some mutant forms of hemoglobin, such as “Zurich” hemoglobin, organic anions such as polycarboxylic acids can be used as allosteric effectors. Furthermore, inorganic anions such as hexacyanoferrate, phosphate or chloride can be used as allosteric effectors.

  Red blood cells encapsulating inositol hexaphosphate according to the present invention can be used to treat a wide range of diseases and disease states. Red blood cells encapsulating IHP produced according to the present invention can be administered to patients with a heart attack, thereby increasing oxygen transport to ischemic heart tissue while simultaneously reducing cardiac output. Erythrocytes encapsulating IHP produced according to the present invention include “hemorrhagic” anemia, surgical complications, stroke, diabetes, sickle cell disease, burns, intermittent claudication, emphysema, hypothermia, peripheral vascular disease, Treatment of any ischemic disease, including but not limited to congestive heart failure, angina pectoris, transient ischemic disease, generalized intravascular coagulation syndrome, adult respiratory distress syndrome (ARDS) and cystic fibrosis Can also be used. A detailed description of the medical application of the composition prepared according to the method of the present invention is also given below.

Continuous Flow Encapsulation Device The method of operation of the device of the present invention, the preferred use of the device, ie the encapsulation of hemoglobin allosteric effectors into red blood cells by electroporation is described below. It is understood that the device can be modified to accommodate other cell populations or vesicles and other biologically active substances. In addition, the device can be modified to use encapsulation methods other than electroporation.

  Briefly, in accordance with the present invention, a blood sample is introduced into a continuous flow encapsulation device. If red blood cells are collected, the blood may be collected directly from the patient or previously collected blood. First, blood is separated into red blood cells, plasma and white blood cells, and waste products. Waste products include dilutions and various blood solutes remaining in the supernatant after centrifugation. Waste products are stored in a waste storage tank inside the device. Plasma and white blood cells are also held in a reservoir inside the system, while red blood cells mix with the encapsulated material. Next, the red blood cell suspension is subjected to electroporation. Following electroporation, the red blood cells are incubated under conditions to reseal the cells. The red blood cells are then treated and washed to remove exogenous unencapsulated biologically active substances. Once the cells have been treated, the red blood cells containing the encapsulated material can optionally be reconstituted with plasma and white blood cells. The reconstituted blood can then be returned directly to the patient or stored for later use. Although described as individual steps, the process is essentially continuous.

  A first embodiment of the present invention will be described with reference to FIG. FIG. 1 schematically shows the structure of a continuous flow encapsulating apparatus of the present invention.

  In accordance with the present invention, a large amount of whole blood is introduced into the supply port 11 of the electroporation system 5. The blood sample may optionally be collected directly from the patient into the electroporation system 5 or may be collected and stored prior to introducing the blood into the system 5. Open valve 12 and place sample into system 5. At the same time, the valve 25 is opened and the anticoagulant is added from the anticoagulant storage tank 27 using the pump 22. The preferred anticoagulant is heparin, although other anticoagulants can be used. A preferred anticoagulant is ACD. Valves 15 and 36 are also opened and pump 40 is used. The mixture of anticoagulant and whole blood passes through a pressure assessment system 19 that monitors the flow through the filter 18 and device and is collected in a blood separation and wash tank 44 that is started when the pump 40 is used. The sensor indicates when the blood separation bath 44 is full of red blood cells. When the blood separation and washing tank is full, the blood supply is stopped. In the stage involving the separation of blood components, a plasma foresis device such as a plasma foresis device manufactured by Haemonetics Corporation (Braintree, Massachusetts, USA) can be used.

  As explained above, when the pump 40 operates clockwise, the blood separation and washing tank 44 is started, the suspension of the anticoagulant and whole blood is centrifuged, and plasma, white blood cells, red blood cells, and waste products are collected. Separated. Valve 87 is opened and plasma and white blood cells are placed in plasma reservoir 89.

  Next, the cells held in the blood separation washing tank 44 are washed optionally and by a cell population to be processed by the apparatus. The valves 33, 15 and 36 are opened, and the physiological saline buffer is added from the diluent storage tank 30 to the blood separation / wash tank 44 containing red blood cells. Pump 40 is still running. The red blood cells are then washed and centrifuged. A preferred saline buffer is a 0.9% sodium chloride solution, although other physiologically isotonic buffers can be used to dilute and wash red blood cells. During the cleaning process, the valve 54 is opened, and unnecessary materials are put into the waste storage tank 57. Again, unnecessary materials are stored in the waste storage tank 57, and red blood cells are held in the blood washing separation tank 44. The washing process is repeated if necessary.

  Through experiments carried out with different pulse lengths and field strengths, it has been found that square pulses have a lower encapsulation efficiency of IHP into human erythrocytes. Even if a large pore is formed in the cell membrane, it is considered that the extracellular IHP is insufficient to enter the erythrocyte. This suggests a more complicated process than the diffusion of IHP into cells after the formation of pores. It is proposed that the electrical pulse must perform two tasks. The first is to create pores in the cell membrane, and the second is to actively move IHP electrophoretically through the pores and into the erythrocytes. This uses a high voltage rectangular pulse (2.13 kV / cm, 2 ms) followed immediately by a lower voltage exponential pulse (1.5 to 1.75 kV / cm, 5 ms). And the encapsulation of IHP in red blood cells is increased to 50% of the encapsulation by the protocol using normal exponential curve type pulses. The exponential pulse itself is much smaller than the electroporation threshold. Both tasks, namely pore formation and electrophoretic migration, can be accomplished most efficiently with the use of exponential curve type pulses. Another embodiment is to subject the IHP more efficiently by subjecting the cells first to a high voltage rectangular pulse and then to a series of lower voltage pulses that tend to advance the IHP into the red blood cells. Encapsulate in cells. In use, cells that travel through the electroporation chamber of the present invention are subjected to a series of pulse trains. The pulse sequence is 80 to 512 pulses, and the preferred number of pulses is 312 pulses. The polarity then changes and the cells are applied to a second series of pulses. When the third pulse sequence is applied, the polarity changes once more. While moving the length of the electroporation chamber, 3-5 pulse sequences are applied to every cell used, and the polarity is reversed during each pulse sequence.

  After separating the red blood cells, the pump 40 is inverted, the pump 22 is stopped, the valves 12, 15, 33, 36, 25, 87 and 54 are closed, and the valves 97 and 64 are opened.

  The IHP solution is pumped from the IHP storage tank 50, and at the same time, the red blood cells are pumped from the blood separation washing tank 44 and sent to the cooling coil 68. Red blood cells and IHP solution are mixed at junction 67 in the tube of the device and then pumped through cooling coil 68. In a preferred embodiment of the invention, the IHP solution and red blood cells may be mixed in the separation wash tank 44 before passing through the cooling coil 68, as will be described in detail below.

  A preferred concentration of IHP in the solution is about 10-100 mM, more preferably about 22.5-50 mM, and most preferably 35 mM. A preferred concentration of potassium chloride in the IHP solution is about 10-5 mM. A preferred concentration of magnesium chloride is about 2-0.5 mM. A preferred concentration of sucrose in the IHP solution is about 67.5-270 mM. It should be understood that other sugars or polymers can be used in place of sucrose.

  The solution used in the present invention is a fluid that increases resistance. It is important that the IHP solution has a high resistivity and contains minimal electrolyte. The IHP of Aldrich Chemical Company or Matrea Chemical Company does not significantly reduce the resistivity of the solution because it does not contain any sodium chloride and minimal other electrolytes. The volume milliosmolarity of the solution is desirably about 300-500. The resistivity is desirably about 87 to 185 Ω · cm. The conductivity is desirably about 4 to 8 nS / cm. The practical salinity is desirably about 4-9 ppt, and the sodium chloride equivalent is desirably about 4.5-9.0 ppt.

The hematocrit of the suspension is preferably about 30-80, most preferably about 40. From the reaction of red blood cells, it was determined that in a fixed cell it is desirable that the high voltage does not exceed 800 V (the gap is 0.4 cm), which corresponds to 2 kv / cm. For a flow cell, the gap is 0.3 cm and the voltage through the cell is limited to 600V (+/− 300V). A number of different electroporation fluid compositions have been tested. Table A shows six samples and their characteristics. A solution of E is preferred as the electroporation solution. Pump 40 is designed to pump both red blood cells and IPH solution and can be adjusted so that the final hematocrit delivered to cooling coil 68 can be measured in advance.

  After mixing, the red blood cell-IHP suspension is then pumped to the cooling coil 68. Cooling can also be accomplished using a water bath or thermoelectric cooling system. For example, the cooling coil 68 is immersed in a cooling bath in the cooling storage tank 69. As the red blood cell-IHP suspension passes through the cooling coil 68, the suspension is cooled to about 1-12 ° C, preferably about 4 ° C. Cooling the red blood cells ensures the persistence of pores formed in the cell membrane during the electroporation process. By using a cooling coil, the surface area of the sample in contact with the cooling element is increased and the cooling rate is increased. Optionally, the cooling coil can be surrounded by a thermoelectric heat pump.

For some applications, it is necessary to heat the cell suspension prior to electroporation. In such a case, the cooling coil 68 is replaced with a heating coil. The maximum temperature that red blood cells can withstand is about 37 ° C.

  Thermoelectric heat pumps work by extracting thermal energy from a particular area, thereby lowering the temperature and shutting off the thermal energy to the higher temperature “heat sink” area. At the cold junction, energy is absorbed by the electrons as they move from a low energy level in the p-type semiconductor element to a higher energy level in the n-type semiconductor element. The power supply provides energy to pass electrons through the system. In the hot junction, energy is expelled to the heat sink as electrons move from the high energy level element (n-type) to the lower energy level element (p-type).

  Thermoelectric elements are generally in a solid state and do not have moving mechanical parts or require a fluid to operate like a vapor-cycle device. However, thermoelectric heat pumps perform the same cooling function as Freon vapor compression or absorption refrigeration equipment. Thermoelectric heat pumps are reliable, small in size and capacity, low cost, light, inherently safer than many other cooling devices, and capable of precise temperature control.

  A preferred thermoelectric heat pump used in the present invention is a product of MELCOR Materials Electronic Products Corp. of Trenton, NJ, USA. Thermocouples are made of high performance crystalline semiconductor materials. The semiconductor material is a quaternary alloy of bismuth telluride, bismuth, tellurium, selenium, and antimony, and an oriented polycrystalline semiconductor having characteristics can be obtained by processing by adding impurities. Pairs connected electrically in series and thermally in parallel are integrated into the module. Modules are placed between plated ceramic plates and have high mechanical strength in compression, optimal electrical insulation and heat conduction. Modules can be placed in parallel to enhance the heat transfer effect and can be stacked in multiple stages to achieve large temperature differences. As the current passes through the heat pump, a temperature difference of more than a maximum rating of 70 ° C. across the thermocouple occurs.

  After cooling, the erythrocyte-IHP suspension is placed in an electroporation chamber 72 where an electric pulse is applied from the pulse generator 75 to the erythrocyte-IHP suspension to form an opening in the cell membrane of erythrocytes. Optionally, an automatic detection device is installed that turns on the pulse generator 75 when the chamber 72 is full of red blood cells. Each time chamber 72 is filled with unencapsulated cells, an electrical pulse is applied to the suspension. A conventional electroporation chamber may be used when the operation of the apparatus is stationary, i.e. when the cells are processed in a discrete batch. In a preferred embodiment of the invention, a flow electroporation chamber is used. In one embodiment, the flow electroporation chamber 72 is made of transparent polyvinyl chloride and includes two opposing electrodes spaced 7 mm apart. The distance between the electrodes varies depending on the flow capacity and field strength. Preferably, the flow electroporation chamber 72 is disposable. The electroporation chamber may also be made of polysolfone, which is preferred for performing some sterilization processes such as autoclave sterilization. A detailed description of the structure and creation of the flow electroporation chamber is given below.

The red blood cell-IHP suspension passes between the two electrodes of the electroporation chamber 72. As the untreated cell suspension enters the chamber 72, an electric field of 1 to 3 KV / cm is formed, which lasts 1 to 4 milliseconds, preferably 2 milliseconds, in a 1.8 ml flow chamber. Preferably, the IHP-erythrocyte suspension is subjected to three high pressure pulses per volume with an electric field strength of about 2600-3200 V / cm per pulse. A pulse of current across the cell membrane causes an electrical dielectric breakdown in the cell membrane, creating pores in the membrane. IHP then diffuses through the pores into the cell.

  After electroporation, the red blood cell-IHP suspension is placed in incubation chamber 78 and incubated at room temperature for about 15-120 minutes, preferably 30-60 minutes. Optionally, the red blood cell-IHP suspension is incubated at about 37 ° C. for about 5 minutes and at room temperature for at least 15 minutes. The incubation chamber 78 may optionally be surrounded by heating means 80. For example, the heating means 80 may be a water bath or a thermoelectric heat pump.

  Optionally, the incubator 78 includes a reseal buffer that assists in resealing and reconstitution of red blood cells. The preferred composition of the reseal buffer is shown in Table B.

In a preferred embodiment of the invention, no reseal buffer is used.
After the incubation, the valve 51 is opened, the pump 40 is operated, and the red blood cell-IHP suspension is returned from the incubation chamber 78 to the blood separation washing tank 44. Excess IHP solution is removed from the red blood cell suspension by centrifugation. The IHP solution that has become unnecessary is sent to the waste storage tank 57. Next, the valves 33, 15 and 36 are opened, and a large amount of diluent is added to the blood separation washing tank 44. Diluents that can be used in the present invention are shown in Table C.

  The red blood cell-IHP suspension is then centrifuged, the supernatant is discarded through a valve 54 into a waste storage tank 57 and the red blood cells are left in the blood separation wash tank 44. Saline buffer from the diluent reservoir 30 is added to the erythrocytes. Cells are washed and after centrifugation, the supernatant is discarded. The washing process is repeated if necessary.

  Optionally, when removing unwanted matter from the separation and wash tank 44, the unwanted matter is passed through an impurity contamination detector 46 to detect free IHP in the waste liquid and is not exogenously encapsulated from the modified red blood cells. Verify that IHP has been removed. The impurity contamination detection system is based on optical changes in the wash buffer. After washing the modified red blood cells and centrifuging them, the supernatant is passed through an impurity contamination detector 46 before being stored in the waste storage tank 57. If exogenous unencapsulated IHP remains in the wash buffer, the discarded solution becomes turbid. Turbidity is due to the reaction of IHP with calcium, which is a component of the wash buffer. The impurity contamination detector 46 uses an optical detection system. Preferably, the light source is an LED and the detector is a photodiode. The voltage difference across the photodiode indicates the amount of IHP in the cleaning solution. The impurity contamination detector 46 is optional.

  After washing, the IHP-erythrocyte product is optionally reconstituted with plasma and white blood cells retained in reservoir 89. The treated red blood cells may be collected in a storage medium or in the patient's autologous plasma in a reinfection bag.

  The resulting IHP encapsulated red blood cells can be administered directly to the patient, or the cells can be stored for later use. IHP in erythrocytes is not released within normal storage periods.

  An example of a preferred embodiment of the present invention will be described with reference to FIG. FIG. 2 schematically shows the structure of the continuous flow encapsulating apparatus of the present invention. Again, regarding the method of operation of the device, the preferred use of the device is described, ie the encapsulation of hemoglobin allosteric effectors into red blood cells by electroporation.

It is understood that the device can be modified to accommodate other cell populations or vesicles and other biologically active substances. In addition, to encompass other encapsulation methods,
It is possible to modify the device.

  In accordance with the present invention, a whole blood sample is placed in the supply port 11 of the electroporation system 10. Open the valve 12 and place the sample into the system 10. At the same time, the valve 25 is opened and the anticoagulant is added from the anticoagulant storage tank 27 using the pump 22. Valves 15 and 36 are also opened and pump 40 is used.

  The mixture of anticoagulant and whole blood passes through the filter 18 and pressure assessment system 19 and is collected in a blood separation and wash tank 44 that is started when the pump 40 is used. The sensor indicates when the blood separation bath 44 is full of red blood cells.

  When the pump 40 operates clockwise, the blood separation washing tank 44 is started, and the suspension of the anticoagulant and whole blood is centrifuged and separated into plasma, white blood cells, red blood cells, and waste products. Valve 87 is opened and plasma and white blood cells are placed in plasma reservoir 89.

  Next, optionally, the cells held in the separation washing tank 44 are washed and centrifuged.

  The valves 33, 35, 15 and 36 are opened, and the physiological saline buffer is added from the diluent storage tank 30 to the blood separation / washing tank 44 containing red blood cells. The valve 12 is closed and the pump 40 is operated.

  During the cleaning process, the valve 54 is opened, and unnecessary materials are put into the waste storage tank 57. Again, unnecessary materials are stored in the waste storage tank 57, and red blood cells are held in the blood washing separation tank 44. The washing process is repeated if necessary. The impurity contamination detection system may optionally be installed between the separation cleaning tank 44 and the waste storage tank 57 in order to control the cleaning process.

  After separating the red blood cells, the pump 40 is inverted, the pump 22 is stopped, the valves 12, 15, 33, 35, 36, 25, 87 and 54 are closed, and the valve 97 is opened. If the cells are washed, the pump 22 is stopped in advance and the valves 12 and 25 are closed. The IHP solution is pumped from the IHP storage tank 50 and sent to the separation and washing tank 44 containing red blood cells. There, erythrocytes and IHP are mixed to form a suspension.

  A preferred concentration of IHP in the solution is about 10-100 mM, more preferably about 23-35 mM, and most preferably 35 mM. A preferred IHP solution consists of the following compounds at the following concentrations:

35 mM neutralized IHP (hexasodium salt) (Matreya Chemical Company)
5 mM KCl
1.0 mM MgCl ₂
135 mM sucrose

  Aldrich Chemical Company's IHP does not significantly reduce the resistivity of the solution because it does not contain any sodium chloride and minimal other electrolytes. It will be appreciated that other solutions with high impedance can be used in the present invention and the components of the solution are not critical. As long as the osmotic properties of the solution are such that cells such as red blood cells are not damaged and the resistivity of the solution is high, the solution is suitable for use in the present invention. The resistivity was measured for several compositions and is shown in FIG. The “CBR solution” is shown in Table A.

  The hematocrit of the suspension is preferably about 30-60, and most preferably about 40. Pump 40 is designed to pump both red blood cells and IPH solution and can be adjusted so that the final hematocrit delivered to cooling coil 68 can be measured in advance.

  After mixing the red blood cells with the IHP solution, the pump 40 is inverted again, the valve 97 is closed and the valve 64 is opened. The red blood cell-IHP suspension is then pumped and passed through the thermoelectric cooling coil 68. A blood bag in a blood warming set, such as a blood bag in a Fenwal Blood Warming Set from Baxter Healthcare Corporation, should be used as a cooling coil 68 Can do. As the red blood cell-IHP suspension passes through the cooling coil 68 in the cold storage tank 69, the suspension is cooled to about 1-12 ° C, preferably about 4 ° C. Optionally, a pump is provided between the cooling coil 68 and the cooling reservoir 69 of the device and in the electroporation chamber 72 to ensure a constant flow rate and to compensate for the volume fluctuations that occur when the cooling coil 68 is full. May be added.

  Optionally, the precooling step may be omitted and the red blood cell-IHP suspension may be sent to the electroporation chamber 72 immediately after mixing. In such an example, the cooling coil 68 and the cooling storage tank 69 are removed from the continuous flow type encapsulating apparatus 10. If the temperature of the electroporation chamber is cold enough to maintain the cell suspension at 4 ° C., it may not be cooled prior to electroporation.

  After cooling, the red blood cell-IHP suspension is placed in the electroporation chamber 72. Chamber 72 is maintained at a temperature of about 4 ° C. As the erythrocyte-IHP suspension passes through the flow electroporation chamber 72, an electrical pulse is applied to the suspension from the pulse generator 75, forming an opening in the cell membrane of erythrocytes.

  The red blood cell-IHP suspension passes between the two electrodes of the electroporation chamber 72. 3-10 illustrate the electroporation chamber. In a preferred embodiment of the present invention, as the untreated cell suspension enters chamber 72, the IHP-erythrocyte suspension is about 3 times per volume at a field strength of about 2600-3200 V / cm per pulse. Of high pressure pulses or pulses. For the introduction of IHP into the blood, it has been confirmed that transferring IHP to red blood cells more efficiently by a series of short pulses instead of a single pulse. The optimum number of pulses is about 10 to 512 pulses per continuous pulse, preferably 312 pulses. It is also advantageous for the polarity of the electric field to change between pulses or between successive pulses. FIG. 28 shows two sequences of representative pulses.

  The electric charge formed across the cell membrane causes dielectric breakdown in the cell membrane and forms pores in the membrane. IHP then diffuses through the pores into the cell. In addition, although not bound by the following hypothesis, it is thought that IHP is actually forcibly introduced into cells in an electric field.

  During electroporation, an electric field of 1 to 3 KV / cm is formed and lasts 1 to 4 milliseconds. The preferred pulse length is 3-4 milliseconds, most preferably 2 milliseconds. The length of the pulse or the continuous length of the pulse is defined as 1 / e. At a flow rate of about 10.6 ml / min, the preferred number of pulses is 3-5 at a preferred pulse rate of 0.29 Hz. The electric field strength is defined as the voltage between the electrodes. The distance between the electrodes is measured in centimeters. Preferred electrical parameters are as follows:

Pulse length or pulse duration = 1.5-2.5 ms
Electric field strength = 2.7-3 KV / cm

  The electroporation chamber can optionally be a sensor in the sense that the resistivity of the cell solution passing through the electroporation chamber is monitored. As the cell solution resistivity changes, there is a feedback circuit that adjusts the pulsing of the cells to maintain optimal electroporation efficiency. For example, when blood is electroporated in an IHP solution, different blood samples may have different resistivity. By monitoring the resistivity of blood, the optimal pulse intensity and pulse timing can be applied based on the resistivity measurement. In addition, if a bubble is introduced into the electroporation chamber, the resistivity changes so that the feedback circuit senses the presence of the bubble and stops pulsing until the bubble leaves the chamber.

  After electroporation, the red blood cell-IHP suspension is placed in incubation chamber 78 and incubated at room temperature for about 10 to 120 minutes, preferably 30 minutes. Optionally, the red blood cell-IHP suspension is incubated at about 37 ° C. for about 5 minutes and at room temperature for at least 15 minutes. Incubation chamber 78 may be surrounded by heating means 80. Any heating means 80 can be used in the practice of the present invention. A preferred heating means 80 is a water bath or a thermoelectric heat pump.

  Optionally, the incubator 78 includes a reseal buffer that assists in resealing and reconstitution of red blood cells. In a preferred embodiment of the invention, no reseal buffer is used.

  After the incubation, the valve 51 is opened and the pump 40 is operated to return the red blood cell-IHP suspension to the blood separation / washing tank 44. Excess IHP solution is removed from the red blood cell suspension by centrifugation. The IHP solution that has become unnecessary is sent to the waste storage tank 57. Next, the valves 33, 37, 15 and 36 are opened, and a large amount of post-washing liquid from the storage tank 31 is added to the blood separation / washing tank 44. In a preferred embodiment of the invention, the post-wash solution consists of a 9% sodium chloride solution containing 2.0 mM calcium chloride and 2.0 mM magnesium chloride. Any physiological saline may be used.

  After the post-wash solution has been added, the red blood cell-IHP suspension is then centrifuged, the supernatant discarded through the valve 54 into the waste storage tank 57 and the red blood cells left in the blood separation and wash tank 44. The cleaning process is repeated until all unencapsulated IHP is removed.

  Optionally, when removing unwanted matter from the separation and wash tank 44, the unwanted matter is passed through an impurity contamination detector 46 to detect free IHP in the waste liquid and is not exogenously encapsulated from the modified red blood cells. Verify that IHP has been removed. The impurity contamination detector 46 is optional.

  After washing, red blood cells containing IHP may be reconstituted with plasma and white blood cells held in reservoir 89. Pump 40 is activated and valves 87, 36 and 92 are opened. Modified red blood cells, and plasma and white blood cells are pumped to a reservoir 96.

  A filter may be installed between reservoir 96 and valve 92 to remove any clots or other impurities from the reconstituted, modified blood.

  Red blood cells encapsulating IHP obtained according to the method of the present invention can be administered directly to the patient, or the cells can be stored for later use. IHP in erythrocytes is not released within normal storage periods.

It is anticipated that the continuous flow encapsulation device of the present invention may be partially modified to utilize other encapsulation methods.

  Furthermore, it is anticipated that the continuous flow encapsulating device may be modified to handle a wide variety of cell populations. Furthermore, this device may be used to encapsulate biologically active substances in synthetic vesicles.

  It is also anticipated that the continuous flow encapsulation device of the present invention may be used to encapsulate a wide range of biologically active substances.

Flow Electroporation Chamber During electroporation, the rate of IHP insertion is linearly dependent on the voltage applied to the cells. In general, as the voltage increases, more IHP is encapsulated, but cell lysis also increases and cell viability decreases. The efficiency of an electroporation system can be assessed by cell viability after electroporation. A low cell viability indicates very low efficiency. The amplitude and duration of the electrical pulse is responsible for the electrical dielectric breakdown of the cell membrane, forming a hole in the pole cap parallel to the electric field. Thus, factors to consider when designing an electroporation system include field strength, pulse length, and number of pulses.

  Since the perfect electroporation target is spherical, its orientation does not affect the efficiency of the applied electric field. If the target is spherical, the target can be 100% electroporated (electroporation) with a single pulse of electric field strength exceeding the threshold. Red blood cells are disk-shaped. Due to its shape and orientation in the electroporation chamber, only about 40% of the cells are electroporated in a single pulse. In order to electroporate the remaining 60%, the electric field strength can be increased. This increases the load on red blood cells that are properly oriented with respect to the electric field and reduces cell viability.

  In order to increase the efficiency of encapsulation while reducing the incidence of cell lysis and cell death, flow electroporation chambers have been developed that utilize multiple short-term pulses. It was confirmed that multiple pulses using steady flow and steady field voltage inserted the maximum amount of IHP with minimal total dissolution from 2 to 24 hours. Multiple pulse systems increase cell viability without increasing the field strength. When using a multiple pulse system, the cell orientation is less critical than when using a single pulse system. The lower field strength is much milder for red blood cells. The multiple pulse system electroporates each cell with multiple pulse systems because the time regulation of the red blood cell flow rate through the chamber and the electroporation pulse and the cell orientation are not as critical as the single pulse system. It is much easier to do. The flow multiple pulse electroporation system also increases the short- and long-term survival of red blood cells when compared to the single pulse method.

FIGS. 11 and 13 show the% of oxygenation of IHP encapsulated red blood cells over a range of oxygen pressures under fixed or flowing conditions, ie, against the P ₅₀ value of IHP encapsulated red blood cells 2 shows the effect of different field strengths on the viability of erythrocytes subjected to electroporation). All readings were taken 24 hours after electroporation. These results show that an optimal encapsulation result is obtained with a large number of pulses with relatively low electric field strength.

A cooled electroporation chamber is preferred to keep the red blood cells at a constant temperature during the electroporation process, thereby improving red blood cell viability. This is accomplished by removing excess heat generated by the electrical pulses during the electroporation process. Excess heat can be removed by cooling the electrodes or cooling the entire flow electroporation chamber. In one embodiment of the invention, the electrode itself is cooled.

  During the electroporation process, blood is pumped to the supply port of the electroporation chamber, and a series of electrical pulses are applied to the red blood cells as they pass through the chamber. Red blood cells exit from the other end of the chamber. The chamber can be made of any type of insulating material including but not limited to ceramic, Teflon®, Plexiglas, glass, plastic, silicon, rubber or other synthetic materials. . Preferably, the chamber is made of glass or polysulfone. Whatever the composition of the chamber, the inner surface of the chamber should be smooth to reduce turbulence of the liquid passing through the chamber. The chamber housing should be non-conductive and biologically inert. For commercial use, it is advantageous for the chamber to be disposable.

  In one example of a preferred embodiment of the present invention, the electrode that is part of the electroporation apparatus comprises brass, stainless steel, gold plated stainless steel, gold plated glass, gold plated plastic, or metal containing plastic. It can be made from any standard, non-limiting, electrically or thermally conductive, hollow material. Preferably, the surface of the electrode is gold-plated. Gold plating reduces oxidation at the electrode and accumulation of hemoglobin and other cell particles. The surface of the electrode should be smooth.

  The electrode may be hollow and consequently cooled by liquid or gas, or may not be hollow, in which case thermoelectric or any other type of conductive cooling is used. In addition to cooling the electrodes, the electroporation chamber itself can also be cooled.

  Preferably, the flow electroporation chamber is disposable. Three embodiments of the electroporation chamber of the present invention will be described in detail below.

  In one example embodiment, the flow electroporation chamber is made of transparent polyvinyl chloride and includes two electrodes facing each other with a spacing of about 7 mm. The electroporation chamber is a partial modification of the chamber made by BTX Electronic Company, San Diego, California. However, when this electroporation chamber is used continuously, the chamber overheats and the viability of cells processed by the device decreases with time. In order to eliminate the overheating problem that occurs when the apparatus is used in a continuous flow process, a continuous flow electroporation chamber was designed. The structure of the continuous flow electroporation chamber is described in detail below.

3-8 illustrate a continuous flow electroporation chamber 72 that is an example of an embodiment of the present invention. As shown in FIG. 3, the flow electroporation chamber 72 includes a housing 100, and two electrodes 102 are fitted into the housing 100 of the flow electroporation chamber 72 on opposite sides thereof. The housing 100 includes a supply channel 104 at one end and a discharge channel 106 at the other. The inlet channel 104 and outlet channel 106 include connectors 108 and 109, respectively, preferably the connectors are male Luer products. Connectors 108 and 109 are hollow and feed channel 1 into the interior of electroporation chamber 72
04 and the outlet channel 106 are formed.

  As can be seen in FIGS. 4 and 5, the inner chamber 110 extends over most of the length of the housing 100 and is sized to accommodate the two electrodes 102. Inner chamber 110 includes a face 111 that is angled to receive the inner end of electrode 102. As described above, the internal chamber 110 is formed by the internal surface of the electrode 102 and the internal surface of the housing 100. The internal chamber 110 is connected to the supply port channel 104 and the discharge channel 106.

  As seen in FIGS. 7 and 8, the electrode 102 of the electroporation chamber 72 of FIGS. 3-6 consists of a flat, elongated, hollow shell. The electrode 102 includes a cooling supply port 112 and a cooling discharge port 114 at the end. As described above, the back surface of the electrode 102, that is, the left side surface in FIG. 7 is in direct contact with the obliquely cut surface 111 of the housing 100.

  The electroporation chamber 72 is designed so that the cell suspension subjected to electroporation enters the electroporation chamber 72 through the supply port 104 and spreads to fill the internal chamber 110. As the red blood cell suspension flows through the inner chamber 110, a pulse or charge is applied across the width of the inner chamber 110.

  In order to maintain a relatively constant temperature during the electroporation process, a coolant or gas is pumped to the cooling supply port 112 and discharged from the cooling discharge port 114 so that the electrode 102 is about 4 ° C. Maintained.

  9 and 10 show a second embodiment, a flow electroporation chamber 172. As seen in FIGS. 9 and 10, the flow electroporation chamber 172 includes a substantially rectangular hollow housing 200. The two electrodes 202 are inserted into the housing 200 so as to face each other and directly contact the wall of the housing 200. In addition, the flow electroporation chamber 172 includes a supply channel 204 at one end of the housing 200 and a discharge channel 206 at the other end. The inlet channel 204 and outlet channel 206 include connectors 208 and 209 that provide a cell suspension supply that supplies a cell suspension or IHP-red blood cell suspension to the electroporation chamber 172. Connected to the device by a tube 216. Connectors 208 and 209, as well as supply channel 204 and discharge channel 206 route cell suspension into or out of housing 200.

  As can be seen in FIG. 10, one end of the supply channel 204 and one end of the discharge channel 206 extend into the interior of the housing 200 to form an internal chamber 210. Thus, the inner chamber 210 is formed by the inner surface of the electrode 202, the inner surface of the housing 200, and the inner surfaces of the supply channel 204 and the discharge channel 206.

  As seen in FIGS. 9 and 10, the electrode 202 of the flow electroporation chamber 172 consists of a flat, elongated, hollow shell. The electrode 202 includes a cooling supply port 212 and a cooling discharge port 214 at the ends, through which the gas or liquid is pumped through the electrode 202 and during electroporation. , Maintain a constant temperature. The electrode 202 is connected to the pulse generator by a cable 220.

Using the chamber described above, the electroporation chamber 1 of FIGS.
72 is designed such that the suspension subjected to electroporation enters the electroporation chamber 172 through the liquid supply port 204 and spreads to fill the internal chamber 210. As the red blood cell suspension flows through the inner chamber 210, a pulse or charge is applied across the width of the inner chamber 210 between the electrodes 202. In order to maintain a relatively constant temperature during the electroporation process, a coolant or gas is pumped, sent to the cooling supply 212 of the electrode 202 through the connectors 208 and 209, and discharged from the cooling discharge 214. As a result, the electrode 202 is maintained at about 4 ° C. Also, the supply channel 204, the discharge channel 206, and the connectors 208 and 209 can be manufactured as an integral, fully integrated glass part rather than as separate components.

  It is anticipated that the electroporation chamber 172 can be made from mechanical drawn glass or any other highly polished material. The inner surface of the electroporation chamber 172 is preferably as smooth as possible to reduce the occurrence of surface turbulence. The components of the machine-drawn plate glass have a nearly ideal surface finish. Furthermore, the components of the mechanically drawn glass are stable and inert to blood components. It is also relatively inexpensive and desirable as a disposable electroporation chamber.

  The electrode may also be composed of mechanically drawn plate glass electroplated with colloidal gold. Also, the surface of the electrode should be highly polished, highly conductive, but biologically inert. Gold electroplating is durable and inexpensive. Fluid coupling can be accomplished using commonly available components.

  The flow electroporation chamber can be made as part of the entire fluid encapsulating device or as a single device. The flow electroporation device may then be connected to a commercially available plasma foresis machine to encapsulate a particular cell population. For example, the flow electroporation chamber may be connected to a commercially available plasma foresis device by electronic or translational hardware or software. Optionally, a pinch valve arrangement and controller operated by a personal computer program can also be used to control the flow electroporation apparatus. Similarly, the power level required to operate a flow electroporation chamber or fluid enveloping device can be established from a power source.

  A third embodiment of a continuous flow electroporation chamber will be described with reference to FIGS. First, referring to FIGS. 14 to 16, the support member 300 is made of a flexible silicone rubber. Support member 300 is essentially diamond-shaped and includes an upper end 301 and a lower end 302. The main portion of the support member 300 has a grid-like “waffle” pattern formed on the support member 300, and consists of a thicker rib portion 303 and a thin portion 304 intermediate the rib 303. Along the edge of the support member 300 are a number of knobs 305, each having a hole 306 formed therethrough.

  The channel 308 extends between the upper end 301 and the lower end 302 of the support structure and is along the main axis of the support structure 300. Channel 308 consists of opposing channel walls 310, 312 connected by a bottom 314. At the upper end 301 of the support member 300, the channel 308 opens into a circular recess 318. A hole 320 is formed in the center of the circular recess 318. An outlet opening 322 is formed at the upper end of the circular recess 318. Similarly, the lower end of channel 308 opens into a circular recess 324 formed in the lower end 302 of support member 300. A hole 326 is formed through the central support structure of the recess 324 and a feed opening 328 is formed at the lower end of the circular recess 324.

  A pair of continuous band electrodes 330 </ b> A and 330 </ b> B made of conductive metal tape or flakes are positioned on the support structure 300. Each of the electrodes 330A, 330B is disposed within the channel 308 and has a portion that extends substantially the entire length of the channel 308. As perhaps best shown in FIG. 16, opposing recesses 332 formed in the side walls 310, 312 of the channel 308 house the electrodes 330A, 330B. Adjacent to the upper and lower ends of channel 308, continuous band electrodes 330A, 330B each exit channel 308 through tight slits formed in channel wall 308. Next, the continuous band electrodes 330A, 330B bend outward and extend substantially parallel to the outer edge of the support member 300 and inwardly spaced therefrom. A slack portion 334 is formed in each of the continuous band electrodes 330A, 330B along the midline of the support structure 300 and adjacent its outer edge for the following purposes.

  A number of roughly rectangular holes 340 are formed on and adjacent to each side of the channel 308. As will be described in more detail below, the holes 340 are optionally arranged to accommodate Peltier thermoelectric elements for cooling purposes. On each side of channel 308, adjacent its upper and lower ends, circular holes 342 are adapted to accommodate capstans for tensioning continuous band electrodes 330A, 330B as shown. Formed. Along the midline of support structure 300 and adjacent to its outer edge, adapted to accommodate electrical contacts that pass through to charge electrodes 330A, 330B, as described in more detail below. A pair of holes 344 are formed.

  Referring now to FIG. 17, the support member 300 is placed on a transparent polycarbonate frame 350. The frame 350 has a flat front wall 352. The inner wall 354 extends rearward from the lateral edge of the flat front wall 352. A rear open channel 356 is formed between the two inner side walls 354. A pair of back walls 358 extend outward from the rear end of the inner side wall 354. The pair of outer side walls 360 extends forward from the outer end of the back wall 358. A forward open channel 362 is formed between the outer side wall 360 and the inner side wall 354. A rod 363 installed so that it can be removed within each front open channel 362 is a convenient means to hang a liquid storage bag in the channel.

  The support structure 300 is disposed on the back surface of the front wall 352 of the polycarbonate frame 350. The support structure 300 is adhesively bonded to the frame 350 and the front wall 352 of the frame 350 seals the open upper end of the channel 308 formed in the front surface of the support structure 300. Channel 308 is thus surrounded to define a liquid passage or “flow cell” 364. In addition, the support structure 300 and associated portions of the frame 350 define an electroporation chamber 366.

  Referring to FIG. 17, the support column 370 has a generally rectangular transverse orientation. The front surface 371 of the support column 370 is formed with a recess 372 that matches the shape and depth of the support structure 300. A number of bismuth telluride Peltier thermoelectric elements 374 are secured to the recesses along the main axis of the recesses 372 at a distance from each side of the recesses 372 and forward from the bottom of the recesses 372. Protrusions. The Peltier thermoelectric element 374 is in thermal communication with a heat sink 375 installed inside the support column 370. An electric fan 376 disposed in the adjacent portion of the support column 370 creates a flow of air through the column and removes heat from the heat sink 375.

There are capstans 377 adjacent to the upper and lower ends of the indentation 372 and spaced on either side of the centerline. There is a pair of electrode contacts 378 adjacent to the outer edge of the recess 372 and located along the major axis of the recess. Eight locator pins 379 are arranged just inside the periphery of the recess 372, and two of the locator pins 379 are located along each of the four walls of the diamond-shaped recess. At the upper and lower ends of the recess 372, a pair of hollow, porous, polymeric cylinders 380 are disposed on the main axis of the recess. The cylinder 380 is preferably formed of an inert foamed polyethylene (such as Porex) having pores sized to allow gas to pass but not liquid. As described in more detail below, these gas permeable and liquid impermeable cylinders function as a means for removing bubbles from the liquid flowing therethrough.

  The volume of the polycarbonate frame 350 is such that the support column 370 is snugly received in an open channel 356 behind the frame. Since the frame 350 is located on the support column 370, the support structure 300 disposed on the back surface of the front wall 352 of the frame 350 fits within a recess 372 formed in the front 371 of the support column 370. The shelf 381 is disposed on the front 371 of the support column 370 directly below the recess 372 and supports the lower edge of the polycarbonate frame 350.

  Using the frame 350 thus disposed on the support column 370, the various elements associated with the recess 372 and the support column 370 cooperate to secure the support structure 300 as shown in FIG. In particular, the thermoelectric cooling element 374 protrudes through the hole 340 in the support structure 300 and contacts the wall of the channel 308. Capstan 377 extends through holes 342 in the upper and lower ends of support structure 300. Electrode contact 378 protrudes through hole 344 in support structure 300. The locator pin 379 fits in the corresponding hole 306 in the knob 305 of the support member 300. A gas permeable, liquid impermeable cylinder 380 then extends through the holes 320, 326 in the recesses 318, 324 at the upper and lower ends 301, 302 of the support structure 300.

  Referring now to FIG. 20, at least one capstan 377 that supports electrodes 330A and 330B, respectively, is tensioned by pulling means 382 or the like to keep the continuous band electrode tensioned. As can also be seen in FIG. 20, each electrode contact 378 has a groove 383 formed in front of it, through which the slack portion 334 of the associated continuous band electrode 330A or 330B passes. The motor 384 that engages and drives each electrode contact 378 can be operated to rotate the electrode contact, thereby winding the electrode 330A or 330B around the contact portion and causing the slack portion to be tight. It is stretched. This winding operation additionally functions to increase the contact surface between the electrode contact 378 and the associated electrode 330A or 330B, thereby enhancing the electrical connection of the electrodes.

  A gas permeable, liquid impervious cylinder 380 at the upper and lower ends of the flow cell 364 (only the top is shown in FIG. 20) is in fluid communication with a vacuum source by a joint 388 and a tube 390. A heat sink 375, shown in FIG. 20, also removes the heat collected by the thermoelectric cooling element 374.

Fluid flow along the appropriate flow path into and out of the flow cell 364 is controlled by a peristaltic pump means 392 located in the support column 350 and a solenoid operated pinch valve 394. Pump means 392 and pinch valve 394 function under the control of appropriate algorithms of computer means (not shown) operably connected thereto.
A cooled plate 396 is placed on the side of the support column 370. A cooling bag 398 held in the channel 362 of the frame 350 is in intimate contact with the plate 396 to cool the liquid being processed following electroporation.

Depending on the ambient environment and the biological material being processed, the plate 396 may optionally be heated to maintain the contents of the bag 398 at a predetermined temperature above ambient temperature.

  FIG. 21 shows a self-contained electroporation apparatus 400. The apparatus 400 includes a cart 402 that serves as a housing and support structure. A support column 370 having an electroporation chamber 366 is disposed in the cart and extends upward therefrom. The cart 402 has a chassis structure 404 that has wheels 406 to facilitate movement of the position of the cart 402. A power storage capacitor 408 is disposed in the chassis structure 404. The power heat sink 410 is in thermal communication with the power storage 408 and removes the heat produced by the power storage.

  Circuit board calculation means 412 is also arranged in the chassis structure 404. The circuit board calculation means 412 is supplied with power by a power supply circuit board 414 arranged in the chassis structure 404 adjacent to the circuit board calculation means. A power heat sink 416 in thermal communication with the power circuit board 414 removes heat produced by the power circuit board. A cooling fan 418 disposed at the lower end of the front panel 420 of the chassis structure 404 sucks air through the chassis structure and removes heat from the heat sink devices 410, 416.

  A system status display 422 operatively associated with the circuit board computing means 412 is located on the front panel 420 of the cart 402. A control switch 424 for setting various parameters of the circuit board calculation means 412 is disposed on the front panel 420 of the cart 402 below the system status display device 422.

  A centrifuge tank 430 is disposed in the top panel 428 of the cart 402. The centrifuge drive motor 432 disposed inside the chassis structure 404 operates in mesh with the centrifuge tank 430. The centrifuge tank 430 includes a rotary connector 434 through which blood is supplied to the centrifuge tank.

  Processing of biological particles in a self-contained electroporation apparatus 400 including an electroporation chamber 366 according to the third embodiment will be described with reference to FIG. The blood supply bag 450 is suspended on one internal bar 363 of the channel 362 of the frame 350. Tube 452 delivers blood to centrifuge tank 430 where the blood is introduced into centrifuge tank through rotary connector 434. The blood is centrifuged to separate red blood cells from plasma, white blood cells, and waste products. Next, the red blood cells are mixed with the substance to be encapsulated. The mixture is routed through tube 454 and is introduced into feed opening 328 at the lower end of cell 364. The mixture flows upward through the flow cell 364 between the electrodes 330A, 330B. The electrodes are charged with pulses as described above for the second embodiment. The gas in the mixture generated by electrolysis is removed by gas permeable, liquid impervious cylinders 380 at the upper and lower ends of the cell. The treated mixture exits the outlet opening 324 at the upper end of the cell, and the outlet tube 456 suspends the treated mixture to a rod 363 inside another chamber 362 of the frame 350 and cools it. To the cooling bag 460 in contact with the prepared plate 396. The liquid is then transferred to a post-process cooling and storage bag 462 that is hung on a rod 363 next to the cooling bag 460.

Pump speed (hereinafter referred to as flow rate), valve operation, centrifugation operation, operation of Peltier thermoelectric element, and pulse charging of the electrode are all controlled by the circuit board calculation means 412. Ideally, the processing speed of the centrifuge tank 430 matches the flow rate of the flow cell 364. However, a storage tank may optionally be installed between the centrifuge tank 430 and the flow cell 364 to accommodate all deviations. In this way, if the centrifuge 430 is faster than the blood processing rate of the flow cell 364 and processes blood, the reservoir will hold all excess mixture until the flow cell “catch up”. Similarly, when the centrifuge 430 is slower in blood processing than the blood processing speed of the flow cell 364, the circuit board calculation means 412 first accumulates the mixture in the storage tank 412. Next, when the centrifuge 430 has processed a sufficient amount of blood, the mixture is sent from the storage tank to the flow cell 364. By the time the mixture volume in the storage tank is exhausted, the centrifuge tank has completed processing the desired amount of blood.

  An optional feature of the electroporation equipment 400 described above is that the cooling element 374 at one point along the flow cell 364 provides a greater or lesser degree of cooling than other cooling elements 374 at other points along the flow cell 364. The series of Peltier thermoelectric cooling elements 374 can be individually controlled so that they can. Because biological particles are heated as they travel along the flow cell 364, it may be necessary to cool the closer to the discharge end of the flow cell 364 than where it is adjacent to the feed end. These variations can be accommodated by individually controlling the various thermoelectric cooling elements 374.

  The various thermoelectric cooling elements 374 install thermal sensors at various points along the flow cell, input the temperature detected by the sensors to the circuit board calculation means 412, and correspond to the temperature detected by the sensors. It can be controlled by controlling various thermoelectric cooling elements. Alternatively, the various thermoelectric cooling elements 374 can be controlled according to the “average” temperature distribution along the flow cell, measured in advance for biological particles. Those skilled in the art will envision other ways to control the various thermoelectric cooling elements 374.

  As will be appreciated by those skilled in the art, there are several reasons why reuse of electroporation cells is undesirable. First, infectious components can be transmitted to other patients. Furthermore, the electrical performance of the electrode surface may be degraded due to the high voltage across the electrode surface, thereby increasing the arc potential. To prevent these and other problems, the features of this third disclosed embodiment provide a means to ensure that the cell is not reused. At the end of the process, before the frame 350 is removed from the support column 370, the motor 384 that engages and operates with the electrode contacts 378 automatically runs and over-rotates to exceed the tensile strength of the electrode 330A, Pull 330B to break the electrode. Since the electrodes 330A and 330B are thus broken, the cell cannot be reused.

  A known risk associated with electroporation devices is that unintended gas is produced by electrolysis. It is known that explosive expression occurs due to excessive pressure due to the generation of such unwanted gases. In order to minimize this possibility, the present invention uses a flow cell 364 defined by three sides with soft silicon rubber. If the pressure is temporarily excessive, the elasticity of the support structure 300 accommodates the expansion of the flow cell 364, thereby reducing the likelihood of an explosion. In addition, the flow cell 364 is sandwiched between the support column 370 and the polycarbonate frame 350 to further protect against the possibility of any explosive phenomenon.

  Although the invention has been described in particular detail with respect to the disclosed embodiments, it will be understood that various changes and modifications can be made within the spirit and scope of the invention as set forth in the appended claims.

Cell Washing Device FIG. 24 is a cutaway schematic diagram of a cell washing device 500 utilizing filtration dialysis, preferably countercurrent filtration dialysis. The complete device has a top and sides and completely encompasses the internal components of the device. Another aspect of the present invention is countercurrent dialysis through porous membranes that remove IHP solutions and alternatives that are compatible with red blood cells, such as but not limited to normal saline. Is a cell washing apparatus using As shown in FIG. 24, the cell washing apparatus 500 includes a first storage tank 505 containing electroporated cells. In the case of cells electroporated in the presence of IHP, these cells pass through the electroporation chamber 72 and are present in a solution containing excess IHP. Next, the electroporated cells are pumped through tube 515 by pump 510 in the direction of the arrow. The cell suspension is introduced into the cell washing device 500 from a tube inlet 520 located in the housing 523 of the cell washing device. The cell flow path inside the device 500 is defined by a cell plate 526 having ridges 525 that form an intricate path through which the cell suspension passes.

  A preferred intricate path is shown in FIG. 25, which shows a side view of a cell plate 526 with ridges on the plate that form the intricate path. The cell plate 526 is pressed against the first side 577 of the semipermeable membrane 575 with sufficient strength to push the cells along the intricate passages formed by the ridges 525.

  It is understood that the intricate path formed by the ridge 525 can be any shape as long as the cell suspension is in contact with the semipermeable membrane 575. Therefore, the cell suspension is in contact with the semipermeable membrane 575 while passing through the cell washing device 500.

  The semipermeable membrane 575 has pores that are sized such that the solution and any components of the solution can pass through the membrane and cells in the solution cannot pass. The semipermeable membrane may be any material that is compatible with the cells in the cell suspension. The semipermeable membrane that can be used in the cell washing apparatus of the present invention includes polypropylene (Travenol Laboratories), cellulose diacetate (Asahi Medical), polyvinyl alcohol (Kuraray), polymethyl methacrylate (Toray), and poly Including but not limited to vinyl chloride (Cobe Laboratories). For erythrocytes, the pores of the semipermeable membrane must be 1 micron or less in diameter, but may be much smaller. The cells move along the intricate path formed by the ridges 525 until the cell suspension exits the device 500 at the outlet tube 530. For cell suspensions with IHP, the cell suspension is then pumped back into the reservoir 500 and recirculated through the device 500 until the IHP value in the wash solution falls to an acceptable value.

  On the other side of the semipermeable membrane 575 is the same saline plate 536 having a ridge 555 that exactly coincides with the ridge on the cell plate 526. The saline plate is pressed against the second side of the semi-permeable membrane 575, thereby forming an intricate path that is symmetrical to the intricate path formed by the ridge 525. For example, a biocompatible cleaning liquid such as physiological saline is sent from the storage tank 540 containing the biocompatible liquid through the tube 567 to the cleaning liquid outlet 550 of the cell cleaning device 500 by the pump 565. .

  It will be appreciated that the wash solution may be any solution that is biocompatible with the cells to be washed. Such washings include isotonic saline, hypertonic saline, hypotonic saline, Krebs-Ringer bicarbonate buffer, Earl's balanced salts, Hanks' balanced salts, BES, BES-Tris, HEPES. , MOPS, TES, and Tricine, but are not limited to these. Cell culture media can be used as washing solutions, including, but not limited to, 199 media, Dulbecco's modified Eagle media, CMRL-1066, minimal essential media (MEM), and PRMI-1640. Absent. In addition, the reseal solution as defined herein can be used as a cleaning solution. Furthermore, any mixture of the above solutions can be used as the cleaning liquid.

The biocompatible solution continues to rise from the outlet 560 until it exits the cell washing device 500.
The device is passed by pump 565 according to the intricate path formed by 5. The biocompatible solution is then discarded through the water pipe 570. It is important that the efficiency of the device 500 is best when the biocompatible solution is pumped in the opposite direction to the solution containing the cells. However, it is anticipated in the present invention that a biocompatible solution can be pumped in the same direction as the solution containing the cells.

  As an example, when using a cell suspension containing IHP obtained from electroporated cells, the cell suspension solution containing IHP is semi-permeable as both solutions are pumped to the cell washer 500. At the same time, the biocompatible solution diffuses through the semipermeable membrane 575 in the opposite direction. As this diffusion continues, the cell suspension solution is gradually diluted and replaced with a biocompatible solution until the IHP value is acceptable.

  The cell washer 500 can optionally have thermoelectric elements 580 attached to the outside of the cell plate 526 and the outside of the wash solution plate 536. It is understood that the thermoelectric element 580 can be attached to one or both of the plates 526 and 536. Thermoelectric element 580 can be used to cool or warm the solution during the wash cycle. Thus, when the cell washer is used with a thermoelectric element attached to it, the incubator 78 is not required because the cells are resealed when warmed in the cell washer that acts as an incubator. It is understood. A biocompatible wash can be a reseal buffer. It will be appreciated that the temperature can be controlled by other methods such as a water bath.

  The shape of the cell washing device 500 may be any shape including a circular container in which the inner part of the circular container contains a cell suspension and is separated from the outer part of the circular container by a semipermeable membrane 575. The circular container 500 can be slowly rotated to help pass the cell-containing solution through the semipermeable membrane 575 and thereby remove contaminating impurities.

  The cell washing device can be composed of any material that is biocompatible with the cells to be washed with the device. The cell plate 526 and the washing plate 536 can be made of elastic silicone rubber.

  Another embodiment of a cell washing apparatus that can be used as an alternative to centrifugation for washing electroporated cells is shown in FIG. In the cell washing apparatus 600 which is the second embodiment of the cell washing apparatus, the central feature of the cell washing apparatus is an elastomer cell 605 made of an elastomer material such as silicone rubber. Referring now to FIG. 27, the elastomer cell 605 is a molded part having a semipermeable membrane 610 at the center of the elastomer cell 605. On both sides of the semipermeable membrane 610 there are horizontal notches 615 that form intricate paths and span the entire length of the elastomer cell.

  As shown in FIG. 26, the elastomer cell 605 includes a supply port 625 for introducing a cleaning solution, a discharge port 630 for removing the cleaning solution, a supply port 635 for introducing cells together with the electroporation solution, And a discharge port 640 for removing cells together with the poration liquid. In this way, the cleaning liquid is introduced to one side of the semipermeable membrane 610 in the elastomer cell 605, circulates through the complicated path, and exits from the discharge port 640. The electroporation solution containing the electroporated cells is introduced on the opposite side of the semipermeable membrane 610, circulates through the complicated path, and exits from the outlet 640.

It will be appreciated that the semi-permeable membrane 610 completely separates both sides, and that all communication means between the two sides pass through the semi-permeable membrane 610. The semipermeable membrane has pores that allow the solution to pass through the membrane 610 but not particles such as cells that pass through the semipermeable membrane 610. The semipermeable membrane may be any material that is biocompatible with the cells in the cell suspension. Semipermeable membranes that can be used in the cell washing apparatus of the present invention include polypropylene (Travenol Laboratories), cellulose diacetate (Asahi Medical), polyvinyl alcohol (Kuraray), polymethyl methacrylate (Toray), and poly Including but not limited to vinyl chloride (Cobe Laboratories). For erythrocytes, the pores of the semipermeable membrane must be 1 micron or less in diameter, but may be much smaller.

  The elastomeric cell can be disposed in the frame 655 and the side 660 holds the elastomeric cell 605 opposite the side 665, thereby placing the elastomeric cell between the side 660 and the side 665. Side 660 can rotate on hinges 665 and 666 to secure. Side 660 is a thermoelectric element that can heat or cool elastomeric cell 610. The side 665 moves over the belt 675 and can subsequently clamp the elastomer cell as the roller moves around the belt 675, followed by an elastic rod 677 that moves vertically to the height of the side 665. This is a pulsation mechanism having a roller 670 for applying pressure.

  In operation, the elastomer cell is placed in the frame 655 and the side 660 (thermoelectric element) contacts the elastomer cell 605. Of course, the side 660 may be a plate without thermoelectric elements. On the first side 615, the supply port is attached to a cleaning liquid tube that is attached to a cleaning liquid storage tank (not shown). The discharge port 630 is connected to a discharge pipe (not shown). On the opposite side of the elastomer cell, a supply port 635 connects to a reservoir (not shown) containing cells and electroporation fluid. The outlet 640 connects to a tube that returns cells and electroporation fluid back to the cell reservoir.

  In operation, peristaltic activator 670 softly pumps the cleaning liquid side, thereby advancing the solution from the supply port to the discharge port. Optionally, similar to the method shown for cell washing device 500, two solutions can be passed through two intricate paths by an external pump. Because the peristaltic activator is pushing the elastomer cell, more liquid moves through the semipermeable membrane 650 due to the mass transfer action. This action continues until the electroporation solution is substantially replaced with the washing solution.

Application of IHP-treated red blood cells The present invention provides a novel method for increasing the oxygen carrying capacity of red blood cells. In accordance with the method of the present invention, IHP binds hemoglobin in a stable manner and shifts the ability of hemoglobin to release oxygen. Red blood cells with IHP-hemoglobin can release more oxygen per molecule than hemoglobin alone, so that more oxygen is diffused into the tissue for each unit of circulating blood. Under normal circumstances, IHP is toxic and cannot be used as a drug. However, linking IHP to hemoglobin in this novel way neutralizes IHP toxicity. In the absence of severe chronic blood loss, treatment with a composition prepared according to the method of the present invention could last a beneficial effect for about 90 days.

  Another advantage of IHP treated red blood cells is that the Bohr effect is not compromised upon storage. Normal erythrocytes stored by conventional methods do not recover their maximum oxygen transport capacity for about 24 hours. This is because the DPG in normal red blood cells must dissociate from the hemoglobin molecules during storage and must be replaced in the body after injection. In contrast, red blood cells treated in accordance with the present invention retain their maximum oxygen carrying capacity during storage and can therefore carry the maximum amount of oxygen to the tissue immediately after being injected into a human or animal.

The use of IHP-treated red blood cells (RBC) is very widespread, including the treatment of many acute and chronic diseases, including inpatients, cardiovascular surgery, chronic anemia, post major surgery Anemia, coronary infarction and related problems, chronic lung disease, cardiovascular patients, self-injection (bleeding, traumatic injury, or surgery) as an encapsulated red blood cell infusion, congestive heart failure, myocardial infarction (heart attack) ), Stroke, peripheral vascular disease, intermittent claudication, circulatory shock, hemorrhagic shock, anemia and chronic hypoxia, respiratory alkaliemia, metabolic alkalosis, sickle cell anemia, lung volume reduction due to pneumonia, surgery , Pneumonia, trauma, chest puncture, gangrene, anaerobic bacterial infection, vascular diseases such as diabetes, alternative or adjuvant treatment with high-pressure chambers, red blood cell collection during surgery, heart failure, anoxia derived from chronic indications , Tissue transplantation, carbon monoxide, nitrogen oxides, and contain cyanide poisoning, but not limited to them.

  A human or animal suffering from one or more of the above disease states is treated by injecting about 0.5-6 units (1 unit = 500 ml) of IHP-treated blood prepared according to the present invention. In some cases, all of the patient's original blood can be substantially completely replaced with IHP-treated blood. The volume of IHP treated red blood cells administered to a human or animal depends on the indication being treated. In addition, the volume of IHP-treated erythrocytes is also dependent on the concentration of IHP-treated erythrocytes in the erythrocyte suspension. It will be appreciated that the amount of IHP red blood cells administered to a patient is not critical and can vary widely and still be effective.

  Red blood cells (RBC) that have been subjected to IHP encapsulation are similar to normal red blood cells except that they can transport 2-3 times more oxygen per unit to the tissue. Thus, the physician will choose to administer 1 unit of IHP encapsulated red blood cells rather than 2 normal red blood cells. Red blood cells treated with IHP will be prepared at a blood processing center in the same manner as current blood processing methods, except for the processing step of encapsulating IHP in cells.

  Although the invention has been described in detail with particular reference to the disclosed embodiments, it will be understood that various changes and modifications may be effected within the spirit and scope of the invention as set forth in the appended claims.

It is the schematic of the 1st embodiment of a continuous flow type encapsulation apparatus. It is the schematic of the 2nd embodiment of a continuous flow type encapsulation apparatus. 1 is a top view of a first embodiment of a flow electroporation chamber having electrodes. FIG. 1 is a top view of a first embodiment of a flow electroporation chamber with electrodes removed. FIG. 1 is a side view of a first embodiment of a flow electroporation chamber. FIG. 1 is an end view of a first embodiment of a flow electroporation chamber. FIG. 1 is a side view of an electrode used with a first embodiment of a flow electroporation chamber. FIG. It is a front view of the electrode of FIG. It is a disassembled perspective view of the 2nd embodiment of a flow electroporation chamber. FIG. 10 is a perspective view of a second embodiment of the flow electroporation chamber of FIG. 9 with the chamber assembled. 6 is a graph comparing the effect of various electric field strengths on% oxygenation of red blood cells encapsulated with IHP under fixed or flowing conditions. 2 is a table comparing the effect of various electric field strengths on P ₅₀ values of red blood cells encapsulated with IHP under fixed or flowing conditions. 2 is a table comparing the viability of erythrocytes subjected to electroporation at various electric field strengths under fixed or flowing conditions. FIG. 6 is a front elevation view of a support member of an electroporation chamber according to a third embodiment of the present invention. FIG. 15 is a cross-sectional view of the support member of FIG. 14 taken along line 15-15. FIG. 16 is an enlarged view of a portion indicated by a circle 16 in FIG. 15. FIG. 6 is an exploded perspective view of an electroporation chamber and a support column to which the chamber is attached according to a third embodiment. FIG. 18 is a perspective view showing the electroporation chamber of FIG. 17 attached to a support column. FIG. 6 is a front elevation view of an electroporation chamber according to a third embodiment attached to a support column. FIG. 20 is a cutaway perspective view of the electroporation chamber and support column of FIG. 19. FIG. 21 is a schematic view of a self-contained electroporation apparatus including the electroporation chamber of FIGS. It is a graph which shows the resistance of several types of IHP solutions. It is the schematic of the 3rd embodiment of a continuous flow type encapsulation apparatus. It is a cutaway figure of a cell washing device. FIG. 7 is a side view of a tube showing cell plate 526 with ridges forming an intricate path and recirculation of the cell suspension. It is a cutaway figure of 2nd embodiment of a cell washing apparatus. FIG. 3 is a cutaway side view of an elastomer chamber. It is a graph which shows the voltage of typical electroporation.

Claims (11)

(A) fluid flow path;
(B) an electrode disposed along the fluid flow path and moving along the fluid flow path is subjected to an electric field suitable for electroporation; and (c) the fluid flow path and fluid communication fault to pump,
Equipped with a,
Computer
i) controlling the speed of the pump so as to establish the flow rate of the fluid flow path in the apparatus for the sample processing rate; and
ii) controlling the pulse charge of the electrodes;
Electroporation device. The apparatus of claim 1, wherein the fluid flow path further comprises at least one wall that is elastically deformable. The apparatus of claim 1 or 2, further comprising an electrical pulse generator operably connected to the electrode. The apparatus according to claim 1, wherein the electrode comprises a continuous band electrode. The apparatus of claim 1, further comprising a cooling element in thermal communication with the fluid flow path. 6. The apparatus of claim 5, further comprising a sensor for measuring the temperature of biological particles moving along the fluid flow path and a computer responsive to the sensor for controlling the cooling element. Apparatus according to claim 5 or 6, wherein the cooling element is an array of thermoelectric cooling elements. 8. A device according to any one of the preceding claims, further comprising a valve arrangement for controlling movement of biological particles through the device. The apparatus according to claim 1, further comprising a support member that is removably attached to the apparatus. The electrode is wrapped around at least a portion of a spindle that rotates the electrode to extend beyond its tension limit, thereby breaking the electrode before the device is removed from the support member Inoperable,
The apparatus according to claim 9. 11. Apparatus according to any one of the preceding claims, wherein the sample processing rate corresponds to the interval of pulses of electrical energy supplied to the electrodes.

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