Method for cryopreserving heart valves

Claim 1

A method of cryopreserving heart valves comprising the sequential steps of: a. dissecting a heart valve from a heart; b. placing the dissected heart valve into an isotonic medium with antibiotics and a cryopreservative; c. freezing the heart valve according to a freezing schedule that will maintain cell viability comprising the sequential steps of: (1) placing the dissected heart valve in a freezing chamber, (2) lowering the temperature of said freezing chamber at a rate of approximately 0.01° C./minute to a sample temperature of approximately +4° C.; (3) lowering the temperature of said freezing chamber at a rate of approximately 1.5° C./minute to a sample temperature of approximately -3° C.; (4) lowering the temperature of said freezing chamber at a rate of approximately 95° C./minute to a chamber temperature of approximately -140° C.; (5) holding the temperature of said freezing chamber at approximately -140° C. for approximately one minute; (6) raising the temperature of said freezing chamber to approximately -100° C. at a rate of approximately 20° C./minute; (7) holding the temperature of said freezing chamber at approximately -100° C. for approximately six minutes; (8) raising the temperature of said freezing chamber to approximately -70° C. at a rate of approximately 10° C./minute; (9) raising the temperature of said freezing chamber to approximately -26° C. at a rate of approximately 20° C./minute; (10) holding the temperature of said freezing chamber at approximately -26° C. for approximately 2 minutes; and (11) lowering the temperature of said freezing chamber at a rate of 1° C./minute until the temperature of the freezing chamber is approximately -80° C.; and d. transferring the heart valve to a storage receptacle maintained at a temperature below -132° C.

Claim 2

A method of cryopreserving heart valves comprising the sequential steps of: a. dissecting a heart valve from a heart; b. placing the dissected heart valve into an isotonic medium with antibiotics and a cryopreservative; c. freezing the heart valve according to a freezing schedule that will maintain cell viability comprising the sequential steps of: (1) placing the dissected heart valve in a freezing chamber, (2) lowering the temperature of said freezing chamber at a rate of approximately 0.01° C./minute to a sample temperature of approximately +4° C.; (3) lowering the temperature of said freezing chamber at a rate of approximately 1.5° C./minute to a sample temperature of approximately -3° C.; (4) lowering the temperature of said freezing chamber at a rate of approximately 95° C./minute to a chamber temperature of approximately -140° C.; (5) holding the temperature of said freezing chamber at approximately -140° C. for approximately one minute; (6) raising the temperature of said freezing chamber to approximately -100° C. at a rate of approximately 20° C./minute; (7) holding the temperature of said freezing chamber at approximately -100° C. for approximately six minutes; (8) raising the temperature of said freezing chamber to approximately -70° C. at a rate of approximately 10° C./minute; (9) raising the temperature of said freezing chamber to approximately -26° C. at a rate of approximately 20° C./minute; (10) holding the temperature of said freezing chamber at approximately -26° C. for approximately 2 minutes; and (11) lowering the temperature of said freezing chamber at a rate of 1° C./minute until the temperature of the freezing chamber is approximately -80° C.; and d. transferring the heart valve to a storage receptacle maintained at a temperature below -132° C.

Claim 6

A method of thawing a previously cryopreserved heart valve, comprising the sequential steps of: a. placing the frozen tissue in a sterile saline bath at a temperature of between approximately 37° C. and 42° C. for approximately 10 to 14 minutes to substantially thaw the frozen tissue; b. transferring the thawed tissue to a medium with approximately 7.5% by volume of cryopreservative for approximately 1 minute; c. transferring the thawed tissue to a medium with approximately 5% by volume of cryopreservative for approximately 1 minute; and d. transferring the thawed tissue to a medium with approximately 0% by volume of cryopreservative.

Claim 7

A method of thawing a previously cryopreserved heart valve, comprising the sequential steps of: a. placing the frozen tissue in a sterile saline bath at a temperature of between approximately 37° C. and 42° C. for approximately 10 to 14 minutes to substantially thaw the frozen tissue; b. transferring the thawed tissue to a medium with approximately 7.5% by volume of cryopreservative for approximately 1 minute; c. transferring the thawed tissue to a medium with approximately 5% by volume of cryopreservative for approximately 1 minute; and d. transferring the thawed tissue to a medium with approximately 0% by volume of cryopreservative.

Claim 8

A method of cryopreserving and thawing heart valves that will maintain cell viability, comprising the sequential steps of: a. dissecting a heart valve from a heart; b. placing the dissected heart valve into a isotonic medium with antibiotics and a cryopreservative; c. freezing the heart valve according to a freezing schedule comprising the sequential steps of: (1) placing the dissected heart valve in a freezing chamber, (2) lowering the temperature of said freezing chamber at a rate of approximately 0.01° C./minute to a sample temperature of approximately +4° C.; (3) lowering the temperature of said freezing chamber at a rate of approximately 1.5° C./minute to a sample temperature of approximately -3° C.; (4) lowering the temperature of said freezing chamber at a rate of approximately 95° C./minute to a chamber temperature of approximately -140° C.; (5) holding the temperature of said freezing chamber at approximately -140° C. for approximately one minute; (6) raising the temperature of said freezing chamber to approximately -100° C. at a rate of approximately 20° C./minute; (7) holding the temperature of said freezing chamber to approximately -100° C. for approximately six minutes; (8) raising the temperature of said freezing chamber to approximately -70° C. at a rate of approximately 10° C./minute; (9) raising the temperature of said freezing chamber to approximately -26° C. at a rate of approximately 20° C./minute; (10) holding the temperature of said freezing chamber to approximately -26° C. for approximately 2 minutes; and (11) lowering the temperature of said freezing chamber at a rate of 1° C./minute until the temperature of the freezing chamber is approximately -80° C.; (d) transferring the heart valve to a storage receptacle maintained at a temperature below -132° C.; and (e) thawing the frozen tissue by the sequential steps of: (1) placing the frozen tissue in a sterile saline bath at a temperature of between approximately 37° C. and 42° C. for approximately 10 to 14 minutes to substantially thaw the frozen tissue; (2) transferring the thawed tissue to a medium with approximately 7.5% by volume of cryopreservative for approximately 1 minute; (3) transferring the thawed tissue to a medium with approximately 5% by volume of cryopreservative for approximately 1 minute; and (4) transferring the thawed tissue to a medium with approximately 0% by volume of cryopreservative.

Claim 9

A method of cryopreserving and thawing heart valves that will maintain cell viability, comprising the sequential steps of: a. dissecting a heart valve from a heart; b. placing the dissected heart valve into a isotonic medium with antibiotics and a cryopreservative; c. freezing the heart valve according to a freezing schedule comprising the sequential steps of: (1) placing the dissected heart valve in a freezing chamber, (2) lowering the temperature of said freezing chamber at a rate of approximately 0.01° C./minute to a sample temperature of approximately +4° C.; (3) lowering the temperature of said freezing chamber at a rate of approximately 1.5° C./minute to a sample temperature of approximately -3° C.; (4) lowering the temperature of said freezing chamber at a rate of approximately 95° C./minute to a chamber temperature of approximately -140° C.; (5) holding the temperature of said freezing chamber at approximately -140° C. for approximately one minute; (6) raising the temperature of said freezing chamber to approximately -100° C. at a rate of approximately 20° C./minute; (7) holding the temperature of said freezing chamber to approximately -100° C. for approximately six minutes; (8) raising the temperature of said freezing chamber to approximately -70° C. at a rate of approximately 10° C./minute; (9) raising the temperature of said freezing chamber to approximately -26° C. at a rate of approximately 20° C./minute; (10) holding the temperature of said freezing chamber to approximately -26° C. for approximately 2 minutes; and (11) lowering the temperature of said freezing chamber at a rate of 1° C./minute until the temperature of the freezing chamber is approximately -80° C.; (d) transferring the heart valve to a storage receptacle maintained at a temperature below -132° C.; and (e) thawing the frozen tissue by the sequential steps of: (1) placing the frozen tissue in a sterile saline bath at a temperature of between approximately 37° C. and 42° C. for approximately 10 to 14 minutes to substantially thaw the frozen tissue; (2) transferring the thawed tissue to a medium with approximately 7.5% by volume of cryopreservative for approximately 1 minute; (3) transferring the thawed tissue to a medium with approximately 5% by volume of cryopreservative for approximately 1 minute; and (4) transferring the thawed tissue to a medium with approximately 0% by volume of cryopreservative.