Chau, G.K.; (2009) Tube-forming device design for the creation of cell-integrated alginate tubes. Doctoral thesis , University of London.

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Abstract

This thesis describes a generic system capable of forming cell-populated alginate tubes either by seeding cells within its lumen, or integrating cells within the tube walls. The applications of an alginate tube are as diverse as applications of alginate beads, and could be used with many types of cells for many different purposes – from cell therapy to tissue engineering. The aim of this work has therefore been on the ability: to reproducibly create alginate tubes with uniformly thick walls of predictable thickness; to be able to monitor and quality control said tubes and a generic cell suspension, including automating aspects of mammalian anchorage-dependent cell culture to improve reliability; and to integrate said cells into an alginate tube without compromise to wall thickness, cell viability and cellular spatial distribution within the alginate tube. This work describes experimental verification a novel fluid dynamics model that predicts with any two fluids used in this reverse dip-coating device that the tube wall thickness will be approximately equal to 2/3 the gap width as gap width becomes negligible. Robustness testing of the tube-forming device prompted two base unit designs and a protocol in order to achieve coefficient of variation (CV) values under 5% of tube length for infusion rates up to 100ml/min and alginate concentrations ranging from 0.50 – 1.00%. Tubes with wall thicknesses between 143.4 – 277.3ìm can be reliably reproduced for tubes of any length. Optical coherence tomography (OCT) at ±10ìm accuracy was adapted to directly monitor alginate wall thickness and rate of shrinkage in real-time through air. This was determined to be ~12 minutes for tube walls to stabilise and a high speed camera showed no spherical regulator spin as the tube is formed, indicating that monitoring at one point is sufficient to determine the overall quality of the tube wall consistency. Cell sample homogeneity monitored by particle sizer revealed two distinct single-celled populations, and smaller peak of cytoplasmic residue. Capillary cytometer was determined the best way to enumerate cell quantity reliably and consistently. Holding time above 3 hours can significantly cause aggregation, but this can be controlled using filtration of known pore size. Kenics static mixers were used to integrate cells into alginate prior to tube formation and showed equally good control to wall thickness as pure alginate tubes at ~CV 7%. Cell viability of above 90% after processing through the static mixer and the tube-forming mark 2 device was achievable using Pronova SLG 100 pre-liquified alginate. The Kenics mixers at 12 elements showed a 49.6% improvement in CV of spatial distribution of cells across alginate, although this could be bettered by increasing number of Kenics static mixing elements, at the cost of increasing dead volume.

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