BACKGROUND Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology. METHODS Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed. RESULTS Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions. CONCLUSION All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.

中文翻译：

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在人类血小板裂解物中人类基质细胞增强增殖过程中，有丝分裂书签因子的上调。

背景技术创新的人类基质细胞疗法需要无异种培养条件。人血小板裂解物（HPL）的各种配方是胎牛血清（FBS）的有效替代品。但是，始终缺乏标准化的制造协议和质量标准妨碍了HPL产品的可比性。这项研究的目的是比较三种不同的HPL制剂与FBS的生化组成，并研究它们对基质细胞生物学的影响。方法从骨髓（BM），白色脂肪组织（WAT）和脐带（UC）中分离基质细胞，并在补充有HPL（pHPL），纤维蛋白原耗尽的血清转化的pHPL（pHPLS），机械纤维蛋白原-耗尽的pHPL（mcpHPL）和FBS。与全血中的标准值相比，分析了生化参数。不同的生长因子和细胞因子通过基于微珠的多重技术进行测量。进行了基质细胞免疫表型的流式细胞术，体外分化以及转录因子SOX2，KLF4，cMYC，OCT4和NANOG的mRNA表达分析。结果在所有pHPL制剂中，生化参数均具有可比性，但在一定程度上与FBS不同。pHPL制剂中总蛋白质，葡萄糖，胆固醇和Na +升高，而FBS中K +和Fe3 +含量更高。与FBS相比，基于pHPL的培养基显着增强了基质细胞的繁殖。在所有四种培养条件下均保持了特征性免疫表型和体外分化潜能。对生长因子和细胞因子的分析揭示了不同的水平，这取决于pHPL的预先存在，基质细胞的消耗或分泌。有趣的是，与添加FBS的培养基相比，在pHPL-培养的基质细胞中，转录和有丝分裂书签因子cMYC和KLF4的mRNA表达以来源依赖的方式显着增强。在所有pHPL培养条件下，所有基质细胞类型的SOX2 mRNA表达均增加。结论所有补充pHPL的培养基均平等地支持WAT和UC衍生的基质细胞的增殖，明显优于FBS。已知能够快速重新进入细胞周期的有丝分裂书签因子在pHPL扩增的细胞中得到显着增强。我们的结果支持基于基质细胞的药用产品更好地表征和标准化人源化培养基。与添加FBS的培养基相比，在pHPL-培养的基质细胞中，转录和有丝分裂书签因子cMYC和KLF4的mRNA表达以来源依赖的方式显着增强。在所有pHPL培养条件下，所有基质细胞类型的SOX2 mRNA表达均增加。结论所有补充pHPL的培养基均平等地支持WAT和UC衍生的基质细胞的增殖，明显优于FBS。已知能够快速重新进入细胞周期的有丝分裂书签因子在pHPL扩增的细胞中得到显着增强。我们的结果支持基于基质细胞的药用产品更好地表征和标准化人源化培养基。与添加FBS的培养基相比，在pHPL-培养的基质细胞中，转录和有丝分裂书签因子cMYC和KLF4的mRNA表达以来源依赖的方式显着增强。在所有pHPL培养条件下，所有基质细胞类型的SOX2 mRNA表达均增加。结论所有用pHPL补充的培养基均同样支持WAT和UC衍生的基质细胞的增殖，明显优于FBS。已知能够快速重新进入细胞周期的有丝分裂书签因子在pHPL扩增的细胞中得到显着增强。我们的结果支持基于基质细胞的药用产品更好地表征和标准化人源化培养基。在所有pHPL培养条件下，所有基质细胞类型的SOX2 mRNA表达均增加。结论所有用pHPL补充的培养基均同样支持WAT和UC衍生的基质细胞的增殖，明显优于FBS。已知能够快速重新进入细胞周期的有丝分裂书签因子在pHPL扩增的细胞中得到显着增强。我们的结果支持基于基质细胞的药用产品更好地表征和标准化人源化培养基。在所有pHPL培养条件下，所有基质细胞类型的SOX2 mRNA表达均增加。结论所有用pHPL补充的培养基均同样支持WAT和UC衍生的基质细胞的增殖，明显优于FBS。已知能够快速重新进入细胞周期的有丝分裂书签因子在pHPL扩增的细胞中得到显着增强。我们的结果支持基于基质细胞的药用产品更好地表征和标准化人源化培养基。