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Simple Summary Extracellular vesicles (EVs) are packages of information released from cells and are often described as mini maps of their cells of origin. EVs have many uses. For example, those found in the blood of cancer patients may inform us about tumor growth, spread, immune suppression, and drug response/resistance. EVs from other sources, inducing certain types of cells grown under laboratory conditions, have potential as therapeutics and drug delivery vehicles. For this reason, a few studies have compared methods of collecting EVs, but typically using only one EV source for the comparison. We had concerns that testing one source and extrapolating to others may not be adequate. To test this, we selected three HER2-positive breast cancer cell lines that grow in exactly the same type of fluid. We collected the fluid and tested two ways of separating the EVs from the fluid. We studied them based on seven characteristics. Both EV separation methods provided reproducible results for any of the given cell lines. However, the characteristics of the EV isolates were cell line- and method-dependent. Thus, we recommend not relying on a single EV source when comparing and selecting separation techniques for fundamental research or exploitation for clinical utility. Abstract To study and exploit extracellular vesicles (EVs) for clinical benefit as biomarkers, therapeutics, or drug delivery vehicles in diseases such as cancer, typically we need to separate them from the biofluid into which they have been released by their cells of origin. For cultured cells, this fluid is conditioned medium (CM). Previous studies comparing EV separation approaches have typically focused on CM from one cell line or pooled samples of other biofluids. We hypothesize that this is inadequate and that extrapolating from a single source of EVs may not be informative. Thus, in our study of methods not previous compared (i.e., the original differential ultracentrifugation (dUC) method and a PEG followed by ultracentrifugation (PEG + UC) method), we analyzed CM from three different HER2-positive breast cancer cell lines (SKBR3, EFM192A, HCC1954) that grow in the same culture medium type. CM from each was collected and equally divided between both protocols. The resulting isolates were compared on seven characteristics/parameters including particle size, concentration, structure/morphology, protein content, purity, detection of five EV markers, and presence of HER2. Both dUC and PEG + UC generated reproducible data for any given breast cancer cell lines’ CM. However, the seven characteristics of the EV isolates were cell line- and method-dependent. This suggests the need to include more than one EV source, rather than a single or pooled sample, when selecting an EV separation method to be advanced for either research or clinical purposes.

Simple Summary Cancer cell lines, grown on plastic dishes i.e., two-dimensional (2D), are routinely used in cancer research, e.g., when evaluating the effectiveness of potential anti-cancer drugs before proceeding to studies in animal models and then human clinical trials. Stop/go decisions are generally made from these initial studies. As only ~10% of potential anti-cancer drugs succeed during clinical development, this suggests that these models are inadequate. Cells grown as three-dimensional (3D) models, akin to a tumor mass and with other cells that would naturally occur in its environment, should be more clinically relevant. We performed a worldwide survey, open to cancer researchers at all stages and in all settings, to find out what models they use; for what purposes, and why they chose those models. The majority reported using 2D models only, mainly due to lack of experience and costs but expressed interest in 3D cultures. Guidelines on how to develop such models cost-effectively are needed. Abstract To develop and subsequently get cancer researchers to use organotypic three-dimensional (3D) models that can recapitulate the complexity of human in vivo tumors in an in vitro setting, it is important to establish what in vitro model(s) researchers are currently using and the reasons why. Thus, we developed a survey on this topic, obtained ethics approval, and circulated it throughout the world. The survey was completed by 101 researchers, across all career stages, in academia, clinical or industry settings. It included 40 questions, many with multiple options. Respondents reported on their field of cancer research; type of cancers studied; use of two-dimensional (2D)/monolayer, 2.5D and/or 3D cultures; if using co-cultures, the cell types(s) they co-culture; if using 3D cultures, whether these involve culturing the cells in a particular way to generate spheroids, or if they use additional supports/scaffolds; techniques used to analyze the 2D/2.5D/3D; and their downstream applications. Most researchers (>66%) only use 2D cultures, mainly due to lack of experience and costs. Despite most cancer researchers currently not using the 3D format, >80% recognize their importance and would like to progress to using 3D models. This suggests an urgent need to standardize reliable, robust, reproducible methods for establishing cost-effective 3D cell culture models and their subsequent characterization.