Poster N2 - Enhancing pro-angiogenic induced pluripotent stem cell-derived mesenchymal stem cell extracellular vesicle production by implementing confinement under flow in a perfusion bioreactor

Introduction: Mesenchymal stem cell extracellular vesicles (MSC EVs) are an emerging biotherapeutic that could serve as an alternative to cell therapies which are often associated with tumorigenicity, undesired differentiation, and low engraftment. In particular, MSC EVs are a promising candidate for regenerative medicine applications such as chronic wound healing and cardiovascular disease due to their inherent pro-angiogenic and anti-inflammatory properties. However, despite pre-clinical promise and growing traction commercially and in clinical trials, there are still zero FDA-approved EV therapeutics. Many factors can be attributed to this including low potency, which necessitates larger and more frequent doses, and lack of scalable EV generation platforms, as most pre-clinical EV research utilizes 2D cell culture. To address low EV potency, approaches such as endogenous and exogenous loading of therapeutic cargo have been explored. However, these methods can be expensive, risk EV degradation, and add labor and cost to both upstream and downstream processes. Using induced pluripotent stem cell-derived MSCs (iMSCs) as an EV source that offers reduced donor variability, we have shown that physically confining the parent cells within pillars as close as 5µm results in enhanced angiogenic bioactivity of the EVs in vitro. While promising, these studies were done in static 2D culture, which is not amenable to large-scale biomanufacturing. Thus, we sought to determine whether mechanical regulation of iMSC EV potency in this manner can be implemented and maintained under flow within a perfusion bioreactor system as a more feasible and scalable EV generation platform.

Materials and

Methods: 5µm confinement devices were made using photolithography and polydimethylsiloxane (PDMS) casting, and the resulting PDMS micropillar arrays were adhered into 3D-printed bioreactor scaffolds. Scaffolds with collagen-coated confinement devices were seeded with iMSCs. The following day, the scaffolds were connected to a pump with a reservoir of EV-depleted media and run at either a 1, 5, or 10 mL/minute flow rate for 24 hours. Control groups consisted of EVs from iMSCs grown in collagen-coated flasks and iMSCs grown in the confinement devices in static culture. All conditioned media underwent three differential centrifugation spins followed by tangential flow filtration to isolate the EVs. EVs were characterized via nanoparticle tracking analysis (NTA). The cells in the devices were fixed and stained using Hoechst, imaged, and total cells were counted using ImageJ. Cells in the flask were trypsinized and counted. Production was quantified as EV per cell using both NTA and cell count data. Finally, EV bioactivity was determined using a tube formation assay as a measure of in vitro angiogenic activity. Briefly, 5E9 EVs/mL in endothelial basal media were mixed with human umbilical vein endothelial cells (HUVECs), seeded in a 48-well plate, and incubated for 3-6 hours before being imaged to quantify tube formation.

Results, Conclusions, and Discussions: Using the 5µm confinement devices within 3D-printed perfusion bioreactor scaffolds since our prior work in static culture indicated that more confinement correlated with higher angiogenic bioactivity, iMSCs were seeded and subjected to flow rates of 1, 5, or 10 mL/minute. The bioactivity of the resulting EVs was assessed in a tube formation assay, where we plated HUVECs with EVs from each group. HUVECs were also plated with EVs from confined iMSCs in static culture to provide insight into potential differing effects from flow and confinement, as well as with EVs from collagen-coated flasks to further analyze differences in capillary-like tube formation induced by EVs from different conditions. We observed that the EVs from confined iMSCs under all flow rates caused significantly more tube formation when compared to EVs from the flask-grown cells and the endothelial basal media treatment control (Figure 1A). Interestingly, flow did not significantly augment EV bioactivity compared to the static 5µm group, suggesting that while angiogenic bioactivity is maintained under flow, it is not further enhanced. We also quantified EV production as EV per cell retained after 24 hours of culture. Our preliminary data here suggests that as flow rate increases, EV yield also increases despite decreased cell retention, which would implicate a change in EV production by each cell when under flow. As such, the 10 mL/minute flow rate on the iMSCs within the confinement devices yielded the greatest number of EVs per cell compared to those from the 0, 1, and 5 mL/minute flow rates and flask culture (Figure 1B). The 1 and 5 mL/minute flow rates also resulted in greater EV production per cell compared to both static culture groups, although less so than the 10 mL/minute group. Taken together, the maintained bioactivity and improved EV production make this bioreactor system, where high degrees of confinement can be implemented under flow, a promising and scalable method for enhanced generation of potent, pro-angiogenic iMSC EVs. This work will help address EV potency and production platform shortcomings, overall contributing to the scalability and potential for clinical translation of iMSC EV therapeutics for regenerative medicine.

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