Skin explant cultures as a source of keratinocytes for cultivation

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1 DOI /s ORIGINAL PAPER Skin explant cultures as a source of keratinocytes for cultivation J. Dragúňová P. Kabát J. Koller Received: 3 July 2012 / Accepted: 13 July 2012 Ó Springer Science+Business Media B.V Abstract Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to reuse pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this passaging yields J. Dragúňová J. Koller Department of Burns and Reconstructive Surgery, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic P. Kabát (&) Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic virupepo@savba.sk P. Kabát Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization. Keywords Skin remnants Explant culture Keratinocytes Dermal fibroblasts Keratinocyte cultivation Keratinocyte sheets Introduction The technique for keratinocyte culturing has been known for over a century. In 1897, Ljunggren reported that fragments of human skin were able to persist in a living state in ascitic fluid at room temperature and subsequently it was possible to graft these explants to a human patient. Medawar (1941) later demonstrated that gentle enzymatic (trypsin) digestion of skin samples both liberated the dermis from the epidermis (along a natural splitting layer) and also disaggregated epidermal cells that could then be propagated and form multilayer sheets that were transplantable onto animals although such cultivation was slow and problematic. Rheinwald and Green (1975a, b) seeded suspensions of disaggregated keratinocytes on a layer of irradiated mouse fibroblast

2 3T3 feeder cells, with this co-culturing method resulting in prolonged proliferation and multilayering. From this advance, Green et al. (1979) were subsequently able to prepare stratified colonies from single keratinocytes that could fuse and form culture-grown epithelium. In noting that formerly uncontrolled fibroblast proliferation in keratinocyte cultures could be supressed using a lethally-irradiated mouse 3T3 fibroblast feeder layer, Leigh and Watt (1994) reported that this understanding of the complex relation of fibroblasts to epidermal cells in culture had opened the way for routine production of cultured epithelium derived from epidermal keratinocytes. Using Rheinwald and Green s feeder-dependent methodology, biologists were now able to prepare viable autologous keratinocyte sheets for application to human patients in treating ulcers, burns and other non-healing wounds (Myers et al. 1995; Smirnov et al. 2003; Moustafa et al. 2007; James et al. 2010; Wood et al. 2006). In 1983, a serum-free, low calcium culture medium for keratinocyte cultivation was developed that avoided the need for use of feeder cells and yet reduced contamination from other cells such as fibroblasts and melanocytes which could potentially overgrow keratinocyte cultures (Boyce and Ham 1983). There are advantages and disadvantages to both feeder-dependent and serum-free methodologies, and many laboratories including the CTB laboratory in Bratislava still opt for cultivation using 3T3 mouse cells or irradiated human fibroblasts (Mujaj et al. 2010; Sun et al. 2004). Feeder fibroblasts secrete extracellular matrix proteins and growth factors which encourage proliferation of keratinocytes and production of multilayer sheets (Navsaria et al. 1994). There remains a lingering problem, however, with cultivation of sufficient amounts of keratinocyte sheets for urgent use with patients. Skin samples available for cultivation are usually very small, especially in the case of heavily burned patients, the consequence of which is a small number of isolated keratinocytes and an unacceptably long time until multisheets are ready for clinical use. There are two possibilities for overcoming this problem: 1. Take an additional skin sample for cultivation This is far from optimal since it leads to repeated traumatisation of the patient. Proliferation of keratinocytes taken some time after an accident is also much slower; the cells take considerably longer to form colonies such that fewer sheets are obtained over an extended cultivation period. A second complication is possible microbiological contamination of the patient. Many hospital patients test positive for highly resistant types of bacteria and these infections mean that cultivation of cells is often unsuccessful. Contamination is not curable using antibiotics, or needs such a high concentration of these medicines that they are toxic for the cells. 2. Use skin remnants after enzymatic preparation of keratinocytes as an explant culture Initial attempts to culture disaggregated epidermal keratinocytes from skin explants as monolayers were limited by short viability and production of mixed cultures of keratinocytes, melanocytes, Langerhans cells, fibroblasts, Merkel cells, nerve cells and glandular cells (Cruickshank et al. 1960). However, explant techniques improved and skin explant cultures have since been used as a model for adult skin epidermal growth and behaviour (Halprin et al. 1979; Hammr 1981). Skin explant cultures have more recently been used to study the response of living skin to chronically implanted materials and devices (Peramo et al. 2009) and topically-applied chemical irritants found in cosmetics, medicines and food (Varani et al. 2007; Nakamura et al. 1990). For experimental studies, skin biopsies serve as a good source of keratinocytes, dermal fibroblasts and other cell types which can be isolated and propagated (Normand and Karasek 1995). After conventional enzymatic isolation of keratinocytes for cultivation, skin biopsy remnants still contain numerous residual aggregated keratinocytes. With heavily burned patients, it is essential to use every available cell for cultivation of keratinocyte sheets, and since 1996 we have made use of the remnants of skin biopsies as explant cultures to amplify the amount of keratinocytes available for transplantation. Materials and methods Preparation of the explant culture Skin biopsies were procured in the operating theatre using a dermatome. Immediately post-procurement, samples were placed in pre-labelled sterile transport bottles containing transport medium and securely sealed. The separation of keratinocytes from each skin biopsy, and subsequent cultivation, was performed according to previously described standard protocol (Fig. 1a; Dragúňová et al. 2011).

3 Fig. 1 a Preparation of the keratinocytes suspension. b Adding medium to an skin explant culture previously dried under laminar flow. c An explant culture ready for cultivation. d Skin explant culture in cultivation medium. e Dermal fibroblasts Following enzymatic treatment and separation of keratinocytes, skin biopsy remnants were placed in a tissue culture grade Petri dish with the epidermal layer presented uppermost (Fig. 1b). The number of Petri dishes seeded with an explant culture depended on the size of the original skin biopsy procured for cultivation. The minimum size of skin fragment used in an explant culture was 1 cm 2. Skin samples were allowed to dry for 20 min under laminar flow to immobilize them on the surface of the culture vessel before adding keratinocyte culture medium (Fig. 1c, d). The quantity of medium required depended on the size of the Petri dish (2 ml for dishes 6 cm in diameter and 5 7 ml growing from explant culture after 2 days of cultivation. f Only keratinocytes grows from this explant culture after 3 days of cultivation for those 10 cm in diameter). No 3T3 feeder cells were used. Dishes were placed in a 37 C CO 2 incubator (7.5 % CO 2 atmosphere) and medium was changed every 2 days during cultivation. The first cells started to migrate out of the explant and grow after 1 5 days. Passaging of keratinocytes growing from the explantate Once cells reached confluency on the surface of the Petri dish, the skin remnants were removed using sterile forceps and reserved to seed a second passage of the explant culture (see below).

4 Cells grown from the skin remnants were a mixture of both keratinocytes and contaminating dermal fibroblasts, making it necessary to separate the two. This was achieved using a two-step trypsinisation process exploiting the difference in time taken to achieve enzymatic separation of these two different cell types: Step 1 Removal of contaminating dermal fibroblasts Trypsin: EDTA solution (1 2 ml) was added to each confluent mixture of keratinocytes and fibroblasts for s, after which the solution was removed by pipette. This took away the bulk of enzymatically detached dermal fibroblasts while subsequent washing with PBS solution removed the majority of any remaining. The longer time needed for trypsinisation of keratinocytes ensured that these cells remained attached to the Petri dish during this short initial digestion. Step 2 Trypsinisation of keratinocytes Fresh Trypsin: EDTA solution (1 2 ml) was added to the keratinocytes for s, after which all but 0.5 ml of digest solution was removed. Incubation of the culture for 5 10 min at 37 C was sufficient to detach keratinocytes from the surface of the Petri dish, after which time 1 2 drops offoetal calf serum were added to stop the enzymatic reaction. Keratinocytes were resupended in 2 4 ml of cultivation medium by pipetting and then seeded on a feeder layer of 3T3 cells for sheet cultivation according to standard protocols (Dragúňová et al. 2011). To maximise the number of cells available for cultivating keratinocyte sheets, the original skin remnants were transfered to new Petri dishes, allowed to dry and cultivated again according to the method described above. This step could be repeated. Indeed, we found we were able to cultivate cells from a single remnant of skin 10 times and still obtain useful numbers of keratinocytes from each of these passages. Results Cells started to grow from the skin remnants 1 5 days after cultures were established (Figs. 1e, f, 2a, b, c, d, e, f), and within days a confluent cell monolayer was formed. (Fig. 3a). It varied whether keratinocytes (Figs. 1f, 2b) or dermal fibroblasts (Figs.1e, 2a) grew out over the surface of the culture vessel first, or if both types of cells started to grow at the same time (Fig. 2c, d). In some cases dermal fibroblasts started to grow from the same part of the skin explant as keratinocytes, whilst in others dermal fibroblasts and keratinocytes grew concurrently from different parts of the same skin explant (Fig. 2e, f). In all cases the population of cells comprised of a mixture of both keratinocytes and dermal fibroblasts. Once a monolayer had been achieved (Fig. 3a), the remnants of skin were transferred to a fresh Petri dish for subsequent rounds of cultivation and trypsinization from the culture vessel surface. Each dish yielded keratinocytes and suspensions of these cells were seeded in cultivation flasks containing mitomycin C-treated (i.e. proliferation inhibited) 3T3 feeder cells and cultivated according to Rheinwald and Green (1975) methodology (Fig. 3c, d). In this way, it was possible to obtain enough keratinocytes from a single Petri dish for 1 2 cultivation flasks (75 cm 2 ) for classical cultivation, while by seeding the rest of the skin into 2 10 Petri dishes (depending on the size of the skin sample) we could subsequently seed a further 4 20 flasks. Contaminating dermal fibroblasts from the first step of trypsinization could also be seeded in cultivation flasks for use in other experiments (Fig. 3b). Biopsy remnants could be transferred to a fresh culture vessel a number times. We managed 10 successful passages, each time obtaining more cells. We observed that the ratio of keratinocytes:dermal fibroblasts varied depending on the number of transfers. The first transfers resultedinamixedcultureconsisting of 90 % keratinocytes:10 % dermal fibroblasts. The percentage of keratinocytes decreased slowly with every transfer so that after 10 transfers only approximately 40 % of the resultant cell population consisted of keratinocytes. However, even a 40 % return is sufficient to initiate keratinocyte cultivation, and when minimal skin is available from a patient, this could be the difference between being able to generate sufficient sheets for transplantation and not being able to(figs. 3e, f, 4a). Keratinocytes isolated from explant cultures grew in culture with inhibited 3T3 cells in the same way as keratinocytes isolated from a skin biopsy, and cells were suitable for passaging to secondary and tertiary cultures and were able to form multilayer sheets for grafting (Fig. 3d). Discussion The use of an explant system as an additional source of keratinocytes has been used in our laboratory since

5 Fig. 2 a Only dermal fibroblasts growing paralelly from explant culture. b Keratinocytes growing from the explant culture after 7 days of cultivation. c Keratinocytes and dermal fibroblasts growing concurrently from the explant culture after 2 days of cultivation. d Keratinocytes and dermal fibroblasts growing concurrently from explant culture after 10 days of cultivation. e, f The dermal fibroblasts and keratinocytes growing from different areas of the same skin explant after 2 days of cultivation During this time we have cultivated keratinocytes from approximately 120 patients. In every case, keratinocyte cultivation from skin explants was successful. The amount of keratinocytes obtained using our methodology depended on the size of the original skin biopsy. After conventional enzymic isolation of the epidermis from the dermis and disaggregation of epidermal cells, remnants of skin were seeded in 2 10 Petri dishes. This translated to seeding of between 4 and 20 culture flasks for subsequent classical Rheinwald and Green cultivation on a 3T3 feeder layer. Each Petri dish (6 cm in diameter) typically yielded cells which corresponded closely with the findings of Guo and Jahoda (2009) who obtained cells from a 3 cm Petri dish. The cultivation period for keratinocytes was also the same in our system as that reported by these authors. The first cells started to grow from our biopsies after 1 5 days. As discussed previously (Dragúňová et al. 2011), cell growth rate was not dependent on the age of the donor, but rather on their metabolic state. Explant cultures always comprised a mixture of dermal fibroblasts and keratinocytes but it was variable whether dermal fibroblasts (Fig. 1e), keratinocytes (Figs. 1f, 2b) or a mixture of both cells (Fig. 2d) grew first. This contrasted with the findings of Guo and

6 Fig. 3 a Confluent keratinocytes and dermal fibroblasts from an explant culture: ready for passaging. b Dermal fibroblasts from an explant culture after isolation by two-step trypsinisation. c Keratinocytes from the explant culture growing in the Rheiwald Green system: normal growth of colonies. d Keratinocytes isolated from the explant culture and subsequently cultivated according to the method of Rheinwald and Green: forming of the colonies-semiconfluent layer. e Dermal fibroblasts and keratinocytes growing from an explant cuture after 4 transfers of the original skin to a fresh culture vessel. f Growing keratinocytes from explant culture after 8 transfers of the original skin to a fresh culture vessel Jahoda, who observed that keratinocytes were always the first to grow from the explant. We suggest that this difference is due to the thickness of the sample prepared for cultivation. Guo and Jahoda routinely used biopsy samples of 2 mm in thickness while adjustments to the dermatome used to procure our biopsies yielded samples of varying thickness. Differences in thickness across a single skin biopsy could explain the fact that we sometimes observed simultaneous growth of dermal fibroblasts and keratinocytes from different points on the same explantate (Fig. 2e, f). Taylor and colleagues showed that keratinocytes migrating from whole skin explants apparently underwent little cell division over the first few days in culture (Taylor et al. 1983). Electron microscopy showed that these cells came from the basal layer of the epidermis (Barrandon and Green 1987). Differing thickness of skin biopsies can thus influence the different type of cells growing from an explant. The decrease in keratinocyte

7 Fig. 4 An explant culture after 10 transfers of the original skin to a fresh culture vessel growth with advancing age of explant culture might be explained by the fact that the basal layer of the epidermis consists of two types of keratinocyte progenitors: keratinocyte stem cells (minor population with high proliferative potential) and transit amplifying cells which display limited proliferation capacity (Morris et al. 1985). Decreasing capacity to proliferate may have led to the reduced number of keratinocytes in the highest passages of our explants. Using the methodology described above, we were able to remove the majority of contaminating dermal fibroblasts by two-step trypsinization. However, it is probable that even after the two-step process a small number of residual dermal fibroblasts were present as contaminants in our keratinocyte cultures. These remaining fibroblasts were inhibited by the 3T3 feeder cells in subsequent cultivation and therefore did not cause us any problems during routine growth of keratinocyte sheets (Fig. 3c, d). We did not experience any instances of dermal fibroblasts overgrowing keratinocytes that we had obtained using the explant culture method. Explant culture-derived keratinocytes were able to grow in the same way as keratinocytes obtained by enzymatic isolation from a primary skin biopsy when transferred to a 3T3 feeder layer. They could be passaged 2 3 times and formed multilayers suitable for harvesting and application to a patient. This is in agreement with the findings of Guo and Jahoda (2009). Keratinocytes obtained from skin biopsies in their system were able to grow successfully in serum free medium. Successful establishment of explant cultures from remnants of skin biopsies is very important since second biopsies from patients hospitalized for a long time tend not to be suitable for cultivation. Firstly, there is the issue of hospital-acquired multi-resistant bacterial infections (Acinetobacter, Staphylococcus, Proteus etc.) which may cover the skin of the patient and be incurable with antibiotics. It is often impossible to cultivate keratinocytes from such a skin sample because resistant bacteria are able to overgrow the cell culture. Antibiotics used to remove such contaminations need to be applied in such a high dose that they are toxic to the keratinocytes. Secondly, patients who have been hospitalized for sustained periods will typically be of metabolically sub-optimal status. Their system will be affected negatively by shock and longterm medication, and this translates into a far-fromideal metabolic status of individual cells. This combination of bacterial contamination and poor metabolic condition means that keratinocytes isolated from such skin biopsies will not grow and divide optimally, making cultivation problematic. Explant cultures can thus serve as an additional source of cells for preparation of sufficient quantities of transplantable sheets without needing to return to the patient. Summary Why use skin explant cultures? Explant cell culture has proved to be a very useful method for obtaining more cells for cultivation. This technique: 1. Makes use of tissue remnants that might normally be discarded after initial enzymatic digestion 2. Avoids the need for repeated biopsies from already-traumatised patients who present a variety of confounding factors (i.e. lack of available skin in cases of substantial burning, high risk of resistant bacterial contamination caused by longterm hospitalisation, and poor metabolic status of individual cells) 3. Addresses the issue of not being able to obtain sufficient amounts of cells for cultivation (biopsy for limbal stem cells) and instances where passaging of cells can lead to a change in phenotype (e.g. limbal cells and chondrocytes). 4. Significantly decreases the time needed to obtain sufficient amounts of cultured cells for clinical application.

8 We suggest that explant cultures can serve as an additional source of cells for any laboratories involved in the cultivation of keratinocytes, as well as limbal stem cells, chondrocytes and osteoblasts. Acknowledgments The authors thank Joanne Martin for critical comments on an early version of the manuscript. References Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84(8): Boyce ST, Ham RG (1983) Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol 81(1 Suppl):33 40 Cruickshank CN, Cooper JH, Hooper C (1960) The cultivation of cells from adult epidermis. J Invest Dermatol 34: Dragúňová J, Kabát P, Koller J, Jarabinská V (2011) Experience gained during the long term cultivation of keratinocytes for treatment of burns patient. Cell Tissue Bank. doi: /s z Green H, Kehinde O, Thomas J (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 76(11): Guo A, Jahoda CA (2009) An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor cells. Exp Dermatol 18(8): Halprin KM, Lueder M, Fusenig NE (1979) Growth and differentiation of postembyonic mouse epidermal cells in explant culture. J Invest Dermatol 72(2):88 98 Hammr H (1981) Stimulated mouse ear epidermis in explant culture the effect of retinoic acid and hexadexane. Arch Dermatol Res 270(4): James SE, Booth S, Dheansa B, Mann DJ, Reid MJ, Shevchenko RV, Gilbert PM (2010) Strayed cultured autologous keratinocytes used alone or in combination with meshed autografts to accelerate wound closure in difficult-to-heal burn patients. Burns 36(3):10 20 Leigh I, Watt FM (1994) Keratinocyte methods. Cambridge University Press, Cambridge Ljunggren CA (1897) Von Fahigkeit des Hautepithels ausserhalb des Organismus sein Leben zu behalten mit Beruchsichtigung der Transplantation. Deutsche Zietschrift fur Chirurgie 47: Medawar PB (1941) Sheets of pure epidermal epithelium from human skin. Nature 148:783 Morris RJ, Fischer SM, Slaga TJ (1985) Evidence that the centrally and peripherally located cells in the murine epidermal proliferative unit are two distinct cell populations. J Invest Dermatol 84(4): Moustafa M, Bullock AJ, Creagh FM, Heller S, Jeffcoate W, Game F, Amery C, Tesfaye S, Ince Z, Haddow DB, Mac- Neil S (2007) Randomized controlled, single blind study of use of autologous keratinocytes on a transfer dressing to treat nonhealing diabetic ulcers. Regen Med 2(6): Mujaj S, Manton K, Upton Z, Richards S (2010) Serum-free primary human fibroblasts and keratinocyte coculture. Tissue Eng Part A 16(4): Myers S, Navsaria H, Sanders R, Green C, Leigh I (1995) Transplantation of keratinocytes in the treatment of wounds. Am J Surgery 170(1):75 83 Nakamura M, Rikimaru T, Yano T, Moore G, Pula PJ, Schofield BH, Dannenberg AM Jr (1990) Full-thickness human skin explants for testing the toxicity of topically applied chemicals. J Invest Dermatol 95(3): Navsaria HA, Sexton C, Bouvard V, Leigh IM, Watt FM (1994) Human epidermal keratinocytes. In: Leigh IM, Watt FM (eds) Keratinocyte methods. Cambridge University Press, Cambridge, pp 5 12 Normand J, Karasek MA (1995) A method for the isolation and serial propagation of keratinocytes, endothelial cells and fibroblasts from a single punch biopsy of human skin. In Vitro Cell Dev Biol Anim 31(6): Peramo A, Marcelo CL, Goldstein SA, Matrin DC (2009) Novel organotypic cultures of human skin explants with an implant-tissue biomaterial interface. Ann Biochem Eng 37(2): Rheinwald JG, Green H (1975a) Formation of a keratinizing epithelium in culture by a cloned cells line derived from a teratoma. Cell 6(3): Rheinwald JC, Green H (1975b) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6(3): Smirnov SV, Kiselev IV, Rogovaya OS, Vasilev AV, Terskikh VV (2003) Skin repair by transplantation of cultured keratinocytes. Bulleten Experimentalnoj Biologii i Meditsiny 135(6): Sun T, Higham M, Layton C, Haycock J, Short R, MacNeil S (2004) Developments in xenobiotic-free culture of human keratinocytes for clinical use. Wound Repair Regen 12(6): Taylor JR, Halprin KM, Levine V, Woodyard C (1983) Effects of methotrexate in vitro on epidermal proliferation. Br J Dermatol 108(1):45 61 Varani J, Perone P, Spahliger DM, Singer L, Diegel KL, Bobrowski WF, Dunstan R (2007) Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage. Toxicol Pathol 35: Wood FM, Kolybaba ML, Allen P (2006) The use of cultured epithelial autograft in the treatment of major burn wounds: eleven years of clinical experience. Burns 32(5):

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