Ma Jason, Hsiung Chung-Chuan, Yang Tzu-Hsien, Sun Hsiu-Yen, Kuo Ming-Ling
Department of Microbiology and Immunology, Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (ROC).
Department of Medical Trial, Bo-ai Clinic, Kaohsiung, Taiwan (ROC).
Stem Cells Int. 2025 Jun 19;2025:5545892. doi: 10.1155/sci/5545892. eCollection 2025.
Mesenchymal stromal cells (MSCs) are recognized for their differentiation and immune regulation capabilities, which enhance their potential for treating various diseases. MSCs can be sourced from diverse tissues, with peripheral blood (PB) serving as a viable alternative to bone marrow. We now present an alternative strategy that eliminates the need for preadministering growth factors, utilizing density gradient methods, and culturing target cells in medium supplemented with autologous serum. PB was collected through venipuncture and then coincubated with glycerin. After incubation, a thin layer of cells above the red blood cells (RBCs) was isolated, showing an increased population of CD34CD45 cells compared to PB mononuclear cell (PBMC) isolation using Ficoll gradient. After culture, adherent spindle-shaped cells were identified and collected to assess MSC surface markers, demonstrating their differentiation potential into adipocytes, osteocytes, and chondrocytes, thus, fulfilling the criteria for MSCs. The population doubling time (PDT) of isolated PB-MSCs was approximately 30-40 h in early passages. These PB-MSCs also exhibited immunomodulatory functions and are capable of suppressing T cell activation. We believe this protocol supports PB as a convenient alternative source for MSC isolation and offers new strategies for acquiring and maintaining PB-MSCs.
间充质基质细胞（MSCs）因其分化和免疫调节能力而受到认可，这增强了它们治疗各种疾病的潜力。MSCs可以从多种组织中获取，外周血（PB）是骨髓的一种可行替代来源。我们现在提出一种替代策略，该策略无需预先施用生长因子，而是利用密度梯度方法，并在补充有自体血清的培养基中培养靶细胞。通过静脉穿刺采集PB，然后与甘油共同孵育。孵育后，分离出红细胞（RBCs）上方的一层薄细胞，与使用Ficoll梯度分离PB单核细胞（PBMC）相比，CD34CD45细胞群体增加。培养后，鉴定并收集贴壁的纺锤形细胞以评估MSC表面标志物，证明它们向脂肪细胞、骨细胞和软骨细胞的分化潜力，因此，符合MSCs的标准。早期传代时，分离的PB-MSCs的群体倍增时间（PDT）约为30-40小时。这些PB-MSCs还表现出免疫调节功能，能够抑制T细胞活化。我们相信该方案支持将PB作为分离MSC的便捷替代来源，并为获取和维持PB-MSCs提供了新策略。
