Investigating the Impact of Various 2D and 3D Cell Culture Platforms on the Production of Extracellular Vesicles.

Cell culture has become the basis for understanding the fundamental mechanisms of cell, tissue and organ function. Although major advancements in uncovering the underlying processes and mechanisms of normal and diseased cell biology have been made by using two-dimensional (2D) cell culture, there is a recent shift in moving towards three-dimensional (3D) culture platforms. The motivation is to better recapitulate the microenvironment of cells in vivo to obtain results that are more indicative to actual cellular processes. In this study, electrospun nano-fibrous scaffolds made of polycaprolactone were used as a 3D culture tool to investigate difference in cell behavior and gene expression in normal breast epithelial cells, 184B5, and breast cancer cells MCF7 and MDA-MB-231. Cells were seeded individually as well as in cocultures on the various platforms and treated with the fluorescent fructose mimic, ManCou-H for 24h and 48h. Differences in cell behavior as well as gene expression was observed amongst the different culture platforms indicating that there are discrepancies when limiting cell culture studies to 2D platforms. The varying gene expression of both GLUT5 and cytokeratin-18 amongst the platforms indicated that 3D culture platforms should be considered in experiment design.

Biomaterial-enabled 3D cell culture technologies for extracellular vesicle manufacturing.

A novel immunoassay for ApoB-100, the main protein component of lipoproteins, enables the development of methods to enrich extracellular vesicles from human plasma while depleting both lipoproteins and free proteins.

Human cancer tissue is a complex ecosystem with heterogeneous cell types that communicate with each other dynamically to promote tumor progression and immune evasion. Hence, experimental systems that destroy or select for cancer cells in culture have often failed to accurately predict clinical responses of new drugs. Therefore, a technology that can retain the intact tumor microenvironment (TME) and accurately predict cancer patient’s response to anti-cancer treatments, including immunotherapies, will greatly benefit patients and dramatically reduce the cost of new drug development. E-slice is a proprietary 3D human tumor culture platform that maintains individual patient tumor’s unique ecosystem and the TME ex vivo for up to 30 days. It is generated by making thin sections of intact, fresh tumor tissues and culturing them in serum-free, defined media. As such, immune components and the TME in E-slices faithfully recapitulate human tumors ex vivo. We demonstrate that E-slices can be used to: 1) measure viability changes upon anti-cancer agent treatment longitudinally; 2) retain the native TME and tissue architecture since E-slices are never dissociated or artificially reconstituted; 3) culture any solid tumor types from patient needle biopsy cores or surgical samples, PDX, or mouse models; 4) perform high content drug screening on human tissues; 5) discover secreted biomarkers and to 6) discover or validate molecular mechanisms of action or therapy resistance; and 7) measure immunotherapy responses ex vivo from human tissues. Importantly, it has been shown to accurately predict individual patient treatment responses to chemotherapies and targeted therapies in 4-8 days, paving the way for evidence-based personalized treatment selections in a clinically actionable time frame. Furthermore, single cell RNA-sequencing analysis demonstrates that E-slices can retain the viability and molecular phenotypes of tumor infiltrating immune cells for up to 8 days ex vivo, providing a unique platform to study human tumor-immune interactions and evaluate efficacy of immune modulators on human tumor tissues ex vivo. Citation Format: Viridiana Leyva-Aranda, Thomas Gallup, Jose Maldonado, Corina Margain, Sang Yun, David Gallup, Min Kim, Kyuson Yun. A novel 3D culture platform, E-slice, retains intact tumor microenvironment and tumor infiltrating immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4200.

In recent years, extracellular vesicles (EVs) have emerged as promising biomarkers, cell-free therapeutic agents, and drug delivery carriers. Despite their great clinical potential, poor yield and unscalable production of EVs remain significant challenges. When using 3D culture methods, such as scaffolds and bioreactors, large numbers of cells can be expanded and the cell environment can be manipulated to control the cell phenotype. This has been employed to successfully increase the production of EVs as well as to enhance their therapeutic effects. The physiological relevance of 3D cultures, such as spheroids, has also provided a strategy for understanding the role of EVs in the pathogenesis of several diseases and to evaluate their role as tools to deliver drugs. Additionally, 3D culture methods can encapsulate EVs to achieve more sustained therapeutic effects as well as prevent premature clearance of EVs to enable more localised delivery and concentrated exosome dosage. This review highlights the opportunities and drawbacks of different 3D culture methods and their use in EV research.

Extracellular vesicle (EV)-based cell-free therapies have emerged as a powerful alternative to stem cell transplantation in regenerative medicine, owing to their ability to promote tissue repair while avoiding safety concerns associated with live-cell therapies. However, traditional two-dimensional (2D) cell cultures used for EV production are constrained by low exosome (Exo) yields and limited biological activity. This study introduces a novel and scalable three-dimensional (3D) culture platform based on a hyaluronic acid (HA) and L-ornithine methyl ester (Orn) hydrogel to enhance the production and therapeutic efficacy of stem cell-derived exosomes. The HA-Orn hydrogel was fabricated via a simple and mild crosslinking strategy, forming a biomimetic matrix that promotes spontaneous spheroid formation. Exosomes derived from 3D cultures (3D-Exo) were compared with those from 2D cultures (2D-Exo) in terms of yield, molecular composition, and biological functions. 3D-Exo exhibited significantly increased yield and superior functional properties, including enhanced stimulation of cell proliferation, migration, angiogenesis, and extracellular matrix remodeling. In vivo, 3D-Exo treatment accelerated wound closure and reduced inflammation in a mouse skin injury model, demonstrating robust therapeutic efficacy and safety. Mechanistic studies revealed distinct miRNA expression profiles and activation of regenerative signaling pathways in 3D-Exo. This work presents a cost-effective, scalable, and bioinspired 3D culture system for high-yield and functionally enhanced Exo production. The HA-Orn hydrogel platform offers significant translational potential for advancing cell-free regenerative therapies, particularly in the context of wound healing.

A major clinical hurdle to translate MSC-derived extracellular vesicles (EVs) is the lack of a method to scale-up the production of EVs with customized therapeutic properties. In this study, we tested whether EV production by a scalable 3D-bioprocessing method is feasible and improves neuroplasticity in animal models of stroke using MRI study. MSCs were cultured in a 3D-spheroid using a micro-patterned well. The EVs were isolated with filter and tangential flow filtration and characterized using electron microscopy, nanoparticle tracking analysis, and small RNA sequencing. Compared to conventional 2D culture, the production-reproduction of EVs (the number/size of particles and EV purity) obtained from 3D platform were more consistent among different lots from the same donor and among different donors. Several microRNAs with molecular functions associated with neurogenesis were upregulated in EVs obtained from 3D platform. EVs induced both neurogenesis and neuritogenesis via microRNAs (especially, miR-27a-3p and miR-132-3p)-mediated actions. EV therapy improved functional recovery on behavioral tests and reduced infarct volume on MRI in stroke models. The dose of MSC-EVs of 1/30 cell dose had similar therapeutic effects. In addition, the EV group had better anatomical and functional connectivity on diffusion tensor imaging and resting-state functional MRI in a mouse stroke model. This study shows that clinical-scale MSC-EV therapeutics are feasible, cost-effective, and improve functional recovery following experimental stroke, with a likely contribution from enhanced neurogenesis and neuroplasticity.

Extracellular Vesicles (EVs) are considered promising nanoscale therapeutics for bone regeneration. To date, EVs are typically procured from cells on 2D tissue culture plastic, an artificial environment that limits cell growth and does not replicate in situ biochemical or biophysical conditions. This study investigated the potential of 3D printed titanium scaffolds coated with hydroxyapatite to promote the therapeutic efficacy of osteoblast-derived EVs. Ti6Al4V titanium scaffolds with different pore sizes (500 and 1000 µm) and shapes (square and triangle) were fabricated by selective laser melting. A bone-mimetic nano-needle hydroxyapatite (nnHA) coating was then applied. EVs were procured from scaffold-cultured osteoblasts over 2 weeks and vesicle concentration was determined using the CD63 ELISA. Osteogenic differentiation of human bone marrow stromal cells (hBMSCs) following treatment with primed EVs was evaluated by assessing alkaline phosphatase activity, collagen production and calcium deposition. Triangle pore scaffolds significantly increased osteoblast mineralisation (1.5-fold) when compared to square architectures (P ≤ 0.001). Interestingly, EV yield was also significantly enhanced on these higher permeability structures (P ≤ 0.001), in particular (2.2-fold) for the larger pore structures (1000 µm). Furthermore osteoblast-derived EVs isolated from triangular pore scaffolds significantly increased hBMSCs mineralisation when compared to EVs acquired from square pore scaffolds (1.7-fold) and 2D culture (2.2-fold) (P ≤ 0.001). Coating with nnHA significantly improved osteoblast mineralisation (>2.6-fold) and EV production (4.5-fold) when compared to uncoated scaffolds (P ≤ 0.001). Together, these findings demonstrate the potential of harnessing bone-mimetic culture platforms to enhance the production of pro-regenerative EVs as an acellular tool for bone repair.

The tumor microenvironment (TME) is a complex 3D cellular system comprised of a diverse amalgam of cells. To characterize the TME, we developed a 3D cell culture platform which facilitates imaging-based analysis and cell viability for extended durations. We used this platform to collect in-situ spatiotemporal measurements of cytokine concentrations in the tumor vicinity, track cellular activity, and model local tumor invasion of glioblastoma (GBM) using fast-scanning confocal microscopy. The mechanism by which we achieve long-term 3D culture is perfusion, modeled as flow through a porous medium. Perfusion facilitates regulated transport of nutrients and waste within the soft microgel medium: Liquid-Like Solids (LLS). The interstitial space is tuned to mimic a capillary bed, and surface bioconjugation of the microgels promotes cellular adhesion and migration. To measure cytokine concentrations, GBM tumoroids were grown in LLS and printed into a dispersion of ELISA beads in LLS. The mechanical stability of LLS ensures the tumor and ELISA beads remain stationary without impeding cellular activity. Cytokine on and off-rates were referenced alongside measured bead fluorescence intensities and positions to fit spatiotemporal reaction-diffusion models. Fast-scanning confocal microscopy facilitated in-situ observation of the evolutionary dynamics of tumor progression. Co-culture of patient-derived tumor explants and autologous PBMCs printed into type I collagen-bioconjugated LLS enabled studies of cancer-immune interactions. In-situ cytokine measurements revealed local IL-8 concentrations reached a maximum value of 2 ng ml−1 after 10 hours. A cellular production rate was estimated at 2 molecules cell−1 s−1. Invasive behavior into the proximal space was determined to be super-diffusive; off-lattice agent-based simulations indicated this behavior is a result of the confinement of invasive fronts to the microgel interstitium. The invading glioblastoma cells used anchorage-dependent migration and were guided by geometric cues to traverse the porous bioconjugated LLS network. Cancer-immune interaction studies revealed average CD8+ speeds greater than 2.8 µm min−1 and both chemotaxis and chemokinetic behavior. CD8+ T-cell killing rates were estimated at approximately 3 cancer cells hr−1 initially, monotonically falling over 12 hours to roughly 1 cell hr−1. The development of the in-situ 3D ELISA assay and imaging-based analysis techniques have enabled the tracking of tumor-immune cell interactions, observation of dynamic tumor progression, and local cytokine concentration profiling at appreciable spatiotemporal resolutions. The combination of a physiologically relevant 3D culture platform with the capacity for in-situ qualitative and quantitative observation may lead to new, powerful, preclinical models that allow for interrogation of the TME and decrease the rate of false discovery. Citation Format: Duy Nguyen, Alexander McGhee, Diego Pedro, Alfonso Pepe, Matthew Schaller, Ryan Smolchek, Jack Famiglietti, Stephanie Warrington, W. Gregory Sawyer. High-throughput 3D tumoroid models for immunotherapy and drug discovery. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4303.

Enhanced extracellular vesicle production and ethanol-mediated vascularization bioactivity via a 3D-printed scaffold-perfusion bioreactor system

Extracellular vesicles (EVs) are essential nanoscale mediators of intercellular communication, holding significant potential as early disease biomarkers and therapeutic agents. Present in biological fluids like blood, EVs and their molecular cargo can be detected in liquid biopsies for diverse diagnostic and therapeutic applications. However, the availability of patient samples is often limited for such research. To tackle this challenge and gain insights into in vivo disease mechanisms, in vitro production of EVs from the cell culture models that closely mimic in vivo conditions has become an essential tool. While 2D cell culture has been the standard for high-throughput studies for decades, 3D cell culture is emerging as a more physiologically relevant in vitro tool for mimicking in vivo environments and providing deeper insights into disease. However, there is currently a lack of literature synthesizing and comparing the effects of 3D versus 2D cell culture models on EV production and analysis. In this review, we examine recent studies that compare the impacts of 3D and 2D cell culture models on EV yield, composition, and functionality. We categorize 3D models into subtypes, including spheroids, hydrogels, rigid scaffolds, and bioreactors. Details of each model's impact on EVs compared to 2D cell culture are presented. Furthermore, we discuss the advantages and limitations of these 3D models as identified in individual studies, offering insights to guide future research directions in this evolving field.

Small extracellular vesicles (sEVs) have great promise as effective carriers for drug delivery. However, the challenges associated with the efficient production of sEVs hinder their clinical applications. Herein, we report a stimulative 3D culture platform for enhanced sEV production. The proposed platform consists of a piezoelectric nanofibrous scaffold (PES) coupled with acoustic stimulation to enhance sEV production of cells in a 3D biomimetic microenvironment. Combining cell stimulation with a 3D culture platform in this stimulative PES enables a 15.7-fold increase in the production rate per cell with minimal deviations in particle size and protein composition compared with standard 2D cultures. We find that the enhanced sEV production is attributable to the activation and upregulation of crucial sEV production steps through the synergistic effect of stimulation and the 3D microenvironment. Moreover, changes in cell morphology lead to cytoskeleton redistribution through cell-matrix interactions in the 3D cultures. This in turn facilitates intracellular EV trafficking, which impacts the production rate. Overall, our work provides a promising 3D cell culture platform based on piezoelectric biomaterials for enhanced sEV production. This platform is expected to accelerate the potential use of sEVs for drug delivery and broad biomedical applications.

Small extracellular vesicles (sEVs) have great promise as effective carriers for drug delivery. However, the challenges associated with the efficient production of sEVs hinder their clinical applications. Herein, we report a stimulative 3D culture platform for enhanced sEV production. The proposed platform consists of a piezoelectric nanofibrous scaffold (PES) coupled with acoustic stimulation to enhance sEV production of cells in a 3D biomimetic microenvironment. Combining cell stimulation with a 3D culture platform in this stimulative PES enables a 15.7-fold increase in the production rate per cell with minimal deviations in particle size and protein composition compared with standard 2D cultures. We find that the enhanced sEV production is attributable to the activation and upregulation of crucial sEV production steps through the synergistic effect of stimulation and the 3D microenvironment. Moreover, changes in cell morphology lead to cytoskeleton redistribution through cell-matrix interactions in the 3D cultures. This in turn facilitates intracellular EV trafficking, which impacts the production rate. Overall, our work provides a promising 3D cell culture platform based on piezoelectric biomaterials for enhanced sEV production. This platform is expected to accelerate the potential use of sEVs for drug delivery and broad biomedical applications.

Mesenchymal stem cells (MSCs) are multipotent cells that have the ability to mediate cellular repair through a combination of soluble paracrine factors, as well as bioactive cargo packaged within extracellular vesicles (EVs). Although MSC-derived EVs have been widely investigated for their regenerative potential, progress toward translational evaluation has been limited in part by challenges in scalable and reproducible manufacturing. We recently reported that human telomerase reverse transcriptase (hTERT)-immortalized MSCs reproducibly produce EVs that retain key characteristics of EVs derived from primary MSCs. Building on this work, three-dimensional (3D) culture systems have emerged as promising platforms for large-scale manufacturing. In this study, we compared the yield, molecular composition, and functional activity of EVs produced from hTERT-immortalized MSCs cultured in either a fixed-bed bioreactor or conventional two-dimensional (2D) flasks. Our data demonstrate that bioreactor culture results in increased EV yield as compared to an equivalent production from 2D cultures. Molecular analyses indicated that bioreactor-derived EVs were associated with a broader spectrum of cargo and were enriched with molecules that may contribute to enhanced reparative function. Importantly, bioreactor-derived EVs also exerted a more pronounced effect in cellular repair assays in vitro. Collectively, these results highlight the potential of fixed-bed bioreactors as scalable platforms for EV production, offering higher yields while preserving molecular composition and functional activity. This approach represents an important step toward achieving the reproducible, high-quality EV production required for research and future translational applications.

Breast cancer (BC) recurrence and metastasis are significant causes of death, mainly due to the emergence of therapy-resistant cells. Secreted factors known as extracellular vesicles (EVs), especially small EVs (SEVs) or exosomes less than 100 nanometers in size, have been implicated in the plasticity associated with drug resistance, BC recurrence, and metastasis. EVs derived from BC cells can transform local or distal non-tumor cells, induce the epithelial-to-mesenchymal transition, and trigger uncontrolled proliferation, angiogenesis, and immune suppression of tumor cells. EVs also mediate resistance to hormone therapy and chemotherapy. Given these oncogenic activities, the production of EVs by BC cells presents an attractive target for inhibition. However, the complexity of EV biogenesis and uptake hampers the effectiveness of most pharmacological inhibitors that target single or specific EV synthetic pathways. Because protein synthesis and sorting are the initial steps in the biogenesis of EVs, we inferred that protein folding is a necessary part of EV synthesis. Previous studies revealed that the protein-folding complex, Chaperonin-Containing TCP-1 (CCT), is upregulated in cancer cells to mitigate the damage from misfolded proteins that accumulate due to increased somatic gene alterations and driver mutations. Using RNA sequencing and optimized methods of EV characterization by flow cytometry and nanoparticle tracking analysis (NTA), we determined that CCT has a role in the production of EVs by BC cells. Inhibition of CCT2, the second subunit of the CCT complex, in BC cells deregulated the gene expression of critical components involved in the early stages of EV biogenesis and decreased the release of CCT2-containing EVs. The exogenous expression of CCT2 in BC cells produced small EVs (SEVs) with increased and selective loading of CCT2 RNA and protein cargo. BC cell culture media, enriched for CCT2-containing SEVs using a 0.1 micromolar filter, increased CCT production and activity in recipient cells. Exogenously expressed CCT2 in BC cells also enriched gene expression associated with extracellular exosomes (GO:0070062). Hence, CCT may be essential for the biogenesis of SEVs with transformative properties and a promising therapeutic target for inhibiting cancer-causing EVs. These studies show that CCT directly regulates components of EV biogenesis in BC cells and advances CCT as an essential driver of BC progression through the production of oncogenic EVs. Citation Format: Annette R. Khaled, Carolyn Dang, Lam Truong, Sydney Laxton, James Velazquez, Shoba Kankipati, Priya Gopalan, SA Litherland. Slamming the breaks on extracellular vesicles that cause breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6584.