Carnevale Gianluca, Pisciotta Alessandra, Riccio Massimo, De Biasi Sara, Gibellini Lara, Ferrari Adriano, La Sala Giovanni B, Bruzzesi Giacomo, Cossarizza Andrea, de Pol Anto
1 Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia , Modena, Italy .
2 Dipartimento Sperimentale Interaziendale, University of Modena and Reggio Emilia , Modena, Italy .
Biopreserv Biobank. 2016 Apr;14(2):138-48. doi: 10.1089/bio.2015.0001. Epub 2016 Feb 1.
Adult mesenchymal stem cells are a promising source for cell therapies and tissue engineering applications. Current procedures for banking of human bone-marrow mesenchymal stem cells (hBM-MSCs) require cell isolation and expansion, and thus the use of large amounts of animal sera. However, animal-derived culture supplements have the potential to trigger infections and severe immune reactions. The aim of this study was to investigate an optimized method for cryopreservation of human bone-marrow fragments for application in cell banking procedures where stem-cell expansion and use are not immediately needed. Whole trabecular fragments enclosing the bone marrow were stored in liquid nitrogen for 1 year in a cryoprotective solution containing a low concentration of dimethyl sulfoxide and a high concentration of human serum (HuS). After thawing, the isolation, colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, cell senescence, apoptosis, and multi-lineage differentiation potential of hBM-MSCs were tested in media containing HuS compared with hBM-MSCs isolated from fresh fragments. Human BM-MSCs isolated from cryopreserved fragments expressed MSC markers until later passages, had a good proliferation rate, and exhibited the capacity to differentiate toward osteogenic, adipogenic, and myogenic lineages similar to hBM-MSCs isolated from fresh fragments. Moreover, the cryopreservation method did not induce cell senescence or cell death. These results imply that minimal processing may be adequate for the banking of tissue samples with no requirement for the immediate isolation and use of hBM-MSCs, thus limiting cost and the risk of contamination, and facilitating banking for clinical use. Furthermore, the use of HuS for cryopreservation and expansion/differentiation has the potential for clinical application in compliance with good manufacturing practice standards.
成人间充质干细胞是细胞治疗和组织工程应用中一个很有前景的来源。目前人类骨髓间充质干细胞（hBM-MSCs）的储存程序需要细胞分离和扩增，因此需要使用大量动物血清。然而，动物来源的培养补充剂有可能引发感染和严重的免疫反应。本研究的目的是研究一种优化的人类骨髓片段冷冻保存方法，用于细胞储存程序，在此程序中干细胞扩增和使用并非急需。将包裹骨髓的整个小梁片段在含有低浓度二甲基亚砜和高浓度人血清（HuS）的冷冻保护溶液中于液氮中储存1年。解冻后，与从新鲜片段中分离的hBM-MSCs相比，在含有HuS的培养基中测试hBM-MSCs的分离、集落形成单位能力、增殖、形态、干性相关标志物表达、细胞衰老、凋亡和多向分化潜能。从冷冻保存片段中分离的人骨髓间充质干细胞在传代较晚时仍表达间充质干细胞标志物，具有良好的增殖率，并表现出向成骨、成脂和成肌谱系分化的能力，类似于从新鲜片段中分离的hBM-MSCs。此外，冷冻保存方法未诱导细胞衰老或细胞死亡。这些结果表明，对于组织样本的储存，最小限度的处理可能就足够了，无需立即分离和使用hBM-MSCs，从而限制了成本和污染风险，并便于临床使用的储存。此外，使用HuS进行冷冻保存和扩增/分化有符合良好生产规范标准的临床应用潜力。
