Abstract
BackgroundMesenchymal stem cells (MSCs) are of great interest in bone regenerative medicine due to their osteogenic potential and trophic effects. However, challenges to large-scale production of MSCs can hinder the translation of MSC therapies. 3D Microcarrier (MC)-based MSC culture presents a scalable and cost-effective alternative to conventional methods of expansion in 2D monolayers. Furthermore, biodegradable MCs may allow for MC-bound MSC delivery without enzymatic harvest for selected applications such as bone healing. However, the effects of cell expansion on microcarriers and enzymatic cell harvest on MSC phenotype and osteogenic differential potential are not well understood. In this study, we characterized human fetal MSCs (hfMSCs) after expansion in 3D microcarrier spinner or 2D monolayer cultures. Following expansion, we compared osteogenic differentiation of cultures seeded with 3D MC-harvested, 3D MC-bound and conventional 2D monolayer (MNL)-harvested cells when cultured in osteogenic induction media on collagen-coated plates.ResultsFetal MSCs expanded on both 3D agitated Microcarriers (MC) and 2D Plastic static monolayer (MNL) cultures express high levels of MSC surface markers. MC-harvested hfMSCs displayed higher expression of early osteogenic genes but slower mineralization kinetics compared to MNL-harvested MSCs during osteogenic induction. However, in the comparison between MC-bound and MC-harvested hfMSCs, osteogenic genes were upregulated and mineralization kinetics was accelerated in the former condition. Importantly, 3D MC-bound hfMSCs expressed higher levels of osteogenic genes and displayed either higher or equivalent levels of mineralization, depending on the cell line, compared to the classical monolayer cultures use in the literature (MNL-harvested hfMSCs).ConclusionBeyond the processing and scalability advantages of the microcarrier culture, hfMSCs attached to MCs undergo robust osteogenic differentiation and mineralization compared to enzymatically harvested cells. Thus biodegradable/biocompatible MCs which can potentially be used for cell expansion as well as a scaffold for direct in vivo delivery of cells may have advantages over the current methods of monolayer-expansion and delivery post-harvest for bone regeneration applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0219-8) contains supplementary material, which is available to authorized users.
Highlights
Mesenchymal stem cells (MSCs) are of great interest in bone regenerative medicine due to their osteogenic potential and trophic effects
Characterization of human fetal MSCs (hfMSCs) expanded in Two dimensional (2D) static monolayer and agitated Three dimensional (3D) MC cultures hfMSCs were expanded on microcarriers in spinner flasks or 2D plastic flasks in monolayer cultures
Flow cytometry analysis of cell surface marker expression showed that 3D MC-harv hfMSCs expressed equivalent high levels of International Society for Cell Therapy (ISCT) MSC markers (CD34, CD105, CD73 and CD90) and lower levels of the endothelial, pericyteassociated marker CD14627,28 and α1 integrin and higher level of the pro-osteogenic α2 integrin, compared to 2D MNL-harv hfMSCs (Fig. 2d)
Summary
Mesenchymal stem cells (MSCs) are of great interest in bone regenerative medicine due to their osteogenic potential and trophic effects. 3D Microcarrier (MC)-based MSC culture presents a scalable and cost-effective alternative to conventional methods of expansion in 2D monolayers. The effects of cell expansion on microcarriers and enzymatic cell harvest on MSC phenotype and osteogenic differential potential are not well understood. Mesenchymal stem cells (MSCs) are cells with the potential to differentiate into multiple cell types including osteoblasts, chondrocytes and adipocytes, and were shown to have trophic effects, modulate immune responses and promote healing in vivo [1]. The commercial viability and clinical translation of MSC therapies, especially those using allogeneic off-the shelf strategies, are hindered by challenges of scalable expansion of these cells [8]. Current methods of MSC expansion and production such as using stacked 2D surfaces as cell attachment substrates have limited scalability, are labor intensive and are costly when large numbers of cells are required [9]
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