WEB Evaluation of microcarriers’ functionalization to promote hMSC amplification.
Several clinical studies have reported the benefit of the administration of Mesenchymal stem cells (MSC) in various cell therapies. However, these studies have also highlighted that their routine applications need urgently new substrates to amplify MSC in vitro in GMP conditions. Indeed, MSC are scarce in the human body compared to the total dose requested in cell therapy. It is therefore critical to amplify MSC in vitro to achieve clinically relevant cell doses. If microcarriers are attractive substrates for this purpose regarding volume to surface area ratio, MSC cultivation on currently available microcarriers raises some concerns due to the use of proteolytic treatments for harvesting cells, i.e. negative impact on cell viability and potential cell phenotype modification.
The overall development strategy relies upon the surface properties’ optimization of microcarriers promoting MSC culture in vitro. To achieve this purpose, two microcarrier families, Dextran-based Cytodex I® and Polystyrene microcarriers have been functionalized by grafting a thin polymer layer either through a two-step reaction (i.e. surface activation prior to polymerization) or physisorption (PS-based microcarriers only). This “smart” polymer layer has been mainly composed of poly N-IsoPropylAcrylaMide (pNIPAM). This polymer has been studied to promote the adhesion and spreading of MSC, but also to control their detachment following a decrease in temperature.
Several analytical techniques have allowed us to analyze in-depth the new matrix generated on the microcarrier, among others ToF-SIMS and NanoSIMS, AFM, NMR and Fluorescence Spectroscopy. These characterizations have confirmed the success of the pNIPAM grafting two-steps reaction on both type of carriers, but also the physical adsorption on the PS-based surface.
In order to optimize the composition of these pNIPAM-microcarriers, we have correlated the structural and chemical modifications made on these particles with the behavior of L929 fibroblasts cultivated on them and use as MSC’s model. Cell viability on microcarriers was measured using the Hoechst nuclei staining and MTT bioassay. Cell thermal detachment, performed at 20°C, was monitored under fluorescent microscopy and image data analysis after digitalization. Preliminary results have indicated that cell culture performance are correlated to the pNIPAM layer specificities i.e. molecular weight (Mw), chains surface density and chemical composition.