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Establishment of Pure Cell Cultures, Cytomorphologic Characteristics, Ultrastructure, Immunophenotype, Biosynthetic Activities, and Generation of Antisera

Sergei A. Grando, MD, PhD, DSc; Olivia M. V. Schofield, MBBS, MRCP; Amy P. N. Skubitz, PhD; David A. Kist; Brian D. Zelickson, MD; Christopher B. Zachary, MBBS, FRCP

Arch Dermatol. 1996;132(10):1185-1193.

Abstract
Background and Design
In this study we developed an in vitro model of nodular basal cell carcinoma (BCC). We obtained pure cultures of BCC cells and compared the morphologic characteristics, ultrastructure, immunophenotype, and behavior of cultured tumor cells with those of their in vivo counterparts. Tumors were excised from patients undergoing Mohs micrographic surgery. We established 69 primary cell cultures from 32 patients with nodular BCC.

Results
Three cell types grew in primary cultures: fibroblasts, normal-appearing keratinocytes, and cells with dual (spindle and epithelioid) morphologic characteristics. Contaminating fibroblasts were removed using 0.125% trypsin—0.02% edetic acid, and normalappearing keratinocytes were cornified and eliminated by temporarily increasing the concentration of calcium in the growth medium. The cells with dual morphologic characteristics remained intact and exhibited relentless growth in pure cultures. That these seemingly immortal cell strains represent true nodular BCC was demonstrated by (1) their biphasic morphologic characteristics and very slow cell growth rate, (2) their capability for anchorage-independent growth in soft agar, (3) their ultrastructural similarities to freshly excised nodular BCC, (4) their ability to generate antibodies selectively labeling nodular BCC tumor nests in vivo, and (5) their immunophenotypic similarities to BCC in vivo on more than 20 different cell markers.

Conclusions
This study provides a simple technique for establishing pure cell cultures of nodular BCC and describes extensively the in vitro parameters of tumor cell growth. The striking differences in behavior of cultured tumor cells in the presence or absence of normal-appearing keratinocytes suggest that normal human epidermal keratinocytes can suppress the growth of BCC cells.

Arch Dermatol. 1996;132:1185-1193

Author Affiliations
From the Departments of Dermatology (Drs Grando, Schofield, Zelickson, and Zachary and Mr Kist) and Laboratory Medicine and Pathology (Dr Skubitz), University of Minnesota Medical School, Minneapolis. Dr Grando is now with the Department of Dermatology, University of California at Davis, Sacramento.

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