In this study, multilayered films of polyethylenimine/poly (sodium-p-styrene sulfonate) (PEI)/(PSS) and type I collagen/heparin sodium (COL)/(HEP) were fabricated using the layer-by-layer technique, and fully characterized using Infrared Variable Angle Spectroscopic Ellipsometry (IRVASE) to simultaneously analyze the chemistry, thickness, and roughness of the multilayers with respect to changes in pH of the washing solution, and changes in temperature. Film topography and Young’s modulus were obtained by atomic force microscopy (AFM) and nanoindentation. Our results show that with IRVASE it is possible to analyze the thickness of the multilayers prepared using a washing solution of pH 5, obtaining values of 71.7 nm and 40.3 nm for three bilayers of PEI/PSS and COL/HEP, respectively. Film roughness varies between multilayer systems, obtaining values of 37.76 nm for three bilayers of PEI/PSS and 33.58 nm for three bilayers of COL/HEP. Increasing the pH of the washing solution for PEI/PSS yielded thinner films that were less susceptible to thermal induced changes in film chemistry in the range of 25 – 150 °C. PEI/PSS films decreased in thickness with increasing temperature up to 75 °C, whereas above 75 °C film thickness increased. Through IRVASE, a transition temperature for the PEI/PSS multilayers was observed at 75 °C. Temperatures above 37 °C drastically alter the chemistry and the thickness of the COL/HEP multilayers indicating a possible degradation of the polymers. We obtained, through nanoindentation, a Young’s modulus of 15000 kPa and 9000 kPa for 12 bilayers of PEI/PSS and COL/HEP, respectively. These results demonstrate that, using IRVASE, we can simultaneously evaluate the physical, chemical, and thermal properties of synthetic and natural multilayered polymeric films.

In this work, we evaluate the enhancing effect of six bilayers of heparin/collagen (HEP/COL)6 layer-by-layer coatings on human Schwann cell (hSCs) adhesion and proliferation in the presence or absence of...

Thin films are of interest in materials design because they allow for the modification of surface properties of materials while the bulk properties of the material are largely unaffected. In this work, we outline methods for the assembly of thin films using a technique known as layer-by-layer (LbL). Furthermore, their interactions with human mesenchymal stromal cells (hMSCs) are discussed. hMSCs are a subject of growing interest because of their potential to treat or cure diseases, given their immunosuppressive properties, multipotent differentiation capabilities, and tissue regeneration capabilities. Numerous improvements and modifications have been suggested for the harvesting, treatment, and culture of hMSCs prior to their administration in human subjects. Here, we discuss methods to assess the interactions of hMSCs with thin LbL-assembled films of heparin and collagen. Three different methods are discussed. The first details the preparation of heparin/collagen multilayers on different surfaces and the seeding of cells on these multilayers. The second method details the characterization of multilayers, including techniques to assess the thickness, roughness, and surface charge of the multilayers, as well as in situ deposition of multilayers. The third method details the analysis of cell interactions with the multilayers, including techniques to assess proliferation, viability, real-time monitoring of hMSC behavior, analysis of hMSC-adhesive proteins on the multilayers, immunomodulatory factor expression of hMSCs, and cytokine expression on heparin/collagen multilayers. We propose that the methods described in this work will assist in the design and characterization of LbL-assembled thin films and the analysis of hMSCs cultured on these thin films.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for cell therapies due to their immunosuppressive capacity that can be enhanced in the presence of interferon-gamma (IFN-γ). In this study, multilayers of heparin (HEP) and collagen (COL) (HEP/COL) were used as a bioactive surface to enhance the immunomodulatory activity of hMSCs using soluble IFN-γ. Multilayers were formed, via layer-by-layer assembly, varying the final layer between COL and HEP and supplemented with IFN-γ in the culture medium. We evaluated the viability, adhesion, real-time growth, differentiation, and immunomodulatory activity of hMSCs on (HEP/COL) multilayers. HMSCs viability, adhesion, and growth were superior when cultured on (HEP/COL) multilayers compared to tissue culture plastic. We also confirmed that hMSCs osteogenic and adipogenic differentiation remained unaffected when cultured in (HEP/COL) multilayers in the presence of IFN-γ. We measured the immunomodulatory activity of hMSCs by measuring the level of indoleamine 2,3-dioxygenase (IDO) expression. IDO expression was higher on (HEP/COL) multilayers treated with IFN-γ. Lastly, we evaluated the suppression of peripheral blood mononuclear cell (PBMC) proliferation when co-cultured with hMSCs on (HEP/COL) multilayers with IFN-γ. hMSCs cultured in (HEP/COL) multilayers in the presence of soluble IFN-γ have a greater capacity to suppress PBMC proliferation. Altogether, (HEP/COL) multilayers with IFN-γ in culture medium provides a potent means of enhancing and sustaining immunomodulatory activity to control hMSCs immunomodulation.