Establishment of a Calcitonin–Producing Rat Medullary Thyroid Carcinoma Cell Line. I. Morphological Studies of the Tumor and Cells in Culture*

Abstract

The calcitonin (CT)-producing transplantable WAG–Rij rat medullary thyroid carcinoma (rMTC) provides a unique model for studies of C cell function. To establish a CTproducing cell line we used the method of alternate culture and animal passage. Primary monolayer cultures of a rMTC (MTC 6–23) were prepared by mincing tissue fragments in phosphate buffer, followed by collagenase digestion. Dissociated single cells were plated in Dulbecco 's Modified Eagle 's Medium (DMEM) supplemented with 15% horse serum and 2.5% fetal calf serum. Cells were maintained in primary culture for 4 weeks; thereafter, subcultures were made using 25% self–conditioned medium, 75% DMEM for the first two passages, and DMEM alone in subsequent subcultures. The resulting cell line (rMTC 6–23) has been serially subcultured more than 25 times over a period of 11 months. The cells grow with a population doubling time of 3–4 days. Cell and culture medium contents of immunoreactive CT were measured by a heterologous RIA employing antiserum to synthetic human CT which cross–reacted quantitatively with rat CT. Basal CT production and cell number increased in parallel during the log phase of cell proliferation, with 0.4–0.7 ng CT accumulating in culture medium per 10⁶ cells every 48 h. Immunoreactive CT was present in approximately 98% of rMTC 6– 23 cells in culture, as determined by immunoperoxidase staining. Ultrastructurally, the cells contained membrane-bound secretory granules which were similar to those observed in the original tumor in vivo. We conclude that rMTC 6–23 is an established cell line that produces CT and retains the morphological characteristics of neoplastic rat C cells.