Comparison of methods for the isolation and culture of Migratory chondroprogenitors from Human articular cartilage

Resident articular stem cells isolated using a migratory assay called Migratory Chondroprogenitors (MCPs) have emerged as a promising cellular therapeutic for the treatment of cartilage pathologies. In-vivo studies using MCPs report their superiority over bone-marrow mesenchymal stem cells and chondrocytes for treating chondral defects. However, there is no consensus on their isolation protocol. This study aimed to compare four reported isolation methods of MCPs and identify the optimal and feasible protocol for future translational work. Human MCPs isolated from osteoarthritic cartilage (n = 3) were divided into four groups: a) MCP1: 8–15 mm cartilage explants, b) MCP2: 8–10 mm explants digested in 0.1% collagenase for 2 hrs. and cultured c) MCP3: 1 mm cartilage explants and d) MCP 4: 25 mm explants with a X tear, 7-day culture, and trypsinization to release migrated cells. The MCPs were subjected to the following analysis: growth kinetics, surface marker expression, mRNA gene expression for markers of chondrogenesis and hypertrophy, and trilineage differentiation. MCPs isolated via the four methods showed similar surface marker profiles, chondrogenic (SOX-9, ACAN, COL2A1) and hypertrophic (COL1, RUNX2) gene expression. The migration time for the MCP3 group was the longest. The MCP1, MCP2, and MCP4 groups produced MCPs with comparable cellular expansion feasibility. MCPs can be preferably isolated by the any of the three above methods based on the investigator’s discretion. In the case of small cartilage samples similar to the MCP3 group, the isolation of MCP is plausible, keeping in mind the additional time required.

采用迁移性软骨祖细胞（Migratory Chondroprogenitors，MCPs）迁移试验分离得到的关节驻留干细胞，已成为治疗软骨病变的极具前景的细胞疗法。采用MCPs开展的体内研究表明，其在治疗软骨缺损方面优于骨髓间充质干细胞（bone-marrow mesenchymal stem cells）与软骨细胞（chondrocytes），但目前学界尚未就其分离方案达成统一共识。
本研究旨在对比四种已报道的MCPs分离方法，筛选出适用于未来转化研究的最优且可行的实验方案。从骨关节炎软骨中分离得到的人源MCPs（n=3）被分为四组：a) MCP1组：采用8–15 mm的软骨外植体；b) MCP2组：将8–10 mm的软骨外植体置于0.1%胶原酶中消化2小时后进行培养；c) MCP3组：采用1 mm的软骨外植体；d) MCP4组：采用带有X形撕裂伤的25 mm软骨外植体，经7天培养后通过胰酶消化释放迁移细胞。
对各组MCPs开展以下分析：生长动力学、表面标志物表达、软骨生成与肥大相关标志物的mRNA基因表达，以及三系分化潜能检测。结果显示，四种方法分离得到的MCPs具有相似的表面标志物谱，以及软骨生成相关（SOX-9、ACAN、COL2A1）和肥大相关（COL1、RUNX2）基因表达模式。其中MCP3组的细胞迁移耗时最长；MCP1、MCP2及MCP4组获得的MCPs具有相当的细胞扩增可行性。研究人员可根据实验需求从上述三种方法中任选其一进行MCPs的分离；若需处理类似MCP3组的小型软骨样本，只要预留额外的实验时长，即可成功分离MCPs。