Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process.

Autor: Heathman TR; Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK., Glyn VA; Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK., Picken A; Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK., Rafiq QA; Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK.; Aston Medical Research Institute, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET., Coopman K; Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK. k.coopman@lboro.ac.uk., Nienow AW; Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK.; Centre for Bioprocess Engineering, University of Birmingham, B15 2TT, UK., Kara B; FUJIFILM Diosynth Biotechnologies, Billingham, TS23 1LH, UK., Hewitt CJ; Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK.; Aston Medical Research Institute, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET.
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2015 Aug; Vol. 112 (8), pp. 1696-707. Date of Electronic Publication: 2015 Apr 20.
DOI: 10.1002/bit.25582
Abstrakt: Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 10(5) cells.mL(-1) in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.
(© 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)
Databáze: MEDLINE
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