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In cell-based therapies, cells are removed from their crowded physiological context and expanded in a dilute culture medium. In this way, the enzymatic reactions involved in the conversion of pro-collagen to collagen are slowed, leading to a decrease in the deposition of extracellular matric (ECM). [1,2] Macromolecular crowding (MMC), is a biophysical approach that can be used to imitates the dense extracellular space, accelerating the deposition of the ECM in vitro and mimicking the in vivo environment. [3,4]
Although, MMC has been shown consistently to enhance and accelerate ECM deposition, the ideal crowding agent is still elusive. Thus, we assessed the potential of carrageenan, a sulfated polysaccharides and Ficoll™ 70 kDa and Ficoll™ 400 kDa cocktail, a neutral polysaccharides as MMC agents.
The crowders effect in vitro was evaluated in human Bone Marrow Derived Stem Cells (hBMSC) after 5, 8 and 11 days of cells culture. A non crowding condition was used as a control group.
The crowders did not affect cell morphology and did not compromise cell viability over the assessed timepoints. Even though, cell metabolic activity using alamarBlue® assay showed a decrease in cell metabolic activity when cells were cultured with carrageenan and Ficoll™ 70 kDa and Ficoll™ 400 kDa cocktail compare to the control group.
SDS-PAGE and corresponding densitometry analysis showed that in all the timepoints both the crowders enhanced the deposition of collagen type I compare to the control group. However, carrageenan induced higher collagen type I deposition compare to the control group and Ficoll™ 70 kDa and Ficoll™ 400 kDa cocktail in all the timepoints. Immunocytochemistry and complementary relative fluorescence intensity analysis for collagen type I and collagen type III showed that both the crowders exibited collagen type I and collagen type III deposition in the timepoints assessed.
The data suggested the importance of the MMC in cell culture and for possible tissue engineering applications and the potential of carrageenan as macromolecolar crowders agents. Further analysis will be conducted to analyse the effect of MMC on the genome and proteome in hBMSC.
1. B. Goldberg, Kinetics of processing of type I and type III procollagens in fibroblast cultures, Proc. Natl. Acad. Sci. U. S. A. 74 (8) (1977) 3322–3325.
2. W.W. Kao, D.J. Prockop, R.A. Berg, Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells, J. Biol. Chem. 254 (7) (1979) 2234–2243.
3. Kumar, P., et al., Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies. Sci Rep, 2015. 5: p. 8729.
4. Chen, C., et al., Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies. Adv Drug Deliv Rev, 2011. 63(4-5): p. 277-90
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