Laboratory of Stem Cells, Institute of Cell Biology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China.
J Biomed Mater Res A. 2010 Mar 1;92(3):817-29. doi: 10.1002/jbm.a.32378.
Human bone marrow mesenchymal stem cells (hMSCs) are promising candidates for cell therapy and tissue engineering. However, the life span of hMSCs during in vitro culture is limited. Human telomerase catalytic subunit (hTERT) gene transduction could prolong the life span of hMSCs and maintain their potential of osteogenic differentiation. Therefore, hMSCs transduced with hTERT (hTERT-hMSCs) could be used as a cell model for in vitro tissue engineering experiment because of its prolonged life span and normal cellular properties. A perfusion culture system for proliferation and osteogenesis of hTERT-hMSCs or primary hMSCs in porous polylactic glycolic acid (PLGA) scaffolds is described here. A cell suspension of hTERT-hMSCs or primary hMSCs (5 x 10(5) cells/250 microL) was seeded and then cultured for 12 days in porous PLGA scaffolds (10 mm in diameter, 3 mm in height) under both static and perfusion culture systems. The seeding efficiency, proliferation, distribution and viability, and osteogenesis of cells in scaffolds were evaluated. The perfusion method generated higher scaffold cellularity and proliferation of cells in scaffolds, and hTERT-hMSCs showed the higher proliferation potential than primary hMSCs. Results from fluorescein diacetate (FDA) staining and scanning electron microscopy (SEM) demonstrated homogeneous seeding, proliferation, and viability of hTERT-hMSCs throughout the scaffolds in the perfusion culture system. On the contrary, the static culture yielded polarized proliferation favoring the outer and upper scaffold surfaces, and resulted in decreasing of cells in the central section of the scaffolds. A flow rate of 0.5 mL/min had an effect on osteogenic differentiation of cells in scaffolds. However, the osteogenic medium promoted the osteogenic efficiency of cells. Scaffolds with hTERT-hMSCs had the higher osteogenesis than scaffolds with primary hMSCs. Thus, these results suggest that the flow condition not only allow a better seeding efficiency and homogeneity but also facilitate uniform proliferation and osteogenic differentiation of hTERT-hMSCs in scaffolds. hTERT-hMSCs could be used as stem cell candidates for bone tissue engineering experiments.
人骨髓间充质干细胞（hMSCs）是细胞治疗和组织工程的有前途的候选者。然而，hMSCs 在体外培养中的寿命是有限的。人端粒酶催化亚单位（hTERT）基因转导可以延长 hMSCs 的寿命并维持其成骨分化的潜能。因此，转染 hTERT 的 hMSCs（hTERT-hMSCs）可以用作体外组织工程实验的细胞模型，因为其寿命延长且具有正常的细胞特性。本文描述了一种用于 hTERT-hMSCs 或原代 hMSCs 在多孔聚乳酸-乙醇酸（PLGA）支架中增殖和成骨的灌注培养系统。将 hTERT-hMSCs 或原代 hMSCs（5 x 10(5)细胞/250 μL）的细胞悬液接种，并在静态和灌注培养系统下在多孔 PLGA 支架（直径 10mm，高度 3mm）中培养 12 天。评估了支架中细胞的接种效率、增殖、分布和活力以及成骨情况。灌注方法可提高支架细胞密度和细胞增殖，并且 hTERT-hMSCs 显示出比原代 hMSCs 更高的增殖潜力。荧光二乙酸酯（FDA）染色和扫描电子显微镜（SEM）的结果表明，在灌注培养系统中，hTERT-hMSCs 在整个支架中均匀接种、增殖和保持活力。相反，静态培养导致支架外表面和上表面的细胞呈极化增殖，导致支架中心部分的细胞减少。0.5mL/min 的流速对支架中细胞的成骨分化有影响。然而，成骨培养基促进了细胞的成骨效率。与原代 hMSCs 相比，hTERT-hMSCs 支架具有更高的成骨能力。因此，这些结果表明，流动条件不仅可以提高更好的接种效率和均匀性，而且还可以促进 hTERT-hMSCs 在支架中的均匀增殖和成骨分化。hTERT-hMSCs 可作为骨组织工程实验的干细胞候选物。
