Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan.
Univ Lille, Inserm, CHU Lille, UMR-S1171. Lille Neuroscience & Cognition, Degenerative and vascular cognitive disorders, F-59000, Lille, France.
J Biomed Sci. 2019 Oct 31;26(1):89. doi: 10.1186/s12929-019-0579-9.
Effective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson's disease models.
Intercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (- 80 °C/37 °C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot.
Platelet lysates contained the expected level of total proteins (ca. 7-14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death.
Outdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models.
必须开发有效的神经退行性疾病神经修复疗法。使用富含神经营养因子的人血小板裂解物作为神经退行性疾病新型治疗策略的研究兴趣日益增加。为确保病毒安全性，应将病原体减少处理纳入用作原料的血小板浓缩物的制备过程中。因此，我们研究了使用一种已许可的光灭活联合光敏剂（氨甲喋呤）和 UVA 照射（拦截）处理灭活病原体的血小板浓缩物（PC）是否可以作为制备在帕金森病模型中具有保留神经保护活性的血小板裂解物的原料。
用拦截处理的 PC 在达到过期日（采集后 7 天）时离心，以去除血浆和血小板添加剂溶液。将血小板沉淀重新悬浮并浓缩在磷酸盐缓冲盐水（PBS）中，进行 3 次冻融循环（-80°C/37°C），然后离心以去除细胞碎片。回收上清液，并根据我们之前的研究，通过热处理（如热处理）进一步纯化或不纯化。用 ELISA、流式细胞术、细胞活力和细胞毒性测定以及 Western blot 分析测定蛋白质和神经营养因子的含量，并评估血小板裂解物对 LUHMES 细胞或原代皮质/海马神经元的毒性和神经保护活性。
血小板裂解物含有预期水平的总蛋白（约 7-14mg/mL）和神经营养因子。病毒灭活和热处理的血小板裂解物对 Lund 人中脑多巴胺能 LUHMES 细胞系和原代神经元均无明显毒性作用。当以 5%和 0.5%的剂量使用时，它们分别增强了 LUHMES 细胞中酪氨酸羟化酶和神经元特异性烯醇化酶的表达，并且对原代神经元中突触蛋白的表达没有显著影响。此外，测试的病毒灭活血小板裂解物对暴露于 erastin（一种诱导铁死亡细胞死亡的诱导剂）的 LUHMES 和原代神经元均表现出很强的神经保护作用。
过期的拦截病原体减少的血小板浓缩物可用于制备安全且高度神经保护的人热处理血小板沉淀裂解物。这些数据为开发有效的神经再生医学用病毒灭活富含功能性神经营养因子的血小板裂解物的生物疗法以及进一步的生物工业发展提供了令人安心的前景。然而，这些数据应在动物模型中得到证实。
