Biology subjects

Jay, S. M.

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

11 recordsLinked to original sources

bioRxiv2025

Perfusion Bioreactor Culture Incorporating Mechanical Confinement Enhances Mesenchymal Stem Cell Extracellular Vesicle Production and Wound Healing Potential

Mesenchymal stem cell extracellular vesicles (MSC EVs) have been widely studied for regenerative medicine and tissue repair applications. However, clinical translation of EV therapeutics has been hampered by low potency and lack of scalable production strategies. This work aims to develop a novel approach that exploits the mechanosensitivity of MSCs to enhance EV potency in the context of enhanced production via bioreactor culture. MSCs are well known to respond to mechanical stimuli such as substrate stiffness and shear stress, and here it is shown that exposing MSCs to another mechanoregulatory parameter, confinement, enhances the pro-angiogenic bioactivity of their EVs. This is consistent across both donor-derived primary MSCs and induced pluripotent stem cell-derived MSCs (iMSCs). The development of a 3D-printed perfusion bioreactor system that enables culture of confined MSCs under flow is also detailed here, resulting in a 67-fold increase in EV production compared to flask culture. iMSC EVs obtained downstream of this confinement-bioreactor culture induce greater vascularization and generally improve wound healing in a diabetic mouse model compared to iMSC EVs from conventional tissue culture. Overall, this work establishes the development of a scalable, bioreactor-based iMSC EV production platform that provides a solution to major translational bottlenecks of therapeutic EVs.

bioengineering↗

bioRxiv2025

Human cell-derived extracellular vesicles exhibit anti-biofilm effects against Pseudomonas aeruginosa

The need for new antimicrobial approaches is one of the most pressing issues in modern medicine. A particular pathogen of concern is multidrug resistant Pseudomonas aeruginosa, which is implicated in over 1 million deaths worldwide yearly. One potential class of novel antimicrobials is extracellular vesicles (EVs), which have been found to have intrinsic antimicrobial and anti-virulence properties. Here, the antimicrobial activity of EVs on P. aeruginosa was explored in the context of biofilms of hyper-virulent strain PA14. We identified the human monocyte cell line THP-1 as a promising source of EVs for this application, inducing reduced PA14 biofilm formation in a dose-dependent manner. THP-1 EVs were not found to affect P. aeruginosa growth planktonically within the biofilm assay or in shaken culture. Additionally, we demonstrate that anti-biofilm effects were conserved with similar efficacy across THP-1 monocyte and differentiated THP-1 macrophage-derived EVs. Further, EVs from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) also reduced PA14 biofilm formation to a similar extent as THP-1 EVs, while EVs from human embryonic kidney cells (HEK293T) had a similar effect to its media control. This work indicates that human cell-derived EVs from several sources possess biological and/or physical properties that reduce PA14 biofilm formation.

microbiology↗

bioRxiv2025

Genetically-programmed Hypervesiculation of Lactiplantibacillus plantarum Increases Production of Bacterial Extracellular Vesicles with Therapeutic Efficacy in a Preclinical Inflammatory Bowel Disease Model

Inflammatory bowel diseases (IBD) affect over 6 million people globally and current treatments achieve only 10-20% rates of durable disease remission. Bacterial extracellular vesicles (BEVs) from probiotic lactic acid bacteria (LAB) are a promising novel therapeutic with mechanisms holding potential to drive increased rates of durable disease remission, including immunomodulation and intestinal epithelial tissue repair. However, translation of these cell-secreted nanovesicles is limited by long standing biomanufacturing hurdles, especially low production yields due to low biogenesis rates from cells. Here, our goal was to identify a candidate probiotic LAB that produces BEVs effective in a preclinical mouse model of IBD, and then genetically engineer the LAB for at least 10-fold increased production yields of BEVs, thereby passing a critical production threshold. We identified Lactiplantibacillus plantarum as a candidate LAB producing BEVs effective in treating acute dextran sulfate sodium (DSS)-induced murine colitis, and with greater efficacy than BEVs from probiotic Escherichia coli Nissle 1917. We then genetically engineered a hypervesiculating L. plantarum strain by inducible expression of a peptidoglycan-modifying enzyme, resulting in a 66-fold increase in BEV productivity. Finally, we confirmed hypervesiculating L. plantarum BEVs were therapeutically effective in the acute DSS mouse model of colitis and found these BEVs were superior in reducing mucosal tissue damage compared to live L. plantarum cells. These findings demonstrate that BEVs from genetically engineered hypervesiculating strain of L. plantarum are a promising preclinical therapeutic candidate for IBD that overcomes historical biomanufacturing limitations of BEV therapeutics.

bioengineering↗

bioRxiv2023

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles (OMVs), have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation co-isolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). E. coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.

bioengineering↗

bioRxiv2023

Induced pluripotent stem cell-derived extracellular vesicles promote wound repair in a diabetic mouse model via an anti-inflammatory immunomodulatory mechanism

Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have recently been widely explored in clinical trials for treatment of diseases with complex pathophysiology. However, production of MSC EVs is currently hampered by donor-specific characteristics and limited ex vivo expansion capabilities before decreased potency, thus restricting their potential as a scalable and reproducible therapeutic. Induced pluripotent stem cells (iPSCs) represent a self-renewing source for obtaining differentiated iPSC-derived MSCs (iMSCs), circumventing both scalability and donor variability concerns for therapeutic EV production. Thus, we initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, we found that their vascularization bioactivity was similar and their anti-inflammatory bioactivity was superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, we employed a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial. In this in vivo model, iPSC EVs more effectively mediated inflammation resolution within the wound bed. Combined with the lack of additional differentiation steps required for iMSC generation, these results support the use of undifferentiated iPSCs as a source for therapeutic EV production with respect to both scalability and efficacy.

bioengineering↗

bioRxiv2023

Differentiation state and culture conditions impact neural stem/progenitor cell-derived extracellular vesicle bioactivity

Extracellular vesicles (EVs) derived from neural progenitor/stem cells (NPSCs) have shown promising efficacy in a variety of preclinical models. However, NPSCs lack critical neuroregenerative functionality such as myelinating capacity. Further, culture conditions used in NPSC EV production lack standardization and identification of optimal conditions for NPSC EV neurogenic bioactivity. Here, we assessed whether further differentiated oligodendrocyte precursor cells (OPCs) and immature oligodendrocytes (iOLs) that give rise to mature myelinating oligodendrocytes could yield EVs with neurotherapeutic properties comparable or superior to those from NPSCs as well as mesenchymal stromal cells (MSCs), as MSC EVs are also commonly reported to have neurotherapeutic activity. We additionally examined the effects of four different extracellular matrix (ECM) coating materials (laminin, fibronectin, Matrigel, and collagen IV) and the presence or absence of growth factors (EGF, bFGF, and NGF) in cell culture on the ultimate properties of EVs. The data show that OPC EVs and iOL EVs performed similarly to NPSC EVs in PC-12 proliferation and RAW264.7 mouse macrophage antiinflammatory assays, but NPSC EVs performed better in a PC-12 neurite outgrowth assay. Additionally, the presence of nerve growth factor (NGF) in culture was found to be maximize NPSC EV bioactivity among the conditions tested. NPSC EVs produced under rationally-selected culture conditions (fibronectin + NGF) enhanced axonal regeneration and muscle reinnervation in a rat nerve crush injury model. These results highlight the impact of culture conditions on NPSC EV neuroregenerative bioactivity, thus providing additional rationale for standardization and optimization of culture conditions for NPSC EV production. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=35 SRC="FIGDIR/small/528366v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@447d23org.highwire.dtl.DTLVardef@73a5cborg.highwire.dtl.DTLVardef@89dbb0org.highwire.dtl.DTLVardef@b62d39_HPS_FORMAT_FIGEXP M_FIG C_FIG Extracellular vesicles (EVs) purified from neural progenitor/stem cells (NPSCs) have been investigated for neurotherapeutic activity, however significant variability in culture conditions limits reproducibility and efficacy of this approach. Here, we examined the impact of extracellular matrix (ECM) components and growth factors in NPSC culture on the bioactivity on the bioactivity of their EVs. The results show that EVs from NPSCs cultured with a rationally-selected ECM type (fibronectin) and growth factor (nerve growth factor (NGF)) enhanced nerve regeneration and muscle recovery in a rat sciatic nerve crush injury model.

bioengineering↗

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↗