低温制剂保存对脂肪间充质干细胞生物学特性的影响

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

背景:脂肪干细胞在GMP洁净车间制备完成后需要做成制剂才能给患者输注。低温制剂相比于冻存制剂有很多优势,然而关于低温短期保存对干细胞生物学特性影响的研究较少。目的:评价用含5%人血白蛋白的复方电解质低温保存24 h对脂肪间充质干细胞生物学特性的影响。方法:取第3-6代人脂肪间充质干细胞,以5×109 L-1的细胞浓度重悬在含5%人血白蛋白的复方电解质注射液中,将细胞悬液转移至冻存管中,置于冷链运输箱,2-8℃低温保存24 h。观察低温保存前后的细胞形态、贴壁能力、细胞活率、细胞直径及细胞免疫表型变化。结果与结论:(1)脂肪间充质干细胞低温保存24 h后,死细胞增多,细胞活率显著下降,但细胞活率仍大于80%,活细胞直径显著增大;(2)脂肪间充质干细胞低温保存24 h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;(3)脂肪间充质干细胞低温保存24 h后,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%,但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;(4)结果表明,低温制剂保存未明显影响脂肪间充质干细胞的贴壁生长能力、存活率及免疫表型等干性生物学特性。

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 of24-hour low-temperature storage using multiple electrolytes containing5%human serum albumin on the biological characteristics of ADSCs.METHODS:ADSCs at passages3-6at a concentration of5×109/L were suspended in multiple electrolytes containing5%human serum albumin.Cell suspension was transferred into cryogenic vials,and then these vials were placed in a cold chain shipping box for2-8℃low-temperature storage for24hours.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 for24hours,the number of dead cells increased.Although cell viability decreased significantly,it was still higher than80%.Cell diameters of living cells increased significantly.(2)After low-temperature storage of ADSCs for24hours,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 for24hours,HLA-DR,CD34and CD45were negatively expressed with a positive rate lower than2%;CD29,CD73and CD105were positively expressed with a positive rate higher than95%.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.