부산대학교 도서관

Bone marrow mesenchymal stem cells (MSC) have been investigated due to its therapeutic effects as multipotent, immunoregulating and anti-apoptotic cells. However, MSC's standard isolation relies on its swiftly adherence to plastic culture flasks, lacking precise cell definition. Therefore, marked heterogeneity persists in the so-called MSC population hampering true multipotent MSC isolation and, consequently, setting back tissue engineering efficiency. The present study proposes a modified adhesion-based protocol to purify bone marrow MSC. MSC were obtained from 24 patients undergoing total hip arthroplasty. We propose a simple additional step to the standard isolation protocol. Normally, marrow cells are cultivated for 3 days and the non-adherant cells are discarded. Instead, we used the non-adherant fraction to initiate a new culture, here named MSC-LA. Primary MSC obtained from standard (MSC-RA) or modified protocol (MSC-LA) were minimally manipulated and compared regarding classical MSC's properties, such as colony formation capacity, in vitro differentiation and cell surface markers. Vascular organization and maintenance capacity was also assessed through tube formation assay. MSC-LA corresponded to a 33% increase in yield, generated fewer but more populated primary colonies and more secondary colonies than MSC-RA. Moreover, MSC-LA displayed equivalent osteogenic but enhanced adipogenic differentiation than MSC-RA. In tube formation assay, MSC-LA demonstrated similar vessel organization capacity to MSC-RA, pruning disorganized tubule formation and assuming perivascular localization and morphology. In cytometry characterization, both MSC groups presented CD45-/CD31-/ CD90+/CD73+/CD44+/CD29+/CD10+/CD54+/CD34- phenotype. While MSC-RA showed greater CD45+, HLA+ and CD184+ cell contamination, MSC-LA displayed enhanced purity of CD24+, CD49d+ and CD146+ cells, markers associated to MSC phenotype. Our adhesion-based isolation protocol promotes greater yield of MSC at very low cost, generating a purified MSC population that may hold greater stemness potential. MSC-LA can optimize tissue engineering therapies through lower contamination of undesired or totally differentiated cell populations supporting autologous MSC use in arthritis patients. Further analysis is necessary to confirm MSC-LA potential in in vivo settings. [ABSTRACT FROM AUTHOR]