Syrian hamster embryo (SHE) cell transformation assay with conditioned media (without X-ray irradiated feeder layer) using 2,4-diaminotoluene, 2,6-diaminotoluene and chloral hydrate

Abstract

The Syrian hamster embryo (SHE) cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The feeder layer of cells consists of X-ray irradiated cells which are still viable but unable to replicate. We have tried seeding the target cells in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer. Three SHE cell isolates were tested to investigate the feasibility of this approach. With freshly prepared conditioned medium (LeBoeuf's Dulbecco's Modified Eagle's Medium with 2 mM l-glutamine and 20% fetal bovine serum), used within 2 weeks of preparation, there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In each experiment, the positive control benzo( a)pyrene [B( a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without feeder cells. Three compounds, 2,4-diaminotoluene (2,4-DAT), 2,6-diaminotoluene (2,6-DAT), and chloral hydrate were tested in the SHE cell transformation assay without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable to those reported in the published literature using the standard methodology with feeder cells, with 2,4-DAT and chloral hydrate eliciting a significant increase in MTF, and 2,6-DAT not eliciting any increase in MTF. The results of this study demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and facilitating the scoring of morphologically transformed colonies.