SUMMARY. Normal human keratinocytes can be serially cultured in vitro and under appropriate culture conditions to give epidermal sheets which may be used to cover deep and large skin defects or burns. The analysis by flow cytometry of epidermal cells in normal human epidermis and cultured human keratinocytes is proposed to predict the optimal time for the grafting of in vitro prepared allogeneic keratinocytes. This analysis regarded their proliferative and immunogenic stage, and the evaluation of antigens whose level of expression is related to these. In 19 skin biopsies and 22 cultured epidermal sheets we analysed: markers of epidermal proliferation/differentiation (31 integrins (CD29) and Kl/K10 intracytoplasmatic keratins; antibodies of immunogenicity anti-HLA-DR and anti-HLA A,B,C, and antibodies which identify other epidermal cells (anti-vimentin and anti-CD45. We made the following observations. First, the phenotype of epidermal cells that were obtained from cultured epidermal sheets was similar to the phenotype of isolated cells from normal human skin in samples treated with lysolecithin. Second, the CD29+ cells increased compared with the CD29-K1/K10+ in normal human skin and cultured sheets, especially the CD29+K1/K10+ group, which was suprabasal but occasionally basal and highly proliferative. This could be due to the high proliferative ability of keratinocytes in culture. Third, the cultured keratinocytes lost nearly all expression of the HLA class II and class I antigens. The cultured epithelial sheets in the laboratory can therefore be grafted successfully and we may suppose that immunological rejection does not occur.

The degree of maturation and the metabolic stage of epidermal cultured sheets prior to grafting is important because there is a relationship between graft survival and its metabolic stage.The ability of keratinocytes to maintain proliferative potential after seeding and their ability to form a properly oriented epithelium in vitro would suggest that autologous keratinocyte grafting may be suitable as a wound dressing
The epidermis has traditionally been divided into three functionally different layers: germinative, differentiated, and cornified. Some evidence suggests that the germinative compartment is heterogeneous. Two groups of antigens are closely related to the microanatomic location and the stage of cellular differentiation in the epidermis: adhesion molecules and keratins.The determination of (32 integrins (CD29) and KI/K10 intracytoplasmic differentiation keratins by flow cytometry, in combination with optical light scatter characteristics of the cells, identifies the proliferative compartments of in vivo human epidermis.
Two different proliferating cell types have been described in human epidermis.One subset of normal epidermis, predominantly basal, is composed of slow cycling, small cells with a primitive cytoplasmic organization (CD29+ Kl/K10-). The other proliferative subset of normal epidermis, which is suprabasal and occasionally basal, is highly proliferative and larger in size, and exhibits a more complex cytoplasmic structure (CD29+ K1/K10+). The cells that express only the K1/K10 marker are the differentiated keratinocytes of skin (CD29K1/K10+).
Human epidermal cells have been successfully grown in serial culture to form keratinizing colonies. The most satisfactory technique, which is based on those of Rheinwald and Green and of Green, uses serum-containing medium and a feeder layer of lethally irradiated murine 3T3 fibroblasts which is required to initiate formation of the keratinocyte colony.
Additional supplements enhance the proliferation, increase the life span of the keratinocyte cultures, and permit a higher proportion of small proliferating cells. The study of (32 integrins Kl/K10 keratins allows the proliferative stages of cultured human keratinocytes to be distinguished.
Although expression-of Kl/K10 is specific for keratinocytes, the anti-CD 29 recognizes the (32 integrin chain and may be expressed on other cells in the epidermis such as melanocytes, Langerhans cells, or infiltrating leukocyte populations. Although the number of these cells in normal epidermis is relatively low compared with that of the keratinocytes, their staining has been realized in order to determine their contribution. Anti-vimentin, anti-HLADR, and anti-HLA A,B,C antibodies have been used to identify them.
Since keratins K1/K10 and vimentin are intracytoplasmatic, it is necessary to make the cells permeable to these antibodies and to allow the appropriate dye molecules to reach the respective antigens in the cell interior. At the same time, the antigens must be preserved in their natural, antigenic conformation, and leakage out of the cell must be prevented. Several protocols for cell preparation and staining are generally applicable, and thus the cells become permeable to lysolecithin and ethanol. Ethanol dramatically alters the cell morphology, cell aggregation is increased, the antigens lose their distribution and antigenicity, and the background staining is usually more apparent. In view of these facts we used lysolecithin.
Lysolecithin (lysophosphatidylcholine) is a lipid with potent membrane-active characteristics. Lysolecithin replaces phospholipids in the membrane bilayer with a resulting loss of integrity of the membrane, thereby allowing the passage of molecules the size of IgG (=150 kDa). At lower concentrations, lysolecithin can serve to make the cell surface membrane permeable without complete lysis of the cell.
The study and quantification of Class 1 and 11 HLA antigens in epidermal cultured sheets enable us to know at what moment epidermal cells diminish their capacity to initiate an allogeneic immune response.
The study of anti-HLA-DR, present in the Langerhans cells, and of anti-HLA A,B,C, present in all epidermal cells at different levels, enables us to evaluate the immunogenicity of epidermal cells extracted from skin biopsies and cultured sheets. Some studies provide evidence that when human epidermal cells are grown in culture, they lose their expression of HLA-DR antigens, but the cells used in organ equivalent grafts express class I MHC antigens.

Epidermal cell suspension

The 41 skin samples - biopsies taken from normal skin and split-thickness skin grafts from burns - were placed in culture medium and transferred to the laboratory. After removal of as much subcutaneous tissue and dermis as possible, the tissue was cut into smaller fragments. These were then digested with 0.17% trypsin solution (Seromed. Biochrom KG) at 4 °C overnight, followed by treatment with trypsin-EDTA (25% - 0.02 mM) solution Seromed. Bliochrom KG) for 10-20 min to ensure dissociation into single cells 2' These cells were studied by flow cytometry or seeded in plastic flasks to obtain epidermal sheets.

Keratinocyte culture method

Cell permeabilizing method

Staining procedure and flow cytometry analysis Cell suspensions were stained using direct or indirect immunofluorescence staining procedures. The mAbs used were:

1. anti-CD29 (Coulter Immunology) and anti-K1/K10 (Cymbus Bioscience Ltd), markers of epidermal proliferation/differentiation;
2. antibodies of immunogenicity anti-HLA-DR (Becton Dickinson Co.) and W6/32 anti-HLA A,B,C conformational (American Type Culture Collection, Rochville, MD);
3. antibodies which identify the other epidermal cells: anti-vimentin (Cymbus Bioscience Ltd) and antiCD45 (Becton Dickinson Co.), using FITC-Goat

F(ab') 2 mouse anti-IgG (H+L) human antibody (Becton Dickinson Co.) for indirect techniques. Flow cytometry was performed using a FACS SCAN (Becton Dickinson Co.).

The cell population obtained was very heterogeneous in size and complexity and the separation of cells into groups would be somewhat arbitrary. Lysolecithin-permeabilized cells had a similar distribution to those that were not permeabilized, except for small-size cells, because treatment with lysolecithin resulted in partial disruption of the plasma membrane, with a resulting loss of cytoplasm and a decrease in the intensity of forward light scatter. However, the nucleus and cytoplasmic structures such as mitochondria, membrane vesicles, and intermediate filaments apparently remained intact, so that cells retained their distinctive differences in size light scatter.
In normal human epidermis, all the DR+, CD45+, and vimentin+ cells showed double staining with anti-CD29, indicating that (31 integrin is indeed present in Langerhans cells, leukocytes, and melanocytes. The number of these cells in the normal epidermis is relatively low compared with the keratinocytes, which comprise about 90% of epidermal cells. The cell percentages that express each marker as the percentage of total epidermal cells from biopsies (Table I) are similar to the percentages described by others.

Fig. 2 shows a secondary culture at least 30 days old which attained thicknesses ranging between four and six cell layers. During the latter stage of growth, secondary cultures were composed of small, rounded basal cells attached to a plastic substrate and suprabasal layers consisting of enlarged, irregularly shaped vacuolated cells resembling the stratum germinativum and spinosum. Although no stratum corneum developed in these cultures, the cells retained the capacity, once grafted, to differentiate terminally. The basal layer was also more evident and organized (CD29+), and the differentiation appeared in the expressivity of intermediate and superficial layers (Kl/K10+). Occasionally a few keratohyaline granules in cells of the intermediate layer and in the most superficial cells were found.

Immunogenicity of epidermal sheets

Cultured cells have similar phenotypes to the epidermal cells of human skin and the cultures could therefore have similar behaviour to that of adult skin. The low values of K1/K10+ cells were consistent with histological and immunohistochemical results, in which cultured epithelial sheets showed a similar structure to normal epidermis although the state of differentiation was not complete. This decrease of K1/K10+ cells was also observed when the basal and suprabasal cells compartments were analysed, especially in the CD29- K1/K10+ group in epidermal sheets. The granulous layer was absent and the superficial cells showed a lower level of keratinization.26 This partially differentiated sheet retained its capacity to differentiate further when grafted onto the wound and could be used as an autograft. This is very important, for establishing a mechanical barrier and for linking the grafted sheet to the underlying connective tissue.
The high percentage of CD29+ cells observed could be due to the use of samples from young donors in the skin biopsies2b because the growth potential of basal keratinocytes declines as the age of the donor increases. In culture the use of biopsies from young donors provides a high percentage of CD29+ cells ,because colony-forming efficiency, which is an expression of the basal cells' capacity to grow, declines with the patients' increasing age. Cell passages in vitro and natural aging in vivo cause a progressive decrease in cells that possess the highest growth potential (holoclones or CD29+ cells) and a progressive increase in cells that possess the lowest growth potential and with a lifetime of no more than 15 generations (paraclones or CD29- cells.
It is important to note that epithelial cells in culture almost entirely lose their immunological ability because of the expression of HLA Class I antigens. At least four cell types in the epidermis may contribute to the immunogenicity of skin:" the Langerhans cells, the thymus-derived dendritic cell, the melanocyte, and the keratinocyte. Some results have shown that human epidermal Langerhans cells also undergo profound morphological and phenotypic changes during in vitro culture of epidermal cells. Some investigators" have observed the Langerhans cell phenotype as early as 24' to 48 h after isolation of epidermal cells and that their class II and class 1 antigenic density dramatically increases.This is important because in the skin initiation of the immune response has been primarily attributed to class II antigen-bearing Langerhans cells and to subsets of immunogenic cells in the dermis (macrophages, lymphocytes, and capillary endothelial cells). The Langerhans cells are probably critical cells for skin graft rejection when skin from one individual is transplanted to another," but the successful grafting of cells across major histocompatibility barriers suggests that grafted cells are either non-immunogenic or so weakly immunogenic that immunological rejection could not be detected." The failure of cultured keratinocytes to be rejected could be associated with the loss of cells expressing class II antigen during culture. It has been demonstrated that cultured epidermal cells are devoid of Langerhans cells and other class II MHC-bearing cells.
Melanocytes grow in the same culture conditions that allow keratinocyte growth, and their growth is specifically induced by epithelial cells. For this reason melanocytes are the most frequently found cells in epidermal cellular suspension;' however, the expression of MHC class I and II antigens has been shown to be deficient in these cells and in Langerhans cells." This may be confirmed by our study.
Keratinocytes and dermal fibroblasts do not consistently express class II antigens" but there are several diseases in which this happens (graft-versus-host disease, mycosis fungoides, allergic contact dermatitis, lichen planus, tuberculoid leprosy)." Morhenn et al." have shown that interferon can induce this keratinocyte class II synthesis. The role of keratinocyte surface class Il in immune responses is not known but Roberts et al." have suggested that keratinocyte class II expression may regulate Langerhans cell traffic in the epidermis.
In view of our current results, with cultured keratinocytes almost entirely losing their immunological capacity, we might suppose that the immunological rejection did not exist. Although several investigators, using a variety of techniques, have failed to demonstrate the survival of cultured keratinocyte allografts in humans, it does not appear that the grafts are rejected, but instead that they are gradually replaced by recipient cells. The complex immunological response to cultured keratinocyte allografts has not been fully elucidated and continues to be an active area of research, and we still need to study the problem of cultured keratinocyte survival in vivo if these results are to be fully confirned.

RESUME. Il est possible de cultiver les kératinocytes humains normaux en série et dans les conditions culturelles appropriées pour produire des lames épidermiques pour couvrir les défauts profonds et étendus de la peau ou les brûlures. Les Auteurs proposent la cytométrie du flux des cellules épidermiques dans l'épiderme humain normal et les kératinocytes humains cultivés pour prédire le temps optimal pour le greffage des kératinocytes allogènes préparés in vitro. Cette analyse concernait leur phase proliférative et immunogène et l'évaluation des antigènes dont le niveau d'expression est corrélé avec eux. Dams 19 biopsies cutanées et 22 lames épidermiques cultivées les Auteurs ont analysé: les marqueurs des intégrines (CD29) (31 de la prolifération/différentiation épidermique et des kératines intracytoplasmatiques Kl/K10; les anticorps de l'immunogénicité anti-HLA-DR et anti-HLA A,B,C, et les anticorps qui identifient d'autres cellules épidermiques (anti-vimentine et anti-CD45). Les Auteurs ont fait les observations suivantes: premièrement, le phénotype des cellules épidermiques obtenues des lames épidermiques cultivées est semblable au phénotype des cellules isolées de la peau humaine normale dans les échantillons traités avec la lysolécithine; deuxièment, les cellules CD+29 sont augmentées par rapport au CD29-Kl/10+ de la peau normale et les lames cultivées, particulierèment dans le groupe CD29+K1/K10+, qui est suprabasal mais quelquefois basal et extrêmement prolifératif (ceci pourrait être causé par la capacité proliférative élévée des kératinocytes cultivés); troisièmement, les kératinocytes cultivés perdent presque toute l'expression des antigènes HLA de classe I e II. Les Auteurs concluent que les lames épithéliales cultivées en laboratoire pourront être greffées avec succès et qu'il est possible que le rejet immunologique ne se manifeste pas.

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