Archs ord Bml Vol. 24. pp. 335 to 341 Pergamon Press Ltd 1979 Prmted m Great Bntain

IN-VITRO CULTIVATION OF HUMAN ORAL KERATINOCYTES

L. TAICHMAN, S. REILLY and P. R. GARANT

Department of Oral Biology and Pathology, School of Dental Medicine, Health Sciences Center, SUNY at Stony Brook, Stony Brook, NY 11794, U.S.A.

Summary-The technique of Rheinwald and Green (1975b) for the serial cultivation of foreskin keratinocytes was modified to facilitate the cultivation of gingival and buccal keratinocytes. We used 7-lo-day epithelial outgrowths from excised tissue fragments as a source of keratino- cytes, whereas Rheinwald and Green used enzymically-dissociated keratinocytes from freshly obtained foreskin. Mitomycin-C treatment as opposed to gamma irradiation was used to suppress the growth of fibroblast feeder layers. Single oral keratinocytes proliferated to form colonies resembling a stratified squamous epithelium. Light and electron microscopy revealed structures usually associated with epithelia, i.e. desmosomes, tonofilaments, tonofibrils, thick- ened cell membranes and a glycocalyx on the free surface of the epithelium. Oral keratinocytes underwent 2@40 doublings before senescence

INTRODUmION

Because of the complexity of the oral environment, in-t&o models for the study of oral mucosal disorders are needed. Oral mucosal cells maintained in vitro would be useful for metabolic studies of epithelial differentiation and keratinization. Oral keratinocytes have been cultivated successfully by Flaxman, Lutzner and Van Scott (1967) and Mlinek and Buchner (1975) but, as in most epithelial cell culture systems, propagation beyond the initial outgrowth proved difficult or impossible (Prunieras, Delescluse and Regnier, 1976).

Rheinwald and Green (1975b) found that human foreskin keratinocytes could be maintained in culture for 20-50 doublings if they were seeded on 3T3 mouse fibroblasts (Todaro and Green, 1963). To prevent overgrowth of the 3T3 cells, they were irradiated with a lethal dose of gamma-rays before being grown with keratinocytes. Single keratinocytes produced macro- scopic colonies of stratified squamous epithelium (Green, 1977).

We applied this technique, with minor modifica- tion, to the cultivation of keratinocytes from gingival and buccal mucosa.

MATERIALS AND METHODS

Gingival and buccal mucosa was obtained from adults undergoing gingivectomy or excisional biopsy. Care was taken to secure material from the margins of the excised tissue that appeared grossly and histo- logically normal. Foreskins from newborn infants were also obtained. Within 12 h after excision, the tissue was minced and fragments placed in 30-mm plastic culture dishes and fed keratinocyte medium consisting of 20 per cent fetal calf serum, 80 per cent Dulbecco minimal essential medium, 0.4 pg/ml hydro- cortisone, 100 units/ml penicillin, 100 pgcglml strepto- mycin, 10 &ml kanamycin, 1 pg/ml Fungizone, and 10mM Hepes buffer. The fragments were held down by a glass coverslip. Incubation was carried out at

37°C in loper cent COz. Kanamycin and Fungizone were omitted from the keratinocyte media after first passage.

After 7-lOdays, the keratinocytes had grown out from the tissue explants and were ready for their first passage. Contaminating fibroblasts were removed as described by Rheinwald and Green (1975a, b) with 0.02 per cent EDTA and adherent keratinocytes dis- lodged with a solution of 0.05 per cent trypsin and 0.01 per cent EDTA. The keratinocytes were collected by low-speed centrifugation, resuspended in fresh ker- atinocyte medium and added to the feeder layers of 3T3 cells prepared as follows: confluent monolayers of 3T3 cells maintained routinely (Rheinwald and Green, 1975a, b) were treated for 3 h in the dark with lOpg/ml mitomycin-C. The 3T3 cells were rinsed thoroughly with fresh keratinocyte medium, tryp- sinized and re-seeded into new culture dishes at 75 cells/mm’ in keratinocyte medium. After 2-3 h, the cells had become attached to the surface and within 24 h could be used as feeder layers.

Mitomycin-C treatment was found to be an effec- tive substitute for gamma-irradiation in conditioning 3T3 cells as feeder layers. The plating efficiency of mitomycin-C-treated cells was measured by seeding known numbers of 3T3 cells onto 60-mm culture dishes and counting toluidine-blue-stained colonies 10days later. The plating effic’ency of mitomycin-C- treated 3T3 cells was less than a .002 per cent and that of untreated controls was 2per cent. Mitomycin-C- treated cells when re-seeded underwent about 1.5 cell doublings while untreated 3T3 cells re-seeded at iden- tical densities underwent about 3.5 cell doublings (Text Fig. 1).

Keratinocytes were fed every 3-4 days and subcul- tured when colony growth covered most of the sur- face of the dish or when the cells began to pile up. The method for sub-culturing was the same as that described above for first passage. Cell counts were made with the Colter Counter (Model ZBI). The clon- ing efficiency of keratinocytes was measured by seed- ing known numbers of single cells on to feeder layers

335

336 L. Taichman, S. Reilly and P. R. Garant

GROWTH OF 3T3 CELLS FOLLOWING MITOMYCIN C TREATMENT

I- MITOMYCIN C

104-_ DAYS

Fig. 1. Growth of 3T3 cells after mitomycin-C treatment. The cells were disaggregated with trypsin and re-seeded in 16 mm wells at 2.5 x lo4 cells/well. Duplicate wells were

counted at each interval.

and staining the colonies with haematoxylin and eosin after 2 weeks of growth.

In preparation for electron microscopy, cultures were fixed for 30 min in a 1: 1 mixture of 0.2 M Kar- novsky fixative, pH 7.2 and 0.1 M cacodylate buffer, and post-fixed for 30min in a 1 :l mixture of 2 per cent 0~0, and 0.2 M collidine buffer, pH 7.4. The cultures were thoroughly rinsed with maleate buffer, pH 5.15, and stained for 30 min with 2 per cent uranyl acetate. The cultures were rinsed again with maleate buffer, dehydrated with ethanol and embedded in Epon. The plastic culture dishes were fractured away from the polymerized Epon after cooling in liquid nitrogen. l-pm thick sections were stained with a 1 per cent solution of toluidine blue for light micro- scopy and ultrathin sections were stained with uranyl acetate and lead citrate for electron microscopy.

RESULTS

One to two days after seeding, the gingival and buccal keratinocytes attached to the substrate and by the fourth to fifth day small colonies of l(r20cells were visible in the spaces between 3T3 cells (Plate Fig. 2). As the keratinocyte colony grew (Plate Fig. 3), 3T3 cells were pushed aside at the periphery until all that remained of the feeder layer was a thin rim of packed cells (Plate Fig. 4). Initially, the colony was formed by a monolayer of cells but, after 7-9 days of growth, stratification became evident in the centre of the colony. Superficial cells were larger in their lateral dimensions, i.e. in a plane parallel to the substratum, than the underlying basal cells and their cytoplasm contained parallel arrays of granular inclu- sions (Plate Fig. 5). In 2-3 weeks, the colonies reached maturity, lateral expansion ceased, borders of adja- cent colonies merged and flattened, irregularly-shaped cells were desquamated from the surface.

Gingival and buccal colonies were similar in cross- section (Plate Figs. 6A, 6B and 7). The number of cell layers ranged from 3 to 7 and nuclei were evident at all levels. Tonofilaments measuring about 8nm were observed in all cells, but were particularly prominent in the superficial cells, where they aggre- gated to form tonofibrils (Plate Fig. 8). The tonofibrils produced the regular array of cytoplasmic granular inclusions seen with phase contrast microscopy in the living keratinocyte (Plate Fig. 5). In our view, fewer tonofibrils were present in the superficial layers of buccal colonies than in gingival or foreskin colonies. Desmosomes were found between all cell layers and in most instances tonofilaments were observed pass- ing into the attachment plaques of the desmosomes (Plate Fig. 9). An electron-dense 12-nm layer was observed on the inner aspect of the plasma membrane of some superficial cells (Plate Fig. 10). This dense material probably represents the “cornified envelope” described by Sun and Green (1977). In cultures of buccal keratinocytes and to a lesser extent in gingival keratinocytes, we observed a glycocalyx along the most superficial aspect (free surface) of the colony (Plate Fig. 11). At no time were keratohyalin granules or membrane-coating granules observed.

After several passages, keratinocytes formed small irregular colonies of stratified cells. There was little lateral growth. Gingival and buccal cells were cul- tured through 24 passages and, based on the gener- ation time for keratinocytes reported by Rheinwald and Green (1975b), we estimate that 20-40 cell doub- lings had occurred. The cloning efficiency of gingival keratinocytes was 1.5 per cent on feeder layers and less than 0.002per cent in the absence of 3T3 cells. If the 3T3 cells were removed with EDTA and the small colonies of adherent keratinocytes fed fresh media, they continued to divide and differentiate. Keratinocytes appear to require 3T3 cells for attach- ment but not for continued growth.

DISCUSSION

The life expectancy of buccal and gingival keratino- cytes corresponds favourably to that reported for foreskin keratinocytes (Rheinwald and Green, 1975b) and fibroblasts (Hayflick, 1965). The growth potential of foreskin keratinocytes can be increased two-fold by the addition of epidermal growth factor to the culture media (Rheinwald and Green, 1977). Prelimi- nary studies in this laboratory (not yet reported) indi- cate that gingival and buccal keratinocytes respond to the growth factor in a similar manner.

Without 3T3 cells, the cloning efficiency of gingival keratinocytes dropped to a very low level. We do not know why this occurs and note with interest the reports of success in culturing human gingival kera- tinocytes (Birkedal-Hansen et al.: 1978) and rat lingual epithelial cells (Jepsen, MacCallum and Lillie, 1978) in the absence of fibroblast support. We have found as J. G. Rheinwald (persona1 communication), that keratinocytes can be cultured in the absence of 3T3 cells if seeded at very high cell densities or as aggregates of undissociated cells. This is possibly the basis of the apparent discrepancies.

We have observed that keratinocytes originating from orthokeratinizing (foreskin), parakeratinizing

In-vitro cultivation of oral keratinocytes 337

(gingiva) and non-keratinizing (buccal mucosa) areas form very similar colonies in culture. Sun and Green (1977) reported similar results with keratinocytes from skin, cornea and conjunctiva, and noted that the kera- tin proteins from each had similar electrophoretic mobilities. It is not clear from these results whether the common pattern of differentiation represents in- ductive effects of the 3T3 feeder cells or an inherent pattern of differentiation of all epithelial cells. Mesen- chymal cells are thought to exert continual inductive effects upon overlying epithelia and in this way modify the basic pattern of differentiation (Flaxman and Maderson, 1976). The fact that keratinocytes cul- tured in the absence of fibroblast support are similar to those cultured with 3T3 cells argues against an inductive effect but does not disprove it. The impor- tant questions are whether keratin~ytes can undergo redifferentiation in culture and what external in- fluences bring this about. Experiments using various types of fibroblasts or non-cellular supports may help to answer these questions. Long-term serial keratino- cyte cultivation provides a new method for the ex- ploration of epidermal differentiation.

Acknowledgements-We thank Drs. J. Sciubba and R. Davis for securing oral biopsies and Ms. M. R. Cullen and Mr. M. I. Cho for their help with the technical aspects of this study. This research was supported by a Biomedical Research Support Grant from N.I.H. and grant No. DE 03745 from N.I.D.R.

REFERENCES

gingival epithelial cell line in vitro. J. dent. Res. 57A, Abstract No. 95.

Fiaxman B. A., Lutzner M. A. and Van Scott E. J. 1967, Cell maturation and tissue organization in epithelial out- growths from skin and buccal mucosa in vitro. J. invest. Derm. 49, 322-329.

Flaxman B. A. and Maderson F. A. 1976. Growth and differentiation of skin. J. invest. Derm. 67, S-14.

Green H. 1977. Terminal differentiation of cultured human epidermal ceils. Ceil 11, 405-416.

Hayflick L. 1965. The limited in vitro life time of human diploid cell strains. Expf Cett Res. 37, 614636.

Jepsen A., MacCallum D. K. and Lillie J. H. 1978. Obser- vations on subcultured stratified squamous epithelium. J. dent. Res. 57A, Abstract No. 97.

Mlinek A. and Buchner A. 1975. In vitro cultivation of adult human gingiva. J. periodont. Res. 10, 73-78.

Prunieras M., Delesciuse C. and Regnier M. 1976. The culture of skin: a review of theories and ex~rimental methods. f. invest. Derm. 67, 58-65.

Rheinwald J. G. and Green H. 1975a. Formation of a kera- tinizing epithelium in culture by a cloned cell line de- rived from a teratoma. Cell 6, 317-330.

Rheinwald J. G. and Green H. 1975b. Serial cultivation of strains of human epidermal keratinocytes: the forma- tion of keratinizing colonies from single ceils. Ceil 6, 331-344.

Rheinwald J. G. and Green H. 1977. Epidermai growth factor and the multiplication of cultured human epider- ma1 keratinocytes. Nature 265, 421-424.

Sun T. T. and Green H. 1977. Cultured epithelial cells of cornea, conjunctiva and skin: absence of marked in- trinsic divergence of their differentiated states. Nature 269, 489493.

Todaro G. J. and Green H. 1963. Quantitative studies on the growth of mouse embryo cells in culture and their development into established lines. J. Cell Biol. 17, 299-313.

Birkedal-Hansen H., Moe K., Dabelsteen E. and Rhein- holdt J. 1978. Propagation of a keratinizing human

Ptates 1 and 2 overleaf

338 L. Taichman, S. Reilly and P. R. Garant

Plate 1.

Fig. 2. A young colony of gingival keratinocytes (K); phase contrast microscopy. The colony contains about 18 cells and is surrounded by 3T3 cells. x 110

Fig. 3. An intermediate-sized colony of gingival keratinocytes (K). x 70

Fig. 4. Mature colonies of gingival keratinocytes (K). 3T3 cells have been pushed into a thin rim. x 120

Fig. 5. Stratification of keratinocytes. The phase contrast microscope was focused on the most super- ficial cells. Cell outlines are indistinct. Note the regular arrays of cytoplasmic granular inclusions

indicated by the arrow. x 200

Plate 2.

Fig. 6(a). Cross-section of gingival keratinocyte colony. The bottom layer corresponds to the basal cells adherent to the culture dish. Epon-embedded colony, 1 pm thick section stained with toluidine

blue. (b) Cross-section of buccal keratinocyte colony. x 1000

Fig. 7. Electron micrograph of gingival keratinocyte colony. The surface attached to the culture dish is at the bottom. Tonofibrils (Tf) are more prominent in the more superficial cells. x 1500

Plate 3.

Fig. 8. Tangential section through a gingival keratinocyte. Dense fibrous aggregates of tonofibrils (Tf) are present in the cytoplasm. The spatial organization of the aggregates resembles the organization

of cytoplasmic granules seen in Fig. 5. Nu, nucleus. x 1200

Fig. 9. Electron micrograph of junction of two buccal keratinocytes showing tonofibrils (Tf) inserting into the attachment plates (AP) of desmosomes x 1200

Fig. 10. The cornified envelope (CE) of gingival keratinocytes. Some keratinocytes in a colony have an electron-dense material deposited on the inner surface of the plasma membrane. Tf, tonofilaments.

x 1200

Fig. 11. Glycocalyx (arrows) on the surface of a buccal keratinocyte. x 1200

In-tiitro cultivation of oral keratinocytes 339

Plate 1.

340 L. Taichman, S. Reilly and P. R. Garant

Plate 2,

In-uifro cultivation of oral keratinocytes 341

Plate 3.