Human Protein Process Sciences, 59 800 Lille, France.
Biotechnol Appl Biochem. 2010 Aug 6;56(4):151-60. doi: 10.1042/BA20100151.
There is emerging interest in the use of standardized virally inactivated human platelet lysate preparations rich in GFs (growth factors) for cell cultures, cell therapy and clinical applications. In the present paper, we report a simple process to prepare a virally inactivated platelet lysate preparation rich in TGF-beta1 (transforming growth factor-beta1), EGF (epidermal growth factor) and IGF (insulin-like growth factor) and depleted of PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor). Apheresis platelet concentrates were treated by the S/D (solvent/detergent) viral inactivation procedure, then subjected to an oil extraction followed by adsorption with activated charcoal and finally sterile-filtered. The resulting preparation contained a mean of 368.4, 2.4 and 54.7 ng/ml of TGF-beta1, EGF and IGF respectively. PDGF-AB and VEGF were essentially completely removed by the charcoal treatment. The mean albumin, IgG, IgM and IgA and fibrinogen contents were approx. 40.0, 8.5, 0.87, 1.66 and 2.65 mg/ml respectively, cholesterol and triglycerides were at 15 and 20.7 mg/ml respectively and TnBP (tri-n-butyl phosphate) and Triton X-45 were at 8.7 and 8.8 p.p.m. respectively. Supplementing MEM (minimum essential medium) with 1-10% of this S/D-treated platelet lysate promoted the proliferation of MG63 and SIRC cell lines as well as, or better than, 10% (v/v) FBS (fetal bovine serum), as based on the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The process used to prepare such S/D-treated platelet lysates is easily scalable for industrial production. Our results open up the possibility to evaluate the potential of this new preparation for stem cell expansion and/or bone tissue engineering and regeneration.
目前人们对使用富含生长因子(growth factors,GFs)的标准化病毒灭活人血小板裂解液制剂来进行细胞培养、细胞治疗和临床应用越来越感兴趣。在本文中,我们报告了一种简单的方法来制备富含 TGF-β1(转化生长因子-β1)、EGF(表皮生长因子)和 IGF(胰岛素样生长因子)且缺乏 PDGF(血小板衍生生长因子)和 VEGF(血管内皮生长因子)的病毒灭活血小板裂解液制剂。用 S/D(溶剂/去污剂)病毒灭活程序处理血小板浓缩物,然后进行油提取,接着用活性炭吸附,最后进行无菌过滤。得到的制剂中 TGF-β1、EGF 和 IGF 的平均含量分别为 368.4、2.4 和 54.7ng/ml。通过活性炭处理基本上可以完全去除 PDGF-AB 和 VEGF。白蛋白、IgG、IgM 和 IgA 的平均含量约为 40.0、8.5、0.87、1.66 和 2.65mg/ml,胆固醇和甘油三酯分别为 15 和 20.7mg/ml,TnBP(三丁基磷酸酯)和 Triton X-45 分别为 8.7 和 8.8ppm。在 MEM(最低必需培养基)中添加 1-10%的经 S/D 处理的血小板裂解液可促进 MG63 和 SIRC 细胞系的增殖,效果与添加 10%(v/v)FBS(胎牛血清)相当,基于 MTS[3-(4,5-二甲基噻唑-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺基苯基)-2H-四唑]测定法。用于制备这种 S/D 处理的血小板裂解液的方法易于扩大到工业生产。我们的结果为评估这种新制剂在干细胞扩增和/或骨组织工程和再生方面的潜力提供了可能性。