the process for making the preferred clc , which is to be cryopreserved by the methods of this invention , is herein briefly described with reference to a flow scheme of the process , fig1 . the process has been described in detail in u . s . pat . nos . 5 , 282 , 859 , re 35 , 399 and 6 , 039 , 859 , to eisenberg , which are entirely incorporated herein by reference . the process for making the clc as described more fully therein and used in this invention comprises : treating a skin sample enzymatically to separate epidermis from dermis ; treating the epidermis enzymatically , preferably with trypsin , to release the keratinocyte cells ; treating the dermis enzymatically , preferably with collagenase , to release the fibroblast cells . one surface of a crosslinked collagen sponge is coated with a layer of high purity nonporous - to - cells , semipermeable collagen , to form a layered collagen substrate ; seeding , i . e . inoculating , the porous , crosslinked collagen sponge with fibroblasts ; culturing , i . e ., incubating , the seeded collagen sponge in the presence of growth medium to allow growth of the fibroblast cells on and within the collagen sponge ; seeding the coated side of the collagen sponge with keratinocytes and culturing in presence of growth medium to yield a clc comprising a collagen matrix with fibroblasts and keratinocytes . various media are used in this invention for operations such as culturing the skin - derived fibroblasts and keratinocytes , seeding and culturing the fibroblasts and keratinocytes , respectively , onto the collagen sponge and the nonporous - to - cells , semipermeable layer of the collagen substrate , and for further culturing of the construct to the point at which it is ready for cryopreservation . such media comprise dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), well - known to those skilled in the art , and supplements such as growth factors and other nutrient components . the preferred growth medium for the culturing of the clc used in this invention , is “ complete dmem ” ( cdmem ) ( see solution a ) which contains , in addition to dmem , components such as fetal bovine serum , recombinant human epidermal growth factor , hydrocortisone , l - glutamine , cholera toxin , nonessential amino acids ( neaa ), hepes ( a buffer ) and glucose , preferably at a concentration of about 4 g / l . after use , the growth medium ( the conditional or spent medium ) is removed and fresh growth medium is added periodically during culturing . a preferred medium for rinsing at the end of culturing is dmem + 2 ( see solution b ), that is , dmem plus l - glutamine plus non - essential amino acids ( neaa ). the process for making the cclc of this invention comprises : equilibrating the clc according to an equilibration program with a cryoprotectant solution comprising at least chondroitin sulfate and dimethyl sulfoxide as previously noted ; lowering the temperature , according to a temperature lowering program , from about ambient temperature to at least about − 90 ° c . and storing at a temperature below about − 150 ° c . it should be noted that details given in the following discussion are meant to serve as examples of how , in general and in preferred modes , the various steps of the processes are carried out and to describe examples of products that result from such processes and are not meant to exclude alternative methods that may logically be used , and the products resulting therefrom , by those skilled in the art who might practice this invention . the following immediate discussion is given with reference to fig2 a flow scheme of the process for making the cclc of this invention . the solutions component conc . solution a cdmem ( hg ) fbs 10 % v / v l - glutamine 5 . 98e + 00 mm rhegf 3 . 04e − 02 ug / ml hc 4 . 01e − 01 ug / ml ct 9 . 69e − 10 mm 1m hepes 3 . 07e − 03 m 1n naoh 1 . 00e − 03 n 45 % glucose 2 . 98e + 00 g / l l - alanine 100 . 2 um l - asparagine . h2o 100 . 2 um l - aspartic acid 100 . 2 um l - glutamic acid 100 . 2 um glycine 100 . 2 um l - proline 100 . 2 um l - serine 100 . 2 um dmem solution b dmem + 2 l - glutamine 5 . 8 um neaa l - alanine 96 . 2 um neaa l - asparagine . h2o 96 . 2 um neaa l - aspartic acid 96 . 2 um neaa l - glutamic acid 96 . 2 um neaa glycine 96 . 2 um neaa l - proline 96 . 2 um neaa l - serine 96 . 2 um dmem solution c primary cryo solution 1m hepes 2 . 50e − 02 m sodium biocarbonate , 7 . 5 % 2 . 00e − 03 w / v folic acid 4 . 00e − 03 mg / ml l - glutamine 4 . 0 mm 1m sodium hydroxide 4 . 40e − 03 m chondroitin sulfate 2 . 50e − 02 w / v dmem solution d secondary cryo solution primary cryo solution + 20 % v / v dmso the clc described above is rinsed with a protein - free dmem + 2 ( solution b ) to remove conditioned medium , agitated , and then incubated for a period of time , e . g ., about 30 minutes . the dmem + 2 in contact with the clc is then replaced with fresh dmem + 2 and agitated . this procedure is repeated , preferably at least about two more times . the clc is then incubated in dmem + 2 , preferably for less then eight hours , until it can be placed in a primary package preparatory to cryopreservation . the rinsed clc is removed from incubation and is placed in a liquid permeable tray between layers of medical grade nonadherent polyethylene net or medical grade gauze . the clc , in its support , is placed within a cryogenically compatible package , the package being fitted with fluid access and removal means . the first cryogenically compatible package , now containing the clc within the support , is sealed by means such as heat . two solutions are prepared in low bicarbonate dmem ( lb - dmem ) comprising dmem , folic acid , l - glutamine , sodium bicarbonate , naoh and hepes . the first solution ( solution c ) is the primary cryoprotectant solution comprising chondroitin sulfate at a basal concentration of about 2 . 5 % with a suitable range of about 2 - 3 %. the second cryoprotectant solution ( solution d ) comprising an approximately 4 : 1 mixture of the primary solution and dimethyl sulfoxide . the solutions are preferably prepared and then stored at temperatures below room temperature to help dissipate the heat of solution formed during the preparation . once prepared , the cryoprotectant solutions are preferably used at room temperature . the primary cryoprotectant solution is admitted through the fluid access means to the package , preferably by a pumping means , while the package is being agitated by means such as a shaking platform , to form an initial equilibrated clc . any non - contaminating pumping means may be used such as a peristaltic pump . the temperature of the primary cryoprotectant solution is introduced into the package at between 5 - 20 ° c ., preferably at room temperature . the secondary cryoprotectant solution ( solution d ) then is admitted through the fluid access means to the package with agitation , in a manner similar to that used for the primary cryoprotectant solution , to form the final equilibrated clc , said clc having a chondroitin sulfate concentration of about 2 . 5 % and dimethyl sulfoxide concentration of about 10 %. the secondary cryoprotectant solution admitted to the package is between about 5 - 20 ° c ., preferably at room temperature . residual air and excess cryoprotectant solution is withdrawn from the package by pumping means , preferably by a peristaltic pump . all fluid access means then are closed or otherwise sealed , e . g ., heat sealing and removed . the package containing the ceclc is sealed , e . g ., by thermal or ultrasonic means , and placed within a cryogenically compatible overwrap to provide a double - packaged clc which is then placed in a heat - conducting , e . g . metal , preferably aluminum , canister . the temperature of the double - packaged clc then is lowered preferably using the vapor phase of liquid nitrogen according to a temperature lowering program . the temperature lowering program may be implemented by means of microprocessor - controlled freezing chambers . a temperature probe is employed to monitor the temperature of the freezing chamber and a second probe monitors the surface of the clc . in practice , it is preferred to monitor the temperature in the ambient space around the clc with the vapor phase of the liquid nitrogen as opposed to the temperature of the clc itself . thus , the temperature recited in the freezing programs herein is that in the freezing chamber unless otherwise stated . the preferred temperature lowering program comprises the steps of : lowering the temperature from ambient temperature to about 4 ° c . ; 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 ; 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 ; lowering the temperature from about − 25 ° c . to about − 70 ° c . at a rate of about 1 ° c ./ minute ; and lowering the temperature from about − 70 ° c . to about − 90 ° c . at a rate of about 5 ° c ./ minute . at this point , it is preferred to hold the temperature of the freezing chamber at about − 90 ° c . until the temperature at the clc surface is between about − 85 ° c . to − 90 ° c . the cclc is then stored below about − 150 ° c . and preferably at about − 170 ° c . to − 190 ° c . in the vapor phase of liquid nitrogen . [ 0048 ] fig3 depicts a flow scheme of the processes for thawing and rinsing the cclc of this invention . the process comprises two steps , the first of which is to allow the frozen cclc to warm up to about − 100 ° c . by allowing it to stand at room temperature . the second step involves thawing the frozen cclc to a temperature above freezing by warming it in room temperature saline or water and thereafter rinsing the cclc substantially free of cryoprotectants . in practice , the double - packaged cclc is removed from vapor phase liquid nitrogen storage in the metal canister and permitted to warm for about four minutes at about ambient temperature , to the first higher temperature usually about − 100 ° c . the double - packaged cclc is further warmed for about five minutes in about ambient temperature water or saline to the second higher temperature , which is in the range from about 4 ° c . to about ambient temperature . the cclc is removed from its outer package and transferred to the sterile or clean field . the cclc is then removed from the primary package while in its liquid permeable support , placed in normal saline at about ambient temperature , with agitation , for about ten minutes , thereby rinsing it to substantially remove the cryoprotectants . alternatively , the rinsing step may be performed in two or more steps by removing the saline rinse solution after five minutes or so of agitation and replacing it with fresh saline and repeating the agitation for another five minutes or less . the following in vitro assays are used to assess the immediate and long - term effects of cryopreservation , thawing and rinsing processes on cclc . the assays include cryoprotectant residuals , cell number , percent cell viability , metabolic activity , histologic examination and cytokine expression . samples for assaying cclc are taken using appropriately sized diameter biopsy punches selected randomly from the cclc device . biopsy punch samples taken from the clc or the cclc are enzymatically digested to release fibroblasts and keratinocytes from the collagen matrix . the percent cell viability and cell number are then determined by a hemocytometer using trypan blue dye exclusion to differentiate living from dead cells . metabolic activity is a measure of the overall physiologic state of viable cells . biopsy punch samples , taken from the clc or the cclc , are incubated with alamar blue dye . the assay measures mitochondrial activity using a non - cytotoxic alamar blue dye which diffuses into the cell mitochondria and undergoes a reduction - oxidation reaction to give a fluorescent product that is read by a fluorescent spectrophotometer . histology provides the following visual assessment of the structure and morphology of both the clc and cclc : 1 . presence and distribution of fibroblasts and keratinocytes within and on the matrix of the collagen substrate . 2 . morphology of the matrix itself , such as the effects of cryopreservation or thawing on the attachment and distribution of keratinocyte cells on the nonporous - to - cells , semipermeable collagen layer , the attachment and distribution of fibroblast cells on and within the collagen sponge layer and 3 . the structural integrity and morphology of the cclc scaffold . the expression of cytokines is a measure of the potential of cclc to stimulate wound healing and tissue regeneration . cytokine expression data is obtained by analyzing spent culture media of the clc prior to cryopreservation and after thawing , rinsing and incubation for about 48 hours in cdmgm - hg . the spent media are assayed for cytokines using appropriate available elisa kits . table 1 3 / 30 comparative results for in vitro functions of cclc and clc number of average ± std dev . cclc / clc samples (× 10 − 5 / cm 2 ) (%) cell number cclc 30 10 . 47 ± 2 . 06 72 . 96 clc 28 14 . 35 ± 2 . 59 number of average ± std dev . cclc / clc samples (%) (%) cell viability cclc 30 95 . 53 ± 1 . 83 98 . 67 clc 28 96 . 82 ± 1 . 56 metabolic activity cclc 30 15 . 87 ± 3 . 24 83 . 05 clc 28 19 . 11 ± 5 . 02 [ 0063 ] table 2 cytokine expression by thawed and rinsed cclc and by clc bfgf gm - csf il - 1α kgf vegf m - csf ( pg / cm 2 / day ) cclc ave ± std dev 128 ± 11 6837 104 ± 49 687 ± 19 3353 ± 579 530 ± 180 clc ave ± std dev 14 ± 7 2908 ± 1408 8 ± 3 291 ± 95 5350 ± 240 986 ± 62 [ 0065 ] fig4 a ) and 4 b ) are pictorial representations of cross - sectional histological views of the clc 10 used in this invention before cryopreservation and cclc 100 obtained from clc 10 after cryopreservation . the two pictures show regions of reference before and after cryopreservation according to the invention . the clc of fig4 a ), beginning from the top of the figure has a nonporous - to - cells , semipermeable collagen layer 14 having normal human keratinocytes 12 on the semipermeable collagen layer 14 , a collagen sponge layer 16 in which spaces 20 are open air spaces of the sponge layer some of which contain normal human fibroblasts 18 within the spaces 20 . boundary lines 22 are the collagen structure of the collagen sponge layer 16 while the thinner boundary lines 24 surround thicker areas of the collagen structure . cclc 100 ( fig4 b ) after cryopreservation has the same sequence of layers and cells top to bottom as is shown in fig4 a ). in all instances the comparison between the various components of the clc and the cclc ( i . e ., before and after cryopreservation ) shows that there is little or no difference in morphologies of the collagen sponge layer 16 and the semipermeable collagen layer 14 , the keratinocytes 12 and the presence and distribution of fibroblasts 18 within the matrix of the collagen sponge 16 , attachment and distribution of keratinocytes 12 on the nonporous - to - cells , semipermeable layer of collagen 14 , and the structural integrity of the boundaries 22 and 24 of the cclc 100 and clc 10 . the following procedure was conducted under aseptic and , unless otherwise noted , ambient conditions : clcs measuring approximately 6 cm × 6 cm × 2 - 3 mm , were produced as described in the eisenberg u . s . pat . nos . 6 , 039 , 760 , 5 , 282 , 859 , and u . s . pat . reissue no . 35 , 399 with the following exceptions : a high glucose culture medium containing about 4 g / l glucose ( instead of 1 g / l ) was used with a feeding schedule for exchanging spent medium with fresh medium of every 2 - 3 days for up to 11 - 13 days as desired instead of the 14 days described in the patents . during the last 3 days of culture ( maturation period ), spent medium samples were collected and frozen in aliquots and held for testing for expression of various cytokines using commercially available elisa assays . on one of the last three days of culturing , the clcs were rinsed in protein - free , serum - free medium and all but one subjected to the cryopreservation step below . the one saved unit was tested for cell number and viability , metabolic activity , and histology testing to be compared with the same parameters for the cclcs prepared below . each rinsed clc was placed in a liquid permeable tray between two opposing sheets of non - adherent medical grade gauze in such a way that the 6 × 6 cm surfaces of the clc were accessible for subsequent treatment by a cryoprotectant solution . the tray was then placed into and heat - sealed within a cryogenic bag having luer lock connectors . the cryogenic bags were then connected to a peristaltic pump via the luerlock connectors and the pump operated to withdraw air from the bags . a primary cryoprotectant solution of 75 cc volume ( solution c ) at or below room temperature containing 2 . 5 % chondroitin sulfate was then introduced using the pump as the bags were continuously agitated . after 9 - 11 minutes of equilibration at room temperature with cyroprotectant solution c under agitation , 75 cc of a secondary cryoprotectant solution ( solution d ) containing 20 % dmso and 2 . 5 % chondroitin sulfate at or below room temperature was introduced using the pump with constant agitation . after a total of 12 - 13 minutes , the pump was reversed , first to withdraw any excess air and then to withdraw 100 cc of solution . the solution introduction ports then were heat - sealed and excess tubing trimmed . each sealed package was then placed into a separate cryogenic overwrap package which then was vacuum heat - sealed . the double - packaged units were then placed into aluminum canisters and placed into a microprocessor - controlled rate freezing chamber . the units were frozen under a microprocessor controlled schedule using the vapor phase of liquid nitrogen and then stored in the vapor phase of liquid nitrogen at below − 150 ° c . until ready for use . the temperature lowering program comprised the steps of : lowering the chamber temperature from ambient temperature to about 4 ° c . ; lowering the chamber temperature from about 4 ° c . to about − 20 ° c ., at a rate of about 1 ° c ./ minute and holding the chamber temperature at about − 20 ° c . for about 15 minutes ; lowering the chamber temperature from about − 20 ° c . to about − 25 ° c ., at a rate of about 0 . 5 ° c ./ minute and holding the chamber temperature at about − 25 ° c . for about 15 minutes ; lowering the chamber temperature from about − 25 ° c . to about − 70 ° c . at a rate of about 1c / minute ; lowering the chamber temperature from about − 70 ° c . to about − 90 ° c . at a rate of about 5 ° c ./ minute ; holding the clc in the freezing chamber until the surface of the clc reached − 85 ° c . to − 90 ° c . ; and storing the cclc below about − 150 ° c . the cryopreserved ( cclc ) units prepared above are suitable for either patient grafting or other studies after subjecting thereto the following procedures . first , the cclc is warmed at room temperature for about four minutes . at the end of the warming time , the overwrapped packaged cclc is placed into 4 liters of room temperature sterile water ( or sterile saline ) bath for thawing in the second stage for about five to seven minutes with periodic manual agitation . the overwrap is cut open , the inner package removed and cut open , and the tray containing the cclc removed . the cclc in the tray is rinsed to remove the cryoprotectant solution by placing it into 3 liters of sterile saline , and agitating for about ten minutes , or alternatively , for five minutes after which the saline is removed and replaced with another 3 liters of saline and agitating for another five minutes . at the end of that time , the cclc can be removed from the tray and applied onto a patient or used for in vitro studies . for in vitro studies , the cclc can be placed back into the high glucose culture medium and incubated and assayed for cytokine expression using commercially available elisa kits . the cclc can also be used immediately for testing , as was done in the pre - cryopreservation step , that is , tested for cell number , viability , metabolic activity , and histology , and testing for comparison with the pre - cryopreservation material . an in vivo grafting study was performed to compare the healing characteristics of full - thickness skin wounds in severe combined immuno - deficient ( scid ) mice grafted with the cclc of example 1 compared to a fresh clc prepared according to the eisenberg u . s . patents referred to in example 1 and used without being subjected to cryopreservation and / or freezing . the study was conducted as two trials of twelve animals each , which were initiated approximately 24 hours apart . a full - thickness wound ( 1 . 7 cm in diameter ) was created using surgical scissors on the dorsum of each mouse just prior to applying a clc or cclc graft of the same size and shape into the wound . six mice in each trial received fresh clc grafts and six mice received cclc grafts . samples of clc and cclc from each of the four groups were assayed in vitro for cell yield , cell viability , and the ability to secrete several wound - healing associated cytokines and growth factors . following placement of the grafts , the wounds were covered with one layer of polyethylene net , then with two layers of paraffin gauze and three circuits of dressing around the trunk of each mouse to secure the grafts . mice were observed and weighed daily . bandages were removed and wounds were examined and photographed macroscopically 8 and 14 days after grafting . the degree of wound contraction was calculated on day 8 and 14 . on day 14 , the mice were sacrificed and the wounds were excised for histological preparation and evaluation . all biopsy samples were placed in fixative , sectioned at 8 um and stained with hematoxylin and eosin , and / or trichrome and / or orcein and methylene blue . the extent of epithelialization , the average dermal and epidermal thickness and the cellularity of the dermis were calculated . other histological features were noted as comments . no significant adverse effects were observed during the course of this study . by day 14 , most of the grafts were not apparent due to the healing processes and resorption of the collagen matrix of the clc and ccls . the fresh clc and cclc grafts produced similar results with respect to macroscopic wound appearance and contraction . microscopically , no significant differences were noted in the healing properties of wounds treated with fresh clc or cclc grafts . by day 14 , 8 / 12 and 7 / 12 of the wounds in the fresh clc and cclc groups , respectively , had undergone complete epithelialization . no difference in epithelialization was observed between fresh clc and cclc grafts and it was concluded , from the results of this study , that cclc grafts of the invention and fresh clc were not substantially different and had similar effects on wound healing parameters when compared in a full - thickness would healing model in scid mice .
processes are described for making a cryopreserved composite living construct 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 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 .