Human mesenchymal stem cells (hMSCs) are a promising candidate for cell-based therapies given their therapeutic potential and propensity to grow in vitro. However, to generate the cell numbers required for such applications, robust, reproducible and scalable manufacturing methods need to be developed. To address this challenge, the expansion of hMSCs in a microcarrier-based bioreactor system was investigated. Initial studies performed in T-flask monolayer cultures investigated the effect of key bioprocess parameters such as dissolved oxygen concentration (dO2), the level of medium exchange and the use of serum-free media. 20 % dO2 adversely impacted cell proliferation in comparison to 100 % dO2, whilst FBS-supplemented DMEM was found to be the most consistent and cost-effective cell culture medium despite the advances in serum-free cell culture media. Several microcarriers were screened in 100 mL agitated spinner flasks where Plastic P102-L was selected as the optimal microcarrier for hMSC expansion given the high cell yields obtained, its xeno-free composition and effective harvest capacity. The findings from the initial small-scale studies culminated in the successful expansion of hMSCs on Plastic P102-L microcarriers in a fully equipped 5 L stirred-tank bioreactor (2.5 L working volume), the largest reported volume for hMSC microcarrier culture to date. A maximum cell density of 1.68 x 105 cells/mL was obtained after 9 days in culture; further growth was limited by the low glucose concentration and lack of available surface area. A novel, scalable harvesting method was also developed, allowing for the successful recovery of hMSCs. Importantly, harvested hMSCs retained their immunophenotype, multipotency and ability to proliferate on tissue culture plastic.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:574219 |
| Date | January 2013 |
| Creators | Rafiq, Qasim Ali |
| Publisher | Loughborough University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://dspace.lboro.ac.uk/2134/12335 |
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