Wolfson STEM Centre, School of Medicine, University of Nottingham, Nottingham NG7 2RD, UK.
Advanced Biomaterials Research Group, Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD, UK.
Int J Mol Sci. 2019 Feb 22;20(4):951. doi: 10.3390/ijms20040951.
Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of transforming growth factor beta (TGF-β) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens.
最近的软骨修复策略强调了间充质祖细胞的潜力，间充质祖细胞是一种可获得的干细胞来源，具有成骨和成软骨的潜力，与生物材料结合用于组织工程。为此，再生医学方法需要强大的模型来确保所选细胞群体能够以可重复和可测量的方式产生所需的细胞类型。体外软骨分化技术已经成熟，但主要是定性的，依赖于样本染色和成像。为了促进体外软骨形成治疗的筛选，已经开发了一种 3D 微球培养物，并结合了三种定量 GAG 测定法，第四种平行测定法测量样品含量以实现归一化。转化生长因子-β (TGF-β) 的作用用于验证这种培养方式，在二维和三维培养配置中，均使蛋白聚糖的产生在平行测定中产生了可测量的增加。与传统微球相比，单层形式似乎不太能够检测到细胞分化的变化，但是在孔内 3D 培养中显示出显著的差异反应。对 2 型胶原表达的影响证实了这些观察结果。基于这些结果，对 3D 培养进行了优化，采用高通量孔内配置的微孔板形式。该模型显示出更高的灵敏度，并证实了 3D 微球在孔内定量测定作为一种有效的分化形式，与简化的高通量软骨筛选兼容。
