Masri, MF; (2015) An ultra scale-down tool set for the predictive design of a membrane separation procedure for preparation of human cell therapies. Doctoral thesis , UCL (University College London).

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Abstract

Tools that allow cost-effective screening of the susceptibility of cell lines to operating conditions which may apply during full-scale processing are central to the rapid development of robust processes for cell-based therapies. In this study, an ultra scale-down (USD) device has been developed for the characterization of the response of human cell lines to membrane-based processing, using just a small quantity of cells that is often all that is available at the early discovery stage. Key operating conditions investigated were cross-membrane flow rate, cell age prior to processing and cell concentration (viscosity). The impact was evaluated by cell damage on completion of membrane processing as assessed by trypan blue exclusion and release of intracellular lactate dehydrogenase (LDH). Similar insight was gained from both methods and this allowed the extension of the use of the LDH measurements to examine cell damage as it occurs during processing by a combination of LDH appearance in the permeate and mass balancing of the overall operation. The main cell line studied was a clinically relevant human fibroblast. As expected, increased shear rates led to significant increases in rate and extent of cell damage. Cells aged (21°C hold for 24 hours) before processing led to a doubling of the extent of damage. Increased cell concentration from 1x106 to 100x106 cells mL-1 gave no change in the proportion of cells damaged. Preliminary studies showed that increased shear stress also led to morphological changes and the appearance of apoptotic cells post-processing. Two other human cell lines were also tested briefly for cell damage; a neural stem cell line and a prostate cancer cell line. These appear to be less robust than the fibroblasts with, for example ~0%, ~18% and ~42% damage being observed at the lowest shear stress (~44 Pa) conditions for fibroblasts, prostate cells and neural stem cells respectively. The effects of increasing shear rate, age of cells or concentration varied for each of the cell lines studied. Overall, this work suggests how membrane processing may be used for the recovery of human cells for therapy and how USD studies can speed the route to manufacture.

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