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We cultured single cell suspensions derived from enzymatically digested rat and mouse brains in the serum-free, hormone-supplemented media originally established for culturing glioma (C6) and neuroblastoma cell lines (B104). We succeeded in selecting different cell types by varying the hormones added to the culture medium. In addition to the medium the attachment factors used for coating the culture dishes proved important for the establishment of a selective culture system. In a comparative morphological study we show that different particular microenvironmental conditions determine the growth and/or survival of different cell types of dissociated brain cells.

The hypothesis that the growth of mammalian cells is regulated by hormones is now supported by considerable evidence. Two rat pituitary cell lines, GH3 and GC, a mouse melanoma, M2R (B16), and a human cervical carcinoma cell, HeLa S-3, have been grown indefinitely in serum-free (SF) hormone-supplemented medium. No visible changes of growth characteristics were observed in the cells grown continuously in the SF condition. However, changes in the activity of a plasma membrane enzyme, alkaline phosphatase, and in the relative intensity of surface proteins that are labeled by the [125I] lactoperoxidase technique were found in HeLa cells grown in the SF condition. To study the role of hormones required in the regulation of cell growth, HeLa cells were grown in the absence of one of the required hormones. The following results were obtained. Epidermal growth factor is probably involved in the regulation of the synthesis of macromolecules such as RNA and of the protein content per cell. Transferrin, the accessory factor in the SF condition, supplies iron for cells. The two basic peptides in this SF system, fibroblast growth factor and insulin, are probably involved in the balance of nutrients and energy inside the cell. The replacement of F12 medium with a better-balanced medium, MCDB 105, can mimic the requirements for these two peptides. The steroid hydrocortisone (HC) is probably involved in alteration of the cell surface. This is indicated by the effects of HC on cell morphology, rate of detachment from the dish, and the pattern of [125I] lactoperoxidase labeling of surface proteins. In addition, it is necessary to change the medium more frequently to maintain the culture in the medium without HC. This observation suggests that HC may be involved in the control of homeostatic properties of the cell surface. The production of rat prolactin by GH3 cells was also studied. GH3 cells in the SF condition produce 1.6 microgram prolactin per 10(5) cells in 24 h, while 2.4 microgram is produced in the presence of serum. Prolactin production in the SF condition is enhanced by the presence of thyrotropin-releasing hormone and inhibited by triiodothyronine (T3). T3 is the major growth factor for these cells. Without it cell growth is severely limited, while prolactin production is elevated. This result suggests that the GH3 cell line in the SF condition may be an ideal system for the study of hormonal regulation of cell growth and specific gene expression.

A number of hormones and factors were found to stimulate growth of cultured rat islet tumor cells, the RIN-r cell line. A serum-free supplemented medium from RIN-r cells was formulated. It consisted of a 1:1 mixture of Ham's F12 and DME media with the addition of insulin, transferrin, triiodothyronine, prolactin, growth hormone, and an extract of proteose peptone (medium IM). The growth rate of RIN-r cells in this medium is as great as it is in 10% serum-supplemented medium. Up to 10 populations doublings occurred over a period of 20 days. Insulin is a very effective mitogen for RIN-r cells and has an effect on concentrations as low as 30 ng/ml. In addition, the insulin-like somatomedin, multiplication stimulating activity (MSA), is a growth factor at 50 ng/ml. It was found that RIN-r cells proliferate and continue to produce immunoreactive insulin in a hormonally and nutritionally defined medium. This medium is derived from medium IM, in which insulin is replaced with MSA and proteose peptone is omitted. Variations of this medium may prove useful in studies on the growth and function of the normal islets in long-term primary culture.

The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [3H]lathosterol to [3H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesterone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells.

Madin Darby canine kidney cells can grow in synthetic medium supplemented with 5 factors--insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine--as a serum substitute. These 5 factors permit growth for one month in the absence of serum, and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl cAMP substitutes for prostaglandin E1 in the medium, suggesting that increased growth of Maden Darby canine kidney cells results from increased intracellular cAMP. Potential applications of the serum-free medium are discussed. The medium permits the selective growth of primary epithelial cell cultures in teh absence of fibroblast overgrowth, and a defined analysis of the mechanisms by which hormones regulate hemicyst formation.