Method for processing and preserving collagen-based tissues for transplantation

Claim 2

The method of claim 1 further comprising the step of fixing said tissue with a cross-linking agent following processing.

Claim 3

The process of claim 1 further comprising the steps of cryopreparing, freezing and drying said tissue after processing.

Claim 4

The method of claim 3 further comprising the step of rehydrating said tissue to a water content of 20% to 70%.

Claim 5

The process of claim 4 further comprising the step of reconstituting said rehydrated tissue with viable cells.

Claim 6

The method of claim 3 further comprising the step of fixing said tissue with a cross-linking agent following drying.

Claim 7

The method of claim 1 wherein said collagen-based tissue is selected from the group consisting of skin, blood vessels, heart valves, ligaments, tendons, bone, cartilage, dura mater, nerves and other similar tissues derived from one or more mammals.

Claim 8

The method of claim 1 further comprising the step of incubating said tissue in a stabilizing solution following procurement.

Claim 9

The method of claim 8 wherein said stabilizing solution contains one or more of the following components: (a) antioxidant agents to prevent hypoxic damage selected from the group consisting of: tertiary butylhydroquinone, alpha tocopherol, mannitol, hydroxyurea, glutathione, ascorbate, ethylenediaminetetraacetic acid, the amino acids histidine, proline and cysteine and combinations thereof; (b) enzymatic agents to minimize damage resulting from hypoxia-related free-radical formation selected from the group consisting of: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and combinations thereof; (c) chemical agents to inhibit hypoxia-related biochemical pathway alterations selected from the group consisting of: allopurinol, lipoxigenase inhibitors, phospholipid methylation inhibitors, calcium channel blocking drugs, calcium binding agents, iron binding agents, metabolic intermediaries, substrates of adenosine triphosphate generation, and combinations thereof; (d) antibiotics selected from the group consisting of: penicillin, streptomycin, gentamicin, kanamycin, neomycin, polymyxin, bacitracin and combinations thereof; (e) antifungal agents selected from the group consisting of: nystatin, vancomycin, amphotericin B and combinations thereof; (f) protease inhibitors selected from the group consisting of: N-ethylmaleimide, phenylmethylsulfonyl fluoride, ethylenediamine tetraacetic acid, ethylene glycol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, ammonium chloride, apoprotinin, hydrogen ions, and combinations thereof; (g) proteoglycan oncotic agents selected from the group consisting of: chondroitin sulfate, heparin sulfate, dermatan sulfate and combinations thereof; (h) buffers selected from the group consisting of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, bicarbonate, potassium phosphate, sodium phosphate, acetate-citrate and combinations thereof; (i) chemical agents to inhibit platelet adhesion, aggregation and activation selected from the group consisting of: heparin, sodium nitroptusside, H7, isobutylmethylzanthine, episilon amino caprioc acid, salicylic acid, dipyridamole, dazoxiben, adenosine, prostacyclin, amiloride, amantadine and combinations thereof; (j) chemical agents to prevent smooth muscle contraction selected from the group consisting of: sodium nitroprusside, isoproterenol, phentolamine, pinacidil, H7, nifedepine, calcitonin gene related peptide, flurazine, papaverine isobutylmethylzanthine, and combinations thereof; and (k) oncotic agents selected from the group consisting of: dextran, glycine, proline and combinations thereof.

Claim 10

The method of claim 1 further comprising the step of incubating said tissue in a processing solution.

Claim 11

The method of claim 10 wherein said processing solution contains one or more of the following components: (a) salts selected from the group consisting of: sodium chloride, magnesium chloride, potassium phosphate and combinations thereof; (b) detergents selected from the group consisting of: polyethylene (100) glycoltert-octyl phenyl ether, polyoxyethylene (20) sorbitan mono-oleate, polyoxyethylene (80) sorbitan mono-oleate, sodium deoxycholate, 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate, octyl-glucoside, sodium dodecyl sulfate and combinations thereof; (c) enzymes selected from the group consisting of: dispase II, trypsin, hyaluronidase, thermolysin and combinations thereof; (d) protease inhibitors selected from the group consisting of N-ethylmaleimide, phenylmethylsulfonyl fluoride ethylenediamine tetraacetic acid, ethylene glycol-bis-(2-aminoethyl ether)-N, N, N', N'-tetraacetic acid leupeptin, ammonium chloride, hydrogen pH, apoprotinin and combinations thereof; and (e) buffers selected from the group consisting of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino) propanesulfonic acid, bicarbonate, potassium phosphate, sodium phosphate acetate-citrate and combinations thereof.

Claim 12

The method of claim 2 wherein said crosslinking agent is glutaraldehyde.

Claim 13

The method of claim 3 wherein said cryopreparation step comprises the incubation of said tissue in a cryopreservation solution followed by freezing of said tissue.

Claim 14

The method of claim 12 wherein said cryopreservation solution includes one or more cryoprotectants.

Claim 15

The method of claim 13 wherein said cryopreservation solution further comprises one or more dry protectants.

Claim 16

The method of claim 13 wherein said cryoprotectant solution contains a combination of an organic solvent and water which does not expand or contract during freezing.

Claim 17

The method of claim 13 wherein said cryoprotectant is selected from the group consisting of: dimethyl sulfoxide, 2-3 butane diol, polyvinylpyrrolidone, propylene glycol, 1,2 propanediol, glycerol, fructose, trehalose, raffinose, hydroxyethyl starch, dextran, sucrose, sorbitol, proline, human serum albumin and combinations thereof.

Claim 18

The method of claim 15 wherein said dry protectant is selected from the group consisting of: sucrose, raffinose, trehalose, zinc, proline, myristic acid, spermine and combinations thereof.

Claim 19

The method of claim 3 wherein said freezing step is achieved by cooling said tissue at rates between - 1°C per minute and -5,000°C per second to a final tissue temperature of less than -20°C.

Claim 20

The method of claim 3 wherein said drying is performed under conditions of temperature, vacuum, condenser surface orientation, condenser surface temperature and heating such that drying occurs below the glass transition of the least thermally stable ice crystal of the frozen tissue and sequentially so that each subsequently more stable ice crystal is dried in a like manner.

Claim 21

The method of claim 20, wherein said drying comprises freeze-drying at temperatures above -70°C.

Claim 22

The method of claim 20 wherein said drying comprises sequential phasic drying in the mid-temperature range of -130°C to -80°C and above.

Claim 23

The method of claim 20 wherein said drying comprises sequential phasic drying by molecular distillation drying at temperatures of -160°C to -90°C and above.

Claim 24

The method of claim 4 wherein said rehydration is achieved by incubating said tissue in a fluid rehydration solution.

Claim 25

The method of claim 24 wherein said rehydration solution includes a buffer selected from the group consisting of: normal saline, Ringer's lactate solution, cell culture media and combinations thereof.

Claim 26

The method of claim 24 wherein said rehydration solution contains agents selected from the group consisting of: (a) antioxidant agents to prevent hypoxic damage selected from the group consisting of: tertiary butylhydroquinone, alpha tocopherol, mannitol, hydroxyurea, glutathione, ascorbate, ethylenediaminetetraacetic acid, the amino acids histidine, proline and cysteine and combinations thereof; (b) enzymatic agents to miniminze damage resulting from hypoxia-related free-radical formation selected from the group consisting of: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and combinations thereof; (c) chemical agents to inhibit hypoxia-related biochemical pathway alterations selected from the group consisting of: allopurinol, lipoxigenase inhibitors, phospholipid methylation inhibitors, calcium channel blocking drugs, calcium binding agents, iron binding agents, metabolic intermediaries, substrates of adenosine triphosphate generation, and combinations thereof; (d) antibiotics selected from the group consisting of: penicillin, streptomycin, gentamicin, kanamycin, neomycin, polymyxin, bacitracin and combinations thereof; (e) antifungal agents selected from the group consisting of: mystatin, vancomycin, amphotericin B and combinations thereof; (f) protease inhibitors selected from the group consisting of: N-ethylmaleimide, phenylmethylsulfonyl fluoride, ethylenediamine tetraacetic acid, ethylene glycolbis-(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, ammonium chloride, apoprotinin, hydrogen ions, and combinations thereof; (g) proteoglycan oncotic agents selected from the group consisting of: chondroitin sulfate, heparin sulfate, dermatan sulfate and combinations thereof; (h) buffers selected from the group consisting of: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, bicarbonate, potassium phosphate, sodium phosphate, acetate-citrate and combinations thereof; (i) agents to inhibit platelet adhesion, aggregation and activation selected from the group consisting of: heparin, sodium nitroprusside, H7, isobutylmethylzanthine, episilon amino caprioc acid, aspirin, dipyridamole, dazoxiben, and adenosine. (j) oncotic agents selected from the group consisting of dextran, glycine and proline.

Claim 27

The method of claim 24 wherein the rehydration of said dried, cryoprepared, processed tissue further comprises the step of the addition of a vaporized rehydration solution followed by incubation in a fluid rehydration solution to form rehydrated tissue.

Claim 28

The method of claim 24 further comprising innoculating said rehydrated tissue with viable cells selected from the group consisting of: autogeneic cells, allogeneic cells or combinations thereof.

Claim 29

The method of claim 24 wherein said rehydration solution is a cross-linking agent.

Claim 30

The method of claim 29 wherein said crosslinking agent is glutaraldehyde.

Claim 32

The method of claim 30 wherein said cryoprotective agents include a vitrification solution, said vetrification solution comprising one or more components selected from the group consisting of: dimethylsulfoxide, propylene glycol, 2-3 butanediol, raffinose, sucrose, polyvinylpyrrolidone, dextran, trehalose and formamide.

Claim 33

The method of claim 31 wherein said rehydrated tissue comprises dermis.

Claim 34

The method of claim 31 wherein said rehydrated tissue comprises one or more vascular conduits of venous or arterial orgin.

Claim 35

The method of claim 31 wherein said rehydrated tissue comprises one or more heart valves.