Identification of IL‐1β and LPS as optimal activators of monolayer and alginate‐encapsulated mesenchymal stromal cell immunomodulation using design of experiments and statistical methods

Abstract

Induction of therapeutic mesenchymal stromal cell (MSC) function is dependent upon activating factors present in diseased or injured tissue microenvironments. These functions include modulation of macrophage phenotype via secreted molecules including prostaglandin E2 (PGE2). Many approaches aim to optimize MSC-based therapies, including preconditioning using soluble factors and cell immobilization in biomaterials. However, optimization of MSC function is usually inefficient as only a few factors are manipulated in parallel. We utilized fractional factorial design of experiments to screen a panel of 6 molecules (lipopolysaccharide [LPS], polyinosinic-polycytidylic acid [poly(I:C)], interleukin [IL]-6, IL-1β, interferon [IFN]-β, and IFN-γ), individually and in combinations, for the upregulation of MSC PGE2 secretion and attenuation of macrophage secretion of tumor necrosis factor (TNF)-α, a pro-inflammatory molecule, by activated-MSC conditioned medium (CM). We used multivariable linear regression (MLR) and analysis of covariance to determine differences in functions of optimal factors on monolayer MSCs and alginate-encapsulated MSCs (eMSCs). The screen revealed that LPS and IL-1β potently activated monolayer MSCs to enhance PGE2 production and attenuate macrophage TNF-α. Activation by LPS and IL-1β together synergistically increased MSC PGE2, but did not synergistically reduce macrophage TNF-α. MLR and covariate analysis revealed that macrophage TNF-α was strongly dependent on the MSC activation factor, PGE2 level, and macrophage donor but not MSC culture format (monolayer versus encapsulated). The results demonstrate the feasibility and utility of using statistical approaches for higher throughput cell analysis. This approach can be extended to develop activation schemes to maximize MSC and MSC-biomaterial functions prior to transplantation to improve MSC therapies.

Donor variability among anti-inflammatory pre-activated mesenchymal stromal cells

“…We previously identified IL-1β and LPS, individually and in combination, as optimal inducers of MSC PGE2 secretion and subsequent attenuation of M1 macrophage TNF-α secretion 56 . In order to optimize the concentration of each of these activating factors for maximal PGE2 secretion, donor 1 MSCs were incubated with increasing doses of IL-1β (0.1, 1, or 10 ng/mL) and/or LPS (10, 100, 1000 ng/mL) and PGE2 secretion was quantified after 6, 24, and 48 hours.…”

“…CD14 + monocytes were isolated from human peripheral blood from healthy adult donors (The Blood Center of New Jersey, East Orange, NJ; New York Blood Center, Long Island City, NY), differentiated to generate macrophages as described previously 56 , and cryopreserved. Macrophages at passage 1 were thawed, seeded into 24-well plates at 5 × 10 4 cells/well, and allowed to attach overnight.…”

“…The nuclei of fixed monolayer MSCs stained with Hoechst and counted via microscopic imaging, as previously described 56 . Alternatively, cell culture supernatants were collected, stored at −80°C, and replaced with fully supplemented RPMI 1640 medium containing Alamar blue (Molecular Probes, Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.…”

“…We previously identified interleukin (IL)-1β and lipopolysaccharide (LPS) as optimal activators of MSC prostaglandin E2 (PGE2) secretion and subsequent attenuation of inflammatory M1 macrophage tumor necrosis factor (TNF)-α secretion using a flexible high throughput design of experiments approach 56 . The current studies were designed to develop a more controlled approach to optimize pre-activating agents for the functional stimulation of MSCs from 6 different donors.…”

The effect of local anesthetic on pro-inflammatory macrophage modulation by mesenchymal stromal cells

“…For comparative purposes, LA and LPS concentrations were selected based on previous in vitro studies performed by our group and others. 6, 7, 15, 18-23 .…”