Long-Term Culture of Murine Epidermal Keratinocytes
1999; Elsevier BV; Volume: 112; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.1999.00605.x
ISSN1523-1747
AutoresBarbara Hager, Philip Fleckman, Jackie R. Bickenbach,
Tópico(s)Silk-based biomaterials and applications
ResumoThe production of transgenic and null mice with skin abnormalities makes it increasingly important to establish cultures of mouse epidermal keratinocytes for in vitro studies. This requires that each cell line be derived from a single mouse and that the cells be carried for multiple passages. Freezing the cells would also be advantageous by allowing comparison of keratinocytes from several mouse lines at the same time. Mouse keratinocytes, however, have been exceedingly difficult to grow as primary cultures, and subculturing these cells has been virtually impossible until now. We describe a gentle dissociation method and a highly supplemented fibroblast conditioned medium that allows us to grow and subculture total mouse keratinocytes for up to 19 subcultures, allowing an increase in cell number of greater than 10 logs. Epidermal keratinocytes from newborn mice were grown on collagen IV coated dishes in murine fibroblast conditioned medium with 0.06 mM calcium and added growth factors. The cells could be passaged, frozen as viable stocks, and induced to differentiate. Morphologically the cultured keratinocytes demonstrated a pattern characteristic of basal cells. Stratified cultures which made mouse keratin 1 and profilaggrin through passage 10 were induced by purging the monolayer cultures of growth factors, then adding medium with 0.15 mM calcium; expression of mouse keratin 1 and profilaggrin was lost by passage 15. The methods explained in detail here should be of great interest to investigators who are now trying to analyze skin phenotypes and expression of markers of epidermal differentiation of their transgenic or knockout mice. The production of transgenic and null mice with skin abnormalities makes it increasingly important to establish cultures of mouse epidermal keratinocytes for in vitro studies. This requires that each cell line be derived from a single mouse and that the cells be carried for multiple passages. Freezing the cells would also be advantageous by allowing comparison of keratinocytes from several mouse lines at the same time. Mouse keratinocytes, however, have been exceedingly difficult to grow as primary cultures, and subculturing these cells has been virtually impossible until now. We describe a gentle dissociation method and a highly supplemented fibroblast conditioned medium that allows us to grow and subculture total mouse keratinocytes for up to 19 subcultures, allowing an increase in cell number of greater than 10 logs. Epidermal keratinocytes from newborn mice were grown on collagen IV coated dishes in murine fibroblast conditioned medium with 0.06 mM calcium and added growth factors. The cells could be passaged, frozen as viable stocks, and induced to differentiate. Morphologically the cultured keratinocytes demonstrated a pattern characteristic of basal cells. Stratified cultures which made mouse keratin 1 and profilaggrin through passage 10 were induced by purging the monolayer cultures of growth factors, then adding medium with 0.15 mM calcium; expression of mouse keratin 1 and profilaggrin was lost by passage 15. The methods explained in detail here should be of great interest to investigators who are now trying to analyze skin phenotypes and expression of markers of epidermal differentiation of their transgenic or knockout mice. Dulbecco’s modified Eagle’s medium with 10% FBS and antibiotics fibroblast conditioned medium from primary fibroblasts MEM Eagle with Earle’s BSS with a calcium level of 0.06 mM, HCM, high calcium medium Eagle with Earle’s BSS with a calcium level of 0.6 mM keratin 10 keratinocyte growth medium with a calcium level of 0.15 mM murine epidermal keratinocyte murine keratin 1 50% EMEM.06 plus 50% fibroblast conditioned medium with 2 ng EGF per ml 0.4 μg hydrocortisone per ml, 0.75 mM aminoguanidine nitrate, and 10–10 M cholera toxin profilaggrin Methods for culture and subculture of human epidermal keratinocytes from a single biopsy have been well documented (Rheinwald and Green, 1975Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-344Abstract Full Text PDF PubMed Scopus (3726) Google Scholar). Furthermore, human skin keratinocytes in these cultures have maintained their division capability for many passages and allogeneic grafts of these cultured cells have been shown to last for years (Rheinwald and Green, 1975Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-344Abstract Full Text PDF PubMed Scopus (3726) Google Scholar;Compton et al., 1989Compton C.C. Gill J.M. Bradford D.A. Regauer S. Gallico G.G. O’Connor N.E. Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting.Lab Invest. 1989; 60: 600-612PubMed Google Scholar;Otto et al., 1993Otto W.R. Nanchahal J. Lu Q-LB N. Dover R. Survival of allogeneic cells in cultured organotypic skin grafts.Plast Reconstr Surg. 1993; 96: 166-176Crossref Scopus (28) Google Scholar). Such has not been the case, however, for mouse epidermal keratinocytes. Traditional culture techniques required that murine keratinocytes be plated at high density, studied in primary culture only, and for some analyses pooled from several mice (Yuspa and Harris, 1974Yuspa S.H. Harris C.C. Altered differentiation of mouse epidermal cell cultures treated with retinyl acetate in vitro.Exp Cell Res. 1974; 86: 95-105Crossref PubMed Scopus (367) Google Scholar;Rosenthal et al., 1991Rosenthal D.S. Steinert P.M. Chung S. Huff C.A. Johnson J. Yuspa S.H. Roop D.R. A human epidermal differentiation-specific keratin gene is regulated by calcium but not negative modulators of differentiation in transgenic mouse keratinocytes.Cell Growth Differ. 1991; 2: 107-113PubMed Google Scholar;Rouabhia et al., 1992Rouabhia M. Germain L. Bélanger F. Guignard R. Auger F.A. Optimization of murine keratinocyte culture for the production of graftable epidermal sheets.J Dermatol. 1992; 19: 325-334Crossref PubMed Scopus (25) Google Scholar). Although these primary cultures have provided much information, with the advent of genetic manipulation resulting in skin phenotypes in both transgenic and knockout mice a method which allows continuous culture of keratinocytes from a single newborn mouse is needed. It is generally known that primary cultures of murine keratinocytes are highly responsive to calcium concentrations, and to maintain proliferation a medium containing less than 0.09 mM Ca2+ must be used (Hennings et al., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regualtion of growth and differentiation in mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1450) Google Scholar). Higher calcium levels initiate terminal differentiation and decrease proliferation (Hennings et al., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regualtion of growth and differentiation in mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1450) Google Scholar;Roop et al., 1983Roop D.R. Hawley-Nelson P. Cheng C.K. Yuspa S.H. Keratin gene expression in mouse epidermis and cultured epidermal cells.Proc Natl Acad Sci USA. 1983; 80: 716-720Crossref PubMed Scopus (129) Google Scholar). It also appears that several growth factors may be required to maintain the cultured keratinocytes (Fischer et al., 1980Fischer S.M. Viaje A. Harris K.L. Miller D.R. Bohrman J.S. Slaga T.J. Improved conditions for murine epidermal cell culture.In Vitro. 1980; 16: 180-188Crossref PubMed Scopus (31) Google Scholar;Hennings et al., 1980Hennings H. Michael D. Cheng C. Steinert P. Holbrook K. Yuspa S.H. Calcium regualtion of growth and differentiation in mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1450) Google Scholar;Bertolero et al., 1984Bertolero F. Kaighn M.E. Gonda M.A. Saffiotti U. Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium.Exp Cell Res. 1984; 155: 64-80Crossref PubMed Scopus (60) Google Scholar, Bertolero et al., 1986Bertolero F. Kaighn M.E. Camalier R.F. Saffiotti U. Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes.In Vitro Cell Dev Biol. 1986; 22: 423-428Crossref PubMed Scopus (42) Google Scholar), although which ones are absolutely necessary has not been clearly determined. To overcome these growth factor deficiencies, investigators have supplemented cultures with fibroblast conditioned medium or grown cells on substrate-coated culture dishes (Yuspa et al., 1981Yuspa S.H. Koehler B.A. Kulesz-Martin M. Hennings H. Clonal growth of mouse epidermal cells in medium with reduced calcium concentration.J Invest Dermatol. 1981; 76: 144-146Abstract Full Text PDF PubMed Scopus (72) Google Scholar;Greenhalgh et al., 1989Greenhalgh D.A. Welty D.J. Strickland J.E. Yuspa S.H. Spontaneous Ha-ras gene activation in cultured primary murine keratinocytes.Mol Carcinogenesis. 1989; 2: 199-207Crossref PubMed Scopus (40) Google Scholar). Although most supplements appeared to aid in the growth of primary murine keratinocytes, no one has reported long-term culture and subculture of these cells with the exception ofBertolero et al., 1986Bertolero F. Kaighn M.E. Camalier R.F. Saffiotti U. Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes.In Vitro Cell Dev Biol. 1986; 22: 423-428Crossref PubMed Scopus (42) Google Scholar and their method has not been used widely. Recently, one of us reported the selection and extended growth of murine epidermal stem cells in culture using a gentle isolation procedure and rapid adherence to collagen IV (Bickenbach and Chism, 1998Bickenbach J.R. Chism E. Selection and extended growth of murine epidermal stem cells in culture.Exp Cell Res. 1998; 244: 184-195Crossref PubMed Scopus (201) Google Scholar). Here we report that the use of this gentle isolation procedure and a highly enriched fibroblast conditioned medium allows growth and subculture of total murine keratinocytes from a single newborn mouse for as many as 19 passages. Expression of biochemical markers of the late stages of epidermal differentiation can be induced by purging the medium of growth factors and increasing the calcium concentration of the medium through 10 passages. This new culture method also allows cultured murine keratinocytes to be kept as frozen stocks, which can then be thawed and grown for several more passages, an advance that should be of benefit to studies that require comparison of cultures of skin keratinocytes from normal, transgenic, or null mice. Newborn (1–2 d old) C57/BL6 mice were used for the preparation of most of the conditioned medium and for most of the keratinocyte culture experiments. Other trials involved inbred C3H/Fej and BALB/C mice, outbred ICR mice, transgenic mice expressing human filaggrin in a C57BL/6J-SJL/J X BALB/C background, 1Presland RB et al. In preparation transgenic mice expressing a dominant negative type II TGFβ receptor (Wang et al., 1997Wang X.-J. Greenhalgh D.A. Bickenbach J.R. Jiang A. Bundman D.S. Derynck R. Roop D.R. Expression of a dominant negative type II TGFβ receptor in the epidermis of transgenic mice documents its role in mediating growth inhibition.Proc Natl Acad Sci USA. 1997; 94: 2386-2391Crossref PubMed Scopus (130) Google Scholar), and LAMA3 null mice in C57/129 background. 2Ryan MC, Lee K, Miyashita Y, Carter WC. Manuscript submitted Several types of media were used: high calcium medium (HCM) consisted of 100 ml MEM Eagle with Earle’s BSS without calcium (EMEM) (BioWhittaker, Walkersville, MD), 8 ml fetal bovine serum (FBS) (Summit, Ft. Collins, CO), 1 ml antibiotic/antimycotic containing 10,000 units per ml penicillin, 10,000 μg streptomycin per ml, and 25 μg Amphotericin B (GIBCO-BRL, Gaithersburg, MD) per ml with CaCl2 added to 0.6 mM. The calcium concentration of FBS was supplied by the vendor. EMEM.06 differed from HCM by the use of 8 ml chelexed FBS (Brennan et al., 1975Brennan J.K. Mansky J. Roberts G. Lichtman M.A. Improved methods for reducing calcium and magnesium concentrations in tissue culture medium: application to studies of lymphoblast proliferation.In Vitro. 1975; 11: 354-360Crossref PubMed Scopus (92) Google Scholar), and a Ca2+ concentration of 0.06 mM. The calcium concentrations of chelexed serum and media were determined by the clinical laboratory at the University of Washington, Seattle, WA using atomic absorption spectroscopy with linearity to 0.009 mM. N-Medium consisted of 50% EMEM.06 plus 50% fibroblast conditioned medium supplemented with 2 ng per ml epidermal growth factor (EGF) (Collaborative Biomedical Products, Bedford, MA), 0.4 μg per ml hydrocortisone (Sigma, St. Louis, MO), 0.75 mM aminoguanidine nitrate (Aldrich, Milwaukee, WI), and 10–10 M cholera toxin (Calbiochem, San Diego CA). DMEM.06 consisted of low glucose Dulbecco’s modified Eagle’s medium made without calcium (GIBCO-BRL), 8 ml chelexed FBS, 1 ml antibiotic/antimycotic, and CaCl2 added to 0.06 mM. COS-FIBRO medium contained 10% FBS and 1% penicillin–streptomycin containing 10,000 units per ml penicillin and 10,000 μg per ml streptomycin (GIBCO-BRL) in low glucose DMEM (GIBCO-BRL). Low calcium freezing medium included 20% chelexed FBS, 8.8% dimethyl sulfoxide (Sigma), in Ca2+ free EMEM; calcium was added to bring to 0.06 mM, if necessary. Keratinocyte growth medium without calcium (Clonetics, San Diego, CA) was supplemented with CaCl2 to bring the medium to the desired calcium concentration. Collagenase solution consisted of 0.35% (wt/vol) crude collagenase, type 1 (GIBCO-BRL) dissolved in M199 (GIBCO-BRL). Newborn mouse pups were killed by decapitation, rinsed in 70% ethanol, limbs and tails amputated, the trunk skin removed (Yuspa and Harris, 1974Yuspa S.H. Harris C.C. Altered differentiation of mouse epidermal cell cultures treated with retinyl acetate in vitro.Exp Cell Res. 1974; 86: 95-105Crossref PubMed Scopus (367) Google Scholar) and rinsed three times in phosphate-buffered saline without Ca2+ or Mg2+ (PBS) (Dulbecco and Vogt, 1954Dulbecco R. Vogt M. Plaque formation and isolation of pure lines with poliomyelitis viruses.J Exp Med. 1954; 99: 167-182Crossref PubMed Scopus (1961) Google Scholar) with 1% antibiotic/antimycotic. The skins were then flattened with the dermal side down on sterile filter paper (Whatman 1) in Petri dishes and incubated overnight at 4°C in 0.25% trypsin (GIBCO-BRL). Dermis was separated from the epidermis and placed in a Petri dish with 5 ml collagenase solution. Dermal pieces were minced with sterile scissors and transferred to an Erlenmeyer flask containing 45 ml of the collagenase solution and stirred in a 36.8°C incubator for 30 min. The solution was then filtered through sterile gauze into centrifuge tubes and spun at 200 × g for 5 min. The supernatant was aspirated, the cells resuspended in HCM and distributed to T150 flasks with one mouse per flask per 25 ml medium. The calcium level of this medium allowed fibroblast proliferation (Boynton et al., 1974Boynton A.L. Whitfield J.F. Isaacs R.J. Morton H.J. Control of 3T3 proliferation by calcium.In Vitro. 1974; 10: 12-17Crossref PubMed Scopus (67) Google Scholar, Boynton et al., 1977Boynton A.L. Whitfield J.F. Isaacs R.J. Tremblay R. The control of human WI-38 cell proliferation by extracellular calcium and its elimination by SV-40 virus-induced proliferative transformation.J Cell Physiol. 1977; 92: 241-248Crossref PubMed Scopus (98) Google Scholar) whereas it hindered keratinocyte growth. Cells were incubated in a humidified atmosphere of 5% CO2, at 36.8°C. After 24 h, the fibroblasts appeared to be 60%–80% confluent and the flasks were thoroughly rinsed with two washes of PBS to remove all traces of calcium. Thirty milliliters of EMEM.06 was added to each flask and the cells were incubated for 48 h. The conditioned medium was poured off, filtered through a 0.45 μm filter (Corning, Corning, NY), aliquoted, and frozen at –20°C. The cells were either discarded or used to produce secondary conditioned medium. Primary fibroblasts were rinsed twice with PBS and incubated in 6 ml 0.25% trypsin at 36.8°C for 5–10 min. Triturated cells were removed to a sterile bottle, 10 ml COS-FIBRO medium per flask was added, and the cells were centrifuged at 200 × g for 5 min. The cells were split 1:2 into 25 ml COS-FIBRO medium per T150 flask. The old flasks were reused along with an equal number of new ones. These secondary fibroblasts reached 60–80% confluence after incubation for 2–3 d. The flasks were then thoroughly rinsed with two washes of PBS to remove all traces of calcium. Thirty milliliters of EMEM.06 was added to each flask and the cells were incubated for 48 h. The secondary conditioned medium was poured off, filtered through a 0.45 μm filter, aliquoted, and frozen at 20°C. The fibroblasts were discarded after this procedure. Collagen IV (Collaborative Biomedical Products) was thawed on ice in a 4°C room and diluted in 0.05 M HCl in order to coat Corning tissue culture dishes with a concentration of either 1 μg per cm2 or 5 μg per cm2 according to the supplier’s instructions. Dishes were allowed to sit for 1 h, followed by two rinses with PBS containing 1% antibiotic/antimycotic, draining well after the last wash. Dishes were then wrapped in stretch-tite plastic food wrap (Polyvinyl Films, Sutton, MA) and stored at 4°C until needed. The collagen solution was either reused immediately or stored at –80°C until needed. We found the collagen solution could be thawed twice and reused up to four times. Newborn mouse pups were processed as above. Epidermis that had been split from dermis after overnight incubation in trypsin was placed in a 50 ml centrifuge tube with EMEM.06 (one to four skins in 7 ml, five or more skins in 10 + ml) and the tube was given 50 firm shakes. The large epidermal pieces were removed, the cells centrifuged at 200 × g for 5 min, resuspended using a plastic pipet in N-Medium, and plated on Collagen IV coated dishes at 5 × 104 cells per cm2. The dishes were placed in a humidified 36.8°C incubator with the CO2 level reduced to 4.5%, refed with N-Medium in 24 h, and again 2–3 d later. On day 7, the cells were ready to be passaged or induced to differentiate. To passage, cultures were rinsed twice with PBS, incubated with 0.25% trypsin for 6–8 min at 36.8°C, triturated using a plastic pipet with an equal amount of cool EMEM.06, centrifuged, and either replated in N-Medium on collagen coated dishes at 2.5 × 104 cells per cm2 or frozen in low calcium freezing medium at 106 cells per ml per vial. Frozen cells were thawed into 5 ml N-Medium in a 60 mm collagen IV coated dish and refed the next day. Passaged and/or thawed cells were fed three times a week and passaged weekly at confluence. Cultures were observed and photographed using an inverted phase contrast microscope. The separation procedure described above for multiple mice was employed for single mice, with one mouse skin per 7 ml N-Medium per 15 ml tube. After 50 firm shakes, the epidermal sheet was removed, the cells counted and plated at 5 × 104 cells per cm2 in N-Medium on collagen IV coated dishes. Cells were fed and passaged or frozen as described above. For the fastest results, primary cells were seeded at 5 × 104 cells per cm2 on collagen IV coated dishes (5 μg per cm2) in keratinocyte growth medium with a calcium concentration of 0.15 mM (KGM.15). They were refed at 24 and 48 h, and harvested at 72 h. Alternatively, cells of any passage were grown on collagen IV coated dishes (1 or 5 μg per cm2) for 1 wk in N-Medium, fed EMEM.06 for 1 d, then KGM.15 for 1–3 d and harvested to detect mouse keratin 1 (MK1), mouse keratin 10 (K10), or profilaggrin (proFG). Unless otherwise specified, 15 μg protein loadings of culture extracts (determined by the dotMETRIC 1 μl Protein Assay, Geno Technology, St. Louis, MO) were resolved on 7.5%–12% or 7.5%–15% polyacrylamide gels, transferred to nitrocellulose, and visualized as previously described (Dale et al., 1987Dale B.A. Gown A.M. Fleckman P. Kimball J.R. Resing K.A. Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins.J Invest Dermatol. 1987; 88: 306-313Abstract Full Text PDF PubMed Google Scholar). Extracts of newborn mouse epidermis were run as controls. MK1 was identified using polyclonal monospecific antibody MK1 (Berkeley Antibody Company, Richmond, CA), K10 was identified by AE1 monoclonal antibody to acidic keratins (a generous gift of T-T. Sun, New York University School of Medicine,Woodcock-Mitchell et al., 1982Woodcock-Mitchell J. Eichner R. Nelson W.G. Sun T.-T. Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies.J Cell Biol. 1982; 95: 580-588Crossref PubMed Scopus (572) Google Scholar), and proFG was identified using polyclonal antiserum (Manabe et al., 1991Manabe M. Sanchez M. Sun T.T. Dale B.A. Interaction of filaggrin with keratin filaments during advanced stages of epidermal differentiation and in ichthyosis vulgaris.Differentiation. 1991; 48: 43-50Crossref PubMed Scopus (89) Google Scholar). The use of 50% conditioned medium with EGF allowed only one or two passages of total murine epidermal keratinocytes (MEK), which is in agreement with another study that is being reported elsewhere (Bickenbach and Chism, 1998Bickenbach J.R. Chism E. Selection and extended growth of murine epidermal stem cells in culture.Exp Cell Res. 1998; 244: 184-195Crossref PubMed Scopus (201) Google Scholar). The technique of Bickenbach and Chism allows long-term culture of murine epidermal stem cells, but growth is delayed over the first 40+ d. To increase the growth potential and the number of passages and decrease the time to passage of total MEK, several additives were tested that we have found helpful in culturing either rat or human keratinocytes. Hydrocortisone and aminoguanidine are necessary for continuous subculture of rat epidermal keratinocytes (Kubilus and Baden, 1983Kubilus J. Baden H.P. The growth and differentiation of cultured newborn rat keratinocytes.J Invest Dermatol. 1983; 80: 124-130Abstract Full Text PDF PubMed Scopus (74) Google Scholar) and continuous expression of profilaggrin (unpublished observations), respectively. Human epidermal keratinocytes grow best with hydrocortisone, EGF, and cholera toxin added to the medium (Rheinwald and Green, 1977Rheinwald J.G. Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.Nature. 1977; 265: 421-424Crossref PubMed Scopus (810) Google Scholar). To determine if these would also be helpful in long-term culture of murine epidermal keratinocytes, a series of trials were undertaken with various combinations of these growth factors (Table 1).Table 1Murine epidermal keratinocyte media trials aEpidermal keratinocytes were cultured from C57/BL6 skin as described in Materials and Methods.Media%CM1 bCM1, conditioned medium from primary fibroblast cultures.% OtherEGF cEGF, epidermal growth factor 2 ng per ml in all but KGM, which was 0.1 ng per ml.Other additions dBullet kit (Clonetics, San Diego, CA) – epidermal growth factor (human recombinant) 0.1 ng per ml, insulin 5 μg per ml, hydrocortisone 0.5 μg per ml, bovine pituitary extract 7.5 mg per ml, gentamicin 50 μg per ml, amphotericin-B 50 ng per ml. AG, aminoguanidine nitrate 0.75 mM; CT, cholera toxin 10–10M; HC, hydrocortisone 0.4 μg per ml.P0 eSubjective observations of growth rate and quality of primary (P0) and first passage (P1) MEK were made, where growth rate was the time necessary for the cultures to reach confluence from identical seeding densities and quality was assessed by the monolayer appearance of the cells, whether complete or irregular with pull-back holes and spaces. Cultures were grown to confluence, the calcium concentration of the medium was increased from 0.06 mM to 0.15 mM, and cells were grown for an additional 2 d before harvesting. No EMEM.06 purge was used (refer to Results). Immunoblots of extracts of primary cultures were analysed as described in Methods and Materials.P1 eSubjective observations of growth rate and quality of primary (P0) and first passage (P1) MEK were made, where growth rate was the time necessary for the cultures to reach confluence from identical seeding densities and quality was assessed by the monolayer appearance of the cells, whether complete or irregular with pull-back holes and spaces. Cultures were grown to confluence, the calcium concentration of the medium was increased from 0.06 mM to 0.15 mM, and cells were grown for an additional 2 d before harvesting. No EMEM.06 purge was used (refer to Results). Immunoblots of extracts of primary cultures were analysed as described in Methods and Materials.K10 fK10, keratin 10 determined by monoclonal antibody AE3.MK1 gMK1, murine keratin 1 determined by monospecific polyclonal antibody.proFG hproFG, murine profilaggrin determined by polyclonal antiserum.A5050% EMEM.06+++++---B3367% EMEM.06++++---C2575% EMEM.06++++---D–100% EMEM.06-++---E–100% DMEM.06-+++---F5050% DMEM.06+++++++---G–100% KGM.15+bullet kit+++++++++++++H5050% EMEM.06+AG++++++---I5050% EMEM.06+CT++++++---J5050% EMEM.06+HC+++++---K5050% EMEM.06+AG, CT+++++--+/-L5050% EMEM.06+AG, HC+++++---M5050% EMEM.06+CT, HC+++++++-+/-N5050% EMEM.06+AG, CT, HC++++++++++a Epidermal keratinocytes were cultured from C57/BL6 skin as described in Materials and Methods.b CM1, conditioned medium from primary fibroblast cultures.c EGF, epidermal growth factor 2 ng per ml in all but KGM, which was 0.1 ng per ml.d Bullet kit (Clonetics, San Diego, CA) – epidermal growth factor (human recombinant) 0.1 ng per ml, insulin 5 μg per ml, hydrocortisone 0.5 μg per ml, bovine pituitary extract 7.5 mg per ml, gentamicin 50 μg per ml, amphotericin-B 50 ng per ml. AG, aminoguanidine nitrate 0.75 mM; CT, cholera toxin 10–10M; HC, hydrocortisone 0.4 μg per ml.e Subjective observations of growth rate and quality of primary (P0) and first passage (P1) MEK were made, where growth rate was the time necessary for the cultures to reach confluence from identical seeding densities and quality was assessed by the monolayer appearance of the cells, whether complete or irregular with pull-back holes and spaces. Cultures were grown to confluence, the calcium concentration of the medium was increased from 0.06 mM to 0.15 mM, and cells were grown for an additional 2 d before harvesting. No EMEM.06 purge was used (refer to Results). Immunoblots of extracts of primary cultures were analysed as described in Methods and Materials.f K10, keratin 10 determined by monoclonal antibody AE3.g MK1, murine keratin 1 determined by monospecific polyclonal antibody.h proFG, murine profilaggrin determined by polyclonal antiserum. Open table in a new tab As can be seen in Table 1, most of the media combinations gave good growth of primary cultures of MEK, and when supplemented with fibroblast conditioned medium (CM1) and EGF, allowed passage of MEK once or twice. In early trials, however, only cells grown in N-Medium (EMEM.06 supplemented with CM1, EGF, aminoguanidine, cholera toxin, and hydrocortisone) when the calcium level was raised to 0.15 mM, and in KGM.15 displayed the differentiation markers MK1, K10, and proFG. Because our interest was not only in the long-term culture of murine keratinocytes from a single newborn pup, but also the expression of biochemical markers of epidermal differentiation, further studies were limited to these two media and to 0.06 mM calcium; other calcium concentrations below 0.09 mM were not tested. N-Medium, but not KGM.15, allowed continuous passage of MEK beyond the second passage. In N-Medium more than 10 passages of MEK were routinely obtained, with an increase in cell number of approximately 105 (Figure 1). One cell line was passaged 19 times, with an increase in cell number of >1010. In N-Medium at typical seeding densities, passaged cultures showed active growth marked by elongated cells for several days (Figure 2a). At confluence a classic basal “pavement” pattern was seen in N-medium. Attempts to subculture before confluence produced insufficient cells, whereas cells could be held at confluence for a few days and still passaged successfully. Early passage cells with a C57/BL6 background produced numerous dark dendritic-like cells, which were probably melanocytes (data not shown). These disappeared around the fourth passage in unfrozen cultures, and sooner in cultures that had been frozen. Cultures from white mice produced no dark cells. Large flattened cells, which ultimately lifted off the dish, were seen in early passages from all mice (Figure 2a). Keratinocytes from several inbred mouse strains, the outbred ICR strain, and from both transgenic and null mice were cultured and passaged successfully using the gentle isolation method and N-Medium. Thus, for continuous growth of MEK N-Medium is recommended. Limited observation of keratinocytes from several strains showed no distinct differences. MEK grew better on collagen type IV coated culture dishes than on uncoated dishes. The concentration of the collagen on the dish made no difference in the growth of the MEK, although it did make a difference in the attachment and adherence of the MEK. Cells appeared to become more firmly anchored to the substrate with higher concentrations of collagen. For subculture, we found that a 1 μg per cm2 collagen concentration worked best, allowing the MEK to be easily passaged. When we tried to purge and raise the calcium lev
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