Transcriptome-wide analysis of human chondrocyte expansion on synoviocyte matrix

Human chondrocytes, expanded and used in autologous chondrocyte implantation techniques, are known to rapidly de-differentiate in culture. These chondrocytes undergo both morphological and phenotypical changes and, eventually, lose the ability to produce hyaline-like matrix when cultured on tissue culture (TC) plastic. Growth on synoviocyte-derived extracellular matrix (SDECM) reduces this de-differentiation allowing for more than double the number of population doublings whilst retaining chondrogenic capacity. The goal of this study was to use RNAseq analysis to examine the differences between TC plastic-expanded and SDECM-expanded human chondrocytes over 4 passages. Human chondrocytes (3 donors) were thawed from primary stocks collected under an IRB approved protocol and cultured on TC plastic flasks or on SDECM-coated flasks at physiological oxygen tension (5%) for 4 passages. RNA was extracted from the cell layer during log expansion (70-90% confluence) phase at passages 1 and 4, column purified and DNAse treated before QC analysis and submitted for next generation RNA sequencing (RNAseq). Significant effects on gene expression were observed due to both growth substrates and passage number. These results may provide insight into the mechanism of how SDECM provides a more chondrogenesis perserving environment for cell expansion.用于自体软骨细胞移植（autologous chondrocyte implantation）技术的扩增人软骨细胞，已知在体外培养过程中会快速发生去分化。此类软骨细胞会同时发生形态学与表型改变，最终在组织培养（TC）塑料表面培养时，丧失合成透明样基质的能力。在滑膜细胞衍生细胞外基质（SDECM）上培养可减轻这种去分化，使群体倍增数提升一倍以上，同时保留成软骨能力。本研究旨在通过RNA测序（RNAseq）分析，对比经TC塑料扩增与SDECM扩增的人软骨细胞在4次传代过程中的基因表达差异。本研究使用3名供者的原代冻存人软骨细胞，这些细胞的原代样本采集符合机构伦理审查委员会（Institutional Review Board, IRB）批准的实验方案。将细胞分别接种于TC塑料培养瓶及SDECM包被培养瓶中，在5%生理氧分压条件下连续传代4次。分别在第1代和第4代的对数增殖期（细胞汇合度70%-90%）提取细胞层RNA，经柱式纯化、脱氧核糖核酸酶（DNAse）处理后进行质量控制（QC）分析，随后提交进行下一代RNA测序（RNAseq）。研究观察到培养底物与传代次数均对基因表达产生显著影响。本研究结果可为阐明SDECM如何为细胞扩增提供更利于维持成软骨表型的培养环境的分子机制提供参考。