IMPLEMENTATION OF A DRUG DISCOVERY TOOL FOR THE EVALUATION OF ANTI-FIBROTIC COMPOUNDS: APPLICATION IN FIBROVASCULAR DISORDERS IRMA ARSIANTI (B. Eng. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements Acknowledgements I would like to express my sincere gratitude to my supervisor, Associate Professor Michael Raghunath, for his supervision and for sharing his invaluable experience during the course of my graduate study. I greatly appreciate his guidance in the research works as well as our informal discussios. Sincere thanks to my co-supervisor, Dr. Phan Toan Thang, for his valuable feedback in this project. I would like to extend my gratitude to the TML members (Ricardo Rodolfo Lareu, Dimitrios Zevgolis, Wong Yuensy, Wang Zhibo, Harve Subramhanya Karthik, Kou Shanshan and the attachment students: Shriju Joshi, Amelia Ann Michael, Natasha Lee, Brenda Lim, Rosanna Chau, Srividia Sundararaman, Zhang Lei) and the Skin Cells Research Group members (Anandaroop Mukhopadhyay and Audrey Khoo). Without their help, support and fruitful discussions, this thesis would not be possible. I would also like to acknowledge the final year students: Lin Gen, Yin Jing, Yanxian, Choo Liling, for making my stay in the laboratory enjoyable. Last but not least, I would like to thank all the support staff in National University of Singapore Tissue Engineering Program (NUSTEP), Tissue Engineering Laboratory and Tissue Repair Laboratory (TRL) for their assistance in this project and Rainbow Instrument (Singapore) for their excellent service for the microplate readers. Graduate Program in Bioengineering i Table of Contents Table of Contents Acknowledgement……………………………………………………………………...i Table of Contents...……………………………………………………………………ii Summary………………………………………………………………………………v List of Tables…………………………………………………………………………vii List of Figures…………………………………………………………………………x Chapter 1. Introduction............................................................................................1 1.1. Project background and significance…………………………………..1 1.2. Objectives……………………………………………………………...3 Chapter 2. 2.1. Literature Review…..………………………………………………….4 Establishment of the cell enumeration assay…………………………..4 2.1.1. Various cell enumeration assays………………………………4 2.1.2. Quantification of cell numbers with DAPI…………………….8 2.2. Enhancement of the collagen matrix in fibroblast cultures…………..10 2.2.1. Collagen properties…………………………………………...11 2.2.2. Collagen biosynthesis………………………………………...12 2.2.3. The challenge in the enhancement of collagen matrix formation in fibroblast culture…………………………………………..16 2.2.4. Macromolecular crowding…………………………………...18 2.2.5. The ideal crowding agent…………………………………….21 2.2.6. Macromolecular crowding in collagen matrix formation…….23 2.3. Chapter 3. 3.1. Exploration of quantitative immunocytochemistry for collagen quantification…………………………………..……………………..24 Experimental Details…………………………………………………25 Equipments…………………………………………………………...25 Graduate Program in Bioengineering ii Table of Contents 3.2. Materials……………………………………………………………...29 3.3. Experimental procedures……………………………………………..33 3.3.1. General methods……………………………………………...33 3.3.2. Cell enumeration assays……………………………………...37 3.3.3. Enhancement of the collagen matrix in fibroblast culture.......38 3.3.4. Collagen quantification assay based on immunocytochemistry ………………………………..………………………………44 Chapter 4. 4.1. Results and Discussion……………………………………………….45 Establishment of the cell enumeration assay…………………………45 4.1.1. Cell enumeration with DAPI…………………………………45 4.1.2. Comparison with MTT cell viability assay…………………..49 4.2. Enhancement of the collagen matrix in fibroblast cultures………….54 4.2.1. Collagen isolation with pepsin digestion…………………….54 4.2.2. The trial of DexS on various fibrogenic fibroblast cell lines...55 4.2.3. Optimization of the DexS concentration in the fibroblast culture………………………………………………………...61 4.2.4. Conversion of procollagen to collagen in the presence of DexS………………………………………………………….66 4.2.5. Collagen crosslinking in the presence of DexS………………68 4.2.6. The cell surface influence on the collagen matrix deposition..69 4.3. Chapter 5. Exploration of quantitative immunocytochemistry for collagen quantification…………………………………………………………75 Conclusions…………………………………………………………..78 References……………………………………………………………………………80 Appendix A. The calculation for Limit of Detection (LOD) and Limit of Quantification (LOQ)………………………………………………...85 Appendix B. The establishment of the cell enumeration assays (the complete results)………………………………………………………………..86 Graduate Program in Bioengineering iii Table of Contents Appendix C. Enhancement of the collagen matrix in fibroblast cultures (the complete results)……………………………………………………...98 Appendix D. Exploration of quantitative immunocytochemistry for collagen quantification (the complete results)………………………………..109 Graduate Program in Bioengineering iv Summary Summary In this project, the principle of macromolecular crowding was applied in a fibroblast culture system to enhance the formation of collagen matrix. We have successfully demonstrated that dextran sulfate (DexS), a polyanionic macromolecule, creates a volume exclusion effect in the culture medium, and thus accelerates the enzymatic processing of procollagen to collagen, and its subsequent deposition. Gel electrophoresis and Western blotting revealed that in normal fibroblast culture, most collagen remained in the culture medium in its unprocessed form. The addition of DexS resulted in the conversion of procollagen to collagen, and the subsequent association of collagen with the cell layer. This observation was confirmed with immunocytochemistry. Remarkably, the crowding effect did not seem to alter the expression level of fibronectin, one of the ECM components. However, we observed re-arrangement of ECM and co-localization of fibronectin with collagen, as compared to conventional culture system without DexS. The optimum concentration of DexS was found to be 50-100 µg/ml. In addition, we were able to show the presence of intensified collagen crosslinking in our culture system. This demonstrated that the specific collagen crosslinking enzyme, lysyl oxidase, was accelerated resulting in the formation of crosslinked collagen matrix in the presence of DexS. This project also aimed to develop an anti-fibrotic drug discovery tool that was fluorometric-based and employed a microplate reader as the quantification device. This tool integrated the cell enumeration assay and the collagen quantification assay on one plate. We have successfully developed a cell enumeration assay that was based Graduate Program in Bioengineering v Summary on the measurement of DAPI-stained nuclei. The fluorescence detected by the microplate readers, FLUOstar and PHERAstar, was then correlated with the cell seeding density. The calibration curves from both readers showed good linearity throughout the tested concentration range; 5000 to 200,000 cells/well for 24-well and 100 to 40,000 cells/well for 96-well plate. In addition, the comparison with MTT assay, an established cell viability assay, showed that the DAPI staining method is comparable or even superior in sensitivity. These results indicated that this method was a suitable cell enumeration assay for the drug discovery tool for adherent cells in monolayer culture in a screening setting. The collagen quantification assay was based on an immunocytochemistry technique. The ECM proteins (collagen and fibronectin) were labeled with specific antibodies that were indirectly conjugated with fluorochromes. The fluorescence measurement showed confirmatory results to that obtained with the gel electrophoresis and immunocytochemistry staining. This preliminary result demonstrated that this method is a potentially suitable collagen quantification assay as a building block of a discovery tool for anti-fibrotic drugs. Graduate Program in Bioengineering vi List of Tables List of Tables Table 3.1 The filter configurations for Olympus IX-71 fluorescence microscope……………………………………………………………27 Table 3.2 3-5% polyacrylamide gel composition……………………………….34 Table 4.1 LOD and LOQ of the cell enumeration assays using DAPI staining and MTT assay……………………………………………………………49 Table 4.2 Comparison between DAPI staining method and MTT assay…….....52 Table 4.3 The densitometry analysis for the optimization of DexS concentration in WI-38 fibroblast culture…………………………………………...64 Table 4.4 The densitometry analysis for the optimization of DexS concentration in HSF culture………………………………………………………..64 Table B.1 The PHERAstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 24-well plate (first experiment)…………………………...86 Table B.2 The PHERAstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 24-well plate (second experiment)………………………...87 Table B.3 The FLUOstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 24-well plate (first experiment)……………………………..88 Table B.4 The FLUOstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 24-well plate (second experiment)………………………….89 Table B.5 The PHERAstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 96-well plate (first experiment)……………………...........90 Table B.6 The PHERAstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 96-well plate (second experiment)………………………...91 Table B.7 The FLUOstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 96-well plate (first experiment)……………………………..92 Table B.8 The FLUOstar reading result for DAPI-stained WI-38 cells plated on a lumox™ 96-well plate (second experiment)………………………….93 Table B.9 The absorbance reading result for WI-38 cell density, plated on 24well plate, as determined with MTT assay (first experiment)………..94 Table B.10 The absorbance reading result for WI-38 cell density, plated on 24well plate, as determined with MTT assay (second experiment)…….95 Graduate Program in Bioengineering vii List of Tables Table B.11 The absorbance reading result for WI-38 cell density, plated on 96well plate, as determined with MTT assay (first experiment)………..96 Table B.12 The absorbance reading result for WI-38 cell density, plated on 96well plate, as determined with MTT assay (second experiment)…….97 Table C.1 The densitometry quantification results for the SDS-PAGE of the cell layer and the medium fraction from pepsin digested HSF culture…...98 Table C.2 The densitometry quantification results for the SDS-PAGE of the cell layer and the medium fraction from pepsin digested WI-38 fibroblast culture………………………………………………………………...99 Table C.3 The FLUOstar reading result for DAPI-stained WI-38 cells after 5 days of culture in the presence of 100 mM AscP and DexS at various concentrations (first experiment)……………………………………100 Table C.4 The FLUOstar reading result for DAPI-stained WI-38 cells after 5 days of culture in the presence of 100 mM AscP and DexS at various concentrations (second experiment)………………………………...101 Table C.5 The densitometry quantification results for the optimization of DexS concentration in WI-38 fibroblast culture…………………………..102 Table C.6 The densitometry quantification results for the optimization of DexS concentration in HSF culture………………………………………..104 Table C.7 The densitometry quantification result for the inhibition of lysyl oxidase with β-APN (Sample 1)……………………………………105 Table C.8 The densitometry quantification result for the inhibition of lysyl oxidase with β-APN (Sample 2)……………………………………106 Table C.9 The densitometry quantification result (based on α1(I) intensity) for the deposition of collagen following the addition of the DexS to the fibroblast culture (Sample 1)………………………………………..107 Table C.10 The densitometry quantification result (based on α1(I) intensity) for the deposition of collagen following the addition of the DexS to the fibroblast culture (Sample 2)………………………………………..108 Table D.1 The PHERAstar reading result for collagen, fibronectin and DAPI staining on WI-38 fibroblasts that were cultured in the presence of 100 µM AscP and DexS at various concentrations for 5 days (first experiment)………………………………………………………….109 Table D.2 The expression level of collagen and fibronectin of WI-38 fibroblasts normalized with the cell population (first experiment)……………..111 Graduate Program in Bioengineering viii List of Tables Table D.3 The PHERAstar reading result for collagen, fibronectin and DAPI staining on WI-38 fibroblasts that were cultured in the presence of 100 µM AscP and DexS at various concentrations for 5 days (second experiment)…………………………………………………………112 Table D.4 The expression level of collagen and fibronectin of WI-38 fibroblasts normalized with the cell population (second experiment)………….114 Graduate Program in Bioengineering ix List of Figures List of Figures Figure 2.1 The molecular structure of MTT and the corresponding reaction product, formazan……………………………………………………...5 Figure 2.2 The chemical structure of DAPI……………………………………….9 Figure 2.3 Excitation and emission profiles of DAPI bound to dsDNA………….9 Figure 2.4 The structure of type I procollagen…………………………………..11 Figure 2.5 The collagen synthesis, processing and assembly……………………16 Figure 2.6 The illustration of the crowding condition in eukaryotic cytoplasm…18 Figure 2.7 The schematic drawing to illustrate the concept of exclusion volume.19 Figure 2.8 Schematic depiction of the predicted dependence of reaction rate on the concentration of crowding agent…………………………………21 Figure 2.9 The structure of DexS with sodium salt……………………………...22 Figure 3.1 The schematic drawing of the first experiment set-up to study the cell surface influence on the collagen deposition………………………...42 Figure 3.2 The schematic drawing of the second experiment set-up to study the cell surface influence on the collagen deposition…………………….44 Figure 4.1 Nuclear staining with DAPI observed under fluorescence microscope……………………………………………………………45 Figure 4.2 The calibration curves for WI-38 cell density, plated on a Lumox™ 24well plate, as quantified with fluorescence microplate readers………46 Figure 4.3 The calibration curves for WI-38 cell density, plated on a Lumox™ 96well plate, as quantified with fluorescence microplate readers………47 Figure 4.4 The calibration curves for WI-38 cell density, plated on 24-well plate, as determined with MTT assay………………………………………50 Figure 4.5 The calibration curve for WI-38 cell density, plated on 96-well plate, as determined with MTT assay………………………………………51 Figure 4.6 Pepsin digested all proteins except collagen…………………………54 Figure 4.7 SDS-PAGE of the cell layer and the medium fraction from pepsin digested HSF culture showing collagen bands and the corresponding densitometry analysis……………………….………………………..55 Graduate Program in Bioengineering x List of Figures Figure 4.8 SDS-PAGE of the cell layer and the medium fraction from pepsin digested WI-38 fibroblast culture showing collagen bands and the corresponding densitometry analysis………………………………...56 Figure 4.9 Immunocytochemistry for collagen I (green) and fibronectin (red) on HDF…………………………………………………………………..58 Figure 4.10 Immunocytochemistry for collagen I (green) and fibronectin (red) on WI-38 fibroblasts……………………………………………………..59 Figure 4.11 WI-38 cell number as quantified with DAPI staining method, after 5 days of treatment with 100µM AscP and DexS at various concentrations………………………………………………………...61 Figure 4.12 SDS-PAGE of the cell layer and the medium fraction of pepsin digested WI-38 culture……………………………………………….63 Figure 4.13 SDS-PAGE of the cell layer and the medium fraction of pepsin digested HSF culture…………………………………………………64 Figure 4.14 The WI-38 fibroblasts morphology observed under phase contrast microscope……………………………………………………………65 Figure 4.15 The HSF morphology observed under phase contrast microscope..…66 Figure 4.16 Western blotting of the medium and the cell layer fraction of HSF culture………………………………………………………………...67 Figure 4.17 The schematic drawing of the effect of volume exclusion in the distribution of procollagen and proteinases in the solution…………..68 Figure 4.18 The inhibition of the lysyl oxidase by β-APN……………………….69 Figure 4.19 The effect of DexS on the deposition of exogenous collagen………..71 Figure 4.20 The deposition of collagen following the addition of the DexS to the fibroblast culture……………………………………………………...73 Figure 4.21 The densitometry analysis and the corresponding graphs of the deposition of collagen following the addition of the DexS to the fibroblast culture……………………………………………………...74 Figure 4.22 The expression level of collagen and fibronectin and the cell population of WI-38 fibroblasts as detected with immunocytochemistry and quantified with PHERAstar microplate reader…………………………………………………………………77 Figure B.1 The calibration curve for WI-38 cell density, plated on a lumox™ 24well plate, as quantified with PHERAstar (first experiment)………...86 Graduate Program in Bioengineering xi List of Figures Figure B.2 The calibration curve for WI-38 cell density, plated on a lumox™ 24well plate, as quantified with PHERAstar (second experiment)……..87 Figure B.3 The calibration curve for WI-38 cell density, plated on a lumox™ 24well plate, as quantified with FLUOstar (first experiment)………….88 Figure B.4 The calibration curve for WI-38 cell density, plated on a lumox™ 24well plate, as quantified with FLUOstar (second experiment)……….89 Figure B.5 The calibration curve for WI-38 cell density, plated on a lumox™ 96well plate, as quantified with PHERAstar (first experiment)………...90 Figure B.6 The calibration curve for WI-38 cell density, plated on a lumox™ 96well plate, as quantified with PHERAstar (second experiment)……..91 Figure B.7 The calibration curve for WI-38 cell density, plated on a lumox™ 96well plate, as quantified with FLUOstar (first experiment)………….92 Figure B.8 The calibration curve for WI-38 cell density, plated on a lumox™ 96well plate, as quantified with FLUOstar (second experiment)……….93 Figure B.9 The calibration curve for WI-38 cell density, plated on 24-well plate, as determined with MTT assay (first experiment)…………………...94 Figure B.10 The calibration curve for WI-38 cell density, plated on 24-well plate, as determined with MTT assay (second experiment)………………...95 Figure B.11 The calibration curve for WI-38 cell density, plated on 96-well plate, as determined with MTT assay (first experiment)…………………...96 Figure B.12 The calibration curve for WI-38 cell density, plated on 96-well plate, as determined with MTT assay (second experiment)………………...97 Figure C.1 The WI-38 cell viability after 5 days of treatment with 100µM AscP and DexS at various concentrations (first experiment)……………..100 Figure C.2 The WI-38 cell viability after 5 days of treatment with 100µM AscP and DexS at various concentrations (second experiment)…………..101 Figure D.1 The expression level of collagen and fibronectin and the cell population of WI-38 fibroblasts as detected with immunocytochemistry and quantified using PHERAstar microplate reader (first experiment)…………………………………………….110 Figure D.2 The expression level of collagen and fibronectin of WI-38 fibroblasts normalized with the cell population (first experiment)……………..111 Figure D.3 The expression level of collagen and fibronectin and the cell population of WI-38 fibroblasts as detected with Graduate Program in Bioengineering xii List of Figures immunocytochemistry and quantified using PHERAstar microplate reader (second experiment)…………………………………………113 Figure D.4 The expression level of collagen and fibronectin of WI-38 fibroblasts normalized with the cell population (second experiment)………….114 Graduate Program in Bioengineering xiii Chapter 1. Introduction 1. Introduction In this chapter, the background and significance of the project is covered in the first section. The second section presents the objectives of the project and the outline of the report. 1.1. Project background and significance Fibrovascular disorders are diseases that are characterized by an increase in the formation of fibrous tissues and its vascularization. This disease can occur in any part of the body, both external and internal, such as skin, eye, joints, lung or liver. The severity of the disease may vary from merely pain and pruritis to functional disability or even fatality. The widespread disposition of this disease thus calls for the search of anti-fibrotic drugs. Fibrovascular disorders are marked with excessive proliferation of fibroblasts that produce massive amount of connective tissues, particularly collagen. It is usually preceded with inflammatory reaction and followed by vascularization of the affected tissues. This phenomenon is similar to that found during wound healing process, which is essentially reflected in the in vitro culture of fibroblasts. Therefore, the assessment of anti-fibrotic drugs usually employs fibroblast culture to determine the effect of the drug on the cell proliferation and collagen deposition. Unfortunately, the assessment of collagen deposition hitherto depends on the quantification of the soluble procollagen (collagen precursor) in the culture medium. This is due to the slow processing of procollagen to collagen in in vitro environment that results in minute amount of collagen matrix and abundance of unprocessed procollagen. The amount of this precursor protein is not necessarily equal to the amount of collagen Graduate Program in Bioengineering 1 Chapter 1. Introduction fibrils in the extracellular matrix that would be the proper measure of fibrosis. Therefore, this project aims to enhance the formation of collagen matrix in in vitro fibroblast culture, to allow an accurate assessment of anti-fibrotic drugs on collagen deposition. As another aspect, the enhancement of the collagen matrix in fibroblast culture may be a valuable tool in tissue engineering. The presence of collagen fibrils in tissue constructs is essential in maintaining the mechanical strength and defining the shape and form of the tissues. Unfortunately, due to the above mentioned procollagen processing setback in vitro, the construction of engineered tissues using fibroblasts has been carried out with suboptimal amounts of endogenous collagen matrix. Therefore, the accomplishment of this project may open an avenue to create fully functional tissue constructs. In addition, collagen has been used as a scaffold or coating in tissue engineering. This collagen mainly comes from various animal origins. In the light of increasing concerns over animal-transmitted diseases, a collagen scaffold fabricated from human fibroblasts might offer an alternative to animal-originated collagen. This project also aimed to develop an anti-fibrotic drug discovery tool that allows the integration of cell enumeration and collagen quantification. Quantification of collagen has been classically done using a radioactive method (metabolic labeling) or colorimetric assays, both having several disadvantages. The first method can be laborious and involves hazardous materials and wastes, whereas the latter is nonspecific to collagen. Therefore, an alternative collagen quantification assay that is based on immunocytochemistry was explored. It was integrated with a cell enumeration method that measures the DNA content of the cell population on test. Graduate Program in Bioengineering 2 Chapter 1. Introduction Both assays are fluorometry-based and the measurement was done using a microplate reader. 1.2. Objectives This project has a main objective of establishing a drug discovery tool that can be applied for the assessment of anti-fibrotic compounds. It can be divided into three sub-aims that provide comprehensive assessments for the fulfillment of the main objective: 1. to establish a rapid cell enumeration assay that is suitable for the quantification of fibroblast population and suitable for a microplate reader analysis, 2. to enhance the formation of collagen matrix on the fibroblast culture, 3. to develop collagen quantification assay that is based on a non-radioactive method and suitable for a microplate reader analysis. Graduate Program in Bioengineering 3 Chapter 2. Literature Review 2. Literature Review This chapter covers the theoretical background for the project, which is divided into three sections; the first is related to the establishment of the cell enumeration assay, the second is related to the enhancement of the collagen matrix on the fibroblast culture, and the third is related to the exploration of the collagen quantification assay. 2.1. Establishment of the cell enumeration assay There are currently many cell enumeration assays, which are based on either cell viability or cell proliferation, available in the market. Much of them will be discussed in the first part of this section. However, despite being convenient, these assays may be implicated with several disadvantages particularly in the application for an antifibrotic drug discovery tool. Therefore, this project aims to establish a reliable cell enumeration assay that is suitable to quantify the fibroblast population and can be incorporated into the anti-fibrotic drug discovery tool. This assay will be based on the quantification of the nuclear content using DNA-binding dye, DAPI, and fluorescent measurement using a microplate reader. 2.1.1. Various cell enumeration assays Conventional cell counting method using a hemacytometer A hemacytometer is a simple device that consists of two fields, each of which is divided into nine 1.0 mm2 squares. A cover glass is placed on top creating a chamber with a depth of 0.1 mm and a volume of 0.1 mm3 (= 10-4 ml) for each square. This method is the most commonly used method to determine the number of viable cells. Usually the dead cells are stained with trypan blue dye, leaving the cells with Graduate Program in Bioengineering 4 Chapter 2. Literature Review uncompromised membrane integrity unstained. The cell suspension is introduced into the hemacytometer chamber and subsequently placed under a microscope for cell counting. Unfortunately, this method has a significant accuracy error due to its subjective nature. Different persons analyzing the same cell population will obtain varying results. This error will be more significant when the number of cells to be counted is small. Therefore this method usually only applies in determination of the cell concentration in batch cultures. Furthermore, counting the cells manually can be laborious and time consuming. Assays that measure metabolic activity Metabolic activity can be an indication of cell viability. There are several metabolic based assays available in the market, MTT assay being the commonly used. This assay is based on the reduction of tetrazolium salts to a colored, water-insoluble formazan that can be quantified in a conventional ELISA plate reader at 570 nm (maximum absorbance) after solubilization. There are currently modified tetrazolium salts, for instance XTT and WST-1 that will be converted by the viable cells to watersoluble formazan, therefore eliminating the solubilization step. Figure 2.1 The molecular structure of MTT and the corresponding reaction product, formazan. (taken from Apoptosis, cell death and cell proliferation, Roche) This assay is relatively simple and convenient. The complete assay starting from the cell culture to the absorbance measurement can be carried out on the same microplate. Graduate Program in Bioengineering 5 Chapter 2. Literature Review However, the cellular metabolic activity is not always equal to the number of viable cells. The metabolic activity of different cell lines may differ resulting in the variation of the cells’ response to tetrazolium salts. In addition, even for a certain type of cell, this response may vary depending on the metabolic state of the viable cell that is influenced by the culture condition, such as pH or D-glucose concentration in the culture medium or the presence of additional substance such as drugs (Shappell, 2003). Therefore, metabolic-based cell viability assay may not be suitable for the drug discovery tool since the drug tested may cause alteration in the metabolic state of the cells. Assays that measure cell proliferation There are several methods to measure cell proliferation, and DNA synthesis is the common method since cellular proliferation requires the replication of cellular DNA. Labeled nucleotides are added to the culture and will be incorporated into the DNA of the dividing cells. Traditionally, this assay involves the use of radiolabeled nucleotide, tritiated thymidine ([3H]-TdR). Alternatively, thymidine analogues, for instance 5bromo-2’-deoxy-uridine (BrdU), are used. The incorporated BrdU is detected immunochemically using a specific ELISA. The complete assay from the start of the cell culture to the ELISA measurement can be performed in the same microplate, making it a convenient assay. Unfortunately, this cell proliferation assay can only capture the cells that replicate within the time window of incubation. This assay fails to include quiescent cells, therefore the result does not represent the whole population of a cell culture. Graduate Program in Bioengineering 6 Chapter 2. Literature Review ATP-based assays The nucleotide adenosine 5'-triphosphate (ATP) plays a dominant role in energy exchange processes in biological systems. The presence of ATP is also a useful marker for cell proliferation. An increase in the ATP level is associated with cell proliferation, whilst cell death exhibits decrease in the ATP level. The commonly used ATP detection method is the chemiluminescent detection of luciferase. Luciferase is the catalyst for the reaction between luciferin and ATP. This reaction produces light as a side product that can be measured using a luminometer. This assay can be performed on the same plate as the culture plate and it is relatively fast. However, this assay has to be completed immediately since ATP does not survive long storage. ATP-based cell proliferation assay might not be suitable when drug treatment is involved. The increase or decrease in the ATP concentration may not necessarily related to the cell number in this case since the drug might alter the biological function of the cells. Assays that measure the cellular DNA quantity The determination of DNA concentration is a reasonable indicator of cell number, since the levels of DNA and RNA in cells are tightly regulated (Frankfurt, 1980). Although the levels of DNA and RNA in individual cells can vary significantly over time, the overall amount of nucleic acid in a given cell population will not change, as long as the cells are asynchronous. Additionally, assays based on nucleic acid binding are generally independent of changes in cellular metabolism. The most common technique to measure nucleic acid concentration is the absorbance determination at 260 nm. This method is simple and easy, however, it is insensitive with a detection Graduate Program in Bioengineering 7 Chapter 2. Literature Review limit of double-stranded DNA (dsDNA) in µg/ml range (Rengarajan et.al., 2002). Moreover, it does not distinguish nucleotides, single-stranded DNA, contaminants and RNA. Recently, the use of DNA-binding dyes to measure DNA concentration has gained popularity recently because it is simple and potentially more sensitive than absorbance measurement (Noites et.al., 1998). There are many DNA-binding dyes available, for instance picogreen, Hoechst, DAPI, ethidium bromide, SYBR and many more. Several studies have shown that picogreen is an ultrasensitive fluorescent nucleic acid stain for quantification of dsDNA in solution, with a detection limit in the range of pg/ml dsDNA (Rengarajan et.al., 2002; Singer et.al., 1997). Unfortunately, DNA quantification method usually involves trypsinization and complete disruption of the cells to get the nuclear contents out. It is not desirable in the anti-fibrotic drug discovery tool, since trypsinization and cell lysing does not allow further processing of the culture such as collagen extraction. 2.1.2. Quantification of cell numbers with DAPI A novel cell enumeration assay based on the DNA quantification method was developed in this project. Nucleic acid dye, 4’,6-Diamidino-2-phenylindole (DAPI), was used to stain the cellular DNA in situ and the fluorescence signal was quantified by a microplate reader. This method is extremely rapid and simple. Moreover, the cell layer remains intact and fixed on the plate allowing further processing of the samples. DAPI is a popular nuclear counterstain when multicolor fluorescent probes are used to stain cellular structures. It emits blue fluorescence that stands out in vivid contrast to Graduate Program in Bioengineering 8 Chapter 2. Literature Review red or green fluorescent probes. The maximum excitation and emission of DAPI when it is bound to dsDNA is 358 nm and 461 nm respectively. Figure 2.2 The chemical structure of DAPI Figure 2.3 Excitation and emission profiles of DAPI bound to dsDNA (taken from MolecularProbes DAPI datasheet). DAPI preferentially stains dsDNA and appears to associate with AT (AdenineThymidine) clusters in the minor groove (Kubista et.al., 1987). Binding of DAPI to dsDNA produces a ~20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove (Barcellona et.al., 1990). DAPI can also bind RNA. However, it is thought that DAPI/RNA binding mode diverse from that of DAPI/dsDNA. The DAPI/RNA involves AU-selective intercalation instead of binding at the AT cluster (Tanious et.al., 1992). In addition, the DAPI/RNA complex exhibits a longer-wavelength fluorescence emission maximum (~500 nm) than the DAPI/dsDNA complex (~460 nm) and a quantum yield that is only about 20% as high (Kapuscinski, 1990). Graduate Program in Bioengineering 9 Chapter 2. Literature Review 2.2. Enhancement of the collagen matrix in fibroblast cultures Among other important properties, the extracellular matrix (ECM) provides a scaffolding structure to which cells are attached within tissues. Collagen is the major component of the ECM and the most abundant protein in human body. Collagen fibrils play an important role in maintaining the mechanical strength in tissues and define the shape and form of tissues in which they occur. Appropriate mechanical strength is also essential for tissue-engineered constructs, especially when it is intended to resist significant mechanical stresses upon implantation into the body, such as tissue-engineered arterial conduit (Johnson and Galis, 2003). In tissues, collagen is synthesized and secreted by fibroblasts forming a matrix of insoluble crosslinked collagen fibrils. These mesenchymal cells are thus tightly surrounded by ECM. In in vitro culture, however, fibroblasts do not produce sufficient collagen matrix. Instead, these cells continuously secrete a large amount of soluble collagen precursors into the culture medium. This culture condition is evidently not an ideal system to investigate the regulation of collagen production by anti-fibrotic drugs. This project therefore aimed to enhance the formation of collagen matrix in fibroblast cultures by using the principle of macromolecule crowding. In this project, a polyanionic macromolecule, dextran sulfate (DexS), was characterized with regards to its potential to facilitate the extracellular collagen deposition. Graduate Program in Bioengineering 10 Chapter 2. Literature Review 2.2.1. Collagen properties Collagen is a molecule that comprises of three polypeptide chains (α-chains). Each chain consists of a repeating glycine-X-Y (Gly-X-Y) triplet, in which X and Y can be any residue, but are usually proline and hydroxyproline respectively. This triplet motif results in left-handed helices that can intertwine with each other forming a righthanded triple-helical structure. N-terminal region Collagen fibril monomer C-terminal region Proα1 Proα1 telopeptide Proα2 Cleavage by N-proteinase Figure 2.4 Cleavage by C-proteinase The structure of type I procollagen (taken from Kielty and Grant, 2002). Fibroblasts synthesize collagen as soluble procollagen, which consists of triple helical section(s) and propeptides at both ends (C- and N-terminals). These propeptides will be cleaved by specific proteinases, leaving the triple-helical domain with non-helical telopeptides at both ends that is a site for collagen crosslinking. The telopeptides are susceptible to proteolytic attack, whereas the intact triple-helical domain is resistant to most proteolytic enzymes. However, it undergoes helix-to-coil transition and becomes susceptible to degradative enzymes when it is heated to above its melting threshold. To date, there are 27 different collagen types that have been identified. Collagen type I, II and III are quantitatively the most important, accounting for over 70% of the total collagens in human body (Kielty and Grant, 2002). The focus of this project is on Graduate Program in Bioengineering 11 Chapter 2. Literature Review collagen type I, which can be found throughout the body except in cartilaginous tissues. It is also synthesized in response to injury and in the fibrous nodules formed as the consequence of fibrotic disease (Kadler et.al., 1996). In type I collagen, the helical molecule is a heterotrimer that consists of two identical α1(I) chains and one α2(I) chain. 2.2.2. Collagen biosynthesis The biosynthesis of collagen begins with the transcription of the individual collagen genes and concludes in the maturation of the collagen fibrils in the ECM. This biosynthesis process is characterized by a number of co- and post-translational modifications, some of which are unique for collagen. • Intracellular processing of procollagen Translation of the mRNA encoding the pre-proα(I) chains occurs on the free ribosomes and begins with the synthesis of the N-terminal. Soon after, these polypeptides begin to fold into appropriate secondary and tertiary structures. As the polypeptide chains are translocated across the endoplasmic reticulum (ER) membrane, intrachain disulfide bonds are formed within the N- and C- terminal propeptides, and hydroxylation of proline and lysine residues occurs within the collagenous domains (Kielty and Grant, 2002). These chains then associate to form heterotrimeric molecules that fold in a C- to N-terminal direction. The C-propeptide plays an important role in the association of the monomeric procollagen chain and in determination of the chain selectivity (Bulleid et.al., 1997; Less et.al., 1997). The stability of the triple-helix formation is dependent not only on the presence of glycine as every third residue, but also requires the presence of 4-hydroxyproline in Graduate Program in Bioengineering 12 Chapter 2. Literature Review the Y position at a high proportion. Enzyme prolyl-4-hydroxylase (P4H) is responsible for the hydroxylation of this imino acid. The presence of hydroxyproline at this specific position appears to favor a specific conformation of the imino acid necessary for the packing of the collagen triple helix (Vitagliano et.al., 2001). In addition, hydroxyproline coordinates an extensive network of water molecules with the triple helix such that water bridges occur within and between the collagen chains (Bella et.al., 1995). The hydroxylation of proline residues also increases the denaturation temperature of procollagen molecules (Berg and Prockop, 1973). There is a positive correlation between the melting temperature of the triple helix and the extent of hydroxylation of proline residues, as well as with the physiological temperature of an organism (Burjanadze, 1979; Privalov, 1982). The hydroxylation process requires ascorbic acid (vitamin C) as a cofactor for P4H. In the absence of this enzyme, procollagens are unable to leave the ER, and therefore new collagen fibrils fail to form. This will result in scurvy, that is associated with a long-term dietary deficiency in vitamin C. There are several other post-translational modification enzymes and chaperones in collagen biosynthesis, such as lysyl hydroxylase, prolyl-3-hydroxylase, chaperone HSP47, etc. However, they will not be discussed in this report. After the procollagen has achieved their correctly folded conformation, this protein will leave ER and will be secreted out to the extracellular space. The mechanism of the procollagen secretion is still poorly understood, however it is known that procollagen follows the classical secretion route for extracellular proteins, passing Graduate Program in Bioengineering 13 Chapter 2. Literature Review through the Golgi complex. Upon release from the Golgi apparatus, the bundles of procollagen form secretory vacuoles that will exit to the plasma membrane. • Extracellular processing of procollagen to form collagen matrix A key step in collagen fibril formation is the specific enzymatic removal of the N- and C-propeptides from procollagen in the extracellular space by N- and C-proteinases. Both enzymes are members of the zinc-binding metalloproteinase family (Prockop et.al., 1998). The N-proteinase has a unique property of only cleaving N-propeptide of procollagen type I that is in native conformation. Under this conformation, Npropeptide is folded back in a hair-pin configuration such that it binds to the first part of the major triple helix of the monomer, and leaving the N-telopeptide in a hair-pin conformation even after the cleavage. The C-proteinase specifically cleaves native and denatured procollagen type I, as well as a precursor of lysyl oxidase. This enzyme cleaves the Ala-Asp bonds, however does not cleave similar bonds in the triple helical domain, suggesting that the specificity depends on the sequences that flank the cleavage site. The subsequent fate of the propeptides has also been studied. These cleaved propeptides have been suggested to play a role in the feedback control of collagen synthesis and there is possibility that they deposit in the matrix (Risteli and Risteli, 1987). Following the cleavage, collagen molecules will self-assemble and align in a quarterstaggered array. This assembly is an entropy-driven process, in which the surface of the protein molecules experiences loss of solvent molecules that will result in Graduate Program in Bioengineering 14 Chapter 2. Literature Review assemblies with a circular cross-section to minimize the surface area/volume ratio of the final assembly (Kadler et.al., 1996). The spontaneous aggregation of collagen molecules into fibrils is followed almost immediately by the formation of covalent cross-links within and between the collagen molecules. These cross-links, that are formed from specific lysine and hydroxylysine residues, are essential in providing the tensile strength and mechanical stability of the collagen fibrils which their structural roles demand (Knott and Bailey, 1998). A specific collagen crosslinking enzyme, lysyl oxidase, plays a main role in the oxidative deamination of lysine and hydroxylysine residues in the nonhelical telopeptide regions to form the corresponding aldehydes (Hong et.al., 2004). These aldehyde residues can spontaneously condense with vicinal peptidyl aldehydes or with peptidyl lysine to generate covalent crosslinkages between the newly formed collagen polymers (Smith-Mungo and Kagan, 1998). It is noteworthy that in collagen I fibers, disulfide bonding does not play a part in the crosslinking because of the absence of cysteine residues (Kielty and Grant, 2002). The catalytic activity of lysyl oxidase is dependent on a strict steric requirement, that is the quarter-staggered alignment of collagen molecules. It also depends on the sequence of amino acids surrounding the target lysyl/hydroxylysyl residues (SmithMungo and Kagan, 1998). Lysyl oxidase binds to the fibril surface and cannot penetrate to its inner domains (Cronlund et.al., 1985). It implies that the oxidation of lysyl groups must occur at the early stage of fibrillogenesis and/or that cross-links are continuously manufactured at the surface of growing fibrils. Graduate Program in Bioengineering 15 Chapter 2. Literature Review Figure 2.5 The collagen synthesis, processing and assembly. 2.2.3. The challenge in the enhancement of collagen matrix formation in fibroblast culture In culture, fibroblasts are maintained in a monolayer culture that is highly unphysiological. Under this condition, fibroblasts have little associated matrix and are bathed in a large volume of culture medium. It is in contrast with the in vivo environment where the cells are tightly surrounded with ECM that is dominated by collagen. The fibroblasts in culture will continuously secrete a large amount of procollagen into the culture medium. However, due to the dilute setting in the Graduate Program in Bioengineering 16 Chapter 2. Literature Review medium, the processing of these precursor proteins to mature collagen fibrils occurs very slowly resulting in the accumulation of procollagen in the culture medium. This issue has been underestimated in both tissue engineering and in anti-fibrotic drug discovery. The construction of engineered tissues using fibroblasts is carried out with sub-optimal amount of endogenous collagen matrix. It may compromise the shape and the mechanical strength of the constructed tissues. Furthermore, the amount of procollagen in the culture medium has been quantified to assess the effect of antifibrotic drugs. This measurement is not satisfactory in this case since it only captures the inhibition of procollagen secretion by the drugs. It does not, however, capture the inhibition of the deposition of collagen on the cell layer, that depends on the procollagen conversion to collagen. This issue becomes significantly challenging when a specific group of anti-fibrotic drugs that prevents the procollagen conversion is involved, for instance C-proteinase inhibitors. Early experiment at BAYER AG showed that it was not possible to demonstrate the effects of C-proteinase inhibitors in fibroblast cultures due to the absence of procollagen conversion. Remarkably, nobody at BAYER was able to solve this conversion problem (Dr Elmar Burchardt, formerly at R&D with BAYER, anti-fibrosis programme, personal communication, March 2005). This project therefore aimed to solve the above mentioned issue by applying the principle of macromolecular crowding in fibroblast cultures to enhance the formation of collagen matrix. Graduate Program in Bioengineering 17 Chapter 2. Literature Review 2.2.4. Macromolecular crowding • The principle In living systems, biochemical processes occur in a medium containing high concentration of macromolecules. Even though one species may not be present in high concentration, the overall species occupy a significant part of the total volume, typically 20-30% (Ellis, 2001a). This fraction is thus physically unavailable to other molecules. Such condition in the living cell has been termed “macromolecular crowding”. Crowded environment occurs in both intracellular and in the extracellular matrix. In Escherichia coli, the concentration of total protein inside the cells is in the range of 200-300 mg/ml, whereas that of RNA is in the range of 75-150 mg/ml, making up the total concentration of 300-400 mg/ml (Ellis, 2001b). In in vitro culture, the concentration of the macromolecule is estimated to be only 1-10 mg/ml. Figure 2.6 illustrates the crowding condition in the eukaryotic cytoplasm. In the ECM of tissues, polysaccharides also contribute to crowding, for instance collagen. Figure 2.6 The illustration of the crowding condition in eukaryotic cytoplasm (taken from Ellis, 2001b). Macromolecular crowding causes an excluded volume effect that leads to a nonspecific repulsive interaction between solute molecules. This interaction is always present regardless of any other attractive or repulsive interactions that may occur Graduate Program in Bioengineering 18 Chapter 2. Literature Review between the solute molecules (Ellis, 2001a). How much of the volume that is unavailable to other macromolecules depends on the numbers, sizes and shapes of all the molecules present in each compartment. The concept of excluded volume is illustrated in figure 2.7. The squares outline the volume containing spherical macromolecules (black) that occupy ~30% of the total volume, a value typical of intracellular compartments. The volume available to another molecule (yellow) is defined as the fraction that can be occupied by the centre of that molecule. If the introduced molecule (red) is small relative to the macromolecules, it can access virtually all of the remaining 70% of the space (figure 2.7a). However, if the introduced molecule (blue) is similar in size to the macromolecule, the available volume is much less than might be expected because the centre of the introduced molecule can not approach the macromolecule less than the distance at which the surfaces of two molecules meet, that is indicated by the open circle around each macromolecule (figure 2.7b). The available volume thus defines an effective concentration of the introduced molecule, which can be much higher than the actual concentration in the total volume. Figure 2.7 The schematic drawing to illustrate the concept of volume exclusion. (a) The introduced molecule (red) is small relative to the macromolecules (black) (b) The introduced molecule (blue) has a similar size to the macromolecules (black) (taken from Ellis, 2001a). Graduate Program in Bioengineering 19 Chapter 2. Literature Review • The consequences of macromolecular crowding The main effect of crowding on biochemical equilibria is to favor the association of macromolecules. Equilibrium constants for macromolecule associations may be increased by two to three orders of magnitude, depending on the relative sizes and shapes of macromolecular reactants and products, as well as the concentration of crowders (Ellis, 2001b). This thermodynamic effect arises from the reduction in excluded volume when macromolecules bind to one another, which leads to a decrease in the total free energy of the solution. The more solute molecules present in a solution and the larger they are, the less randomly they can be distributed. Thus, as the total concentration of macromolecule rises, the configurational entropy of each macromolecule species becomes smaller and its contribution to the total free energy of the solution increases. In other words, the most favored state excludes the least volume to the other macromolecules present. This conclusion also applies to all biochemical processes in which a change of excluded volume occurs, for instance the formation of oligomeric structures such as fibrin, collagen and multienzyme complexes in metabolic pathways (Ellis, 2001a). It is important to note that crowding only enhances the inherent tendency of macromolecules to bind to one another, but it does not create this tendency de novo. The effect of crowding on reaction rate is complex and may depend on hydrodynamic as well as thermodynamic properties of the system. Consider a substrate (S) and enzyme (E) association to form a product (P) with SE* as the transition state: S+E SE* P+E If the overall rate-limiting step is the encounter rate of S with E, the reaction is diffusion-limited. This encounter rate is proportional to the sum of the translational Graduate Program in Bioengineering 20 Chapter 2. Literature Review diffusion coefficients of the associating species, which are generally reduced in the presence of substantial concentration of crowding agents (Minton, 2005). Therefore, crowding is expected to decrease the rate of diffusion-limited association (shortdashed curve in figure 2.8). In contrast, if the rate of association is limited by the rate of conversion of SE* to P, such reaction is referred to as chemically rate-limited. In this case, the association of S and E to SE* can be treated as being at equilibrium (Ellis, 2001b). But crowding increases association such that this equilibrium is displaced to the right and the overall reaction rate will increase as the concentration of crowding agent rises (long-dashed curve in figure 2.8). However, even if the reaction is chemically rate limited, the rate of encounter of reactants decreases monotonically with increasing volume occupancy of crowder. Therefore, ultimately crowding will reach a point at which the reaction is no longer chemically rate limited, and further increases in crowder concentration are expected to result in a decrease in association rate (continuous curve in figure 2.8). diffusion –limited reaction chemically –limited reaction Overall reaction rate Figure 2.8 Schematic depiction of the predicted dependence of reaction rate on the concentration of crowding agent (taken from Ellis, 2001b). 2.2.5. The ideal crowding agent To be an ideal crowder, the macromolecule should have a molecular weight in the range of 50 – 200kDa, be highly water-soluble and not be prone to self-aggregation. Graduate Program in Bioengineering 21 Chapter 2. Literature Review The molecular shape has to be globular, rather than extended, to prevent solutions becoming too viscous to handle (Chebotareva et.al., 2004). The agent should be easily available in highly purified form so that the use of high concentrations does not introduce problems associated with contaminants. Most importantly, the agent should not interact with the system under test, except via steric repulsion. This requirement is the most difficult to meet. It is essential to establish that any effects observed when using crowding agents are not the result of inadvertent changes in other factors, such as pH, ionic strength or redox potential (Ellis, 2001a). This requirement abolishes the possibility of using highly concentrated cell extracts as crowding agent, since any interpretation will be complicated by specific interactions, hydrolase activity and the presence of denatured proteins. Commonly used synthetic crowding agents include Ficolls, dextrans, polyethylene glycol and polyvinyl alcohol (Ellis, 2001a). In this project, we have chosen dextran sulfate (DexS) with a molecular weight of 500 kDa. This macromolecule is a polyanionic derivative of dextran and mimics natural mucopolysaccharide (for example chondroitin sulphate, dermatan sulphate). It is freely soluble in water forming clear solutions, readily degradable by ecological systems, possesses high purity and good stability. Figure 2.9 The structure of DexS with sodium salt. Graduate Program in Bioengineering 22 Chapter 2. Literature Review DexS has a widespread application in medical industry as an anti-coagulant agent and as ingredients of cream and ointment for treating thrombophlebitis and for cosmetic applications. It is also used to accelerate the hybridization rate of DNA fragments and to stabilize proteins such as fibroblast growth factor, alcohol oxidase and yeast alcohol dehydrogenase when stored in solution. 2.2.6. Macromolecular crowding in collagen matrix formation The principle of macromolecular crowding has been tested for the collagen matrix formation. Dermal fibroblasts that are cultured in the presence of neutral polymers, such as dextran T-40, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG), show association of collagen on the cell layer, as detected by gel electrophoresis (Bateman et.al., 1986; Jukkola et.al., 1991). It is in contrast to the conventional fibroblast culture where procollagen is found accumulating in the medium. The processing of type I collagen in the presence of macromolecules occurs by the initial removal of the C-propeptide that results in the transient accumulation of a pNcollagen intermediate. It is then followed by a slower cleavage of the N-propeptide to produce completely processed collagen molecules (Bateman et.al., 1986; Bateman and Golub, 1990). The increase of C- and N-proteinase activity has also been observed in the presence of DexS and PEG (Hojima et.al., 1994). In a cell-free system, the rate of procollagen cleavage by C-proteinase from chick embryo tendons is increased by 10- to 15-fold, whereas N-proteinase activity is increased by 2- to 4-fold in the presence of DexS. With PEG, the C-proteinase activity is increased by 5- to 20-fold, whereas the Nproteinase is increased by 2- to 5-fold. Graduate Program in Bioengineering 23 Chapter 2. Literature Review 2.3. Exploration of quantitative immunocytochemistry for collagen quantification Until recently, tritiated proline (3H-Pro) incorporation to collagen protein has been a widely used method for quantifying collagen synthesis in the cell culture. However, despite being very sensitive, this quantification assay is based on a radioactive method that can be laborious and time-consuming. In addition, this method produces radioactive wastes that require special treatment. A colorimetric assay that offers an alternative to the radioactive method has therefore gained popularity. This assay uses Sirius red, a collagen-binding dye, that reacts with the side chain groups of the basic amino acids present in collagen. The bound-dye can then be quantified with spectrophotometer or colorimeter. This assay is rapid and can be completed within an hour. However, our laboratory has shown that the dye binds non-specifically to proteins derived from fetal calf serum in the culture medium (Lareu et.al., in press). This will thus lead to overestimation of the collagen content. In this project, a collagen quantification assay that is based on immunocytochemistry was explored. The collagen associated with the cell layer was detected with a specific antibody that was indirectly conjugated with a fluorochrome. The fluorescence signal, that reflected the amount of protein, could then be quantified using a microplate reader. In contrast to the colorimetric assay, this explored assay directly measures the collagen content in the ECM, not in the culture medium, without disrupting the cell layer. Multi-staining of intra- and extracellular proteins are possible with this assay, allowing quantification of several proteins on one plate. Furthermore, it can be integrated with the DAPI staining method to quantify the cell number (section 2.1.2). Graduate Program in Bioengineering 24 Chapter 3. Experimental Details 3. Experimental Details The equipments and materials used in the research are presented in the first two sections of the chapter. In the last section, the experimental procedures are discussed. 3.1. Equipments 1) FLUOstar OPTIMA and PHERAstar microplate reader (BMG Labtechnologies GmbH, Offenburg, Germany) FLUOstar OPTIMA and PHERAstar microplate readers can perform a wide variety of reading for fluorescence intensity, absorbance and luminescence. Both of them use xenon flash-lamp as the light source. These readers are fully automated and their associated software is version 1.30-0 for FLUOstar and 1.50-0 for PHERAstar. For the purpose of this project, the FLUOstar is fitted with 355, 485, 550 nm excitation filters and 460, 520, 590 nm emission filters. The PHERAstar has three filter modules installed, each of which has one excitation filter and one emission filter. In this project, filter modules with excitation/emission wavelength of 340/460nm, 485/520nm and 540/590nm are used. These filters are bandpass filters with a length of 10 nm. The settings of the instrument for the fluorescence measurement: Reading direction: FLUOstar offers the flexibility in the reading direction: top-top, top-bottom, bottombottom, bottom-top (the first referred to the excitation source point and the latter referred to the emission reading point). After optimization, top-bottom and top-top reading direction offered the best reading for the experimental setup in this project. Graduate Program in Bioengineering 25 Chapter 3. Experimental Details PHERAstar, on the other hand, only offers top-top reading direction. Therefore, for the consistency of the results, top-top reading direction was used throughout the project. Scanning mode: There are three scanning modes that these microplate readers offer: none, matrix and orbital. In the “none” mode, the measurement is taken only at one point in the centre with an area equal to the size of the probe. FLUOstar has a measurement probe with a diameter of 9 mm, whereas the probe for PHERAstar has a diameter of 0.8 mm. In the “matrix” mode, the well is divided into n×n reading spots; the instrument will take a reading from each spot and average them. In the “orbital” mode, the instrument takes reading from several spots along a circle with n diameter and averages them. In this study, the “matrix” mode was used in 24-well plate format since it represented the population of the whole area of the well. For the FLUOstar, “matrix” 3×3 was used, whereas “matrix” 15×15 was used for the PHERAstar. In 96-well plate format, “none” mode was used for FLUOstar since the size of the measurement probe was close to the size of the well, whereas “matrix” 5×5 was used for PHERAstar. Gain: The instrument is equipped with an amplifier to enhance the reading signal. The instrument will automatically adjust the gain of the amplifier based on the highest signal detected from the sample plate. Alternatively, the user can manually set the gain. Focal height: This setting is only available for PHERAstar. After spotting the well that gives the highest signal, the instrument will scan this well for the highest signal along its height Graduate Program in Bioengineering 26 Chapter 3. Experimental Details to find the optimum focal height. This focal height is maintained throughout the reading on a particular plate. The focal height was normally 6.4 mm for lumox™ 96well plate and 10.1 mm for 24-well plate. 2) Tecan SUNRISE absorbance microplate reader Tecan SUNRISE microplate reader is used to measure absorbance on 96-well plates. The associated software is Magellan version 5.00. 3) Olympus IX-71 inverted fluorescence microscope (Olympus Corp., Tokyo, Japan) Olympus IX-71 microscope is fitted with TH4 halogen lamp power supply unit and DP70 microscope digital camera. This digital camera is automated using DP controller software version 1.1.1.65. The images are analyzed using DP manager version 1.1.1.71. For the purpose of this project, this microscope was fitted with filters that allow visualization in blue, red and green spectrum. Table 3.1 Visualization spectrum Blue Green Red The filter configurations for Olympus IX-71 fluorescence microscope Excitation filter Emission filter Note 330-385 nm (BP) 460-490 nm (BP) 510-550 nm (BP) 420 nm (LP) 515-560 nm (BP) 560-620 nm (BP) BP : bandpass filter LP : longpass filter 4) GS-800 Calibrated Densitometer (Bio-Rad Laboratories, Inc., CA, USA) GS-800 calibrated densitometer is used to scan the protein gels. It converts transparent and opaque electrophoretic samples into digital data. This data is then analyzed with Quantity One® software (version 4.5.2). Graduate Program in Bioengineering 27 Chapter 3. Experimental Details 5) Gel electrophoresis and Western transfer apparatus Mini-PROTEAN® 3 Cell was utilized to perform gel electrophoresis of 8 × 7.3 cm gels. Mini Trans-Blot® electrophoretic transfer cell was then utilized to perform wet Western transfer. Both cells were purchased from Bio-Rad Laboratories, Inc., CA, USA. The gel electrophoresis of 16 × 14 cm gels was performed in the Hoefer system (Hoefer, Inc., CA, USA). 6) LAS-1000 Luminescent Image Analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan) LAS-1000 system is used to capture chemiluminescent images. This system consists of a CCD camera system and a dark box. This system is controlled using an Image reader LAS-1000 Pro version 2.51. Graduate Program in Bioengineering 28 Chapter 3. Experimental Details 3.2. Materials 1) Cells Hypertrophic scar fibroblasts (HSF) (primary cells) were obtained from Dr Phan Toan Thang (Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore). Human dermal fibroblasts (HDF) (primary cells) were obtained from A/P Dietmar W. Hutmacher, Tissue Engineering laboratory (Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore). Human lung fibroblasts WI-38 were purchased from American Type Culture Collection (ATCC), VA, USA. 2) Cell culture reagents The cell culture reagents; Dulbecco’s Modified Eagle Media (DMEM), fetal bovine serum (FBS), penicillin streptomycin (PS), trypsin-EDTA (10x) and Hank’s Balanced Salt Solution (HBSS), were purchased from Gibco (Invitrogen Corporation, CA, USA). The DMEM contained high glucose (4500mg/L D-glucose), 110 mg/L sodium pyruvate, GlutaMAX™-I (a substitute of L-glutamine) and phenol red. 3) Cell culture apparatus Culture flasks, multiwell plates and Lab-Tek™ chamber slides were from Nalge Nunc International, Denmark. Lumox™ 24- and 96-well plates (Greiner Bio-One, Germany) were chosen based on its features; a black polystyrene body with an optically clear, gas permeable, 50 Graduate Program in Bioengineering 29 Chapter 3. Experimental Details µm fluorocarbon film base. The film’s gas-permeability promoted homogenous heat transfer and reliable oxygen supply as well as CO2 disposal. In addition, its black body and optically clear bottom offered low autofluorescence, making it suitable for fluorescence quantification purposes. 4) Antibodies and fluorescence dye Rabbit-α-human collagen I was purchased from Chemicon (Chemicon International, Inc., CA, USA). Mouse-α-human fibronectin was purchased from Sigma (SigmaAldrich, Inc., MO, USA). Both antibodies were used as primary antibodies in the immunocytochemistry. The fluorochrome-conjugated secondary antibodies (AlexaFluor546 goat-α-mouse and -α-rabbit and AlexaFluor488 chicken-α-mouse and -α-rabbit) were purchased from Molecular Probes (Invitrogen Corp., CA, USA). The primary antibody in Western blotting, mouse-α-human collagen I,II,III, was purchased from Monosan, The Netherlands. The secondary antibody, goat-α-mouse HRP conjugated was purchased from Pierce (Pierce Biotechnology, Inc., IL, USA). 4’,6-Diamidino-2-phenylindole (DAPI) was purchased from Molecular Probes (Invitrogen Corp., CA, USA). 5) Gel electrophoresis and Western blotting chemicals The chemicals for polyacrylamide gels; 30% acrylamide/bis 37.5:1 (2.6%C), ammonium persulfate and TEMED (N,N,N’,N’-Tetra-methyl-ethylenediamine), Bromophenol blue, β-mercaptoethanol (β-ME), Precision Plus Protein™ Dual Graduate Program in Bioengineering 30 Chapter 3. Experimental Details Color Standards, Tween-20, non-fat dry milk, and nitrocellulose membrane (0.45 µm pore size) were from Bio-Rad (Bio-Rad Laboratories Inc., CA, USA). Collagen type I from human placenta and glycine were from Sigma (SigmaAldrich, Inc., MO, USA). Tris (JT Baker, Mallinckrodt Baker, Inc., NJ, USA). Sodium Dodecyl Sulfate (SDS) (Usb Corp., OH, USA). Super Signal® West Dura extended duration substrate (Pierce Biotechnology, Inc., IL, USA). 6) Gel staining The silver staining kit, Silver Quest, was from Invitrogen (Invitrogen Corp., CA, USA) and used according to the manufacturer’s instruction. 7) Other chemicals Pepsin from porcine gastric mucosa and Complete protease inhibitor 25x (Roche Diagnostics GmbH, Mannheim, Germany) L-Ascorbic acid Phosphate Magnesium salt n-hydrate (AscP) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) Dextran Sulfate sodium salt (Mw ~ 500kDa) (DexS) (Amersham Biosciences, Denmark). Sodium Chloride (NaCl) and Sodium Hydroxide (NaOH) were from QReC. Triton-X and ethylene diaminetetraacetic acid (EDTA), disodium salt dehydrate were from Bio-Rad (Bio-Rad Laboratories, Inc., CA, USA). Graduate Program in Bioengineering 31 Chapter 3. Experimental Details Bovine serum albumin (BSA), MTT, paraformaldehyde (PFA) and 3Aminopropionitrile fumarate salt (β-APN) were from Sigma (Sigma-Aldrich, Inc., MO, USA). Polyvinyl alcohol mounting medium with DABCO, antifading agent, was from Fluka (Sigma-Aldrich, Inc., MO, USA). 8) Solvents All the solvents used were analytical grade. The water that was used to make up solutions was deionised water from an ultrapure Millipore water generator. N,N-Dimethyl Formamide (DMF) and phosphate buffer solution (PBS) tablets were from Sigma (Sigma-Aldrich, Inc., MO, USA). To make 1x PBS, 1 tablet of PBS was dissolved in 200 ml water. Glacial acetic acid and 36.5-38% HCl were from JT Baker (Mallinckrodt Baker, Inc., NJ, USA) Methanol (Lab-Scan Asian Co., Ltd., Thailand) Ethanol (Hayman Ltd., England) Glycerol (Asia Pacific Specialty Chemicals Ltd., Australia) Graduate Program in Bioengineering 32 Chapter 3. Experimental Details 3.3. Experimental procedures 3.3.1. General methods 1) Cell culture Fibroblasts were cultured in DMEM containing 10% FBS and 1% PS at 37ºC with 5% CO2. Before use, FBS was heat inactivated at 56ºC for 30 min. The medium was changed every 2-3 days. After reaching 90% confluency, the culture was trypsinized and replated at a ratio of 1:5. Trypsinization was performed by incubating the cells in a minimal amount (1ml for T-75 flask) of Trypsin-EDTA (1x) for 2 min at 37ºC. The trypsin was then inactivated by adding excess amount of complete medium. TrypsinEDTA (1x) was prepared from the stock solution that was diluted 10 times in HBSS. Before seeding the cells onto multiwell plates, the cell number was determined using hemacytometer. 10 µl of the cell suspension was mixed with 10 µl of 0.4% trypan blue to exclude the dead cells. 10 µl of this mixture was loaded onto the hemacytometer chamber. The cell density was determined according to the following formula: Cell density (cells/ml) = total number of viable cells in 4 corner squares × 2 × 104 4 Graduate Program in Bioengineering 33 Chapter 3. Experimental Details 2) Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis (SDS-PAGE) The 3-5% polyacrylamide gel was made according to the formula below: Table 3.2 3-5% polyacrylamide gel composition 5% Separation Gel (1mm thickness) 30% Acrylamide/Bis (37.5 : 1) 1.875M Tris (pH 8.8) 10% SDS ddH2O APS (100mg/ml) TEMED Total 3% Stacking Gel (1mm thickness) 30% Acrylamide/Bis (37.5 : 1) 1.25M Tris (pH 6.8) 10% SDS ddH2O APS (100mg/ml) TEMED Total Hoefer system (Hoefer) 3.15 ml 3.8 ml 0.19 ml 11.8 ml 0.038 ml 0.019 ml 19 ml Hoefer system (Hoefer) 0.75 ml 0.75 ml 0.12 ml 5.8 ml 0.062 ml 0.019 ml 7.5 ml Mini-PROTEAN® 3 system (Bio-Rad) 0.83 ml 1 ml 0.05 ml 3.07 ml 0.042 ml 0.005 ml 5 ml Mini-PROTEAN® 3 system (Bio-Rad) 0.2 ml 0.2 ml 0.033 ml 1.55 ml 0.017 ml 0.003 ml 2 ml The separation gel was dispensed first, overlayed with 10% ethanol and left to polymerize. Subsequently, the ethanol was decanted and the stacking gel was dispensed on top of the separation gel with the well-comb inserted to form the sample reservoirs. The running buffer was made from 3.02 g tris, 14.4 g glycine, and 1 g SDS in 1 L water. This buffer was cooled overnight at 4˚C prior to gel electrophoresis. The samples were prepared in 5x blue sample buffer (5x SB). This sample buffer was made from 0.625 ml of 1.25M tris (pH 6.8) and 0.25 g SDS that were dissolved in 2 ml water. This solution was topped up with glycerol to 5 ml and 1.25 mg Graduate Program in Bioengineering 34 Chapter 3. Experimental Details bromophenol blue was added to the buffer. The prepared samples were then denatured by heating at 95˚C for 5 min. 3) Pepsin digestion to retrieve collagen The culture medium from the fibroblast culture plated on a 24-well plate was aspirated and stored in Eppendorf tubes (~ 450 µl). Subsequently, 50 µl of 1 mg/ml pepsin (in 1 N HCl) was added to each tube and the digestion proceeded for 2 hours on an orbital shaker. 60 µl of 1N NaOH was then added to each tube to neutralize the solution and to stop the enzyme activity. The total volume for each sample was thus ~560 µl. The stock solution of pepsin (1mg/ml in 1 N HCl) was diluted 1:10 with HBSS to make up a final concentration of 100 µg/ml in 0.1N HCl. After washing the cell layer once with HBSS, 250 µl of this pepsin solution was added to each well and the digestion proceeded for 2 hours on an orbital shaker. After transferring the well content to eppendorf tubes, 30 µl of 1N NaOH was added to each tube to stop the pepsin activity. The total volume for each sample was thus 280 µl. The collagen concentration in the cell layer fraction was twice as concentrated as that in the corresponding medium fraction. The pepsin digested samples were then subjected to SDS-PAGE under non-reducing condition. 60 µl of each sample was mixed with 20 µl of 5x SB. The collagen I standard was collagen type I from human placenta. 2 µg of this collagen was mixed with 3 µl of 1N NaOH and 50 µl 5x SB. The molecular weight standard was 1 µl of Precision Plus Protein™ Dual Color Standards mixed with 50 µl 5x SB. The gel Graduate Program in Bioengineering 35 Chapter 3. Experimental Details electrophoresis was carried out in a Hoefer system and the protein bands were visualized using the Silver Quest silver staining kit. 4) Densitometry The silver stained gel was scanned using GS-800 calibrated densitometer. This digital data was then analyzed using Quantity One®. Each collagen band was framed with a rectangular box that indicated one volume field. Subsequently, the collagen band was quantified using a ‘local background subtraction method’. Under this method, each volume field had its own mean background intensity that was calculated from the total intensity of the pixels in a 1-pixel border around the volume boundary divided by the total number of border pixels. This mean background was subtracted from the intensity of each pixel inside the volume boundary. Any pixel inside the boundary that had the same intensity as the mean background intensity was eliminated from the quantification. The quantification results were displayed as adjusted volume and % volume. The calculation was done as follow: Volume = the total pixel intensity inside the volume boundary × the area of a single pixel (mm2) Background volume = the total background intensity × the area of a single pixel (mm2) Adjusted volume = volume – background volume % volume = volume / (summation of all the volumes in the image) × 100% Graduate Program in Bioengineering 36 Chapter 3. Experimental Details 5) Statistical analysis Statistical comparisons made were performed using an unpaired t-test assuming equal variances with two-tailed distribution (Microsoft Excel software). Differences were considered significant if P[...]... implicated with several disadvantages particularly in the application for an antifibrotic drug discovery tool Therefore, this project aims to establish a reliable cell enumeration assay that is suitable to quantify the fibroblast population and can be incorporated into the anti- fibrotic drug discovery tool This assay will be based on the quantification of the nuclear content using DNA-binding dye, DAPI,... enhance the formation of collagen matrix in in vitro fibroblast culture, to allow an accurate assessment of anti- fibrotic drugs on collagen deposition As another aspect, the enhancement of the collagen matrix in fibroblast culture may be a valuable tool in tissue engineering The presence of collagen fibrils in tissue constructs is essential in maintaining the mechanical strength and defining the shape... consuming Assays that measure metabolic activity Metabolic activity can be an indication of cell viability There are several metabolic based assays available in the market, MTT assay being the commonly used This assay is based on the reduction of tetrazolium salts to a colored, water-insoluble formazan that can be quantified in a conventional ELISA plate reader at 570 nm (maximum absorbance) after... coating in tissue engineering This collagen mainly comes from various animal origins In the light of increasing concerns over animal-transmitted diseases, a collagen scaffold fabricated from human fibroblasts might offer an alternative to animal-originated collagen This project also aimed to develop an anti- fibrotic drug discovery tool that allows the integration of cell enumeration and collagen quantification... number in this case since the drug might alter the biological function of the cells Assays that measure the cellular DNA quantity The determination of DNA concentration is a reasonable indicator of cell number, since the levels of DNA and RNA in cells are tightly regulated (Frankfurt, 1980) Although the levels of DNA and RNA in individual cells can vary significantly over time, the overall amount of nucleic... quantification Quantification of collagen has been classically done using a radioactive method (metabolic labeling) or colorimetric assays, both having several disadvantages The first method can be laborious and involves hazardous materials and wastes, whereas the latter is nonspecific to collagen Therefore, an alternative collagen quantification assay that is based on immunocytochemistry was explored It was... was integrated with a cell enumeration method that measures the DNA content of the cell population on test Graduate Program in Bioengineering 2 Chapter 1 Introduction Both assays are fluorometry-based and the measurement was done using a microplate reader 1.2 Objectives This project has a main objective of establishing a drug discovery tool that can be applied for the assessment of anti- fibrotic compounds. .. significance Fibrovascular disorders are diseases that are characterized by an increase in the formation of fibrous tissues and its vascularization This disease can occur in any part of the body, both external and internal, such as skin, eye, joints, lung or liver The severity of the disease may vary from merely pain and pruritis to functional disability or even fatality The widespread disposition of this... can be divided into three sub-aims that provide comprehensive assessments for the fulfillment of the main objective: 1 to establish a rapid cell enumeration assay that is suitable for the quantification of fibroblast population and suitable for a microplate reader analysis, 2 to enhance the formation of collagen matrix on the fibroblast culture, 3 to develop collagen quantification assay that is based... DAPI/RNA binding mode diverse from that of DAPI/dsDNA The DAPI/RNA involves AU-selective intercalation instead of binding at the AT cluster (Tanious et.al., 1992) In addition, the DAPI/RNA complex exhibits a longer-wavelength fluorescence emission maximum (~500 nm) than the DAPI/dsDNA complex (~460 nm) and a quantum yield that is only about 20% as high (Kapuscinski, 1990) Graduate Program in Bioengineering ... salt Graduate Program in Bioengineering 22 Chapter Literature Review DexS has a widespread application in medical industry as an anti- coagulant agent and as ingredients of cream and ointment for. .. was trypsinized and replated at a ratio of 1:5 Trypsinization was performed by incubating the cells in a minimal amount (1ml for T-75 flask) of Trypsin-EDTA (1x) for at 37ºC The trypsin was then... potentially suitable collagen quantification assay as a building block of a discovery tool for anti- fibrotic drugs Graduate Program in Bioengineering vi List of Tables List of Tables Table 3.1 The filter