| Co-Investigator(Kenkyū-buntansha) |
WATANABE Kazuto DOKKYO UNIV., SCH. MED., Dept. Physiol. Assist. Professor, 医学部, 助手 (80146167)
ENAMI Jumpei ZENYAKU KOGYO Co, LTD., Dept. Biotech. Res. Lab., Manager, 研究所バイオテク研究室, 室長 (30112634)
KOHMOTO Kaoru UNIV. TOKYO, FAC. AGR. Dept. Animal Breeding, Prof., 農学部, 教授 (30011894)
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| Research Abstract |
In this study, we developed the three-dimensional cell culture system for various types of cells using collagen gel matrix, and found that this culture system was better to maintain their physiological functions than monolayer culture system as shown by the following results.
1. To examine whether androgens directly stimulate the growth of prostate epithelial cells, a serum-free culture system was developed. The isolated mouse prostate epithelial cells were embedded in a collagen gel matrix and cultured a serum-free medium. Under these culture conditions fibroblastic growth was rare. Addition of EGF to this basal medium stimulated the growth of the epithelial cells. Further addition of dihydrotestosterone or testosterone further stimulated growth ; whereas under the monolayer cell culture conditions, androgens did not stimulate the epithelial growth.
2. The rat hepatocytes embedded in collagen gel matrix and in alginate-coated collagen gel beads maintained the secretion of albumin during
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long term period, and showed the elevation of liver specific enzyme activity including tyrosine aminotransferase. These phenomena were not observed in monolayer culture system. On the other hand, the abilities of DNA synthesis in collagen gel-embedded culture was similar to monolayer culture system, and showed the cell density-dependent inhibition of growth.
3. To grow nerve cells in suspension culture we have developed an alginate-coated collagen gel fiber culture method. Nerve cells from cfdck embryo dorsal root ganglia grew along the longitudinal axis of the fiber when the fiber width was narrow.
4. To establish the in vitro system of circadian pacemaker model in mammal, we applied the long term cell culture system of dissociated suprachiasmatic nucleus(SCN)neurons. Dispersed cells obtained from the SCN of 2 to 6 days Sprague-Dawley rats were plated on collagen-coated plastic dishes. Within 4 days after plating, cells formed three dimensional network structures and neural fibers were spread to connect each structure. The arginine vasopressin concentration in all of cultured dishes showed a clear free-running circadian rhythm with 23.5 hr mean period. While the dispersed S CN cells in collagen gel-embedded culture did not form such a network structure and showed low vasopressin secretion without circadian rhythmicity.
5. It is well known that the extremally fertilized mouse ova or one-cell-stage fertilized ova from mouse oviduct cannot continue development and that the majority of these ova stop at 2-cell-stage(2-cell block). To establish the extemal culture method for full development of fertilized ova, we applied various culture systems. The 93% of onecellrstage ovum developed to blastula stage when they were cultured inside the resected oviduct, ind the 46% of ova developed to the same stage on the incised oviduct. The only 13% of ova developed to blastula stage when they were cultured alone on collagen gel matrix, or in parallel culture widi oviduct.
From these results, it is concluded that the collagen gel-embedded culture provides good environment for various epithelial cells. To establish the culture conditions for SCN neuron and fertilized ovum, further examinations are needed. Less
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