Burns (1992)18,Supplement 1, SII-S15
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Culture techniques for human keratinocytes M. L. Tenchini, Dipartimento
C. Ranzati and M. Malcovati
di Biologia e Genetica per le Scienze mediche, dell’Universiti
Introduction In vitro culture of human epithelial cells has attracted a remarkable interest in the last decade, not only in view of the importance of their practical applications, but also because of the wide range of basic investigations which can be performed on them. The most striking practical application is undoubtedly the successful use of in vitro cultured epithelial sheets as autografts or allografts on patients with extensive tegumental losses (Green et al., 1979; Gallico et al., 1984; Thivolet et al., 1986; Cuono et al., 1986; Faure et al., 1987). Basic research applications are reviewed in a following paper in this supplement. Keratinocytes, particularly human keratinocytes, are difficult cells to grow. The success of the culture depends upon the solution of two main interconnected problems. On one side these cells display nutritional requirements at least quantitatively different from those of other cell types; on the other, their in vitro growth can be easily overcome by the growth of different cell types, such as fibroblasts from connective tissue. Two approaches are currently in use for the in vitro culture of keratinocytes: one is based on the technique of Rheinwald and Green (1975a, b) and Green (1978), utilizing a serum-containing medium and a feeder-layer of murine 3T3 fibroblasts; the other relying on serum-free media, in the absence of a feeder-layer. In this paper both approaches will be reviewed and the respective advantages and possible applications discussed.
Techniques of in vitro culture of keratinocytes Dissociation
of skin keratinocytes
Human keratinocytcs for in vitro culture arc generally obtained from a cutaneous biopsy (Z-J cmL), which should be used for setting up the culture within 24 h. The biopsy, (
I992 Butterwurth~Heintmann
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cut into smaller fragments, is digested with trypsin to ensure separation of epidermis from dermis. This is a crucial step, since an effective separation of keratinocytes from dermal fibroblasts is necessary to avoid overgrowth of the latter during culture. Epidermis is dissociated into single cells by proteolytic treatment. Single cells are then washed, counted and seeded for a primary culture in plastic flasks. From a l-cm2 biopsy, approximately 3 x IO6 cells are obtained. This population contains all epidermal cell types, but keratinocytes from the basal layer (and melanocytes) are the sole cells able to proliferate, and therefore to give rise to colonies; cells from the other epidermal layers, having undergone a terminal differentiation process, are no longer able to divide (Watt, 1988). Clonogenic cells represent only 1 per cent of the total cell population: the plating efficiency of the primary explant is therefore very low. Culture
of keratinocytes
on a feeder-layer
of murine
fibroblasts
This method, described by Rheinwald and Green, is based on the co-culture of keratinocytes with irradiated, nonproliferating fibroblasts. Such cells, although themselves unable to proliferate, favour the growth of keratinocytes through a complex and still unclear mechanism and, at the same time, inhibit the growth of contaminating fibroblasts. Murine as well as human fibroblasts have been tested in this perspective: murine fibroblasts of the in vitro stabilized cell line NIH 3T3 turned out to be the most effective for this purpose (Rheinwald and Green, 1975a). The most widely used procedure to obtain nonproliferating NIH 3T3 cells is based on their exposure to high doses (6000rad) of gamma rays. Alternatively, treatment with mitomycin-C can be used (Rheinwald, 1980; Blacker et al., 1987), but, since this compound is a mutagen, irradiation is generally preferred, Murine fibroblasts are easily cultured in a medium (D-mem) containing IO per cent bovine serum. A suitable number of cells can be irradiated either attached to culture flasks or in suspension. After control of the efficacy of irradiation in blocking proliferation, 3T3 cells can be used as a direct support for the culture of keratinocytes. Keratinocytes are plated at a suitable density, with 3T3 cells, in a medium (Lb/e I) derived from the mixture of two basal media, currently used for in vitro culture of several cell types, with the addition of fetal bovine serum and of a series of supplements sustaining keratinocyte proliferation. Epidermal growth factor (EGF) (Rheinwald and Green, 1977). which enhances growth rate, and cholera toxin,
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Burns (1992) Supplement I
which stimulates the activity of adenylate cyclase, thus increasing intracellular levels of cyclic AMP (Green, 1978) are the most relevant among these additives. Hormones, such as hydrocortisone, triiodothyronine and insulin, provide a further increase in the rate of proliferation and stimulate the appearance of a regular, multilayered epithelial aspect of the culture (Green et al., 1979). Several substrates for growth of keratinocytes, other than the feeder-layer of irradiated murine fibroblasts, have been tested. These include different molecules associated with basal membranes, synthetic membranes, surfaces conditioned by different cell types and dermal substrates (Karasek, 1983; Bemstam et al., 1986; Gilchrest et al., 1980; Stenn et al., 1983; Kubo et al., 1987; Vaughan et al., 1986; Tinois et al., 1987; Regnier et al., 1988; Grinnell et al., 1987; Boyce and Hansbrough, 1988). Although satisfactory results have been obtained in several cases, the feeder-layer technique developed by Rheinwald and Green remains the most satisfactory solution, at least from a practical point of view. In the presence of the feeder-layer of 3T3 fibroblasts and of the medium described in T&&l, colonies, as shown in F~&re I, can be obtained from single keratinocytes. As colonies grow, through cycles of cell division, 3T3 fibroblasts are displaced and form a ‘crown’ all around them. The size of the colonies increases with time until they fuse and give rise, in approximately 10 days, to a continuous, multilayered sheet of keratinocytes, free of murine fibroblasts. At this time, proliferation stops. Further in vitro propagation of confluent primary keratinocytes can be obtained by detaching them from the surface of the culture flasks by proteolytic treatment. The cell suspension is plated in new flasks together with lethally irradiated 3T3 cells, in order to obtain secondary cultures. Since cells in culture are less differentiated than in native skin, the percentage of clonogenic cells in the population obtained from primary cultures is much higher than from skin explants (plating efficiency is approximately 30 per cent). It must be stressed that keratinocytes have a limited life-span: generally they stop growth after some 50 cell divisions, corresponding to five to six in vitro passages (Rheinwald and Green, IW5b). For grafts on patients, epithelial sheets at the second passage are generally used. Up to 30 secondary cultures can be set up from a primary culture. Since the surface of each flask is 75 cmz, the epithelial surface obtained is approximately 2250 cm’ (75 x 30). HOWever, since after detachment of sheets from the plastic surface by treatment with dispase (see below) a contraction
TableI. Medium for culture of human keratinocytes on a 3T3 feeder-layer A. Medium
for keratinocytes
Basal medium
D-mem Ham F12
75% 25%
Supplements
Fetal bovine serum Adenine Epidermal growth factor Hydrocortisone Insulin Cholera toxin Transferrin 3.3’,5-Triiodothyronine
10%
B. Medium
24pgiml 10 ng/ml 0.5 ng/ml 5 pg/ml 6 ng/ml lOpg/ml 1.3 ng/ml
for 3T3
Basal medium
D-mem
Supplements
Newborn bovine serum Glutamine
10% 0.29 mg/ml
Figure 1. Phase-contrast micrographs of human keratinocytes growing in the presence of a feeder-layer of lethally irradiated 3T3 mouse fibroblasts, at different times of culture. a, 5 days; b, 8 days; c, II days; d, 15 days (magnification x 212).
of approximately 50 per cent is observed, the total available surface is reduced to approximately 1000cm’. In about 3 weeks it is therefore possible to obtain a thousand-fold expansion from 1 cmZ of skin. The time required for expansion depends upon several factors, the most important of which is the age of the donor: the delay necessary to obtain a given in vitro expansion is proportional to the age of the donor (Stanulis-Praeger et al., 1988). Preparation of epithelial sheets for clinical use Through this procedure continuous, multilayered, partially keratinized epithelial sheets can be obtained. In order to use them as grafts to reconstruct tegumental losses, it is necessary to detach them from the culture flasks without damaging their integrity. This is achieved by treatment with the neutral protease dispase (Green et al., 1979; Kitano and Okada, 1983). This enzyme breaks emidesmosomes linking the basal layer of the sheet to the plastic surface of the flask, without affecting desmosomes and other cell junctions. In order to extract the sheets from the flasks, these must be opened by means of a hot wire: no flasks with sliding covers or suitable square or rectangular Petri dishes are commercially available. During treatment with Dispase, sheets contract and thicken, as indicated above. It has been shown that epiderma1 cells maintain their viability after this treatment (Green et al., 1979). Epithelial sheets are then mounted on a suitable gauze. They can be directly grafted onto patients or stored frozen for later use. Culture of keratinocytes in serum-free media The technique of Rheinwald and Green offers the advantage of allowing an excellent cellular growth in a relatively short time, as required for clinical use. It presents, however, several intrinsic disadvantages due to the presence of the feederlayer and of serum in the medium. This stimulated a long series of investigations aimed at identifying completely defined culture conditions. A two-fold experimental approach has been used to formulate new media. On one side the composition of the
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Tenchini et al.: Culture techniques for human keratinocytes basal medium was varied, both qualitatively and quantitatively, as far as ions and nutrients (amino acids, other organic compounds, vitamins, etc.) were concerned. These investigations led to the proposal of a few media (Ml99, Fl2, MCDB150) relatively specific for keratinocytes. They allowed reasonable growth and required lower serum levels than ‘traditional’ media (Eisinger et al., 1979; Barnes and Sato, 1980; Peehl and Ham, 1980a). In particular, the macromolecular component of serum (dialysed serum) was still required. Further adjustments in the concentrations of nutrients led to the formulation of medium MCDB151 (Peehl and Ham, 198ob), which was transformed into medium MCDB152 by the addition of a cocktail of trace elements. Finally, Boyce and Ham (1983, 1986) proposed basal medium MCDB153, which is presently used. On the other side, several supplements to the basal medium have been tested in the formulation of the final culture medium. Among them, a mixture of EGF, hydrocortisone, insulin, transferrin, monoethanolamine, phosphoethanolamine and bovine pituitary extract (Table 10 allowed clonal growth of keratinocytes in the complete absence of serum (Tsao et al., 1982; Wille et al., 1984). In these conditions, cells can undergo up to 40 generations before the onset of senescence. Several additional supplements, such as progesterone (Tsao et al., 1982), bovine brain extract (Maciag et al., 1981), fraction IV of Cohn from human serum (Maciag et al., 198 l), bovine thymus extract (Stanulis-Praeger et al., 1988), human placental extract (O’Keefe et al.‘, 1985), bovine hypothalamus extract (Gilchrest et al., 1984) and triiodothyronine (Maciag et al., 1981) have been successfully tested although they are not currently used for practical or economic reasons. The correct choice of the concentration of two components is probably responsible for the satisfactory inhibition of proliferation of dermal fibroblasts (always present, to some extent, in the initial culture of keratinocytes): that of adenine (which is 2O-fold higher than that for optimal growth of fibroblasts) and that of calcium ions (0.3 mM, a value well below the levels necessary for fibroblasts). One of the most interesting conclusions drawn from these nutritional studies is that keratinocytes do not present qualitatively specific requirements, with the possible exception of monoethanolamine and phosphoethanolamine. It has been suggested, on the basis of the relatively high concentrations of both compounds necessary for growth of these cells, that they might be incorporated into phospholipids, although a possible regulatory role has not been ruled out (Kane-Sueoka
Amino acids, the concentration of which seems to be a limiting fac to r for the growth of cultures (Pittelkow and Scott, 1986; Shipley and PiRelkow, 1987), deserve a particular mention. They are isoleucine, and to a lesser extent histidine, methionine, phenylalanine, tryptophan and tyrosine. It is interesting to recall that, in the rare clinical
cases of isoleucine deficiency, a form of dermatitis has been described among the symptoms. It is worthwhile discussing the level of the chemical definition of this serum-free medium, also indicated as ‘defined’ medium. Obviously, the level of definition depends upon the purity of water and of the compounds used in the preparation of the basal medium, as well as upon the nature of the materials (bottles, filters, etc.) used. However, supplements to the basal medium introduce a higher degree of uncertainty. Among them, two proteins (EGF and insulin, added in relatively small amounts) and two low molecular weight compounds (monoethanolamine and phosphoethanolamine) are commercially available in a relatively pure form. Bovine
pituitary
extract,
on the contrary,
It is very effective in promoting
and Errick, 1981).
Factors affecting in vitro growth differentiation of keratinocytes Table II. Composition
MCDB
Basal medium Epldermal growth factor Bovine pituitary extract
*For composttion (1986)
and
of serum-free medium for stock cultures of
human keratinocytes
Supplements
is chemically
growth of both primary and long-term cultures and, in contrast to serum, does not stimulate proliferation of fibroblasts nor terminal differentiation of keratinocytes (Boyce and Ham, 1983). Attempts to purify one or more substances responsible for these effects have been unsuccessful so far. If strictly necessary, bovine pituitary extract can be omitted, but in this situation the concenbation of calcium ions must be significantly increased in order to obtain a satisfactory growth rate (Boyce and Ham, 1986). Transferrin is a second supplement, the purity of which is unsatisfactory (Tsao et al., 1982). It- has, however, been shown that it can be omitted, provided that the concentrations of iron and zinc in the basal medium are correspondingly increased (Boyce and Ham, 1985). In spite of the above uncertainties in the chemical definition of the medium, this system offers the best balance, so far obtained, between definition of the medium and the growth rate of keratinocytes. Human keratinocytes at different stages of growth in serum-free medium are shown in F@lre2. Melanocytes, which are co-cultured with keratinocytes in both culture conditions, can be easily observed in serum-free cultures, where they are not masked by mouse fibroblasts (F@ire 3). It has been demonstrated that melanocytes, which are present in the basal layer of in vitro cultured epithelium, transfer melanosomes to keratinocytes (De Luca et al., 1989). undefined.
153'
10 ng/ml 140 llg/ml
Ethanolamine Phosphoethanolamine Hydrocortisone lnsulln (Transferrin)
500 ng/ml 5 pg/ml lO~~g/ml
Calcium chloride
1x10
of this basal medium,
6 llglml 14llg/ml
see Boyce and Ham
4M
This aspect of t-he biology of in vitro cultured keratinocytes has been mainly studied on cells growing in serum-free media, which offer the possibility of avoiding the uncertainties due to unknown factors present in serum or produced by the feeder-layer. Growth
factors
and hormones
Two hormones (insulin and hydrocortisone) and a growth factor (EGF) regulate the growth of keratinocytes in serumfree media. Among them, EGF and insulin are necessary for clonal growth, while hydrocortisone affects colony size, which, in its absence, is considerably reduced (Wille et al., 19841.
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Figure 2. Phase-contrast
micrographs ot human keratinocytes growing in serum-free medium, at different times of culture. a, 8 days; b, 12 days; c, 18 days; d, after induction to terminal differentiation with CA+ + (magnification x 190).
Figure 2 shows keratinocytes at different stages of growth in a medium containing the above-mentioned factors; colonies progressively grow until they fuse giving rise to a continuous layer. In these experiments, the calcium concentration was kept at low levels (0.1 mM), in order to allow cells to multiply without undergoing differentiation (which would cause a progressive loss of proliferative potential). Induction of terminal differentiation was obtained by increasing the level of Ca+ + to 1 KIM (see below): this shift is accompanied by a dramatic change in cell morphology (Figure zd). Role of calcium ions Calcium ions play a crucial role in the growth and differentiation of keratinocytes. The most important calcium-dependent function is the formation of desmosomes, which is directly proportional to the concentration of these ions (Iones et al., 1982). No desmosomes are observed in vitro in the absence of Ca+ + . It has been suggested that the calcium-dependent formation of such structures is a key step in the regulation of epidermal stratification and differentiation (Boyce and Ham, 1983). More recently, it has been demonstrated that calciuminduced differentiation of desmosomes can be mediated through a direct action of these ions on the activity of phospholipase C (Jaken and Yuspa, 1988). Phenotypic effects subsequent to the addition of Ca’ ’ to medium MCDB153 differ according to the concentration of the ions. At concentrations of 0.3 mM, maximal growth rate is reached and intermediate filaments and keratohyaline granules begin to appear, while the growth rate of fibroblasts is highly reduced. At lower Cat ’ concentrations (0.1-0.03 mM), stratification is absent, cells are flattened and progressively lesser and lesser differentiated as concentration of the ion decreases. Finally, at high calcium concentrations (1 mM), stratification and differentiation occur (Figure2d). In this experimental system, therefore, differentiation can be modulated simply by varying the calcium concentration.
Figure 3. Micrographs of melanocytes present in cultures of human keratinocytes growing in serum-free medium (magnification x 367).
As described above, calcium ions influence both keratinocyte growth and differentiation. Different mechanisms are involved in the two processes. Recently, it has been observed that strontium has mitogenic effects on keratinocytes in culture and can therefore substitute for calcium in stimulating proliferation of these cells. However, in contrast to calcium, strontium does not induce terminal differentiation (Praeger et al., 1987; Furukawa et al., 1988). Therefore, the use of strontium may facilitate the study of the different mechanisms underlying growth and differentiation of keratinocytes. Cultures at a&liquid interface When cultured according to conventional techniques, keratinocytes grow submerged in the liquid medium, attached to the surface of plastic flasks. Their differentiation, in these conditions, is blocked at a stage corresponding to the granular layer of epidermis. In order to create conditions closer to the physiological ones, cultures can be ‘lifted in such a way that cells of the basal layer are directly or indirectly (through a dermis-like substrate) in contact with the culture medium, while cells in the most superficial layers are exposed to air. Substrates used for this purpose are collagen, laminin or fibrin gels floating on the surface of the medium. In these conditions complete differentiation, including a komeous layer, is obtained (Prunieras et al., 1983; Bemstam et al., 1986).
Conclusions The main advantage of the feeder-layer technique is the excellent cellular growth. In fact this technique is currently used in the large majority of laboratories involved in the preparation of epithelial sheets for grafting. Nevertheless the presence of the feeder-layer and of serum can represent a
Tenchini
et al.: Culture
techniques
for human
keratinocytes
considerable drawback because metabolic activities of 3T3 fibroblasts and unknown components of serum can mask metabolic activities of keratinocytes or interfere with the activity of added substances. For these reasons the serumfree approach is preferred for studies on the biology of keratinocytes
under
controlled
conditions.
Acknowledgements This work was supported by grants from Consiglio Nazionaie delle Richerche, Rome (Italy), contract no. 88.03121.26 and by Progetto Finalizzato ‘Biotecnologie e biostrumentazione’, contract no. 8900240.70.
References Barnes D. and Sato G. (1980) Methods for growth of cultured cells in serum-free medium. Analyt. Biochem. 102, 255. Bemstam L. I., Vaughan F. L. and Bernstein I. A. (1986) Keratinocytcs grown at the air-liquid interface. In Vitro Cell Develop. Biol. 22, 695. Blacker K. L., Williams M. L. and Goldyne M. (1987) Mitomycin C-treated 3T3 fibroblasts used as feeder layers for human keratinocyte culture retain the capacity to generate eicosanoids. /. Invest. Demalol. 89, 536. Boyce S. T. and Ham R. G. (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. 1. Invest. Derrnatoi. 81, 33. Boyce S. T. and Ham R. G. (1985) Cultivation, frozen storage and clonal growth of normal human epidermal keratinocytes in serum-free medium. /, &sue Culttire Meth. 9, 83. Boyce S. T. and Ham R. G. (1986) Normal human epidermal keratinocytcs. In: Weber M. M. and Sekely L. (eds), In Vitro Models for Cancer Research, vol. 3. CRC: Boca Raton, p. 245. Boyce S. T. and Hansbrough J. F. (1988) Biological attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Sttrgely 103, 421. Cuono C., Langdon R. and McGuire J. (1986) Use of cultured epidermal autografts and dermal allografts as skin replacement after bum injury. Luncet i, 1123. De Luca M., Franzi i\. T., D’Anna F. et al. (1988) Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium. Etcr.1, Cell Biol. 46, 176. Eisinger M., Soho Lee J., Hefton J. M. et al. (1979) Human epidermal cell cultures: growth and differentiation in the absence of dermal components or medium supplements. koc. Nat/. Acad. Sri. US,4 76, 5340. Faure M., Mauduit G., Schmitt D. et al. (1987) Growth and differentiation of human epidermal cultures used as auto- or allografts in humans. Br. 1. Dewnafol. 116, 161. Furukawa F., Huff J. C., Lyons M. B. et al. (1988) Characterization and practical benefits of keratinocytes cultured in strontiumcontaining serum-free medium. I. Invest. Derrnatol. 5, 690. Gallico G. G., O’Connor N. E., Compton C. C. et al. (1984) Permanent coverage of large bum wounds with autologous cultured human epithelium. N. Engl. 1. Med. 331. 448. Cilchrest B. A., Nemore R. E. and Maciag T. (1980) Growth of human keratinocytes on fibronectin-coated plates. Cell. Biol. Intern. Reports 4, 1009.
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Gilchrest B. A., Marshall W. L., Karassik R. L. et al. (1984) Characterization and partial purification of keratinocyte growth factor from the hypothalamus. 1. Cell. Physiol. 120, 377. Green H. (1978) Cyclic AMP in relation to proliferation of the epidermal cell. A new view. Cell 15, 801. Green H., Kehinde 0. and Thomas J. (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Nad. Acad. Sci. USA 76, 5665. Grinnell F., Toda K. I. and Seymour C. L. (1987) Reconstitution of human epidermis in vitro is accompanied by transient activation of basal keratinocyte spreading. Erp. Cell Res. 172, 439. Jaken S. and Yuspa S. H. (1988) Early signal for keratinocyte differentiation: role of Ca’+ -mediated inositol lipid metabolism in normal and neoplastic epidexmal cells. Carcinogenesis 9, 1033. Jones J. C. R., Goldman A. I., Steinart P. M. et al. (1982) Dynamic aspects of the supramolecular organisation of intermediate filament networks in cultured epidermal cells. Cell Moflilu 2, 197. Kano-Sueoka T. and Errick J. E. (1981) The effects of phosphoethanolamine and ethanolamine on growth of mammary carcinoma cells in culture. Lrp. Cell Res. 136, 137. Karasek M. A. (1983) Culture of human keratinocytes in liquid medium. I. Inoest. Dermatol. 81, 245. Kitano J. and Okada N. (1983) Separation of the epidermal sheets by dispase. Br. J Dermatoi. 108, 555. Kubo M., Kan M., Isemura M. et al. (1987) Effects of extracellular matrices of human keratinocyte adhesion and growth and on its secretion and deposition of fibronectin in culture. 1. Invest. Dematol. 88, 594. Maciag T., Nemore R. E., Weinstein R. et al. (1981) An endocrine approach to the control of epidermal growth: serum-free cultivation of human keratinocytes. Science 211, 1452. O’Keefe E. J., Payne R. E. and Russel N. (1985) Keratinocyte growth-promoting activity from human placenta. 1. Cell Physiol. 124, 439. Peehl D. M. and Ham R. G. (1980a) Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium. In Vitro Cell Develop. Biol. 16, 516. Peehl D. M. and Ham R. G. (198Ob) Clonal growth of human keratinocytes with small amounts of dialyzed serum. In Vitro Cell Develop. Biol. 16, 526. Pittelkow M. R. and Scott R. E. (1986) New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive bums. Mayo Clin. Proc. 61, 771. Praeger F. C., Stanulis-Praeger B. M. and Gilchrest B.A. (1987) Use of strontium to separate calcium-dependent pathways for proliferation and differentiation in human keratinocytes. 1, Cell Physiol. 132, 81. Prunieras M., Regnier M. and Woodley D. (1983) Methods for cultivation of keratinocytes with an air-liquid interface. 1, Invest. Dennabl. 81, 280. Regnier M., Desbas C., Bailly C. et al. (1988) Differentiation of normal and tumoral human keratinocytes cultured on dermis: reconstruction of either normal or tumoral architecture. In Vitro Cell Develop. Biol. 24, 625. Rheinwald J. G. (1980) Serial culfivation of normal human epidermal keratinocytes. In: Harris C. C., Trump B. F. and Stoner G. D. (eds), Methods in Cell Biology, vol. 21A. New York: Academic Press, p. 229. Rheinwald J. G. and Green H. (1975a) Formation of a keratinizing epit-helium in culture of a cloned cell line derived from a teratoma. Ceil 6, 317.
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Rheinwald J. G. and Green H. (1975b) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331. Rheinwald J. G. and Green H. (1977) Epidemal growth factor and
the multiplication of cultured human epidemal keratinocytes. Nature 265, 421. Shipley G. D. and Pittelkow M. R. (1987) Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium. Arch. Dermalol. 123, 1541. Stanulis-Praeger B. M., Yaar M., Redziniak G. et al. (1988) An extract of bovine thymus stimulates human keratinocytes growth in vitro. 1, Imesf. Dermafol. 90, 749. Stenn K. S., Madri J. A., Tinghitella T. et al. (1983) Multiple mechanism of dissociated epidennal cell spreading. 1. Cell Biol. 96, 63. Thivolet J., Faure M., Demidem A. et al. (1986) Long-term survival and immunological tolerance of human epidermal allografts produced in culture. Transplankakion 42, 274. Tinois E., Faure M., Chatelain P. et al. (1987) Growth and differentiation of human keratinocytes on extracellular matrix. Arch. Demakol. Res. 279, 241.
Tsao M. C., Walthall B. J. and Ham R. G. (1982) Clonal growth of normal human epidermal keratinocytes in a definied medium. I. Ce// Physiol. 110, 219. Vaughan F., Gray R. H. and Bernstein I. A. (1986) Growth and differentiation of primary rat keratinocytes on synthetic membranes In Vitro Cell Develop. Biol. 22, 141. Watt F. M. (1988) Epidermal stem cells in culture. 1. Cell. Sri. Suppl. 10, 85. Wille J. J., Pittelkow M. R., Shipley G. D. et al. (1984) Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics and cell cycle studies. 1. Cell Physiol. 121, 31.
Correspondence should be addressed to: Professor Maria Luisa Tenchini, Dipartimento di Biologia e Genetica per le Scienze mediche, via Viotti 5, I - 20133 Milano, Italy.
Permanent coverage of full skin thickness burns with autologous cultured epidermis and re-, epithelialization of partial skin thickness lesions induced by allogetieic cultured epidermis: a multicentre study in the treatment of children R. Cancedda”‘, A. M. Tamisani3, C. Di Noto3, L. Muller’, M. De Lucalfz, S. Bondanzal”, D. Dioguardl “, E. Brienza4, A. Calvario4, R. Zermani5, D. Di Mascio5 and F. Papadia5 ‘IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, ‘Istituto di Oncologia Clinica e Sperimentale, Universita’ di Genova, Genoa, 3Servizio Chirurgico di Pronto Soccorso, Istituto Giannina Gaslini, Genoa, *Cattedra di Chirurgia Plastica, Facolta’ di Medicina e Chirurgia, Universita’ di Bari, Bari and Ystituto di Chirurgia Plastica e Centro Ustioni, Facolta’ di Medicina e Chirurgia, Universita’ di Par-ma, Parma, Italy
Introduction When burn wounds cover large areas of the body surface, remaining donor sites might not be sufficient to prepare enough split-thickness mesh grafts. This problem becomes more critical in children since the use of large donor sites increases the skin defect and, especially in patients sustaining large burn wounds, might actually increase the mortality rate. Moreover, hypertrophic scars develop frequently on the donor sites in children. Skin-derived human keratinocytes can be cultured in vitro (Rheinwald and Green, 1973, starting from a 1-2 cm2 biopsy. With the appropriate culture conditions, the initial cell population present in the skin $” 1992 Butterworth-Heinemann 0305-4179/92/OSOS1&03
Ltd
biopsy can be amplified in secondary cultures and can generate coherent epithelial sheets sufficient to cover the entire body surface in a period of 2-4 weeks (Green et al., 1979; De Luca et al., 1989). Autologous epithelial sheets (autografts) can be detached from the surface vessel and successfully used for permanent coverage of large burn wounds (O’Connor et al., 1981; Gallico et al., 1984; Cuono et al., 1986; Faure et al., 1987; Herzog et al., 1988; De Luca et al., 1989; Teepe et al., 1990a). Recovery of children sustaining full skin thickness bums covering up to 98 per cent of the body surface has been reported (Gallico et al., 1984). However, levels of ‘take’ (i.e. the presence of viable epithelium at the removal of the petroleum jelly gauze)
Privhd
it? Great
Brihk
Sll
Culture techniques for human keratinocytes M. L. Tenchini, Dipartimento
C. Ranzati and M. Malcovati
di Biologia e Genetica per le Scienze mediche, dell’Universiti
Introduction In vitro culture of human epithelial cells has attracted a remarkable interest in the last decade, not only in view of the importance of their practical applications, but also because of the wide range of basic investigations which can be performed on them. The most striking practical application is undoubtedly the successful use of in vitro cultured epithelial sheets as autografts or allografts on patients with extensive tegumental losses (Green et al., 1979; Gallico et al., 1984; Thivolet et al., 1986; Cuono et al., 1986; Faure et al., 1987). Basic research applications are reviewed in a following paper in this supplement. Keratinocytes, particularly human keratinocytes, are difficult cells to grow. The success of the culture depends upon the solution of two main interconnected problems. On one side these cells display nutritional requirements at least quantitatively different from those of other cell types; on the other, their in vitro growth can be easily overcome by the growth of different cell types, such as fibroblasts from connective tissue. Two approaches are currently in use for the in vitro culture of keratinocytes: one is based on the technique of Rheinwald and Green (1975a, b) and Green (1978), utilizing a serum-containing medium and a feeder-layer of murine 3T3 fibroblasts; the other relying on serum-free media, in the absence of a feeder-layer. In this paper both approaches will be reviewed and the respective advantages and possible applications discussed.
Techniques of in vitro culture of keratinocytes Dissociation
of skin keratinocytes
Human keratinocytcs for in vitro culture arc generally obtained from a cutaneous biopsy (Z-J cmL), which should be used for setting up the culture within 24 h. The biopsy, (
I992 Butterwurth~Heintmann
1~.305~-4
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Ltd
degli Studi di Milano, Italy
cut into smaller fragments, is digested with trypsin to ensure separation of epidermis from dermis. This is a crucial step, since an effective separation of keratinocytes from dermal fibroblasts is necessary to avoid overgrowth of the latter during culture. Epidermis is dissociated into single cells by proteolytic treatment. Single cells are then washed, counted and seeded for a primary culture in plastic flasks. From a l-cm2 biopsy, approximately 3 x IO6 cells are obtained. This population contains all epidermal cell types, but keratinocytes from the basal layer (and melanocytes) are the sole cells able to proliferate, and therefore to give rise to colonies; cells from the other epidermal layers, having undergone a terminal differentiation process, are no longer able to divide (Watt, 1988). Clonogenic cells represent only 1 per cent of the total cell population: the plating efficiency of the primary explant is therefore very low. Culture
of keratinocytes
on a feeder-layer
of murine
fibroblasts
This method, described by Rheinwald and Green, is based on the co-culture of keratinocytes with irradiated, nonproliferating fibroblasts. Such cells, although themselves unable to proliferate, favour the growth of keratinocytes through a complex and still unclear mechanism and, at the same time, inhibit the growth of contaminating fibroblasts. Murine as well as human fibroblasts have been tested in this perspective: murine fibroblasts of the in vitro stabilized cell line NIH 3T3 turned out to be the most effective for this purpose (Rheinwald and Green, 1975a). The most widely used procedure to obtain nonproliferating NIH 3T3 cells is based on their exposure to high doses (6000rad) of gamma rays. Alternatively, treatment with mitomycin-C can be used (Rheinwald, 1980; Blacker et al., 1987), but, since this compound is a mutagen, irradiation is generally preferred, Murine fibroblasts are easily cultured in a medium (D-mem) containing IO per cent bovine serum. A suitable number of cells can be irradiated either attached to culture flasks or in suspension. After control of the efficacy of irradiation in blocking proliferation, 3T3 cells can be used as a direct support for the culture of keratinocytes. Keratinocytes are plated at a suitable density, with 3T3 cells, in a medium (Lb/e I) derived from the mixture of two basal media, currently used for in vitro culture of several cell types, with the addition of fetal bovine serum and of a series of supplements sustaining keratinocyte proliferation. Epidermal growth factor (EGF) (Rheinwald and Green, 1977). which enhances growth rate, and cholera toxin,
s12
Burns (1992) Supplement I
which stimulates the activity of adenylate cyclase, thus increasing intracellular levels of cyclic AMP (Green, 1978) are the most relevant among these additives. Hormones, such as hydrocortisone, triiodothyronine and insulin, provide a further increase in the rate of proliferation and stimulate the appearance of a regular, multilayered epithelial aspect of the culture (Green et al., 1979). Several substrates for growth of keratinocytes, other than the feeder-layer of irradiated murine fibroblasts, have been tested. These include different molecules associated with basal membranes, synthetic membranes, surfaces conditioned by different cell types and dermal substrates (Karasek, 1983; Bemstam et al., 1986; Gilchrest et al., 1980; Stenn et al., 1983; Kubo et al., 1987; Vaughan et al., 1986; Tinois et al., 1987; Regnier et al., 1988; Grinnell et al., 1987; Boyce and Hansbrough, 1988). Although satisfactory results have been obtained in several cases, the feeder-layer technique developed by Rheinwald and Green remains the most satisfactory solution, at least from a practical point of view. In the presence of the feeder-layer of 3T3 fibroblasts and of the medium described in T&&l, colonies, as shown in F~&re I, can be obtained from single keratinocytes. As colonies grow, through cycles of cell division, 3T3 fibroblasts are displaced and form a ‘crown’ all around them. The size of the colonies increases with time until they fuse and give rise, in approximately 10 days, to a continuous, multilayered sheet of keratinocytes, free of murine fibroblasts. At this time, proliferation stops. Further in vitro propagation of confluent primary keratinocytes can be obtained by detaching them from the surface of the culture flasks by proteolytic treatment. The cell suspension is plated in new flasks together with lethally irradiated 3T3 cells, in order to obtain secondary cultures. Since cells in culture are less differentiated than in native skin, the percentage of clonogenic cells in the population obtained from primary cultures is much higher than from skin explants (plating efficiency is approximately 30 per cent). It must be stressed that keratinocytes have a limited life-span: generally they stop growth after some 50 cell divisions, corresponding to five to six in vitro passages (Rheinwald and Green, IW5b). For grafts on patients, epithelial sheets at the second passage are generally used. Up to 30 secondary cultures can be set up from a primary culture. Since the surface of each flask is 75 cmz, the epithelial surface obtained is approximately 2250 cm’ (75 x 30). HOWever, since after detachment of sheets from the plastic surface by treatment with dispase (see below) a contraction
TableI. Medium for culture of human keratinocytes on a 3T3 feeder-layer A. Medium
for keratinocytes
Basal medium
D-mem Ham F12
75% 25%
Supplements
Fetal bovine serum Adenine Epidermal growth factor Hydrocortisone Insulin Cholera toxin Transferrin 3.3’,5-Triiodothyronine
10%
B. Medium
24pgiml 10 ng/ml 0.5 ng/ml 5 pg/ml 6 ng/ml lOpg/ml 1.3 ng/ml
for 3T3
Basal medium
D-mem
Supplements
Newborn bovine serum Glutamine
10% 0.29 mg/ml
Figure 1. Phase-contrast micrographs of human keratinocytes growing in the presence of a feeder-layer of lethally irradiated 3T3 mouse fibroblasts, at different times of culture. a, 5 days; b, 8 days; c, II days; d, 15 days (magnification x 212).
of approximately 50 per cent is observed, the total available surface is reduced to approximately 1000cm’. In about 3 weeks it is therefore possible to obtain a thousand-fold expansion from 1 cmZ of skin. The time required for expansion depends upon several factors, the most important of which is the age of the donor: the delay necessary to obtain a given in vitro expansion is proportional to the age of the donor (Stanulis-Praeger et al., 1988). Preparation of epithelial sheets for clinical use Through this procedure continuous, multilayered, partially keratinized epithelial sheets can be obtained. In order to use them as grafts to reconstruct tegumental losses, it is necessary to detach them from the culture flasks without damaging their integrity. This is achieved by treatment with the neutral protease dispase (Green et al., 1979; Kitano and Okada, 1983). This enzyme breaks emidesmosomes linking the basal layer of the sheet to the plastic surface of the flask, without affecting desmosomes and other cell junctions. In order to extract the sheets from the flasks, these must be opened by means of a hot wire: no flasks with sliding covers or suitable square or rectangular Petri dishes are commercially available. During treatment with Dispase, sheets contract and thicken, as indicated above. It has been shown that epiderma1 cells maintain their viability after this treatment (Green et al., 1979). Epithelial sheets are then mounted on a suitable gauze. They can be directly grafted onto patients or stored frozen for later use. Culture of keratinocytes in serum-free media The technique of Rheinwald and Green offers the advantage of allowing an excellent cellular growth in a relatively short time, as required for clinical use. It presents, however, several intrinsic disadvantages due to the presence of the feederlayer and of serum in the medium. This stimulated a long series of investigations aimed at identifying completely defined culture conditions. A two-fold experimental approach has been used to formulate new media. On one side the composition of the
s13
Tenchini et al.: Culture techniques for human keratinocytes basal medium was varied, both qualitatively and quantitatively, as far as ions and nutrients (amino acids, other organic compounds, vitamins, etc.) were concerned. These investigations led to the proposal of a few media (Ml99, Fl2, MCDB150) relatively specific for keratinocytes. They allowed reasonable growth and required lower serum levels than ‘traditional’ media (Eisinger et al., 1979; Barnes and Sato, 1980; Peehl and Ham, 1980a). In particular, the macromolecular component of serum (dialysed serum) was still required. Further adjustments in the concentrations of nutrients led to the formulation of medium MCDB151 (Peehl and Ham, 198ob), which was transformed into medium MCDB152 by the addition of a cocktail of trace elements. Finally, Boyce and Ham (1983, 1986) proposed basal medium MCDB153, which is presently used. On the other side, several supplements to the basal medium have been tested in the formulation of the final culture medium. Among them, a mixture of EGF, hydrocortisone, insulin, transferrin, monoethanolamine, phosphoethanolamine and bovine pituitary extract (Table 10 allowed clonal growth of keratinocytes in the complete absence of serum (Tsao et al., 1982; Wille et al., 1984). In these conditions, cells can undergo up to 40 generations before the onset of senescence. Several additional supplements, such as progesterone (Tsao et al., 1982), bovine brain extract (Maciag et al., 1981), fraction IV of Cohn from human serum (Maciag et al., 198 l), bovine thymus extract (Stanulis-Praeger et al., 1988), human placental extract (O’Keefe et al.‘, 1985), bovine hypothalamus extract (Gilchrest et al., 1984) and triiodothyronine (Maciag et al., 1981) have been successfully tested although they are not currently used for practical or economic reasons. The correct choice of the concentration of two components is probably responsible for the satisfactory inhibition of proliferation of dermal fibroblasts (always present, to some extent, in the initial culture of keratinocytes): that of adenine (which is 2O-fold higher than that for optimal growth of fibroblasts) and that of calcium ions (0.3 mM, a value well below the levels necessary for fibroblasts). One of the most interesting conclusions drawn from these nutritional studies is that keratinocytes do not present qualitatively specific requirements, with the possible exception of monoethanolamine and phosphoethanolamine. It has been suggested, on the basis of the relatively high concentrations of both compounds necessary for growth of these cells, that they might be incorporated into phospholipids, although a possible regulatory role has not been ruled out (Kane-Sueoka
Amino acids, the concentration of which seems to be a limiting fac to r for the growth of cultures (Pittelkow and Scott, 1986; Shipley and PiRelkow, 1987), deserve a particular mention. They are isoleucine, and to a lesser extent histidine, methionine, phenylalanine, tryptophan and tyrosine. It is interesting to recall that, in the rare clinical
cases of isoleucine deficiency, a form of dermatitis has been described among the symptoms. It is worthwhile discussing the level of the chemical definition of this serum-free medium, also indicated as ‘defined’ medium. Obviously, the level of definition depends upon the purity of water and of the compounds used in the preparation of the basal medium, as well as upon the nature of the materials (bottles, filters, etc.) used. However, supplements to the basal medium introduce a higher degree of uncertainty. Among them, two proteins (EGF and insulin, added in relatively small amounts) and two low molecular weight compounds (monoethanolamine and phosphoethanolamine) are commercially available in a relatively pure form. Bovine
pituitary
extract,
on the contrary,
It is very effective in promoting
and Errick, 1981).
Factors affecting in vitro growth differentiation of keratinocytes Table II. Composition
MCDB
Basal medium Epldermal growth factor Bovine pituitary extract
*For composttion (1986)
and
of serum-free medium for stock cultures of
human keratinocytes
Supplements
is chemically
growth of both primary and long-term cultures and, in contrast to serum, does not stimulate proliferation of fibroblasts nor terminal differentiation of keratinocytes (Boyce and Ham, 1983). Attempts to purify one or more substances responsible for these effects have been unsuccessful so far. If strictly necessary, bovine pituitary extract can be omitted, but in this situation the concenbation of calcium ions must be significantly increased in order to obtain a satisfactory growth rate (Boyce and Ham, 1986). Transferrin is a second supplement, the purity of which is unsatisfactory (Tsao et al., 1982). It- has, however, been shown that it can be omitted, provided that the concentrations of iron and zinc in the basal medium are correspondingly increased (Boyce and Ham, 1985). In spite of the above uncertainties in the chemical definition of the medium, this system offers the best balance, so far obtained, between definition of the medium and the growth rate of keratinocytes. Human keratinocytes at different stages of growth in serum-free medium are shown in F@lre2. Melanocytes, which are co-cultured with keratinocytes in both culture conditions, can be easily observed in serum-free cultures, where they are not masked by mouse fibroblasts (F@ire 3). It has been demonstrated that melanocytes, which are present in the basal layer of in vitro cultured epithelium, transfer melanosomes to keratinocytes (De Luca et al., 1989). undefined.
153'
10 ng/ml 140 llg/ml
Ethanolamine Phosphoethanolamine Hydrocortisone lnsulln (Transferrin)
500 ng/ml 5 pg/ml lO~~g/ml
Calcium chloride
1x10
of this basal medium,
6 llglml 14llg/ml
see Boyce and Ham
4M
This aspect of t-he biology of in vitro cultured keratinocytes has been mainly studied on cells growing in serum-free media, which offer the possibility of avoiding the uncertainties due to unknown factors present in serum or produced by the feeder-layer. Growth
factors
and hormones
Two hormones (insulin and hydrocortisone) and a growth factor (EGF) regulate the growth of keratinocytes in serumfree media. Among them, EGF and insulin are necessary for clonal growth, while hydrocortisone affects colony size, which, in its absence, is considerably reduced (Wille et al., 19841.
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Bums (1992)Supplement 1
Figure 2. Phase-contrast
micrographs ot human keratinocytes growing in serum-free medium, at different times of culture. a, 8 days; b, 12 days; c, 18 days; d, after induction to terminal differentiation with CA+ + (magnification x 190).
Figure 2 shows keratinocytes at different stages of growth in a medium containing the above-mentioned factors; colonies progressively grow until they fuse giving rise to a continuous layer. In these experiments, the calcium concentration was kept at low levels (0.1 mM), in order to allow cells to multiply without undergoing differentiation (which would cause a progressive loss of proliferative potential). Induction of terminal differentiation was obtained by increasing the level of Ca+ + to 1 KIM (see below): this shift is accompanied by a dramatic change in cell morphology (Figure zd). Role of calcium ions Calcium ions play a crucial role in the growth and differentiation of keratinocytes. The most important calcium-dependent function is the formation of desmosomes, which is directly proportional to the concentration of these ions (Iones et al., 1982). No desmosomes are observed in vitro in the absence of Ca+ + . It has been suggested that the calcium-dependent formation of such structures is a key step in the regulation of epidermal stratification and differentiation (Boyce and Ham, 1983). More recently, it has been demonstrated that calciuminduced differentiation of desmosomes can be mediated through a direct action of these ions on the activity of phospholipase C (Jaken and Yuspa, 1988). Phenotypic effects subsequent to the addition of Ca’ ’ to medium MCDB153 differ according to the concentration of the ions. At concentrations of 0.3 mM, maximal growth rate is reached and intermediate filaments and keratohyaline granules begin to appear, while the growth rate of fibroblasts is highly reduced. At lower Cat ’ concentrations (0.1-0.03 mM), stratification is absent, cells are flattened and progressively lesser and lesser differentiated as concentration of the ion decreases. Finally, at high calcium concentrations (1 mM), stratification and differentiation occur (Figure2d). In this experimental system, therefore, differentiation can be modulated simply by varying the calcium concentration.
Figure 3. Micrographs of melanocytes present in cultures of human keratinocytes growing in serum-free medium (magnification x 367).
As described above, calcium ions influence both keratinocyte growth and differentiation. Different mechanisms are involved in the two processes. Recently, it has been observed that strontium has mitogenic effects on keratinocytes in culture and can therefore substitute for calcium in stimulating proliferation of these cells. However, in contrast to calcium, strontium does not induce terminal differentiation (Praeger et al., 1987; Furukawa et al., 1988). Therefore, the use of strontium may facilitate the study of the different mechanisms underlying growth and differentiation of keratinocytes. Cultures at a&liquid interface When cultured according to conventional techniques, keratinocytes grow submerged in the liquid medium, attached to the surface of plastic flasks. Their differentiation, in these conditions, is blocked at a stage corresponding to the granular layer of epidermis. In order to create conditions closer to the physiological ones, cultures can be ‘lifted in such a way that cells of the basal layer are directly or indirectly (through a dermis-like substrate) in contact with the culture medium, while cells in the most superficial layers are exposed to air. Substrates used for this purpose are collagen, laminin or fibrin gels floating on the surface of the medium. In these conditions complete differentiation, including a komeous layer, is obtained (Prunieras et al., 1983; Bemstam et al., 1986).
Conclusions The main advantage of the feeder-layer technique is the excellent cellular growth. In fact this technique is currently used in the large majority of laboratories involved in the preparation of epithelial sheets for grafting. Nevertheless the presence of the feeder-layer and of serum can represent a
Tenchini
et al.: Culture
techniques
for human
keratinocytes
considerable drawback because metabolic activities of 3T3 fibroblasts and unknown components of serum can mask metabolic activities of keratinocytes or interfere with the activity of added substances. For these reasons the serumfree approach is preferred for studies on the biology of keratinocytes
under
controlled
conditions.
Acknowledgements This work was supported by grants from Consiglio Nazionaie delle Richerche, Rome (Italy), contract no. 88.03121.26 and by Progetto Finalizzato ‘Biotecnologie e biostrumentazione’, contract no. 8900240.70.
References Barnes D. and Sato G. (1980) Methods for growth of cultured cells in serum-free medium. Analyt. Biochem. 102, 255. Bemstam L. I., Vaughan F. L. and Bernstein I. A. (1986) Keratinocytcs grown at the air-liquid interface. In Vitro Cell Develop. Biol. 22, 695. Blacker K. L., Williams M. L. and Goldyne M. (1987) Mitomycin C-treated 3T3 fibroblasts used as feeder layers for human keratinocyte culture retain the capacity to generate eicosanoids. /. Invest. Demalol. 89, 536. Boyce S. T. and Ham R. G. (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. 1. Invest. Derrnatoi. 81, 33. Boyce S. T. and Ham R. G. (1985) Cultivation, frozen storage and clonal growth of normal human epidermal keratinocytes in serum-free medium. /, &sue Culttire Meth. 9, 83. Boyce S. T. and Ham R. G. (1986) Normal human epidermal keratinocytcs. In: Weber M. M. and Sekely L. (eds), In Vitro Models for Cancer Research, vol. 3. CRC: Boca Raton, p. 245. Boyce S. T. and Hansbrough J. F. (1988) Biological attachment, growth, and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-sulfate substrate. Sttrgely 103, 421. Cuono C., Langdon R. and McGuire J. (1986) Use of cultured epidermal autografts and dermal allografts as skin replacement after bum injury. Luncet i, 1123. De Luca M., Franzi i\. T., D’Anna F. et al. (1988) Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium. Etcr.1, Cell Biol. 46, 176. Eisinger M., Soho Lee J., Hefton J. M. et al. (1979) Human epidermal cell cultures: growth and differentiation in the absence of dermal components or medium supplements. koc. Nat/. Acad. Sri. US,4 76, 5340. Faure M., Mauduit G., Schmitt D. et al. (1987) Growth and differentiation of human epidermal cultures used as auto- or allografts in humans. Br. 1. Dewnafol. 116, 161. Furukawa F., Huff J. C., Lyons M. B. et al. (1988) Characterization and practical benefits of keratinocytes cultured in strontiumcontaining serum-free medium. I. Invest. Derrnatol. 5, 690. Gallico G. G., O’Connor N. E., Compton C. C. et al. (1984) Permanent coverage of large bum wounds with autologous cultured human epithelium. N. Engl. 1. Med. 331. 448. Cilchrest B. A., Nemore R. E. and Maciag T. (1980) Growth of human keratinocytes on fibronectin-coated plates. Cell. Biol. Intern. Reports 4, 1009.
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Gilchrest B. A., Marshall W. L., Karassik R. L. et al. (1984) Characterization and partial purification of keratinocyte growth factor from the hypothalamus. 1. Cell. Physiol. 120, 377. Green H. (1978) Cyclic AMP in relation to proliferation of the epidermal cell. A new view. Cell 15, 801. Green H., Kehinde 0. and Thomas J. (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Nad. Acad. Sci. USA 76, 5665. Grinnell F., Toda K. I. and Seymour C. L. (1987) Reconstitution of human epidermis in vitro is accompanied by transient activation of basal keratinocyte spreading. Erp. Cell Res. 172, 439. Jaken S. and Yuspa S. H. (1988) Early signal for keratinocyte differentiation: role of Ca’+ -mediated inositol lipid metabolism in normal and neoplastic epidexmal cells. Carcinogenesis 9, 1033. Jones J. C. R., Goldman A. I., Steinart P. M. et al. (1982) Dynamic aspects of the supramolecular organisation of intermediate filament networks in cultured epidermal cells. Cell Moflilu 2, 197. Kano-Sueoka T. and Errick J. E. (1981) The effects of phosphoethanolamine and ethanolamine on growth of mammary carcinoma cells in culture. Lrp. Cell Res. 136, 137. Karasek M. A. (1983) Culture of human keratinocytes in liquid medium. I. Inoest. Dermatol. 81, 245. Kitano J. and Okada N. (1983) Separation of the epidermal sheets by dispase. Br. J Dermatoi. 108, 555. Kubo M., Kan M., Isemura M. et al. (1987) Effects of extracellular matrices of human keratinocyte adhesion and growth and on its secretion and deposition of fibronectin in culture. 1. Invest. Dematol. 88, 594. Maciag T., Nemore R. E., Weinstein R. et al. (1981) An endocrine approach to the control of epidermal growth: serum-free cultivation of human keratinocytes. Science 211, 1452. O’Keefe E. J., Payne R. E. and Russel N. (1985) Keratinocyte growth-promoting activity from human placenta. 1. Cell Physiol. 124, 439. Peehl D. M. and Ham R. G. (1980a) Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium. In Vitro Cell Develop. Biol. 16, 516. Peehl D. M. and Ham R. G. (198Ob) Clonal growth of human keratinocytes with small amounts of dialyzed serum. In Vitro Cell Develop. Biol. 16, 526. Pittelkow M. R. and Scott R. E. (1986) New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive bums. Mayo Clin. Proc. 61, 771. Praeger F. C., Stanulis-Praeger B. M. and Gilchrest B.A. (1987) Use of strontium to separate calcium-dependent pathways for proliferation and differentiation in human keratinocytes. 1, Cell Physiol. 132, 81. Prunieras M., Regnier M. and Woodley D. (1983) Methods for cultivation of keratinocytes with an air-liquid interface. 1, Invest. Dennabl. 81, 280. Regnier M., Desbas C., Bailly C. et al. (1988) Differentiation of normal and tumoral human keratinocytes cultured on dermis: reconstruction of either normal or tumoral architecture. In Vitro Cell Develop. Biol. 24, 625. Rheinwald J. G. (1980) Serial culfivation of normal human epidermal keratinocytes. In: Harris C. C., Trump B. F. and Stoner G. D. (eds), Methods in Cell Biology, vol. 21A. New York: Academic Press, p. 229. Rheinwald J. G. and Green H. (1975a) Formation of a keratinizing epit-helium in culture of a cloned cell line derived from a teratoma. Ceil 6, 317.
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Burns (1992) IS, Supplement
1, S16-S18
Printed in Great Briiain
Rheinwald J. G. and Green H. (1975b) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331. Rheinwald J. G. and Green H. (1977) Epidemal growth factor and
the multiplication of cultured human epidemal keratinocytes. Nature 265, 421. Shipley G. D. and Pittelkow M. R. (1987) Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium. Arch. Dermalol. 123, 1541. Stanulis-Praeger B. M., Yaar M., Redziniak G. et al. (1988) An extract of bovine thymus stimulates human keratinocytes growth in vitro. 1, Imesf. Dermafol. 90, 749. Stenn K. S., Madri J. A., Tinghitella T. et al. (1983) Multiple mechanism of dissociated epidennal cell spreading. 1. Cell Biol. 96, 63. Thivolet J., Faure M., Demidem A. et al. (1986) Long-term survival and immunological tolerance of human epidermal allografts produced in culture. Transplankakion 42, 274. Tinois E., Faure M., Chatelain P. et al. (1987) Growth and differentiation of human keratinocytes on extracellular matrix. Arch. Demakol. Res. 279, 241.
Tsao M. C., Walthall B. J. and Ham R. G. (1982) Clonal growth of normal human epidermal keratinocytes in a definied medium. I. Ce// Physiol. 110, 219. Vaughan F., Gray R. H. and Bernstein I. A. (1986) Growth and differentiation of primary rat keratinocytes on synthetic membranes In Vitro Cell Develop. Biol. 22, 141. Watt F. M. (1988) Epidermal stem cells in culture. 1. Cell. Sri. Suppl. 10, 85. Wille J. J., Pittelkow M. R., Shipley G. D. et al. (1984) Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics and cell cycle studies. 1. Cell Physiol. 121, 31.
Correspondence should be addressed to: Professor Maria Luisa Tenchini, Dipartimento di Biologia e Genetica per le Scienze mediche, via Viotti 5, I - 20133 Milano, Italy.
Permanent coverage of full skin thickness burns with autologous cultured epidermis and re-, epithelialization of partial skin thickness lesions induced by allogetieic cultured epidermis: a multicentre study in the treatment of children R. Cancedda”‘, A. M. Tamisani3, C. Di Noto3, L. Muller’, M. De Lucalfz, S. Bondanzal”, D. Dioguardl “, E. Brienza4, A. Calvario4, R. Zermani5, D. Di Mascio5 and F. Papadia5 ‘IST, Istituto Nazionale per la Ricerca sul Cancro, Genoa, ‘Istituto di Oncologia Clinica e Sperimentale, Universita’ di Genova, Genoa, 3Servizio Chirurgico di Pronto Soccorso, Istituto Giannina Gaslini, Genoa, *Cattedra di Chirurgia Plastica, Facolta’ di Medicina e Chirurgia, Universita’ di Bari, Bari and Ystituto di Chirurgia Plastica e Centro Ustioni, Facolta’ di Medicina e Chirurgia, Universita’ di Par-ma, Parma, Italy
Introduction When burn wounds cover large areas of the body surface, remaining donor sites might not be sufficient to prepare enough split-thickness mesh grafts. This problem becomes more critical in children since the use of large donor sites increases the skin defect and, especially in patients sustaining large burn wounds, might actually increase the mortality rate. Moreover, hypertrophic scars develop frequently on the donor sites in children. Skin-derived human keratinocytes can be cultured in vitro (Rheinwald and Green, 1973, starting from a 1-2 cm2 biopsy. With the appropriate culture conditions, the initial cell population present in the skin $” 1992 Butterworth-Heinemann 0305-4179/92/OSOS1&03
Ltd
biopsy can be amplified in secondary cultures and can generate coherent epithelial sheets sufficient to cover the entire body surface in a period of 2-4 weeks (Green et al., 1979; De Luca et al., 1989). Autologous epithelial sheets (autografts) can be detached from the surface vessel and successfully used for permanent coverage of large burn wounds (O’Connor et al., 1981; Gallico et al., 1984; Cuono et al., 1986; Faure et al., 1987; Herzog et al., 1988; De Luca et al., 1989; Teepe et al., 1990a). Recovery of children sustaining full skin thickness bums covering up to 98 per cent of the body surface has been reported (Gallico et al., 1984). However, levels of ‘take’ (i.e. the presence of viable epithelium at the removal of the petroleum jelly gauze)
