PMID- 30975604 OWN - NLM STAT- MEDLINE DCOM- 20200504 LR - 20200505 IS - 1477-2566 (Electronic) IS - 1465-3249 (Linking) VI - 21 IP - 6 DP - 2019 Jun TI - Sub-confluent culture of human mesenchymal stromal cells on biodegradable polycaprolactone microcarriers enhances bone healing of rat calvarial defect. PG - 631-642 LID - S1465-3249(19)30040-4 [pii] LID - 10.1016/j.jcyt.2019.03.004 [doi] AB - In the current emerging trend of using human mesenchymal stromal cell (MSCs) for cell therapy, large quantities of cells are needed for clinical testing. Current methods of culturing cells, using tissue culture flasks or cell multilayer vessels, are proving to be ineffective in terms of cost, space and manpower. Therefore, alternatives such as large-scale industrialized production of MSCs in stirred tank bioreactors using microcarriers (MCs) are needed. Moreover, the development of biodegradable MCs for MSC expansion can streamline the bioprocess by eliminating the need for enzymatic cell harvesting and scaffold seeding for bone-healing therapies. Our previous studies described a process of making regulated density (1.06 g/cm(3)) porous polycaprolactone biodegradable MCs Light Polycarprolactone (LPCL) (MCs), which were used for expanding MSCs from various sources in stirred suspension culture. Here, we use human early MSCs (heMSCs) expanded on LPCL MCs for evaluation of their osteogenic differentiation potential in vitro as well as their use in vivo calvarial defect treatment in a rat model. In summary, (i) in vitro data show that LPCL MCs can be used to efficiently expand heMSCs in stirred cultures while maintaining surface marker expression; (ii) LPCL MCs can be used as scaffolds for cell transfer for transplantation in vivo; (iii) 50% sub-confluency, mid-logarithmic phase, on LPCL MCs (50% confluent) exhibited higher secretion levels of six cytokines (interleukin [IL]-6, IL-8, Vascular endothelial growth factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), growth-regulated oncogene-alpha (GRO-alpha) and stromal cell-derived factor-1alpha (SDF-1alpha)) as compared with 100% confluent, stationary phase cultures (100% confluent); (iv) these 50% confluent cultures demonstrated better in vitro osteogenic differentiation capacity as compared with 100% confluent cultures (higher levels of calcium deposition and at earlier stage); the improved bone differentiation capacity of these 50% confluent cultures was also demonstrated at the molecular level by higher expression of early osteoblast genes Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP), collagen type I, osterix and osteocalcin); and (v) in vivo implantation of biodegradable LPCL MCs covered with 50% heMSCs into rats with calvarial defect demonstrated significantly better bone formation as compared with heMSCs obtained from monolayer cultures (5.1 +/- 1.6 mm(3) versus 1.3 +/- 0.7 mm(3)). Moreover, the LPCL MCs covered with 50% heMSCs supported better in vivo bone formation compared with 100% confluent culture (2.1 +/- 1.3 mm(3)). Taken together, our study highlights the potential of implanting 50% confluent MSCs propagated on LPCL MCs as optimal for bone regeneration. This methodology allows for the production of large numbers of MSCs in a three-dimensional (3D) stirred reactor, while supporting improved bone healing and eliminating the need for a 3D matrix support scaffold, as traditionally used in bone-healing treatments. CI - Copyright (c) 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved. FAU - Lam, Alan Tin-Lun AU - Lam AT AD - Stem Cell Group 2 Bioprocessing Technology Institute, Agency of Science, Technology and Research (A*STAR), Singapore. Electronic address: alan_lam@bti.a-star.edu.sg. FAU - Sim, Eileen Jia-Hui AU - Sim EJ AD - Stem Cell Group 2 Bioprocessing Technology Institute, Agency of Science, Technology and Research (A*STAR), Singapore. FAU - Shekaran, Asha AU - Shekaran A AD - Stem Cell Group 2 Bioprocessing Technology Institute, Agency of Science, Technology and Research (A*STAR), Singapore. FAU - Li, Jian AU - Li J AD - Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), Singapore. FAU - Teo, Kim-Leng AU - Teo KL AD - Stem Cell Group 2 Bioprocessing Technology Institute, Agency of Science, Technology and Research (A*STAR), Singapore. FAU - Goggi, Julian L AU - Goggi JL AD - Isotopic Molecular Imaging Laboratory, Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), Singapore. FAU - Reuveny, Shaul AU - Reuveny S AD - Stem Cell Group 2 Bioprocessing Technology Institute, Agency of Science, Technology and Research (A*STAR), Singapore. FAU - Birch, William R AU - Birch WR AD - Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), Singapore. FAU - Oh, Steve Kah-Weng AU - Oh SK AD - Stem Cell Group 2 Bioprocessing Technology Institute, Agency of Science, Technology and Research (A*STAR), Singapore. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20190408 PL - England TA - Cytotherapy JT - Cytotherapy JID - 100895309 RN - 0 (Biocompatible Materials) RN - 0 (Cytokines) RN - 0 (Polyesters) RN - 24980-41-4 (polycaprolactone) SB - IM MH - Animals MH - Biocompatible Materials/*chemistry MH - Bioreactors MH - Bone Regeneration/*physiology MH - Cell Count MH - Cell Culture Techniques/instrumentation/*methods MH - Cell Differentiation MH - Cell- and Tissue-Based Therapy/methods MH - Cells, Cultured MH - Cytokines/metabolism MH - Humans MH - Male MH - Mesenchymal Stem Cell Transplantation/methods MH - Mesenchymal Stem Cells/*cytology/physiology MH - Osteogenesis/*physiology MH - Polyesters/chemistry MH - Rats, Nude MH - Skull OTO - NOTNLM OT - bioprocessing OT - bone formation OT - cell confluence OT - cytokines OT - mesenchymal stromal cells OT - osteogenic OT - polycaprolactone OT - porous microcarrier EDAT- 2019/04/13 06:00 MHDA- 2020/05/06 06:00 CRDT- 2019/04/13 06:00 PHST- 2018/12/05 00:00 [received] PHST- 2019/02/20 00:00 [revised] PHST- 2019/03/13 00:00 [accepted] PHST- 2019/04/13 06:00 [pubmed] PHST- 2020/05/06 06:00 [medline] PHST- 2019/04/13 06:00 [entrez] AID - S1465-3249(19)30040-4 [pii] AID - 10.1016/j.jcyt.2019.03.004 [doi] PST - ppublish SO - Cytotherapy. 2019 Jun;21(6):631-642. doi: 10.1016/j.jcyt.2019.03.004. Epub 2019 Apr 8.