Development, characterisation and application of a novel nanofiber-based 3D technique for isolation, enrichment and purification of extracellular vesicles from mesenchymal stem cells

Alfred, Kingsley Isaac

Abstract

Extracellular vesicles (EVs) are nanoscale, lipid-bound particles carrying bioactive molecules produced by cells. Mesenchymal stem cells (MSC) are preferred sources for producing therapeutically relevant EVs (MSC-EVs). Nonetheless, harnessing MSC-EVs for preclinical and clinical applications encounters significant obstacles: the traditional 2D MSC culture systems limit MSC proliferation and functional activity, leading to reduced EVs yield; existing methods for isolating EVs lack consistency, are time-intensive and difficult to scale, and can adversely impact the biological integrity of EVs. To address these challenges, this research effectively developed, tested and successfully applied an innovative 3D nanofiber based devices dubbed “the super inserts” that allow both 3D in vitro culture of MSC and direct isolation/purification of EVs from conditioned media during culture. The super inserts consisted of a top chamber and a bottom chamber partitioned by nano-porous 3D biomimetic PLA nanofiber membrane with refined surface modification. The in situ isolated EVs (3D MSC-EVs) underwent comprehensive characterization, and were compared to EVs derived from traditional 2D MSC cultures isolated via ultracentrifugation (2D MSC-EVs). These analyses included morphological assessments, standard size profiling, and expression of CD9, CD63, and CD81 tetraspanins. Additionally, nucleic acid and lipid membrane staining were performed using complementary methods such as Cryo-EM imaging, dynamic light scattering, and nano-flow cytometry, all in accordance with ISEV guidelines. Two therapeutically relevant in vitro models, myocardial ischemia and reperfusion injury and macrophages inflammation, were used to assess the EVs’ efficacy on regulation of cardiomyocytes and macrophages’ responses. MSC cultivated in the super insert grew 68-94% faster as compared to culturing in 2D TCP. Using the different variations of the super inserts, EVs of sizes 73.87 nm, 77.08 nm and 88 nm were isolated. The isolated EVs exhibited typical lipid bilayer and round morphology. Compared to 2D MSC-EVs obtained ultracentrifugation, in situ isolation led to a fivefold increase in EVs yield. 3D MSC-EVs displayed significantly greater abundance of nucleic acids (75% vs 72.2%), lipid membranes (67% vs 29%) and tetraspanins (CD9, CD63 and CD81) markers (69-93% vs 7.7%) compared to 2D MSC-EVs. Additionally, 3D MSC-EVs exhibit superior therapeutic potential in attenuating myocardial ischemia and reperfusion injuries compared to 2D MSC-EVs and were more efficient at suppressing LDH release from cardiomyocytes following I/R simulation by 69% and 84% at 24 and 48 hours respectively, in comparison to 53% and 78% with 2D MSC-EVs. The enhanced therapeutic potential of 3D MSC-EVs over 2D MSC-EVs was attributed to elevated concentrations of cytokines VEGF and IGF-1 with ~ 2.8 and 2 fold higher in 3D MSC-EVs than in 2D MSC-EVs respectively. Conditioned media from MSC grown in the super inserts (3D-CDM) showed greater anti-inflammatory effects on M1 macrophages than conditioned media from MSC grown on 2D TCP (2D-CDM). The total protein yield was 6.9 fold higher in 3D-CDM than 2D-CDM. Specifically, 3D-CDM markedly reduced the release of pro-inflammatory cytokines IL-8, IL-6, and IFNγ, and caused a 2.49 fold and 3.33 fold increase in the secretion of IL-10 and IL-4 respectively by M1 macrophages compared to 2D-CDM. Overall, this research indicates that 3D-MSC-EVs could surpass the traditional 2D MSC-EVs in clinical effectiveness. The introduction of the innovative super inserts emerges as a promising resolution to the persistent challenges in EVs isolation, offering a method that does not only reduces time and cost but also enhances the yield, integrity and purity of EVs.

Citation

Alfred, K. I. Development, characterisation and application of a novel nanofiber-based 3D technique for isolation, enrichment and purification of extracellular vesicles from mesenchymal stem cells. (Thesis). Keele University. Retrieved from https://keele-repository.worktribe.com/output/1015802

• Thesis Type: Thesis
• Deposit Date: Dec 13, 2024
• Public URL: https://keele-repository.worktribe.com/output/1015802
• Additional Information: Embargo on access until 23 October 2026 - The thesis is due for publication, or the author is actively seeking to publish this material.
• Award Date: 2024-12