The Blood Bank, The National University Hospital of Iceland, Reykjavik, Iceland.
Faculty of Medicine, Biomedical Center, University of Iceland, Reykjavik, Iceland.
PLoS One. 2020 Apr 15;15(4):e0220163. doi: 10.1371/journal.pone.0220163. eCollection 2020.
Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation.
METHODOLOGY/PRINCIPAL FINDINGS: Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates.
CONCLUSION/SIGNIFICANCE: These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing.
间充质基质细胞（MSCs）的临床应用通常需要体外扩增步骤才能达到临床相关数量。体外细胞扩增需要在基础哺乳动物细胞培养基中添加生长因子。为避免使用含有动物物质的补充剂，可以用人血小板裂解物（hPL）代替胎牛血清，该物质量取自过期且已灭活病原体的血小板浓缩物。然而，目前全球大多数输血单位都没有进行病原体灭活。由于血库是生产 hPL 的血小板浓缩物的唯一来源，因此确保产品安全和标准化生产方法非常重要。在这项概念验证研究中，我们评估了在血小板裂解后应用病原体灭活（通过阿米洛利/紫外线 A 处理）从过期血小板浓缩物中生产 hPL 的可行性，通过评估生长因子、细胞因子的保留情况以及支持 MSC 增殖和三系分化的能力来评估。
方法/主要发现：骨髓来源的 MSC（BM-MSCs）通过在血小板裂解后应用阿米洛利/紫外线 A 处理进行病原体灭活，从过期血小板裂解物中（hPL-PIPL）中进行扩增和分化。结果与使用常规过期病原体灭活血小板浓缩物（hPL-PIPC）后进行病原体灭活的结果进行了比较。 hPL-PIPL 处理的可溶性生长因子和细胞因子浓度低于 hPL-PIPC 处理。当用作细胞培养的补充剂时，hPL-PIPL 中的 BM-MSCs 增殖速度较慢，但更一致。支持三系分化的能力在裂解物之间具有可比性。
结论/意义：这些结果表明，通过在血小板裂解后应用病原体灭活（通过阿米洛利/紫外线 A 处理），可以从过期且未经处理的血小板裂解物中生产出功能性 hPL。在过期后进行病原体灭活可能为进一步标准化全球 hPL 生产方法、增加起始材料的数量以及满足未来在人类细胞培养中使用无动物补充剂的需求提供了宝贵的解决方案。
