US 6,638,709 B2 · 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.

Claims10

Dependent claims

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 in a chamber, 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.

3 more elements

Dependencies are inferred from the claim text.

Processes for making cryopreserved composite living constructs and products resulting therefrom

Life of this patent

Processes for making cryopreserved composite living constructs and products resulting therefrom

Life of this patent

Abstract

Claims10