Extraction and Purification of Extracellular Vesicles from Porcine Tissue and Fetal Bovine Serum
Board Location: #155
Discipline: Technology and Engineering
Subcategory: Chemical/Bimolecular/Process Engineering
Session: 3
Neha Jayarajan - The University of Texas at Austin
Co-Author(s): 1. Jesús Jiménez, The University of Puerto Rico at Mayagüez, Mayagüez, Puerto Rico2. Dr. Camilo Mora, The University of Puerto Rico at Mayagüez, Mayagüez, Puerto Rico
Mesenchymal stromal cells (MSCs) are a promising source of stem-cell-based therapies, and there is a necessity for a large number of MSCs to fulfill the clinical demand. This study aims to contribute to a scaled bioreactor system by investigating extracellular vesicles (EVs), a key element in understanding MSC communication and cell proliferation status. Specifically, EV extraction and purification methods were explored using two EV sources: extracellular matrix (ECM) and fetal bovine serum (FBS). We hypothesize that the selected sources, ECM and FBS, will serve as valuable suppliers of EVs for optimizing and enhancing the EV extraction methods. This optimization is essential before applying the technology to extract EVs from MSC-conditioned media. For ECM-based EV extraction, decellularized ECM was combined and stirred with collagenase, followed by a series of centrifugations and ultrafiltration. FBS-based EV extraction began by administering a TiO2 adsorption protocol and then conducting particle analysis using dynamic light scattering (DLS). From the ECM extraction, an SDS-PAGE was conducted and revealed distinct 100 kDa bands, within the potential size of EVs, in the collagenase-treated sample that were absent in the control. As the purification process progressed, these bands became more clear and opaque, indicating clearance of other proteins and concentration of potential EVs. In the FBS-based extraction, the DLS revealed a reduced number of particles around 100 nm, within the potential size of EVs, post-treatment. However, analysis also showed that larger particles, close to 400 nm, emerged after TiO2 treatment, indicating possible particle impurities. In the future, studies could focus on refining the extraction techniques to maximize EV yield and quality. Overall, this research represents the promise of ECM and FBS as sources of EVs, facilitating deeper exploration of MSC communication in scaled bioreactor systems.
Funder Acknowledgement(s): 1. NSF Engineering Research Center for Cell Manufacturing Technologies (CMaT)2. The University of Puerto Rico at Mayagüez3. Biomaterials and Biomacromolecules Research & Technologies Lab (BioMa2RT Lab)
Faculty Advisor: Dr. Camilo Mora, camilo.mora@upr.edu
Role: In this project, I independently conducted the digestion of the ECM as well as the SDS-PAGE and its analysis. I also independently administered the TiO2 protocol and conducted the DLS analysis. The decellularization of the ECM from porcine tissue was conducted by my colleague, but upon receiving the decellularized ECM, I independently conducted the remainder of the experiments.