Abstract 4
Introduction
Microglia, the primary resident immune cells in the brain, are activated in neurodegenerative diseases. Activated microglia release pro‐inflammatory cytokines that can further damage the central nervous system. Due to their immunomodulatory/immunosuppressive properties, mesenchymal stromal cells (MSCs) are being widely tested as a cellular therapy for neuro‐inflammatory conditions. However, due to the unique characteristics of each MSC line, it is important to establish robust assays to demonstrate potency of each lot manufactured for clinical use.
Objective
We set out to establish robust and reliable methods to evaluate the immunosuppressive capacity of human umbilical cord tissue‐derived MSC lines. Using Immortalized MicroGlial Cell Line (IMG), we developed a simple and reliable assay to determine MSC effectiveness in suppressing microglial activation.
Methods
For IMG culture assays, MSCs were first plated in a 24‐well plate and incubated for 24 hours. Activated IMG were then added, and the cells were incubated for another 24 hours. The supernatant was then collected for ELISA to detect the release of mouse tumor necrosis factor alpha (TNFα). For primary microglial culture assays, cells were plated in a 24‐well plate for 2 days before treating with lipopolysaccharide for 1 hour. MSCs were then added. The supernatant was collected after 24 hours for ELISA for the detection of TNFα. For slice cultures, 12 hours of lysophosphatidylcholine (LPC) treatment induced extensive microglia activation. After washing off LPC, MSCs were added. Five hours after the MSC treatment, changes in microglia morphology were evaluated using confocal microscopy.
Results
MSCs significantly reduced the production of mTNFα in IMG cultures (P < .0001) and in primary microglia cultures (P < .0001) (Fig. 1). In slice cultures, LPC treatment resulted in microglia activation. MSC treatment rescued microglia from amoeboid morphology back to a calm, ramified resting stage. Sholl analysis confirmed this observation, demonstrating a significantly increased number of microglia projections after MSC treatment compared with the LPC group (P < .001).
Figure 1.
MSCs suppress microglia activation in IMG assays. MSCs reduced the release of TNFα after LPS stimulation (t‐test)
Abbreviations: IMG, Immortalized MicroGlial Cell Line; LPS, lipopolysaccharide; MSCs, mesenchymal stromal cells; TNFα, tumor necrosis factor alpha
Discussion
All three assays were effective in quantifying the inhibitory effects of MSCs on activated microglia. Among them, the IMG assay is least time‐consuming and, thus, is most suitable for large‐scale screening. Additionally, IMG cells can be expanded and cryopreserved, creating a reproducible source of material for screening MSC lines.