Investigating Bioengineering Approaches to Produce Immuno-modulatory Mesenchymal Stromal Cells for Therapy

Abstract

Human mesenchymal stromal cells (hMSCs) possess potent anti-inflammatory and pro-repair properties that underpin their regenerative potential. However, current clinical trial protocols require transfusion of a substantial number of cells for a positive therapeutic benefit. To meet demand, this rare population of tissue progenitors requires extensive in-vitro expansion.Conventional two-dimensional tissue culture systems are poorly suited to generating clinically relevant cell numbers, motivating the development of dynamic bioprocesses such as stirred tank bioreactors (STRs).

STRs utilising microcarrier growth surfaces have been employed in laboratory settings to support higher hMSC densities and better control over physicochemical parameters necessary for optimal cell growth. However, current cell harvesting methodologies from microcarriers post expansion subject hMSCs to intense mechanical and proteolytic enzymatic stress, resulting in low recovery rates, reduced cell viability and a loss of immunomodulatory properties.

This thesis aimed to optimise STR bioprocessing methodologies using degradable microcarriers that promote the expansion of naïve, immunomodulatory hMSCs as well as reducing harvesting time. This research demonstrates that the enzyme-mediated degradation of Cytodex 3 microcarriers significantly enhances the recovery of hMSCs from STR systems, without affecting viability. Indeed, an almost complete recovery of cells which retain a naïve, stem like phenotype as well as functional immunomodulatory properties and sustained their lineage differentiation potential was observed.

Importantly, hMSCs from the optimised STR-Cytodex 3 cultures can be further expanded within a second STR showing their proliferation is not affected, can be upscaled into 1L STRs to numbers required for a clinical dose and recover from long-term storage. This demonstrates the potential of this refined bioprocessing approach to generate large numbers of therapeutically active progenitors required for cell banks and clinical therapeutics. The resultsprovide a simplified pipeline to address some of the main downstream challenges in the largescaleexpansion and isolation of therapeutically viable hMSCs.

Date of Award	Sept 2025
Original language	English
Awarding Institution	Aston University
Supervisor	Ewan Ross (Supervisor) & Emma Louise Shepherd (Supervisor)