METHOD FOR PRESERVING TRANSPLANTABLE SHEETS OF EPITHELIUM CULTURED IN VITRO

Abstract: A process for preserving cultured epithelial sheets by incubating the sheets at room temperature in media with a glycerol or dimethylsulfoxide as cryopreservant for a predetermined period of time and then freezing the cultured sheets. The cooling gradient is characterized as having a lower initial rate of cooling followed by a higher rate of cooling.

Description: Background of the Invention: 1. Field of the Invention The present invention generally relates to the cryologic preservation of cellular tissue. More particularly, the present invention relates to the cryologic preservation of epithelial sheets obtained by cellular culture. 2. Brief Description of the Background Art Despite recent progress in treating burnt tissue (i.e., tissue which is characterized by sufficient amounts of denatured protein to result in cell injury or death) the death-rate of burn victims still remains unacceptably high. Such fatality levels are, of course, highly dependent on the patient's age and on the percentage of burnt body area. The death-rates are, in fact, dramatically high for third degree burns (i.e., when full-thickness dermis has been destroyed) of 50-60% or more body area. In these cases, especially, in addition to the immediate problems of advanced cardio-vascular shock and the threats of somewhat less-imminent sepsis or hydro-electrolytic impairment, yet another (slightly delayed) problem is caused by the absence of a cutaneous covering over a large body area. The difficulty with such missing coverings, or open areas, is that they tend to not spontaneously recover, since they have been completely damaged or destroyed throughout their whole thickness. When the normally closed epithelial barrier is opened to the outside environment, a chronic pathological condition is created which, together with the above identified infective and metabolic complications, progressively worsen the patient's local and systemic conditions. The sum total of these problems often overwhelms the burn victim , who frequently dies. It is therefore evident, once the emergency phase of treatment has been overcome, that the main medical problem is to cover the cutaneous deficit as quickly and as effectively as possible. According to conventionally accepted medical practice, wound areas are covered by transplanting split-thickness tissue pieces removed from healthy unburnt regions (autografts). However, when the burnt area exceeds, for example, 60-70%, there is usually insufficient donor tissue available for grafting. Moreover, the autograft donor site itself provides yet additional area which lack whole cutaneous coverage. These drawbacks must be considered together with the facts that autograft donor sites also tend to recover slowly and may yield deleterious healing residues. It is also medically acceptable to cover the cutaneous deficit by the trasplantion of homologous cadaver skin, lyophilized heterologous skin (from, for example, pig), synthetic artificial cutis and the like. However, these alternate meterials, being allogenic will slough spontaneously within 1-6 months, and they must eventually be replaced with autologous tissue and are thus, at best, temporary in nature. Methods recently disclosed in U.S. Patents Nos. 4,016,036; 4,304,866 and 4,456,657 teach methods for culturing human epithelial films obtained from cutaneous keratinocytes. These films, of course, may be autologous and therefore would allow burnt areas to be covered without being subject to later rejection or sloughing. These methods in these U.S. patents are based on the removal of a small (2-3 cm²) piece from a healthy donor area. An epithelial cellular suspension obtained using the donor tissue is then cultured and expanded through subsequent inoculations of the primary culture. When the secondary and tertiary cultures become confluent, multi-layered sheets of epithelium can be obtained. Thus, several serious problems are potentially overcome, such as the poor availability of residual healthy skin in the seriously burnt patient or the need to use expensive pig, cadaver, or artificial cutis, each of which may not be readily available and whose use may be strictly regulated because of antibody reaction. On the other hand, the keratinocytes cultures obtained according to the above methods require from 3-4 weeks until they are suitably expanded so that film sheets may be obtained. In other words, the burnt patient must remain without protection of the burnt areas for the initial 3-4 weeks. This initial period is, of course, the most critical and so, that period during which the graft of healthy cutis would be most highly beneficial. Additionally, the cultured epithelial sheets taught in these U.S. patents must be used within a few hours of harvesting since it has not been possible to develop a conservation method which can maintain expanded epidermis sufficiently to allow their travel to distant user centers. The literature contain many reports of attempts to cryopreserve skin samples. See, for example, May et al., "Cryopreservation of Skin Using An Insulated Heat Sink Box Stored At -70°C", Cryobiology, 22:205-14 (1985); May et al., "Recent Developments in Skin Banking and The Clinical Use of Cryopreserved Skin", 73(4) J. Med. Assoc. Ga., 75:233-36 (1984); Yang et al. "Growth of Human Mammary Epithelial Cells on Collagen Cell Surfaces", Cancer Research, 41(10)4093-4100 (1981); Biagini et al. "Skin Storage in Liquid Nitrogen, An Ultrastructural Investigation", Journal of Cutaneous Pathologhy, 6:5-17 (1979); Taylor, "Cryopreservation of Skin: Discussion and comments", Cryobiology, 3 (2) 1966; and Baxter et al., "Cryopreservation of Skin: A Review", Transplantation Proceedings, Vol. 17 (6) Suppl 4, 112-120 (1985). This and other literature demonstrates that a substantial fraction of the cells in frozen epithelial tissue after thawing are capable of ongoing metabolism. However, epithelial sheets intended for use as autografts or allografts and to function as a burn dressing must contain a relatively high content of mitotically competent cells after cryopreservation. The provision of sheets with consistent bioproperties requires that they be capable of mitosis and stratified differentiation. This capability heretofore has not been demonstrated for frozen and thawed cultured epithelial samples. One cannot predict with certainty the effect of freezing and thawing on the fate of cells in cultured confluent sheets which lack supporting dermal material. The sheet comprises cells connected together by surface protein and are only a few cells thick. They are not differentiated in the form of a fully stratified epidermis. Freezing and thawing involves intracellular and intercellular ice crystal formation, expansion, and contraction, each having unpredictable effects on individual cultured cells and on the sheet as a whole. The availability of frozen cultured skin graft materials would be advantageous to permit storage and shipment and also for those patients that require several transplantation, since it would facilitate a better coordination between culture and surgical schedules. Were such products to be developed, it would be possible to realize the institution of "tissue banks" or other depositories of allogenic expanded epidermis which could be relied upon in the event of emergency. Readily available cultured allografts in a frozen state can have several clinical applications. For example the present inventors have evidence that deep second degree burns heal much faster when grafted into cultured allografts. Limited full thickness lesions clinically appearing as third-degree burns were reepithelialized by the recipient keratinocytes, whose growth and probably migration were strongly stimulated by cultured allografts. Cultured allografts could also be applied in most of donor sites utilized for split-thickness mesh-grafts. This results in a nice healing of those areas even after 4 days. The method described herein can be successfully utilized also for cryologic preservations of mucosa expanded in vitro . In this regard, the present inventors have determined that expanded mucosa can be obtained in vitro starting from a punch biopsy following the methodology described for production of epithelial films from cutaneous keratinocytes. The present inventors have also determined that the in vitro expanded mucosa can be successfully transplanted in patients that require oral mucosa transplant. It is an object of this invention to provide a method of producing cultured, cryopreserved, epithelial sheets which function when thawed as a permanent autograft or temporary, wound-healing allograft; and to provide a process enabling the banking, distribution, and subsequent consistent use of cultured epidermal sheets in a form which facilitates manipulation by the physician, and is useful in the treatment of skin burns, ulcers, and other wounds. Another object is to provide storage stabilized epithelial wound dressings capable of mitosis and stratified differentiation to produce a permanent autograft or temporary allograft. These and other objects and features of the invention will be apparent from the description, drawings and claims which follow. Summary of the invention The present invention provides a process for preserving transplantable sheets of confluent epithelial cells which have been cultured in vitro . The preferred sheets comprise cultured, confluent keratinocyte cells, most preferably epidermal keratinocytes. The present invention also enables the transport of preserved cultivated epidermis and therefore, the institution of a bank of cryopreserved expanded allogenic epidermis. According to the present invention, cultivated epithelial sheets which are otherwise ready to be grafted are incubated in media containing conventional nutritive agents and a cryopreservative agent. The sheets are incubated for a predetermined period of time and thereafter frozen to a final temperature of about -100°C. The cooling gradient utilized is characterized by a slower initial phase and a faster final phase. The process results in a storage-stabilized epithelial wound dressing consisting of a frozen confluent sheet of cultured epithelial cells disposed on a non-adherent support backing. When thawed, and applied to the surface of a wound, the sheet of cells is metabolically competent and capable of mitosis and stratified differentiation to produce a permanent autograft or temporary allograft. That is, if the sheets are produced from epithelial cells sampled from a patient, application results in a permanent autograft. Sheets cultured from human epithelial cells make an excellent, metabolically competent temporary, wound-healing allograft when applied to other human patients. Brief Description of the Drawings Figure 1a is an optical micrograph of a cultured human epithelial sheet before being treated in accordance with the present invention; Figure 1b is an optical micrograph of the cultured epithelial sheet of Figure 1 which has been treated in accordance with the present invention and then thawed; and Figure 2 is an electron micrograph of the cultured epithelial sheet of Figure 1b. Detailed Description of the Invention The present invention utilized sheets of epithelial films, and the like, such as those which are produced in accordance with U.S. Patents Nos. 4,016,036; 4,304,866 and 4,456,657, discussed above, which patents are hereby expressly incorporated herein. The sheets are first stabilized by being incubated in media, preferably nutrient media, containing a cryopreservative agent. The intracellular cryopreservant concentration is an important factor in the success of the procedure. The cryopreservative agent is preferably contained at from 8-15 wt.%, and is preferably glycerol or dimethylsulfoxide. Most preferably, the cryopreservative is glycerol which is used at about 10 wt.%. The sheets are incubated at about room temperature, preferably for not more than 15 minutes. In this regard, the incubation time has been found to be somewhat critical since the present inventors have determined that the length of such periods dramatically affects cell viability and the ability of transplanted keratinocytes to form colonies. In particular, tests carried out which vary the period of incubation reveal a rather remarkable decrease in the ability of the keratinocytes to form colonies after both very short incubation times (of less than about 2 minutes) and fairly long times (of more than about 15 minutes), whereas the ability to form colonies greatly increases at times of about 4-7 minutes and is maximized at periods of about 5-7 minutes. Equivalent intracellular concentrations of the cryopreservative may be introduced into the cells using higher concentrations and shorter incubation periods, or lower concentrations and longer incubation periods. After incubation, the epithelial sheets are subjected to a freezing procedure wherein temperature is strictly controlled, such that the rate of the temperature drop is slower at the start of the procedure than at the end of procedure. The slow cooling gradient, e.g., no more than -2°C/min. and preferably about -1°C/min., is used through the range of temperature immediately above, through, and immediately below 0°C. Further cooling below the freezing point may be done faster. It is preferred to suspend cooling for a few minutes before the cells have been cooled below the freezing point. At the culmination of freezing, the now-frozen sheets have preferably reached at most -80°C, and most preferably at most -100°C. In this state, the treated epithelial sheet may be maintained for at least about 3 months without substantially changing their morphological and functional characteristics. In fact, pieces of epithelial sheets obtained by treatment in accordance with the present invention are comparable in terms of both morphology and graftability to those which are otherwise obtainable from fresh cultures which have not been subjected to cryopreservation. The sheets preferably are disposed in face-to-face contact with a non adherent backing material prior to freezing. Conventional sterile gauze treated with petroleum jelly works well. The backing acts as an aid in manipulating the fragile sheets while frozen and after they are thawed. The backing also serves as a convenient temporary covering for the sheet after application to the surface of a skin wound. In this regard, Figure 1a illustrates a sheet before being subjected to the process of the present invention, whereas the Figure 1b refers to a sheet which has been subjected to the freezing process of the present invention and has also been defrosted. The sheet of Figure 1b is, therefore, ready for use as a transplant. Both sheets of Figures 1a and 1b have been fixed, incorporated, cut and dyed with hematoxylin eosine. Figure 2 is an electron micrograph of the in vitro cultivated epidermal sheet of Figure 1b. From these micrographs, it is evident that the present invention results in good cell conservation of the basal layer as well as the good conservation of intercellular structure. When the treated sheets are to be used, it is sufficient to incubate them about at 37°C for a few minutes, followed by washing in physiological saline or in suitable culture media. In addition to the coverage of burnt areas, the preserved epidermis sheets should find useful applications in other fields such as, for instance, in oncologic plastic or reconstructive surgery. The preserved in vitro expanded mucosa can also be utilized in oral surgery. The following example further illustrates one preferred embodiment of the invention, but is neither intended nor to be considered as limitative in scope. EXAMPLE The keratinocytes cultures were obtained utilizing the procedures of the U.S. patents identified above which were incorporated herein. In particular, the confluent secondary keratinocyte cultures obtained according to U.S. Patent No. 4,016,036 were detached from their culture bottles, transferred on vaseline coated gauze and anchorred by means of metallic vascular surgery clips according to the method disclosed in U.S. Patent No. 4,304,866. These cultivated tissue pieces were then transferred under sterile conditions in 12 x 20 sterile polyester/polyethylene/aluminum bags (Gambro S.A. - 18, rue de Calais - 75885 Paris) (3 grafts in each bag), 100 ml of media was then added to the bags, wherein the media comprised: Dulbecco modification of Eagle's medium 54 wt.% Ham's F12 27 wt.% fetal calf serum (FCS) 9 wt.% glycerol 10 wt.% glutamine 4 mM adenine 1.8 x 10⁻⁴ M insulin 5 mcg/ml transferrine 5 mcg/ml triiodothyronine 2 x 10⁻⁹ M hydrocortisone 0.4 mcg/ml epidermal growth factor 10 ng/ml penicillin-streptomycine 50 U/ml The bags were thermally sealed and incubated at room temperature for between 5-7 minutes. The bags were placed in a suitable container and transferred into a suitable programmable freezer (one such freezer is known as the Programmable Temperature Controller PTC-300 and is commercially available from Planer Products, Ltd.). The tissue pieces were then frozen using following freezing program. Starting temperature: +25°C; -5°C/min. to + 3°C; pause for 4 min.; - 1°C/min. to - 7°C; -25°C/min. to -40°C; +l5°C/min. to -25°C; - 2°C/min. to -40°C; and - 3°C/min. to -100°C. When the final temperature of -100°C was attained, the plastic bags were transferred in a suitable metal container which was previously cooled to -80°C at least 2 hours earlier. The metal container was then rapidly transferred to a -80°C freezer. The bags containing the frozen epithelial sheets may be sent to long distances provided only that temperature increases are avoided. The frozen sheets may be preserved to -80°C for at least 3 months, while maintaining their morphological and functional characteristics. When it is necessary to use the frozen films, the bags are removed from the freezer, immediately immersed in a water-bath at +37°C and incubated for about 10 minutes. The bags are then immersed for a few seconds in 70% ethanol. The bags are then opened in sterile conditions, the epithelial sheets are removed, transferred in sterile containers, thoroughly washed with culture medium or physiological saline and used. Use involved conventional wound bed preparation to present to the cells of the thawed sheet a substrate suitable for intimate contact. The sheets are applied by placing them in surface-to-surface contact with the wound area with the backing facing outward, and then gently pressing the sheet through the backing to conform to the shape of the wound surface. Multiple sheets may be used for larger areas. It will be appreciated, of course, that various modifications of the foregoing procedures are within the purview of those skilled in the art and that such modifications and the like are intended to be covered by the claims which follow.

Claims: A method for preserving epithelial sheets comprising the steps of: a) incubating the epithelial sheets at room temperature in media containing a cryopreservant for a period of time from 2 minutes to 15 minutes; b) freezing said epithelial sheets using a cooling rate of no more than -2°C/min through the range of temperature immediately above and immediately below 0°C. A method according to claim 1 wherein the cryopreservant is glycerol or dimethylsulfoxide. A method according to claim 1 wherein the cryopreservant is added to the nutrient media at a concentration of from 8-15 wt%. A method according to any one of the previous claim wherein the room temperature incubation lasts for a period of time from 4 minutes to 7 minutes. A method according to any one of the previous claims wherein the epithelial sheets have been obtained from cellular culture. The method according to any one of the previous claims wherein said sheets are frozen at most to -80°C. The method of preserving epithelial sheets of any of the previous claims, wherein said media substantially comprises: Dulbecco modification of Eagle's medium 54 wt.% Ham's Fl2 27 wt.% fetal calf serum (FCS) 9 wt.% glycerol 10 wt.% glutamine 4 mM adenine 1.8 x 10⁻⁴ M insulin 5 mcg/ml transferrine 5 mcg/ml triiodothyronine 2 x 10⁻⁹ M hydrocortisone 0.4 mcg/ml epidermal growth factor 10 ng/ml penicillin-streptomycine 50 U/ml The method of preserving epithelial sheets of any of the previous claims, wherein substantially the following freezing program is used: starting temperature: +25°C; -5°C/min. to + 3°C; pause for 4 min.; - 1°C/min. to - 7°C; -25°C/min. to -40°C; +15°C/min. to -25°C; - 2°C/min. to -40°C; and - 3°C/min. to -100°C. The method for preserving epithelial sheets according to any one of the previous claims, further comprising the step of thawing said frozen sheets. A wound dressing obtainable by the method of claims 1-9 comprising a cryopreserved, thawed, cultured, confluent sheet comprising differentiable, metabolically and mitotically competent, stratified epithelial cells having conserved intercellular structure. The dressing of claim 10 further comprising a backing disposed in contact with a surface of the sheet adapted for removal from the surface. The dressing of claim 10 wherein the epithelial cells are keratinocytes. A frozen, cultured, stratified epithelial sheet obtainable by the method of claims 1-9 which, when thawed, is useful as a skin wound dressing, exhibits conserved intercellular structure, and comprises mitotically and metabolically competent epithelial cells. A frozen cultured epithelial sheet obtainable by the process of claims 1-9. A frozen cultured epithelial sheet according to claim 14 wherein the epithelial cells are keratinocytes. A frozen cultured epithelial sheet according to claim 14 or 15 further comprising a backing disposed in contact with a surface of the sheet. A frozen cultured epithelial sheet according to claim 16 wherein the backing comprises a material adapted for removal from the surface of said sheet.

Inventors: CANCEDDA, RANIERI, DE LUCA, MICHELE
Applicants: IST NAZ RIC SUL CANCRO, ISTITUTO NAZIONALE PER LA RICERCA SUL CANCRO
Application year: 1988
Main classification: A01N1
IPC codes: C12N5, A61L27, A01N25, A01N1