Objectives: Large-scale expansion of epidermal keratinocytes is essential in the application of these cells for severe burn treatment in patients. Therefore, this study was designed to evaluate various conditions in the expansion of human epidermal keratinocytes.

Materials and Methods: The epidermis was separated from the dermis of skin samples using dispase. The epidermis was trypsinized for keratinocyte isolation. Keratinocytes were cultured in various conditions, with or without a human dermal fibroblast feeder layer, mitomycin C treatment, and different culture media.

Results: Our results suggest that keratinocytes cultured on a human dermal fibroblast feeder layer were grown for several passages. Extensive deformation and rapid deterioration were observed in the cultured cells without a feeder layer and in serum-free medium.

Conclusions: Human dermal fibroblasts treated with mitomycin C can provide optimal conditions for proliferation of keratinocytes.

Key words : Culture medium, Dermal fibroblasts, Feeder layer, Mitomycin C

Introduction

The epidermis is an excellent source of keratinocytes, and application of these cells is an invaluable strategy for skin engineering. Accessibility to appropriate keratinocytes is necessary for skin generation in the treatment of chronic ulcers and burn patients.^1-3 The culture of epidermal keratinocytes under incompatible conditions leads to differentiation and growth arrest at an early passage in vitro.^4,5 It has been possible to culture keratino­cytes on feeder layers or by methods using defined serum-free and low-calcium media.^5,6 To culture keratinocytes, xenotropic feeder layer cells were repeatedly used to improve cell survival, attachment, and proliferation.⁷

Extensive application of autologous keratinocytes in the fields of tissue engineering, burn treatment, and research necessitates successful cultivation of keratinocytes.^8,9 There is evidence that stimulation of fibroblasts to produce growth factors affects keratinocyte-fibroblast interactions, and this may lead to keratinocyte growth by paracrine signaling.¹⁰ Coculture of keratinocytes with a feeder layer of 3T3 cells has been discussed for application in the treatment of skin wounds.^11,12 The use of animal-derived feeder layers has disadvantages because of potential contamination with pathogens. Elimination of animal-derived materials in cell culture remains an important aim for epithelial remodeling.

Application of human feeder layers has been evaluated in a few studies.^13-16 Irradiated human dermal fibroblasts and mouse fibroblasts (3T3 cells) can improve the life span of human keratinocytes. Therefore, the present study was performed to evaluate various conditions such as culture medium and feeder layer use on keratinocyte morphology and proliferation in our cell culture laboratory.

Materials and Methods

Separation of epidermis and dermis from skin samples
The separation of skin layers and cell culture were performed as previously described with a few variations.¹⁷ All studies involving human subjects were approved by the ethics committee of Shiraz University of Medical Sciences in accordance with the ethical guidelines of the 1975 Declaration of Helsinki.

Twenty skin samples were collected from Plastic Surgery Department with informed consent, which were washed in 70% isopropanol. The skin was placed in Dulbecco phosphate buffered saline (DPBS) containing 20 μg/mL gentamicin for 1 hour. The skin was minced into small pieces and placed in 1.5 mg/mL dispase (Roche, Basel, Switzerland) in culture medium that contained 5 μg/mL gentamicin, overnight at 2ºC to 8ºC. The skin pieces were removed from the dispase solution, and the epidermis was separated from the dermis with fine forceps.

Isolation and culture of fibroblasts
The dermis was digested with 0.3% collagenase type I (Gibco/BRL, Grand Island, NY, USA) for 4 hours in a water bath at 37ºC. The cell suspension was centrifuged at 1200 rpm for 5 minutes. The cell pellet was rinsed twice with DPBS. The cells were cultured in F12-Dulbecco Modified Eagle Medium (F12:DMEM, 1:1) (Gibco/BRL) supplemented with 15% fetal bovine serum (FBS) and 1 μg/mL gentamicin. The cells were incubated at 37ºC in 5% carbon dioxide. The culture medium was replaced every 2 to 3 days.

Treatment of fibroblasts with mitomycin C as a feeder layer
The confluent fibroblasts were treated with mitomycin C for induction of the postmitotic state. To treat the cells, human dermal fibroblasts were incubated in DMEM containing 10% FBS and 8 μg/mL mitomycin C for 4 hours at 37ºC. The cells were rinsed 3 times with DPBS and replated in new culture flasks in F12-DMEM containing 10% FBS.

Isolation and culture of keratinocytes
The epidermal pieces were digested with 0.05% trypsin/ethylenediaminetetraacetic acid (Gibco/BRL) for 5 to 10 minutes at 37ºC. After cell dissociation, an equal volume of 1 mg/mL soybean trypsin inhibitor (Gibco/BRL) in DPBS was added to stop trypsin activity. The cell suspension was centrifuged at 1200 rpm for 5 minutes, and the pellet was washed with keratinocyte medium. The keratinocytes were counted using trypan blue and a hemocytometer and plated at a density of 4 to 8 × 10⁴ cells/cm². Keratinocytes were cultured in different conditions, with or without the human dermal fibroblast feeder cells in keratinocyte medium (Sigma-Aldrich, St. Louis, MO, USA) with or without FBS.

Keratinocyte freezing and thawing
Keratinocytes from primary cultures were frozen. The cells were counted and 1 × 10⁶ cells were suspended in cryopreservation medium containing 90% FBS and 10% dimethyl sulfoxide. Freezing was done with slow cooling.

For thawing, the cryovial was immersed in a water bath at 37ºC. The cell suspension was diluted with 5 mL keratinocyte medium and centrifuged. The cell pellet was counted and cultured in 3 different conditions including (1) culture on feeder cells in keratinocyte serum-free medium, (2) culture without feeder cells in keratinocyte serum-free medium, and (3) keratinocyte medium supp­lemented with 10% FBS.

Analysis of morphology and expansion of cells in various conditions
During the culture of keratinocytes and fibroblasts, microscopic evaluation was done to inspect the morphologic and proliferative changes.

Results

Fibroblasts at primary culture in F12-DMEM and 15% FBS started attaching to the culture surface on day 2 and reached confluence on day 7 (Figure 1). The keratinocytes that were attached to the culture surface were visible 3 days after primary seeding in all groups, and the mean keratinocyte size and morphology were similar for all groups.

The cultured keratinocytes in conditions without serum or a feeder layer showed limited proliferation, but supplementing the medium with 10% FBS led to increased proliferation rate. In the condition without serum and feeder layer, only 3 of 10 cases reached confluence. Keratinocytes in serum-free and serum-supplemented medium showed deformation with culture continuation (Figure 2).

The keratinocytes were cultured on feeder layer cells treated with mitomycin C with or without serum (Figure 3). With these conditions, colonies showed typical morphology of epithelial cells. In the application of serum-supplemented medium, the proliferation rate of cells was increased. Culture of keratinocytes after cryopreservation showed that growth of cells with normal morphology was possible on feeder cells in serum-free medium (Figure 4).

Discussion

Cultivation of human epidermal keratinocytes is an important method for skin engineering. Although keratinocytes can experience few passages, they provide an important source of epithelial cells with high proliferation rate and efficacy for many biological studies and wound therapies.¹⁸ It is important to obtain expansion of keratinocytes on a large scale and avoid the differentiation of these cells for clinical and experimental applications.

Culture of human keratinocytes from the epidermis has been evaluated by several researchers.^19,20 The key to obtaining suitable keratinocytes is optimization of the culture system to support keratinocyte growth. We compared various conditions such as the presence of a feeder layer and type of medium on the growth of these cells. We also evaluated the growth of keratinocytes after cryopreservation in different conditions. We reported that application of feeder layer cells is effective for keratinocyte culture. Using serum in the culture medium led to increased proliferation rate, but with prolonged application of this medium, keratinocytes changed morphology. Keratinocytes showed normal size and morphology in conditions without serum or feeder layer cells, but growth rate was decreased and the cells had extensive apoptosis. Keratinocytes usually are cultured for 3 to 4 passages for clinical applications.¹⁹ We could culture these cells up to 3 passages. When the keratinocytes could be cultivated after cryopreservation, it was evident that growth of cells with normal morphology was possible on feeder cells in serum-free medium.

It was reported that the best method to obtain adequate numbers of skin keratinocytes for clinical applications is to use a fibroblast feeder layer. Epidermal-dermal interactions by soluble factors may cause migration and proliferation of keratinocytes that leads to remodeling of wounded skin.^21,22 It is better to use a human than mouse fibroblast feeder layer because the risk of contamination with infections and animal antigens may be decreased with the human feeder layer. Detachment of 3T3 cells from the culture surface occurs after 7 to 10 days, but the human feeder layer remains constant up to several weeks after cessation of proliferation.²³

Irradiated human dermal fibroblasts provide a suitable human feeder layer that has similar efficiency as mouse fibroblasts for effective expansion of keratinocytes in vitro, but keratinocytes grown without a feeder layer may stop growth after a few passages.¹⁹ Researchers showed that coculture of follicular keratinocytes with dermal fibroblasts treated with mitomycin C supported the proliferation of keratinocytes. They noted that it is easier to handle human fibroblasts than 3T3 cells, and homologous fibroblasts better mimic the physiologic state.²³ In addition, cocultures of keratinocytes with murine 3T3 cells are considered xenogeneic products, but the use of autologous dermal fibroblasts as a feeder layer can be classified as an autologous product for clinical applications.²⁴

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