E\p DvriiuiinI 1995: 4: I,S3-I9I Primed in Dcununk • All rii;hl.s rcscrvccl Copyrighi O Munk.sgaard 1995 Experimental Dermatology tSSN 0906-6705 Calcium: a crucial consideration in serum-free keratinoeyte culture Daniels J T, Harris I R, Kearney J N, Itigham E, Calcium: a crucial eonsideration in serum-tVee keratitiocyte culture. Exp Dermatol 1995; 4; 183-191. © Munksgaafd, 1995 Abstfact: This investigatioti was conducted when previously repeatable experimental data became impossible to reproduce when using keratino- cytes cultured iti serutni-ft'ce medium. Differenees in calcium tnolarity be- tween batches of medium were identified as a source of variation in cultured keratinoeyte populations. The susceptibility of cultured keratinocytes to even small alterations iti calcium molarity has been detnonstrated. 2 regular medium batches were compared with a special preparatioti of medium, devoid of calciutn chloride then suppletnented with a known coneen- tration of calciutn ions. Culture progress in each medium was assessed by: tnorphological observation, % cells expressing involucrin and prolif- erating eell nuclear antigen, cell attaehment, growth rate and colotiy forming efficiency. In order to control the phenotype of cultured kera- tinocytes, it! a repodueible system, it is recotnmended that serum-free keratinoeyte tnedium is purchased with the otnission of calciutn chloride. Supplementation of this tnediutn tnay then be tnade by the itivestigator to suit individual culture requirements. J. T. Daniels', I. R. Harris', J. N. Kearney' and E. Ingham^ ^Yorkshire Regional Tissue Bank, Pinderfields General Hospital, Aberford Road, Wakefield: ^Department of Microbiology, The University ot Leeds, Leeds, UK Key words: keratinoeyte - calcium - proliferation J. T Danieis, Yorkshire Regionai Tissue Bank, Pinderfields General Hospital, Aberford Road, Wakefieid, UK. Accepted tor publication 14 December 1994 Introduction The development of serum-free media has permit- ted the culture of keratinocytes under defined con- ditions (1-4), One of the major advantages is the ability to selectively culture and study proliferat- ing, migratory keratinoeyte populations, rather than terminally differentiating epithelial cells. In the epidermis, al the onset of terminal differen- tiation, basal cells are triggered to move towards the skin surface. Throughout their transit, basal cells undergo biochemical and morphological changes resulting in the formation of dead, flat- tened enucleated squames (5), The formation of an epithelium in vitro has been achieved (6), Keratinocytes cultured in low calcium (0,03 mM), serum-free medium do not undergo terminal differentiation. The colonies comprise large, flat cells which form a 'crazy paving' pattern. Under phase contrast microscopy, the keratinocytes have bright halos due to their loose apposition to one another. The cells towards to colony edge are ruffled (7) and take on an 'activated', migratory phenotype similar to keartinocytes in a wound situation. This activated state is thought to be due to the expression of a5 integrin subunits and the re-organisation of pi integrin subunits (8), This is in co'ntrast to the morphology of differentiating keratinoeyte cultures produced by the Rheinwald and Green technique (9), The colonies produced by the latter method have tightly apposed cells which do not tend to keep their halos. Until recently human keratinocytes had been successfully cultured in keratinoeyte serum-free medium (Gibco Life Technologies) in this labora- tory. The culture technique, source of skin samples and product suppliers had not been changed, yet the cultures produced had taken on a differentiat- ing appearance. Some of the other problems ex- perienced are described elsewhere (Daniels et al. in preparation). After surveying other users of the same medium, it became apparent that ours was not an isolated problem. Since an increase in cal- cium concentration in the medium could act as a trigger for differentiation, calcium molarity was measured by flame photometry and was found to be variable amongst medium batches. In co-oper- ation with this work, Gibco prepared a special or- der batch of medium without the addition of cal- cium chloride dihydrate, Tn this investigation kera- 183