Effect of 2D and 3D Culture Microenvironments on Mesenchymal Stem Cell-Derived Extracellular Vesicles Potencies

Abstract

Therapeutic benefits of mesenchymal stem cells (MSCs) are now widely believed to come from their paracrine signalling, i.e. secreted factors such as cytokines, chemokines, and extracellular vesicles (EVs). Cell-free therapy using EVs is an active and emerging field in regenerative medicine. Typical 2D cultures on tissue culture plastic is far removed from the physiological environment of MSCs. The application of 3D cell culture allows MSCs to adapt to their cellular environment which, in turn, influences their paracrine signalling activity. In this study we evaluated the impact of 3D MSCs culture on EVs secretion, cargo proteome composition, and functional assessment in immunomodulatory, anti-inflammatory and anti-fibrotic properties.MSC-EVs from 2D and 3D cultures expressed classical EV markers CD81, CD63, and CD9 with particle diameter of <100 nm. There were distinct changes in immunomodulatory potencies where 3D cultures exhibited reduced indoleamine 2,3-dioxygenase (IDO) activity and significantly reduced macrophage phagocytosis. Administration of 2D and 3D EVs following double dose bleomycin challenge in aged mice showed a marked increase of bodyweight loss in 3D group throughout days 7–28. Histopathological observations of lung tissues in 3D group showed increased collagen deposition, myofibroblast differentiation and leukocytes infiltrations. Assessment of lung mechanics showed 3D group did not improve lung function and instead exhibited increased resistance and tissue damping. Proteome profiling of MSC-EV composition revealed molecular enrichment of EV markers (compared to parental cells) and differential proteome between EVs from 2D and 3D culture condition associated with immune-based and fibrosis/extracellular matrix/membrane organization associated function.This study provides insight into distinct variation in EV protein composition dependent on the cellular microenvironment of the parental cells, which could have implications in their therapeutic effect and potency. Overall, this work suggests that EVs produced from 3D MSC cultures did not enhance typical MSC-EV properties expected from 2D cultures (immunomodulation, anti-fibrotic, anti-inflammatory). The outcome highlights critical differences between MSC-EVs obtained from different culture microenvironments, which should be considered when scaling up MSC culture for clinical manufacturing.

A quick pipeline for the isolation of 3D cell culture‐derived extracellular vesicles

“…Critically in the EV field, recent studies comparing 2D and 3D culture ‐derived EVs have demonstrated culture condition –dependent impact in the EV properties including size (Rocha et al., 2018 ; Villasante et al., 2016 ), cargo (Palviainen et al., 2019 , Millan et al., 2021 ; Rocha et al., 2018 ; Thippabhotla et al., 2019 ; Villasante et al., 2021 ), therapeutic potential (Cha et al., 2018 ), secretion efficiency (Palviainen et al., 2019 ; Millan et al., 2021 ; Rocha et al., 2018 ; Thippabhotla et al., 2019 ), and drug‐delivery potential (Liang et al., 2019 ). Predominantly, the 3D cultures as a source of EVs have been seen to be beneficial when compared to 2D, but opposite findings exist (Kusuma et al., 2022 ). This highlights the importance of testing and applying an appropriate in vitro cell culture model for each EV research application, which requires setting up and optimizing novel models.…”

Investigating the Consistency of Extracellular Vesicle Production from Breast Cancer Subtypes Using CELLine Adherent Bioreactors

“…These differences suggest that the 3D high-density culture affects EV cargo, however, a direct comparison is needed to confirm these assumptions. It would also be interesting to assess whether the growth conditions affect the function of the EVs as well as their cargo, as recently reported for mesenchymal stem cells [19, 73]. Furthermore we note that although high culture cell viability is recommended for EV preparations in the MISEV2018 guidelines [45], the conditions in the bioreactor system may be more physiologically relevant due to the 3D structure and natural cell turnover which is abundant in tumours.…”

Mesenchymal stem cell culture within perfusion bioreactors incorporating 3D-printed scaffolds enables improved extracellular vesicle yield with preserved bioactivity

“…The observed similarity in angiogenic activity between the flask- and bioreactor-derived MSC EVs were supported by similar cellular uptake of the EVs by HUVECs and comparable surface protein fingerprints ( Figure 6B, D ). Previous works have shown that cell culture conditions (e.g., bioreactor vs. flask or 2D vs. 3D culture) can enhance EV efficacy, alter EV uptake by recipient cells, and change the EV proteome profile [50, 63, 64]. Importantly, proteomic profiling of EVs utilizing the technique used in the current investigation (i.e., MALDI-TOF MS) can be sensitive.…”