Speaker
Description
Background: Extracellular vesicles (EVs) are spherical, bilayer-enclosed particles that are released in a constitutive manner by nearly all cell types and which play a key role in the intercellular communication and the regulation of both physiological and pathological processes. As non-replicating agents with nanometric sizes and loading capacities, interesting biochemical properties and unequalled biocompatibility, EVs have positioned themselves as key agents for numerous therapeutic applications. They can be used as drug delivery vehicles or disease biomarkers, for instance. An emerging domain is that of bacterial EVs (BEVs). As key mediators of the microbiota-host crosstalk, they offer exciting additional biomedical prospects in particular in the development of novel therapies for inflammatory bowel diseases (IBD). However, other than standard protocols, there are only few studies of novel stimuli to enhance the secretion of BEVs to meet these therapeutic needs.
Methods: Here, we introduce a patented high-throughput production method, supported by a published article, that relies on the introduction of hydrodynamic shear stress on producing cells to promote their vesicles release. The probiotic strain Escherichia coli Nissle 1917 was used for the proof-of-concept and was cultured while being submitted to high-speed rotations up to 24 hours in a specialized rotating vessel. Produced BEVs were characterized by cryo-EM imaging and the impact of hydrodynamic and standard conditions on the protein cargo of EVs was explored through proteomic analysis and functional in vitro assays. Their therapeutic potential was further studied using a biomimetic Gut-on-chip model featuring a functional epithelial layer (Caco-2/TC7 and HT29-MTX at a 6:1 ratio) interfaced with a collagen-based matrix. Fluorescently labeled BEVs were perfused into the epithelial tubule to study their internalization.
Results: Overall, the results showed a clear increase both in bacterial growth and EV-yield when using our technology compared to control conditions, with an increase of produced EVs linked to the rotation speed of the stimulation. Observed EVs using cryo-EM show typical morphological types of BEVs including mainly outer-membrane vesicles for all conditions. Interestingly, proteomic analysis revealed an hydrodynamically-induced protein signature at the cargo level as shown through PCA and clustering analysis. Importantly, the produced EVs demonstrated potential therapeutical properties, with demonstrated effects in pro-inflammatory responses, exhibiting different efficacy when produced under hydrodynamic stimulation. Preliminary microfluidic assays further confirmed that these BEVs are efficiently internalized by the intestinal epithelial cells within the Gut-on-chip device.
Conclusion & Perspectives: This integrated approach demonstrates that our novel shear-stress technology significantly outperforms traditional BEV production methods in terms of yield and speed, reducing production time from several days to only 24 hours. The produced EVs offer similar morphological characteristics as standard condition while also differing through cargo content and potential therapeutical properties. The Gut-on-chip model now serves as a high-resolution platform to investigate the functional impact of these "hydrodynamically-primed" vesicles. Ongoing studies aim to evaluate how these BEVs modulate the epithelial functionality and integrity by studying the expression of tight junction proteins both at the mRNA and protein level by RT-qPCR and fluorescence microscopy respectively.