Survival of porcine mesenchymal stem cells over the alginate recovered cellular method

Abstract

Although previous studies have established alginate as a three-dimensional scaffold for chondrogenic differentiation of mesenchymal stem cells (MSCs), little is known about the cytotoxic effects of the polymerizing and chelating reagents used in the alginate recovered cellular method. Swine MSCs (sMSC) were suspended in the polymerizing agent, CaCl(2), and viability was assessed using a trypan blue exclusion assay at intervals over 2 h. MSCs were also suspended in two different chelating agents, 55 mM sodium citrate and 50 mM EDTA, and viability was calculated. Finally, sMSCs and human MSCs were encapsulated and cultured in vitro. The sMSC were collected at day 4 and the cells were recovered by chelation. Encapsulated hMSCs were cultured with TGFβ3 and IGF-1 and assayed by qRT-PCR for collagen types I and II and sox9 with encapsulated human dermal fibroblasts and chondrocytes as controls recovered on days 0, 7, and 14. Only 53% of sMSCs were viable after 10 min in CaCl(2), the standard polymerization period, whereas 94% of encapsulated sMSCs in polymerized alginate were alive at 15 days. After 20 min in chelating agents, sodium citrate and EDTA, only 46% and 17% of the cells, were viable, respectively. The alginate recovered sMSCs yielded 12% survival. Human MSCs upregulated cartilage-specific collagen type II over the 14-day culture. Collagen I remained unchanged in the hMSCs. Alginate is a suitable agent for chondrodifferentiation of MSCs and can be dissolved by chelation, but the agents involved in hydrogel polymerization and cell recovery should be altered to improve MSC survival.

A three-dimensional dynamic coculture system enabling facile cell separation for chondrogenesis of mesenchymal stem cells

“…This is consistent with our previous finding, wherein coculture of MSCs with ACs downregulated production of cartilaginous ECM [29]. Additionally, cells could be recovered from alginate gel biocompatibly with sodium citrate for additional analysis and other applications [38]. When this coculture system was applied to induce the chondrogenic differentiation of rMSCs, a better chondrogenesis was achieved based on GAG quantification.…”

Encapsulated mesenchymal stromal cells for in vivo transplantation

“…Proliferation was assessed on days 2, 5, 7, 10, 14, 18 and 21 post encapsulation as previously described (Cohen et al 2010). Briefly, capsules from three samples per condition were counted, subsequently dissociated in 1% EDTA (Sigma Aldrich) for 10 min and centrifuged at 400 g for 5 minutes.…”

The stimulation of the cardiac differentiation of mesenchymal stem cells in tissue constructs that mimic myocardium structure and biomechanics

“…0.047″, charged with +18 kV) into a T-25 flask that was placed on an aluminum plate charged with -10 kV. The electrosprayed MSCs were divided into three parts for 1) staining with Trypan Blue to investigate if electrical treatment under high voltage causes cell death [39]; 2) seeding in a 96-well culture plate to determine growth rate and gene expression of the as-electrosprayed cells; and 3) seeding into a flask to expand cells for evaluating if electrospraying affects longer-term cell growth and multipotency. MSCs followed the same treatment process but without applying an electrical field, were used as the control.…”