Casajuana Ester, Mar; (2025) Investigating the Effect of Different Cell Microcarriers on EV Secretion. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Extracellular vesicles (EVs) are promising therapeutic agents due to their safety, stability, and clearance advantages over cell-based therapies. However, their clinical translation faces significant challenges, including low yields and difficulties in scalable production. EVs are often isolated from anchorage-dependent cells, which can undergo multiple changes, including cargo alterations, when exposed to shear stress, cell aggregation, and substrate changes in 3D cultures. Microcarriers enable these cells to be cultured in dynamic environments, optimising EV production conditions. This work compares EV production from human embryonic kidney (HEK-293) cells and adipose-derived mesenchymal stem cells (ADMSC) grown on various commercially available microcarriers (Cytodex-1, Cytodex-3, Corning Enhanced, Collagen, FACT-III, and Cytopore-1), which differ in composition. Multiple techniques were used to compare the surface topography, stiffness, and culture medium protein absorption of the different microcarriers. Hydrophilic microcarriers (Cytodex-1 and -3, and Cytopore-1) had lower stiffnesses and adsorbed more protein. The pores in Cytopore-1 microcarriers created an uneven surface, resulting in the highest roughness values. Cell growth kinetics, attachment, and distribution on the microcarrier surfaces varied by microcarrier and cell type. For HEK-293 cells, Cytodex-1 and -3 outperformed others, offering higher seeding efficiency, more homogenous cell spreading, and a lower proliferation-to-cytotoxicity ratio. For ADMSC, Collagen microcarriers achieved the highest cell expansion and provided more uniform cell distribution along with a lower proliferation-to-cytotoxicity ratio. HEK-293 and ADMSC-EVs from all microcarriers expressed EV markers CD81, CD63, and CD9, displayed classical cup-shaped morphology, and had diameters mostly between 50-250 nm. Culturing HEK-293 cells on Cytodex-1, Corning Enhanced, and Collagen microcarriers resulted in higher EV productivity (EV concentration normalised to cell number). ADMSC-EV productivity was highest with Cytodex-1 microcarriers. ADMSC-EVs promoted human umbilical vein endothelial cells (HUVEC) migration dose-dependently, with those from Cytodex-1 microcarriers showing the greatest effect. This is the first study to compare the effect of different commercially available microcarriers on EV production. The results indicate that cell substrate and culture conditions influence EV production, a key consideration for large-scale EV manufacturing.

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