The inhibitory effects of IMRCs on activated PBMC (CD3/28 and IL-2 costimulated) proliferation at ratios of 1:2, 1:20 and 1:100 in vitro.ĭifferences in cell characteristics of IMRCs cultured on monolayers and different microcarriers in stirred bioreactors. Real-time qPCR for IDO1 mRNA in IMRCs harvested from monolayers and HAMCs before and after IFN-γ (0, 50 and 100 ng/mL) treatment. Morphology of IMRCs harvested from monolayers and HAMCs before and after IFN-γ stimulation. Representative immunofluorescence staining of IMRC-laden microcarriers in situ after they were induced to undergo osteogenic differentiation (osteocalcin), adipogenic differentiation (FABP-4), and chondrogenic differentiation (aggrecan). Viability of IMRCs harvested from monolayers or HAMC in clinical injection buffer over time at 4 ☌. Distribution of cell diameters of IMRCs harvested from monolayer culture or HAMCs expansion. Karyotyping of IMRCs harvested from HAMC. Isotype antibodies were used as controls for gating.

MSC-specific markers of IMRCs harvested from HAMCs (100–200 μm HAMC100). IMRCs expanded on HAMCs maintain cell stemness and immunomodulatory capabilities in stirred bioreactors. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Attachment efficiency of IMRCs cultured for 24 h on HAMC with different diameter ranges in bioreactors. Green indicates live cells, and red indicates dead cells. Live/dead staining of IMRCs on HAMC with different diameter ranges after 1 day. Cumulative population doublings of IMRCs cultured on HAMC20, HAMC100 and HAMC200 after 6 days. Cell density of IMRCs cultured on HAMC20, HAMC100 and HAMC200 after 6 days in bioreactors. Growth curves of IMRCs cultured on HAMC20, HAMC100 and HAMC200 in bioreactors. Attachment efficiency of IMRCs cultured for 24 h on HAMC20, HAMC100 and HAMC200. Cytoskeleton, focal adhesion and nucleus were imaged by rhodamine phalloidin (green), vinculin (red) and DAPI (blue). Live/dead staining of IMRCs on HAMC20, HAMC100 and HAMC200 after 1 day, 3 days, 5 days and 7 days.

Young's modulus of HAMC modified with different collagen concentrations (HAMC20, HAMC100 and HAMC200) analysed by AFM. Pictures of the 3-blade impeller (right). Photographs of the DASbox system and individual bioreactor filled with culture suspension (left). IMRCs expanded on HAMC coated with different collagen concentrations or particle sizes in bioreactors. In summary, we have established a scalable culture system for hESC-MSCs, providing novel approaches for future cell therapies.īioreactor Culture system Human embryonic stem cells Mesenchymal stem cells Microcarrier. In addition, the harvested cells exhibited high viability, immunomodulatory and regenerative therapeutic promise comparable to monolayer cultured MSCs while showing more increased secretion of extracellular matrix (ECM), particularly collagen-related proteins. We used optimal microcarriers with a coating collagen concentration of 100 μg/mL or concave-structured surface (cHAMCs) for IMRC amplification in a stirred bioreactor, expanding IMRCs within six days with the highest yield of over one million cells per milliliter. In this study, we developed a new microcarrier with hyaluronic acid (HA) as the core material, which allowed scalable serum-free suspension culture of hESC-derived MSCs (IMRCs). However, a scalable culture system for hPSC-derived MSCs is urgently needed to meet the cell quantity and quality requirements of practical clinical applications. Mesenchymal stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are a desirable cell source for cell therapy owing to their capacity to be produced stably and homogeneously in large quantities.

Mesenchymal stem cells culture