Extracellular Vesicles Facilitate Large-Scale, Homogenizing Dynamic Exchange of Proteins and RNA Among Cultured Chinese Hamster Ovary (CHO) and Human Cells

Abstract

Cells in culture are viewed as unique individuals in a large population communicating through extracellular molecules and, more recently extracellular vesicles (EVs). Our data here paints a different picture: the homogenizing effect of large-scale exchange of cellular material through EVs. We show that Chinese Hamster Ovary (CHO) cells dynamically produce and uptake EVs, to exchange proteins and RNAs at large-scale. To visualize the dynamic production and cellular uptake of EVs, we used correlative confocal microscopy and scanning electron microscopy, as well as flow cytometry to interrogate labeled cells. We employed cells expressing fluorescent proteins (GFP, miRFP703) and tagged cells with protein and RNA dyes. Flow cytometry was used to quantify the exchange of cellular RNA between cells through EVs. This EV-mediated dynamic exchange observed in CHO cultures was also observed in cultures of the human CHRF-288-11 cell line and of primary human hematopoietic stem and progenitor cells. This study demonstrates an underappreciated native cell communication and protein/RNA exchange mechanism mediated by EVs spanning cell type and lines, suggesting the proximity of cells in normal and tumor tissues may also result in prolific cellular exchange. This exchange would be expected to homogenize the cell-population cytoplasm and dynamically regulate cell proliferation and cellular state.

Kinetic and functional analysis of abundant microRNAs in extracellular vesicles from normal and stressed cultures of Chinese Hamster Ovary (CHO) cells

“…CHO EVs have only recently garnered significant attention. In particular, we have found that CHO EVs exchange large quantities of RNA cargo between their parent cells (Belliveau & Papoutsakis, 2022. The nature of that cargo, which has been investigated by several groups (Belliveau & Papoutsakis, 2023;Busch et al, 2022;Keysberg et al, 2021), is now known to vary significantly as a function of metabolite (ammonia and osmotic) stress (Belliveau & Papoutsakis, 2023).…”

“…CHO cells were obtained and cultured as described previously (Belliveau & Papoutsakis, 2022. Briefly, a CHO-K1 cell line expressing the VRC01 antibody was cultured in HyClone ActiPro media (Cytiva, Marlborough, MA, USA) supplemented with 6 mM L-glutamine.…”

“…CHO EVs were isolated as described (Belliveau & Papoutsakis, 2022. As noted, only large CHO EVs (i.e., "CHO MPs") were collected and analyzed for this study.…”

“…Chinese hamster ovary (CHO) cells have long served as mainstays of industrial protein production. Recently, we have found that CHO cells in culture participate in a massive and continuous exchange of extracellular vesicles (EVs) (Belliveau & Papoutsakis, 2022), thus impacting culture performance by a hitherto unknown and unexplored mechanism . EVs are submicron-sized particles enclosed by a lipid bilayer which originate from either multivesicular bodies or the plasma membrane.…”

“…The focus of this work is the larger EVs originating largely from the plasma membrane (i.e., CHO "microparticles," or "CHO MPs"), which have been the focus of fewer studies, but which are easier to isolate-a boon for their potential as a diagnostic tool-and capable of carrying larger cargo loads, thus suitable for a broader range of synthetic applications using DNA, RNA, protein, nucleoprotein (such as CRISPR systems) and other organic molecules as payload. Our previous publications (Belliveau & Papoutsakis, 2022 have provided detailed CHO MP characterization that meets or exceeds the MISEV2018 guidelines from the International Society for Extracellular Vesicles (Thery et al, 2018). Here, we use the same isolation methods as before, expanding our CHO MP characterization by focusing primarily on various miR cargoes and their potential functions in CHO cell culture.…”