Serum‐free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential

Abstract

There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several indications such as graft-versus-host disease, acute myocardial infarction, Crohn’s disease, and multiple sclerosis. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the indications further exacerbates this manufacturing challenge. In contrast, spheroid MSC culture does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In this present study, we developed and optimized serum free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components. The optimization yielded two prototype media that could allow MSCs to form aggregates and proliferate in both static cultures and dynamic cultures. The expanded MSCs expressed the expected surface markers for mesenchymal cells (CD73, CD90 and CD105). In addition, the expanded cells demonstrated multipotency and differentiated to the osteocyte, chondrocyte and adipocyte lineages, which showed similar or enhanced differentiation levels compared with serum-containing adherent cultures.

Cellular spheroids obtained from mesenchymal stem cells derived from bone marrow and limb muscle of early human embryo

“…In spheroids derived from tibia bone, CD44, CD73, and CD90 expression is lower, and CD105 expression is much lower, than in monolayer cultures (Frith et al, 2010). CD105 expression in cells derived from human bone marrow MSCs depends on serum content (Alimperti et al, 2014).…”

“…It has been demonstrated that MSC spheroids exhibit increased anti inflammation effect, increased differentiation potential, and enhanced expression of cytokines and pluripotent genes. It facilitates the understanding of organogenesis mechanisms and their application in the regenerative medicine (Alimperti et al, 2014;Bogdanova Jatniece et al, 2014;Guo et al, 2014;Murphy et al, 2014;Xiao et al, 2014;Li et al, 2015).…”

Characterization and Angiogenic Potential of Human Neonatal and Infant Thymus Mesenchymal Stromal Cells

“…We also evaluated the angiogenic characteristics of thymus MSCs using an established 3D sprouting assay [41][42][43] because 3D culture is known to enhance the regenerative abilities of MSCs and more closely approximates the in vivo setting [44][45][46][47]. In multicellular spheroids, HUVECs and thymus MSCs are in a 3D, highcell-density aggregate during initial formation, facilitating direct contact and paracrine cell interactions.…”

CDH2 and CDH11 act as regulators of stem cell fate decisions

“…The use of MSC for tissue repair requires the migration and homing to the site of damaged tissue and it has been shown that both the migratory and proliferation potential of these cells are affected by CDH2 and CDH11 [100, 101]. MSCs have also been shown not only to have differentiation potential but also potent anti-inflammatory effects [102], which are enhanced when cultured as 3D spheroid aggregates [103, 104]. Interestingly, both CDH2 and CDH11 were shown to be critical in the response of synovial fibroblasts to inflammation [105, 106], suggesting that cadherins may also be important in mediating the anti-inflammatory effects of MSC.…”