ISOLATION AND CULTURE OF MESENCHYMAL STEM CELLS DERIVED FROM ADIPOSE TISSUE AND BONE MARROW OF DOGS

Abstract The objective of this study was to establish a protocol for the isolation and culture of mesenchymal stem cells (MSC) from bone marrow and adipose tissue of dogs. Three 6-month-old male dogs were used. Approximately 3.0 cm3 of adipose tissue and 3.0 mL of bone marrow were collected. The samples were processed and the isolated cells were cultured in DMEM. The cells were subjected to phenotypic characterization and to osteogenic, adipogenic, and condrogenic differentiation to confirm the isolation of the MSC. The cells showed elongated and fusiform morphology and they were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Phenotypic characterization revealed high expression of the MSC markers CD90(80.04%) and CD29(96%) in the cells from bone marrow and high expression of CD90(60.94%) and CD29(77.08%) in the cells from adipose tissue. In addition, phenotypic characterization revealed low expression of hematopoietic markers CD45(1.45%) and CD34(1.53%) in the cells from bone marrow and low expression of CD45(1.45%) and CD34(1.53%) in the cells from adipose tissue. Based on these results, the modifications applied to classical protocols simplified the process and proved to be efficient in the isolation, culture, and expansion of MSC isolated from the bone marrow and adipose tissue of dogs.

摘要 本研究旨在建立一套从犬骨髓与脂肪组织中分离培养间充质干细胞（mesenchymal stem cells, MSC）的实验方案。实验选用3只6月龄雄性犬，采集约3.0 cm³脂肪组织与3.0 mL骨髓样本。对样本进行处理后，将分离得到的细胞置于DMEM培养基中培养。随后对细胞开展表型鉴定，并通过成骨、成脂及成软骨分化实验验证所分离细胞确为MSC。结果显示，所获细胞呈长梭形形态，可定向分化为成骨细胞、脂肪细胞与软骨细胞。表型鉴定结果表明，骨髓来源的细胞高表达MSC标志物CD90(80.04%)与CD29(96%)；脂肪来源的细胞高表达CD90(60.94%)与CD29(77.08%)。此外，骨髓与脂肪来源的细胞均低表达造血系标志物CD45(1.45%)与CD34(1.53%)。基于上述结果，本研究对经典实验方案所做的优化简化了操作流程，且证实该方案可高效分离、培养并扩增犬骨髓与脂肪组织来源的MSC。