血小板裂解液替代胎牛血清培养间充质干细胞

Human platelet lysates as an alternative to fetal bovine serum for the culture of mesenchymal stemcells

目的观察人血小板裂解液（HPL）对脐带间充质干细胞（UC-MSCs）的形态、增殖、表面标志、诱导分化能力的影响。方法取正常足月妊娠产妇脐带血，离心后收集血沉棕黄层及血清按4：1混合后-20℃冻存，反复冻融2次后制成HPL，按10％比例加入培养基。分离获得UC-MSCs，在含10％HPL或10％胎牛血清（FBS）的培养基中培养，比较培养30d所获得的累积倍增级、最大传代代数所获得的细胞数及第3代细胞6孔板中培养6d所获得的细胞量；通过流式细胞仪检测HPL对培养细胞的表面标志的影响；观察HPL对培养细胞的成骨、成脂及成软骨诱导分化能力的影响。结果HPL或FBS培养的UC-MSCs形态相同，均为长梭形，传至第3代形态无明显改变。在HPL或FBS中培养30d所获得的累积倍增级比较差异无统计学意义（P〉0．05）；HPL培养的细胞在第10代的细胞量[（7．5±0．4）X10^6个]明显多于10％FBS组[（4．34±0．2）×10^6个]；第3代UC-MSCs在6孔板培养6d后，HPL组所得细胞数明显多于FBS组（P〈0．05）。流式结果显示，UC-MSCs表面高表达CD44、CD73、CD90、CD105，不表达或低表达CD11b、CD19、CD31、CD34、CD45及人类白细胞抗原DR基因（HLA-DR）。与10％FBS培养的UC—MSCs比较，10％HPL培养并未影响其表面标记的表达。HPL或FBS培养的UC-MSCs均能进行三系诱导分化，其分化能力无明显差异。结论HPL可以代替FBS来进行培养和扩增UC-MSCs。在保持其形态、表面标志、分化能力不变的情况下，UC-MSCs在HPL环境下增殖更快。

Objective To investigate the impact of human platelet lysates （HPL） on the morphol- ogy, proliferation, cell surface markers, and differentiation capacity of umbilical cord-derived mesenchymal stem cells （UC-MSCs）. Methods Cord blood was collected from umbilical cord after the delivery of pla- centa. Four buffy coat units and one serum unit were pooled and frozen at -20 ℃. After two fi＇eeze-thaw cycles, at least 10 units of freeze-thaw lysed human plateiets were further pooled to yield HPL. UC-MSCs were isolated and cultured in medium containing either 10% HPL or fetal bovine serum （FBS）. To com pare the effect of HPL on MSCs expansion, the 30 d cumulative population doublings were determined, UC-MSCs were maximally expanded, and the number of cells cultured in six-well plate for 6 d was coun ted. Cell surface antigen pbenotyping was performed with UC-MSCs cultured in either 10% HPL or 10% FBS at passage 3. The effect of HPL on the differentiation capacity of UC-MSCs was also examined. Re- suits HPL has no effect on the morphology of UC-MSCs ; as shown by spindle-like cells at passage 3. The 30 d cumulative population doublings were statistical comparable between HPL and FBS. However, the number of cells maximally expanded at passage ten in HPL [ （7. 5 ±0.4） x l0^6 ] ceils was much greater than in FBS [ （4. 3 ±0. 2） x 10^6] cells. After culturing for 6 d, cell number/well in the HPL group was greater than the FBS group （ P 〈 O. 05 ）. UC-MSCs were strongly positive against CD44, CD73, CD90, and CDI05; whereas they were negative for CDllb, CD19, CD 31, CD34, CD45, or Human leukocyte antigen-DR （HLA-DR）. No significant differences between HPL and FBS were detected. UC-MSCs de rived from both HPL and FBS demonstrated differentiation toward the osteogenic, adipogenic, and chondro genic lineages. Conclusion HPL may be considered as an alternative to FBS for the culture and expan sion of UC-MSCs in vitro. HPL favors very rapid expansion while maintaining the morphology, cell surface markers, and differentiation capacity.