Methods in Cell Science 23: 189–196 (2002) 2001 Kluwer Academic Publishers. Printed in the Netherlands.
An improved method for culture of epidermal keratinocytes from newborn mouse skin Lari Häkkinen, Leeni Koivisto & Hannu Larjava Laboratory of Periodontal Biology, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada Accepted in revised form 14 November 2001
Abstract. Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture
medium containing low Ca2+ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin αvβ6. The expression level of αvβ6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.
Key words: Cell culture, Mouse skin keratinocytes
1. Introduction
2. Materials
Recent advances in the generation of transgenic mouse lines have increased the need to use mouse skin keratinocytes in cell culture studies. Previously, several methods to isolate and culture epidermal keratinocytes from mouse skin have been described [1]. However, isolation and long term culture of keratinocytes has remained difficult with complex culture protocols sometimes requiring use of fibroblast feeder layers, and with limited reproducibility of the cell isolation process and difficulty to maintain the cells for more than three to five passages in culture. Here we describe for the first time a detailed method to isolate putative epidermal keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. This method is further improved and simplified from the previously published methods [2–5]. By using the protocol described below we have been able to isolate skin keratinocytes with a high success rate and managed to maintain them in culture for at least 26 passages without changes in cell morphology, proliferation rate or viability. Additionally, keratinocytes obtained by using the described method were able differentiate to produce a stratified epithelium in an organotypic culture model.
A. Culture media and dishes 01. EMEM, Ca2+-free culture medium, can be purchased from BioWhittaker, Inc.,1 Walkersville, MD, Cat. No. 06-174G. 02. Fetal bovine serum (FBS) can be purchased from Life Technologies,2 Rockville, MD, Cat. No. 16140-071. 03. DMEM can be purchased from Life Technologies,2 Rockville, MD, Cat. No. 31600-075. 04. HEPES can be purchased from Sigma Chemical Co.,3 St. Louis. MO, Cat. No. H-9136. 05. Cell culture dishes (diameter 60 mm) and flasks (T25 and T175) can be purchased from Falcon, Becton Dickinson,4 Franklin Lakes, NJ, Cat. No. 353004, 353108 and 353112, respectively. B. Chemicals and accessories 01. Calcium and magnesium-free phosphate buffered saline (PBS) can be purchased from Life Technologies,2 Rockville, MD, Cat. No. 21600-069. 02. 100× Antibiotics mixture (penicillin 10,000 units/ml, streptomycin sulphate 10 mg/ml,
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amphotericin B 25 µg/ml in 0.85% saline) can be purchased from Life Technologies,2 Rockville, MD, Cat. No. 15240-062. Trypsin 0.25%/EDTA 1 mM solution can be purchased from Life Technologies,2 Rockville, MD, Cat. No. 25200-072. Chelex can be purchased from Bio-Rad Laboratories,5 Hercules, CA, Cat. No. 1422842. Dimethyl sulfoxide (DMSO) can be purchased from Sigma Chemical Co.,3 St. Louis, MO, Cat. No. D-8779. CaCl2 dihydrate can be purchased from JT. Baker,6 Phillipsburg, NJ, Cat. No. 1336-01. Stock solution (1 M) can be prepared in sterile distilled H2O and filtered using a 25 mm Acrodisc Syringe filter with 0.2 µm pore size (PALL Corporation, Gelman Labs, Ann Arbor, MI,7 Cat. No. 4192) before use. Type I collagen from bovine skin (Vitrogen100; 3 mg/ml stock solution), can be purchased from Cohesion,8 Palo Alto, CA. Purified bovine plasma fibronectin can be purchased from Chemicon International,8 Temecula, CA, Cat. No. FC 014. Human recombinant epidermal growth factor (EGF) can be purchased from Life Technologies,2 Rockville, MD, Cat. No. 13247-010. Teflon mesh (Spectra/Mesh Fluorocarbon, 70–100 µm) for cell filtering can be purchased from Spectrum,10 Rancho Dominguez, CA, Cat. No. 145904. PES 500 ml bottle top filter (0.2 µm pore size) for filtering fibroblast conditioned medium can be purchased from Nalge Nunc International,11 Rochester, NY, Cat. 2953320. Bard-Parker rib-back carbon steel surgical blades (no. 15) can be purchased from Beckton Dickinson AccurateCare, Franklin Lakes, NJ, Cat. 371115.
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2. Procedure A. Keratinocyte isolation from newborn mouse skin 01. Sacrifice the newborn mice (0–3 days old) by asphyxiation using CO2. For starting the keratinocyte culture, pooled skins from three newborn mice are required. 02. Remove the trunk skin in one piece by using surgical scissors and a scalpel with a no. 15 surgical blade (Bard-Parker, Beckton Dickinson AccurateCare, Franklin Lakes, NJ). 03. Wash the skin immediately with: 1) PBSantibiotics solution [PBS (Life Technologies,
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Rockville, MD) containing 1% antibiotics mixture (from the 100× stock solution; Life Technologies, Rockville, MD)] for 2 min; 2) distilled H2O for 2 min; 3) 70% ethanol for 1 min; 4) fresh PBS-antibiotics solution for 2 min. In the laminar flow cell culture hood, place the skin the dermal side facing up in a sterile culture dish keeping the tissue moist in the PBS-antibiotics solution. Using sterile instruments (forceps and a scalpel with a no. surgical 15 blade), remove subcutaneous fat by scraping carefully with the side of the scalpel blade. Avoid pressing too hard because that may cause damage to the epidermal layer. After removing fat, skin should look almost transparent when examined against a light source. However, if the skin is trimmed too much the keratinocytes will be lost. This is a critical step and may require practice. Cut the skin into 10 mm wide strips with a sharp scalpel and place the tissue strips into a new sterile dish covered with PBS-antibiotics solution to keep the tissue moist. Once all the mice are done, place the skin stripes epdermis side up on an empty 60 mm cell culture plate, and then carefully add 4 ml of trypsin/EDTA (0.25%/l mM; Life Technologies, Rockville, MD) to the culture plate to float the skins on the trypsin/EDTA solution. Do not allow trypsin/EDTA solution to leak on top of the skin to avoid disintegration of the epidermis leading to difficulty in the separation procedure of the dermal and epidermal tissues (see below). After overnight incubation at 4 °C, place the tissues epidermis side up on an empty 60 mm culture plate and separate epidermis from dermis by gently pulling the tissues apart by using a pair of sterile forceps. Place the epidermises into a sterilized Ehrlenmayer flask with a sterilized magnetic mixing bar containing enough complete keratinocyte growth medium (see below) to completely cover the tissues and mixed on a magnetic stirrer with a medium speed at room temperature for 1 h. Then, filter the cell suspension through a sterile teflon mesh (70–100 µm; Spectrum, Rancho Dominguez, CA) attached to a sterile 50 ml syringe to remove remaining tissue pieces. Collect the cells from the filtrate by centrifugation (500 g) for 10 min at 4 °C. Count the cells (expected yield is about 4–5 × 106 cells per three mice) and seed 1 × 106 cells in a fibronectin-collagen precoated (see below) 60 mm cell culture plate
191 (Falcon, Becton Dicinson, Franklin Lakes, NJ) in 4 ml of the complete keratinocyte growth medium (see below). 12. Allow the cells to attach in the cell culture incubator with 5% CO2 at 37 °C. After 10 min, remove the unattached cells by a gentle wash of the cell layer with the medium and then add 4 ml of the complete keratinocyte growth medium (see below) to the cells. It is expected that about 10–20% of the total amount of cells remain attached after 10 min incubation. 13. Culture the cells in the cell culture incubator with 5% CO2 at 37 °C for 4 days before replacing the medium with a fresh growth medium. Thereafter, change the medium every other day. Keratinocytes start to grow slowly and first colonies will appear after 10–16 days. Cell growth speeds up once the cell density becomes greater. 14. At early confluence (80–90% confluent) subculture the cells by detaching them by a brief treatment (1–2 min) with the trypsin/EDTA solution (Life Technologies, Rockville, MD), inactivate the trypsin with EMEM (BioWhittaker, Inc., Walkersville, MD) containing CaCl2 (0.05 mM), Chelex (Bio-Rad Laboratories, Hercules, CA) treated FBS (9%; Life Technologies, Rockville, MD) and 1% antibiotics mixture (from 100× stock solution; Life Technologies, Rockville, MD). For routine subculturing, seed 5 × 105 cells in a T25 cell culture flask (Falcon, Becton Dickinson, Franklin Lakes, NJ) in 5 ml of the complete keratinocyte growth medium (see below). For subculturing, coating of the culture substrate with fibronectin-collagen solution is not required. When needed, cells can be frozen for storage in liquid nitrogen by suspending 5 × 105 cells in 1 ml of the complete keratinocyte growth medium containing 10% DMSO (Sigma Chemical Co., St. Louis, MO) before freezing by routine protocol. B. Preparation of collagen-fibronectin coated culture substrates For coating, incubate 1 ml of the collagenfibronectin solution containing 10 µg/ml of bovine plasma fironectin (Chemicon International, Temecula, CA), and 30 µg/ml of type I collagen (Vitrogen 100, 3 mg/ml stock solution; Cohesion, Palo Alto, CA) in DMEM (Life Technologies, Rockville, MD) containing 25 mM HEPES (Sigma Chemical Co., St. Louis, MO) in a 60 mm cell culture plate for 1 h at room temperature. Then, vacuum-aspirate the fibronectin-collagen solution and wash the substrate three times with PBS (Life Technologies, Rockville MD) before seeding the cells.
C. Preparation of complete keratinocyte growth medium Procedure (adapted from Bickenbach and Chism [4]): In order to prepare complete keratinocyte growth medium, mix the following ingredients in Ca2+free EMEM (BioWhittaker, Inc., Walkersville, MD) to give the indicated final concentrations: 1. 0.05 mM CaCl2 (from sterile 1 M stock solution in sterile distilled H2O). 2. 9% Chelex (Bio-Rad Laboratories, Hercules, CA) treated FBS (see below). 3. 1% antibiotics mixture (penicillin 100 units/ml, streptomycin sulphate 100 µg/ml, amphotericin B 0.25 µg/ml; from 100× stock solution, Life Technologies, Rockville MD). 4. 4 ng/ml EGF (Life Technologies, Rockville MD). 5. 50% fibroblast conditioned medium (see below). D. Chelex (Bio-Rad Laboratories, Hercules, CA) treatment of fetal bovine serum to remove Ca2+ should be done exactly as described by Swierenga and MacManus [6]. E. Isolation of newborn mouse skin fibroblasts by using the explant culture method. Procedure: 1. Collect and wash a newborn mouse skin and then remove the subcutaneous fat tissue as above. 2. Place the skin in a sterile cell culture dish containing DMEM with 10% FBS and antibiotics mixture to keep the tissue moist. Cut the skin into small pieces (less than 1 mm × 1 mm × 1 mm) using sterile sharp surgical scissors and a scalpel. 3. Place in total 6–12 tissue pieces into a 60 mm cell culture dish (Falcon, Becton Dickinson, Franklin Lakes, NJ) using sterile forceps. Under the laminar flow hood, allow the tissue pieces to air dry shortly (about 15 min) to allow attachment of the tissue to the dish. Then, carefully add 4 ml of the fibroblast growth medium [DMEM (Life Technologies, Rockville, MD) containing 10% FBS (Life Technologies, Rockville, MD), 25 mM HEPES (Sigma, St. Louis, MD) and 1% antibiotics mixture (from 100× stock solution; Life Technologies, Rockville, MD)] to cover the tissue pieces without detaching them and incubate the dishes at 37 °C in the presence of 5% CO2 in a cell culture incubator. 4. Replace the medium with fresh medium for the first time after 4 days and every third day thereafter. Migration of fibroblasts with spindle cell morphology can usually be detected for the first time about 2–3 days after starting the culture.
192 5. At early confluence, detach the cells by a brief treatment with the trypsin/EDTA solution (Life Technologies, Rockville, MD) followed by a short wash with the fibroblast growth medium to inhibit the trypsin activity. 6. For subculturing, seed 2.5 × 105 cells in a T25 cell culture flask (Life Technologies, Rockville, MD) in 5 ml of the fibroblast growth medium. Subculturing up to passage 3 will promote selective growth of fibroblasts over other cell types, including endothelial cells, resulting in a pure fibroblast culture. 7. Cells can be frozen for storage in liquid nitrogen in the growth medium containing 10% DMSO (Sigma, St. Louis, MD) by using routine methods. F. Preparation of conditioned medium from newborn mouse skin fibroblast cultures Procedure: For collecting conditioned medium, use newborn mouse skin fibroblasts at passage 3–5. Seed fibroblasts (2 × 106 cells) in a T175 cell culture flask (Falcon, Becton Dickinson, Franklin Lakes, NJ) in their normal growth medium and allow them to grow to about 60% confluence at 37 °C in the presence of 5% CO2. Then, wash the cell layer once with PBS before adding 30 ml of the low-Ca2+ medium (see below) for collecting fibroblast conditioned medium. Culture cells at 37 °C in the presence of 5% CO2. After 48 h, collect the conditioned medium and immediately freeze it for storage. After collecting the conditioned medium, culture the fibroblasts in their normal growth medium for at least two days to allow the cells to recover before they can be used for conditioned medium collection again. We have successfully collected conditioned medium from the same culture for up to 3–4 times before subculturing the cells into lower density to start a new culture. However, do not use over-confluent cultures for the medium collection because cell viability and metabolism may be changed at very high density. Before use, thaw the conditioned medium at 4 °C overnight, centrifuge at 5000 g for 10–15 min at + 4 °C and filtrate using a 500 ml bottle top filter with 0.2 µm pore size (Nalge Nunc International, Rochester, NY) to remove cell particles. For preparing the low-Ca2+ medium for fibroblast conditioned medium collection, mix the following ingredients in Ca2+-free EMEM (BioWhittaker, Inc., Walkersville, MD) to give the indicated final concentrations: • 0.05 mM CaCl2 (from the 1 M stock solution in sterile distilled H2O); • 9% Chelex treated FBS (see above); • 1% antibiotics mixture (penicillin 100 units/ml, streptomycin sulphate 100 µg/ml, amphotericin
B 0.25 µg/ml; from 100× stock solution, Life Technologies, Rockville MD).
4. Results and discussion The proliferating cells in the epidermis are located in the basal epithelial cell layer and can be categorized into stem cells and transit amplifying cells [7]. The epidermal stem cells are characterized by unlimited capacity for self-renewal, high expression of β1 integrins and rapid adhesion to extracellular matrix proteins [7]. The stem cells can give rise to either new stem cells or transit amplifying cells [8]. The transit amplifying cells are programmed to undergo terminal differentiation after few cell divisions. They also express lower levels of β1 integrins and adhere more slowly on extracellular matrix proteins than the stem cells [4, 7, 9]. In the present study, we have taken advantage of the property of the keratinocyte stem cells to rapidly attach on extracellular matrix proteins to isolate keratinocytes from mouse skin for extended cell culture. For this purpose, we enzymatically separated epidermal keratinocytes from newborn mouse skin. The putative stem cells were then selected by allowing the keratinocytes to attach on an extracellular matrix composed of type I collagen and fibronectin. Cells that were not attached in 10 minutes were discarded to separate the rapidly attaching stem cells from the slower adhering, more differentiated keratinocytes. Subsequently, the rapidly attached cells were allowed to grow in a specified keratinocyte growth medium. We have used the described culture protocol to isolate newborn mouse skin keratinocytes from wild type and three transgenic mouse lines of the FVB/NHsd background. The success rate of the cultures has been about 70% (about 7 out of ten tissue culture plates inoculated with mouse keratinocytes gave rise to continuous keratinocyte cultures). To date, the cell cultures recovered from frozen stocks have been subcultured at least up to 10 passages. The highest number of subcultures is at the moment 26 in a keratinocyte line from the wild type mouse. A previous report by Hager et al. [5] reported subculturing of mouse keratinocyte stem cells for 19 times. One of the advantages of the described protocol as compared to previous keratinocyte culture methods [1] is that the keratinocyte culture does not require a fibroblast feeder layer to support cell growth. To promote keratinocyte growth, we have used the specified keratinocyte growth medium [4] that was supplemented with 0.05 mM CaCl2, 50% fibroblast conditioned medium, 9% fetal bovine serum and EGF (4 ng/ml). Furthermore, we have used a composite matrix composed of type I collagen and fibronectin to selectively promote adhesion and growth of keratinocyte stem cells. Keratinocyte stem cells express higher levels of the collagen receptor
193 α2β1 than the transit amplifying cells [9]. Therefore, collagen matrix supports rapid adhesion of the stem cells over more differentiated cells [9]. Interestingly, freshly isolated keratinocytes are not able to attach to fibronectin [10] suggesting that they do not express functional fibronectin receptors α5β1 and αvβ6 integrins. Similar to wound healing, expression of α5β1 [10]) and αvβ6 [11–13] integrins is induced in keratinocytes when they are placed in cell culture [7–14]. Both α5β1 and αvβ6 integrins can promote cell proliferation [15–17]. Therefore, it is possible that, while the collagen matrix supports the initial adhesion of keratinocyte stem cells expressing high levels of α2β1 integrin, fibronectin promotes cell proliferation through α5β1 and αvβ6 integrins after initial adhesion when expression of these fibronectin receptors is induced. Unlike the protocols described by Bickenbach and Chism [4] and Hager et al. [5] who used type IV collagen-coated tissue culture plastic to promote cell adhesion during subculturing, we have been able to omit the coating of the culture plates after the first subculturing of the cells. This suggests that collagen-fibronectin matrix may be beneficial for the initial adhesion and growth of the keratinocyte stem cells but once selected and activated, the cells do not require these exogenous molecules for adhesion and growth. In the present study, the cells showed the typical keratinocyte cell morphology (Figure 1A) and this
morphology remained unchanged during subculturing (Figures 1B and 1C). To confirm the epithelial cell phenotype we analyzed the cell surface expression of αvβ6 integrin that is exclusively expressed by epithelial cells [11]. Immunocytochemical staining showed that the cell cultures expressed αvβ6 integrin and that it localized to the focal adhesions of migrating cells (Figure 1D). To compare the cell surface expression of αvβ6 integrin in the mouse keratinocytes with a human skin keratinocyte line (HaCaT; a spontaneously immortalized skin keratinocyte line that has retained the property to differentiate) the cells were immunocytochemically stained with a monoclonal antibody that recognizes both human and mouse αvβ6 integrin (10D5; Chemicon, Temecula, CA), and the level of integrin expression was analyzed by fluorescentactivated cell sorting (FACS) as we have described previously [18]. In a parallel experiment, we also compared the expression of αvβ6 integrin in normal human keratinocytes from newborn skin (NHEK; Clonetics, San Diego, CA) with HaCaT cells by using a monoclonal antibody against human αvβ6 integrin (E7P6; Chemicon, Temecula, CA). The results showed that all cell lines expressed comparable levels of αvβ6 integrin (Figures 2A, 2B, 2C) confirming the epithelial cell phenotype of mouse keratinocyte cultures. The expression of αvβ6 integrin in mouse keratinocyte cultures remained
Figure 1. Cell morphology (A–C) in mouse skin keratinocyte cultures from wild type FVB/NHsd mice after subculturing three times (A), six times (B), or 20 times (C). During increasing number of subculturing, morphology of keratinocytes remains unchanged. Cells were stained with crystal violet and photographed using a 20× objective. D: Immunocytochemical staining of αvβ6 integrin in the keratinocytes from wild type FVB/NHsd mice that have been subcultured twelve times. In migrating kerationocytes, αvβ6 integrin localizes at focal adhesions at the edge of the lamellar cytoplasm and in the filopodia (arrows). Cells were photographed by using a 63× objective. Representative samples of two parallel cell strains are shown.
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Figure 2. Comparison of cell surface expression of αvβ6 integrin in newborn human skin keratinocytes (NHEK) and HaCaT keratinocytes (A), and HaCaT keratinocytes and mouse skin keratinocytes (mKC) from wild type FVB/NHsd mice (B) by using fluorescence-activated cell sorting analysis. NHEK, HaCaT and mouse keratinocytes were assayed at P2, P19 and P6, respectively. A: Cells were reacted with a monoclonal antibody against human β6 integrin (E7P6; Chemicon) before incubation with the FITC-conjugated secondary antibody and FACS analysis. Result of a single experiment is shown. B: Cells were reacted with a monoclonal antibody recognizing both human and mouse β6 integrin (10D5 Chemicon) before incubation with the FITC-conjugated secondary antibody and FACS analysis. Results show mean ± SD of two experiments. C: Representative histograms of the HaCaT cell and mouse keratinocyte (mKC) FACS analyses from one of the two experiments shown in B. Control: cells were reacted with the FITC-conjugated secondary antibody only before FACS analysis; beta-6: cells were reacted with the antibody recognizing both human and mouse β6 integrin (10D5; Chemicon) followed by incubation with the FITC-conjugated secondary antibody before FACS analysis. Results showed that HaCaT cells and newborn human skin keratinocytes expressed comparable levels of the epithelial cell-specific integrin αvβ6 and that there was no statistically significant difference (Student's t-test) in the expression level of αvβ6 integrin between HaCaT and mouse skin keratincoytes.
unaltered between early (P6) and late (P24) passage (data not shown). In order to compare the growth of mouse keratinocytes at passage 5 and 26, we seeded equal numbers of cells in triplicate in a 96-well tissue culture plate in the complete keratinocyte growth medium and analyzed the number of viable cells after
18, 96 and 144 h by using a CellTiter96 cell proliferation assay (Promega, Madison, WI) as we have described previously [19]. The results showed that the cells at passage 6 and passage 26 proliferated at the same rate (Figure 3) indicating that extended subculturing did not affect the growth and viability of the cells. The ability of the keratinocytes to pro-
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Figure 3. Analysis of mouse keratinocyte growth after the cells were subcultured five (P5) and 26 (P26) times. For the experiment, equal number of keratinocytes from wild type FVB/NHsd mice were seeded in the complete mouse keratinocyte culture medium in triplicate in a 96-well culture plate and the number of viable cells was measured after 18, 96 and 144 h by using CellTiter96 cell proliferation assay (Promega). Results show mean ± SD of triplicate wells from three parallel experiments. There were no statistically significant differences (Student's t-test) in the relative numbers of cells at corresponding time points between cells at P26 as compared with P6.
cell culture medium [20]. In this system, mouse keratinocytes differentiated and formed epidermal tissue closely resembling that of normal mouse skin (Figures 4A and 4B). The epidermis in the organotypic culture, as in the normal skin, was composed of a basal cell layer and 2–3 suprabasal cell layers that showed the typical morphological characteristics of cell differentiation and epidermal stratification (Figures 4A and 4B). Interestingly, in the organotypic culture, mouse keratinocytes produced a thick keratin layer (Figure 4A). The protocol described in this paper provides a simplified and effective method to isolate and maintain in long term culture epidermal keratinocytes from newborn mouse skin. This method would be valuable to isolate and study keratinocytes from various transgenic mouse lines that are currently available.
Acknowledgements We thank Dr. Christopher Hildebrand for performing the organotypic culture of the mouse keratinocytes. This study was supported by CIHR (grant MOP12589) and Finnish Cultural Foundation.
Notes on suppliers liferate in culture for several successive subcultures suggests that they represent the keratinocyte stem cells. Epithelial stem cells have the ability to differentiate to form epidermal-like tissue in organotypic cultures [4, 7, 9]. Therefore, we studied the property of the mouse keratinocyte cultures to undergo terminal differentiation and to produce epidermal tissue by using an organotypic culture model where the cells were cultured on a cell-free connective tissue matrix at the air-liquid interface in a specified
01. BioWhittaker, Inc, Walkersville, 8830 Biggs Ford Road, MD 21793-0127, USA 02. Life Technologies, Rockville, 9800 Medical Center Drive, PO Box 6482, MD, USA 03. Sigma Chemical Co, PO Box 14508, St. Louis, MO 63178, USA 04. Falcon, Becton Dickinson, Franklin Lakes, 1 Becton Drive, NJ 07417-1886, USA 05. Bio-Rad Laboratories, Hercules, 2000 Alfred Nobel Drive, CA 94547, USA 06. JT. Baker, Phillipsburg, 222 Red School Lane, NJ 08865, USA
Figure 4. Hematoxylin and eosin staining of an organotypic culture of mouse keratinocytes from wild type FVB/NHsd mice (A) and a histological section from a skin biopsy from an adult wild type FVB/NHsd mouse (B). In the organotypic culture, mouse keratinocytes differentiated to form an epidermis composed of a basal cell layer (arrowheads) and 2–3 suprabasal cell layers which is histologically similar to mouse skin shown in B. Mouse keratinocytes also produced a thick keratin layer (K) in the organotypic culture. Result of a single experiment is shown. CT: connective tissue; E: epithelium. Histological sections were photographed by using a 20× objective.
196 07. PALL Corporation, Gelman, Labs, Ann Arbor, 600 S. Wagner Road, MI 48103-9019, USA 08. Cohesion, 2500 Faber Place, Palo Alto, CA 94303, USA 09. Chemicon International, Temecula, 28835 Single Oak Drive, CA 92590, USA 10. Spectrum, Rancho Dominguez, 18617 Broadwick Street, CA 90220-6435, USA 11. Nalge Nunc International, Rochester, 75 Panorama Creek Drive, PO Box 20365, NY 14602-0356, USA 12. Bard-Parker, Beckton Dickinson AccurateCare, Franklin Lakes, 1 Becton Drive, NJ 07417-1886, USA
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11. Breuss JM, Gallo, J. DeLisser HM, et al. (1995). Expression of the β6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci 108: 2241–2251. 12. Haapasalmi K, Zhang K, Tonnesen M, et al. (1996). Keratinocytes in human wounds express αvβ6 integrin. J Invest Dermatol 106: 42–48. 13. Häkkinen L, Hildenbrand HC, Berndt A, et al. (2000). Immunolocalization of tenascin-C, α9 integrin subunit and αvβ6 integrin during wound healing in human oral mucosa. J Histochem Cytochem 48: 985–998. 14. Huang X, Wu J, Spong S, et al. (1998) The integrin αvβ6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin. J Cell Sci 111: 2189–2195. 15. Agrez M, Chen A, Cone RI, et al. (1994). The αvβ6 integrin promotes proliferation of colon carcinoma cells through a unique region of the β6 cytoplasmic domain. J Cell Biol 127: 547–556. 16. Bata-Csorgo Z, Cooper KD, Ting KM, et al. (1998). Fibronectin and α5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis. J Clin Invest 101: 1509–1518. 17. Pivarcsi A, Szell M, Kemeny L, et al. (2001). Serum factors regulate the expression of the proliferationrelated genes α5 integrin and keratin 1, but not keratin 10, in HaCaT keratinocytes. Arch Dermatol Res 293: 206–213. 18. Koivisto L, Larjava K, Häkkinen L, et al. (1999). Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin. Cell Adhes Commun 7: 245–257. 19. Häkkinen L, Strassburger S, Kähäri VM, et al. (2000). A role for decorin in the structural organization of periodontal ligament. Lab Invest 12: 1869–1880. 20. Hildebrand HC, Häkkinen L, Wiebe C, et al. (in press). Characterization of organotypic keratinocyte cultures on de-epithelialized tongue mucosa. Histology & Histopathology.
Address for Correspondence: Dr. Lari Häkkinen, Laboratory of Periodontal Biology, Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, 2199 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 Phone: (Office) +604-822-0096, (Lab): +604-822-0744, Fax: +604-822-3562; E-mail:
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