ESTABLISHING GOVERNING EQUATIONS FOR 3D CELL CULTURE IN PERFUSION BIOREACTORS By JAGDEEP THRIBHUVAN PODICHETTY Bachelor of Technology in Chemical Engineering Jawaharlal Nehru Technological University Anantapur, Andhra Pradesh, India 2009 Master of Science in Chemical Engineering Oklahoma State University Stillwater, Oklahoma 2011 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY May, 2014 ESTABLISHING GOVERNING EQUATIONS FOR 3D CELL CULTURE IN PERFUSION BIOREACTORS Dissertation Approved: Dr. Sundar Madihally Dissertation Adviser Dr. AJ Johannes Dr. Gary Foutch Dr. Hasan Atiyeh ii ACKNOWLEDGEMENTS It’s my pleasure to thank each and every one who helped make this dissertation possible. I would like to express my deepest gratitude to my family for their love and support throughout my life. I would like to thank my advisor Dr. Sundar Madihally for his guidance and support throughout my research, studies and time at Oklahoma State University. His passion and enthusiasm for research has been a great motivator, he has been a good mentor and role-model. I really appreciate his time and effort in reviewing and providing input to not only improve this dissertation but countless other manuscripts and abstracts. Additionally, I would like to extend my thanks to my colleagues Jimmy Walker, Abdu Kalf, Kumar Singarapu, Christian Tormos, Swapneel Deshpande, Carrie German, Kevin Roehm, Leigh Krause, Lukasz Witek, and Yang Shi for their direct and indirect help with this research and reviewing the many versions of this dissertation. Thanks to my committee members, Dr. AJ Johannes, Dr. Gary Foutch and Dr. Hasan Atiyeh for their guidance, assistance, and feedback. I would like to thank my friends for the wonderful graduate school experience. Lastly, I would like to thank Oklahoma State University and the School of Chemical Engineering for providing a great learning opportunity. iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: JAGDEEP PODICHETTY THRIBHUVAN Date of Degree: MAY, 2014 Title of Study: ESTABLISHING GOVERNING EQUATIONS FOR 3D CELL CULTURE IN PERFUSION BIOREACTORS Major Field: CHEMICAL ENGINEERING Abstract: Culturing cells and regenerating tissues in vitro on 3D scaffolds involves several challenges, such as efficient nutrient transportation, uniform stress distribution, and the removal of wastes. Bioreactors not only allow reproducibility but also provide a controlled environment for production of tissues. The objective of this study was to establish fundamental governing equations for the design of tissue engineering bioreactors and scaffolds. The governing equations related to nutrient permeability, mechanical and structural properties of the scaffolds, as well as nutrient consumption kinetics were tested. Large scaffolds with a high aspect ratio were utilized so that the obtained experimental measurements have high signal-to-noise ratio. This allowed the validation of the governing equations used in the computational models with high fidelity. Three different scaffold preparation techniques, freeze drying, salt leaching, and electrospinning were used to fabricate scaffolds with different microarchitecture. Chitosan, gelatin, and polycaprolactone polymers were used to prepare scaffolds. Two types of bioreactor configurations, flow-through and axial-flow, were used in this study. Both were designed to hold same sized scaffolds, but differ in flow configuration, which made them suitable for evaluating and validating the equations. Bioreactors of appropriate flow configuration were constructed in-house for experimental analysis. Computational Fluid Dynamics (CFD) simulations were performed to predict pressure drop, shear stress, deformation, nutrient distribution profile and exit concentration at various operating conditions. Additionally, non-ideal distribution models such as segregation and dispersion were combined with residence time distribution to predict the exit concentration. The model predictions were validated using an experimental setup with metabolically active liver cells. The results show that the scaffold permeability can be calculated using scaffold pore characteristics and deformation could be predicted using simulation. The axial-flow bioreactor performed better than flow-through bioreactor with superior nutrient distribution, lower shear stress, and deformation. Comparison of the outlet oxygen concentrations between the simulation and experimental results showed good agreement with the dispersion model. However, outlet oxygen concentrations from segregation model were lower. These insights help monitor in vitro tissue regeneration, understand the effect of mechanical stimulus on 3D cell culture, and improve quality of the regenerated tissue. iv TABLE OF CONTENTS Chapter Page 1. INTRODUCTION .....................................................................................................1 Aim1: To model porous scaffold deformation induced by medium perfusion ........3 Aim 2: To modeling pressure drop using generalized scaffold characteristics in an axial-flow bioreactor for soft tissue regeneration. ..............................................4 Aim 3: To evaluate nutrient consumption profile and validate consumption predictions with cell culture experiments ................................................................5 Summary ..................................................................................................................6 2. BACKGROUND ......................................................................................................7 2.1 The field of tissue engineering ...........................................................................7 2.2 Biomaterials and bioscaffolds ............................................................................8 2.2.1 Scaffold preparation techniques ..............................................................11 2.2.2 Biomaterial and scaffold properties ........................................................12 2.3 Bioreactors in tissue engineering .....................................................................15 2.4 Flow through porous media .............................................................................20 2.5 Nutrient consumption in porous media ............................................................22 2.6 Non-ideal fluid distribution models for predicting exit concentration ............23 2.7 Computational fluid dynamics in bioreactor design ........................................26 2.8 Three-dimensional cell culture in perfusion bioreactors .................................26 3. MODELING MEDIA INDUCED POROUS SCAFFOLD DEFORMATION .......29 3.1 Introduction ......................................................................................................29 3.2 Materials and methods .....................................................................................31 3.2.1 Sources of materials ................................................................................31 3.2.2 Preparation of porous scaffolds. .............................................................31 3.2.3 Estimation of pore size and porosity of scaffolds ...................................32 3.2.4. Estimation of elastic modulus and Poisson ratio ...................................32 3.2.5. Pressure drop measurement ...................................................................34 3.3. Computational modeling .................................................................................35 3.4. Results and discussion ....................................................................................41 3.4.1 Scaffold characterization ........................................................................41 v Chapter Page 3.4.2. Poisson’s ratio measurement..................................................................42 3.4.3. Darcy to Brinkman transition in the flow-through bioreactor ...............43 3.4.4. Validation of permeability equation ......................................................44 3.4.5. Validation of fluid flow-structural mechanics coupling approach ........47 3.4.6. Effect of scaffold deformation on shear stress. ......................................50 3.4.7. Effect of permeability on pressure drop and deformation. ..................52 3.4.8. Effect of scaffold mechanical properties on structural deformation ......54 3.5 Acknowledgements ..........................................................................................56 4. MODELING PRESSURE DROP IN AXIAL-FLOW BIOREACTOR FOR SOFT TISSUE ENGINEERING ......................................................................................57 4.1 Introduction ......................................................................................................57 4.2. Materials and methods ....................................................................................58 4.2.1 Preparation of porous scaffolds ..............................................................58 4.2.2 Evaluation of mechanical properties. ......................................................60 4.2.3 Estimation of scaffold permeability. .......................................................60 4.2.4 Pressure drop measurement ....................................................................62 4.3. Computational fluid dynamics modeling ........................................................63