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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.degree. C.; and storing the CCLC at about -150.degree. 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.

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