FR2589165A1 - Process for producing a cultured human epithelium, the epithelial tissue obtained and its application in allografts - Google Patents

Abstract

The process makes it possible to obtain a cultured human epithelium lacking class II D/DR antigens which are responsible for rejections caused by the immune response. This epithelium is produced in vitro from epithelial cells derived from a primary culture which were frozen and then recultured. The cultures are preferably carried out in the presence of irradiated fibroblasts. The epithelial tissues obtained can be grafted, immediately or after cryopreservation, onto recipients without specific compatibility with the donor.

Description

 The present invention relates to a process for obtaining human epithelia by cell culture in vitro, such that the epithelia obtained do not contain class II D / DR antigens responsible for the immune rejection of allografts, as well as epithelial tissues obtained by this process and their applications.

 It is known that the allograft of a tissue originating from a genetically different individual and which has not undergone any treatment, is rejected and it is generally accepted that this rejection is linked to the presence in the tissue, natural or resulting from a culture. , dendritic cells which carry class II D / DR antigens in humans.

In patent application WO85 / 00042, JM HEFTON describes a method for obtaining a culture of human epidermis cells not comprising immunocompetent cells which consists in culturing the cells after having dissociated them and treated either with an enzyme, such as that the
DNase, to selectively destroy immunocompetent cells, either by a protein which binds selectively on immunocompetent cells, such as tobacco glycoprotein or rutin, and then separate the cells thus labeled from other epidermal cells.

 Furthermore, methods for obtaining a tissue reconstituted by culturing human epithelium cells have been described. H. Green and Colt. in US 4,016,036 have shown that the culture of human keratinocytes in a nutritive medium containing fibroblasts of murine origin, previously treated to limit their multiplication power, makes it possible to obtain confluent colonies of cells and even a stratified epithelium. Such a particularly interesting method has been successfully applied to the preparation of tissues of human origin which were not immediately rejected after their transplant to mice; the grafted epithelial layer had been isolated from the culture medium after treatment with the dispase, as described in US 4,304,866.

 However, even if the number of dendritic cells associated with the keratinocytes obtained in this type of culture is limited, there remains, most of the time, a sufficient quantity so that one cannot consider using the tissue thus prepared for an allograft, in a recipient who does not have good HLA compatibility with the donor of the cultured cells.

 The culture method according to the invention makes it possible to obtain epithelial tissues, devoid of dendritic cells and therefore of which the class II antigens are absent; these tissues will advantageously be used as allografts for covering extensive wounds of the skin and other mucous membranes in burned or injured patients but may also be used to study, in vitro, the dermatological activities of various compounds.

 This process consists in cultivating under conventional conditions human epithelial cells, in dissociating the cells thus obtained, then in freezing and thawing them before multiplying them in a nutritive medium, comprising fibroblasts which do not multiply, until the obtaining cellular tissue.

 The means which can be used to obtain the cells which will be frozen are numerous, as long as they make it possible to obtain cells which can grow and divide.

 In the case of human skin, the epidermis is separated from the underlying dermis beforehand, and then the keratinocytes are dissociated, for example by trypsinization before putting them to the skin. This trypsinization is advantageously carried out in the presence of EDTA (ethylenediamine tetraacetic acid) , according to a known method.

 The cells of the other mucous membranes, such as the gingival mucosa, the larynx or the pharynx, will also be dissociated.

 The culture before freezing is carried out under conventional conditions, known to allow the multiplication of human epithelial cells in culture. The nutritive medium, which contains the essential amino acids and vitamins, can for example be that known under the name of modified Eagle medium or of Eagle medium modified by Dolbecco (DMEM).

 Preferably, fetal calf serum and epidermal growth factor (EGF) are added to these media. We know that the conditions of pH, temperature and humidity are important for a good culture, and different solutions have been proposed to date.

 As it has been found that, when the culture was carried out in the presence of fibroblasts previously treated to block their multiplication, or of extracts of these fibroblasts, the cultures were faster and the confluence earlier, it is preferred to operate under these conditions.

 We know that the multiplication power of fibroblasts is destroyed by exposure of cells to ionizing radiation, X or gamma rays, to ultraviolet rays or by treatment of cells with chemical compounds that will damage DNA, such as alkylating agents. , and it suffices to introduce into the culture medium a given quantity of fibroblasts having undergone one of these treatments.

 After about two weeks, when the epithelial cell culture arrives at confluence, or some time before, the cells are separated from the medium and dissociated before being suspended in a medium suitable for the next stage of freezing / thawing. . This stage, the two parts of which can be more or less separated in time, is fundamental; in its absence, a certain quantity of dendritic cells would remain even in the subcultures of epithelial cells and therefore significant risks of rejection in the case of allografts.

 It is one of the merits of the invention to have demonstrated how to suppress all traces of cells carrying histocompatibility markers, making it possible to graft the tissues obtained in culture to any individual, without any relationship with the donor of the first cells originally cultured.

 Freezing and thawing conditions are not critical as they preserve cell viability. The cooling of the cells which have been dissociated in the event of confluence of the culture, for example by trypsination, or even mechanically, must be rapid. It is sufficient that the suspension of cells be brought at least four hours to -200C for the dendritic cells to disappear, but it is preferable to keep at least 24 hours at -700C. The cells can also be kept for several weeks, and even several months before thawing; in this case, they will be stored at the temperature of liquid nitrogen, around -1500C to -1950C. The advantage of keeping the cells for extended periods is another advantageous consequence of the method according to the invention. One can thus build up stocks of epithelial cells which can be used in the form of tissues which will be obtained after only a few days of culture.

 Among the liquid media suitable for freezing cell suspensions, a particularly preferred solution for keratinocytes is a DMEM solution containing 50% (v / v) of fetal calf serum (SVF) and 10% (v / v) of dimethyl sulfoxide ( DMSO).

 The cell suspensions are frozen in sealed ampoules from 1 ml to 5 ml; their heating will be carried out in a conventional manner, fairly quickly, for example by immersion in a bath at 370C.

 The thawed cells are separated from the preservation medium, by centrifugation, then sown on a nutritive medium, optionally enriched in fetal calf serum and epidermal growth factor and containing fi fibroblasts which have undergone a treatment suppressing their multiplication power; the number of fibroblasts, preferably of the 3T3 type, of murine origin is from 1 to 20 times that of the thawed cells seeded. The standard and seeding density of 10 4 to 10 5 cells per cm2 and the atmosphere of the culture chambers is enriched with water vapor and C02, at a rate of approximately 10% by volume of C02 and H20 as long as the culture is not confluent, then 5% of C02 ensued.

 The time to reach the confluence of the culture depends on the initial seeding density and the nature of the nutrient medium. In general, confluence is obtained within a few days, but it is possible to remove the layer of cells formed for use, even before the confluence phase. The very short time then elapsing between thawing and obtaining a graftable tissue is another advantage of the invention. In the absence of a freezing step between the primary culture and the subculture, the number of dendritic cells expressing class II antigens decreases only very slowly over time, without completely disappearing therefrom; thus, after 4 weeks of a subculture of keratinocytes, in the presence of irradiated fibroblasts, there is still approximately 0.5% of dendritic cells. If these keratinocytes are frozen and thawed before subculture, a sample of the culture put in contact with allogenic lymphocytes in the system of mixed lymphoepidermal culture does not trigger any immune reaction, from the first week of culture as afterwards.

The cell tissue is detached from the surface of the culture vessel by any means capable of separating it without dissociating the epithelial cells. The separation is facilitated by using the action of a proteolytic enzyme and especially the dispase, as described in the patent.
US 4,304,866 The transfer of the tissue to the place of application is advantageously carried out by placing it on a rectangle of vaselina gauze, which mechanically consolidates it and prevents its dehydration. I1 can be stored at 370C in an atmosphere enriched with C02 for a few hours but its prolonged storage is also possible at temperatures below -1500C, and it is another merit of the invention to have demonstrated that the tissues thus preserved could, immediately after reheating, be grafted on recipients without specific histo-compatibility with the initial donor.

The fabric is placed on a support to be frozen, which facilitates handling. Vaseline gauze is preferred as a support, but a collagen or biomaterial support would also be suitable. The whole reconstituted tissue and support is introduced into a solution whose composition will prevent the bursting of the cells subjected to the cold. Several mixtures suitable for this purpose are known; one can quote for example the solution of DMEM with 50% (v / v) of SVF and 10% (v / v) of DMSO or a solution of phosphate buffer (ph = 7.2) containing 3% (v / v) albumin and 10% (v / v) of
DMSO.

 It is preferred to cool the fabric in stages; thus closed enclosures, such as welded plastic bags containing the freezing solution in which the epithelial tissue has been immersed on a support, are brought quickly to -500C, by introduction into an enclosure at this temperature and then after 1 hour at 2 hours, cooled to a temperature below -700C. It is preferred to cool to -1500C, given the ease of obtaining and maintaining such a temperature with liquid nitrogen baths. Under these conditions, the tissues can be stored for at least twelve months, without any apparent alteration of their properties.

 In the following, an example of a particular implementation of the invention is described, from the stage of removal of the epithelium to the allograft.

A - EPITHELIUM COLLECTION AND CELL DISSOCIATION.

2
A piece of skin of approximately 2 cm 2, taken from a donor, is introduced into sterile phosphate buffer (pH = 7.2). In this medium, the dermis is eliminated and the epidermis is washed several times with a phosphate buffered saline, then dilacerated in 10 ml of 0.1% aqueous solution of trypsin (w / v).

 The suspension of the f-ragments is then introduced into a trypsinization flask with 10 ml of 0.1% trypsin solution and 10 ml of 0.02% (w / v) aqueous EDTA solution at 370C. The dissociated cells are harvested every thirty minutes by centrifugation of the supernatant, while 15 to 18 ml of trypsin solution (0.1%) + EDTA (0.02%) are poured onto the residual fragments.

B - PRIMARY CULTURE.

 The epidermal cells thus obtained are sown in cell culture flasks at the rate of 0.5 × 2 10 5 to 2 × 10 6 cells per 75 cm on a layer of irradiated 3T3 fibroblasts, with 20 ml of culture medium. The medium is composed of a mixture of DMEM and Ham's F12 (75/25) media with 10% (v / v) FCS, 1% (w / v) penicillin / streptomycin and an amount less than 1% of hydrocortisone, cholera toxin, insulin, adenine, transferrin, triiodothyronine, EGF. The culture is carried out at 370C in an atmosphere enriched in CO2 (10%) and H2O (10%). The nutrient medium is changed every two days.

The fibroblast irradiation was carried out with a cesium source (dose 6000 rads) on a culture at confluence, or almost, carried out on a nutritive medium based on DMEM + 10% FCS; this irradiation was carried out a few hours before the introduction of 3T3 into the primary culture medium 6, where they were seeded at the rate of 1.5 to 2 × 10 6
2 cells per 75 cm, in 20 ml of nutrient medium.

 When the culture of epidermal cells arrives at confluence, or almost, that is to say after 10 days, the cells are harvested and dissociated by bringing them into contact with a trypsin solution (0.1%) containing EDTA (0.02%) for 15 minutes at 370C. The cells are then washed with culture medium before being suspended in the freezing medium.

C - FREEZING / DEFROSTING.

 2 x 106 cells are introduced per 2 to 4 ml glass ampoule with a DMEM solution containing penicillin / streptomycin (1%), SVF (50%), DMSO (10%).

 The sealed ampoules are brought to -700C in a few minutes and kept 24 hours at this temperature.

They can then be thawed or brought to -1500C, by immersing them in vapors of liquid nitrogen, for a prolonged conservation. Defrosting is carried out in a few minutes by immersing the ampoules directly in a 370C bath.

D - SUBCULTURES.

 The thawed cells are seeded in culture flasks under conditions identical to those described in step B, but at this stage, the seeding density is such that there can be up to 10 times more fibroblasts than epidermal cells. The culture is carried out as before in an atmosphere enriched with C02 and H20, at 370C.

For a seeding density corresponding to 5 thawed epidermal cells out of 75 2 2 x 105 thawed epidermal cells out of 75 cm, the confluence is obtained in 10 to 13 days; whereas for 4 x 105 cells, confluence is obtained in 8 to 11 days and for 106 in 7 days
E - ISOLATION OF THE EPITHELIAL TISSUE.

When the confluence has been obtained, the cultures are washed with a DMEM solution (10 ml per 75 cm dish) and added 15 ml of 2.5 mg / ml solution of the protease, called "dispase II", marketed by Boehringer Mannheim. 45 to 60 minutes after the addition, the culture which has been maintained at 370C is separated from the dispase, rinsed several times with
DMEM and detached from the substrate is then placed on the rectangle of epidermal culture thus obtained, a piece of vaseline gauze, it is made to adhere and the fabric is deposited on the gauze by inversion. The whole can be kept for four hours in an atmosphere enriched with C02 before applying the graft to a wound.

 Its prolonged conservation requires a new stage of freezing.

F - EXTENDED CONSERVATION AND APPLICATION.

2
20 to 30 cm of reconstituted epidermal tissue, on its vaselined gauze support, are introduced into 30 to 50 ml of DMEM solution or phosphate buffer (pH = 7.2) containing penicillin / streptomycin and 10% DMSO in a metal enclosure with watertight closure, in a sterile atmosphere.

 The enclosures are suddenly cooled to -500C and left for 1 hour 30 minutes at this temperature before being immersed in liquid nitrogen.

 For thawing, the chambers are taken out of liquid nitrogen and introduced into a bath at 370C. After thawing, in a few minutes, the strips of epidermal tissue fixed on the gauze are removed from the preserving liquid and after 10 minutes at 370C in a sterile atmosphere, they are applied to the wounds to be covered.

 Skin flaps thus prepared, obtained either after stage E or after prolonged storage, were grafted into individuals genetically different from the initial donor, with no compatibility either for blood groups or for class I or II antigens. . No sign of clinical rejection has been observed over a period of more than six months, although the identity of the grafted skin remains that of the donor. The in vitro controls, carried out by mixed cultures between the keratinocytes of the graft and the lymphocytes of the recipients, confirmed the absence of response of the latter to the presence of the foreign cells even after 2 months.

Claims (11)

 1. Method for obtaining epithelial tissues by culture of dissociated cells, taken from a donor, comprising a primo culture and a subculture, characterized in that, between the two culture stages, freezing is carried out until at least - 2O0C then the thawing of the cells in suspension in a nutritive medium.  2. Method according to claim 1, characterized in that the subculture is carried out on a nutritive medium comprising fibroblasts, which are no longer liable to multiply.  3. Method according to claim 1, characterized in that the primary culture and the subculture are carried out on a nutritive medium comprising fibroblasts, which are no longer liable to multiply.  4. Method according to one of claims 2 or 3, wherein the fibroblasts have been irradiated.  5. Method according to any one of the preceding claims, in which the dissociated cells are frozen in a DMEM solution containing fetal calf serum and dimethyl sulfoxide.  6. Epithelial tissues obtained by a process according to any one of claims 1 to 5.  7. Culture epithelial tissues devoid of class II immunocompetent dendritic cells.  8. Frozen epidermal cells for obtaining cultured epithelial tissue.  9. Human skin allograft obtained by a method according to any one of claims 1 to 5.  10. An allograft according to claim 9, originating from the thawing of an epithelial tissue, in accordance with one of claims 6 or 7, which has been stored at a temperature below -150 C.  11. An allograft according to claim 9 or 10, prepared by the process which consists in  1) take a skin fragment and isolate the cells of the epidermis by trypsination,  2) cultivate these cells on a nutritive medium in the presence of fibroblasts having lost their multiplication power, in an atmosphere containing CO 2 and H 2 O  3) freezing a suspension of the cells obtained,  4) thaw the cells quickly;  5) cultivate the cells under the same conditions as in step 2 until an epithelial tissue is obtained,  6) separate the tissue from the culture vessel by treatment with a protease.