Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: Effects on in vitro and in vivo osteochondrogenesis
Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: Effects on in vitro and in vivo osteochondrogenesis
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DOI：
10.1002/jcp.1081
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发表时间：
2001-06-01
影响因子：
5.6
通讯作者：
Caplan, AI
中科院分区：
文献类型：
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作者：
Lennon, DP;Edmison, JM;Caplan, AI
大鼠骨髓间充质干细胞(RMSCs)是骨髓间质中的一小部分细胞，具有向骨、软骨、脂肪和纤维组织分化的潜能。这些间充质祖细胞作为原代分离细胞和传代细胞保存在单独的封闭式模块化培养箱中，分别用95%的空气和5%的二氧化碳(20%或对照氧气)或5%的氧气、5%的二氧化碳和90%的氮气(5%或低氧)净化。在第一代，将来自每种氧气条件的一些细胞加载到多孔陶瓷载体中，并在体内实验中植入同基因宿主动物体内进行骨软骨形成的实验。其余的细胞在体外继续保持与原代培养相同的氧分压或切换到另一种条件。对第一代细胞进行体外成骨实验，包括碱性磷酸酶活性、钙和DNA含量的测定，以及von Kossa染色检测矿化情况。保持在低氧中的培养物有更多的菌落作为初代分离物，并且在整个体外时间内增殖更快，如初代培养结束时的血细胞计数和第一代细胞的DNA值增加所表明的那样。此外，在5%氧气中培养的rMSCs比在20%氧气中培养的细胞产生更多的骨，当采集并加载到多孔陶瓷立方体中并植入同基因宿主动物中时。最后，成骨的标志物，包括碱性磷酸酶活性、钙含量和von Kossa染色，在整个培养过程中一直处于低氧环境中的培养物中升高。这些标记物的表达通常在第一代细胞从对照切换到低氧时高于基础水平，而在细胞从低氧切换到对照时表达降低。我们得出结论，培养中的rMSCs在还原氧气的气氛中发挥最佳功能，这种气氛更接近于体内记录的氧分压。J.细胞。物理。187：345-355,2001。(C)2001年Wiley-Liss，Inc.
Rat mesenchymal stem cells (rMSCs) represent a small portion of the cells in the stromal compartment of bone marrow and have the potential to differentiate into bone, cartilage, fat, and fibrous tissue. These mesenchymal progenitor cells were maintained as primary isolates and as subcultured cells in separate closed modular incubator chambers purged with either 95% air and 5% CO2 (20% or control oxygen) or 5% oxygen, 5% CO2, and 90% nitrogen (5% or low oxygen). At first passage, some cells from each oxygen condition were loaded into porous ceramic vehicles and implanted into syngeneic host animals in an in vivo assay for osteochondrogenesis. The remaining cells were continued in vitro in the same oxygen tension as for primary culture or were switched to the alternate condition. The first passage cells were examined for in vitro osteogenesis with assays involving the quantification of alkaline phosphatase activity and calcium and DNA content as well as by von Kossa staining to detect mineralization. Cultures maintained in low oxygen had a greater number of colonies as primary isolates and proliferated more rapidly throughout their time in vitro, as indicated by hemacytometer counts at the end of primary culture and increased DNA values for first passage cells. Moreover, rMSCs cultivated in 5% oxygen produced more bone than cells cultured in 20% oxygen when harvested and loaded into porous ceramic cubes and implanted into syngeneic host animals. Finally, markers for osteogenesis, including alkaline phosphatase activity, calcium content, and von Kossa staining, were elevated in cultures which had been in low oxygen throughout their cultivation time. Expression of these markers was usually increased above basal levels when cells were switched from control to low oxygen at first passage and decreased for cells switched from low to control oxygen. We conclude that rMSCs in culture function optimally in an atmosphere of reduced oxygen that more closely approximates documented in vivo oxygen tension. J. Cell. Physiol. 187: 345-355, 2001. (C) 2001 Wiley-Liss, Inc.