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.
Interferon-gamma (IFN-γ) plays a vital role in modulating the immunosuppressive properties of human mesenchymal stem/stromal cells (hMSCs) used in cell therapies. However, IFN-γ suffers from low bioavailability and degrades in media, creating a challenge when using IFN-γ during the manufacturing of hMSCs. Metal−organic frameworks (MOFs), with their porous interiors, biocompatibility, high loading capacity, and ability to be functionalized for targeting, have become an increasingly suitable platform for protein delivery. In this work, we synthesize the MOF PCN-333(Fe) and show that it can be utilized to immobilize and deliver IFN-γ to the local extracellular environment of hMSCs. In doing so, the cells proliferate and differentiate appropriately with no observed side effects. We demonstrate that PCN-333(Fe) MOFs containing IFN-γ are not cytotoxic to hMSCs, can promote the expression of proteins that play a role in immune response, and are capable of inducing indoleamine 2,3-dioxygenase (IDO) production similar to that of soluble IFN-γ at lower concentrations. Overall, using MOFs to deliver IFN-γ may be leveraged in the future in the manufacturing of therapeutically relevant hMSCs.
Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN‐γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance (QCM), and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring the level of indoleamine 2,3-dioxygenase (IDO) secretion. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.
1,2 This grew into an interest in the 1970s by Friedenstein and contemporaries after studying the bone marrow. 3 MSCs are an excellent candidate for cell therapy because of having intrinsic differentiation potentials into bone, cartilage and fat cells not found previously in other cells. They can also be isolated and expanded easily in vitro, and produce abundant useful growth factors and cytokines. 2,4 Mesenchymal stem cells have become the top used stem cell type for clinical application with encouraging results. 5,6 Significant progress has been made in stem cell research in recent years. The main therapeutic effects of MSCs are now attributed to the stimulation of several innate repair processes in injured tissues in vivo by secreted factors as well as the immunomodulation response. Therefore, MSC therapy is expected to find clinical application in human diseases. 6,7 However, there are some critical issues that need to be addressed before MSCs can be used for clinical therapy in humans which will help determine the efficiency of cells administered to the patients as a therapeutic approach, most important of which is immunosuppressive properties. 8,9 MSCs can be affected by tumor support that may affect potential tumorigenesis after MSCs transplantation, including different donors and tissues, inconsistent protocols, varying dosages and differing transfusion patterns, and mechanisms that control the behavior of the MSCs at the target site. 10,11 Because of their unique features, stem cells are undoubtedly a great hope for the treatment of many diseases. More research is developing on the potential long-term risks associated with MSC therapy. However, Additional studies would also be a major contribution to stem cell biology in general as well as their transplantation.
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