3D cell culture stimulates the secretion of in vivo like extracellular vesicles

Abstract

For studying cellular communications ex-vivo, a two-dimensional (2D) cell culture model is currently used as the "gold standard". 2D culture models are also widely used in the study of RNA expression profiles from tumor cells secreted extracellular vesicles (EVs) for tumor biomarker discovery. Although the 2D culture system is simple and easily accessible, the culture environment is unable to represent in vivo extracellular matrix (ECM) microenvironment. Our study observed that 2D culture-derived EVs showed significantly different profiles in terms of secretion dynamics and essential signaling molecular contents (RNAs and DNAs), when compared to the three-dimensional (3D) culture-derived EVs. By performing small RNA next-generation sequencing (NGS) analysis of cervical cancer cells and their EVs compared with cervical cancer patient plasma EV-derived small RNAs, we observed that 3D culture-derived EV small RNAs differ from their parent cell small RNA profile which may indicate a specific sorting process. Most importantly, the 3D culture derived EV small RNA profile exhibited a much higher similarity (~96%) to in vivo circulating EVs derived from cervical cancer patient plasma. However, 2D culture derived EV small RNA profile correlated better with only their parent cells cultured in 2D. On the other hand, DNA sequencing analysis suggests that culture and growth conditions do not affect the genomic information carried by EV secretion. This work also suggests that tackling EV molecular alterations secreted into interstitial fluids can provide an alternative, non-invasive approach for investigating 3D tissue behaviors at the molecular precision. This work could serve as a foundation for building precise models employed in mimicking in vivo tissue system with EVs as the molecular indicators or transporters. Such models could be used for investigating tumor biomarkers, drug screening, and understanding tumor progression and metastasis.

Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes

“…Most of the studies about EVs from human cells have been made with cell lineages, including HeLa [ 29 , 30 ], THP-1 [ 18 , 31 ], HEK [ 32 , 33 ], HMC-1 [ 34 , 35 ], intestinal cell lines [ 36 ], or brain endothelial cells lines [ 37 , 38 ], and few with primary cells, such as dendritic cells (DCs) [ 39 – 41 ] or neutrophils [ 42 ]. However, detailed protocol for isolation, characterization, and analysis of interaction with recipient cells of sEVs released by primary human macrophages derived from circulating monocytes are barely available.…”

Human Plasma-Derived 3D Cultures Model Breast Cancer Treatment Responses and Predict Clinically Effective Drug Treatment Concentrations

“…It should be clarified that the differences between the number of particles/mL and the EV protein concentration are reflecting the measurement of any particle isolated by ultracentrifugation in comparison to the actual protein measurement. Enhanced secretion of EVs in 3D models has previously been reported [ 67 ]. Finally, human plasma was found to be an excellent reservoir for cytokines involved in key cancer hallmarks including: pro-inflammatory cytokines (IL-β1, MIP-1a, TNFα, and RANTES) [ 68 , 69 ], chemokines influencing the invasiveness and migration (SDF-1) [ 70 ], cytokines supporting tissue repair/ECM degradation and remodeling (TIMP1, TIMP2, MMP1, and MMP9) [ 71 , 72 ], and cytokines promoting cell growth (INF-γ, PDGF-AB, IL-2, and LIF) [ 69 , 73 ].…”

Engineered Extracellular Vesicles: Tailored-Made Nanomaterials for Medical Applications

“…However, the majority of studies elucidating EVs composition and hence their mechanism of action, isolate these nanoparticles from 2D TCP, a highly artificial system compared to the cells’ native microenvironment [ 139 ]. For example, Thippabhotla et al demonstrated that EVs derived from cervical cancer cells cultured on TCP displayed a significantly altered RNA and DNA content when compared to that of EVs derived from 3D models, with the later exhibiting ~96% RNA similarity to in vivo derived EVs (compared to ~80% similarity of TCP derived EVs) [ 140 ]. Improving the biomimicry of EVs isolated from in vitro systems allows for more accurate native replication using various engineering strategies utilised.…”