Processes for making cryopreserved composite living constructs and products resulting therefrom

Abstract

Processes are described for making a cryopreserved Composite Living Construct (CCLC) as well as a corresponding thawed and rinsed CCLC, comprised of separated layers of cultured fibroblasts and cultured keratinocytes, wherein the percent of cells that are viable, i.e., the cell viability, of such CCLC is at least about 70%. The viable cell density in the CCLC is at least about 50% of that before cryopreservation. The storage stability of the CCLC is at least about 12 months. Additionally, the metabolic activity of thawed and rinsed CCLC is at least about 50% of the Composite Living Construct (CLC) before cryopreservation. The structural integrity of CCLC is substantially the same as the CLC before cryopreservation. The process for making the CCLC comprises the steps of: providing a collagen substrate comprised of a collagen sponge layer and a nonporous to cells, semipermeable collagen layer; seeding and culturing, in the presence of a cell growth medium, fibroblasts on and within the collagen sponge layer and keratinocytes on the nonporous to cells, semipermeable collagen layer, thereby providing a CLC; equilibrating the CLC, according to a defined equilibration program with a cryoprotectant solution comprising at least chondroitin sulfate and dimethylsulfoxide; lowering the temperature, according to a programmed rate, to about −90° C.; and storing the CCLC at about −150° C. or lower. The process for preparing the CCLC to treat wounds in humans and in animals additionally comprises programmed thawing as well as a rinsing sequence to substantially remove the cryoprotectants.

We claim:  
     
         1 . In the method for the cryopreservation of fibroblast cells and keratinocyte cells present in a matrix holding said cells wherein solutions comprising a non-cell-penetrating component and a cell-penetrating component are used as the cryoprotectant solutions and wherein said cells are equilibrated with said solutions and further wherein the cryoprotectant solution-equilibrated cells are subjected to freezing temperatures, the improvement which comprises the steps of 
 a) lowering the temperature from ambient temperature to about 4° C.,    b) lowering the temperature from about 4° C. to about −20° C. at a rate of about 1 ° C./minute and holding the temperature at about −20° C. for about 15 minutes,    c) lowering the temperature from about −20° C. to about −25° C. at a rate of about 0.5° C./minute and holding the temperature at about −25° C. for about 15 minutes,    d) lowering the temperature from about −25° C. to about −70° C. at a rate of about 1° C./minute,    e) lowering the temperature from about −70° C. to about −90° C. at a rate of about 5° C./minute,    f) storing at a temperature of about −150° C. or below.    
     
     
         2 . The method of  claim 1  wherein said non-cell-penetrating component is selected from the group consisting of a polysaccharide and glycosaminoglycans and the cell-penetrating component is selected from the group consisting of glycerol and dimethyl sulfoxide and wherein said equilibration takes place at about ambient temperature and further wherein the first step of equilibration comprises adding to said cells a first solution containing the non-cell-penetrating component and allowing the cells to equilibrate therewith followed by adding a second solution comprising the cell-penetrating component and the non-cell penetrating component to said cells and allowing the cells to equilibrate therewith.  
     
     
         3 . The method according to  claim 2  wherein the non-cell-penetrating component is chondroitin sulfate or dextran and the cell-penetrating component is dimethyl sulfoxide.  
     
     
         4 . The method according to  claim 3  wherein the first cryoprotectant solution comprises chondroitin sulfate at a basal concentration of about 2-3% and wherein the second cryoprotectant solution comprises a mixture of the first solution and dimethyl sulfoxide in the approximate ratio of 4:1.  
     
     
         5 . The method according to  claim 1  wherein following the chamber temperature lowering step at from about −70° C. to about −90° C., an additional step is performed which comprises holding the temperature of the freezing chamber at approximately −90° C. until the temperature of the matrix surface is between about −85° C. and −90° C. and thereafter storing the CCLC at a temperature of about −150° C. or lower.  
     
     
         6 . The method according to  claim 5  wherein the CCLC is stored at approximately −170° C. to −190° C.  
     
     
         7 . The method according to  claim 4  wherein the first cryoprotectant solution contains approximately 2-3% chondroitin sulfate and after equilibration therewith at room temperature an equal amount of a secondary cryoprotectant solution containing approximately 20% DMSO and 2-3% chondroitin sulfate is introduced into the system and equilibrated therewith.  
     
     
         8 . The method according to  claim 5  wherein the temperature lowering program is achieved through the use of the vapor phase of liquid nitrogen.  
     
     
         9 . The method according to  claim 1  wherein the CLC structure comprises a collagen sponge layer containing cultured human fibroblasts and a collagen layer that is semi-permeable and non-porous to biological cells and permeable to gases and non-cellular components upon which a cultured layer of human keratinocytes is present.  
     
     
         10 . The method according to  claim 5  wherein the CCLC comprises 
 a) a sponge first layer comprising a cross-linked collagen sponge, said first layer having upper and lower surfaces, said sponge containing cultured fibroblast cells therein,  
 b) a non-porous second layer comprising a high purity collagen essentially free of exogenous glycosaminoglycans, said second layer having upper and lower surfaces, the lower surface thereof being in contact with the upper surface of said first layer, and  
 c) a layer comprising cultured keratinocyte cells in contact with the upper surface of said non-porous collagen second layer.  
 
     
     
         11 . A cryopreserved composite living matrix prepared from an uncryopreserved matrix comprising fibroblasts and keratinocytes, said cryopreserved matrix comprising separated layers of cultured fibroblasts and cultured keratinocytes wherein said cells have a cell viability expressed as the percent of total number of viable cultured fibroblasts and keratinocytes of at least about 70% of the original number of viable cells originally present in the uncryopreserved matrix and a total number of all fibroblasts and keratinocytes that is at least about 50% of the original number of fibroblasts and keratinocytes originally present in the uncryopreserved matrix, a metabolic activity of viable cells that is at least 50% matrix of the original metabolic activity of the viable cells originally present in the uncryopreserved matrix structural integrity substantially familiar to that of the original matrix before cryopreservation and a storage stability of about at least twelve months.