Implementation of a drug discovery tool for the evaluation of anti fibrotic compounds application in fibrovascular disorders

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.

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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.

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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.

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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

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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.

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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.

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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

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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

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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).

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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.

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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

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