Beitr. Path Vol. 161, 122-130 (1977)
In Vitro Pathogenesis Section, Experimental Pathology Branch, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland, U.S.A., and American Red Cross Blood Laboratory, Bethesda, Maryland, U.S.A. (R.F.E.)
Morphology of Mouse Epidermal Cells in Vitro: A Scanning Electron Microscopy Study Morphologische Kennzeichen epidermaler Zellen neugeborener Mallse in Gewebekultur K. ELG]Ol, R. FOLLING ELG]O, and H. HENNINGS
Summary Scanning electron microscopy of isolated epidermal cells from newborn mice grown in vitro showed that the cultures consisted of several morphologically different types of cells. Young cultures had many smooth, round cells while older cultures contained more cells with rough or ruffled surface and a varying number of flat, irregular cells. Probably, the various cell types corresponded to different stages of differentation (keratinization). Cultures that were grown in the presence of retinyl acetate (12.5 fLg /ml) had more round and smooth cells after several days in vitro than the controls. This could indicate that retinyl acetate delayed or altered cell differentiation. Scanning electron microscopy of the cultures consistently showed that the cells were situated at different layers. The apparent monolayer seen by phase contrast microscopy therefore seems to be an optical phenomenon due to projection of the cells onto the same plane.
Light microscopy studies of epidermal cells grown in vitro have shown that proliferation and differentiation (keratinization) mainly occur after the cells have formed aggregates (Briggaman et al., 1967; Fusenig, 1971; Fusenig and Worst, 1974; Yuspa and Harris, 1974). From the periphery of such aggregates cells proliferate and form a more or less continuous sheet that appears as a monolayer when examined by phase contrast microscopy, or by ordinary light microscopy after fixation and staining. 1 This work was done during the tenure of an American Cancer Society Eleanor Roosevelt International Cancer Fellowship awarded by the International Union Against Cancer.
Morphology of Mouse Epidermal Cells in Vitro .
I2 3
To examine in more detail the in vitro growth pattern of epidermal cells, we have studied cultures of epidermal cells from newborn mice by means of scanning electron microscopy (SEM). This technic has the advantage that it gives a three-dimensional illustration of the cells and the cell aggregates. It also permits a closer examination of the morphologic details of the surface of proliferating and differentiating cells. We shall also indicate how this technic can be helpful in evaluating some morphologic alterations occurring when cells are grown in the presence of vitamin A (retinyl acetate), which modifies epidermal growth and differentiation in vivo and in vitro.
Materials and Methods Cell cultures
Cultures of epidermal cells from newborn BALB/c mice were made as described previously (Yuspa and Harris, r974). The cells were seeded in 35 mm Falcon plastic cell culture dishes containing round cover slips (0.8 X r06 cells per ml). Medium r99 plus r r% fetal calf serum (Rehatuin Fetal Bovine Serum) and r% antibiotic-antimycotic mixture (GIBCO, Grand Island, N. Y.) was used as growth medium. The medium was changed every 24 hours after seeding. Some cultures were grown in medium containing trans-~-retinyl acetate (RA). This medium was prepared immediately before use and the treatment started 24 hours after plating. RA was dissolved in DMSO and added to the medium to give a final concentration of 12.5 iJ-g/ml and 1.25% DMSO. RA dissolved in DMSO was either used immediately after preparation or stored as a I mg/m! solution of RA in DMSO at - 70° C for not more than ro days. All handling of RA-containing solutions was made in indirect lighting. The control cultures in this series of experiments were grown in medium containing 1.25°/0 DSMO. Scanning electron microscopy
The glass coverslips were removed from the plastic dishes, washed in cold PBS and fixed in buffered 1.24% glutaraldehyde with 4% sucrose (pH 7.2) for 24 hours. After washing in phosphate buffer they were postfixed in r% OS04 for r hour, dehydrated in increasing concentrations of ethanol up to roo%. The cells were then passed through 50°/0 amylacetate/50% ethanol, and rooO/o amylacetate. The covers lips were processed through a Denton vacuum (DCP r) critical-point drying apparatus, and finally coated with palladiumgold. All specimens were examined in a mini-SEM ICI Scanning Scope.
Results Light microscopy The cultures were observed daily by phase contrast microscopy and the findings were essentially similar to those described by Yuspa and Harris
124 . K. Elgjo, R. Foiling Elgjo, and H . Hennings
Fig. 1. SEM picture of 48-hour culture showing a part of the central core where keratin-like material (right half) covers the underlying cells. At the periphery of the aggregate (left half) many Type I cells are see n. X 2,000.
(1974), who carried out experiments with the same in vitro system. Small aggregates of epidermal cells were seen after about 12 hours in vitro. Dur-
ing the following days cells grew out from the periphery and formed what appeared to be a monolayer of cells. Birefringent material was usually observed in the center of the aggregates. This material probably represents keratin (Yuspa and Harris, 1974).
Scanning electron microscopy SEM revealed that the aggregates had a more complex structure than could be seen by phase contrast microscopy, or in fixed and stained slides of the cultured epidermal cells. Even the early and small aggregates had one or more flat cells in the center that probably represented keratinized cells (Fig. I). Some aggregates had flat and irregular sheets of material that was interpreted as keratin, and such material could completely cover the underlying cells (Fig. 2).
Fig. 2. In this 48-hour culture most of the cells in the aggregate are covered by keratin-like material and only a few cells are seen at the periphery of the aggregate. X 3,000.
Fig. 3. lo-day culture with cluster of Type round shape. X 5,000. 9 8eilr. Path. Vol. 161
2
cells, with creased of ruffled surface and
126 .
K. Elgjo, R. Foiling Elgjo, and H. Hennings
Fig. 4. 6o-hour culture, demonstrating a Type 2 cell in the upper left corner. The other cells represent Type 3 cells with rough, "ulcerated" surfaces, resembling sand paper. X 7,000.
After about 24 hours in culture, the cells started to spread from the periphery of the aggregates. The cells in this peripheral growth zone seemed to form a monolayer when examined by light microscopy, but SEM clearly showed that even the peripheral cells spread out on different levels from the central aggregates like multilayered clusters of cells. Mitoses were seen only among these peripherally situated cells. After 1-2 days in culture it was possible to distinguish between several types of cells. Transitions between the various types were frequently found, but most cells could be classified as follows: I. Round cells with smooth or finely ruffled surface (Fig. I). 2. Round cells with markedly ruffled or corrugated surface (Fig. 3). 3. Round cells with rough, sometimes granular surface with "ulcerations" (Fig. 4). 4. Irregular cells with heavily creased surface and with one or more craters on the surface. Some of these cells were still more or less spherical, while others were flat. No sharp distinction could be drawn between
Fig. 5. This picture of a 69-hour culture shows a Type 4 cell with a crater in the surface (left of center). Fully keratinized cells are situated underneath this cell. A smaller cell (right of center) is probably in the process of being keratinized (Type 4 cell). X 5,000.
Fig. 6. Io-day culture treated with RA. The cells grow as aggregates with a few keratinized cells in the center. The majority of the cells represent Type I and 2 cells. A mitosis (telophase) is seen near the upper right corner. X 700.
128 . K. Elgjo, R. FoIling Elgjo, and H. Hennings
such cells and sheets of keratin-like material that probably represented fully keratinized cells (Fig. 5). In all cultures Type 3 and 4 cells were prevalent in the centers of the aggregates, while Type I and 2 cells were found in peripheral growth zones. In older cultures, the number of Type I cells decreased even in the peripheral zone, and after 9-10 days the majority of the cells belonged to Type 3 and 4. A continuous monolayer of cells was not found by SEM at any time or in any culture. The cells that were grown in medium containing RA could not be distinguished qualitatively from the routinely treated cells. After about 3 days in culture there were, however, some quantitative differences between RA-treated cells and those grown in control medium. RA-treated cells had less keratin-like material in the center of the aggregates, and more Type I cells were seen in the peripheral zone of the cell aggregates. In the RAtreated cultures, several Type I and 2 cells were found even after 10 days in culture, and mitoses were observed among these cells (Fig. 6).
Discussion Epidermal cells usually require the presence of fibroblasts, collagen, or a fibroblast-conditioned medium to grow and differentiate (Wessels, 1967; Briggaman and Wheeler, 1968; Karasek, 1968; Karasek and Charlton, 1971; Rheinwald and Green, 1975). Epidermal cells from newborn mice seem to be different in this respect, as they will grow and differentiate at least for a limited period of time in the absence of cells of mesenchymal origin and of collagen (Fusenig and Worst, 1974; Yuspa and Harris, 1974). Like other epidermal cells in culture, they consistently grow as aggregates of cells, and keratinization mainly takes place where several cells are in contact with each other. It thus seems to be a kind of "contact communication" between the epidermal cells that directs some cells to continue proliferation and others to differentiate. This phenomenon has been discussed by Rheinwald and Green (1975). Single, isolated keratinized cells (Type 3 and 4 cells) were observed in early cultures. Attachment of differentiated cells from the primary cell preparation has been observed with phase contrast microscopy; thus these cells were not the result of rapid differentiation of some isolated cells in vitro. The cells observed in the periphery of the aggregates (Type I and 2 cells) probably represented young cells capable of division, as mitotic figures were often seen among these cells on the first days after seeding.
Morphology of Mouse Epidermal Cells in Vitro . I29
Most likely, these cells correspond to epidermal basal cells. The cells at the bottom of the aggregates could not be seen by means of SEM. Other investigators have, however, made sections of other types of epidermal cells growing as aggregates in vitro, and have found a layer of cells underneath the keratinizing cells (Rheinwald and Green, I 97 5). This bottom layer was interpreted as "epidermal basal layer", and the cells were morphologically similar to basal layer cells in vivo (Rheinwald and Green, 1975)·
When the cultured epidermal cells were examined by phase contrast microscopy, the peripheral cells in a given culture all looked similar and the center was covered by ill-defined keratinlike material and cell debris. SEM demonstrated a great diversity of cell types among the peripheral cells and the central cells. With increasing age of the cultures, more and more of the peripheral cells changed into Type 2, 3, and 4 cells. To a certain extent, this penomenon may represent degenerative alterations in the cell population. However, many of the peripheral cells showed changes that seemed to indicate keratinization, such as increasing ruffling of the cell surface. Such surface alterations characterized the cells found at the center of the aggregates where keratin-like material usually was present. Craters (Fig. 5) were seen only in older cultures, where they quite frequently occurred. We have interpreted this phenomenon as a surface alteration related to keratinization. We have never observed such craters in other cell types prepared in the same way as the epidermal cells, and we therefore do not think it is likely that it is an artifact caused by the fixation and preparation procedures. Phase contrast microscopy of older cultures usually showed a monolayer of cells between the aggregates, but no such monolayer was seen by SEM. This phenomenon was not due to artifacts produced during the preparation of cells for SEM, since phase contrast microscopy of cultures prepared for SEM still showed a monolayer of cells between the aggregates in older cultures. The most likely explanation of this discrepancy is that phase contrast microscopy creates an optical impression of a monolayer as the cells will be projected onto a plane even when they actually are not in contact with each other and are growing at different levels above the glass surface. In RA-treated cultures the cells seemed to remain morphologically "young" for a longer period of time than those growing in control medium. This is in good agreement with the findings of Yuspa and Harris (1974), They found in their experiments that RA-treated, newborn epidermal cells in vitro probably had a longer average life span and less tendency to keratinize than cells grown in control medium.
13 0
.
K. Elgjo, R. Hilling Elgjo, and H. Hennings
References Briggaman, R. A., Abele, D. c., Harris, S. R., and Wheeler, C. E.: Preparation and characterization of a viable suspension of postembryonic human epidermal cells. J. invest. Derm. 48,159-168 (1967) Briggaman, R. A., and Wheeler, C. E.: Epidermal-dermal interactions in adult human skin: Role of dermis in epidermal maintenance. ]. invest. Derm. fl, 454-465 (1968) Fusenig, N. E.: Isolation and cultivation of epidermal cells from embryonic mouse skin. Naturwissenschaften 58, 421-422 (1971) Fusenig, N. E., and Worst, P. K. M.: Mouse epidermal cell cultures. I. Isolation and cultivation of epidermal cells from adult mouse skin. J. invest. Derm. 63, 187-193 (1974) Karasek, M. A.: Growth and differentiation of transplanted epithelial cell cultures. J. invest. Derm. fl, 247-252 (1968) Karasek, M. A., and Charlton, M. E.: Growth of postembryonic skin epithelial cells on collagen §els. J. invest. Derm. 56, 205-210 (1971) Rheinwald, ]. G., and Green, H.: Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331-344 (1975 ) Wessels, N. K.: Differentiation of epidermis and epidermal derivatives. New Engl. J. Med. 277, 21-33 (1967) Yuspa, S. H., and Harris, C. c.: Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp. Cell Res. 86, 95-105 (1974)
Received January 24, 1977 . Accepted in revised form June 6, 1977
Key words: Mouse epidermal cells - Tissue culture - Scanning electron microscopy - Retinyl acetate - Newborn Mice KjeIl, Elgjo, M. D., Institute of Pathology, Rikshospitalet, Oslo I, Norway Ragna FoIling Egljo, M. D., The Norwegian Radium Hospital, Montebello, Oslo 3, Norway Henry Hennings, Ph. D., National Cancer Institute, Room 3A2I, Building 37, Bethesda, Maryland 20014, U.s.A.
In Vitro Pathogenesis Section, Experimental Pathology Branch, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland, U.S.A., and American Red Cross Blood Laboratory, Bethesda, Maryland, U.S.A. (R.F.E.)
Morphology of Mouse Epidermal Cells in Vitro: A Scanning Electron Microscopy Study Morphologische Kennzeichen epidermaler Zellen neugeborener Mallse in Gewebekultur K. ELG]Ol, R. FOLLING ELG]O, and H. HENNINGS
Summary Scanning electron microscopy of isolated epidermal cells from newborn mice grown in vitro showed that the cultures consisted of several morphologically different types of cells. Young cultures had many smooth, round cells while older cultures contained more cells with rough or ruffled surface and a varying number of flat, irregular cells. Probably, the various cell types corresponded to different stages of differentation (keratinization). Cultures that were grown in the presence of retinyl acetate (12.5 fLg /ml) had more round and smooth cells after several days in vitro than the controls. This could indicate that retinyl acetate delayed or altered cell differentiation. Scanning electron microscopy of the cultures consistently showed that the cells were situated at different layers. The apparent monolayer seen by phase contrast microscopy therefore seems to be an optical phenomenon due to projection of the cells onto the same plane.
Light microscopy studies of epidermal cells grown in vitro have shown that proliferation and differentiation (keratinization) mainly occur after the cells have formed aggregates (Briggaman et al., 1967; Fusenig, 1971; Fusenig and Worst, 1974; Yuspa and Harris, 1974). From the periphery of such aggregates cells proliferate and form a more or less continuous sheet that appears as a monolayer when examined by phase contrast microscopy, or by ordinary light microscopy after fixation and staining. 1 This work was done during the tenure of an American Cancer Society Eleanor Roosevelt International Cancer Fellowship awarded by the International Union Against Cancer.
Morphology of Mouse Epidermal Cells in Vitro .
I2 3
To examine in more detail the in vitro growth pattern of epidermal cells, we have studied cultures of epidermal cells from newborn mice by means of scanning electron microscopy (SEM). This technic has the advantage that it gives a three-dimensional illustration of the cells and the cell aggregates. It also permits a closer examination of the morphologic details of the surface of proliferating and differentiating cells. We shall also indicate how this technic can be helpful in evaluating some morphologic alterations occurring when cells are grown in the presence of vitamin A (retinyl acetate), which modifies epidermal growth and differentiation in vivo and in vitro.
Materials and Methods Cell cultures
Cultures of epidermal cells from newborn BALB/c mice were made as described previously (Yuspa and Harris, r974). The cells were seeded in 35 mm Falcon plastic cell culture dishes containing round cover slips (0.8 X r06 cells per ml). Medium r99 plus r r% fetal calf serum (Rehatuin Fetal Bovine Serum) and r% antibiotic-antimycotic mixture (GIBCO, Grand Island, N. Y.) was used as growth medium. The medium was changed every 24 hours after seeding. Some cultures were grown in medium containing trans-~-retinyl acetate (RA). This medium was prepared immediately before use and the treatment started 24 hours after plating. RA was dissolved in DMSO and added to the medium to give a final concentration of 12.5 iJ-g/ml and 1.25% DMSO. RA dissolved in DMSO was either used immediately after preparation or stored as a I mg/m! solution of RA in DMSO at - 70° C for not more than ro days. All handling of RA-containing solutions was made in indirect lighting. The control cultures in this series of experiments were grown in medium containing 1.25°/0 DSMO. Scanning electron microscopy
The glass coverslips were removed from the plastic dishes, washed in cold PBS and fixed in buffered 1.24% glutaraldehyde with 4% sucrose (pH 7.2) for 24 hours. After washing in phosphate buffer they were postfixed in r% OS04 for r hour, dehydrated in increasing concentrations of ethanol up to roo%. The cells were then passed through 50°/0 amylacetate/50% ethanol, and rooO/o amylacetate. The covers lips were processed through a Denton vacuum (DCP r) critical-point drying apparatus, and finally coated with palladiumgold. All specimens were examined in a mini-SEM ICI Scanning Scope.
Results Light microscopy The cultures were observed daily by phase contrast microscopy and the findings were essentially similar to those described by Yuspa and Harris
124 . K. Elgjo, R. Foiling Elgjo, and H . Hennings
Fig. 1. SEM picture of 48-hour culture showing a part of the central core where keratin-like material (right half) covers the underlying cells. At the periphery of the aggregate (left half) many Type I cells are see n. X 2,000.
(1974), who carried out experiments with the same in vitro system. Small aggregates of epidermal cells were seen after about 12 hours in vitro. Dur-
ing the following days cells grew out from the periphery and formed what appeared to be a monolayer of cells. Birefringent material was usually observed in the center of the aggregates. This material probably represents keratin (Yuspa and Harris, 1974).
Scanning electron microscopy SEM revealed that the aggregates had a more complex structure than could be seen by phase contrast microscopy, or in fixed and stained slides of the cultured epidermal cells. Even the early and small aggregates had one or more flat cells in the center that probably represented keratinized cells (Fig. I). Some aggregates had flat and irregular sheets of material that was interpreted as keratin, and such material could completely cover the underlying cells (Fig. 2).
Fig. 2. In this 48-hour culture most of the cells in the aggregate are covered by keratin-like material and only a few cells are seen at the periphery of the aggregate. X 3,000.
Fig. 3. lo-day culture with cluster of Type round shape. X 5,000. 9 8eilr. Path. Vol. 161
2
cells, with creased of ruffled surface and
126 .
K. Elgjo, R. Foiling Elgjo, and H. Hennings
Fig. 4. 6o-hour culture, demonstrating a Type 2 cell in the upper left corner. The other cells represent Type 3 cells with rough, "ulcerated" surfaces, resembling sand paper. X 7,000.
After about 24 hours in culture, the cells started to spread from the periphery of the aggregates. The cells in this peripheral growth zone seemed to form a monolayer when examined by light microscopy, but SEM clearly showed that even the peripheral cells spread out on different levels from the central aggregates like multilayered clusters of cells. Mitoses were seen only among these peripherally situated cells. After 1-2 days in culture it was possible to distinguish between several types of cells. Transitions between the various types were frequently found, but most cells could be classified as follows: I. Round cells with smooth or finely ruffled surface (Fig. I). 2. Round cells with markedly ruffled or corrugated surface (Fig. 3). 3. Round cells with rough, sometimes granular surface with "ulcerations" (Fig. 4). 4. Irregular cells with heavily creased surface and with one or more craters on the surface. Some of these cells were still more or less spherical, while others were flat. No sharp distinction could be drawn between
Fig. 5. This picture of a 69-hour culture shows a Type 4 cell with a crater in the surface (left of center). Fully keratinized cells are situated underneath this cell. A smaller cell (right of center) is probably in the process of being keratinized (Type 4 cell). X 5,000.
Fig. 6. Io-day culture treated with RA. The cells grow as aggregates with a few keratinized cells in the center. The majority of the cells represent Type I and 2 cells. A mitosis (telophase) is seen near the upper right corner. X 700.
128 . K. Elgjo, R. FoIling Elgjo, and H. Hennings
such cells and sheets of keratin-like material that probably represented fully keratinized cells (Fig. 5). In all cultures Type 3 and 4 cells were prevalent in the centers of the aggregates, while Type I and 2 cells were found in peripheral growth zones. In older cultures, the number of Type I cells decreased even in the peripheral zone, and after 9-10 days the majority of the cells belonged to Type 3 and 4. A continuous monolayer of cells was not found by SEM at any time or in any culture. The cells that were grown in medium containing RA could not be distinguished qualitatively from the routinely treated cells. After about 3 days in culture there were, however, some quantitative differences between RA-treated cells and those grown in control medium. RA-treated cells had less keratin-like material in the center of the aggregates, and more Type I cells were seen in the peripheral zone of the cell aggregates. In the RAtreated cultures, several Type I and 2 cells were found even after 10 days in culture, and mitoses were observed among these cells (Fig. 6).
Discussion Epidermal cells usually require the presence of fibroblasts, collagen, or a fibroblast-conditioned medium to grow and differentiate (Wessels, 1967; Briggaman and Wheeler, 1968; Karasek, 1968; Karasek and Charlton, 1971; Rheinwald and Green, 1975). Epidermal cells from newborn mice seem to be different in this respect, as they will grow and differentiate at least for a limited period of time in the absence of cells of mesenchymal origin and of collagen (Fusenig and Worst, 1974; Yuspa and Harris, 1974). Like other epidermal cells in culture, they consistently grow as aggregates of cells, and keratinization mainly takes place where several cells are in contact with each other. It thus seems to be a kind of "contact communication" between the epidermal cells that directs some cells to continue proliferation and others to differentiate. This phenomenon has been discussed by Rheinwald and Green (1975). Single, isolated keratinized cells (Type 3 and 4 cells) were observed in early cultures. Attachment of differentiated cells from the primary cell preparation has been observed with phase contrast microscopy; thus these cells were not the result of rapid differentiation of some isolated cells in vitro. The cells observed in the periphery of the aggregates (Type I and 2 cells) probably represented young cells capable of division, as mitotic figures were often seen among these cells on the first days after seeding.
Morphology of Mouse Epidermal Cells in Vitro . I29
Most likely, these cells correspond to epidermal basal cells. The cells at the bottom of the aggregates could not be seen by means of SEM. Other investigators have, however, made sections of other types of epidermal cells growing as aggregates in vitro, and have found a layer of cells underneath the keratinizing cells (Rheinwald and Green, I 97 5). This bottom layer was interpreted as "epidermal basal layer", and the cells were morphologically similar to basal layer cells in vivo (Rheinwald and Green, 1975)·
When the cultured epidermal cells were examined by phase contrast microscopy, the peripheral cells in a given culture all looked similar and the center was covered by ill-defined keratinlike material and cell debris. SEM demonstrated a great diversity of cell types among the peripheral cells and the central cells. With increasing age of the cultures, more and more of the peripheral cells changed into Type 2, 3, and 4 cells. To a certain extent, this penomenon may represent degenerative alterations in the cell population. However, many of the peripheral cells showed changes that seemed to indicate keratinization, such as increasing ruffling of the cell surface. Such surface alterations characterized the cells found at the center of the aggregates where keratin-like material usually was present. Craters (Fig. 5) were seen only in older cultures, where they quite frequently occurred. We have interpreted this phenomenon as a surface alteration related to keratinization. We have never observed such craters in other cell types prepared in the same way as the epidermal cells, and we therefore do not think it is likely that it is an artifact caused by the fixation and preparation procedures. Phase contrast microscopy of older cultures usually showed a monolayer of cells between the aggregates, but no such monolayer was seen by SEM. This phenomenon was not due to artifacts produced during the preparation of cells for SEM, since phase contrast microscopy of cultures prepared for SEM still showed a monolayer of cells between the aggregates in older cultures. The most likely explanation of this discrepancy is that phase contrast microscopy creates an optical impression of a monolayer as the cells will be projected onto a plane even when they actually are not in contact with each other and are growing at different levels above the glass surface. In RA-treated cultures the cells seemed to remain morphologically "young" for a longer period of time than those growing in control medium. This is in good agreement with the findings of Yuspa and Harris (1974), They found in their experiments that RA-treated, newborn epidermal cells in vitro probably had a longer average life span and less tendency to keratinize than cells grown in control medium.
13 0
.
K. Elgjo, R. Hilling Elgjo, and H. Hennings
References Briggaman, R. A., Abele, D. c., Harris, S. R., and Wheeler, C. E.: Preparation and characterization of a viable suspension of postembryonic human epidermal cells. J. invest. Derm. 48,159-168 (1967) Briggaman, R. A., and Wheeler, C. E.: Epidermal-dermal interactions in adult human skin: Role of dermis in epidermal maintenance. ]. invest. Derm. fl, 454-465 (1968) Fusenig, N. E.: Isolation and cultivation of epidermal cells from embryonic mouse skin. Naturwissenschaften 58, 421-422 (1971) Fusenig, N. E., and Worst, P. K. M.: Mouse epidermal cell cultures. I. Isolation and cultivation of epidermal cells from adult mouse skin. J. invest. Derm. 63, 187-193 (1974) Karasek, M. A.: Growth and differentiation of transplanted epithelial cell cultures. J. invest. Derm. fl, 247-252 (1968) Karasek, M. A., and Charlton, M. E.: Growth of postembryonic skin epithelial cells on collagen §els. J. invest. Derm. 56, 205-210 (1971) Rheinwald, ]. G., and Green, H.: Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331-344 (1975 ) Wessels, N. K.: Differentiation of epidermis and epidermal derivatives. New Engl. J. Med. 277, 21-33 (1967) Yuspa, S. H., and Harris, C. c.: Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp. Cell Res. 86, 95-105 (1974)
Received January 24, 1977 . Accepted in revised form June 6, 1977
Key words: Mouse epidermal cells - Tissue culture - Scanning electron microscopy - Retinyl acetate - Newborn Mice KjeIl, Elgjo, M. D., Institute of Pathology, Rikshospitalet, Oslo I, Norway Ragna FoIling Egljo, M. D., The Norwegian Radium Hospital, Montebello, Oslo 3, Norway Henry Hennings, Ph. D., National Cancer Institute, Room 3A2I, Building 37, Bethesda, Maryland 20014, U.s.A.
