Dhurjati Ravi, Liu Xiaomei, Gay Carol V, Mastro Andrea M, Vogler Erwin A
Department of Materials Science and Engineering, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Tissue Eng. 2006 Nov;12(11):3045-54. doi: 10.1089/ten.2006.12.3045.
A specialized bioreactor is used to grow mineralizing, collagenous tissue up to 150 microm thick from an inoculum of isolated murine (mouse calvaria MC3T3-E1, American Type Culture Collection (ATCC) CRL-2593) or human (hFOB 1.19 ATCC CRL-11372) fetal osteoblasts over uninterrupted culture periods longer than 120 days (4 months). Proliferation and phenotypic progression of an osteogenic-cell monolayer into a tissue consisting of 6 or more cell layers of mature osteoblasts in the bioreactor was compared with cell performance in conventional tissue-culture polystyrene (TCPS) controls. Cells in the bioreactor basically matched results obtained in TCPS over a 15-day culture interval, but loss of insoluble extracellular matrix and an approximate doubling of apoptosis rates in TCPS after 30 days indicated that progressive instability of cultures maintained in TCPS with periodic refeeding but without subculture. In contrast, stable cultures were maintained in the bioreactor for more than 120 days, suggesting that extended-term tissue maintenance is feasible with little or no special technique. Transmission electron microscopy ultramorphology of tissue derived from hFOB 1.19 recovered from the bioreactor after only 15 days of culture showed evidence of osteocytic-like processes and gap junctions between cells like those observed in vivo, in addition to elaboration of the usual osteoblastic markers such as alkaline phosphatase activity and mineralization (alizarin red). Thus, the bioreactor design based on the principle of simultaneous growth and dialysis was shown to create an extraordinarily stable peri-cellular environment that better simulates the in vivo condition than conventional tissue culture. The bioreactor shows promise as a tool for the in vitro study of osteogenesis and osteopathology.
一种专门的生物反应器用于从分离的小鼠（小鼠颅骨MC3T3-E1，美国典型培养物保藏中心（ATCC）CRL-2593）或人（hFOB 1.19，ATCC CRL-11372）胎儿成骨细胞接种物中培养矿化的胶原组织，厚度可达150微米，培养周期超过120天（4个月）且不间断。将生物反应器中成骨细胞单层增殖并向由6层或更多层成熟成骨细胞组成的组织的表型进展与传统组织培养聚苯乙烯（TCPS）对照中的细胞性能进行比较。生物反应器中的细胞在15天的培养间隔内基本与TCPS中获得的结果相符，但30天后TCPS中不溶性细胞外基质的丧失和凋亡率近似翻倍，表明在定期换液但不进行传代培养的情况下，TCPS中维持的培养物逐渐不稳定。相比之下，生物反应器中可维持稳定培养超过120天，这表明无需特殊技术或只需极少特殊技术即可实现长期组织维持。仅培养15天后从生物反应器中回收的源自hFOB 1.19的组织的透射电子显微镜超微结构显示，除了具有碱性磷酸酶活性和矿化（茜素红）等常见成骨细胞标志物外，还存在骨细胞样突起和细胞间间隙连接的证据，类似于在体内观察到的情况。因此，基于同步生长和透析原理设计的生物反应器被证明能创造出一种极其稳定的细胞周围环境，比传统组织培养能更好地模拟体内条件。该生物反应器有望成为骨生成和骨病理学体外研究的工具。
