Biology subjects

Jay, S. M.

Publications and source records attributed to Jay, S. M..

5 recordsLinked to original sources

bioRxiv2022

Mesenchymal stem cell culture within perfusion bioreactors incorporating 3D-printed scaffolds enables improved extracellular vesicle yield with preserved bioactivity

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here we assessed effects of a 3D-printed scaffold-perfusion bioreactor system on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type heavily implicated in generating EVs with therapeutic potential. Our results indicate that perfusion bioreactor culture results in an ~40-80-fold increase, depending on measurement method, in MSC EV production compared to conventional cell culture. Additionally, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to major EV translational issues (i.e., scalability and low potency) with potential for adaptation to various EV-based therapeutics and capacity for improvement alongside the continuous advancements in 3D-printing technologies.

bioengineering↗

bioRxiv2022

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination

Extracellular vesicles (EVs) are widely implicated as novel diagnostic and therapeutic modalities for a wide range of diseases. Thus, optimization of EV biomanufacturing is of high interest. In the course of developing parameters for a HEK293T EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static vs dynamic) and isolation techniques (i.e., ultracentrifugation vs tangential flow filtration vs size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established LPS-stimulated mouse macrophage model. We unexpectedly found that, depending on culture condition and isolation strategy, HEK293T EVs appeared to significantly suppress the secretion of pro-inflammatory cytokines (i.e., IL-6, RANTES) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to fetal bovine serum (FBS) EV contamination in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.

bioengineering↗

bioRxiv2022

Combinatorial microRNA loading into extracellular vesicles for anti-inflammatory therapy

Extracellular vesicles (EVs) have emerged as promising therapeutic entities in part due to their potential to regulate multiple signaling pathways in target cells. This potential is derived from the broad array of constituent and/or cargo molecules associated with EVs. Among these, microRNAs (miRNAs) are commonly implicated as important and have been associated with a wide variety of EV-induced biological phenomena. While controlled loading of single miRNAs is a well-documented approach for enhancing EV bioactivity, loading of multiple miRNAs has not been fully leveraged to maximize the potential of EV-based therapies. Here, an established approach to extrinsic nucleic acid loading of EVs, sonication, was utilized to enable controlled loading of multiple miRNAs in HEK293T EVs. Combinations of carefully chosen miRNAs were compared to single miRNAs with respect to anti-inflammatory outcomes in assays of increasing stringency, with the combination of miR-146a, miR-155, and miR-223 found to have the most potential amongst tested groups.

bioengineering↗

bioRxiv2022

The Impact of Storage Condition and Duration on Function of Native and Cargo-Loaded Mesenchymal Stromal Cell Extracellular Vesicles

As evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect to EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake, and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are scarcely reported. Here, EV outcomes following storage at different temperatures (room temperature, 4{degrees}C, -20{degrees}C, -80{degrees}C) for various durations as well as after lyophilization were assessed. Mesenchymal stem/stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4-6 weeks at -20{degrees}C, -80{degrees}C, and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model respectively, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA (miRNA) as well as long non-coding RNA (lncRNA) cargo in MSC EVs. These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storage of MSC EVs for both basic research and translational purposes.

bioengineering↗

bioRxiv2021

Sustained Released of Bioactive Mesenchymal Stromal Cell-Derived Extracellular Vesicles from 3D-Printed Gelatin Methacrylate Hydrogels

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate therapeutic potential of MSC EV-laden GelMA bioinks.

bioengineering↗