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Title: Bioprocessing of mammalian cells for tissue engineering and cell therapies
Author: Zoro, Barnaby James Henry
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2005
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It is envisaged that in order to achieve the stringent quality requirements of future human cell and tissue manufacturing processes, implementation of automated processing systems will be required. As automated systems are capable of operating at high speeds, throughput is likely to be constrained by sensitivity of cellular materials to mechanical stress. The effect of laminar capillary shear stress (pipe flow) on rat aortic smooth muscle cells was investigated. Initial studies showed that repeated exposure to high shear stress (>100 Pa) causes substantial cell damage, with surviving cells able to grow normally. A flow cytometry assay for cell membrane integrity was developed and exhibited high assay resolution, precision and speed. Further shear studies indicated minimal cell damage or loss in the shear stress range 5-25 Pa, with some evidence of cell damage at high shear stresses (35-75 Pa). Cell loss at very low shear stress ( 2 Pa) was attributed to surface adhesion. Manufacture of cell based therapeutics requires the generation and processing of concentrated cell suspensions. Volume mean cell diameter for rat aortic smooth muscle cells was measured to be 22.4 mum and enabled conversion between cell concentration and cell volume fraction in suspensions. Concentrated cell suspensions behaved as 'power law' (shear thinning) fluids and showed similarity with reported rheology of concentrated yeast and plant cell suspensions. Predictions based on literature values for oxygen uptake rate of mammalian cells indicated that oxygen depletion is likely to occur during processing of cell pellets and concentrates. Two semi-automated cell concentration protocols (centrifuge-resuspend) were developed and compared. Both protocols achieved cell volume fraction of around 0.3-0.4 (wet pellet) and the superior protocol exhibited intact cell yield of ~75%. Apparent loss of cells was mainly attributed to formation of large aggregates during concentration. A 'windows of operation' analysis was constructed to examine process feasibility in the context of experimental results. Three different processing scenarios were predicted to be feasible and it was shown that processing yield is a critical factor in determining overall process feasibility and manufacturing costs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available