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

Abstract

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.

Keywords: Microcapsules; alginate; inflammation; intrathecal; mesenchymal stem cells.

Fig. 1

MRI visualization of ChMNP capsules in rat spinal cords without and with contusion. Noncontused rats without (a) or with (b)–(e) intrathecal injection of ChMNP capsules were imaged at indicated times after injection. In the axial image, without ChMNP capsules note the gray region in panel A (arrow) corresponding to the spinal cord and the bony vertebral body, which is marked by a thin arrow (a) and at higher magnification in the insert. Signals (black) were detected with ChMNP capsules after 2 days (b) and they persisted for 42 days (c) in axial images. Horizontal images at 7 days (d1–d7) show localization of ChMNP capsules along the spinal axis (bracket in d1) localized in serial sections from dorsal to ventral encompassing ~ 2.2 mm. A 3D reconstruction of super-threshold MRI signals superimposed on an X-ray (e) shows a ventrolateral distribution, which is within the ventral distribution of ChMNP particles in brown (e′) in a bright field image of the same spinal cord after dissection from the spine. SCI Rats at 42 days after intrathecal injection of ChMNP capsules without (f, f′) or with (g, g′) eMSC were imaged (n = 2, 3, respectively). A bracket marks the SCI site at T9-10 in the horizontal image (f) with signals of ChMNP particles in dorsal regions of T13- L2; the signal extends more caudally to L3–4 (f′) but was primarily ventral (not shown). Horizontal image (g) with signals of ChMNP particles in eMSC in ventral regions of L3–L5; note the signal is in the spine above the vertebral body (g′).

Fig. 2

Effects of small eMSC on recovery after SCI. (a) Post-injury locomotor function assessed using BBB scoring was done weekly for 8 weeks by trained observers in SCI rats injected with 5 × 10⁴ free human MSC (F-MSC), small encapsulated MSC (3 × 104), or saline 1 day after SCI. Data is represented as mean ± SD. The eMSC (n = 5) showed consistently higher scores than F-MSC (n = 5) and saline group (n = 6) and attained significance at week 7 and 8 (*, p < 0.05 ANOVA repeated measures with Tukey HSD test). (b) Percent preserved white matter at 8 weeks after SCI was calculated by measuring ECR staining in cross sections from rats treated with saline (n = 4), F-MSC (n = 5) and eMSC (n = 3) at the indicated positions relative to the injury epicenter; (NC, normal control). Significant differences were detected in the percent of preserved white matter between eMSC versus saline groups (*, p < 0.05) or versus F-MSC (#, p < 0.05).

Fig. 3

Efficiency of eMSC ejection through narrow bore needles. Graph shows the effect of capsule size on eMSC ejection yield (%, mean ± SD) in 2–3 experiments for three different eMSC sizes (small ~ 0.2 mm, medium ~ 0.35 mm and large ~ 0.5 mm). The ejection yield with larger capsules was much lower than the small and medium size capsules due to aggregation (see text). The inserts show tips of 0.17 inner diameter needles with blunt or beveled ends, which gave large- and medium-sized capsules, respectively.

Fig. 4

Different sizes of eMSC reduce TNF-α secretion when cultured with activated macrophages. Culture supernatants collected from transwell co-cultures of LPS-activated macrophages with eMSC at 1:1 ratio were assayed for TNF-α by ELISA and normalized to levels in LPS-activated macrophages supernatants without added eMSC. Differences between the LPS only control and each of the three different sized eMSC were significant (*, p < 0.05) and the difference between the medium-sized capsules and the large and small capsules was also significant (#, p < 0.05).

Fig. 5

Effect of medium-size eMSC transplantation on activated macrophages in SCI. Tissues from SCI rats one week after injection with ~ 0.35 mm eMSC (d)–(f) or saline as a control (a)–(c) were cryosectioned in cross sections and were incubated with Isolectin IB4 Alexa-488 conjugate. Confocal imaging showed more robust staining in control than eMSC treated sections in white matter and gray matter regions. Quantitation (g) of IB4 staining regions in equivalent dorsal midline circles drawn in spinal cord cross sections (for example, C′ and F′ are magnified images from C and F, respectively) were measured as super-threshold areas at ~2.2 mm caudal to the injury epicenter. IB4 levels decreased significantly (*, p < 0.05) in the eMSC group (n = 3) as compared to the control (n = 4) group. Scale bars are 500 μm in panels (a)–(f) and 100 μm in panels C′ and F′.

Fig. 6

Medium-size eMSC increased white matter sparing. Spinal cord cross sections in regions adjacent to those analyzed in Fig. 5 from spinal cord contusion control (a)–(c) and ~0.35 mm eMSC (d)–(f) injected groups were stained for ECR. The percent of white matter (g) is significantly higher in eMSC group (n = 7) as compared to control group (n = 3) (*, p < 0.05).