Development of matrix-culture system for osteoblastic cells using active-form ascorbic acid

成骨是一个包含多种细胞的复杂过程，这些细胞与生长因子或分化因子相互作用。体外细胞培养系统是研究这一过程的有用工具。我们试图建立一种大规模的成骨细胞样细胞培养体系。以小鼠成骨样细胞克隆株MC3T3-E1为材料。作为初步研究，该细胞系在常规塑料培养皿中培养。细胞增殖，呈多层堆积，形成结节，最后钙化。活性抗坏血酸用于维持细胞在血清缺乏条件下的生长。细胞培养在直径0.5~1.0 mm的胶原凝胶球形小球上。细胞在含有4种交联剂的胶原凝胶上培养，细胞增殖覆盖整个珠体表面，最多形成7层细胞。在这些试剂中，六亚甲基二异氰酸酯(HMDIC)和长链环氧剂被发现是制备无收缩凝胶用于培养的有效方法。细胞在凝胶上增殖并钙化。第三种方法是将细胞包埋在胶原凝胶中进行培养。在此条件下，细胞增殖并表现出碱性磷酸酶活性增强，这是成骨细胞的特征。当骨的鸟嘌呤/盐酸提取物，即粗骨形成蛋白，加入到这个系统中时，细胞的碱性磷酸酶活性显著增加。

Osteogenesis is a complex process containing several kinds of cells, which interact each other with growth factors or differentiation factors. In vitro cell culture system is a useful tool for the research on this process. We attempted to establish a large-scale culture system of osteoblast-like cells. A clonal oste*oblast-like cell line (of mouse), MC3T3-E1, was used as a material. As a preliminary study, the cell line was cultured on convensional plastic dishes. The cells proliferated and were stacked in mult-layers, then, formed noduli and finally calcified. The active-form ascorbate was usefui in sustaining cell growth under serum dificient condition.The cells were cultured on spherical beads of collagen gel having a diameter of 0.5-1.0 mm. The cells proliferated and covered the entire surface of the beads forming at most 7 cellular layers.The cells were cultured on collagen gels cross-linked with 4 cross-linking agents. Among these agents, hexamethylenediisocyanate ( HMDIC ) and a long chain epoxy agent were found to be effective to prepare uncontracted gels for culture. The cells pro-liferated and were calcified on the gels.The third method was to culture cells embeded in collagen gels. Under this condition, the cells proliferated and exhibited to increase alkaline phosphatase activity, which is a characteristics of osteoblastic cells. When guanidin/ HCL extracts of bone, crude bone morphogenetic proteins, were incorporated in this system, the cells responded with significant increase in alkaline phosphatase activity.