Thesis-2017-Robinson.pdf (7.07 MB)
Low-temperature pausing: an alternative short-term preservation method for use in cell therapies
thesisposted on 2017-05-30, 16:19 authored by Nathalie J. Robinson
With encouraging advancements in cell therapies, there is a requirement for an effective short-term cell preservation method, enabling time for quality assurance testing and transport to their clinical destination. This project aims to pause cells at ambient temperatures, whilst maintaining viability and function post-preservation. Ambient cell preservation bypasses ice crystal exposure and toxic solute concentrations experienced with cryogenic storage. Storage in ambient conditions also avoids use of toxic cryoprotectants and aims to greatly reduce costs and reliability on specialist machinery. Early work used HOS TE85 cells (derived from an osteosarcoma) as a model. When atmospheric factors were controlled, HOS TE85 cells demonstrated effective recovery in terms of morphology, membrane integrity (viability >90%) and fold growth expansion when paused at ambient temperature for up to 144 hours. Without atmospheric control, addition of the buffering agent HEPES (25mM) to cell medium was required to keep viability above 70%, as well as to maintain yield and continual passage following 144 hours pausing. The pausing potential of therapeutically relevant human mesenchymal stem cells (hMSCs) from three individual donors (M2, M3 and M4) was tested by keeping cells in suspension for up to 72 hours. Using standard medium with the addition of 25mM HEPES, average membrane integrity was maintained above 70%. Following pausing for between 24 72 hours, hMSC attachment efficiency, immunophenotype and tri-lineage differentiation capacity (osteogenesis, adipogenesis and chondrogenesis) remained similar to non-paused cells. Apart from a short lag phase on the first passage, hMSC fold growth expansion level was consistent with the control for all three donors over 3 x 6 day passages. The colony forming unit (CFU) efficiency of paused cells was significantly reduced when compared with non-paused M2 and M4 lines, whilst M3 retained a similar CFU efficiency to its non-paused counterpart. On return to normal culture conditions, hMSCs had comparable metabolic activity rates with non-paused cells for up to 9 hours. Stable pH is vital during pausing and additional antioxidants or apoptotic inhibiters may be required to keep average viability well-above the 70% threshold, set by the US Food and Drug Administration. Collectively, results have been encouraging and show potential for the movement towards using ambient temperature preservation as an option for the short-term storage and transport of cells for therapy.
- Aeronautical, Automotive, Chemical and Materials Engineering
- Chemical Engineering
Publisher© Nathalie Jayne Robinson
Publisher statementThis work is made available according to the conditions of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) licence. Full details of this licence are available at: https://creativecommons.org/licenses/by-nc-nd/4.0/
NotesA Doctoral Thesis. Submitted in partial fulfilment of the requirements for the award of Doctor of Philosophy of Loughborough University.