Apparatus and Method For Electroporation of Biological Samples

Abstract

The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations in blood by electroporation to achieve therapeutically desirable changes in the physical characteristics of the various cell populations in blood.

1 . An electroporation chamber comprising a chamber for containing a suspension of cells to be electroporated; the chamber being at least partially defined by opposing oppositely chargeable electrodes; and wherein the thermal resistance of the chamber is less than approximately 10° C. per Watt. 
     
     
         2 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 9.5° C. per Watt. 
     
     
         3 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 9° C. per Watt. 
     
     
         4 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 8.5° C. per Watt. 
     
     
         5 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 9.5° C. per Watt. 
     
     
         6 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 8° C. per Watt. 
     
     
         7 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 7.5° C. per Watt. 
     
     
         8 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 7° C. per Watt. 
     
     
         9 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 6.5° C. per Watt. 
     
     
         10 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 6° C. per Watt. 
     
     
         11 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 5.5° C. per Watt. 
     
     
         12 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 5° C. per Watt. 
     
     
         13 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 4.5° C. per Watt. 
     
     
         14 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 4° C. per Watt. 
     
     
         15 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 3.5° C. per Watt 
     
     
         16 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 3° C. per Watt. 
     
     
         17 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 2.5° C. per Watt. 
     
     
         18 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 2° C. per Watt. 
     
     
         19 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is less than approximately 1.5° C. per Watt. 
     
     
         20 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is approximately 0.1° C. per Watt to 4° C. per Watt. 
     
     
         21 . The electroporation chamber of  claim 1 , wherein the thermal resistance of the chamber is approximately 1.5° C. per Watt to 2.5° C. per Watt. 
     
     
         22 . A method of electroporating a cell comprising flowing a suspension of cells to be electroporated into an electroporation chamber in accordance with  claim 1 , and electroporating said cells. 
     
     
         23 . The method of  claim 22 , wherein the thermal resistance of the electroporation chamber is less than approximately 9.5° C. per Watt. 
     
     
         24 . The method of  claim 23 , wherein the thermal resistance of the electroporation chamber is less than approximately 9° C. per Watt. 
     
     
         25 . The method of  claim 24 , wherein the thermal resistance of the electroporation chamber is less than approximately 8.5° C. per Watt. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 25 , wherein the thermal resistance of the electroporation chamber is less than approximately 8° C. per Watt. 
     
     
         28 . The method of  claim 27 , wherein the thermal resistance of the electroporation chamber is less than approximately 7.5° C. per Watt. 
     
     
         29 . The method of  claim 28 , wherein the thermal resistance of the electroporation chamber is less than approximately 7° C. per Watt. 
     
     
         30 . The method of  claim 29 , wherein the thermal resistance of the electroporation chamber is less than approximately 6.5° C. per Watt. 
     
     
         31 . The method of  claim 30 , wherein the thermal resistance of the electroporation chamber is less than approximately 6° C. per Watt. 
     
     
         32 . The method of  claim 31 , wherein the thermal resistance of the electroporation chamber is less than approximately 5.5° C. per Watt. 
     
     
         33 . The method of  claim 32 , wherein the thermal resistance of the electroporation chamber is less than approximately 5° C. per Watt. 
     
     
         34 . The method of  claim 33 , wherein the thermal resistance of the flow chamber electroporation chamber is less than approximately 4.5° C. per Watt. 
     
     
         35 . The method of  claim 34 , wherein the thermal resistance of the electroporation chamber is less than approximately 4° C. per Watt. 
     
     
         36 . The method of  claim 35 , wherein the thermal resistance of the electroporation chamber is less than approximately 3.5° C. per Watt. 
     
     
         37 . The method of  claim 36 , wherein the thermal resistance of the electroporation chamber is less than approximately 3° C. per Watt. 
     
     
         38 . The method of  claim 37 , wherein the thermal resistance of the electroporation chamber is less than approximately 2.5° C. per Watt. 
     
     
         39 . The method of  claim 38 , wherein the thermal resistance of the electroporation chamber is less than approximately 2° C. per Watt. 
     
     
         40 . The method of  claim 39 , wherein the thermal resistance of the flow chamber electro oration chamber is less than approximately 1.5° C. per Watt. 
     
     
         41 . The method of  claim 40 , wherein the thermal resistance of the electroporation chamber is approximately 0.1° C. per Watt to 4° C. per Watt. 
     
     
         42 . The method of  claim 41 , wherein the thermal resistance of the electroporation chamber is approximately 1.5° C. per Watt to 2.5° C. per Watt. 
     
     
         43 . An electroporation device comprising the electroporation chamber of  claim 1 , the chamber further defined as comprising:
 walls defining a flow channel having an electroporation zone configured to receive and to transiently contain a suspension of cells to be electroporated;   an inlet flow portal in fluid communication with the flow channel, whereby the suspension can be introduced into the flow channel through the inlet flow portal;   an outlet flow portal in fluid communication with the flow channel, whereby the suspension can be withdrawn from the flow channel through the outlet portal;   the walls defining the flow channel within the electroporation zone comprising a first electrode forming a substantial portion of a first wall of the flow channel and a second electrode forming a substantial portion of a second wall of the flow channel opposite the first wall, the first and second electrodes being such that when placed in electrical communication with a source of electrical energy an electric field is formed therebetween through which the suspension can flow.   
     
     
         44 .- 46 . (canceled) 
     
     
         47 . The device of  claim 43 , wherein the first and second electrodes are spaced from each other at least 1 mm. 
     
     
         48 . The device of  claim 43 , wherein the flow chamber has a ratio of combined electrode surface in contact with buffer to the distance between the electrodes of approximately 1 to 100. 
     
     
         49 . The device of  claim 43 , wherein the cells electroporated in the flow channel are not substantially thermally degraded thereby. 
     
     
         50 . An electroporation device comprising:
 a chamber for containing a suspension of cells to be electroporated;   the chamber being at least partially defined by opposing oppositely chargeable electrodes; and   wherein the chamber has a ratio of combined electrode surface in contact with buffer to the distance between the electrodes of approximately 1 to 100.   
     
     
         51 . The device of  claim 50 , wherein the ratio is approximately 1 to 90. 
     
     
         52 . The device of  claim 50 , wherein the ratio is approximately 1 to 80. 
     
     
         53 . The device of  claim 50 , wherein the ratio is approximately 1 to 70. 
     
     
         54 . The device of  claim 50 , wherein the ratio is approximately 1 to 60. 
     
     
         55 . The device of  claim 50 , wherein the ratio is approximately 1 to 50. 
     
     
         56 . An electroporation device comprising an electroporation chamber in accordance with  claim 1 , the chamber comprising:
 walls defining a flow channel configured to receive and to transiently contain a suspension of cells to be electroporated;   an inlet flow portal in fluid communication with the flow channel, whereby the suspension can be introduced into the flow channel through the inlet flow portal;   an outlet flow portal in fluid communication with the flow channel, whereby the suspension can be withdrawn from the flow channel through the outlet portal;   the walls defining the flow channel comprising a first electrode forming at least a portion of a first wall of the flow channel and a second electrode forming at least a portion of a second wall of the flow channel opposite the first wall, the first and second electrodes being such that when placed in electrical communication with a source of electrical energy an electric field is formed therebetween.   
     
     
         57 . The device of  claim 56 , wherein the thermal resistance of the flow channel is less than approximately 4° C. per Watt. 
     
     
         58 . The device of  claim 56 , wherein the thermal resistance of the flow channel is approximately 0.1° C. per Watt to 10° C. per Watt. 
     
     
         59 . The device of  claim 56 , wherein the thermal resistance of the flow channel is approximately 1.5° C. per Watt to 2.5° C. per Watt 
     
     
         60 . The device of  claim 56 , wherein the first and second electrodes are spaced from each other at least 1 mm. 
     
     
         61 . The device of  claim 56 , wherein the first and second electrodes are spaced from each other at least 3 mm. 
     
     
         62 . The device of  claim 56 , wherein the flow channel has a ratio of combined electrode surface in contact with buffer to the distance between the electrodes of approximately 1 to 100. 
     
     
         63 . The device of  claim 56 , wherein the flow channel has a ratio of combined electrode surface in contact with buffer to the distance between the electrodes of approximately 1 to 100 and wherein the first and second electrodes are spaced from each other at least 1 mm. 
     
     
         64 . The device of  claim 56 , wherein the flow channel has a ratio of combined electrode surface in contact with buffer to the distance between the electrodes of approximately 1 to 100 and wherein the first and second electrodes are spaced from each other at least 3 mm. 
     
     
         65 . The device of  claim 56 , wherein the flow channel has a ratio of combined electrode surface in contact with buffer to the distance between the electrodes of approximately 1 to 100 and wherein the first and second electrodes are spaced from each other approximately 3 mm to approximately 2 cm. 
     
     
         66 . The device of  claim 56 , wherein the cells electroporated in the flow channel are not substantially thermally degraded thereby. 
     
     
         67 . An electroporation device comprising an electroporation chamber in accordance with  claim 1 , the chamber comprising:
 walls defining a flow channel configured to receive and to transiently contain a a suspension comprising particles;   an inlet flow portal in fluid communication with the flow channel, whereby the suspension can be introduced into the flow channel through the inlet flow portal;   an outlet flow portal in fluid communication with the flow channel, whereby the suspension can be withdrawn from the flow channel through the outlet flow portal;   the walls defining the flow channel comprising a first electrode plate forming a first wall of the flow channel and a second electrode plate forming a second wall of the flow channel opposite the first wall; wherein the area of the electrodes contact with the suspension, and the distance between the electrodes is chosen so that the thermal resistance of the flow channel is less than approximately 4° C. per Watt.   the paired electrodes placed in electrical communication with a source of electrical energy, whereby an electrical field is formed between the electrodes;   whereby the suspension of the particles in the flow channel can be subjected to an electrical field formed between the electrodes.   
     
     
         68 . The device of  claim 67 , wherein the electrode plates defining the flow channel further comprises: a gasket formed from an electrically non-conductive material and disposed between the first and second electrode plates to maintain the electrode plates in spaced-apart relation, the gasket defining a channel therein forming opposed side walls of the flow channel. 
     
     
         69 . The device of  claim 67 , wherein the gasket forms a seal with each of the first and second electrode plates. 
     
     
         70 . The device of  claim 67 , wherein the device comprises a plurality of flow channels, and wherein the gasket comprises a plurality of channels forming opposed side walls of each of the plurality of channels. 
     
     
         71 . The device of  claim 67 , wherein one of the inlet flow portal and the outlet flow portal comprises a bore formed in one of the electrode plates and in fluid communication with the flow channel. 
     
     
         72 . The device of  claim 67 , wherein the other of the inlet flow portal and the outlet flow portal comprises a bore formed in the one of the electrode plates and in fluid communication with the flow channel. 
     
     
         73 . The device of  claim 67 , wherein the other of the inlet flow portal and the outlet flow portal comprises a bore formed in the other of the electrode plates and in fluid communication with the flow channel. 
     
     
         74 . The device of  claim 67 , further comprising a cooling element operatively associated with the flow channel to dissipate heat. 
     
     
         75 . The device of  claim 67 , wherein the cooling element comprises a thermoelectric cooling element. 
     
     
         76 . The device of  claim 67 , wherein the cooling element comprises a cooling fluid flowing in contact with the electrode. 
     
     
         77 . The device of  claim 67 , wherein the cooling element comprises a heat sink operatively associated with the electrode. 
     
     
         78 . The device of  claim 67 , wherein the heat resistance of the flow channel is less than approximately 3° C. per watt. 
     
     
         79 . The device of  claim 67 , wherein the heat resistance of the flow channel is less than approximately 2° C. per watt. 
     
     
         80 . The device of  claim 67 , wherein the heat resistance of the flow channel is between approximately 0.5° C. per Watt and 4° C. per Watt. 
     
     
         81 . The device of  claim 67 , wherein the heat resistance of the flow channel is between approximately 1° C. per Watt and 3° C. per Watt. 
     
     
         82 . The device of  claim 67 , wherein the heat resistance of the flow channel is between approximately 1.5° C. and 2.5° C. 
     
     
         83 . The device of  claim 67 , wherein the first electrode comprises an elongated, electrically conductive structure,
 wherein the second electrode comprises a tubular, electrically conductive structure;   wherein the electrodes are concentrically arranged such that the second, tubular electrode surrounds the first electrode in spaced-apart relation thereto; and   wherein the flow channel is disposed within an annular space defined between the first and second electrodes.   
     
     
         84 . The device of  claim 83 , wherein the electrodes form at least a portion of the walls defining the flow channel. 
     
     
         85 . The device of  claim 83 , further comprising concentric annular spacers for maintaining the first and second electrodes in spaced-apart, concentric relation. 
     
     
         86 . The of  claim 83 , wherein the device is arranged in series with a second, like device. 
     
     
         87 . The of  claim 83 , wherein the device is arranged in parallel with a second, like device. 
     
     
         88 . A method of transfecting a cell comprising providing a desired therapeutic agent, protein or peptide, or nucleic acid or an expression vector coding for a desired protein or peptide and introducing the therapeutic agent, protein, peptide, nucleic acid or expression vector and the cell into an electroporation chamber in accordance with  claim 1 , and subjecting them to electroporation. 
     
     
         89 .- 103 . (canceled) 
     
     
         104 . The method of  claim 88 , where and the desired nucleic acid or protein is b-cell differentiation factor, b-cell growth factor, mitogenic cytokine, chemotactic cytokine, colony stimulating factor, angiogenesis factor, cadherin, selectin, integrin, NCAM, ICAM, L1, t-cell replacing factors, differentiation factor, transcription factor, mRNA, heat shock protein, nuclear protein complex, RNA/DNA oligomer, IFN-alpha, IFN-beta, IFN-omega, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, leptin, myostatin, macrophage stimulating protein, platelet-derived growth factor, TNF-alpha, TNF-beta, NGF, CD40L, CD137L/4-1BBL, human lymphotoxin-beta, TNF-related apoptosis-inducing ligand, monoclonal antibody, fragments of monoclonal antibody, G-CSF, M-CSF, GM-CSF, PDGF, IL1-alpha, IL1-beta, FGF IFN-gamma, IP-10, PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, fibroblast growth factor, VEGF, or soluble receptor and any fragments or combinations thereof. 
     
     
         105 . The method of  claim 88 , wherein the desired nucleic acid or protein is erythropoietin or fragments thereof. 
     
     
         106 . The method of  claim 88 , wherein the desired nucleic acid or protein is endostatin or fragments thereof. 
     
     
         107 . The method of  claim 88 , wherein the desired nucleic acid or protein is angiostatin or fragments thereof. 
     
     
         108 . The method of  claim 88 , wherein the desired nucleic acid or protein is IL12 or fragments thereof. 
     
     
         109 . The method of  claim 88 , wherein the desired nucleic acid or protein is IL2 or fragments thereof. 
     
     
         110 . The method of  claim 88 , comprising the further step of administering the transfected cells to a patient. 
     
     
         111 .- 115 . (canceled) 
     
     
         116 . The method of  claim 88 , wherein the therapeutic agent is AGM-1470 (TNP-470), MetAP-2; growth factor antagonists, antibodies to growth factors; growth factor receptor antagonists; TIMP, batimastat, marimastat; genistein SU5416; alphaVbeta3/5, retinoic acid fenretinide, 11-epihydrocortisol, corteloxone, tetrahydrocortisone and 17-hydroxyprogesterone; staurosporine, MDL 27032; 22-oxa-1 alpha, and 25-dihydroxyvitamin D3; indomethacin and sulindac; minocycline; thalidomide and thalidomide analogs and derivatives; 2-methoxyestradiol; tumor necrosis factor-alpha; interferon-gamma-inducible protein 10 (IP-10); interleukin 1 and interleukin 12; interferon alpha, beta or gamma; angiostatin protein or plasminogen fragments; endostatin protein or collagen 18 fragments; proliferin-related protein; group B streptococcus toxin; CM 101; CAI; troponin I; squalamine; L-NAME; thrombospondin; wortmannin; amiloride; spironolactone; ursodeoxycholic acid; bufalin; suramin; tecogalan sodium; linoleic acid; captopril; irsogladine; FR-118487; triterpene acids; castanospermine; leukemia inhibitory factor; lavendustin A; platelet factor-4; herbimycin A; diaminoanthraquinone; taxol; aurintricarboxylic acid; DS-4152; pentosan polysulphite; radicicol; fragments of human prolactin; erbstatin; eponemycin; shark cartilage; protamine; Louisianin A, C and D; PAF antagonist WEB 2086; auranofin; ascorbic ethers; or sulfated polysaccharide D 4152. 
     
     
         117 .- 126 . (canceled) 
     
     
         127 . An electroporation device comprising an electroporation chamber, the chamber comprising:
 walls defining a flow channel configured to receive and to transiently contain a continuous flow of a suspension of cells to be electroporated;   an inlet flow portal in fluid communication with the flow channel, whereby the suspension can be introduced into the flow channel through the inlet flow portal;   an outlet flow portal in fluid communication with the flow channel, whereby the suspension can be withdrawn from the flow channel through the outlet portal;   the walls defining the flow channel comprising a first electrode forming at least a portion of a first wall of the flow channel and a second electrode forming at least a portion of a second wall of the flow channel opposite the first wall, the first and second electrodes being such that when placed in electrical communication with a source of electrical energy an electric field is formed there between through which the suspension can flow; and   wherein said first electrode is spaced from said second electrode by a distance greater than 3 mm.   
     
     
         128 . The electroporation device of  claim 127 , wherein said first electrode is spaced from said second electrode by a distance of approximately 4 mm to 2 cm. 
     
     
         129 . The electroporation device of  claim 127 , wherein said first electrode is spaced from said second electrode by a distance of approximately 5 mm to 1 cm.