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.

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].…”

3D Cell Cultures as Prospective Models to Study Extracellular Vesicles in Cancer

“…Another example of the physiological environment effect has been observed in cervical cancer 3D cultures. In this case, the EVs were loaded with a small RNA profile comparable (~96% similarity) to in vivo circulating plasma-derived EVs from cervical cancer patients [ 64 ].…”

Therapeutic Potential of Extracellular Vesicles for the Treatment of Corneal Injuries and Scars

“…Extrinsic factors such as culture medium composition and the extracellular matrix may influence the quantity and potency of EVs. For example, a three-dimensional system has been shown to stimulate EV production, 82 and treatment with proinflammatory cytokines enhances the release of anti-inflammatory EVs from MSCs. 72 , 83 , 84 Overexpression of the bioactive molecules in parental cells could result in more potent EVs.…”