Extracellular vesicles from organoids and 3D culture systems

Abstract

When discovered, extracellular vesicles (EVs) such as exosomes were thought of as junk carriers and a means by which the cell disposed of its waste material. Over the years, the role of EVs in cell communication has become apparent with the discovery that the nano‐scale vesicles also transport RNA, DNA, and other bioactive components to and from the cells. These findings were originally made in EVs from body fluids of organisms and from in vitro two‐dimensional (2D) cell culture models. Recently, organoids and other 3D multicellular in vitro models are being used to study EVs in the context of both physiologic and pathological states. However, standard, reproducible methods are lacking for EV analysis using these models. As a step toward understanding the implications of these platforms, this review provides a comprehensive picture of the progress using 3D in vitro culture models for EV analysis. Translational efforts and regulatory considerations for EV therapeutics are also briefly overviewed to understand what is needed for scale‐up and, ultimately, commercialization.

Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions

“…Whereas, the cultivation of hUC-MSCs in a microcarrier-based culture was demonstrated to increase the yield of EVs up to 20-fold compared to 2D cultures [ 52 ]. Additional studies describe similar findings with culture systems such as a 3D-printed scaffold-perfusion bioreactor [ 83 , 84 ], spheroid/aggregate/organoid culture [ 85 ], and 2.5D surfaces (e.g. microcarriers).…”

The Roles of Extracellular Vesicles and Organoid Models in Female Reproductive Physiology

“…Spheroids are 3D cell clusters that do not require an extracellular matrix to form and cannot self-assemble or proliferate long term ( Figure 1 D) [ 6 ]. Similar to explants, spheroids often form a necrotic core, which impacts studies involving EVs, as apoptotic bodies are released by these dying cells, which can confound data because the size of apoptotic bodies overlaps with other EV types (see below) [ 7 ]. Developing spheroids with endometrial glandular tissue gave rise to a new generation of 3D cell cultures in domestic animals, which included the first canine 3D endometrial gland and stromal cell co-cultures [ 8 ].…”

“…Most research evaluating EVs secreted by organoids has focused on cancer, cardiac repair, and stem cells [ 7 ], but the data from these studies may be extrapolated for use in reproductive organoids and EVs. For example, while evaluating stem cells of the oral cavity, the authors compared 2D and 3D cell culture models and concluded that 3D cell cultures secrete a significantly higher amount of EVs [ 90 ].…”

In vivo imaging of EVs in zebrafish: New perspectives from “the waterside”

“…As for spheroids, organoids, and assembloids, 44 the community is still mostly focussing on the isolation of EVs from these structures followed by a posteriori analyses, for example by proteomics or RNA‐omics. Interestingly, compared to EVs derived from cells grown in 2D monolayers, acquisition of a complex 3D structure by the exact same cell type is known to modify the protein and nucleic acid cargo content of the released EVs, as well as their size distribution 45‐48 . This indicates that the spatial and geometric organization, and therefore the complex cell‐cell/cell‐ECM interactions impact the (sub)type of EVs that is secreted.…”

“…Interestingly, compared to EVs derived from cells grown in 2D monolayers, acquisition of a complex 3D structure by the exact same cell type is known to modify the protein and nucleic acid cargo content of the released EVs, as well as their size distribution. 45 , 46 , 47 , 48 This indicates that the spatial and geometric organization, and therefore the complex cell‐cell/cell‐ECM interactions impact the (sub)type of EVs that is secreted. It is clear that these 3D systems are a qualitative improvement over 2D systems and therefore rightly deserve more attention in EV research.…”