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Optimization of Enzyme Dissociation Process Based On OPTIMIZATION OF ENZYME DISSOCIATION PROCESS BASED ON REACTION DIFFUSION MODEL TO PREDICT TIME OF TISSUE DIGESTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Bhavya Mehta, B.E. ***** The Ohio State University 2006 Dissertation Committee: Dr. Jeffrey J. Chalmers Approved By Dr. David L. Tomasko Dr. Deborah M. Grzybowski ------------------------------------------------------ Adviser Chemical Engineering Graduate Program ABSTRACT With the advent and improvement of highly sensitive and accurate molecular analysis technologies, it is possible to study increasingly complex cellular processes. These technologies have played a significant role in cancer diagnosis. However, a common complaint of studies using these technologies is the heterogeneity of the original sample. The lack of homogeneity is a challenge when one works with non-cultured, three-dimensional tissue, which normally consists of a variety of different cell types, in different states. Therefore, the ability to analyze and separate a heterogeneous cell population based on cellular characteristics is a significant analytical and preparative resource for molecular analysis techniques. The sample preparation techniques in case of a tissue sample are rudimentary and opportunities are ample. The cellular components of a tissue sample are bonded tightly together by extracellular material (ECM). The main components of ECM are proteins (collagen and elastin), which are broken down using enzymes such as collagenase, elastase, metalloproteinases etc. The enzyme dissociation process is an integral part of life sciences laboratory and is designed predominantly based on empirical experience of each research group. There is little or no fundamental understanding available about the enzyme dissociation process to produce single cell suspension from a tissue. We believe that enzyme dissociation technique can be predicted and improved by applying ii fundamentals of enzyme kinetics of ECM degradation and diffusion of enzyme molecule through ECM. This research utilized breast tissue (normal and malignant) as a model, but the principles are applicable to other tissue digestion process as well. This study focused on understanding each ECM component in breast tissue and concluded that collagen concentration and distribution are the rate controlling component in breast tissue digestion. Based on high collagen concentration we hypothesized that actual time of tissue digestion can be modeled based on digestion time of collagen. To model the digestion time of collagen in tissue; primary variables like collagen concentration in the tissue, the solution kinetics of collagen degradation and the effect of mass transfer of collagenase in the breast tissue was determined. It was quantified that average collagen concentration in breast tissue is 53 ± 37 µg of collagen per mg of wet breast tissue weight. This concentration provided the basis for the substrate concentration in enzyme digestion process. The kinetic parameters of collagenase was quantified based on fluorescence measurement as 5.9 ± 3.1 µg of collagen/U.min (kcat) and 7.2 ± 1.7 µg of collagen/ml (KM). These kinetic parameters along with the collagen concentration were utilized to calculate the time of digestion based on analytical solution of Michaelis – Menten kinetics. This time was an order of magnitude less than observed by other researchers, which ranged from 1 hour to 16 hours for breast tissue. This discrepancy in the time of digestion was attributed to mass transfer of enzyme in the tissue matrix. The diffusion of enzyme was modeled based simple reaction diffusion model and the predictions were confirmed with empirical studies. Based on these results, it was confirmed that time of breast tissue digestion can be predicted based on time of collagen digestion. iii iv Dedicated to my grandmother, my parents and my brother v ACKNOWLEDGMENTS This exploratory study would not have been possible without the guidance, motivation and challenges presented by my adviser Dr. Jeff Chalmers. I would also like to thank my co-advisor Dr. Deb Grzybowski for her valuable insights and inspirational support. I also wish to express my gratitude to Dr. David Tomasko and Dr. S.T. Yang for being part of my committee and providing constructive criticism on my work. I am indebted to Ms. Kristie Melnik (Research Associate, Dr. Chalmers Lab) and Dr. Xiaodong Tong (Post doc, Dr. Chalmers Lab) for their important recommendations. It was a privilege to work and discuss challenges with highly motivated colleagues like Mr. David Holman, Dr. Shubhayu Basu, Mr. Luis G. Velazquez-Vargas, Mr. Shunahshep Shukla, Dr. Mei Shao, Dr. Mike Mollet, Dr. Huading Zhang and Dr. Oscar Lara. Mr. Ruben Godoy, Mr. Weiwei Hu, Mr Burr Zimmerman and Dr. Supaporn Suwannakham have been constant source of help in and around our laboratory. I consider myself fortunate to enjoy memorable graduate research experience with all my lab mates and fellow graduate students. I am also grateful to Mr. Brian Kemmenoe and Ms. Kathy Wolkan at Campus Microscopy and Imaging Facility at The Ohio State University for their helpful nature and technical know how in matters of imaging. Also, it was an honor to mentor and work with Mr. Andrew Galusha during my research. vi I also want to extend my gratefulness to Dr. Thomas Abraham, Ms Leena Ukil, Ms Tahera Zabuawala and my circle of friends who have never let me miss my family. I also thank Mr. Suvankar Sengupta and all my other team members for helping me to pursue my love for cricket. I am indebted to Mr. Rakesh Jain and Ms Neha Jain for knowledgeable discussions on teachings in Jainism. Lastly, I thank my grandmother, Ms. Pushapaben Mehta; my father, Mr. Chandrakantbhai Mehta; and my brothers, Mr. Somil Mehta and Mr. Nirav Vora and the rest of my family members for their unconditional love and immense faith in me. vii VITA April 18, 1979 Born, Bombay, India June 2000 B.E. Chemical Engineering, University of Mumbai, India October, 2000 – June, 2001 Trainee Engineer, NemOrganics Ltd., Bombay September, 2001 – Present Graduate Research Associate, The Ohio State University PUBLICATIONS Research Publication Holman DW, Grzybowski DM, Mehta BC, Katz SE, Lubow M. 2005. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue. Cerebrospinal Fluid Res 2:9. FIELD OF STUDY Major Field: Chemical Engineering Minor Field: Bioseparation, Cell Culture and Immunological techniques viii TABLE OF CONTENTS Abstract…………………………………………………………………….. …… ii Dedication……………………………………………………………………….. v Acknowledgments………………………………………………………………. vi Vita……………………………………………………………………………… viii List of Tables…………………………………………………………………… xiii List of Figure…………………………………………………………………… xvi Chapters: 1. Introduction...……………………………………………………………….. 1 1.1 Research Motivation……………………………………………………. 1 1.2 Overview of sample preparation methods for molecular diagnosis.......... 3 1.3 Research Objective……………………………………………………… 5 1.4 Contribution and impact of research……………………………………. 6 1.5 Dissertation organization……………………………………………….. 8 2. Literature Review……………………………………………………………. 12 2.1 Epidemiology of breast cancer………………………………………….. 12 2.2 Cancer detection and molecular diagnosis……………………………… 13 2.3 Sample preparation techniques for molecular analysis of tissue…........... 14 2.3.1 Freeze thaw, tissue pulverization and reagent based methods........... 14 2.3.2 Microdissection…………………………………………………….. 14 2.3.3 Single cell suspension method………………………………... ……. 16 2.3.3.1 Mechanical dissociation……………………………………… 17 2.3.3.2 Enzyme digestion………………………………………. ……. 18 ix 2.4 Problem definition……………………………………………………….. 20 2.5 Research plan……………………………………………………………. 22 2.5.1 Description of extracellular matrix…………………………………. 22 2.5.2 Types of collagenase, enzyme kinetics and measurement of kinetic parameter……………………………………………………………. 24 2.5.3 Influence of reaction rate and diffusion rate on enzyme digestion…. 28 2.6 Culture of arachnoidal cells in 3D matrix to study of cerebrospinal fluid (CSF) outflow……………………………………………………... 33 2.6.1 Background of Cerebrospinal fluid………………………………… 33 2.6.2 Circulation of CSF around brain…………………………………… 34 2.6.3 Structure of Arachnoid granulation………………………………… 35 2.6.4 In vitro model of CSF outflow……………………………………... 38 3. A Simple Method of Characterizing The Kinetics Of Bacterial Collagenase Using Dye Quenched Fluorescence Substrate…………………………. …… 55 3.1 Introduction………………………………………………………... …… 55 3.2 Materials and methods…………………………………………….. …… 60 3.2.1 Materials…………………………………………………………… 60 3.2.2 Methods……………………………………………………………. 60 3.2.2.1 Preparation of standard curve………………………….......... 60 3.2.2.2 Preparation of Collagen and enzyme solutions……………… 61 3.2.2.3 Time course measurements…………………………….......... 61 3.2.2.4 Data analysis and comparison………………………….. …… 62 3.3 Results…………………………………………………………………... 63 3.3.1 Preparation of standard curve………………………………………. 63 3.3.2 Measurement of increase in fluorescence as a measure of reaction rate………………………………………………………………….. 63 3.3.3 Determination of kinetic parameters……………………………….. 64 3.3.4 Reproducibility of assay and determination Michaelis Menten kinetic parameter for DQTM gelatin from porcine skin and DQTM human collagen type IV…………………………………………… 65 3.4 Discussion………………………………………………………………. 65 4. Optimization of Enzyme Dissociation Protocol Based on Reaction Diffusion Model to Predict Time of Tissue
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