Immunomodulatory human mesenchymal stromal cells (hMSC) have been incorporated into therapeutic protocols to treat secondary inflammatory responses post-spinal cord injury (SCI) in animal models. However, limitations with direct hMSC implantation approaches may prevent effective translation for therapeutic development of hMSC infusion into post-SCI treatment protocols. To circumvent these limitations, we investigated the efficacy of alginate microencapsulation in developing an implantable vehicle for hMSC delivery. Viability and secretory function were maintained within the encapsulated hMSC population, and hMSC secreted anti-inflammatory cytokines upon induction with the pro-inflammatory factors, TNF-α and IFN-γ. Furthermore, encapsulated hMSC modulated inflammatory macrophage function both in-vitro and in-vivo, even in the absence of direct hMSC-macrophage cell contact and promoted the alternative M2 macrophage phenotype. In-vitro, this was evident by a reduction in macrophage iNOS expression with a concomitant increase in CD206, a marker for M2 macrophages. Finally, Sprague-Dawley rat spinal cords were injured at vertebra T10 via a weight drop model (NYU model) and encapsulated hMSC were administered via lumbar puncture 24 hours post- injury. Encapsulated hMSC localized primarily in the cauda equina of the spinal cord. Histological assessment of spinal cord tissue 7 days post SCI indicated that as few as 5×104 encapsulated hMSC yielded increased numbers of CD206-expressing macrophages, consistent with our in-vitro studies. The combined findings support the inclusion of immobilized hMSC in post-CNS trauma tissue protective therapy, and suggest that conversion of macrophages to the M2 subset is responsible, at least in part, for tissue protection.

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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.

Anti-fibrotic and tissue regenerative mesenchymal stromal cell (MSC) properties are largely mediated by secreted cytokines and growth factors. MSCs are implanted to augment joint cartilage replacement and to treat diabetic ulcers and burn injuries simultaneously with local anesthetics, which reduce pain. However, the effect of anesthetics on therapeutic human MSC secretory function has not been evaluated. In order to assess the effect of local anesthetics on the MSC secretome, a panel of four anesthetics with different potencies — lidocaine, procaine, ropivacaine and bupivacaine — was evaluated. Since injured tissues secrete inflammatory cytokines, the effects of anesthetics on MSCs stimulated with tumor necrosis factor (TNF)-α and interferon (IFN)-γ were also measured. Dose dependent and anesthesia specific effects on cell viability, post exposure proliferation and secretory function were quantified using alamar blue reduction and immunoassays, respectively. Computational pathway analysis was performed to identify upstream regulators and molecular pathways likely associated with the effects of these chemicals on the MSC secretome. Our results indicated while neither lidocaine nor procaine greatly reduced unstimulated cell viability, ropivacaine and bupivacaine induced dose dependent viability decreases. This pattern was exaggerated in the simulated inflammatory environment. The reversibility of these effects after withdrawal of the anesthetics was attenuated for TNF-α/IFN-γ-stimulated MSCs exposed to ropivacaine and bupivacaine. In addition, secretome analysis indicated that constitutive secretion changes were clearly affected by both anesthetic alone and anesthetic plus TNFα/IFNγ cell stimulation, but the secretory pattern was drug specific and did not necessarily coincide with viability changes. Pathway analysis identified different intracellular regulators for stimulated and unstimulated MSCs. Within these groups, ropivacaine and bupivacaine appeared to act on MSCs similarly via the same regulatory mechanisms. Given the variable effect of local anesthetics on MSC viability and function, these studies underscore the need to evaluate MSC in the presence of medications, such as anesthetics, that are likely to accompany cell implantation.