Controlled Presentation of Cytokines within 3D Mesenchymal Stem Cell Constructs Enhances Immunomodulatory Activity

            Mesenchymal
stem cells (MSCs) offer a potent cell therapy for the treatment of inflammatory
and immune disorders due to their ability to modulate components of innate and
adaptive immunity. Although MSCs have been investigated as cell therapies for diseases
including graft-versus-host, type I diabetes, and inflammatory bowel disease
(IBD), experimental results thus far have been inconsistent. Recent studies
have suggested that MSC immunomodulation can be greatly influenced by the
cellular microenvironment and the concentration of inflammatory cytokines,
including IFNγ and TNFα, which stimulate MSC immunomodulation. The
low levels of cytokines observed in chronic inflammation may not be sufficient
to induce MSC immunomodulation, limiting the ability of MSCs to regulate
inflammation in these disease states. Engineering the local microenvironment
within MSC constructs via biomaterial-based presentation of inflammatory
cytokines may serve as a novel means of regulating MSC immunomodulatory
paracrine secretion in vivo. Therefore, the objective of this study was to regulate human MSC
immunomodulation via the incorporation of immobilized-cytokine microparticles
within 3D MSC spheroids.

            TNFα was
immobilized on the surface of microparticles by incubating streptavidin-coated
polystyrene microparticles (~3μm diameter) with 100 ng biotinylated TNFα per 9x10⁵
particles overnight based on the number of streptavidins on the surface of the
polystyrene microparticles (1.2x10⁶ streptavidin-molecules per
microparticle). After 18 hours of incubation, the supernatant was analyzed via
ELISA for remaining TNFα to quantify binding to the particles. Microparticles
with immobilized TNFα were subsequently suspended in PBS and incubated for
9 days to determine the stability of cytokine immobilization. Additionally, microparticles
with or without immobilized-TNFα were incorporated into 3D MSC spheroids
(6:1 microparticles-to-MSCs) by addition of particles and cells into Aggrewell?
microwells to form 500-cell MSC spheroids. After 18 hours of formation, microparticle-incorporated
MSC spheroids were transferred into rotary orbital suspension culture (65 rpm) and
cultured for 4 days. A subset of spheroids without microparticles were also
treated with soluble TNFα (10ng/mL). Immunomodulatory factor release (PGE2,
TGFβ1, IL6) from MSC spheroids was quantified by ELISAs and normalized to
cell numbers. Finally, MSC spheroids were co-cultured with IFNγ- and lipopolysaccharide-activated
macrophages in 0.4 µm pore transwells for 16 hours and conditioned media was
collected to determine macrophage expression of TNFα.

The loading
efficiency of streptavidin-microparticles was determined to be approximately 47%
(1.7x10^-4 ng TNFα per microparticle). Furthermore, less than 3%
of the microparticle bound TNFα was released from the microparticles over
9 days of culture. Incorporation of microparticles with immobilized cytokines
within MSC spheroids increased the secretion of the immunomodulatory factors PGE2
(2.2-fold, p < 0.001) and IL-6 (3.6-fold, p < 0.001) compared to
non-treated controls. This increase was comparable to treatment with soluble
TNFα, demonstrating the cytokine remained bioactive after immobilization
on the surface of the microparticles. Finally, greater suppression of the
inflammatory response of IFNγ and LPS-activated macrophages was observed
in co-cultures of MSC spheroids incorporated with immobilized-cytokine
microparticles (71% reduction of macrophage TNFα secretion) compared to
untreated spheroids (42% reduction). These results demonstrate the ability to
immobilize bioactive cytokines to the surface of microparticles that can be
stably incorporated in MSC aggregates to enhance immunomodulation.