Extracellular vesicles facilitate large‐scale dynamic exchange of proteins and RNA among cultured Chinese hamster ovary 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 paint a different picture: large-scale exchange of cellular material through EVs. 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. Using cells expressing fluorescent proteins (GFP, miRFP703) and cells tagged with protein and RNA dyes, we show that Chinese hamster ovary (CHO) cells dynamically produce and uptake EVs to exchange proteins and RNAs at a large scale. Applying a simple model to our data, we estimate, for the first time, the per cell-specific rates of EV production (68 and 203 microparticles and exosomes, respectively, per day). This EV-mediated massive exchange of cellular material observed in CHO cultures was also observed in cultured human CHRF-288-11 and primary hematopoietic stem and progenitor cells. This study demonstrates an underappreciated massive protein and RNA exchange between cells mediated by EVs spanning cell type, suggesting that the proximity of cells in normal and tumor tissues may also result in prolific exchange of cellular material. This exchange would be expected to homogenize the cell-population cytosol and dynamically regulate cell proliferation and the cellular state.

The microRNomes of Chinese Hamster Ovary (CHO) cells and their extracellular vesicles, and how they respond to osmotic and ammonia stress

“…The morphology of cells in stressed cultures was markedly different from cells in standard cultures. As previously presented (Belliveau & Papoutsakis, 2022), in exponential phase of standard cultures, cells exhibit long microvilli that cover a large portion of the cell surface and EV-like structures attached to the cell as shown in two representative SEM images in Figure 1e. Cells from ammonia and osmotic stressed cultures lack the long microvilli structure and the cell surface appear to be covered in EV-like structure.…”

“…Cells from ammonia and osmotic stressed cultures lack the long microvilli structure and the cell surface appear to be covered in EV-like structure. Microvilli are likely important in the ability of cells to capture and integrate EVs (Belliveau & Papoutsakis, 2022; Jiang et al, 2017). The lack of microvilli on stressed cells might suggest a reduced cellular capacity to capture and integrate EVs from the surrounding medium.…”

“…EVs were isolated with differential ultracentrifugation following the protocol previously described (Belliveau and Papoutsakis (2022)). Briefly, cells were first removed from the culture by centrifugation at 180 g for 5 minutes, then cellular debris and apoptotic bodies removed by centrifugation at 2000 g for 10 minutes.…”

“…We had previously shown that CHO cells exchange massively cytoplasmic material through EVs (Belliveau & Papoutsakis, 2022), thus suggesting that EVs have a homogenizing effect of cellular content, and notably of small non-coding RNAs, including miRs, in which EVs are highly enriched in. miRs regulate many cellular programs and phenotypes, including cell growth, metabolism, protein expression, and secretion and thus culture performance.…”

“…These loading mechanisms may differ for microparticles and exosomes and among cell types, and overall are not well understood (Corrado et al, 2021). We have demonstrated in in vitro cultures of CHO cells and human primary and immortalized cells that there is a massive, dynamic process whereby EVs are continuously produced and taken up by cells (Belliveau and Papoutsakis (2022)) leading to widespread exchange of proteins and RNAs via EVs between the cells in the same culture. In view of the extensive exchange of RNAs between cells in culture through EVs, and the central role of miRs in the cellular adaptive responses to stress (Anthony K. L. Leung & Sharp, 2007; A.…”