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.
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Heather Branscome, MS, PhD
Senior Scientist, ATCC
Dr. Heather Branscome is a Senior Scientist with ATCC. Throughout her 17-year career she has gained broad experience working in both academic and industry settings. She has extensive experience in cell and molecular biology and completed her graduate training in Biosciences from George Mason University. While at ATCC she has held positions in manufacturing, quality control, and technology transfer to support the production and qualification of cell lines and other critical biological reagents to support the scientific community. In her current role she manages a team of biologists to support the CDC’s International Reagent Resources (IRR) program, as well as other government contracts. Since 2018, she has played a key role in establishing and maintaining ATCC’s extracellular vesicle (EV) portfolio. In this role she was responsible for developing and validating large-scale EV manufacturing protocols and performing various EV biochemical and functional assays. Her current research is focused on advanced methods for EV purification, characterization of novel EV subtypes, and mechanistic studies of stem cell-derived EVs in different models of cellular repair. She currently serves as director and instructor for two local Bio-Trac® biotechnology training programs and maintains an active affiliation with George Mason University.