Unidad de Producción y Reprogramación Celular (UPRC), Red Andaluza de Diseño y Traslación de Terapias Avanzadas (RAdytTA), Fundación Progreso y Salud, 41092, Seville, Spain.
Programa de Doctorado en Biología Molecular, Biomedicina e Investigación Clínica, Universidad de Sevilla, Seville, Spain.
Stem Cell Res Ther. 2023 Apr 7;14(1):69. doi: 10.1186/s13287-023-03300-z.
There remains much interest in improving cryopreservation techniques for advanced therapy medicinal products (ATMPs). Recently, human platelet lysate (hPL) has emerged as a promising candidate to replace fetal bovine serum (FBS) as a xeno-free culture supplement for the expansion of human cell therapy products. Whether hPL can also substitute for FBS in cryopreservation procedures remains poorly studied. Here, we evaluated several cryoprotective formulations based on a proprietary hPL for the cryopreservation of bioengineered tissues and cell therapy products.
We tested different xenogeneic-free, pathogen-inactivated hPL (ihPL)- and non-inactivated-based formulations for cryopreserving bioengineered tissue (cellularized nanostructured fibrin agarose hydrogels (NFAHs)) and common cell therapy products including bone marrow-derived mesenchymal stromal cells (BM-MSCs), human dermal fibroblasts (FBs) and neural stem cells (NSCs). To assess the tissue and cellular properties post-thaw of NFAHs, we analyzed their cell viability, identity and structural and biomechanical properties. Also, we evaluated cell viability, recovery and identity post-thaw in cryopreserved cells. Further properties like immunomodulation, apoptosis and cell proliferation were assessed in certain cell types. Additionally, we examined the stability of the formulated solutions. The formulations are under a bidding process with MD Bioproducts (Zurich, Switzerland) and are proprietary.
Amongst the tissue-specific solutions, Ti5 (low-DMSO and ihPL-based) preserved the viability and the phenotype of embedded cells in NFAHs and preserved the matrix integrity and biomechanical properties similar to those of the standard cryopreservation solution (70% DMEM + 20% FBS + 10% DMSO). All solutions were stable at - 20 °C for at least 3 months. Regarding cell-specific solutions, CeA maintained the viability of all cell types > 80%, preserved the immunomodulatory properties of BM-MSCs and promoted good recovery post-thaw. Besides, both tested solutions were stable at - 20 °C for 18 months. Finally, we established that there is a 3-h window in which thawed NFAHs and FBs maintain optimum viability immersed in the formulated solutions and at least 2 h for BM-MSCs.
Our results show that pathogen-inactivated solutions Ti5 allocated for bioengineered tissues and CeA allocated for cells are efficient and safe candidates to cryopreserve ATMPs and offer a xenogeneic-free and low-DMSO alternative to commercially available cryoprotective solutions.
人们仍然热衷于改进先进治疗药物产品（ATMP）的冷冻保存技术。最近，人血小板裂解液（hPL）已成为一种很有前途的候选物，可以替代胎牛血清（FBS）作为人细胞治疗产品的无动物来源培养补充物。hPL 是否也可以替代 FBS 用于冷冻保存程序，这方面的研究仍很欠缺。在这里，我们评估了几种基于专有的人血小板裂解液（hPL）的冷冻保护剂配方，用于冷冻保存生物工程组织和细胞治疗产品。
我们测试了不同的无动物来源、病原体灭活的 hPL（ihPL）和非灭活的基于 hPL 的配方，用于冷冻保存生物工程组织（细胞化纳米结构化纤维蛋白琼脂糖水凝胶（NFAHs））和常见的细胞治疗产品，包括骨髓来源的间充质基质细胞（BM-MSCs）、人真皮成纤维细胞（FBs）和神经干细胞（NSCs）。为了评估 NFAHs 解冻后的组织和细胞特性，我们分析了它们的细胞活力、特性和结构及生物力学特性。此外，我们还评估了冷冻保存细胞解冻后的细胞活力、恢复和特性。在某些细胞类型中还评估了免疫调节、细胞凋亡和细胞增殖等其他特性。此外，我们还检查了配方溶液的稳定性。这些配方正在与 MD Bioproducts（苏黎世，瑞士）进行招标，属于专有配方。
在组织特异性溶液中，Ti5（低 DMSO 和 ihPL 基）可保持 NFAHs 中嵌入细胞的活力和表型，并保持基质完整性和与标准冷冻保存溶液（70% DMEM+20% FBS+10% DMSO）相似的生物力学特性。所有溶液在-20°C 下至少稳定 3 个月。对于细胞特异性溶液，CeA 保持了所有细胞类型的活力>80%，保持了 BM-MSCs 的免疫调节特性，并促进了良好的解冻后恢复。此外，两种测试溶液在-20°C 下可稳定 18 个月。最后，我们确定解冻的 NFAHs 和 FBs 在配方溶液中浸泡时保持最佳活力的时间窗口为 3 小时，而 BM-MSCs 至少为 2 小时。
我们的结果表明，用于生物工程组织的病原体灭活溶液 Ti5 和用于细胞的 CeA 是冷冻保存 ATMP 的有效且安全的候选物，为商业上可获得的冷冻保护剂溶液提供了无动物来源和低 DMSO 的替代方案。
