# Hydrogel Embedding of Mesenchymal Stem Cells Supports Extracellular Vesicle Production

## TL;DR

Using a hydrogel to grow mesenchymal stem cells increases extracellular vesicle production, which could improve regenerative medicine therapies.

## Contribution

A scalable 3D hydrogel method for producing extracellular vesicles from mesenchymal stem cells is proposed and validated.

## Key findings

- Hydrogel-encapsulated MSCs produced more extracellular vesicles per volume than traditional 2D cultures.
- EVs from 3D cultures showed improved functionality in a wound healing assay.
- EVs from both 3D and 2D cultures had similar morphological and molecular properties.

## Abstract

Extracellular vesicles (EVs) derived from adherent cells are promising therapeutics for a wide variety of diseases. Previous studies have shown that mesenchymal stem cell (MSC)‐derived EVs have many applications in wound healing and regenerative medicine. Specifically, MSC‐derived EVs are safer than cell‐based therapies because EV treatments do not involve the administration of live cells to patients. However, a lack of scalable workflows for producing EVs from 2D adherent sources is a major current limitation of the field. One proposed method for culture scale‐up is to encapsulate MSCs in a gelatin methacryloyl (GelMA) hydrogel, which provides a 3D matrix that better mimics the in vivo microenvironment of human cells and can be shaped into spherical beads or thin films to support the growth of shear‐sensitive cells inside bioreactors. To establish proof of concept, we embedded MSCs in a layer of GelMA hydrogel to assess the production rate, molecular properties, and functional characteristics of EVs collected from 3D cultures. Hydrogel‐encapsulated MSCs yielded a greater number of EVs per volume of culture compared to traditionally grown unencapsulated MSCs, and 3D cultures produced EVs with improved functionality in a scratch assay relative to vehicle treatment. These findings support the hypothesis that GelMA can be used to support scalable manufacturing of bioactive EVs from adherent cell sources.

This study investigates the effects of 3D culture conditions on extracellular vesicle (EV) production by mesenchymal stem cells (MSCs) in a gelatin‐based hydrogel matrix (GelMA). The EVs from both conditions were found to have similar morphological, molecular, and functional properties, but 3D cultures produced more total EVs per volume.