Evaluation of methods for cell harvesting and the biological properties at successive passages of canine bone marrow stromal cells
Evaluation of methods for cell harvesting and the biological properties at successive passages of canine bone marrow stromal cells
复制标题
DOI：
10.2460/ajvr.73.11.1832
复制
发表时间：
2012-11-01
影响因子：
1
通讯作者：
Inaba, Toshio
中科院分区：
文献类型：
--
作者：
Nishida, Hidetaka;Shoji, Yuki;Inaba, Toshio
目的：比较犬骨髓基质细胞（BMSCs）体外连续传代的制备方法和生物学特性。样本-从9只比格犬的股骨中采集的骨髓间充质干细胞。采用成纤维细胞实验比较两种获取骨髓间充质干细胞的方法：抽吸法和灌注法。流式细胞术分析细胞表面标记物。通过检测3-胸腺嘧啶氢的放射性，分析了增殖活性的变化。通过衰老相关的p-半乳糖苷酶染色研究细胞衰老，并评估与传代相关的分化特性（成骨和脂肪生成）。结果：抽吸法产生的成纤维细胞明显多于灌注法。两种方法采集的细胞CD44和CD90阳性，CD34和CD45阴性。诱导后，细胞具有成骨和成脂表型。通过抽吸法获得的骨髓间充质干细胞的生物学特性与传代相关。随着传代次数的增加，细胞增殖活性降低，p -半乳糖苷酶染色细胞比例增加。分化能力在传代3时减弱。结论及临床意义：抽吸法是一种较好的骨髓间充质干细胞采集方法。在传代早期，犬骨髓间充质干细胞具有增殖活性和成骨、成脂分化的潜力，但随着传代次数的增加，增殖活性降低。考虑通道对于任何寻求通过使用骨髓间充质干细胞来再生组织的策略的成功都是重要的。（Am J Vet Res 2012;73:1832-1840）
Objective-To compare methods for harvesting canine bone marrow stromal cells (BMSCs) and determine the biological properties of canine BMSCs at successive passages in vitro.Sample-BMSCs collected from the femurs of 9 Beagles.Procedures A fibroblast assay was performed to compare 2 methods for harvesting BMSCs: the aspiration and perfusion method. Flow cytometric analysis was performed to evaluate the cell surface markers. Changes in proliferative activity were analyzed by examining radioactivity of hydrogen 3-thymidine. Cell senescence was studied via senescence-associated p-galactosidase staining, and differentiation properties (osteogenesis and adipogenesis) were estimated in association with passage.Results-The aspiration method yielded significantly more fibroblasts than the perfusion method. The cells harvested by both methods gave positive results for CD44 and CD90 and negative results for CD34 and CD45. After induction, the cells had osteogenic and adipogenic phenotypes. The biological properties of BMSCs harvested by the aspiration method were estimated in association with passage. With increasing number of passages, the proliferative activity was reduced and the proportion of cells with senescence-associated P-galactosidase staining was increased. The capacity of differentiation was reduced at passage 3.Conclusions and Clinical Relevance-The aspiration method was superior for collection of BMSCs. In early passages, canine BMSCs had the proliferative activity and potential of osteogenic and adipogenic differentiation, but this decreased with increased number of passages. Consideration of passage will be important to the success of any strategy that seeks to regenerate tissue though the use of BMSCs. (Am J Vet Res 2012;73:1832-1840)