Abstract
Introduction
The immense interest in extracellular vesicles (EVs) underlies a significant need for both the isolation of high-quality EVs from large-scale batches and the development of industry standards for the characterization and quality control testing of EVs. While traditional methods, which include the use of ultracentrifugation and density gradients, are suitable for small-scale studies, the development of scalable and robust processes for the isolation of EVs is essential to meet the growing needs of the scientific community. Here, we report the large-scale isolation and characterization of functional EVs from both cancer cell lines and stem cells.
Methods
EVs were isolated from the supernatants of commercially relevant ATCC cell types using Tangential Flow Filtration (TFF). Production of EVs was carried out in biological replicates. The morphology and size distribution of EVs were evaluated through Nanoparticle Tracking Analysis and electron microscopy. EV-associated cargo was evaluated by multiple strategies including western blot, mass spectrometry, RNA sequencing, and multiplex assays. EV functionality was demonstrated in several in vitro cell based assays using both 2D and 3D cultures.
Results
Our optimized TFF protocols resulted in high yields of EVs (ie, ≥ 1.0 × 1010 EVs/mL) and greater than 80% of EV populations fell within the range of 50-200 nm in size. We also observed cell-dependent expression levels of different EV cargo (ie, tetraspanins, lncRNAs) between cancer EVs and stem cell EVs. Importantly, our quantitative data showed minimal lot-to-lot variation. Our functionality assays showed that stem cell EVs can enhance cell migration, promote angiogenesis, and exert anti-inflammatory and anti-apoptotic effects in different recipient cell types. On the other hand, cancer EVs were found to promote cell transformation in an anchorage independent growth assay.
Conclusion
Collectively, we demonstrate our ability to reproducibly manufacture production-scale batches of high-quality EVs from multiple different cell types. Our EVs are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. Therefore, they can serve as ideal reference materials that can support a wide range of different EV-based research applications.
Download the presentation to learn about our method for isolating high-quality extracellular vesicles from large-scale batches
DownloadPresenter
Heather Branscome, MS, PhD
Supervisor, Laboratory Operations, ATCC
Heather Branscome is a Lead Biologist in Manufacturing Science and Technology at ATCC. She has over 13 years of cross-functional experience working in both cell and molecular biology laboratories. In her current role she leads technology transfer activities for a wide range of products including exosomes/extracellular vesicles, CRISPR/Cas9 engineered cell lines, and induced pluripotent stem cells. She earned her MS in Cell and Molecular Biology from George Mason University and recently earned a PhD in Biosciences. Her primary research interests surround the advanced purification of EVs and the functional analysis of stem cell EVs in the context of CNS repair.
