Taylor couette devices for bioreactor applications

TAYLOR-COUETTE DEVICES FOR BIOREACTOR APPLICATIONS QIAO JIAN NATIONAL UNIVERSITY OF SINGAPORE 2013 TAYLOR-COUETTE DEVICES FOR BIOREACTOR APPLICATIONS QIAO JIAN (B. Eng., Tsinghua University) A THEIES SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _______________________________ Qiao Jian Acknowledgments i ACKNOWLEDGEMENTS First and foremost, I would like to show my deepest gratitude to my supervisor professor Wang Chi-Hwa, who has provided me with valuable guidance on my research. Then I shall extend my thanks to Dr Deng Rensheng for his kind help with my research and useful advice on writing this report. In the following, I would like to show my thanks to my colleagues including Dr. Nie Hemin, Sudhir Hulikal Ranganath, Alireza Rezvanpour, Cheng Yongpan, Mr. Xu Qing Xing Noel, Pooya Davoodi, Zhang Wenbiao, Miss Lei Chenlu, Cui Yanna for their kind help with my experiments and the lab officer of WS2, Miss Li Fengmei, Li Xiang, Lim Hao Hiang Joey and Tan Evan Stephen for facilitating me with the administrative matters in the lab. Especially, I would like to thank Miss Jiang Yuwei for her strong support for finishing this report and valuable advice on revising this report. Finally I would like to appreciate the National University of Singapore for providing me the research scholarship to support my study and research. ii Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS . i TABLE OF CONTENTS . ii SUMMARY v LIST OF FIGURES ix Chapter Introduction . 1.1 Background 1.2 Objectives 1.3 Organization of thesis Chapter Literature review . 2.1 Angiogenesis 2.2 PEX protein . 2.3 QM7 cell line . 2.4 NIH/3T3 cell line . 2.5 Polymeric porous PLGA Scaffolds 2.6 Cell seeding and culture . 11 2.7 Taylor vortex flow . 12 Table of Contents iii Chapter Particle-liquid Flow in a Taylor-Couette Device in the Presence of Mobile light Particle . 19 3.1. Introduction 19 3.2 Materials and methodology . 22 3.3 Result and discussion . 28 3.4 Conclusions . 42 Chapter 4: Study of Oxygen Transport in a Taylor-Couette Bioreactor . 45 4.1. Introduction 45 4.2 Material and method 48 4.3 Result and discussion . 52 4.4 Conclusions . 71 Chapter Production of PEX Protein from QM7 Cells Cultured in Polymer Scaffolds in a Taylor-Couette Bioreactor 73 5.1 Introduction . 73 5.2 Materials and Methods 76 5.3 Result and discussion . 85 5.4 Conclusions . 99 Chapter Droplet behavior in a Taylor vortex . 102 6.1 Introduction . 102 6.2 Materials and methodology . 104 6.3 Result and discussion . 110 6.4 Conclusions . 123 Chapter Conclusions and recommendations 125 Table of Contents iv 7.1 Conclusions . 125 7.2 Recommendations . 128 REFERENCES 130 LIST OF JOURNAL PUBLICATIONS 139 LIST OF CONFERENCE PRESENTATIONS 140 v Summary SUMMARY With research and development for almost one century, the Taylor-Couette device is now applied in many practical applications such as reaction, filtration, extraction and bioreactor. We intend to use the Taylor-Couette bioreactor to culture cells that are seeded in a biodegradable porous scaffold. Therefore, the behavior of light porous particle, oxygen transport and cell proliferation was measured in this study. Firstly, we present a study on the behavior of a very light non-spherical particle in the Taylor vortex. The particle used (a cube with the edge length of mm and the density of 0.13 g/cm3) was introduced into a working fluid of mineral oil (density of 0.86 g/cm3 and viscosity of 0.066 Pa.s) contained in a Taylor-Couette device with an aspect ratio of and a radius ratio of 0.67. The interaction between the floating particle and Taylor vortices was investigated using a high speed camera and a particle image velocimetry (PIV) system. Moreover, computational fluid dynamics simulation was performed to calculate the liquid flow pattern and analyze the particle motion. Our results show that the particle behavior in the Taylor-Couette device is strongly dependent on the Reynolds number. With the increasing Reynolds number, four types of particle trajectories were sequentially identified, including a circular trajectory on the surface of the inner cylinder, random shifting between the circular trajectory and oval orbit, a stable oval orbit in the annulus, and a circle along the vortex center. Several unreported particle Summary vi behaviors were also observed, such as the self-rotation of the particle when it moves along the above trajectories. In addition, the PIV measurements show that the trapped particle can only influence the flow pattern locally around the particle. The study can help understand the particle behavior in a Taylor vortex better and therefore benefit applications of particle-laden Taylor vortex devices. Oxygen concentration is always the most significant constraint in a bioreactor and can limit the cell proliferation rate. It is therefore important to know the mass transfer phenomenon and oxygen transport pattern inside the system. However, most studies on the mass transfer properties of the Taylor-Couette bioreactor were focused on the conventional Taylor-Couette device (which has a higher aspect ratio) and rarely on that with a short aspect ratio. In this study, the equilibrium oxygen concentrations at different Reynolds numbers and operation conditions were measured and the mass transfer coefficients were also calculated. CFD simulation was carried out to compare with the experimental results. Both experimental and simulation results showed that the equilibrium oxygen concentration and mass transfer coefficient increased with Reynolds number. To further improve the mass transfer efficiency, air bubble was introduced to the bottom of the rotating inner cylinder and the vortex center. It was proven that the mass transfer coefficient of oxygen could be significantly increased with the trapped bubble. After the study of oxygen transport in the Taylor-Couette bioreactor, the bioreactor was used to culture cells seeded in a biodegradable porous scaffold and produce PEX protein. Two different cell lines (NIH/3T3 and QM7) were seeded Summary vii into PLGA sponges, which were fabricated using a solvent-free supercritical gas foaming method, and then cultured in the Taylor-Couette bioreactor. Cell proliferation was characterized using Quant-iT™ PicoGreen® dsDNA assay and the results indicated that high mass transfer rate in the Taylor-Couette bioreactor enhanced cell proliferation. Qualitative distribution of live/dead cells was characterized using LIVE/DEAD® Viability/Cytotoxicity assay and SEMand the results showed that cells cultured in static control mainly proliferated on the outer surface while the cells of Taylor-vortex bioreactor group could penetrate into the scaffold. The production yield of PEX protein, from QM7 cells transfected with pM9PEX, was quantified using PEX ELISA and the results showed a much higher PEX mass per scaffold for bioreactor than the control. As such, there is potential for the use of Taylor-Couette bioreactor in the mass production of PEX protein. Besides the application as bioreactor, the interesting phenomenon of droplet behavior was observed. The droplets (water or ethanol with the volume of 15-30 μL) were introduced into a Taylor-Couette device with an aspect ratio of and a radius ratio of 0.67, in which a mineral oil (density of 0.86 g/cm3 and viscosity of 0.066 Pa.s) was used as the working fluid. This configuration ensures a laminar Taylor vortex flow with no occurrence of wavy vortex in the entire operating range. The behavior of the droplets was investigated with a high speed camera and a phase Doppler interferometer (PDI) system. The water droplet can be trapped in the vortex center at low Reynolds numbers, corresponding to a circular trajectory, which gradually develops into a three-dimensional toroidal motion with Chapter 125 Chapter Conclusions and recommendations 7.1 Conclusions In this project, the production of PEX protein from transfected QM7 cells cultured in a porous scaffold was studied in a Taylor-Couette bioreactor. in order to have a better understanding of biomedical application of the Taylor-Couette device, several parts of study were carried out, which consist of the particle behavior in a Taylor vortex, oxygen transport in a Taylor-Couette bioreactor, cell proliferation in porous scaffolds in a Taylor-Couette bioreactor as well as the droplet behavior in a Taylor vortex. In the first part, which is study of the behavior of very light particle (used as the scaffold for cell culture for the third part), it was found that the particle behavior depended on the Reynolds number; therefore, the particle trajectory could be precisely controlled by adjusting the Reynolds number. At low Reynolds number, the particle moved around the surface of inner cylinder. When the Reynolds number was increased, the particle showed unstable trajectories: it could move randomly between the circular trajectory and oval orbit. At moderate high Reynolds number regime, the particle showed a stable oval orbit; however, when Reynolds number was further increased, the particle could be trapped at the vortex center and turned to a circular orbit. Besides the particle behavior, the flow field with the existence of particle was also investigated. The results showed that when the particle was trapped at the vortex center, it only had very trivial local Chapter 126 influence to the flow field, which implied that the Taylor vortex flow field was well maintained even when the scaffold was introduced, therefore, the advantage of the Taylor vortex could still be preserved. After the confirmation of stable flow field in the Taylor-Couette device, the oxygen transport in the device was further studied through both experiments and simulation in the second part. It was notable that increasing the Reynolds number at low Reynolds number regime could significantly increase the mass transfer coefficient of oxygen. Moreover, the introduction of new mass transfer surface, bottom bubbles and trapped bubbles, could further increase the mass transfer coefficient, thus the equilibrium oxygen concentration was enhanced significantly. Superior to the conventional bioreactor, the shear stress in Taylor bioreactor could be reduced by avoiding sparging. Last but not least, the mass transfer correlation was also derived for the analysis of the reactor behavior as well as scale-up study in future. The preliminary study of the cell culture was carried out and the results showed that high proliferation rate could be achieved in the scaffold with high mass transfer and suitable shear stress. In the third part, the Taylor-Couette bioreactor was then applied to improve the QM7 cell proliferation rate and the production yield of PEX protein. This involved culturing recombinant QM7 cells, which was seeded in porous polymer scaffolds in a Taylor-Couette bioreactor with a lower aspect ratio and a bigger annular gap. The s cell seeded constructs were suspended in growth medium in the annular gap of the Taylor-Couette reactor for the production of PEX protein. The results indicated a notable higher cell proliferation and PEX protein production owing to the high mass transfer rate and moderate shear stress Chapter 127 environment in the Taylor-Couette bioreactor. The QM7 cell proliferation was studied qualitatively by the live/dead assay staining and confocal microscopy imaging and it discovered that the viable cells were evident both in the interior and exterior of the scaffolds cultured in the reactor. In comparison, the static control group exhibited viable cells only in the outer surface, which proved that the dynamic culture in the Taylor bioreactor helps the cell penetration as well as proliferation in the porous scaffold. On the other hand, PEX ELISA results indicated a much higher PEX mass production rate per scaffold for bioreactor group than static control group, to be more precise, there was a 50 fold increase in dynamic bioreactor group. This gives a promising potential of Taylor-Couette bioreactor in the mass production of the PEX protein, this highly improved production efficiency might reduce the high price of the cancer treatment drugs. Besides the useful applications as a bioreactor, some interesting phenomena were observed in the Taylor-Couette device. The behavior of insoluble liquid phase droplet in the Taylor-Couette device was measured. The results show that the droplets behavior is strongly dependent on the Reynolds number, interfacial tension and density of dispersed phase. The water droplet could be trapped at vortex center at low Reynolds number regime; but if the Reynolds number is increased, it has the trend to escape from the vortex and perform a “dancing” behavior with the increasing Reynolds number. However, the ethanol droplet was trapped inside the vortex at high Reynolds number regime and performs a dancing behavior at low Reynolds number regime, which acted reversely as compared with the water. Furthermore, the ethanol droplet could be destroyed by the shear Chapter 128 stress caused by the rotation of inner cylinder; however, the small droplets from the broken ethanol droplet could coalesce into big droplet again due to the Taylor vortex. The study may contribute to understand the droplet behavior in a Taylor vortex and therefore benefit the application of Taylor vortex device in liquidliquid extraction. 7.2 Recommendations Due to the time constrain, some studies are not yet fully explored. There are some recommendations for future works. 1. Scale-up of the Taylor-Couette bioreactor: the experiment result shows that the Taylor-Couette bioreactor could improve the yield of PEX protein significantly; however, it is just a prototype within a laboratory scale. The study of scale-up should be carried out for the future applications of the Taylor-Couette bioreactor. 2. Purification of PEX protein: in this study, the product of PEX protein was detected by ELISA; however, due to the time constrain, we are not able to carry out the experiments for purification of the PEX protein. With the high yield of PEX production, the cost of purification could also be reduced; therefore in total the price of PEX protein will be reduced and it could be a good benefit for application to the clinic treatment of tumor cells 3. Application of Taylor-Couette device as an extractor: our study demonstrated the breakup-coalescence of the droplet with low interfacial tension in a Taylor vortex. This spontaneous process could have the great potential application in the extraction process. The small droplets after the breakup could increase the surface Chapter 129 area of mass transfer and therefore increase the extraction efficiency; while the spontaneous coalescence behavior reduced the time of separation of two phases after extraction. 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Biotechnol Bioeng. 2008; 100: 998 - 1009 Publications 139 LIST OF JOURNAL PUBLICATIONS Qiao J, Deng R, Wang CH. Particle Motion in a Taylor Vortex. submitted to International Journal of Multiphase Flow (2014) Qiao J, Wang CH. Study of Oxygen Transport in a Taylor-Couette Bioreactor. Submitted to Biochemical Engineering Journal (2014) Qiao J, Lew CMJ, Karthikeyan A, Wang CH. Production of PEX Protein from QM7 Cells Cultured in Polymer Scaffolds in a Taylor-Couette Bioreactor. Biochemical Engineering Journal. 2014; 88: 179-187. Qiao J, Deng RS, Wang CH. Droplet Behavior in a Taylor Vortex. International Journal of Multiphase Flow. 2014. DOI: 10.1016/j.ijmultiphaseflow.2014.08.011 Publications 140 LIST OF CONFERENCE PRESENTATIONS Qiao J, Lim EWC, Wang CH. Bubble Behavior and Oxygen Transport in a Taylor-Couette Bioreactor. AIChE annual meeting, Minneapolis, 2011 Qiao J, Wang CH. Flow Characterization in a Taylor-Couette Bioreactor in the Presence of Mobile Scaffolds. AIChE annual meeting, Minneapolis, 2011 Qiao J, Deng R, Wang Ch. Particle-Liquid Flow in a Taylor-Couette Device in the Presence of Mobile Porous Particles. 5th Asian Particle Technology Symposium, Singapore, 2012 Qiao J, Deng R, Wang CH. Droplet behavior in a Taylor vortex. AIChE annual meeting, Pittsburgh, 2012 Qiao J, Wang CH. Study of Cell Proliferation in Porous Scaffold in a TaylorCouette Bioreactor. AIChE annual meeting, Pittsburgh, 2012 [...]... behaviors in a Taylor vortex and therefore benefit certain applications like liquid-liquid extraction conducted in Taylor vortex devices Keywords: Taylor vortex, Taylor- Couette bioreactor, particle behavior, oxygen transport, cell culture, PEX protein, droplet behavior ix List of Figures LIST OF FIGURES Figure 2.1 Lab scale bioreactors: (A) Spinner-flask bioreactor, (B) Direct perfusion bioreactor, (C)... in the stirred and aerated bioreactor can cause high Chapter 1 4 shear stress and lead to break-up of fragile mammalian cells; therefore, different bioreactors were designed to overcome these limitations, and the Taylor- Couette bioreactor was one of them Taylor- Couette flow is a classic topic in fluid dynamic studies (Taylor, 1923; Davey, 1962) Conventional Taylor- Couette devices have the composition... transfer Because the Taylor- couette bioreactor is not suitable for suspended cells, Santiago et al (2011) cultured the cells in micro-carriers and showed that the Taylor- Couette device could provide effective oxygen transfer and mass transfer The conventional Taylor- Couette device applied as a bioreactor is a device with long aspect ratio; however, it is believed that the Taylor- Couette reactor with... development of almost one century, the Taylor- Couette device has been applied in several engineering areas, such as filter, extraction and reactor (Wereley and Lueptow, 1999; Davis and Weber, 1960; Sczechowski, 1995) A recent application for Taylor- Couette device is the bioreactor Haut et al (2003) cultured the suspended cells in a conventional Taylor- Couette bioreactor; however, suspended cells are... light particle in a Taylor vortex was studied In Chapter 4, the oxygen transport in a Taylor- Couette bioreactor is presented In Chapter 5, the production of PEX Protein from QM7 cells cultured in polymer scaffolds in a Taylor- Couette Bioreactor was studied In Chapter 6, the droplet behavior in a Taylor vortex is presented Finally, Chapter 7 gave general conclusions and recommendations for future studies... cell viability in a Couette -Taylor bioreactor (Curran and Black, 2004; 2005) The mass transfer of oxygen could be greatly enhanced in the presence of Taylor vortex, which suggests that the Taylor- Couette bioreactor could be a potential device for the long term and mass production of cell culture (Curran and Black, 2004; 2005) Dusting and Balabani (2009) studied the mixing effect of Taylor vortex in non-... immobilized porous scaffold in this type of Taylor- Couette bioreactor and proved that the moderate Chapter 1 5 shear stress (0.02–0.19Pa) generated in the bioreactor improved the proliferation of cells 1.2 Objectives High shear stress and low mass transfer rate are always constraints of conventional bioreactor To overcome these limitations, the Taylor- Couette bioreactor was applied to cultivate mammalian... showed that the moderate shear stress provided by the Taylor vortex could be helpful to improve the proliferation of the rBMS cells (Zhu et al., 2009) 2.7.3 Taylor- Couette bioreactor The Taylor- Couette bioreactor was developed from the conventional TaylorCouette device The culture parameters could be precisely controlled by altering the rotation speed of the rotating cylinder because the Reynolds number... Some commonly device for both dynamic seeding and culture includes the spinner-flask bioreactor (Figure 2.1A), the direct perfusion bioreactor (Figure 2.1B) and the rotating wall vessel (Figure 2.1C) (Martin et al 2004) 12 Chapter 2 A B C Figure 2.1 Lab scale bioreactors: (A) Spinner-flask bioreactor, (B) Direct perfusion bioreactor, (C) Rotating wall vessel (Martin et al., 2004) 2.7 Taylor vortex flow... Stephanopoulo, 1993; Liu and Hong, 2001) The synthesis of bio-product is carried out in a bioreactor Conventional bioreactors can be classified as three major types, include no stirred non aerated bioreactor, no stirred aerated bioreactor, and stirred and aerated reactor The former two types of bioreactor are applied for cultivation of anaerobic organism, while the latter is used to culture the microbes . TAYLOR- COUETTE DEVICES FOR BIOREACTOR APPLICATIONS QIAO JIAN NATIONAL UNIVERSITY OF SINGAPORE 2013 TAYLOR- COUETTE DEVICES FOR BIOREACTOR. behaviors in a Taylor vortex and therefore benefit certain applications like liquid-liquid extraction conducted in Taylor vortex devices. Keywords: Taylor vortex, Taylor- Couette bioreactor, particle. per scaffold for bioreactor than the control. As such, there is potential for the use of Taylor- Couette bioreactor in the mass production of PEX protein. Besides the application as bioreactor,