title: Enhanced in vitro osteogenic differentiation of human fetal MSCs attached to 3D microcarriers versus harvested from 2D monolayers.
abstract: BACKGROUND
  Mesenchymal 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.
  
  
  RESULTS
  Fetal 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).
  
  
  CONCLUSIONS
  Beyond 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.
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.forename: Asha
authors.initials: A
authors.lastname: Shekaran
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.forename: Eileen
authors.initials: E
authors.lastname: Sim
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.forename: Kah Yong
authors.initials: KY
authors.lastname: Tan
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore.|Cancer & Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore, 169857, Singapore.|Department of Reproductive Medicine, KK Women's and Children's Hospital, Singapore, 229899, Singapore.
authors.forename: Jerry Kok Yen
authors.initials: JK
authors.lastname: Chan
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119228, Singapore.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Choolani
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore.
authors.forename: Shaul
authors.initials: S
authors.lastname: Reuveny
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore. steve_oh@bti.a-star.edu.sg.
authors.forename: Steve
authors.initials: S
authors.lastname: Oh
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005333:Fetus / Q000166:cytology / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds