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 the 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 the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve 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 a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

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 human embryonic kidney cells (HEK293T) EV production platform, we examined the combinatorial effects of cell culture conditions (i.e., static versus dynamic) and isolation techniques (i.e., ultracentrifugation versus tangential flow filtration versus size-exclusion chromatography) on functional characteristics of HEK293T EVs, including anti-inflammatory bioactivity using a well-established lipopolysaccharide-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., interleukin-6, RANTES [regulated upon activation, normal T cell expressed and secreted]) in the stimulated mouse macrophages. Further examination revealed that these results were most likely due to non-EV fetal bovine serum components in HEK293T EV preparations. Thus, future research assessing the anti-inflammatory effects of EVs should be designed to account for this phenomenon.

Combinatorial microRNA Loading into Extracellular Vesicles for Increased Anti-Inflammatory Efficacy.

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 load multiple miRNAs in HEK293T EVs. Combinations of 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 the tested groups.

Extracellular Vesicle Loading Via pH-Gradient Modification.

Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication and consequently have the potential to be potent therapeutic vectors. Beyond their endogenous function, EVs are also being harnessed as drug delivery vehicles with possible benefits over synthetic nanoparticle systems. Despite advances in loading exogenous molecules into extracellular vesicles, efficient incorporation of nucleic acids remains a challenge due to aggregation and degradation. In this chapter, we detail a method to load EVs with negatively charged cargo, in particular nucleic acids, by modifying the internal pH of the vesicles to be acidic. This approach demonstrates that pH modification of EVs enables efficient loading of nucleic acids with functional cargo.

Therapeutic Potential of Extracellular Vesicles for Sepsis Treatment.

Sepsis is a deadly condition lacking a specific treatment despite decades of research. This has prompted the exploration of new approaches, with extracellular vesicles (EVs) emerging as a focal area. EVs are nanosized, cell-derived particles that transport bioactive components (i.e., proteins, DNA, and RNA) between cells, enabling both normal physiological functions and disease progression depending on context. In particular, EVs have been identified as critical mediators of sepsis pathophysiology. However, EVs are also thought to constitute the biologically active component of cell-based therapies and have demonstrated anti-inflammatory, anti-apoptotic, and immunomodulatory effects in sepsis models. The dual nature of EVs in sepsis is explored here, discussing their endogenous roles and highlighting their therapeutic properties and potential. Related to the latter component, prior studies involving EVs from mesenchymal stem/stromal cells (MSCs) and other sources are discussed and emerging producer cells that could play important roles in future EV-based sepsis therapies are identified. Further, how methodologies could impact therapeutic development toward sepsis treatment to enhance and control EV potency is described.

Extracellular miR-146a-5p Induces Cardiac Innate Immune Response and Cardiomyocyte Dysfunction.

Previous studies have demonstrated that transient myocardial ischemia leads to release of cellular nucleic acids such as RNA. Extracellular RNA reportedly plays a pivotal role in myocardial inflammation and ischemic injury in animals. RNA profiling has identified that numerous microRNA (miRNAs), such as ss-miR-146a-5p, are upregulated in plasma following myocardial ischemia, and certain uridine-rich miRNAs exhibit strong proinflammatory effects in immune cells via ssRNA-sensing mechanism. However, the effect of extracellular miRNAs on myocardial inflammation and cardiac cell function remains unknown. In this study, we treated adult mouse cardiomyocytes with miR-146a-5p loaded in extracellular vesicles and observed a dose- and TLR7-dependent production of CXCL-2, IL-6, and TNF-α. In vivo, a single dose of myocardial injection of miR-146a-5p induced both cytokine expression (CXCL2, IL-6, and TNF-α) and innate immune cell activation (CD45+ leukocytes, Ly6Cmid+ monocytes, Ly6G+ neutrophils), which was significantly attenuated in the hearts of TLR7 KO mice. We discovered that conditioned media from miR-146a-treated macrophages stimulated proinflammatory cytokine production in adult cardiomyocytes and significantly inhibited their sarcomere shortening. Finally, using an electric cell impedance-sensing assay, we found that the conditioned media from miR-146a-treated cardiac fibroblasts or cardiomyocytes impaired the barrier function of coronary artery endothelial cells. Taken together, these data demonstrate that extracellular miR-146a-5p activates multiple cardiac cells and induces myocardial inflammation and cardiomyocyte dysfunction via intercellular interaction and innate immune TLR7 nucleic acid sensing.

Enhanced Loading of Functional miRNA Cargo via pH Gradient Modification of Extracellular Vesicles.

Based on their identification as physiological nucleic acid carriers in humans and other organisms, extracellular vesicles (EVs) have been explored as therapeutic delivery vehicles for DNA, RNA, and other cargo. However, efficient loading and functional delivery of nucleic acids remain a challenge, largely because of potential sources of degradation and aggregation. Here, we report that protonation of EVs to generate a pH gradient across EV membranes can be utilized to enhance vesicle loading of nucleic acid cargo, specifically microRNA (miRNA), small interfering RNA (siRNA), and single-stranded DNA (ssDNA). The loading process did not impair cellular uptake of EVs, nor did it promote any significant EV-induced toxicity response in mice. Cargo functionality was verified by loading HEK293T EVs with either pro- or anti-inflammatory miRNAs and observing the effective regulation of corresponding cellular cytokine levels. Critically, this loading increase is comparable with what can be accomplished by methods such as sonication and electroporation, and is achievable without the introduction of energy associated with these methods that can potentially damage labile nucleic acid cargo.

The effects of red tide (Karenia brevis) on reflex impairment and mortality of sublegal Florida stone crabs, Menippe mercenaria.

The Florida stone crab, Menippe mercenaria, is a major commercial fishery that occurs primarily along Florida's west coast, where harmful algal blooms of Karenia brevis frequently develop. To determine sublethal and lethal effects of K. brevis on M. mercenaria, we exposed sublegal stone crabs to three seawater treatments in laboratory conditions: no K. brevis (control), a low-toxin K. brevis strain (Wilson LT), and a toxic K. brevis (New Pass strain). Total food consumed, reflex impairment and survivorship of each crab was monitored throughout the nine-day experiment. Crabs in the toxic treatment consumed 67% less food. The probability of an individual losing a reflex significantly increased with time (days), and there was a 42% decrease in survivorship in the toxic treatment. This is the first study to demonstrate negative effects of K. brevis on the stone crab, presenting the critical need of further investigation to fully understand how red tide may impact sustainability of the fishery.