The Development of the Methods for Mass Culture, Maintenance and Transplantation of Various Cells Using Extracellular Matrix

在本研究中，我们利用胶原凝胶基质开发了各种类型细胞的三维细胞培养体系，并发现该培养体系比单层培养体系更能维持细胞的生理功能，结果如下：1。为了研究雄激素是否直接刺激前列腺上皮细胞的生长，我们建立了无血清培养系统。将分离的小鼠前列腺上皮细胞包埋于胶原凝胶基质中，并在无血清培养基中培养。在这种培养条件下，成纤维细胞很少生长。在基础培养基中加入EGF可刺激上皮细胞的生长。进一步添加双氢睾酮或睾酮进一步刺激生长；而在单层细胞培养条件下，雄激素对上皮细胞的生长没有刺激作用。包埋于胶原凝胶基质和海藻酸包被胶原凝胶珠的大鼠肝细胞在更长时间内维持白蛋白的分泌，并表现出酪氨酸转氨酶等肝脏特异性酶活性的升高。这些现象在单层培养体系中没有观察到。另一方面，胶原凝胶包埋培养的DNA合成能力与单层培养系统相似，并表现出细胞密度依赖性的生长抑制作用。为了培养悬浮培养的神经细胞，我们建立了海藻酸包被胶原凝胶纤维培养方法。3 .当纤维宽度较窄时，胚背根神经节神经细胞沿纤维的纵轴方向生长。为了在体外建立哺乳动物昼夜节律起搏器模型，我们采用分离的视交叉上核（SCN）神经元长期细胞培养系统。从2 ~ 6天大的Sprague-Dawley大鼠的SCN中获得分散的细胞，将其置于胶原包被的塑料培养皿中。镀后4天内，细胞形成三维网络结构，神经纤维扩散连接各网络结构。所有培养皿精氨酸抗利尿素浓度均表现出明显的自由运行的昼夜节律，平均周期为23.5小时。而分散的S - CN细胞在胶原凝胶包埋培养中没有形成这样的网络结构，且抗利尿激素分泌低，无昼夜节律性。众所周知，极受精卵或来自小鼠输卵管的单细胞期受精卵不能继续发育，其中大多数卵在2细胞期停止发育（2细胞阻滞）。为了建立受精卵充分发育的体外培养方法，我们应用了多种培养体系。单细胞期卵子在切除的输卵管内发育为囊胚期的比例为93%，在切开的输卵管内发育为囊胚期的比例为46%。在胶原凝胶基质上单独培养或在输卵管内平行培养时，只有13%的卵子发育到囊胚期。由此可见，胶原凝胶包埋培养为各种上皮细胞提供了良好的生长环境。为了建立SCN神经元和受精卵的培养条件，还需要进一步的研究。少

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 … More 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