(19h) Layer-By-Layer Assemblies of Collagen/Heparin Towards the Manufacturing of Human Mesenchymal Stem Cells

Stem cell transplantation is a promising treatment for multiple diseases or disorders. However, challenges associated with large-scale manufacturing severely limit these therapies. Therefore, there is a need for alternatives to promote the production of stem cells with high therapeutic value. In this project, multilayers of collagen/heparin (COL/HEP) were used as a bioactive surface to enhance human mesenchymal stem cell behavior. Multilayers varying the final layer between COL (6 bilayers) and HEP (6.5 bilayers) were fabricated. The effect of interferon gamma (IFN-g) in the culture medium was also evaluated. In-vitro studies consisted on evaluating the cellular adhesion and morphological characteristics, proliferation response, and protein expression of human mesenchymal stem cells (hMSCs) seeded onto the multilayers. Infrared Variable Angle Spectroscopic Ellipsometry confirmed the physical-chemical properties of the multilayers fabricated on the substrate. Characteristic peaks for COL and HEP could be observed in the spectrums obtained at 25 °C (temperature of fabrication of multilayers) and 37°C (temperature of incubation). IRVASE reported an average thickness of 129 nm for 6 bilayers and 178 nm for 6.5 bilayers. For roughness, 6 bilayers presented an average of 168 nm and 6.5 bilayers a value of 230 nm. The cells’ morphological characteristics evaluated by confocal microscopy showed that the multilayers support a more well developed and spread morphology. Addition of IFN-g promoted cell elongation. For the proliferation response, an EDU Imaging Kit was used. The results % of positive EDU cells showed that hMSCs seeded onto multilayers ending in HEP (6.5 bilayers) reported a 34%, 6 bilayers a 33% and tissue culture plastic (TCP) a 30%. In the conditions supplemented with IFN-g, a decrease in proliferation was observed, obtaining a decrease of 3% in multilayers ending in HEP, 30% in multilayers ending in COL and 27% to hMSCs seeded on TCP. Luminex® assay was used to detect multiple human biomarkers including vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF-2), colony stimulating factor 1 (M-CSF), interleukin 10 (IL-10) and interleukin 6 (IL-6). Multilayers ending in HEP supplemented with IFN-g in the culture medium exhibited a greater stimulation on the protein expression of hMSCs. In conclusion, 6.5 bilayers of collagen/heparin (bilayers ending in HEP) is a surface that promotes better manufacturing of healthy cells than those seeded in conventional surfaces like TCP, presenting the possibility of offering regenerative therapies using cells with better quality to maximize safety and efficacy in clinical trials.