Associate Professor Union College Schenectady, New York, United States
Introduction: Mesenchymal stromal cells (MSCs) have emerged as promising therapeutics for immune diseases due to their modulation of immune cells. Their therapeutic effects are mediated through the secretion of immunomodulatory factors packaged in extracellular vesicles (EVs). Control of MSC growth and EV production are critical components of EV therapeutic development, but there is limited understanding of how different aspects of the manufacturing process impact EV reproducibility and production. Studies have shown the effectiveness of traditional methods including 2D flask "stacks" and bioreactors for large-scale production of MSCs and EVs. However, these methods lack precise control over the microenvironment. 3D hydrogels emerge as a promising alternative due to their ease of modification and their capacity to better mimic the physiological conditions inside the human body. Since hydrogels are fabricated from modified polymers, their biomechanical properties are easy to tune, enabling versatility in the resultant cellular responses. Specifically, this study investigates the influence of the 3D hydrogel microenvironment on MSC-EV immunomodulatory function.
Materials and
Methods: The overall hydrogel design employed in the current work is shown in Figure 1A. MSCs are encapsulated through Michael-Type addition reactions, with densities ranging from 2.5% to 4% weight percent. Then, TNF-alpha and IFN-Gamma cytokines are fed to the MSCs to induce EV and immunomodulatory factor production. At predetermined intervals, media samples containing factors are stored for later testing with IDO assay, while hydrogels are stained with Calcein for cell viability assessment. The IDO assay determines immunomodulatory factor production across different time points. Confocal microscopy is utilized to gather slice samples of the hydrogels. These samples are then processed to quantify cell density, cell morphology, and gel size. Cell count is manually determined across several slices of a stack sample, distinguishing live and dead cells based on the Calcein signal intensity. For cell morphology analysis, ImageJ is used for the image pre-processing, utilizing background subtraction, intensity-based thresholding, and binary image conversion. Form factor is the quantification of the roundness of the cell in 2D, as observed in Figure 1B. Form factor is calculated with CellProfiler after the ImageJ image processing. Gel volume is determined by measuring the area of each slice in the image stack using the free-hand selection tool in ImageJ. Previous work has shown that matrix metalloproteinase (MMP) degradable hydrogels allow cell-mediated degradation over time, which is corroborated in Figure 1C.
Results, Conclusions, and Discussions: MSCs encapsulated in adhesive-degradable hydrogels that allow cell-mediated degradation and spreading exhibit increased EV production compared to cells in 2D environments, as observed in Figure 2. Adhesive-degradable peptides enable MSCs to spread and divide over time by allowing MSCs to break the MMP-degradable crosslinks as they grow, thereby better mimicking the conditions inside the human body compared to traditional 2D culture methods. There is no apparent relationship between cell morphology, measured by FF, and immunomodulatory function, as shown in Figure 3. Microenvironment control is achieved by tuning the peptide formulation, which allows toggling the density of the hydrogel, subsequently affecting the cell-mediated degradation rate. The speed at which the hydrogel degrades significantly influences the quantity of factors produced, as shown in previous work, highlighting the influence of MSCs’ microenvironment on their factor production performance. Currently, multiple adhesive-degradable hydrogel formulations are undergoing testing to evaluate their ability to support the prolonged spreading of MSCs and identify the formulation that best maximizes factor production. Furthermore, the manufacturing process is continuously refined to improve reproducibility and decrease the variability of hydrogel morphologies. Future work will focus on conducting tests on hydrogel cryopreservation to determine the viability of the MSCs after undergoing freezing and thawing cycles. The factor secretion effectiveness of the hydrogels will be assessed for post-thaw applications, to allow rapid deployment if the waiting period for priming is too lengthy, thereby ensuring long-term storage capabilities.
Acknowledgements (Optional): Special thanks to the Union College Electrical, Computer, and Biomedical Engineering Department, as well as to Dr. Khetan for this opportunity.
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