Novel Strategy to Enhance Human Mesenchymal Stromal Cell Immunosuppression: Harnessing Interferon-Gamma Presentation in Metal-Organic Frameworks Embedded on Heparin/Collagen Multilayers.

The immunomodulatory potential of human mesenchymal stromal cells (hMSCs) can be boosted when exposed to interferon-gamma (IFN-γ). While pretreating hMSCs with IFN-γ is a common practice to enhance their immunomodulatory effects, the challenge lies in maintaining a continuous IFN-γ presence within cellular environments. Therefore, in this research, we investigate the sustainable presence of IFN-γ in the cell culture medium by immobilizing it in water-stable metal-organic frameworks (MOFs) [PCN-333(Fe)]. The immobilized IFN-γ in MOFs was coated on top of multilayers composed of combinations of heparin (HEP) and collagen (COL) that were used as a bioactive surface. Multilayers were created by using a layer-by-layer assembly technique, with the final layer alternating between collagen (COL) and heparin (HEP). We evaluated the viability, differentiation, and immunomodulatory activity of hMSCs cultured on (HEP/COL) coated with immobilized IFN-γ in MOFs after 3 and 6 days of culture. Cell viability, compared to tissue culture plastic, was not affected by immobilized IFN-γ in MOFs when they were coated on (HEP/COL) multilayers. We also verified that the osteogenic and adipogenic differentiation of the hMSCs remained unchanged. The immunomodulatory activity of hMSCs was evaluated by examining the expression of indoleamine 2,3-dioxygenase (IDO) and 11 essential immunomodulatory markers. After 6 days of culture, IDO expression and the expression of 11 immunomodulatory markers were higher in (HEP/COL) coated with immobilized IFN-γ in MOFs. Overall, (HEP/COL) multilayers coated with immobilized IFN-γ in MOFs provide a sustained presentation of cytokines to potentiate the hMSC immunomodulatory activity.

Delivery of Immobilized IFN-γ With PCN-333 and Its Effect on Human Mesenchymal Stem Cells.

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.

Crosslinked Layered Surfaces of Heparin and Poly(L-Lysine) Enhance Mesenchymal Stromal Cell Behavior in the Presence of Soluble Interferon Gamma.

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, 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 significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. 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.

Immunomodulatory functions of human mesenchymal stromal cells are enhanced when cultured on HEP/COL multilayers supplemented with interferon-gamma.

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.

Methods for the Assembly and Characterization of Polyelectrolyte Multilayers as Microenvironments to Modulate Human Mesenchymal Stromal Cell Response.

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.

Optimization, synthesis, and characterization of coaxial electrospun sodium carboxymethyl cellulose-graft-methyl acrylate/poly(ethylene oxide) nanofibers for potential drug-delivery applications.

In this study, nanofiber drug carriers were fabricated via coaxial electrospinning, using a new, degradable core-shell nanofiber drug carrier fabricated via coaxial electrospinning. Fabrication of the shell was carried out by graft polymerization of sodium carboxymethyl cellulose (NaCMC) with methyl acrylate (TCMC) and poly(ethylene oxide) (PEO). Tetracycline hydrochloride (TCH) was used as a drug model incorporated within the nanofibers as the core, and their performance as a drug carrier scaffold was evaluated. The loading of TCH within PEO nanofibers and the loading of TCH within the TCMC nanofibers were characterized via different techniques. The structure morphology of the obtained nanofibers was viewed under scanning electron microscope (SEM). The changes in the polymer structure before and after grafting and confirmation of incorporation of the drug in the fibers were characterized by Fourier transform infrared spectroscopy (FT-IR). Response surface methodology (RSM) was applied to predict the optimum conditions for fabrication of the nanofibers. The cell viability of the optimized samples was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The TCH loaded into the optimized core-shell sample of TCMC 3% (w/v)/PEO 1% (w/v) had a smooth and beadless morphology with a diameter of 86.12nm, slow and sustained drug release, and excellent bactericidal activity against a wide range of bacteria. This shows promise for use as an antibacterial material in such applications as tissue engineering and pharmaceutical science.

Electrospinning of thermoplastic carboxymethyl cellulose/poly(ethylene oxide) nanofibers for use in drug-release systems.

Polymeric delivery systems using electrospun nanofibers have been developed for many applications, notably for drug delivery and tissue engineering in the healthcare industry. In this study, a nanofiber drug carrier was fabricated via two different electrospinning processes, blend and coaxial, using a new degradable thermoplastic carboxymethyl cellulose (TCMC) and poly(ethylene oxide) (PEO). The TCMC was synthesized by graft copolymerization with methyl acrylate. Tetracycline hydrochloride served as a drug model incorporated within these nanofibers and their performance as a drug-carrier scaffold was evaluated. The structure morphology of the obtained nanofibers was viewed under scanning electron microscope, and the changes of the structural of polymer before and after graft and confirmation of incorporation of the drug on the fibers was characterized by Fourier transform infrared spectroscopy. Response surface methodology was applied to predict the optimum condition for fabrication of the nanofibers. The drug-delivery profile showed that drug release from the optimized core-shell sample TCMC 3% (w/v)/PEO 1% (w/v) was much slower than for the blend nanofibers and was sustained for 72h with only 26.06% burst release within the first 30min. The drug-loaded TCMC 3% (w/v)/PEO 1% (w/v) core-shell sample nanofibers showed excellent bactericidal activity against a wide range of bacteria, indicating their potential as antibacterial materials in various applications such as tissue engineering and pharmaceutical science.