Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles.

BACKGROUND AIMS: 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 of 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 rarely reported. METHODS: The authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, -20°C, -80°C) for various durations as well as after lyophilization. RESULTS: Mesenchymal 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°C and -80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs. CONCLUSIONS: These findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes.

Extracellular vesicles as therapeutics for inflammation and infection.

Extracellular vesicles (EVs) are an emergent next-generation biotechnology with broad application potential. In particular, immunomodulatory bioactivity of EVs leading to anti-inflammatory effects is well-characterized. Cell source and culture conditions are critical determinants of EV therapeutic efficacy, while augmenting EV anti-inflammatory bioactivity via diverse strategies, including RNA cargo loading and protein surface display, has proven effective. Yet, translational challenges remain. Additionally, the potential of direct antimicrobial EV functionality has only recently emerged but offers the possibility of overcoming drug-resistant bacterial and fungal infections through novel, multifactorial mechanisms. As discussed herein, these application areas are brought together by the potential for synergistic benefit from technological developments related to EV cargo loading and biomanufacturing.

Enhanced Extracellular Vesicle Cargo Loading via microRNA Biogenesis Pathway Modulation.

Extracellular vesicles (EVs) are physiological vectors for the intercellular transport of a variety of molecules. Among these, small RNAs, and especially microRNAs (miRNAs), have been identified as prevalent components, and there has thus been a robust investigation of EVs for therapeutic miRNAs delivery. However, intrinsic levels of EV-associated miRNAs are generally too low to enable efficient and effective therapeutic outcomes. We hypothesized that miRNA localization to EVs could be improved by limiting competing interactions that occur throughout the miRNA biogenesis process. Using miR-146a-5p as a model, modulation of transcription, nuclear export, and enzymatic cleavage steps of miRNA biogenesis were tested for impact on EV miRNA loading. Working in HEK293T cells, various alterations in the EV biogenesis pathway were shown to impact miRNA localization to EVs. The system was then applied in induced pluripotent stem cells (iPSCs), a more promising substrate for therapeutic EV production, and EVs were separated and assessed for anti-inflammatory efficacy in vitro and in a murine colitis model, where the preservation of function was validated. Overall, the results highlight necessary considerations when designing a cell culture system for the devoted production of miRNA-loaded EVs.

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.

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 explored in clinical trials for treatment of diseases with complex pathophysiologies. 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, it is initially sought to evaluate the therapeutic potential of iMSC EVs. Interestingly, while utilizing undifferentiated iPSC EVs as a control, it is found that their vascularization bioactivity is similar and their anti-inflammatory bioactivity is superior to donor-matched iMSC EVs in cell-based assays. To supplement this initial in vitro bioactivity screen, a diabetic wound healing mouse model where both the pro-vascularization and anti-inflammatory activity of these EVs would be beneficial is employed. In this in vivo model, iPSC EVs more effectively mediate 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.

Radioiodination of extravesicular surface constituents to study the biocorona, cell trafficking and storage stability of extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) are produced by all cell types and serve as biological packets delivering a wide variety of molecules for cell-to-cell communication. However, the biology of the EV extravesicular surface domain that we have termed EV 'biocorona' remains underexplored. Upon cell secretion, EVs possess an innate biocorona containing membrane integral and peripheral constituents that is modified by acquired constituents post secretion. This distinguishes EVs from synthetic nanoparticulate biomaterials that are limited to an adsorption-based, acquired biocorona. METHODS: The EV biocorona molecular constituents were radiolabeled with 125I to study biocorona constituents and its surface dynamics. As example toolset applications, 125I-EVs were utilized to study EV cell trafficking and the stability of the EV biocorona during storage. RESULTS: The biocorona of EVs consisted of proteins, lipids, DNA and RNA. The cellular uptake of 125I-EVs was temperature dependent and internalized 125I-EVs were rapidly recycled by cells. When 125I-EVs were stored in a purified state, they exhibited time and temperature dependent biocorona shedding and proteolytic degradation that was partially inhibited in the presence of serum. CONCLUSION: The EV biocorona is complex and dynamic. Radiolabeling of the EV biocorona enables a unique platform methodology to study the biocorona and will facilitate unlocking EV's full clinical translation potential. GENERAL SIGNIFICANCE: The EV biocorona affects EV mediated biological processes in health and disease. Acquiring knowledge of the EV biocorona composition, dynamics, stability and structure not only informs the diagnostic and therapeutic translation of EVs but also aids in designing biomimetic nanomaterials for drug delivery.