(J-382) Effects of storage time and temperature on mesenchymal stromal cell extracellular vesicle yield and modulation of microglia morphology

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are small lipid-bound structures secreted by MSCs. They have been found to facilitate tissue repair and regulate the immune responses of various disease-associated cell types. Despite their therapeutic potential, challenges in clinical translation arise due to functional variations from unstandardized manufacturing, storage and characterization practices. To advance the clinical application of MSC-EVs, it is crucial to identify the impact of storage conditions, such as temperature and time, on their yield and potency.

Disease-specific potency assays are critical for the comprehensive functional assessment of MSC-EVs. In this context, we have shown that microglia, the primary brain cells that respond to and drive neuroinflammation, demonstrate morphological shifts that reflect changes in their functionality. By treating microglia in the disease-associated morphology with MSC-EVs and comprehensively profiling their morphological shifts, we can assess the effects of different storage temperatures and times on the potency of MSC-EVs in a neuroinflammatory disease-specific context.

MSC-EVs were derived from MSCs (RoosterBio) cultured in serum-free media. Two rounds of ultracentrifugation (94,000 x g and 140,000 x g) were used to harvest the MSC-EVs from the media and the pellet was resuspended in PBS++ (Ca2+ and Mg2+). Fresh and boiled MSC-EVs (95°C at 15 minutes) were tested to determine baseline yield and potency. The remaining samples were aliquoted and stored at RT, 4°C, -20°C, and -80°C. Each MSC-EV formulation (at each temperature/time point) was characterized in terms of protein content, particle size and count, as well as microglia modulation (morphological potency assay). 

A MicroBCA protein assay (Thermofisher) was used to evaluate changes in protein concentration every week. C20 immortalized human microglia cells were seeded at a density of 480 cells/well in 96-well plates. After 24 hours, cells underwent a media change either with or without 5ng/mL of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) to mimic neuroinflammation. Activated (+CTL) cells were then simultaneously treated with MSC-EVs stored at the 4 different temperatures. Twenty-four hours post-treatment, the cells were fixed with 4% paraformaldehyde (PFA) and stained with Hoechst (nucleus), wheat germ agglutinin (WGA) and phalloidin (cell plasma and actin). Cells were imaged using the Cytation5 fluorescence microscope and analyzed in CellProfiler to measure single-cell morphological features. Using principal component analysis (PCA) on 21 cell and nuclear features, an overall morphological score (PC1) was developed to assess the shift in microglia morphology (Figure 1).

Results and Discussion: Boiling MSC-EVs increased their protein concentration significantly. This can be associated with protein denaturization resulting from MSC-EV degradation at high temperatures. MSC-EV protein concentration increased compared to fresh MSC-EVs for all temperatures after one week of storage followed by reduced protein content after 2 and 3 weeks of storage (Figure 2A). There were no observed differences in protein concentration between storage temperatures at a given time point. 

Morphological assessment of microglia revealed significant changes between the control groups (-CTL vs +CTL) and after treatment with MSC-EVs stored at RT and 4°C (Figure 2B). Lower PC1 values indicate ‘higher potency’ of MSC-EV groups as the overall microglia shifts from IFN-γ/TNF-α activated microglia controls (+CTL) to inactivated microglia controls (-CTL). Moreover, boiling of MSC-EVs effectively abrogated the observed microglia modulation of fresh MSC-EVs (#p< 0.05, Figure 2B). MSC-EVs stored at –20°C and -80°C (for 1 or 2 weeks) had no significant effect on microglia morphology. The results indicate that MSC-EV potency decreased over time and with decreasing temperatures. No correlation was found between microglia morphology and MSC-EV protein concentration (Figure 2C).

Conclusion: Relying solely on an assay that determines protein concentration is insufficient to determine the effect of storage temperature or time on MSC-EV function. We have shown that it does not provide clear evidence of functional changes based on our microglia morphological potency assay. Assays assessing MSC-EV yield need to be supplemented with functional assays for determining the optimal storage temperature to preserve MSC-EVs. To overcome this limitation, the use of disease-specific potency assays becomes essential. High-throughput, single-cell assessment of microglia morphological changes quantifies differences between temperature conditions and storage time. This valuable information is crucial for optimizing storage protocols and ensuring the preservation of MSC-EV functionality for effective clinical applications. This approach also enables the screening of other parameters like the effect of storage in different buffers and lyophilization. Furthermore, it can be extended to different cell types and disease-specific models.

References:

1. Levy, D et al., (2023). Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles. Cytotherapy, 25(5), 502–509. https://doi.org/10.1016/J.JCYT.2022.11.006


2. Marklein et al., (2019). Morphological profiling using machine learning reveals emergent subpopulations of interferon-γ–stimulated mesenchymal stromal cells that predict immunosuppression. Cytotherapy, 21(1), 17–31. https://doi.org/10.1016/J.JCYT.2018.10.008


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