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.

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.

Abstract: This invention describes a collagen construct uniquely suited as a substrate for production of biologically active wound dressings, skin equivalents or skin substitutes. The collagen construct of the invention comprises a porous collagen sponge and a cell-impermeable transitional layer between the sponge and the boundary surface of the cell-impermeable transitional layer. Optionally, a further layer conducive to the attachment of cells, e.g. keratinocytes, may be coated as a third layer on the cell-impermeable layer. The sponge layer may be seeded with dermal cells, or other suitable cells and the boundary surface or the third layer may be seeded with epithelial cells, e.g. keratinocytes. The process used for the production of this construct also is described.

Abstract: Skin equivalents and processes for preparing them are described. The living skin equivalent comprises a layer of cultured keratinocyte cells, a layer of high purity, non-porous collagen and a dermal layer of cultured fibroblast cells in a porous, cross-linked collagen sponge. Processes are described for producing the skin equivalent with descriptions on how to obtain and treat skin precursor materials to yield suitable fibroblasts and keratinocytes.The collagen layers, the physical forms thereof, and treatments thereof are also described. Preferably, the non-porous, highly-purified collagen is selected from Type 1, Type 3, or mixtures of Type 1 and Type 3 collagen. The collagen is purified ideally by treatment with pepsin, to remove antigenic substances.The collagen sponge used can be any suitable form of collagen sponge which will support fibroblast growth.The keratinocytes used in the invention are preferably prepared by the "drop" method spotted evenly on culture media and incubated to coalescence.