Objective

The study is aimed to verify the possibility of using humanized alternatives to fetal bovine serum (FBS) such as umbilical cord blood plasma (CBP) and AB⁺ plasma to support the long-term growth of mesenchymal stromal cells (MSCs) derived from the umbilical cord. We hypothesized that umbilical CBP would be a potential substitute to FBS, especially for small scale autologous clinical transplantations.

Methods

The MSCs were cultured for six consecutive passages to evaluate xeno-free media's ability to support long-term growth. Cell proliferation rates, colony-forming-unit (CFU) efficiency and population doublings of expanded MSCs, were investigated. Ex vivo expanded MSCs were further characterized using flow cytometry and quantitative PCR. The impact of cryopreservation and composition of cryomedium on phenotype, viability of MSC was also assessed.

Results

Our results on cell proliferation, colony-forming unit efficiency suggested that the expansion of the cells was successfully carried out in media supplemented with humanized alternatives. MSCs showed lower CFU counts in FBS (~ 25) than humanized alternatives (~ 35). The gene expression analysis revealed that transcripts showed significant differential expression by two to three folds in the FBS group compared with MSCs grown in medium with humanized alternatives (p < 0.05). In addition, MSCs grown in a medium with FBS had more osteogenic activity, a signature of unwanted differentiation. The majority of ex vivo expanded MSCs at early and late passages expressed CD44⁺, CD73⁺, CD105⁺, CD90⁺, and CD166⁺ in all the experimental groups tested (~ 90%). In contrast to the other MSC surface markers, expression levels of STRO-1⁺ (~ 21–10%) and TNAP⁺ (~ 29–11%) decreased with the increase in passage number for MSCs cultured in a FBS-supplemented medium (p < 0.05).

Conclusion

Our results established that CBP supported culture of umbilical cord tissue-derived MSCs and is a safer Xeno free replacement to FBS. The use of CBP also enables the storage of umbilical cord tissue derived MSCs in patient-specific conditions to minimize adverse events if cells are delivered directly to the patient.

中文翻译：

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人源化胎牛血清替代品用于脐带间充质基质细胞临床应用的批判性评价

客观的

该研究旨在验证使用人源化替代胎牛血清 (FBS) 的可能性，例如脐带血浆 (CBP) 和 AB ⁺血浆，以支持源自脐带的间充质基质细胞 (MSC) 的长期生长。绳索。我们假设脐带 CBP 将成为 FBS 的潜在替代品，特别是对于小规模自体临床移植。

方法

MSC 连续培养 6 代，以评估无异种培养基支持长期生长的能力。研究了细胞增殖率、集落形成单位 (CFU) 效率和扩增的 MSC 的群体倍增。使用流式细胞术和定量 PCR 进一步表征体外扩增的 MSC。还评估了冷冻保存和低温培养基的组成对 MSC 的表型和活力的影响。

结果

我们关于细胞增殖、集落形成单位效率的结果表明，细胞的扩增在补充有人源化替代品的培养基中成功进行。MSC 在 FBS (~ 25) 中的 CFU 计数低于人源化替代品 (~ 35)。基因表达分析显示，与在具有人源化替代物的培养基中生长的 MSC 相比，FBS 组中的转录本显示出 2 到 3 倍的显着差异表达（p < 0.05）。此外，在含有 FBS 的培养基中生长的 MSC 具有更多的成骨活性，这是不需要的分化的标志。大多数体外扩增的 MSC 在早期和晚期传代表达 CD44 ⁺、 CD73 ⁺、 CD105 ⁺、 CD90 ⁺和 CD166 ⁺在所有测试的实验组中 (~ 90%)。与其他 MSC 表面标志物相比，STRO-1 ⁺ (~ 21–10%) 和 TNAP ⁺ (~ 29–11%) 的表达水平随着在 FBS 补充培养基中培养的 MSC 的传代数增加而降低（ p < 0.05)。

结论

我们的结果表明，CBP 支持脐带组织来源的 MSC 的培养，并且是 FBS 的更安全的无异种替代品。CBP 的使用还能够在患者特定条件下储存脐带组织衍生的 MSC，以最大程度地减少细胞直接递送给患者时的不良事件。