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.
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