Cryopreservation of cultured skin or cornea equivalents with agitation

Claim 1

A method for vitrifying a cultured skin or cornea equivalent, comprising: (a) serially immersing said cultured skin or cornea equivalent in a series of cryoprotectant solutions which increase in concentration and decrease in temperature to produce a cryoprotected cultured skin or cornea equivalent; (b) cooling said cryoprotected cultured skin or cornea equivalent to a temperature at or below -110° C., at a cooling rate at -10° C. per minute or faster to produce a vitrified cultured skin or cornea equivalent; and (c) storing said vitrified cultured skin or cornea equivalent at a temperature of -110° C. or below to produce a stored cultured skin or cornea equivalent, wherein one or more members of said series of cryoprotectant solutions of step (a) which increase in concentration and decrease in temperature containing the immersed cultured skin or cornea equivalent is agitated under conditions sufficient to achieve effective penetration of said cryoprotectant solution into said cultured skin or cornea equivalent prior to storage.

Claim 2

A method for vitrifying a cultured skin or cornea equivalent, comprising: (a) serially immersing said cultured skin or cornea equivalent in a series of cryoprotectant solutions which increase in concentration and decrease in temperature to produce a cryoprotected cultured skin or cornea equivalent; (b) cooling said cryoprotected cultured skin or cornea equivalent to a temperature at or below -110° C., at a cooling rate at -10° C. per minute or faster to produce a vitrified cultured skin or cornea equivalent; and (c) storing said vitrified cultured skin or cornea equivalent at a temperature of -110° C. or below to produce a stored cultured skin or cornea equivalent, wherein one or more members of said series of cryoprotectant solutions of step (a) which increase in concentration and decrease in temperature containing the immersed cultured skin or cornea equivalent is agitated under conditions sufficient to achieve effective penetration of said cryoprotectant solution into said cultured skin or cornea equivalent prior to storage.

Claim 3

A method for vitrifying a cultured skin or cornea equivalent, comprising: (a) serially immersing said cultured skin or cornea equivalent in a series of cryoprotectant solutions which increase in concentration and decrease in temperature to produce a cryoprotected cultured skin or cornea equivalent at a final temperature of from -10° C. to -30° C.; (b) cooling said cryoprotected cultured skin or cornea equivalent to a temperature at or below -110° C. at a cooling rate at of -10° C. per minute or faster to produce a vitrified cultured skin or cornea equivalent; and (c) storing said vitrified cultured skin or cornea equivalent at a temperature of -110° C. or below to produce a stored cultured skin or cornea equivalent, wherein one or more members of said series of cryoprotectant solutions of step (a) which increase in concentration and decrease in temperature containing the immersed cultured skin or cornea equivalent is agitated under conditions sufficient to achieve effective penetration of said cryoprotectant solution into said cultured skin or cornea equivalent prior to storage.

Claim 4

A method for vitrifying a cultured skin or cornea equivalent, comprising: (a) serially immersing said cultured skin or cornea equivalent in a series of cryoprotectant solutions which increase in concentration and decrease in temperature to produce a cryoprotected cultured skin or cornea equivalent at a final temperature of from -10° C. to -30° C.; (b) cooling said cryoprotected cultured skin or cornea equivalent to a temperature at or below -110° C. at a cooling rate at of -10° C. per minute or faster to produce a vitrified cultured skin or cornea equivalent; and (c) storing said vitrified cultured skin or cornea equivalent at a temperature of -110° C. or below to produce a stored cultured skin or cornea equivalent, wherein one or more members of said series of cryoprotectant solutions of step (a) which increase in concentration and decrease in temperature containing the immersed cultured skin or cornea equivalent is agitated under conditions sufficient to achieve effective penetration of said cryoprotectant solution into said cultured skin or cornea equivalent prior to storage.

Claim 5

A method for vitrifying a cultured skin or cornea equivalent, comprising: (a) immersing said cultured skin or cornea equivalent in a first cryoprotectant solution having a concentration of from 15% to 35% cryoprotectant solution in base media at room temperature for a period of time of from 10 to 20 minutes to produce a first cryoprotected cultured skin or cornea equivalent; (b) immersing said first cryoprotected cultured skin or cornea equivalent of step (a) in a second cryoprotectant solution having a concentration of from 40% to 60% cryoprotectant solution in base media at a temperature of from 10° C. to -2° C. for a period of time of from 10 to 20 minutes to produce a second cryoprotected cultured skin or cornea equivalent; (c) finally immersing said second cryoprotected cultured skin or cornea equivalent of step (b) in a third cryoprotectant solution having a concentration of 100% cryoprotectant solution at a temperature of from -10° C. to -30° C. for a period of time of from 10 to 20 minutes to produce a third cryoprotected cultured skin or cornea equivalent; (d) cooling said third cryoprotected cultured skin or cornea equivalent of step (c) to a temperature at or below -110° C. at a cooling rate of -10° C. per minute or faster to produce a vitrified cultured skin or cornea equivalent; and (e) storing said vitrified cultured skin or cornea equivalent at a temperature at or below -110° C. to produce a stored cultured skin or cornea equivalent, wherein said cultured skin or cornea equivalent of at least step (c) and said cryoprotectant solution are agitated under conditions sufficient to achieve effective penetration of said cryoprotectant solution into said cultured skin or cornea equivalent.

Claim 6

A method for vitrifying a cultured skin or cornea equivalent, comprising: (a) immersing said cultured skin or cornea equivalent in a first cryoprotectant solution having a concentration of from 15% to 35% cryoprotectant solution in base media at room temperature for a period of time of from 10 to 20 minutes to produce a first cryoprotected cultured skin or cornea equivalent; (b) immersing said first cryoprotected cultured skin or cornea equivalent of step (a) in a second cryoprotectant solution having a concentration of from 40% to 60% cryoprotectant solution in base media at a temperature of from 10° C. to -2° C. for a period of time of from 10 to 20 minutes to produce a second cryoprotected cultured skin or cornea equivalent; (c) finally immersing said second cryoprotected cultured skin or cornea equivalent of step (b) in a third cryoprotectant solution having a concentration of 100% cryoprotectant solution at a temperature of from -10° C. to -30° C. for a period of time of from 10 to 20 minutes to produce a third cryoprotected cultured skin or cornea equivalent; (d) cooling said third cryoprotected cultured skin or cornea equivalent of step (c) to a temperature at or below -110° C. at a cooling rate of -10° C. per minute or faster to produce a vitrified cultured skin or cornea equivalent; and (e) storing said vitrified cultured skin or cornea equivalent at a temperature at or below -110° C. to produce a stored cultured skin or cornea equivalent, wherein said cultured skin or cornea equivalent of at least step (c) and said cryoprotectant solution are agitated under conditions sufficient to achieve effective penetration of said cryoprotectant solution into said cultured skin or cornea equivalent.

Claim 7

The method of any one of claims 1, 2 or 3, wherein said cryoprotectant solution comprises one or more agents selected from the group consisting of: a cell-penetrating, glass-forming agent and a non cell-penetrating, glass-forming agent.

Claim 13

The method of any one of claims 1, 2, or 3, wherein said stored skin or cornea equivalent of step (c) is stored in liquid nitrogen at a temperature of -196° C.

Claim 15

The method of any one of claims 1, 2 or 3, further comprising: thawing said stored cultured tissue equivalent at a rate effective to produce a thawed viable cultured tissue equivalent.

Claim 18

The method of any one of claims 1, 2 or 3, wherein said cooling rate is from -10° C. per minute to -30° C. per minute.

Claim 19

The method of any one of claims 1, 2 or 3, wherein said cultured tissue equivalent is sterilely vacuum sealed in a protective pouch prior to said step of cooling.