Hydrodynamic Shear Sensitivity of Suspension Cultured Plant Cells
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1i.o1 Hydrodynamic Shear Sensitivity of Suspension Cultured Plant Cells by WONG Vai Tak Victor Thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide Department of Chemical Engineering Faculty of Engineering July 2001 ) AcxxowLEDGMENTS of several individuals and I would like to acknowledge the invaluable contributions like to thank my supervisors Dr David organisations towards this work. Firstly, I would their guidance and williams, Dr Christopher colby and Dr David saint for taking the time to edit this thesis and encouragement throughout the project, and for thank Prof Eric Dunlop for his advice and suggest useful changes. I would also like to help in the early periods of the project' setting up the Plant Cell Culture I am grateful to Mr Chris Mansell for his help with well as to Mrs Mary Barrow and Mrs laboratory and for his technical assistance, as I am particularly indebted to the Elaine Minerds for their wonderful secretarial support' Peter Kay, for providing their practical departmental workshop staff, especially Mr viscometer' I would also like to thank expertise in the construction of the novel Couette Department of Environmental Biology at Dr Joe v/iskich and Mz Lidia Mischis of the DO probes and their invaluable advice The University of Adelaide, for the loan of the regarding the oxygen uptake rate measurements' FinancialsupportforthisworkwasprovidedbyanAustralianResearchCouncil scholarship and for contributing grant. I am grateful for their award of a postgraduate towards the operating expenses incurred' IwouldliketothankmyfellowpostgraduatestudentswithintheDepartmentof and discussion' Being an overseas student Chemical Engineering for their camaraderie friends, both in and outside the university' in a foreign land, the emotional support of periods during the project' Although I cannot was essential for getting through difficult to thank Rev. Father Maurice Shinnick. name everyone, I would especially like thanks to my parents' for their Finally, I would like to expfess my deepest, heartfelt my Their unconditional support during my support and encouragement throughout life' them that I would like to dedicate the spirit of education has been invaluable and it is to this work. lt Acknowledgments SuvrvrARY There is growing interest in the industrial cultivation of plant cells in bioreactors for the production of valuable bio-products, including drugs, food flavours and dyes. However, some types of plant cells are sensitive to hydrodynamic shear stresses present in commercial bioreactors, which may significantly inhibit cell growth and product expression achievable. Recent studies have shown that various chemical compounds that disrupt cellular ion-signalling pathways may alleviate similar inhibitory effects on whole plants caused by mechanical and osmotic stresses. This thesis investigates whether the same compounds might also alleviate the negative effects of fluid shear stress on suspension cultured plant cells. The ultimate goals behind this work are: (i) to consider whether the addition of these compounds or other measures could be used to improve the productivity and performance of suspension cultured plant cells grown in industrial bioreactors; and (ii) elucidate the biochemical mechanisms that cause a biological response to fluid shear in suspension cultured plant cells. The effects of four chemical compounds on the biological response of Daucus carota (carrot) cells exposed to laminar shear in a Couette viscometer were studied. The four compounds were: ruthenium red, an intracellular Ca2* channel bìocker; verapamil, a voltage-operated Ca2* channel blocker; 5-nitro-2,3- phenylpropylaminobenzoic acid (NPPB), a Cl- channel blocker; and N-(6-aminohexyl)- 5-chloro-l-napthalene-sulfonamide (V/7), a calmodulin inhibitor. These compounds have previously been shown to alleviate the effects of mechanical and osmotic stresses in whole plants. The carrot cultures were subjected to defined, controlled and reproducible laminar shear stresses in a novel Couette viscometer with O2-permeable walls. The Couette viscometer was designed with the aid of mathematical modelling to ensure sufficient oxygenation. The carrot cells were exposed to different levels of shear stress in the viscometer (38-100 N/m2) and shake flasks in factorial experiments designed to establish the effects of the chemicals on various indicators of biological response. The indicators used were mitochondrial activity, coupled and uncoupled oxygen uptake rate (OUR) and membrane integrity. Additional experiments were conducted in shake flasks over 24 hours to study the effect of long-term exposure of these chemicals on the biological response of the carrot cells in low fluid shear Summary lú conditions. Patch-clamp studies were also performed separately to monitor changes in ion channel behaviour in response to cell membrane stretching, since membrane stretching caused by fluid shear forces may significantly affect the activity of ion channels involved in the signalling process. Results from the factorial experiments found that only W7 and ruthenium red were able to mitigate the negative effects of fluid shear on the carrot cells. W7 reduced the effect of shear on the coupled OUR of the carrot cells by approximately 50Vo, while ruthenium red almost eliminated the effect of shear on mitochondrial activity. However, shake flask experiments suggest that long-terrn exposure of the carrot cells to ruthenium red inhibits mitochondrial activity by approximately 50Vo, counteracting the protective effect observed against fluid shear. The patch-clamp measurements found multiple channels on the cell membrane. One of the channels was likely a Cl- channel but no sensitivity to membrane stretching was detected for this channel. The other channels could not be identified due to their infrequent appearance in the patches, which could not always be successfully patch- clamped. The above results imply that calmodulin and calcium are involved in mediating the biological response of carrot cells to shear. Furthermore, addition of chemicals that suppress calmodulin activity (W7) anüor reduce calcium (ruthenium red) in the cytoplasm of the plant cells could be used to diminish stress effects on OUR (W7) and increase mitochondrial activity (ruthenium red), which could prevent fluid shear from inhibiting the growth rate of the cells in suspension cultures, but only where such chemicals are not toxic to the plant cell itself (ruthenium red). An alternative approach to chemical addition could be to reduce expression of calmodulin and/or activity of calcium channels by genetic engineering. These findings affect our current understanding of the biochemical mechanism by which plant cells perceive and respond to fluid shear forces. Summary lv TAnr-,n op CoNTENTS Chapter L Introduction I l.l Background 1 1.2 Purpose of study 4 1.3 Organization of thesis 5 Chapter 2 Literature Review 8 2.1 Shear sensitivity of plant cell cultures 8 2.2 Biological indicators of shear effects l2 2.3 Shear devices t1 2.4 Mechano-sensitivity of plants 20 2.5 Ion channels in mechanical signal transduction 22 2.6 Conclusions 25 Chapter 3 Methodology 27 3.1 Shear viscometer based investigations 28 3.2 P atch-clamp investigations 31 Chapter 4 Experimental Apparatus 32 4.I Shear device configuration JJ 4.2 Considerations for Couette viscometer 35 4.3 Range of shear stress 35 4.4 Flow stability 4t 4.5 Oxygen transfer 44 4.5.1 Introduction 44 4.5.2 Mathematical model 45 4.5.3 Model solution - Numerical solution strategy 50 Table of Contents 4.5.4 Model parameters 52 4.5.5 Simulation results 54 4.5.6 Alternative viscometer design 56 4.6 Temperature control 60 4.7 Sterility 63 4.8 Description of apParatus 64 Chapter 5 Experimental Methods 67 5.1 Cell culture and medium 67 5.2 Chemicals 68 5.3 Viscometer shear tests 68 5.3.1 Sample PreParation 68 5.3.2 Shear tests in viscometer 68 5.3.3 Biological assays 70 5.3.3.1 Mitochondrial activitY 70 5.3.3.2 Membrane integritY 7l 5.3.3.3 Dissolved oxygen measurement and calculation of oxYgen uPtake rate 7I 5.3.3.4 Regrowth abilitY 72 5.3.3.5 Precautions to limit variability in assay results 13 5.4 Patch-clamPing t4 5.4.1 Protoplast isolation for patch-clamping 14 5.4.2 Solutions for patch-clamping 75 5.4.3 Patch formation and clamping 75 5.4.4 Data acquisition and analysis 71 vt Table of Contents Chapter 6 Factorial Design and Statistical Analysis Methods 78 6.1 Definitions 19 6.2 The half-fraction factorial design,2s-l 81 6.3 Statistical analysis methods 84 6.3.1 Normal scores plot of effects 84 6.3.2 Analysis of variances and F-tests 85 6.3.3 Analysis of residuals 86 Chapter 7 Preliminary Studies 88 7.1 Comparison of growth in shake flasks at different speeds 89 7 .2 Comparison of DO in viscometers 93 7.3 Shear tests in oxygen-perrneable viscometer 96 7.3.1 Effect of shear on biological response 96 7 .3.2 Kinetics of cell damage 108 1.3.3 Variation of biological activity with energy dissipation in the viscometer tt2 7.3.4 Comments on coupled OUR lt4 7.3.5 Correlation between mitochondrial activity and uncoupled OUR TI7 7.4 Discussion 119 Chapter 8 Results and Discussion 124 8.1 Effects of ion channel blockers and calmodulin inhibitor 125 8.1.1 Effects on coupled OUR 125 8.1.2 Effects on uncoupled OUR 138 8.1.3 Effects on mitochondrial activity 145 8.1.4 Effects on membrane integrity 150 8.1.5 Summary of effects for factorial experiments 153 8.1.6 Effects of long-term exposure 155 8.2 Patch-clamp investigations r57 Table of Contents vll 8.3 Discussion 163 8.3.1 Effects of ion-signalling disruption on biological response to shear t63 8.3.2 Implications for industrial plant cell culturing r6l 8.3.3 Possible mechanism for perception and response to fluid shear by plant cells 170 Chapter 9 Conclusions .