While three‐dimensional spheroids outperform traditional two‐dimensional monolayer culture for human adipose‐derived stem cells (hASCs), there is not a consensus on the most successful method for enhancing their adipogenic differentiation and minimizing the loss of physiologically relevant, fatty spheroids during culture. To this end, we compared three culture methods, namely, elastin‐like polypeptide‐polyethyleneimine (ELP‐PEI) coated surfaces, ultra‐low attachment static culture, and suspension culture for their ability to form and retain productive hASC spheroids. The ELP‐PEI coatings used the ELP conjugated to two molecular weights of PEI (800 or 25,000 g/mol). FTIR spectroscopy, atomic force microscopy, and contact angle goniometry revealed that the ELP‐PEI coatings had similar chemical structures, surface topography, and hydrophobicity. Time‐lapse microscopy showed that increasing the PEI molecular weight resulted in smaller spheroids. Measurement of triglyceride content showed that the three methods induced comparable differentiation of hASCs toward the adipogenic lineage. DNA content and morphometric analysis revealed merging of spheroids to form larger spheroids in the ultra‐low attachment static culture and suspension culture methods. In contrast, the retention of hASC spheroid sizes and numbers with a regular spheroid size (~100 μm) were best atop the ELP‐PEI800 coatings. Overall, this research shows that the spheroid culture atop the ELP‐PEI coatings is a suitable cell culture model for future studies involving long‐term, three‐dimensional culture of mature adipocytes derived from hASCs.

中文翻译：

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比较无支架培养技术中人脂肪干细胞球体的形成、脂肪成熟和保留。

虽然三维球体优于传统的二维单层培养人脂肪干细胞 (hASCs)，但对于在培养过程中增强其成脂分化和最大程度减少生理相关脂肪球体损失的最成功方法尚未达成共识. 为此，我们比较了三种培养方法，即弹性蛋白样多肽-聚乙烯亚胺 (ELP-PEI) 涂层表面、超低附着静态培养和悬浮培养，以了解它们形成和保留生产性 hASC 球体的能力。ELP-PEI 涂层使用与两个分子量的 PEI（800 或 25,000 g/mol）共轭的 ELP。FTIR 光谱、原子力显微镜和接触角测角法表明 ELP-PEI 涂层具有相似的化学结构、表面形貌和疏水性。延时显微镜显示增加 PEI 分子量会导致球体更小。甘油三酯含量的测量表明，这三种方法诱导 hASCs 向成脂谱系的可比分化。DNA 含量和形态计量分析表明，在超低附着静态培养和悬浮培养方法中，球体合并形成更大的球体。相比之下，在 ELP-PEI800 涂层上，hASC 球体尺寸和数量与规则球体尺寸（~100 μm）的保留最佳。总体而言，这项研究表明，ELP-PEI 涂层顶部的球体培养物是未来研究的合适细胞培养模型，涉及来自 hASC 的成熟脂肪细胞的长期三维培养。