Department of Reproduction, Obstetrics and Herd Health, Ghent University, Merelbeke, Belgium.
Tissue Eng Part C Methods. 2011 Nov;17(11):1061-70. doi: 10.1089/ten.tec.2011.0052. Epub 2011 Aug 26.
Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation methods. The mononuclear cell recovery after Percoll separation, however, resulted in significantly more putative MSC colonies; and, therefore, this isolation method was used for all further experiments. Culture conditions such as cell density and medium or serum coating of the wells did not significantly affect putative MSC recovery. Isolated MSC using Percoll were subsequently differentiated toward the osteogenic, chondrogenic, and adipogenic lineage. In addition, MSC were phenotyped by multicolor flow cytometry based on their expression of different cell protein markers. Cultured MSC were CD29, CD44, and CD90-positive and CD79α, Macrophage/Monocyte and MHC II-negative. In conclusion, this study reports optimized protocols to isolate, culture, and characterize solid equine MSC from UCB.
间充质基质细胞（MSC）代表了支持细胞治疗新临床概念的有前途的细胞群体。已经描述了从脐血（UCB）分离 MSC 的多种分离程序。相比之下，马的数据很少。在当前的研究中，使用羟乙基淀粉的沉淀法和使用氯化铵（NH 4 Cl）裂解红细胞的方法与两种密度梯度分离方法（Ficoll-Paque 和 Percoll）进行了比较。所有四种分离方法都可以建立贴壁细胞集落。然而，Percoll 分离后的单个核细胞回收导致了更多的潜在 MSC 集落；因此，这种分离方法用于所有进一步的实验。细胞密度和孔中培养基或血清涂层等培养条件不会显著影响潜在 MSC 的回收。使用 Percoll 分离的 MSC 随后向成骨细胞、软骨细胞和成脂细胞系分化。此外，基于不同细胞蛋白标志物的表达，通过多色流式细胞术对 MSC 进行了表型分析。培养的 MSC 为 CD29、CD44 和 CD90 阳性，CD79α、巨噬细胞/单核细胞和 MHC II 阴性。总之，本研究报告了从 UCB 分离、培养和表征固体马 MSC 的优化方案。
