Trombi Luisa, Danti Serena, Savelli Sara, Moscato Stefania, D'Alessandro Delfo, Ricci Claudio, Giannotti Stefano, Petrini Mario
Dept. of Clinical and Experimental Medicine, University of Pisa; OtoLab, Azienda Ospedaliero-Universitaria Pisana (AOUP);
OtoLab, Azienda Ospedaliero-Universitaria Pisana (AOUP); Dept. of Civil and Industrial Engineering, University of Pisa.
J Vis Exp. 2016 Dec 8(118):54845. doi: 10.3791/54845.
Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions - namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.
人骨髓间充质基质细胞（hMSCs）在体外使用不同培养基进行培养。然而，其在临床应用中的限制主要取决于用于培养的异种营养物质所带来的潜在生物危害和炎症风险。人源衍生物或重组材料是减少这些反应的首选候选物。因此，自体来源的培养补充剂和材料是最佳的营养物质和最安全的产品。在此，我们描述了一种在自体条件下分离和培养骨髓hMSCs的新方案——即使用患者来源的血清作为培养基补充剂，使用纤维蛋白作为hMSC给药的支架。实际上，hMSC/纤维蛋白凝块构建体在多种临床应用中可能极为有用。特别是，我们专注于其在骨科手术中的应用，其中来自供体自身血液的纤维蛋白凝块可实现有效的细胞递送以及营养物质/废物交换。为确保最佳安全条件，避免hMSC转化和组织过度生长的风险至关重要。出于这些原因，本文所述方法还表明要进行最小程度的体外hMSC扩增，以减少细胞衰老和形态变化，并在植入前进行短期成骨分化，以诱导成骨谱系特化，从而降低后续不受控制增殖的风险。
