Methods for the in vitro cultivation of cells from the tissues of salmonid fishes Public Deposited

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  • Methods examined for the preparation of cells from tissues of salmonid fishes for primary cultivation were the fragment (explant) and enzyme dispersion techniques. Both methods were employed for the cultivation of a variety of tissues from Pacific salmon, Oncorhynchus spp., and the steelhead trout, Salmo gairdneri gairdneri. A technique was devised whereby embryonic cells of these fishes were prepared in primary culture and maintained with periodic fluid changes and transfers over long periods of time. These preparations, referred to as stock cultures, were used as a cell source for much of the experimental work reported here. The optimum concentration of cell inoculum for primary culture of embryonic cells was found to be one to one and one-half million per ml of culture medium. The optimum concentration of cell inoculum for subcultures of the primary cultures was found to be 600,000 per ml of medium. Several enzymes were compared with trypsin for the preparation of primary cell cultures. All were able to cause separation of cells from the tissue mass; however, these test enzymes were found to be more toxic to the cells than trypsin. The first of a series of experiments to determine factors influencing cell growth involved the comparison of five tissue culture media. Eagle's minimum essential medium supplemented with 20 percent agamma calf serum was found to be more growth stimulatory for embryonic cells than any of the other preparations tested. The addition of 25 percent by volume of Eagle's basal medium with 20 percent agamma calf serum removed from an actively growing culture to 75 percent by volume of this same medium failed to stimulate more cell growth than did Eagle's basal medium prepared with all fresh components. There appeared to be no growth stimulatory factors for these cells present in the fluid of actively growing cultures. Antibiotics have been employed at concentrations as high as 1000 units of penicillin, 1000 micrograms of streptomycin and 100 units of Mycostatin per ml of medium for the control of microorganisms without any indication of toxicity. Cultures were routinely carried for long periods of time at concentrations of 100 units penicillin, 100 micrograms streptomycin and 25 units Mycostatin without obvious harmful effects. Vitamin B₁₂ was found to inhibit growth of steelhead trout embryonic cells at concentrations of 0.5, 1.0, and 2.0 mg per liter in Eagle's basal medium supplemented with 20 percent agamma calf serum. Oxaloacetic acid was also incorporated into this same medium at 2.5, 5.0, and 10.0 millimolar concentrations. This compound was found to inhibit the growth of coho salmon embryonic cells. The use of blood serum to supplement media was investigated. Homologous fish serum was toxic for the steelhead embryonic cell cultures tested. Horse serum failed to support growth of these same cells. Human and calf serum, with and without gamma globulin, were compared and the agamma calf serum was superior to the other three as a growth stimulant for fish cells. The growth of coho salmon embryonic cells was inhibited when incubated in an atmosphere containing concentrations of one and three percent CO₂. Steelhead trout embryonic cells were found to grow best at 18° and 23° C. Twenty-eight and 35° C. were both lethal for these cells. Cultures incubated at 4° and 13° C. failed to grow. Twenty-five degrees centigrade was believed to be inhibitory for these cultures but not lethal. Observations indicated that the pH of the medium used to culture chinook salmon embryonic cells increased in alkalinity shortly after planting, followed by a slow decrease requiring approximately 28 days to reach values near neutrality. Cells of hepatoma tissue excised from adult rainbow trout were cultured and studied during the course of this work. A number of cultures have been grown under conditions of continuous cultivation. Five of these preparations are still viable and two have exceeded one year in culture.
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