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Biological activity of a standardized freeze-dried platelet derivative to be used as cell culture medium supplement.

机译：用作细胞培养基补充剂的标准化冻干血小板衍生物的生物活性。

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Serum of animal origin and in particular fetal bovine serum is the most commonly utilized cell culture medium additive for in vitro cell growth and differentiation. However, several major concerns have been raised by the scientific community regarding the use of animal sera for human cell-based culture applications. Among the possible alternatives to the animal serum, platelet-derived compounds have been proposed since more than 10 years. Nevertheless, the high degree of variability between the different platelet preparations, and the lack of standardized manufacturing and quality control procedures, made difficult to reach a consensus on the applicability of this novel cell culture medium supplement. In this study, we describe the preparation of a standardized platelet-rich plasma (PRP) derivative obtained starting from human-certified buffy coat samples with a defined platelet concentration and following protocols including also freeze-drying, gamma irradiation and biological activity testing prior the product release as cell culture medium additive. Biological activity testing of the different preparations was done by determining the capability of the different PRP preparations to sustain human bone marrow mesenchymal stem cell (MSC) clone formation and proliferation. Taking advantage of a developed MSC in vitro clonogenicity test, we also determined biological activity and stability of the freeze-dried gamma-sterilized PRP preparations after their storage for different times and at different temperatures. The PRP effects on cell proliferation were determined both on primary cell cultures established from different tissues and on a cell line. Results were compared with those obtained in "traditional" parallel control cultures performed in the presence of bovine serum [10% fetal calf serum (FCS)]. Compared to FCS, the PRP addition to the culture medium increased the MSC colony number and average size. In primary cell cultures and in cell line cultures, the PRP promoted cell proliferation also in conditions where the FCS had not a proliferation stimulating effect due to either the nature of the cells and the tissue of origin (such as human articular chondrocytes from elderly patients) or to the critical low density cell seeding (such as for HeLa cells). In summary, the standardized PRP formulation would provide an "off-the-shelf" product to be used for the selection and expansion of several cell types also in critical cell culture conditions.
机译：动物来源的血清，尤其是胎牛血清是用于体外细胞生长和分化的最常用的细胞培养基添加剂。然而，关于将动物血清用于基于人类细胞的培养应用中，科学界提出了几个主要问题。在动物血清的可能替代品中，自十多年来以来就已经提出了血小板衍生的化合物。然而，不同血小板制品之间的高度可变性，以及缺乏标准化的生产和质量控制程序，使得很难就这种新型细胞培养基补充剂的适用性达成共识。在这项研究中，我们描述了从人体认证的血沉棕黄层样品开始，从具有规定的血小板浓度的标准血沉棕黄层样品中制备标准化富血小板血浆（PRP）衍生物的方法，该方案还包括冷冻干燥，伽马射线辐照和生物活性测试。产品释放作为细胞培养基的添加剂。通过确定不同的PRP制剂维持人骨髓间充质干细胞（MSC）克隆形成和增殖的能力，进行了不同制剂的生物活性测试。利用已开发的MSC体外克隆性测试，我们还确定了冷冻干燥的伽马灭菌的PRP制剂在不同时间和不同温度下保存后的生物活性和稳定性。在从不同组织建立的原代细胞培养物中以及在细胞系中都确定了PRP对细胞增殖的影响。将结果与在牛血清[10％胎牛血清（FCS）]存在下进行的“传统”平行对照培养中获得的结果进行比较。与FCS相比，向培养基中添加PRP可增加MSC菌落数和平均大小。在原代细胞培养和细胞系培养中，由于细胞的性质和来源组织（例如，来自老年患者的人关节软骨细胞），FCS也没有刺激增殖的条件下，PRP也会促进细胞增殖或临界低密度细胞接种（例如用于HeLa细胞）。总而言之，标准化的PRP配方将提供“现成的”产品，用于在关键细胞培养条件下选择和扩增几种细胞类型。

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