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

ABSTRACT: Extracellular vesicles (EVs) have garnered significant interest in the biotechnology field due to their intrinsic therapeutic properties as well as their ability to serve as vehicles for bioactive cargo. However, the lack of an established biomanufacturing platform and limited potency of EVs in vivo remain critical bottlenecks for clinical translation. In this study, we utilized a 3D-printed scaffold-perfusion bioreactor system to assess the response of dynamic culture on extracellular vesicle production from endothelial cells (ECs). We also investigated whether ethanol conditioning, which was previously shown to enhance vascularization bioactivity of EC-derived EVs produced in standard 2D culture conditions, could be employed successfully for the same purpose in a 3D production system. Our results indicate that dynamic culture in a perfusion bioreactor significantly enhances EV production from human ECs. Moreover, the use of ethanol conditioning in conjunction with dynamic culture induces pro-vascularization bioactivity of EC-derived EVs that is correlated with increased EV levels of pro-angiogenic lncRNAs HOTAIR and MALAT1. Thus, this study represents one of the first reports of rationally-designed EV potency enhancement that is conserved between static 2D and dynamic 3D EV production systems, increasing the potential for scalable biomanufacturing of therapeutic EC-derived EVs for a variety of applications. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) have substantial therapeutic potential in a variety of applications. However, translation of EV-based therapies may be hindered by biomanufacturing challenges. EV production to date has predominantly involved the use of tissue culture flasks. Here, we report, for the first time, the use of a tubular perfusion bioreactor system with an integrated 3D-printed biomaterial scaffold for EV production from human endothelial cells. This system increases EV yield by over 100-fold compared to conventional tissue culture systems. Further, we show that an ethanol-conditioning approach that our group previously developed in 2D culture for enhancing EV potency is compatible with this new system. Thus, potency enhancement of EVs for vascularization applications is possible even with significantly increased production rate.

SUBMITTER: Patel DB

PROVIDER: S-EPMC6531369 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Patel Divya B DB Luthers Christopher R CR Lerman Max J MJ Fisher John P JP Jay Steven M SM

Acta biomaterialia 20181122

Extracellular vesicles (EVs) have garnered significant interest in the biotechnology field due to their intrinsic therapeutic properties as well as their ability to serve as vehicles for bioactive cargo. However, the lack of an established biomanufacturing platform and limited potency of EVs in vivo remain critical bottlenecks for clinical translation. In this study, we utilized a 3D-printed scaffold-perfusion bioreactor system to assess the response of dynamic culture on extracellular vesicle p ...[more]

PMID: 30471476

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Project description:Development of systems allowing the maintenance of native properties of mesenchymal stromal cells (MSC) is a critical challenge for studying physiological functions of skeletal progenitors, as well as towards cellular therapy and regenerative medicine applications. Conventional stem cell culture in monolayer on plastic dishes (2D) is associated with progressive loss of functionality, likely due to the absence of a biomimetic microenvironment and the selection of adherent populations. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow cells within the pores of 3D scaffolds in a perfusion-based bioreactor system, followed by enzymatic digestion for cell retrieval. The 3D-perfusion system supported MSC growth while maintaining cells of the hematopoietic lineage, and thus generated a cellular environment mimicking some features of the bone marrow stroma. As compared to 2D-expansion, sorted CD45- cells derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) maintained a 4.3-fold higher clonogenicity and exhibited a superior differentiation capacity towards all typical mesenchymal lineages, with similar immunomodulatory function in vitro. Transcriptomic analysis performed on MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability as well as a significant upregulation of multipotency-related gene clusters following 3D-perfusion as compared to 2D expansion. The described system offers a model to study how factors of a 3D engineered niche may regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems. Nucleated cells were isolated from 5 fresh human bone marrow aspirates by means of red blood cells lyses buffer and then were seeded into a 3D perfusion bioreactor system using a pure hydroxyapatite 3D scaffold and in conventional Petri dishes (2D). After culture for 19 days, cells from both systems were enzymatically retrieved and sorted using anti-CD45-coated magnetic beads. Total RNA was extracted from CD45- cells, QCed and hybridized to Affymetrix microarrays.

Dataset Information

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

Publications

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

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