Spheroid model for functional osteogenic evaluation of human adipose derived stem cells.
Spheroid model for functional osteogenic evaluation of human adipose derived stem cells.
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DOI：
10.1002/jbm.a.35974
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发表时间：
2017-04
期刊：
影响因子：
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通讯作者：
Janorkar AV
中科院分区：
文献类型：
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作者：
Gurumurthy B;Bierdeman PC;Janorkar AV
3D培养系统具有通过允许更好的细胞-细胞相互作用来模拟天然微环境的能力。我们已经制备了体外三维成骨细胞培养模型，使用人脂肪来源的干细胞（hASCs）培养的重组弹性蛋白样多肽（ELP）共轭带电荷的聚乙烯亚胺（PEI）。我们证明了在ELP-PEI包被的组织培养聚苯乙烯（TCPS）上培养的hASC形成3D球状体，并且与在未包被的TCPS上形成的传统二维（2D）单层相比，表现出向成骨谱系的上级分化。活/死存活力测定证实在3周培养期结束时>90%活细胞。在相同的培养期间，在2D单层中观察到比3D球状体更高的蛋白质含量，因为2D环境允许继续增殖，而3D球状体经历接触抑制生长停滞。标准化碱性磷酸酶（ALP）活性，这是早期成骨分化的指标是三维球体较高。标准化骨钙素（OCN）的生产，这是成骨成熟的指标也较高的3D球体，而2D单层没有明显的OCN生产。在第22天，增加茜素红吸收的3D球状体表现出更大的矿化活性比2D单层。总之，这些结果表明在ELP-PEI涂覆的TCPS表面上的3D球状体培养物中hASC的成骨分化上级在未涂覆的TCPS表面上形成的2D单层。三维球体干细胞培养中这种增强的成骨作用可以通过为骨组织工程中增强的细胞功能和相互作用提供更好的微环境来作为二维培养的替代方案。
3D culture systems have the ability to mimic the natural microenvironment by allowing better cell-cell interactions. We have prepared an in vitro 3D osteogenic cell culture model using human adipose derived stem cells (hASCs) cultured atop recombinant elastin-like polypeptide (ELP) conjugated to a charged polyelectrolyte, polyethyleneimine (PEI). We demonstrate that hASCs cultured atop the ELP-PEI coated tissue culture polystyrene (TCPS) formed 3D spheroids and exhibited superior differentiation toward osteogenic lineage compared to the traditional two dimensional (2D) monolayer formed atop uncoated TCPS. Live/dead viability assay confirmed >90% live cells at the end of the 3-week culture period. Over the same culture period, higher protein content was observed in 2D monolayer than 3D spheroids, as the 2D environment allowed continued proliferation, while 3D spheroids underwent contact-inhibited growth arrest. The normalized alkaline phosphatase (ALP) activity, which is an indicator for early osteogenic differentiation was higher for 3D spheroids. The normalized osteocalcin (OCN) production, which is an indicator for osteogenic maturation was also higher for 3D spheroids while 2D monolayer had no noticeable OCN production. On day 22, increased Alizarin red uptake by 3D spheroids showed greater mineralization activity than 2D monolayer. Taken together, these results indicate a superior osteogenic differentiation of hASCs in 3D spheroid culture atop ELP-PEI coated TCPS surfaces than the 2D monolayer formed on uncoated TCPS surfaces. Such enhanced osteogenesis in 3D spheroid stem cell culture may serve as an alternative to 2D culture by providing a better microenvironment for the enhanced cellular functions and interactions in bone tissue engineering.