Method and package design for cryopreservation and storage of cultured tissue equivalents

Abstract

This invention is directed to an efficient cryopreservation package design of harvested mammalian tissues and living cultured tissue equivalents made by in vitro technology. The invention involves immersing a mammalian tissue or cultured tissue equivalent in a cryoprotectant solution, agitating the cryoprotectant solution and the immersed tissue to achieve effective penetration of the cryoprotectant solution into the tissue, and then freezing the tissue at a very slow freezing rate. In the freezing step, extracellular ice formation is initiated by seeding. The cryopreserved tissue may be stored for indefinite periods of time prior to use. The cultured tissue equivalent is an in vitro model of the equivalent human tissue, such as skin or cornea, which, when retrieved from storage can be used for transplantation or implantation in vivo or for screening compounds in vitro.

Description

technical field

[0001] This application is a divisional application of Chinese patent application 96191673.7 (method and packaging design for cold storage and storage of cultured tissue equivalent) submitted on January 30, 1996.

[0002] The present invention relates to cryopreservation of harvested tissues and equivalents of cultured tissues obtained by in vitro techniques. The invention also relates to a refrigerated packaging design for harvested tissue and cultured tissue equivalents that is low cost, easy to handle, and maximizes the viability of the refrigerated tissue or tissue equivalent, with which refrigeration technology, whether refrigerated harvested Tissue or refrigerated cultured tissue, which may be stored indefinitely until use, is an in vitro specimen of the same human tissue that, when retrieved from storage, may be used for in vivo transplantation or implantation, or for in vitro screening of compounds. Background technique

[0003] In Vitro Techniques T...

Examples

example 1 to example 3

[0078] Examples 1 to 3 are refrigeration and thawing techniques with optimal packaging design of the present invention.

example 1

[0079] Example 1: Living skin with packaging design

[0080] Refrigeration of identical (LSE)

[0081] Live skin equivalent (LSE) constructs can be prepared according to US Patent Application Serial No. 08 / 193,809. Nine to 10 days after airlift, LSEs and ligated carrier inserts (TRANSWELL(R), Costar, Cambridge), were placed in 100 mm Petri dishes (Costar). Immerse the construct and transwell in 25 ml of cold storage solution, 2M glycerol in DMEM, in a 100 mm Petri dish for 1 hour to flood the LSE construct with the cold storage solution. During filling, place the construct in a 10% CO 2 Shake at 70 rpm for 1 hour on an orbital shaker (Bellco) in an air chamber. Shaking results in more complete filling and better reproducibility of the refrigeration method. After the LSE was filled with cryogenic fluid, the Petri dish containing LSE, vector insert, and extracellular cryogenic fluid (2M glycerol and DMEM) was placed in a refrigerated box and heat-sealed. In a co-...

example 2

[0085] Example 2: Thawing refrigerated LSE

[0086] The refrigerated LSE was removed from the vapor-phase liquid nitrogen storage, transferred to dry ice and heated to approximately -75°C. After equilibrating at -75°C, transfer the cultured tissue alike to a 37°C water bath; preferably a 4°C water bath; best left at room temperature. Once all of the cold storage solution is seen to be in the liquid phase, the cultured tissue equivalent packaging unit is preferably transferred to a biosafety cabinet, or other sterile location, and washed with ethanol. The lid of the Petri dish is removed by peeling off or the seat of the lid is cut from the bottom of the packaging unit to remove the lid. Excess refrigeration fluid is then poured off and the carrier containing the cultured tissue equivalent is transferred to a Petri dish. Add 25 ml of washing solution (room temperature), preferably DMEM, to the Petri dish containing the cultured tissue equivalent for about 30 minutes to wash aw...

technical field

[0001] This application is a divisional application of Chinese patent application 96191673.7 (method and packaging design for cold storage and storage of cultured tissue equivalent) submitted on January 30, 1996.

[0002] The present invention relates to cryopreservation of harvested tissues and equivalents of cultured tissues obtained by in vitro techniques. The invention also relates to a refrigerated packaging design for harvested tissue and cultured tissue equivalents that is low cost, easy to handle, and maximizes the viability of the refrigerated tissue or tissue equivalent, with which refrigeration technology, whether refrigerated harvested Tissue or refrigerated cultured tissue, which may be stored indefinitely until use, is an in vitro specimen of the same human tissue that, when retrieved from storage, may be used for in vivo transplantation or implantation, or for in vitro screening of compounds. Background technique

[0003] In Vitro Techniques T...

Examples

example 1 to example 3

[0078] Examples 1 to 3 are refrigeration and thawing techniques with optimal packaging design of the present invention.

example 1

[0079] Example 1: Living skin with packaging design

[0080] Refrigeration of identical (LSE)

[0081] Live skin equivalent (LSE) constructs can be prepared according to US Patent Application Serial No. 08 / 193,809. Nine to 10 days after airlift, LSEs and ligated carrier inserts (TRANSWELL(R), Costar, Cambridge), were placed in 100 mm Petri dishes (Costar). Immerse the construct and transwell in 25 ml of cold storage solution, 2M glycerol in DMEM, in a 100 mm Petri dish for 1 hour to flood the LSE construct with the cold storage solution. During filling, place the construct in a 10% CO 2 Shake at 70 rpm for 1 hour on an orbital shaker (Bellco) in an air chamber. Shaking results in more complete filling and better reproducibility of the refrigeration method. After the LSE was filled with cryogenic fluid, the Petri dish containing LSE, vector insert, and extracellular cryogenic fluid (2M glycerol and DMEM) was placed in a refrigerated box and heat-sealed. In a co-...

example 2

[0085] Example 2: Thawing refrigerated LSE

[0086] The refrigerated LSE was removed from the vapor-phase liquid nitrogen storage, transferred to dry ice and heated to approximately -75°C. After equilibrating at -75°C, transfer the cultured tissue alike to a 37°C water bath; preferably a 4°C water bath; best left at room temperature. Once all of the cold storage solution is seen to be in the liquid phase, the cultured tissue equivalent packaging unit is preferably transferred to a biosafety cabinet, or other sterile location, and washed with ethanol. The lid of the Petri dish is removed by peeling off or the seat of the lid is cut from the bottom of the packaging unit to remove the lid. Excess refrigeration fluid is then poured off and the carrier containing the cultured tissue equivalent is transferred to a Petri dish. Add 25 ml of washing solution (room temperature), preferably DMEM, to the Petri dish containing the cultured tissue equivalent for about 30 minutes to wash aw...

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