Silva Couto, Pedro; (2022) Development of a scalable and consistent manufacturing process for human mesenchymal stromal cells from different sources for therapeutic applications. Doctoral thesis (Ph.D), UCL (University College London).

Text PhD Thesis PSC.pdf - Accepted Version Access restricted to UCL open access staff until 1 October 2025. Download (6MB)

Abstract

Over 1500 clinical trials using human mesenchymal stem/stromal cells (hMSCs) have been registered worldwide during the last two decades. Although hMSCs can be isolated from multiple sources, little is known about the biological differences. To address this challenge, a multi-source screening study was conducted using both adult (adipose tissue-AT, bone marrow-BM) and perinatal (umbilical cord tissue-UCT) sources of hMSCs. UCT-hMSCs demonstrated higher proliferative ability than AT- and BM-hMSCs. Moreover, higher transduction efficiencies were achieved with UCT-hMSCs when transduced with a 2nd generation lentiviral vector (LV) system. With respect to VEGF production, BM-hMSCs were shown to result in the highest concentrations, followed by AT-hMSCs with UCT-hMSCs showing negligible production levels. Microcarrier screening studies were also established and these differences did not appear to impact cell to matrix interactions. The same two microcarriers (Cytodex 1 and Spherecol) led to the highest cell concentrations when a screening study was performed. This work extended existing hMSC-microcarrier work by investigating the feasibility of using microcarrier and suspension culture to expand gene-edited hMSCs. An LV system was used to generate two genetically modified hMSC populations, one carrying a GFP gene and a VEGF gene. These populations were successfully expanded in a 100 mL total volume spinner flask reaching comparable yields with an untransduced control (≈1.8x105 cell/mL). Neither the genetic modification step nor the expansion step changed the cellular immunophenotype. Finally, this thesis investigated the generation of a novel, suspension-adapted hMSC cell line with the potential for EV production. An immortalised AT-hMSC cell line was adapted to be cultured in suspension without the use of microcarriers reaching concentrations close to 3.0x106 cell/mL. Although CD73 and CD105 expression levels remained close to 90%. CD90 was down regulated during the adaptation process.

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