Wang Tsung-Jen, Chen Ming-Sheng, Chou Ming-Li, Lin Hsiu-Chen, Seghatchian Jerard, Burnouf Thierry
Department of Ophthalmology, Taipei Medical University Hospital, Taipei, Taiwan; Department of Ophthalmology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan.
Transfus Apher Sci. 2017 Oct;56(5):769-773. doi: 10.1016/j.transci.2017.08.021. Epub 2017 Aug 31.
Human platelet lysates (HPLs) are emerging as the new gold standard supplement of growth media for ex vivo expansion of cells for transplant. However, variations do exist in the way how HPLs are prepared. In particular, uncertainties still exist regarding the type of HPL most suitable for corneal endothelium cells (CEC) expansion, especially as these cells have limited proliferative capacity.
Three distinct HPL preparations were produced, with or without calcium chloride/glass beads activation, and with or without heat treatment at 56°C for 30min. These HPLs were used to supplement basal D-MEM growth medium, each at a protein concentration equivalent to that of 10% fetal bovine serum (FBS; control). Impact on CEC (BCE C/D-1b cells) in vitro morphology, viability and capacity to express Zonula occludens-1 (ZO-1) tight junction marker was assessed by Western blotting.
BCE C/D-1b cells grown in all HPL supplements exhibited four of essential characteristic properties: adhesion capacity, microscopic morphology and viability similar to that observed when using 10% FBS. In addition, Western blots analysis revealed an expression of the ZO-1 marker by BCE C/D-1b cells in all conditions of culture.
CECs can expand ex vivo in a basal medium supplemented with the three HPLs without noticeable difference compared to FBS supplement. These data support further studies to evaluate the potential to use HPLs as a clinical-grade xeno-free supplement of CEC for corneal transplant.
人血小板裂解物（HPLs）正成为用于细胞体外扩增以进行移植的生长培养基的新金标准补充剂。然而，HPLs的制备方式确实存在差异。特别是，关于最适合角膜内皮细胞（CEC）扩增的HPL类型仍存在不确定性，尤其是因为这些细胞的增殖能力有限。
制备了三种不同的HPL制剂，有或没有氯化钙/玻璃珠激活，以及有或没有在56°C热处理30分钟。这些HPLs用于补充基础D-MEM生长培养基，每种的蛋白质浓度相当于10%胎牛血清（FBS；对照）。通过蛋白质印迹法评估对CEC（BCE C/D-1b细胞）体外形态、活力和表达紧密连接蛋白-1（ZO-1）紧密连接标志物能力的影响。
在所有HPL补充剂中生长的BCE C/D-1b细胞表现出四个基本特征：黏附能力、微观形态和活力与使用10%FBS时观察到的相似。此外，蛋白质印迹分析显示在所有培养条件下BCE C/D-1b细胞均表达ZO-1标志物。
与FBS补充剂相比，CECs可以在补充有三种HPLs的基础培养基中进行体外扩增，且无明显差异。这些数据支持进一步研究以评估使用HPLs作为角膜移植中CEC的临床级无动物源补充剂的潜力。
