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Sizes and Sufficient Quantities of MSC Microspheres for Intrathecal Injection to Modulate Inflammation in Spinal Cord Injury.

Microencapsulation of mesenchymal stem cells (MSC) in alginate facilitates cell delivery, localization and survival, and modulates inflammation in vivo. However, we found that delivery of the widely used ~0.5 mm diameter encapsulated MSC (eMSC) by intrathecal injection into spinal cord injury (SCI) rats was highly variable. Injections of smaller (~0.2 mm) diameter eMSC into the lumbar spine were much more reproducible and they increased the anti-inflammatory macrophage response around the SCI site. We now report that injection of small eMSC >2 cm caudal from the rat SCI improved locomotion and myelin preservation 8 weeks after rat SCI versus control injections. Because preparation of sufficient quantities of small eMSC for larger studies was not feasible and injection of the large eMSC is problematic, we have developed a procedure to prepare medium-sized eMSC (~0.35 mm diameter) that can be delivered more reproducibly into the lumbar rat spine. The number of MSC incorporated/capsule in the medium sized capsules was ~5-fold greater than that in small capsules and the total yield of eMSC was ~20-fold higher than that for the small capsules. Assays with all three sizes of eMSC capsules showed that they inhibited TNF-α secretion from activated macrophages in co-cultures, suggesting no major difference in their anti-inflammatory activity in vitro. The in vivo activity of the medium-sized eMSC was tested after injecting them into the lumbar spine 1 day after SCI. Histological analyses 1 week later showed that eMSC reduced levels of activated macrophages measured by IB4 staining and increased white matter sparing in similar regions adjacent to the SCI site. The combined results indicate that ~0.35 mm diameter eMSC reduced macrophage inflammation in regions where white matter was preserved during critical early phases after SCI. These techniques enable preparation of eMSC in sufficient quantities to perform pre-clinical SCI studies with much larger numbers of subjects that will provide functional analyses of several critical parameters in rodent models for CNS inflammatory injury.