IJicJ¢'hbnh'a e! 17i~physic.a Acta, 1133(Iqq2) 153 15q

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Coculture of newborn rat skin epidermal cells with Swiss 3T3 cells: effect of the phases of 3T3 cells on attachment, growth and

keratin synthesis of epidcrmal cells Kazuto Ohkura and Hiroshi Terada Facrtlry of Pharl~tacet~ticzrl Scle~we~. L',irersity ~( T~kI~'h+ma. ~ku.~hh+ul ~'lapan ~

(Rec•ivetl 27 Seplcmbcr It)till

Key ~,,ords" Skin; Epidermal cell: Cell zuhure: Cotlugel~

Swiss albino mouse 31'3 cells in vzrious states were inoculated onto one side of Millipore filters. The other side of the filter was then coated with type I collagen and inoculated with newborn rat skin epidermal cells. On cocnllure of these cells, the attachment, growth and keratin synthesis of epidermal cells were found to depend on the slate of the 3"1"3 cellS: 3"1"3 cells in the stationary phase of growth were the most effective, followed by those in the logarithmic growth phase, those in the lag phase and plasmolyzed fibroblasls being only slightly effective. The effects of 3T3 cells in different slates correlated well with their abilities tu synthesize type IV collagen, but not type I collagen: with an increase in type IV collagen synthesis by the 3"I'3 cells, attachment of epidermal cells to the cell support, and their growth and synthesis of keratins increased. This culture system is concluded to mimic conditions in skin in viva, an,l tkcrefore to be sttitable for studies on the effects of fibrobtasts on the growth of el~idermal cells.

Introduction In the skin, fibroblasts in the dermis are important for the maintenance and regulation of epidermal cell proliferation. In a cell culture system also, fibroblasts are n e c e s s a ~ for proliferation of epidermal cells, and without them, epidermal cells are not able to grow or to become keratinized. However, on eoeulture of epidermal cells with flbroblasts, the epidermal cells are lost due to over-growth of the fibroblasts. Thus, mouse fibroblast 3T3 cells that have lost the ability to grow by ray-irradiation are usually used as a feeder layer [I]. However, in ~ueh culture systems, the mechanisms regulating growth and differentiation of epidermal cells by fibroblasts cannot be clearly determined, As a first step ia understanding the role of fibroblasts, in this study we examined the effect of the state of intact fibroblasts on proliferation of skin epidermal cells; i.e,, their viability, growth, attachment to a. sup-

Correspondence: H. Terada. Faculty of Pharmaceutical Sciences. Universityof Tokushima.Shomachi-I,Tokushima770, Japan.

porting matrix and synthesis of keratins. We reported previousty that epidermal cells from newborn rat skin attach much more effectively to Millipore filters coated with type IV collagen than those coated with type I collagen, and that addition of ascurbie acid increases their attachment to type I collagen to their level of attachment to type [V co[lagen by enhancing their synthesis of type IV collagen [2]. Type IV collagen is thought to facilitate attachment of skin epidermal cells tO each other as well as to cell matrices. Thus, it was of interest to examine the effect of type IV collagen synthesis by fibroblasts on the proliferation of skin epidermal cells+ Mouse fibroblasts such as Swiss 3T3 [3-5] and BALB 31"3 [6] cells are reported to produce type IV collagen as welt as entaetin, laminin, fibronectin and types I and I11 collagens, In this study, we examined the effect of the state of Swiss 3T3 cells on attachment and growth of epidermal cells in relation to type IV collagen synthesis by fibroblasts. We also examined its effect on keratin synthesis by epidermal ceils, because synthesis of keratin is regarded as the most imi:ortant factor for growth of sk:in epidermal cells. We used type ! collagen gel as a support for skin

154 epidermaI ceils, because its u,~e should faeilitate direction of improvement of cell atlachment, as it has lower affina.~ than type IV collagen for the cells. Materials and Methods

Reagems. The sources of the reagents used in this study wcre as folIows: Eagle's minimum esscntia[ medium (MEM), Flow Laboratories, McLean, U.K.; fetal bovine serum (FBS), Wittaker M.A. Bioproducts, Walkersville, MD, U.S.A,; dispase. Godo-Shusei Co., Tokyo, Japan; MillicelI-CM (]2 mm diameter), Millipore Products, Bedford, MA, U,S.A.; type ! collagen (bovine skin dermis), Kokcn, Tokyo, Japan; cellgrosserP, Sumilomo Pharmaceutical, Osaka, Japan; anti-human placenta type IV collagen serum (rabbit), Medac, Hamburg, Germany; horse-radish peroxidase conjugated anti-rabbit lgG fraction (goat), Cooper Biomedical, Malvem, PA, U,S.A.; anti-keratin serum, Biomcda, Foster City, CA, U.S.A.; fluorescein isothiocyanate conjugated anti-rabbit IgG (goat), Organon Tekunika, West Chester, PA, U.S.A. Swiss albino mouse 3T3 eells were kindly supplied by Drs. Ryaji Konishi and Takcru Fujii, Teikoku Seiyaku Co., Kagawa, Japan. Preparatioa of epiderntat cells ft~m newborn rat skht. Epidermal cells were prepared as described previously [2]. 3-day-old Wistar rats were decapitated, and then their whole skin was removed and cut into pieces of 5 × 5 mm z. These pieces of skin were digested first with dispase tiff00 U / m l ) at 4°C for 24 h and then with trypsin (0.25%)-EDTA (0.02%) at room temperature for 11] rain. 37"3 cell culture. Swiss 3T3 cells were suspended in Eagle's MEM buffered with 23.8 m'M sodium bicarbonate and 20 mM Hepes (pH 7.4) and supplemented with 10% FBS (Eagle's MEM-FBS) ~r serum-free complete synthetic medium cctgrosscr-P. A volume of 100 p,I of cell suspension at initial density of 1.8.10 ~ cells/ml was seeded onto one side of a 12 mm Millipure filler in a 100 mm dish (Falcon ltltl5) (Fig. IA). This side is termed the basal side hereafter. The cells were cultured at 37~C in a C O , incubator for 1, 5 and 9 days to obtain ceils in the lag phase, logarithmic growth phase and stationary phase, respectively. When the 3T3 cells were cultured in serum-free complete synthetic medium celgrosser-P for 4 days, they did not grow and became plasmolyzed. These eelIs were used as plasmolyzed 3T3 cells. Coath~g of MillipoJe filte~ with type I collagen. Type ! collagen dissolved at 3 m g / m l in 0.01 M acetic acid was diluted with Ca 2÷- and Mg2÷-free phosphatebuffered saline (PBS) (pH 7+4) to a final concentration of 0.18 (w/v)%, and applied at l(kq pA/well to the opposite side of the Millipore filters to the basal side. The filters were then replaced in the well with the collagen-coated surface exposed to the atmosphere and

A) Culture of Swiss 3T3 ceils 3T'd.cell suspension apie~~ido

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