Determination of colony numbers in pig epidermis as an estimate for radiosensitivity. A rapid assay based on in vitro BrdU-labelling

Abstract

A rapid assay has been developed for the quantitation of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. The number of surviving clonogenic cells per unit area was related to the epidermal in vivo response of moist desquamation. After irradiation with single doses, ranging from 20 to 36 Gy, skin biopsies were taken and incubated in dispase for enzymatic separation of the epidermis and dermis. Full thickness epidermal sheets were labelled with bromodeoxyuridine (BrdU) in vitro. Proliferating cells were visualized using standard immunohistochemical procedures. Cell groups containing > or = 16 cells were counted as colonies. These colonies were first seen on day 14/15 after irradiation. The number of colonies per cm2, as a function of skin surface dose, yielded a cell survival curve with a D0 (+/- SE) of 3.87 +/- 0.57 Gy. The ED50 for the epidermal in vivo reaction of moist desquamation corresponded with a colony density of 2.7 colonies per cm2. After higher doses, abundant smaller colonies of 4-8 BrdU-positive cells were seen and these were more radioresistant, as represented by higher D0 values