Effect of low-temperature preparation on the biological characteristics of adipose-derived mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.33.002

Abstract

Abstract:

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) that are manufactured in good manufacturing practice (GMP) clean rooms should be made into stem cell preparations before administration. Low-temperature preparation has many advantages over cryopreservation preparation; however, little is reported on the effect of short-term low-temperature storage on the biological characteristics of stem cells.
OBJECTIVE: To evaluate the effect of 24-hour low-temperature storage using multiple electrolytes containing 5% human serum albumin on the biological characteristics of ADSCs.
METHODS: ADSCs at passages 3-6 at a concentration of 5×10⁹/L were suspended in multiple electrolytes containing 5% human serum albumin. Cell suspension was transferred into cryogenic vials, and then these vials were placed in a cold chain shipping box for 2-8 ℃ low-temperature storage for 24 hours. Cell morphology, adhesion ability, cell viability, cell diameters and cell immunophenotyping before and after the storage were observed.
RESULTS AND CONCLUSION: (1) After low-temperature storage of ADSCs for 24 hours, the number of dead cells increased. Although cell viability decreased significantly, it was still higher than 80%. Cell diameters of living cells increased significantly. (2) After low-temperature storage of ADSCs for 24 hours, few cells which were circle-shaped lost adhesion ability, and most cells could adherently grow, with the spindle-shaped morphology similar to the cells before preservation. (3) After low-temperature storage of ADSCs for 24 hours, HLA-DR, CD34 and CD45 were negatively expressed with a positive rate lower than 2%; CD29, CD73 and CD105 were positively expressed with a positive rate higher than 95%. However, the cell cluster was clearly divided into two parts after the preservation. Cells with enlarged diameters moved right in the FSC/SSC dot-plot. These results show that low-temperature preparation storage has no significant effect on the stemness of ADSCs, such as adhesion ability, cell viability and cell immunophenotype.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Cryopreservation, Serum Albumin, Tissue Engineering

CLC Number:

R394.2

Zhang Feng-li, Shao Xiao-hu, Ren Huai-juan, Chen Yan-tian, Qi Nian-min. Effect of low-temperature preparation on the biological characteristics of adipose-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5255-5261.

Figures/Tables 2

2.1 脂肪间充质干细胞的形态如图1所示，用Ⅰ型胶原酶法消化脂肪组织得到的原代脂肪间充质干细胞接种在6孔板上，1 d后，观察到部分细胞贴壁生长，由圆形向梭形伸长生长，也有部分细胞已经呈现梭形形态；5 d后，贴壁的细胞为典型的梭形形态，贴壁生长状态良好，细胞分裂多次，形成明显的克隆；10 d后，细胞达到80%左右汇合，形态均一，无杂质细胞，可进行传代。 2.2 脂肪间充质干细胞的生长动力学如图2所示，细胞接种至24孔板后，生长状况良好，增殖迅速，第5天荧光值即达到最高，此后，细胞生长进入平台期，荧光值呈现略微下降趋势，至第8天，培养板内细胞过多卷起，结束实验。 2.3 脂肪间充质干细胞的多向分化如图3所示，实验组脂肪间充质干细胞添加成骨诱导培养基培养21 d，茜素红染色后发现有明显的钙结节沉积，对照组无钙盐沉积现象；成骨分化半定量分析结果看出，实验组吸光值显著性高于对照组(P < 0.05)。如图3所示，实验组脂肪间充质干细胞添加成脂诱导培养基培养21 d，油红O染色后发现细胞胞浆内有大量红色油滴；对照组无红色油滴出现，且细胞密集，呈梭形生长；成脂分化半定量分析结果看出，实验组吸光值显著性高于对照组(P < 0.05)。结果说明脂肪间充质干细胞具有多向分化潜能。 2.4 低温制剂保存后脂肪间充质干细胞的活率如图4A，B所示，低温保存24 h后，被PI染成红色的亮点增多，即死细胞增多，细胞活率显著下降，但细胞活率仍大于80%；被AO染成绿色的亮点变大，活细胞直径显著增大(图4C)。 2.5 低温制剂保存后脂肪间充质干细胞的贴壁能力如图5所示，细胞保存后少量细胞为圆形，失去贴壁能力，大部分细胞能贴壁生长，形态为梭形，与细胞保存前贴壁形态相似。 2.6 低温制剂保存后脂肪间充质干细胞的免疫表型低温制剂保存后，脂肪间充质干细胞HLA-DR、CD34和CD45均为阴性表达，阳性率低于2%；CD29、CD73和CD105均为阳性表达，阳性率高于95%(图6)，符合间充质干细胞的鉴定标准[11]，保存未显著降低脂肪干细胞的“干性”。然而，保存后的细胞显著地由一群分为两群，部分细胞体积增大向右移动(图7)，与上述计数仪测量的结果一致。"

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