title: Biodegradable poly-ε-caprolactone microcarriers for efficient production of human mesenchymal stromal cells and secreted cytokines in batch and fed-batch bioreactors.
abstract: Large numbers of human mesenchymal stromal cells (MSCs) used for a variety of applications in tissue engineering and cell therapy can be generated by scalable expansion in a bioreactor using microcarriers (MCs) systems. However, the enzymatic digestion process needed to detach cells from the growth surface can affect cell viability and potentially the potency and differentiation efficiency. Thus, the main aim of our study was to develop biocompatible and biodegradable MCs that can support high MSC yields while maintaining their differentiation capability and potency. After cell expansion, the cells that covered MCs can be directly implanted in vivo without the need for cell harvesting or use of scaffold. Poly-ε-caprolactone (PCL) is known as a biocompatible and biodegradable material. However, it cannot be used for generation of MCs because its high density (1.14 g/cm3) would exclude its applicability for suspension MCs in stirred reactors. In this article, we describe expansion and potency of MSCs propagated on low-density (1.06 g/cm3) porous PCL MCs coated with extracellular matrices (LPCLs) in suspended stirred reactors. Using these LPCLs, cell yields of about 4 × 104cells/cm2and 7- to 10-fold increases were obtained using four different MSC lines (bone marrow, cord blood, fetal and Wharton's jelly). These yields were comparable with those obtained using non-degradable MCs (Cytodex 3) and higher than two-dimensional monolayer (MNL) cultures. A fed-batch process, which demonstrated faster cell expansion (4.5 × 104cells/cm2in 5 days as compared with 7 days in batch culture) and about 70% reduction in growth media usage, was developed and scaled up from 100-mL spinner flask to 1-L controlled bioreactor. Surface marker expression, trilineage differentiation and clonogenic potential of the MSCs expanded on LPCL were not affected. Cytokine secretion kinetics, which occurred mostly during late logarithmic phase, was usually comparable with that obtained in Cytodex 3 cultures and higher than MNL cultures. In conclusion, biodegradable LPCL can be used to efficiently expand a variety of MSC lines in stirred scalable reactors in a cost-effective manner while maintaining surface markers expression, differentiation capability and high levels of cytokine secretion. This study is the first step in testing these cell-biodegradable porous MC aggregates for tissue engineering and cell therapy, such as bone and cartilage regeneration, or wound healing.
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore. Electronic address: alan_lam@bti.a-star.edu.sg.
authors.forename: Alan Tin-Lun
authors.initials: AT
authors.lastname: Lam
authors.affiliation: Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Jessica Pei-Wen
authors.initials: JP
authors.lastname: Toh
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Eileen Jia-Hui
authors.initials: EJ
authors.lastname: Sim
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Allen Kuan-Liang
authors.initials: AK
authors.lastname: Chen
authors.affiliation: Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Department of Reproductive Medicine, Kandang Kerbau Women's and Children's Hospital, 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.
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), Singapore.
authors.forename: Shaul
authors.initials: S
authors.lastname: Reuveny
authors.affiliation: Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: William R
authors.initials: WR
authors.lastname: Birch
authors.affiliation: Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore. Electronic address: steve_oh@bti.a-star.edu.sg.
authors.forename: Steve Kah-Weng
authors.initials: SK
authors.lastname: Oh
mesh_terms: D020341:Absorbable Implants; D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D003911:Dextrans / Q000737:chemistry; D006801:Humans; D008422:Materials Testing; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D055616:Microtechnology / Q000295:instrumentation; D011091:Polyesters / Q000737:chemistry*; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D003470:Culture Media; D016207:Cytokines; D003911:Dextrans; D011091:Polyesters; C016240:polycaprolactone; C052839:Cytodex
keywords: Mesenchymal stromal cells; bioreactors; cytokines; microcarriers