The development of in vitro techniques to facilitate the study of hemopoiesis in the rainbow trout, Oncorhynchus mykiss

Abstract

As a first step in improving knowledge of factors controlling and influencing blood cell formation in fish, three general types of cell cultures have been developed as tools to study hemopoiesis in rainbow trout. The first of these is long-term hemopoietic cultures from the major hemopoietic organs, the spleen and head kidney. These cultures are initiated from spleen or kidney tissue and form a complex stromal layer of cells on the tissue culture surface. Like mammalian long-term bone-marrow cultures, these cultures produce numerous types of progeny cells for a period of several months, without the addition of exogenous growth factors other than serum. The products are difficult to characterize due to the lack of definitive markers for fish leukocytes, but the primary products of spleen cultures seem to be macrophages and dendritic cells, while the kidney produces a broader array of cells, including macrophages, granulocytes and occasionally lymphocytes. The dendritic cell progeny of spleen cultures are morphologically similar to dendritic cells found in mammals, and exhibit similar patterns of movement.

From these spleen cultures, two cell lines of different types have been developed. The first of these, RTS11, is representative of progeny cells, and is a non-adherent cell line consisting mainly of small, round cells with a small percentage of larger, granular, macrophage-like cells. The round cells appear to be an earlier stage of monocyte/macrophage development. They respond with increased growth to several crude extracts of rainbow trout origin, including PHA-LCM and cell-line conditioned medium, but not to cell-line conditioned medium of mammalian cells known to secrete cytokines influencing mammalian hemopoiesis. The second cell line, RTS34st, is representative of the stromal cell layer found in long-term hemopoietic cultures. It is made up of fibroblastic and epithelial cells, and is able to provide a hemopoietic inductive microenvironment (HIM) capable of supporting in vitro hemopoiesis. When suspensions of head kidney leukocytes or RTS11 cells are added, they adhere selectively to the fibroblastic stromal cell population, where they form proliferative foci, increase in number, and release non-adherent cells into the medium.

Both of the cell lines developed produce conditioned medium that stimulates ^3H-thymidine incorporation by freshly isolated rainbow trout leukocytes. Their response to fish extracts and production of factors stimulating trout leukocyte proliferation suggests that fish may respond to fish specific factors. These cell lines may thus be a potential source of novel fish cytokines or growth factors.

Finally, cultures in semi-solid media were initiated with isolated head kidney leukocytes. Of four semi-solid media tested, colony formation was best in 1.05% methylcellulose. Colony formation was greatly stimulated by rainbow trout serum, which promoted the growth of macrophage-like cell colonies, suggesting that trout serum may also contain growth factors stimulating hemopoiesis. The methylcellulose colony assay developed is potentially useful in scoring the types and numbers of cells formed in response to growth factors and cytokines, and will be useful in screening the activity of crude extracts to help identify important modulators of hemopoiesis in rainbow trout.