Micropatterned Cell Arrays for Detecting DNA Danmage
By
Sukant Mittal
B.S. Biomedical Engineering
University of California, Irvine, 2005
Submitted to the Department of Electrical Engineering and Computer Science
in Partial Fulfillment of the Requirements for the Degree of
Master of Science in Electrical Engineering and Computer Science
at the Massachusetts Institute of Technology
June, 2008
©2008 Massachusetts Institute of Technology. All rights reserved.
Author
De~abtment of Electrical Engineering and Computer Sciences
Certified by_
Sangeeta %. Bha 4a, MD, PhD. Professor of Health Sciences and
Technology and Electrical Engineering and Computer Sciences
Accepted by
Terry P. Orlando,
rofessor, Electrical Engineering and Computer
Sciences
MA•CHUSETS INS
OF TEOHNOOGY
JUL 0 1 2008
LIBRARIES
-- _------~31.
Micropattemed Cell Arrays for Detecting DNA Damage
by
Sukant Mittal
Submitted to the
Department of Electrical Engineering and Computer Science
June, 2008
In Partial Fulfillment of the Requirements for the Degree of
Master of Science in Electrical Engineering and Computer Science
Abstract
Numerous agents are capable of interacting with DNA and damaging it. Permanent changes in
the DNA structure can be both mutagenic and cytotoxic; therefore, methods to measure the
susceptibility of cells to mutations are important for risk assessment and identifying therapeutic
interventions. One classical method for assessing DNA damage at a global level is the COMET
assay, based on electrophoretic extension of the nuclear DNA in single cells embedded in
agarose. In this assay, the size and shape of the extended 'comet tail' can be correlated to breaks
in the DNA. This assay was first developed in the mid 1980s for nonadherent cells such as
lymphocytes; however, it has been plagued by technical difficulties, low throughput, and lab-tolab variation. These challenges have been exacerbated in adhesion-dependent cells as DNA
damage accrues variably over time as they are enzymatically detached from their
microenvironment.
This thesis explores whether the COMET assay can be improved by micropatterning adherent
cells prior to agarose embedding. Hepatocytes were chosen as a model cell type and x-ray
radiation was chosen as a model DNA damaging agent. In order to establish the feasibility of
measuring x-ray induced damage on hepatocyte DNA, standard curves were first generated for
hepatocytes suspended in agarose. These experiments revealed a minimum detectable threshold
of 1 Gy and displayed a monotonic increase in DNA damage in response to exposures up to 10
Gy. In comparison, adherent hepatocytes overlaid with agarose and irradiated in situ displayed
similar levels of mean damage but lower levels of variability than suspended cells. We
hypothesize that the decreased variability could be due to a reduction in programmed cell death
incurred by detachment of adherent cells.
Finally, we explored the feasibility of performing the comet assay by in situ irradiation of a
micropatterned array of adherent cells. Single cell hepatocyte patterning was achieved by
photolithographic patterning of collagen I on glass and optimization of seeding conditions.
Gradiations of x-ray exposure were achieved by employing localized domains of lead shielding
between the cells and the source. As a proof of principle, we obtained two domains of
differential x-ray exposure and the resulting DNA damage was similar in the micropatterned
format to the randomly-organized adherent format. Several challenges emerged from these
experiments including potential interactions of DNA with the glass surface leading to 'streaking'
artifacts. Nonetheless, with increased resolution of x-ray exposure, and further technical
improvements, this assay has the potential to offer both reduced variability for adherent cells as
well as assay multiplexing due to spatial encoding of x-ray dosage.
Thesis supervisor: Sangeeta N. Bhatia, MD, PhD
Title: Professor of Electrical Engineering and Computer Sciences and Health Sciences and
Technology
Acknowledgements
I would like to express my thanks to a number of individuals for their contributions
to this thesis.
First, I would like to thank my advisors, Sangeeta Bhatia and Bevin Engelward, for their
guidance, encouragement and patience during the completion of this thesis. It has been an
amazing experience working under their supervision, and I will be forever thankful for this
opportunity.
My gratitude extends to Jaquelyn Yanch, Rachel Batista and Kurt Broderick, who helped
tremendously towards shaping the end of the project. I am also very thankful to my colleagues
and friends at professor Engelward's lab, especially, James Mutamba and Werner Olipitz, who
spent numerous hours explaining and performing the 'comet assay' with me.
Justin Lo and Scott Pollom, star undergraduates at MIT who helped me finish my work and were
always up for challenging and time consuming experiments were a pleasure to work with. I would also like to thank my friends Dipanjan Sen, Srujan Linga, Srikanth Patala, Sandeep
Sethuraman,Vivek Sharma and Qian Wang, with whom I have had some wonderful experiences,
and whose friendship I will value for life.
I owe my deepest thanks to my parents, and my brother, to whom I look upto as a role model for
their unconditional support, and their belief in me all these years. They have done the best job of
being family when family was needed. I owe them more than I would be able to express.
Table of Contents
A cknowledgem ents ...................................................................................................................................................
4
List of Figures ..............................................................................................................................................................
List of Tables ................................................................................................................................................................
....................................
Chapter 1: Introduction ..................................................................................................
8
10
1.1 Background and Motivation ............................................................................................................................. 10
1.2 Objectives ...................................................................................................
. ..........................................
Chapter 2: Introduction to DNA damage and Review of the underlying Detection Techniques ....................
2.1 Introduction to DNA Damage.............
..............................................................................................
12
13
13
2.2 Methods to detect DNA damage ....................................................................................................................... 15
2.2.1 Alkaline Single Cell Gel Electrophoresis.................................................................................................. 17
2.2.2 Neutral Single Cell Gel Electrophoresis ........................................................................ ..................... 17
............................ 18
2.2.3 Enzym e Specific Base Lesions ...............................................................................
2.2.4 Unscheduled DN A Synthesis (UD S) .......................................................................... ........................ 18
19
2.2.5 M icronucleus Assay (M N ) .. ....................................................................................................
2.2.6 Sister Chrom atid Exchanges (SCEs)......................................................................................................... 19
2.2.7 y-H 2AX A ssay .......................................................................................................................................... 19
2.2.8 Fluorescence in situ Hybridization (FISH) A ssay.....................................................20
2.3 Comet Image Analysis ...................................................................................................
.............................. 21
2.4 Motivationfor ChoosingPatternedComet Assay as a Method to Detect DNA Damage .............................. 22
2.5 Review of CellularMicropatterningTechniques ........................................ ......... .................................... 24
2.5.1 Photolithography.......................................................................................................................................25
2.6.2 Soft Lithography ............................................................................................
..................................... 25
....................................... 26
2.6.3 M icrospotters ...............................................................................................
2.7 Radiation.............................................................................................................................................
Chapter 3: DNA Damage induced in Primary Rat Hepatocytes by X-ray Radiation .....................................
3.1 Introduction................................................
...........................................................................................
3.2 X-ray Radiation Setup.........................................................
.............................
28
30
30
............................. 31
3.3 Materials and Methods ........................................
....................................
3.3.1 Type-I Collagen Preparation from Rat Tails .................................................................... ...................
3.3.2 Alkaline Com et Assay Buffer Preperation............................................... ...........................................
........................................
3.3.2.1 Lysis Buffer.........................................................................................
3.3.2.2 Alkaline/Electrophoresis Buffer .................................................... ..............................................
3.3.2.3 Neutralizing Buffer..................................................................................
....................................
3.3.3 Primary Rat H epatocyte Isolation ...................................................... .................................................
3.3.4 Photolithographic M icropatterning ............................................................................ .........................
3.3.5 Collagen Island Fabrication ..................................................................................
..............................
3.3.6 Com et Assay on suspended Hepatocytes ................................................ ............................................
32
32
32
32
33
33
33
34
34
36
3.4 Results.........................................................................................................................................................
37
3.4.1 Gel Percentage Optimization for Comet Assay...................................................................................37
3.4.2 X-ray Radiation Dose Response on Primary Rat Hepatocytes Suspended in Agarose Gel ................... 39
3.4.3 Comet Assay on Timecourse Study of Hepatocytes ......................................
41
3.4.4 Primary Rat Hepatocyte DNA Damage Dose Response to X-ray radiation at 24 h Post Culture .......... 44
Chapter 4: Comet Assay on Adherent Hepatocytes ......................................................................................... 47
4.1 Introduction .............................................................................................................................................
47
4.2 Methods............................................................................................................................................................
47
4.2.1 Optimization of the Collagen Island Size for Single Hepatocyte Capture ......................................
47
4.2.2 Optimization of Hepatocyte seeding concentration for Single Cell Capture............................
... 48
4.2.3 Modified Protocol for Patterned Adherent Comet Assay on Primary Rat Hepatocytes ......................... 49
4.3 R esults...............................................................................................................................................................
50
4.3.1 Dose Response using Comet Assay on Adherent Primary Rat Hepatocytes..........................
... 50
4.3.2 Dose Response Comparison between Adherent Primary Rat Hepatocytes and Suspended Primary Rat
Hepatocytes using Comet Assay .....................................................
51
4.3.3 Rationale for using Patterned Comet Assay........................................................................................54
4.3.4 Collagen Island Fabrication Characterization ..................................................................................... 55
4.3.5 Optimization of Single Hepatocyte Capture on Photolithographically Microfabricated Collagen Islands
...........
............. ..........................
................................ .................................. ..........................
......
.............
56
4.3.5.1 Optimization of Hepatocyte seeding concentration for Single Cell Capture .................................. 57
4.3.5.2 Hepatocyte Single Cell Capture Distribution ..................................... .........
........ 57
4.3.4 Characterization of X-ray Radiation Setup for Patterned Comet Assay ................................................ 60
4.3.5 Comet Assay on Patterned Hepatocytes.............................................................................................. 61
Chapter 5: Conclusions ...........................................................................................................................................
64
Bibliography ...............................................................................................................................................................
66
List of Figures
Figure 1. DNA damage response. DNA damage is recognized by sensor proteins that then initiate a
network of signal transduction pathways. This ultimately results in the activation of effector proteins that
execute the functions of the DNA damage response, including recruitment of DNA repair proteins, cell
cycle arrest, damage induced transcription, or the induction of apopotosis [89].
Figure 2.DNA damaging measuring techniques. (I) UDS, as assessed by autoradiography, in HepG2
cells after exposure to 500 pM MMS for 4 h. A, Cells in S phase; B, Cells in G1/M/G2 phase without
DNA repair; C, Cells in repair. [17]. (I1) A, B: Photomicrographs of typical mononuclei in human
lymphocytes C,D: Photomicrographs of typical binucleated cells with micronuclei in human lymphocytes
[20]. (III) Chromatids in which only one strand of DNA incorporated, BrdU show a normal Giemsa stain
(dark), whereas, ones with two substituted chromatids stain less darkly [73]. (IV) Immunocytochemical
staining for phosphorylated histone H2AX in human glioma cell after 2 Gy irradiation [25].
Figure 3. Comet assay protocol description. (A) Figure describing the possible cell sources for the
comet assay. (B) Standard comet assay Protocol.
Figure 4. Analysis of a typical comet. The green curve represents the head intensity whereas the purple
curve represents the tail intensity [60].
Figure 5. Surface micropatterning techniques. (A) Immobilization/patterning the biomolecules of
interest using microcontact printing (gCP). The stamp is inked with an alkanethiol and printed onto a gold
substrate. The alkanethiol has terminal groups that help immobilize the biomolecule of interest [75]. (B)
Immobilization/patterning of biomolecules using stencil method. The stencil (PDMS mold) acts either as
barrier to the biomolecule in the regions where the stencil contacts the substrate [74].
Figure 6. Schematic representation of the direct and indirect actions of ionizing radiation on DNA.
Direct mechanism is usually followed by particulate radiation, whereas the indirect damage mechanism
route is mostly a feature of electromagnetic radiation (X-rays, Trays) [75].
Figure 7. X-ray radiation setup for irradiating hepatocyte samples.
Figure 8. Microfabrication of spherical collagen islands using photolithography.
Figure 9. Comparing the effect of agarose gel percentage on olive tail moment as a function of Xray radiation dose. ( indicates the different OTM's between cells irradiated at 0 Gy and 10 Gy
in 0.7 % agarose, +indicates the different OTM's between cells irradiated at 0 Gy and 10 Gy in
1 % agarose p < 0.05. * indicates the different OTM's of cells in 1%agarose and 0.7% agarose
irradiated at 10 Gy, p < 0.05.)
Figure 10-1. Raw data representation of the X-ray irradiation dose response on primary rat hepatocytes
suspended in agarose gel (0.7% w/v) using alkaline comet assay.
Figure 10-2. X-ray dose response on primary hepatocytes suspended in agarose gel (0.7% w/v).
Figure 11. Frequency distribution of cells as a function of Olive Tail Moment at time
To= Ohr,T= lhr,T2=3hrs,T 3=4hrs,T4=21hrs and T5=24 hrs. The distributions shift left with increasing time
of culture indicating repair of DNA damage that might have occurred during isolation.
Figure 12 X-ray Dose response on Primary Rat hepatocytes comparing the Mean OTMs at 0 h and 24 h.
Figure 13 X-ray setup for patterned, adherent hepatocytes. To incorporate multiple radiation doses on the
same slide, selective blocking using lead sheets is used.
Figure 14 X-ray dose response on adherent hepatocytes.
Figure 15-1. Dose response comparison of OTM of suspended and adherent hepatocytes.
Figure 15-2. Compasion of standard deviations in Mean OTMs between suspended and adherent
hepatocytes.
Figure 16. Cell morphology and the corresponding comet morphology.
Figure 17. Quantitation of the collagen islands fabricated using photolithography and comparison to the
starting mask size and the intermediate photoresist mold. The difference in the sizes of the mask hole,
photoresist hole size and the collagen antibody island size was insignificant for all categories (p < 0.05).
Figure 18-1. Large patterned areas of hepatocytes at 2X magnification.
Figure 18-2. Single Cell hepatocyte Patterning at 10X Magnification elucidating single cell patterning.
Figure 18-3. Hepatocyte cell capture as a function on island size. Only areas on the slide with an
array of 10x 10 patterned cells were considered.
Figure 18-4. Percentage single cell hepatocyte capture as a function of island size.
* indicates
indicates statistical significance between 20 pm
statistical significance between 15 pm and 20 pm, 4
and 25 pm and *ndicates statistical significance between 15 pm and 25 pm (p < 0.05).
Figure 19. Intensity profile of the region exposed to radiation (Left, 10 Gy) and the region shielded by a
1cm thick lead block placed 0.5 cm from the plate (Right).
Figure 20-1. Patterned heptocyte microscope slide with marked resions of comet analysis (0 Gy, 10 Gy).
The analysis region was chosen at a distance greater than 200 pm from the edge of the lead sheet. The
distance between each comet - 250 pm.
Figure 20-2. Dose response comparison between patterned ( 0 Gy: 3.17 + 2.19 , 10 Gy: 12.84 + 4.034 )
and unpatterned ( 0 Gy: 1.69 + 1.48, 10 Gy: 13.94 + 3.04) adherent primary rat hepatocytes.
List of Tables
Table 1. Types of DNA damage [70].
Table 2. OTMs of 0.7% and 1% Agarose gels at radiation dose 0 Gy and 10 Gy
Table 3. Dose response values of hepatocytes at Oh, 24h.
Table 4. Dose response Mean OTM values for adherent hepatocytes.
Table 5. Mean OTM and standad deviation X-ray dose response values for suspended and adherent
hepatocytes.
Abbreviations
OTM
SCGE
Gy
DNA
SSB
DSB
Olive Tail Moment
Single Cell Gel Electrophoresis
Gray
Deoxyribonucleic Acid
Single Strand Break
Double Strand Break
Chapter 1: Introduction
1.1 Background and Motivation
We are continuously exposed to a variety of harmful (e.g. genotoxic) and beneficial (e.g.
antioxidants) agents in our everyday life. Exposure to external harmful agents (radiation, toxic
chemicals) as well as deficient endogenous processes can lead to irreversible DNA damage.
DNA damage has been reported to be the initial event in carcinogenesis, neurological disorders
and ageing. Further, if unrepaired these irreversible chemical changes in the structure of the
DNA could be hereditary in nature [81]. Detection and quantitation of DNA damage is therefore
a problem of sizeable importance.
Various assays have been developed over the past fifty years to score DNA damage and repair.
Most common of these assays are Unscheduled DNA Synthesis test (UDS), Micronuclear Assay
(MN), Sister Chromatid Exchange Test (SCE), y-H2AX assay and the Comet assay (Section
2.2). The comet assay is grounded on a remarkably simple principle that involves embedding the
cells of interest in agarose and lysing them leaving the unbroken DNA in a supercoiled state;
strand breaks relax the DNA supercoil and are revealed on electrophoresis, the free loops of
DNA extending to the anode to form a structure that geometrically resembles a comet, hence the
name comet assay. The DNA is then fluorescently tagged and classified by analyzing
morphological parameters obtained by image analysis and integration of intensity profiles or by
visual scoring [42-44].
Comet assay (single cell gel electrophoresis, SCGE) and its variants, offers myriad advantages to
the conventional methods of detecting DNA damage. Besides being simple, cheap and a
sensitive indicator of DNA damage, the comet assay offers versatility in the end points measured
through, the ability to extract important information from a minority population of cells and the
potential to be modified into a high throughput assay.
Previously, the comet assay has been used for cells that are biologically adherent and nonadherent in nature. Cells used for the comet assay can be primary cells obtained from
disaggregating a animal tissue or from cell line of interest.
10
However, for adherent cells like
primary hepatocytes the process of suspending in agarose and running a comet assay on them
poses several problems. Primary hepatocytes show excessive damage if they are taken through
the comet assay immediately after isolation from the source. This may be due to the physical or
chemical trauma experienced by the cells during isolation. Further, to get an accurate estimate of
the damage induced in the DNA of cells at a certain radiation dose, requires the cells to be
processed according to the comet assay protocol after the radiation insult. However,
trypsinization of the irradiated cells from the culture surface takes a significant amount of time
before they are suspended in agarose which may reduce cell viability and allow partial repair of
DNA. This may confound the levels of initial DNA damage induced at a certain radiation dose.
In addition, it has previously been shown that there is additional DNA damage is induced in
fibroblasts due to trypsinization from the surface before suspending in agarose. This could
confound the DNA damage radio-sensitivity of the adherent cells and result in lower signal to
noise ratio for studies evaluating DNA damage at low levels [83]. Culturing the primary adherent
cells on a suitable surface for a certain length of time (optimized for that particular cell) after
isolation may allow the cells to recover from the trauma suffered during isolation.
Here we describe a modified comet assay protocol in which the DNA damage in adherent cells is
quantified using the comet assay without detaching the cells from the surface. Briefly, the cells
are seeded onto the collagen coated microscope, overlaid with agarose gel at 370C, incubated in
media (with serum) in order to allow for stabilization of DNA damage repair mechanisms and
recovery, irradiated with the desired X-ray dose to, treated with the required comet assay buffers
and taken through gel electrophoresis. This process eliminates the need to trypsinize adherent
cells, thus opening doors to more versatile applications.
This thesis studies the DNA damage dose response of adherent primary rat hepatocytes to X-ray
radiation using comet assay for the first time. The use of radiation holds promise in research as
well as clinical settings. In basic research, radiation is used as a DNA damaging insult due to its
controlled and reproducible dose nature. Clinically, hepatocytes are the parenchymal liver cells
and their radio-sensitivity to X-rays is of fundamental importance in planning radio-therapeutic
strategies for treatment of primary and metastatic lesions of the liver. Normal cells are
responsible for dose limiting of the conventional radiotherapy of tumors, however, very little
information with respect to the radiation sensitivity of primary human epithelial cells obtained
from normal tissues is available. It is therefore required that the prescribed dose to the tissue be
calculated by assessing the dose limits of radiation to the surrounding normal tissues, as well as
the dose control for the tumors.
1.2 Objectives
This thesis is aimed at developing a modified comet assay protocol for cells adherent in nature.
We chose primary rat hepatocytes due to the adherent nature of hepatocytes, their clinical
importance and the lack of studies in the past on the effect of radiation on hepatocytes. The first
objective of the thesis is to compare effects of exposure to a model DNA damaging agent (X-ray
radiation) on hepatocytes when they are in their native attached state as opposed to when they are
suspended in agarose gel. Secondly, in an effort to explore the potential benefits of patterning
adherent hepatocytes on the surface of a regular glass slide we find the optimum conditions
(collagen island size and hepatocyte seeding concentration) for single hepatocyte cell patterning
on the surface of a microscope slide. Patterning adherent cells on small circular collagen islands
would help in maintaining spherical cell morphology by confining the region of attachment of
cells on the surface. Maintaining spherical morphology is important in order to use commercial
software for standard comet assay analysis. Further, through surface patterning a desired spatial
resolution on positioning the hepatocytes can be obtained, which would reduce comet overlap
and hence make them analyzable. Lastly, we characterize the dose response of adherent primary
rat hepatocytes (using the modified comet assay protocol for hepatocytes) and extend the
quantitation by using hepatocyte spatial arraying as a tool to multiplex X-ray radiation dose on
the same slide. This shows proof of concept for high throughput processing of the comet assay.
Chapter 2: Introduction to DNA damage and review
of the underlying detection techniques
2.1 Introduction to DNA Damage
DNA is hereditary information storehouse contained in every mammalian cell and is under
constant attack from various inherent endogenous factors and exogenous factors. DNA damage
has been established to be associated with cancer and other hereditary diseases; however DNA
damage is also used as a means to cure certain cancers through chemotherapy and radiotherapy
and is also responsible for the side effects that show up due to these treatments [71, 72]. In this
view, the balance of life must be maintained by having a control of the avoidance of mutations
by the DNA repair mechanisms and other responses to cellular DNA damage that effect the
stability of the DNA and generation of the mutations.
DNA can be damaged in a number of different ways and nature has found sophisticated ways to
repair these damages in the DNA. Despite the thousands of random changes created in the DNA
every day due to heat and metabolic reactions, only a few stable changes accumulate in the DNA
sequence of a cell permanently.
The type of DNA damage depends on the source and the
environment under which the DNA was exposed to that source (Table 1).
The cellular response to DSBs is a complex process that involves a network of interacting signal
transduction pathways [88]. This process is initiated by as yet unidentified proteins that detect or
sense DNA damage and subsequently transmit a signal by activating a cascade of
phosphorylation events. This ultimately results in the initiation of a number of cellular responses,
which help to ensure the maintenance of genomic stability, including cell cycle arrest,
transcriptional activation, recruitment and activation of DNA repair proteins and, in some cases,
induction of cell death by apoptosis (Figure 1). The importance of this response is evidenced by
the fact that mutations that alter any aspect of the process have significant effects on DSB repair.
DNA damage
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Figure 1. DNA damage response. DNA damage is recognized by sensor proteins that then initiate a network of
signal transduction pathways. This ultimately results in the activation of effector proteins that execute the functions
of the DNA damage response, including recruitment of DNA repair proteins, cell cycle arrest, damage induced
transcription, or the induction of apopotosis [89].
Exposure to Ionizing radiation
abasic sites, oxidized bases, nicks,
fragmentation
Exposure to Heat
fragmentation, abasic sites, nicks,
cyclopurine lesions
Mechanical Shearing
fragmentation, nicks
Exposure to Light (UV)
thymine dimers
Exposure to aqueous solution
abasic sites, oxidized bases, deaminated
cytosine, nicks
Table 1. Types of DNA damage [Modified from 70]
2.2 Methods to detect DNA damage
Several methods have been developed to study DNA damage in various eukaryotic cells in the
past. In this section we will review some of the methods of detecting DNA damage.
The comet assay is used to elucidate the DNA damage (SSBs, DSBs) using fluorescence
microscopy. Comet assay is a gel electrophoresis method which in its simplest form requires the
cells under investigation to be embedded in agarose on a microscopic slide. Cells for the assay
are obtained by tissue disaggregation and purifying the relevant primary cell type or culturing
and trypsinizing primary cells or modified cell lines from a cell culture dish before embedding in
agarose. A lysis solution is used to degrade lipids and proteins from the extra-nuclear and nuclear
regions and exposed to electric fields. Damage in the form of strand breaks relax the supercoiled
DNA and are revealed on electrophoresis, the free loops of DNA extending to the anode to form
a structure that morphologically resembles a comet, hence the name comet assay. The agarose
gel used is a natural hydrogel obtained from sea weed or cell walls of algae that provide a
scaffold for immobilizing the cells once solidified. After performing the electrophoresis the
DNA is stained with a fluorescent dye (Ethidium bromide, SYBR green etc.) and viewed using a
fluorescent microscope. Commercial software is then used in order to analyze the images and
quantify the data on the basis of informative properties such as the distance migrated by the
DNA towards the anode and the percentage that has migrated. These parameters are indicative of
the number of strand breaks that are present in the DNA of the cell. The popularity of the comet
assay is primarily due to its ability to quantify the DNA damage in any cell population of monodispersed cells. Under a given set of tested/standardized conditions the assay can provide
information about the heterogeneity in the DNA damage and the repair of individual cells in the
given population of cells. Further, consistent and reproducible conditions to induce DNA damage
can equip us with the ability to study quantify the repair capability of cells on a single scale
resolution. This can have far reaching applications in the field of chemotherapy and early
detection of cancer [33,34,35]. In addition, the comet provides versatility with the end point
information (SSB's, DSB's and lesion specific sites) that can be achieved by simply changing a
few conditions during buffer incubations. Comet assay is also flexible towards altering its
sensitivity to detect DNA damage. Change in variables like agarose gel percentage and
temperature can affect the sensitivity of the assay considerably [33, 45]. However, the promise
of using the comet assay is plagued by the sensitivity of the endpoints due to the lack of
standardization of the experimental protocol and laborious sample processing nature. The
schematic elucidating the procedure of the comet assay is shown in Figure 3.
A
+ / -exposure
Primary cells
+ / - exposure
11111 1 1 1il ii l
L.- __ _______
.
Trysin treatment
_•_
,sintreatment
Single cell sample
Adherentcells
+1/exposure
LLU
UU1
Cells embedded in agarose gel
exposed to radiation
B
Single Cell Sample Preparation
Slide Preparation ( Agarose
precoatslides, cell-gel
suspension)
Neutral Lysis
COMET ASSAY PROTOCOL
Neutrai Wash
Neutraf Electrophoresis
Stain
Image Analysis
Figure 3. Comet Assay Protocol description. (A) Figure describing the possible cell sources for the
comet assay. (B) Standard comet assay protocol
2.2.1 Alkaline Single Cell Gel Electrophoresis
The alkaline version of the comet assay is used to detect a combination of DNA single strand
breaks, double strand breaks and alkali-labile sites in the DNA. The alkaline solution unwinds
the DNA from its supercoiled structure and exposes single strand breaks, double strand breaks
and the alkali labile sites in the DNA. The electrophoresis buffer helps conduct current through
the electrophoresis chamber and migration of DNA through the agarose gel towards the anode.
The standard protocol followed is as follows: The cells are diluted to the required concentration
in 0.7 % agarose gel (w/v). The microscope slides are prepared by either precoating the slides
with industrial grade agarose gel or fixing a commercial sheet of Gelbond (Lonza Inc.) in order
to keep the gel attached to the microscope slide. The gel and cell mixture (500 pL) is gently
poured over the slide and gelled at 40 C. The slide can be exposed to the insult of interest
(chemical, radiation etc.) at this point. A more common alternative is to expose the tissue and
disaggregate the primary cells from the animal or treat a modified cell line before adding to the
gel. The slide is then submerged in lysis buffer for 1 h, alkaline buffer for 40 min,
electrophoresis for 30 min at 0.55 V/cm and neutralization buffer for 20 min in separate coplin
jars. The slides are stained with EtBr (10 pg/ml) for 10 min and the slides were visualized under
the microscope.
2.2.2 Neutral Single Cell Gel Electrophoresis
The neutral version of the comet assay is used to primarily detect DNA double strand breaks.
Protocol details for this version of the comet assay are described as follows: The cells are diluted
to the required concentration in 0.7 % agarose gel (w/v). The microscope slides are prepared by
either precoating the slides with industrial grade agarose gel or fixing a commercial sheet of
Gelbond (Lonza Inc.) in order to keep the gel attached to the microscope slide. The gel and cell
mixture (500 pL) is gently poured over the slide and gelled at 40 C. The slide can be exposed to
the insult of interest (chemical, radiation etc.) at this point and submerged in lysis solution (30
mm EDTA, 0 .5% SDS, pH 8) for 4h at 500 C. A more common alternative is to expose the tissue
and disaggregate the primary cells from the animal or treat a modified cell line before adding to
the gel. Slides are then washed free of detergent in a large volume of TBE buffer (90 mm Tris, 2
mm EDTA, 90 mm boric acid, pH 8. 5) for 2-16 h followed by electrophoresis in TBE buffer for
30 min at 0 .55 volts/cm. The slides are stained with EtBr (10 Vg/ml) for 10 min and the slides
were visualized under the microscope.
2.2.3 Enzyme Specific Base Lesions
The comet assay is both highly sensitive and highly versatile, rivaling even the most advanced chemical
techniques for detecting extremely rare DNA lesions [86, 87].
By combining the comet assay with
enzymes that convert damaged bases into single strand breaks, it is possible to directly assess the extent to
which conditions lead to formation of certain classes of base lesions. One enzyme commonly used in this
application is the Fpg glycosylase, which has both glycosylase activity that removes a broad range of
oxidized bases and lyase activity that introduces a single strand break 3' to the original base lesion. Thus,
'Fpg-sensitive-sites' provide a useful gauge of the levels of oxidized bases. it has previously been shown
that the comet assay is as sensitive as HPLC combined with either electrochemical detection [86, 87].
This study not only showed not only that the comet assay is highly sensitive, but also that by creating
standard curves, they could quantify the lesions levels using a modified comet assay.
2.2.4 Unscheduled DNA Synthesis (UDS)
Unscheduled DNA synthesis is a term that describes the replication of the DNA during the
nucleotide excision repair. As such, it is different from the replication mechanism that takes
place in eukaryotic cells only in the S phase. The method involves culturing of the cells on slides
and exposing the cells to the DNA damaging agent of interest in the presence of medium that
contains [3 H]-thymidine specific radioactive marker that is incorporated into the cells that are
replicating outside of the S phase of the cells. The amount of DNA replication in 'S' phase is
much greater than in UDS and it is therefore easy to eliminate the S phase cells because of their
high labeled indices (Figure 2-I).An indicator of the unscheduled DNA synthesis is the increase
in the levels of [3H]-thymidine in the DNA of the cultured mammalian cells during the repair of
damage. Autoradiography is usually used to detect this type of damage. While the assay serves
as a good indicator for assessing repair it is not useful in detecting SSB's/DSB's, unlike the
comet assay. Further, UDS specific to long patch repair pathways and is therefore not useful in
detecting base excision repair during oxidative damage, which constitutes an important field of
study in DNA damage and repair.
2.2.5 Micronucleus Assay (MN)
The micronucleus assay relies on detection of small vesicles that contain chromatin yet are
separate from the nucleus. The micronucleus is formed during the anaphase/metaphase of the cell
mitosis (cell division). This can happen if there is an entire lagging chromosome (aneugenic
event) or an acentric fragment detaching from the chromosome (clastogenic event)
is not
integrated into the daughter nuclei (Figure 2-II) [19]. The MN assay has been used with high
sensitivity for detecting DNA fragments of nuclei; however, unlike the comet assay it cannot
detect DNA base lesions.
2.2.6 Sister Chromatid Exchanges (SCEs)
The sister chromatid exchange DNA test involves the exchange of genetic material between
homologous chromosomes by breakage and reunion. This occurs during pairing of chromosomes at
meiosis, and in some organisms even during mitosis. This type of DNA damage usually occurs
during the S phase of the cell cycle and is usually induced by mutagenic factors that can interfere
with DNA replication or can form DNA adducts. To allow for a differential staining which
allows for distinguishing appearance of the chromatids, Bromo-deoxy-uridine (BrdU) is added to
the culture medium for the duration in which two complete cell cycles are completed.
Chromatids in which only one strand of DNA is incorporated, shows a normal Giemsa stain
(dark), whereas, ones with two substituted chromatids stain less darkly (Figure 2-III).
2.2.7 y-H2AX Assay
An early event after the introduction of the double strand breaks in the DNA is phosporylation of
the histone variant, histone 2A. H2AX is the part of 10% of all nucleosomes in a cell [20-22].
The DNA activated kinases, ATM, ATR and DNA-PK are responsible for the amplification of
the phosphorylated H2AX in a 2 Mbp sequence of DSB, within minutes after its formation [10,
23-27] (Figure 2-IV). This localized formation of Gamma H2AX allows for microscopical
detection of distinct foci by fluorescent gamma H2AX-specific antibodies that possibly detect a
single DSB [28-30]. It is possible to detect a single locus within a nucleus which makes this
method very sensitive towards detecting DSBs in cells. However, the assay is not optimal for
cells in 'S' phase, where there is an increase in serine 139 of the H2AX protein in the cell.
Increased levels of serine 139 interfere with the signal seen due to the phosphorylation at sites
19
where DSBs exist making analysis of the signal from the y-H2AX technically challenging [3132]. Although DSBs are an important for of DNA damage, the inability of the y-H2AX assay to
measure DNA damage such as SSBs and base modifications makes it less attractive when
compared to other assays like the comet assay. A modification of this y-H2AX assay by
incorporating the principle of flow cytometry has made the assay faster and high throughput,
though expensive [85]. Immunohistochemcial detection of phosphorylated H2AX is the most
sensitive method for detecting DNA double strand breaks in resting cells [13,14]. However, the
inability of the Gamma H2AX assay to detect DNA damage for cells in their 'S' phase, makes it
cumbersome to use. In addition exposure to many environmental factors lead to instability during
the 'S' phase of cell cycle [15, 16]. In contrast, the comet assay is able to detect DNA damage in
all phases of the cell cycle
2.2.8 Fluorescence in situ Hybridization (FISH) Assay
Fluorescence in situ Hybridization (FISH) has been at the forefront in locating specific
chromosomes, specific genes or regions of chromosomes [38]. When used in concert with the
comet assay FISH makes it possible to determine the spatial distribution of chromosome-specific
DNA sequences at the level of the individual nucleus (nonelectrophoresed) as well as in
chromatin fibers of comets (electrostretched
chromosomal DNA). Using probes of
oligonucleotides or cDNA the sequences of interest can be identified in the DNA. An example of
this methods utility is to measure gene-specific repair rates after low insult dose. This
methodology is likely to bring new insights into the field of interphase nuclear ultrastructure.
The technical challenges encountered due to high hybridization temperatures which would lead
to melting of the agarose gel have been solved to enable this method [39, 40].
MOn ot~lnlUccS
II
onucldeus
Figure 2. DNA damage measuring techniques. (I) UDS, as assessed by autoradiography, in HepG2
cells after exposure to 500 pM MMS for 4 h. A, Cells in S phase; B, Cells in Gl/M/G2 phase without
DNA repair; C, Cells in repair. [17]. (II) A, B: Photomicrographs of typical mononuclei in human
lymphocytes C,D: Photomicrographs of typical binucleated cells with micronuclei in human lymphocytes
[20].(111) Chromatids in which only one strand of DNA incorporated, BrdU show a normal Giemsa stain
(dark), whereas, ones with two substituted chromatids stain less darkly [73]. (IV) Immunocytochemical
staining for phosphorylated histone H2AX in human glioma cell after 2 Gy irradiation [25].
2.3 Comet Image Analysis
Once the cells have been agarose they are fluorescently labeled (Etbr, SYBR green etc.). In the
traditional comet protocol, the cells are found in the agarose gel manually and a region of interest
is defined and overlaid onto the fluorescent nucleus (Figure 4) and the software automatically
assesses the area, labels the region of highest intensity as nucleus. Alternatively, high end
automated stage microscopes
equipped with advanced
comet analysis softwares
can
automatically locate gel comets in agarose gel by setting intensity threshold values background
and fluorescent DNA. Using automated microscoped reduces the manual labor involved during
analysis at the expense of higher probability of false detection of a comet and the high cost of
equipment. Assuming that the nucleus is symmetrical, the edges of the nucleus are defined. A
density plot for the nucleus is generated. The program then defines a region outside of this area
and marks it as background and subtracts it from the intensity plot of the nucleus. After the
nucleus is excluded, the program searches the area for where the tail of the comet is expected to
be located and calculated the intensity of the pixels in the region of interest that have intensity
greater than the background levels of fluorescence. A variety of parameters are generated such as
Tail length, Head Intensity, Tail Intensity and the Olive Tail Moment (OTM). OTM described by
the relative fraction of the head and tail intensities and multiplied by the tail length is the most
widely used parameter in reporting the comet assay results.
Olive Tail Moment (OTM) =
Tail Intensity
x Tail Length
Head Intensity
Figure 4. Analysis of a typical comet. The comet image analysis program measures the intensity of each
pixel in the head and tail region and calculates the desired parameters like OTM [60].
2.4 Motivation for choosing patterned comet assay as a method to detect DNA
damage
Myriad sensitive DNA damage detection assays are available. Some of the most sensitive assays
used today include separation of nucleotides by Gamma H2AX, chromatography, mass
spectrometry of breaks/nicks surrounding nucleotides damaged in low concentration and
digesting DNA to single nucleotides [12]. Even though mass spectrometry is the most precise in
terms of estimating the concentration of strand breaks in the DNA it is still not easily available to
'cottage laboratories' across the globe. In addition mass spectrometry cannot be scaled up for
epidemiological studies because of large sample requirement. In view of these observations we
find it fitting to use the single cell gel electrophoresis to detect DNA damage with high
sensitivity in a relatively inexpensive manner. Many biochemical assays require that the cells be
homogenized in order for the DNA to be extracted and analyzed. In such a situation, it becomes
impossible to pinpoint which cell type is most damaged in the population. Moreover, the signal
from high number of cells with low damage will overwrite the damage associated with high
damage in small number of cells. However, information about the minority cell population can
give us important information, since it only takes one cell to develop into a full blown cancer.
This concept is an attractive feature of the comet assay as it allows single cell analysis as
opposed to most other techniques that rely on the averaged signal from the population.
Despite the huge potential of the assay has not been used to its full potential. Some of the major
reasons for the underutilization of the comet assay are a) labor intensive and time consuming
procedure b) expensive nature of the assay in terms low sample processing to reagents used ratio
c) lack of standardization of the assay procedure leading to inter laboratory variability. One
approach to solve the above mentioned problems would be to pattern cells in an array. Patterning
cells in an array would enable exposure of different rows/ columns of the array to different insult
conditions. Briefly, exposing different rows of arrayed cells to different radiation exposure times,
while masking the other cells, would result in a range DNA damaging conditions on the same
slide. One can think of extending the same principal to single cell resolution and chemical DNA
damaging agents. Processing multiple conditions on the same slide would reduce the quantity of
reagents used post insult exposure rendering the process high throughput as opposed to the
current procedure, where a single condition is processed on one slide.
Patterning cells on a slide promises other features such as automation during data collection by
using a motorized microscope thereby reducing the current intensive manual labor associated
with comet analysis. Spatial organization of cells in an array allows us to pin point location of
every cell in the array. By setting the initial point of data capture on the microscope and
incrementing it a fixed distance in x- and y- direction, we can use a motorized microscope to
capture data without manual vigilance or interference. Previously, attempts have been made to
automate the image comet assay image analysis [41, 42, 43]. However, either these methods are
expensive to implement or use imaging techniques that rely on complex algorithms which
provide imprecise information on the comets due to lack of proper auto-focusing on the comet
image or failure to distinction between single cell comet assay versus overlapping comets and
their background (i.e. EtBr crystals). In this setting it would be extremely useful to have single
cells in the same z- plane in order to set the proper focus at the beginning of the experiment in
the same plane and pattern single cells in an array to avoid loss of information due to
overlapping of comets from different cells. This points us in the direction of implementing a
protocol for patterned and surface adherent single cell comet assay protocol.
Furthermore, there is a need to establish a quantitative, high throughput protocol for comet assay
on adherent cells. To date there have been only a few reports of comet assay performed on
adherent cells [46]. The DNA damage in adherent comets has been quantified using a visual
scoring system, a method which is highly dependent on the user's understanding of classification
of comets. This introduces variability in the results. A more quantitative study is required to
make the protocol robust and independent of individual bias. An automated comet analysis
system would remove the variability due to the user. We therefore focus on developing an
adherent patterned single cell protocol. Besides the lack of information on effects of DNA
damaging agents to cells adherent in nature, surface adherent cells would provide a single plane
of focusing for image analysis. Patterning cells would help with high throughput and possible
automation of the assay.
2.5 Review of cellular micropatterning techniques
As described earlier, patterning adherent cells would be an asset to the comet assay as it would
potentially make the assay high throughput, reduce variability and reduce the manual labor.
Some widely used strategies to micropattem cells are described below.
Over the past two decades techniques have been borrowed from the semiconductor industry for
application in biology to enable capability of positioning cells in controlled areas and exclude
them from other regions on a substrate. The techniques were initially developed in the IC
industry (mostly photolithography) to pattern various thin metal lines on a substrate. Today, the
same technique has helped develop research tools in the study of role of heterotypic cell
interactions on organ specific functions [47], nerve growth cone guidance [48,49], study of cell
shape regulation of growth and apoptosis [50] and cell-cell interaction regulation of cell cycle
[51]. Myriad patterning techniques, including, microfluidics [53], polymeric stencils [54] and
self assembled monolayers [55] have been explored in the past decade to pattern adhesive
proteins or blocking chemicals cells on a 2-D substrate.
2.5.1 Photolithography
Photolithography uses a thin layer of photoresist which can be permanently cross linked to the
substrate or used as a sacrificial layer to form blocking regions (known as hard lithography), or
for making molds (eg. PDMS used in 'Soft Lithography' [57-62]) that can deposit biological
molecules [52].
Photolithograhy has been used for patterning adhesive/non-adhesive protein
regions by acting as a barrier to the protein being adsorbed, photoablating proteins that are preadsorbed onto the surface [63],covalently linking proteins to photosensitive groups [64] and
immobilizing proteins on thiol terminated siloxane films patterned using UV lights [65].
Although photolithography requires expensive equipment, this method reproducibly produces
feature sizes less than 1 pm [65] (Figure 8).
2.6.2 Soft Lithography
Another equivalent approach (microcontact priniting, RCP) makes use of 'soft lithographic'
methods in which an elastometric stamp is formed by a cast of an elastomeric prepolymer
substance against a relief structure. The relief structure is usually formed using photolithography.
The relief structures of the mold are 'inked' with an alkanethiol which are stamped on a gold
sputtered surface. The alkanethiol is transferred to the gold substrate only in regions where the
PDMS stamp contacts the substrate. Subsequent exposure of the remaining bare gold substrate to
a second alkanethiol group generates a surface that presents terminal groups that might aid in
immobilizing a different adhesive/non-adhesive biomolecule. Unlike photolithography, which
requires individual substrate modification, the stamping process produces a template a generation
of reusable stamps can be produced. However, some of the problems involved with stamping is
the need to sputter every substrate and the inherent hetergenous nature of stamping. The process
is illustrated briefly (Figure 5B). The polymeric stamps (eg. PDMS) have also been used as
stencils where the biomolecule of interest adsorbs physico-chemically in the regions exposed by
the stencil and precluded where the stencil is sealed to the substrate. An alternate version of this
method is to first physico-chemically adsorb the biomolecule onto the substrate and then seal the
stencil on the substrate by using a mechanical load. The substrate is then exposed to high energy
plasma which removes the biomolecule from the regions where the stamp was not in contact
with the substrate. This would create an inverse pattern to the method mentioned above. The
stencil procedure is a cheaper and quicker alternative to stamping and photolithography
procedures, but compromises on the resolution of the patterned biomolecule (Figure 5C).
2.6.3 Microspotters
The latest development in the field of rapid patterning of biomolecules on a 2-D substrate is the
development of high throughput micro spotters [66]. Spotted microarrays of proteins or DNA are
printed by dipping the spotting pins into the source well of either and then depositing the protein
or DNA on a chemically derivatized glass substrate [67, 68]. The pins of the spotter are filled up
by capillary action and the surface tension between the spotting buffer and substrate then acts to
deposit the spots [69]. Various factors affect the spot size. Optimization of the contact time of the
pin with the substrate, surface chemistry, viscosity of the protein of the DNA solution to be
spotted and the relative humidity of the environment in which the experiment is being performed
are important factors [69].
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Figure 5. Surface micropatterning techniques (A) Immobilization/patterning the biomolecules of
interest using microcontact printing (gCP). The stamp is inked with an alkanethiol and printed onto a
gold substrate. The alkanethiol has terminal groups that help immobilize the biomolecule of interest [75].
(B) Immobilization/patterning of biomolecules using stencil method. The stencil (PDMS mold) acts either
as barrier to the biomolecule. in the regions where the stencil contacts the substrate [74].
.
.
....
2.7 Radiation
Radiation can be classified as directly ionizing or indirectly ionizing. Particulate radiation e.g.
fast moving neutrons etc. is usually classified as direct ionizing radiation, i.e., a charged particle
with the appropriate kinetic energy can directly disrupt the atomic structure of the absorbing
material through which it passes and therefore produce potentially injurious chemical and
biological alterations. Electromagnetic radiation (eg. X rays, y radiation) on the other hand are
indirect ionization radiations. They do not produce biological or chemical changes directly.
During passage through the absorbing material electromagnetic radiations produce fast moving
charged by particles by giving up energy. X-rays and y rays are more penetrating than the
ionizing, particulate radiation. Therefore the relative influence of direct and indirect ionization
due to electromagnetic or particulate radiation respectively on biological tissue will differ
significantly [75].
A schematic depiction of radiation striking DNA and irreversibly damaging it is depicted in
Figure 6. This can either happen by a direct "hit" or by a two step mechanism involving the
ionization of an intermediary such as water and the production of high reactive species such as
free radicals. Free radicals possess an unpaired electron in their outermost reactive shell.
Roughly two-thirds of the electromagnetic radiation damage due to X-rays or y rays is due to the
action of the OH-radical produced during the reaction below.
H 20
H 20
+
H 20 + + e
-
H 20 ---
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Figure 6. Schematic representation of the direct and indirect actions of ionizing radiation on DNA.
Direct mechanism is usually followed by particulate radiation, whereas the indirect damage mechanism
route is mostly a feature of electromagnetic radiation (X-rays, yrays) [75].
Chapter 3: DNA Damage induced in Primary Rat
Hepatocytes by X-ray Radiation
3.1 Introduction
In addition to the natural ionizing radiation from the surroundings and outer space, humans
are exposed to variable amounts of radiation from artificial sources. The largest component
of artificial radiation is from exposures during medical diagnosis and treatment of disease
[77]. As mentioned earlier, deleterious effects of the biological tissue are a result of the
energy absorbed from radiation and radiation-DNA interaction. As a clinical application, it
is therefore important to study the effect of radiation on normal tissue surrounding tumors
before a dose limit can be prescribed for radiotherapy. Even though the advent of 3D-CRT
and Intensity modulated radiation therapy (IMRT) has improved the precision of irradiating
cancers to minimize damage to the surrounding organs it still poses threat to the healthy
cells in the cancer affected tissue [78]. Currently, little information with respect to the
radiation sensitivity of hepatocytes, parenchymal liver cells, from normal tissues is
available even though the radiation response of these normal cells is responsible for dose
limiting of the conventional radiotherapy of tumors. Lack of information is due to the
unavailability of an assay that reliably queries the initial DNA damage induced in primary
cells that are adherent in nature.
This chapter probes the important parameters required to obtain a radiation-DNA damage
dose response for primary rat hepatocytes. Further in this chapter we optimize parameters
related to single cell patterning. These parameters are used to implement a new comet
assay protocol for patterned adherent hepatocytes in chapter 4.
3.2 X-ray Radiation Setup
To date there have been no studies that show the effects of radiation on DNA damage in
hepatocytes using comet assay. Radiation is used as a model DNA damaging agent that reliably
exposes the cells to the desired insult dose and therefore helps us understand the effects of
radiation on initial DNA damage. The reproducible dosing nature of radiation eliminates any
variable DNA damage due to the source. To implement the exposure of hepatocytes with X-ray
radiation we design a setup as shown in Figure 7.
The hepatocytes were suspended in gelled agarose and placed in a tissue culture dish filled with
media (with serum). The samples were irradiated at room temperature using a 250 KVp X-ray
machine (Siemens Stabilapan 2) calibrated at 1Gy/min.
X-ray Source
/F·
t
5iches 1I
Cell Culture Dish with
Sample
Adjustable Stand
Figure 7. X-ray radiation setup for irradiating hepatocyte samples.
3.3 Materials and Methods
3.3.1 Type-I Collagen Preparation from Rat Tails
Type I collagen has been established as an adhesive protein for primary rat hepatocyte
attachment. The collagen was extracted from the tendons in the tails of Lewis rats. The protocol
is described by Dunn et al. [7]. Briefly, tendons were dissected from the rat tail and were
dissolved in a stir bath containing 200 ml of 3%(v/v) acetic acid overnight at 40C. The solution
was filtered using four layers of cheesecloth and centrifuged at 12000g for 2h. The supernatant
was collected and 40 ml of 30% (w/v) NaCl was added to precipitate the supernatant. The
precipitated pellet was collected by centrifuging at 4000g for 30 min.
The pellet was dissolved in 50 ml of 0.6% (v/v) acetic acid and the solution was dialyzed five
times against ImM hydrochloric acid. To sterilize, 0.15 mL of chloroform was added to the
solution. The solution was stirred for 2 days and the cap was loosely screwed to allow
evaporation of chloroform from the solution. The preparation yields type I collagen mostly in its
non cross-linked, native and triple helical form [8].
3.3.2 Alkaline Comet Assay Buffer Preperation
The protocol for the comet assay requires a set of buffers which are used during the experiment.
Here we describe the formulations of the stock and working solutions for the buffers used during
the alkaline comet assay. All the solutions were consistently made according to the formulations
described below.
3.3.2.1 Lysis Buffer
The lysis buffer is responsible for dissolving the cellular and the nuclear membranes of the cell
and exposing the cellular DNA for electrophoresis. The pH-9.8 of the lysis stock solution (2.5
M NaCl, 100mM Na 2EDTA, 10mM Tris) was adjusted during the preparation by adding NaOH
pellets and measuring the pH with a measuring meter. The stock solution was stored at 40C.
3.3.2.2 Alkaline/Electrophoresis Buffer
The formulation for the Alkaline and Electrophoresis buffers is the same. The stock solution (0.3
M NaOH, ImM Na2EDTA) was carefully adjusted for pH -13 by adding NaOH pellets. The
Stock solution was stored at 40C.
3.3.2.3 Neutralizing Buffer
The neutralizing buffer serves the purpose of washing off the alkaline/electrophoresis solution
content from the gel. The neutralizing buffer (IM Tris) was carefully adjust for pH-7.5 using
35 %HCl and stored at 40C.
3.3.3 Primary Rat Hepatocyte Isolation
Hepatocytes were isolated from 2-3 month old female Lewis rats (Charles River, MA) weighing
180-220 g, by a modified procedure described by Seglen [8, 9,10]. Briefly, the animals were
anesthetized in a chamber containing saturated ether. The liver was first perfused through the
portal vein with 400 ml of perfusion buffer followed by a ImM ethylenediamineetraacetic acid
(EDTA) at 30 ml/min. The perfusate was then equilibrated with 1 L/min 95 % 02 and 5 %CO2
through a silicone tubing that was maintained at 370C using a heat exchanger. Subsequently, 200
ml of 0.05% collegenase in perfusion buffer was perfused with 5 mM of CaCl 2 at a flow rate of
20ml/min for 10 minutes. At this stage the swollen liver was torn from the ligaments and was
transferred to culture dish. The liver capsule was torn apart and the cell suspension was passed
through two nylon filter mesh grids sized 250 pm and 62 pm respectively. The cells suspension
was centrifuged for 3 min. at 500 rpm at 40C. Non-parenchymal cells (i.e. stellate, kupffer,
endothelial) cells are more buoyant and float in the supematant which is aspirated to leave a
pellet of primary rat hepatocytes. The pellet is then resuspended in Krebb's Buffer A cell count
of 150 million - 250 million cells was obtained per isolation and a repeated viability of 88% 93% was calculated using trypan blue exclusion. Non-parenchymal cells based on the
morphology and size (-10pm) constituted < 1%of the cells after filtering. Hepatocyte culture
medium consisted of Dulbecco's Modified Eagle Medium with high glucose, 0.5 U/mL insulin
1% (v/v) penicillin-streptomycin, 7 ng/mL glucagon, 7.5g/mL hydrocortisone, and 10% (v/v)
fetal bovine serum.
3.3.4 Photolithographic Micropatterning
Detailed procedures for microfabrication of substrates and the subsequent modification have
been previously described [79]. Briefly, 60 mm x 50 mm x Imm glass slides (Fisher Scientific
Inc.) were spin coated (2 pm) with positive photoresist (S1813, Shipley Corportaion). The slides
were then baked to evaporate excess solvents and exposed to UV light for 90 sec in a bottom side
mask aligner (Karl Suss, Waterbury Center, VT) through transparency photo-masks printed at
8000 d.p.i (Fineline Imaging). Exposed photoresist was then developed (Microposit 321
Developer, Shipley), rinsed in deionized water and baked for 90 sec to complete curing. The UV
exposure time and the bake time were optimized to allow easy "lift off' of the photoresist during
sonication with acetone. To ensure complete removal of UV-exposed photoresist down to bare
glass, slides were exposed to oxygen plasma (oxygen pressure 250 mTorr, base vacuum 80
mTorr, 200 Watts for 10 minutes). Slides were subsequently rinsed with water and immersed in a
100 gg/mL solution of collagen type-I for 45-60 min at 370C. Collagen undercuts the photoresist
if the slides are incubated in the collagen-I solution for longer than 1 hour under the optimized
time of UV-light exposure and bake time. Substrates were then sonicated (Fisher, Pittsburg, PA)
in acetone for 10 seconds to remove the residual photoresist, rinsed several times with water,
dried under a stream of air, and stored dry at 40C for upto 2 weeks prior to use. Uniform
collagen-modified substrates to be used for randomly distributed cultures were generated by
exposing the entire photoresist-coated slide with UV light, and subsequently following the
processing procedures outlined above for micropattemed wafers (Figure 8). One drawback of
this method is the necessity of exposing proteins to acetone, or a similar solvent, in order to
remove the residual photoresist. In spite of this theoretical limitation, this method has been
successfully utilized in the past for the micropatterning of collagen I-IV, fibronectin, Bovine
Serum Albumin and fibronectin.
3.3.5 Collagen Island Fabrication
The patterned photoresist slide is incubated with the collagen extracted from the tendons of the
rat tail (section 3.3.1) for 2.5 hr at 370 C. The collagen physicochemically adsorbs to the glass.
The collagen is then aspirated and the slide is washed gently using DDH 2 0 to remove the excess
collagen in the dish containing the slide. The slide is then sonicated in a glass bath containing
acetone to lift of the photoresist and rinsed gently with water 3-4 times leaving behind islands of
collagen (Figure 8). The collagen islands were visualized by tagging the patterned collagen glass
slides with a primary antibody, specific for type 1 collagen and a secondary antibody to amplify
the signal (Alexa Fluor 488). Briefly, antibody solution was prepared in 1% BSA and Tris
Buffered Saline (v/v) (Tris - pH 7.4, 150mM NaCl) and the patterned collagen slide was
incubated in the solution for 3 hrs. The slide was washed 2-3 times with DDH 20 and incubated
for 1 hr with the secondary antibody and viewed using Fluorescien Isothiocyanate (FITC) filter
cube. A control slide (cleaned glass slides, no pattern) incubated with the primary and the
secondary antibodies was used to detect the non specific adhesion of the antibody to the glass
surface and antibody clumping. A control slide incubated with just the secondary antibody was
used as an indicator for the background due the secondary antibody. The mask pattern size used
to pattern the photoresist and the resulting collagen island size was imaged using a imaging
software (Metamorph 5.0) on a automated stage microscope (Nikon Eclipse E200).
Spin photoresist
m
UV expose
through mask
Develop resi9t
Incubate with
Coagen
Sonkate in Acetone,
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Figure 8. Microfabrication of spherical collagen islands using photolithography.
3.3.6 Comet Assay on suspended Hepatocytes
Isolated rat hepatocytes were seeded on a Falcon 150 x25 mm (#353025) style polystyrene
dishes that were incubated with 500gg/ml (v/v) collagen-I and held for 24 hours after isolation at
370 C and 5% CO 2 in an incubator. The dishes were incubated with 5 mL trypsin for 10 min at
370 C to lift off the cells and were quenched with C+H medium with serum. The cells were resuspended in 0.7 % gel at a concentration of 1x10 5 cells/mL in a 1 mL eppendorf tubes. The cellgel suspension was pipetted onto regular slides (VWR 25mmx75mm) treated by dipping into
industrial grade agarose and dried. This provides adhesion to the cell-gel suspension on
solidification. The gels were solidified by keeping in a cold room environment at 40 C for 10 min.
36
The cells embedded in solidified agarose gel were irradiated with 0 Gy, 1 Gy, 2 Gy, 4 Gy and 10
Gy and were immediately put into lysis after irradiation to prevent any cellular repair. Each
radiation dose group was replicated on three slides to validate the results.
3.4 Results and Discussion
3.4.1 Gel Percentage Optimization for Comet Assay
Agarose gel percentage is an important parameter that determines the sensitivity of observed
DNA damage in cells in the comet assay. We need to find the appropriate agarose gel percentage
that does not tear during the comet assay and also improves the sensitivity of the assay. An
increase in sensitivity is desired in order to detect the lowest DNA damaging insult dose.
Previously, the use of 0.7% and 1% agarose gel (w/v) has been reported [44, 45]. We tested 0.5
%, 0.7 % and 1% agarose gels for adequate gel strength to sustain the shear forces through the
comet assay and the role of gel percentage on the comet tails. Results from the experiments
performed are summarized below (Table 2).
Agarose percentage is directly proportional to the gel strength and inversely proportional to its
matrix pore size. An increase in the agarose concentration leads to an increase in gel strength but
results in smaller agarose gel pore size. Smaller pore size of the agarose gel deters the DNA to
electrophorese towards the anode in the electrophoresis chamber. This results in smaller comet
tails, if all other parameters such as buffer incubation times, electrophoresis strength and time are
kept the same. The OTM's for the unexposed hepatocytes (0 Gy) are statistically not different in
0.7 % and 1 % agarose gel. Exposure to radiation at 10 Gy shows a statistical difference ( p <
0.05 ) in the OTM's between hepatocytes in 1% and 0.7% agarose gel. The OTM statistical
insignificance for hepatocytes in 1% and 0.7 % agarose gels at 0 Gy can be explained due to the
presence of low damage in cell DNA indicating that the pore size at both gel concentrations was
similar in terms of allowing electrophoresis of DNA through it. The OTM was statistically
different ( p < 0.05) between unexposed cells and the cells irradiated at 10 Gy for both agarose
concentrations. This indicated that a significant amount of DNA damage was induced due to the
radiation.
X Ray Radiation Dose
0.7 % Agarose Gel
(Olive Tail Moment + S.D)
0 Gy
10 Gy
8.4 + 5.1
30.5 + 6.5
1 %Agarose Gel
(Oilve Tail Moment + S.D)
4.9 ± 3.3
18.1 ±3.8
Table 2. OTMs of 0.7% and 1%agarose gels at radiation dose 0 Gy and 10 Gy.
Effect of Gel Percentage on Olive Tail
Moment
40o
T
3s
SOGy
ao V
exon0
y
30
+I
15
10
-
0 S-1
1%
Agarose Gel Percentage
0.7 %
Figure 9. Comparing the effect of agarose gel percentage on olive tail moment as a function of X-ray
radiation dose. ( * indicates the different OTM's between cells irradiated at 0 Gy and 10 Gy in
0.7 % agarose, +indicates the different OTM's between cells irradiated at 0 Gy and 10 Gy in 1
% agarose p < 0.05. * indicates the different OTM's of cells in 1% agarose and 0.7% agarose
irradiated at 10 Gy, p < 0.05.)
We chose 0.7% agarose gel as our standard for all the experiments as it is strong enough to
withstand the fluid shear forces and successfully finish running the comet assay as opposed to
0.5 % agarose gel, which breaks during the assay. Also, the 0.7 % agarose gel increases the
sensitivity of OTM at the same dose as compared to the 1 % agarose gel (Figure 9, Table 2). The
OTM shows statistically different results (p < 0.05) between cells in 1 % and 0.7 % agarose gels.
This might be due to the pore size at 0.7 % agarose gel allowing lesser impedance to the
damaged DNA as compared to the 1% agarose gel.
I
3.4.2 X-ray Radiation Dose Response on Primary Rat Hepatocytes Suspended in Agarose
Gel
The purpose of these first set of experiments on hepatocytes was to obtain, using the comet
assay, a set of X-ray radiation dose to which the primary rat hepatocytes respond, i.e., induce
significant damage in the parenchymal liver cells that can be picked up by the alkaline comet
assay protocol. We use these doses to compare the modified comet assay protocol for adherent
cells with the current comet assay protocol later in the thesis. In a previous study, it has been
observed that hepatocytes suspended in 0.7 % (w/v) agarose gel respond to a radiation dose of
0 Gy, 1 Gy and 4 Gy, showing astatistically significant increase in damage with increase in
radiation dose [88]. Here, we tested to determine if the primary rat heptocytes responded
similarly.
In order to prepare the slide with suspended cells so that the cells can be exposed to X-ray, fresh
isolated primary rat hepatocytes were immediately mixed with 0.7 % Agarose (w/v) in PBS after
isolation. The cell and gel mixture was diluted to lx105 cells/ml in C+H media without serum.
The cell concentration was optimized in order to reduce overlapping comets due to overcrowding
of cells. The cell suspension was gently pipetted onto a clean glass slide with a gasket made out
of a commercial product (GELBOND, Lanzo Inc.), to help the adhesion of agarose onto the glass
slide. The slide was then allowed to sit on ice to allow the gel and cell suspension and trap the
cells. Five radiation conditions were chosen ( 0 Gy, 1 Gy, 2 Gy, 4 Gy and 10 Gy at a dose rate of
1 Gy/min) and each condition was replicated using three slides. The slides were then subjected to
the alkaline comet assay protocol mentioned in section 2.3.1.
Primary rat hepatocytes were suspended in agarose gel and exposed to radiation. The 0 Gy
radiation data represents the baseline damage in the isolated hepatocytes (control group). There
is an increase in the DNA damage in the primary hepatocytes with the increase in the radiation
dose (Figure 10-2), the minimum being at 1 Gy (Mean OTM ± Std. Dev.: 5.21 ± 4.79) and the
maximum at 10 Gy (Mean OTM ± Std. Dev.: 12.2 + 6.1). This indicates that the hepatocytes are
responsive to the selected dose range ( 1Gy, 2 Gy, 4 Gy and 10 Gy). The raw data for each
radiation dose is shown in Figure 10-1. The large standard deviation in the OTM's is an indicator
of the heterogenous response of each cell towards the insult (Figure 10-2). We also observe that
the standard deviation of the OTM increased with the increase in radiation dose insult. This can
be explained on the basis of the presence of a subpopulation of cells that are radio-resistant to Xrays at these doses. The results of the experiment are summarized below (Table 4).
X-ray Dose Responseon Primary Rat
Hepatocytes (Raw Data)
40 1
35
30
25
E
20,n
10
0
1
2
3
4
5
6
7
8
9
10 11 12
x-ray Rfaiation Dose (Gy)
Figure 10-1. Raw data representation of the X-ray irradiation dose response on primary rat hepatocytes
suspended in Agarose gel ( 0.7% w/v) using alkaline comet assay.
X-ray Dose response on Primary Rat
Hepatocytes
j
20 .
"18I
16
14
12
12
4
2
OGy
1Gy
2Gy
4Gy
10Gy
X-ray Radiation Dose (OV)
Figure 10-2. X-ray dose response on primary hepatocytes suspended in Agarose gel (0.7% w/v).
X-ray Radiation Dose
0 Gy
1 Gy
2 Gy
Mean Olive Tail Moment + S.D
3.2 + 3.3
5.2 + 4.8
6.4 + 5.3
4 Gy
7.4 + 5.8
10 Gy
12.2 + 6.1
Table 4. X-ray dose response OTM values on primary Hepatocytes suspended in Agarose gel (0.7%
w/v).
The X-ray DNA dose response on hepatocytes suggests that the cells were radiosensitive and
The OTM increased with the increase in X-ray dose. The dose response values obtained were in
accordance with previous studies on the DNA damaging effects of X-ray on hepatocytes [90].
The mean OTM values indicating the DNA damage due to radiation were not statistically
significant suggesting that radiosensitivity of each hepatocyte is different.
3.4.3 Comet Assay on Timecourse Study of Hepatocytes
During isolation the hepatocytes are subjected to physical and chemical trauma which might
cause the damage levels to present higher than usual [83]. It is therefore essential for the
hepatocytes to recover in order to gauge the amount of damage induced at a particular dose of Xray radiation. Conversely, over time hepatocytes might lose their viability in an in vitro culture
environment. We perform this experiment in order to determine the optimum time for culturing
hepatocytes after isolation.
The experimental groups for this experiment included hepatocytes that were taken through the
comet assay immediately and hepatocytes suspended in agarose permeated with media in a tissue
culture incubator were incubated on a collagen coated microscope slide for 1 hr, 3 hrs, 4 hrs, 21
hrs and 24 hrs. Hepatocytes taken through the comet assay were and suspended in agarose gel
(370C) at a concentration of 1x10 5 cells/ml in eppendorf tubes, immediately after isolation. The
cell-gel suspension (1ml) was pipetted onto slides pre-coated with industrial grade agarose to
prevent the cell-gel suspension to slip off after solidification during the alkaline comet assay
protocol.
Hepatocytes used after 1 hr, 3 hrs, 4 hrs, 21 hrs, 24 hrs were cultured in hepatocyte media with
serum on pre-treated polystyrene dishes and kept in an incubator at 370C for the time length after
isolation. The dishes were coated with rat collagen-I before the hepatocytes are plated on them.
Cells were seeded at a near-confluent concentration of 1x10 5 cells/ml in C+H serum with
medium on a collagen coated surface. Cells for comet analysis at the appropriate time points, i.e.,
T=I hr, 3 hr, 4 hr, 21 hr, 24 hr were trypsinized and quenched with C+H serum medium. A
volume of the trypsinized cells was collected and the cell concentration was determined using a
coulter counter. A total of 15x103 cells were spun down and the supernatant was removed and
resuspended in low melting point (LMP) Agarose. The cell-gel suspension was pipetted on the
Agarose precoated glass slide and the comet assay protocol was carried out as described in
section 3.3.1. The same protocol was carried out for all the time points. Buffers/incubation
solutions were made at the beginning of the of the time-course experiment, thus ensuring that the
cells are exposed to the same solutions.
The purpose of this experiment is to determine the optimum time for culturing hepatocytes after
isolation. During isolation the hepatocytes are subjected to physical and chemical trauma which
might cause the damage levels to present higher than usual [83]. It is therefore essential for the
hepatocytes to recover in order to gauge the amount of damage induced at a particular dose of Xray radiation. Firstly, evaluating the OTM from time points To (Cells processed immediately after
isolation) and TI(Cells processed 1 hr after incubation on an attached surface) we observe that
there exists a subpopulation of cells in the sample that have abnormal OTM (OTM > 6, based on
the dose response). However, this trait seems to disappear at time point T2 (Cells processed 2 hrs
after incubation) and higher (Figure 11).
100
Distribution ofcell damage (T0 hr)
Distribution of cell damage (T 1 hr)
70,
610 "
70 1
0
2
4
8
6
10
12
E
16
24
0
2
6
4
•
m
•
10
12
16
10
Olve TadMeet (OTM)
Owe TaMIant oM)
m
I~~~~
8
,t=&.zL.&=:
L..
.
ml Im
.---1J.--...,.
-
Distribution of cell damage 1T
=3
oit n
o
ce
A L.,.
IT(
amqe
rs)
hrs)
6o
0
so
WI
'70
8•0
60
.so
40
30
20
10
0
----
0
2
4
8
6
10
I
12
1U
0
18
4
2
r--·---y-----r---~"p"---·-"-~--l~
6
8
10
12
ribution
of cell da
14
18
16
le
h
Distribution ofcell damage (T= 24 hrs)
Distributionof call dam e ( T= 21 hrs)
'I
................
..
U
0
2
4
6
abo
10
12
TapiAnrm
a
* Imun
4
6
18
0
2
-
4
-
6
..
8
.
12
I4
.
16
18
cAmETanM-a mm IInTaM
Figure 11. Frequency distribution of cells as a function of Olive Tail Moment at time
To= Ohr,T 1=lhr,T2=3hrs,T 3=4hrs,T 4=2lhrs and T5=24 hrs. The distributions shift left with increasing time
of culture indicating repair of DNA damage that might have occurred during isolation.
During isolation the hepatocytes are subjected to physical and chemical trauma which might
cause the damage levels to present higher than usual [83]. A frequency distribution of the
number of cells as a function of olive tail moment revealed that the most optimum time, as
quantified by OTM, for culturing primary rat hepatocytes after isolation is 24 h. Most of the cells
show OTM concentrated at a value of 2 or 4, indicating that there might be repair mechanisms
involved that shift the distributions shown in Figure 11 to the left with time. Another explanation
is that only the healthy cells are analyzed at later time points whereas the severely damaged or
dead cells are excluded from the analysis due to their detachment from the surface over time.
Even though OTM distributions at 3 hrs of hepatocyte incubation and thereafter are similar, we
choose to incubate for 24hrs due to convenience in resuming the experiment.These findings are
consistent with the previously reported culture time for adherent cells in the literature [91].
3.4.4 Primary Rat Hepatocyte DNA Damage Dose Response to X-ray radiation at 24 h Post
Culture
It was evident from the timecourse experiment that primary rat hepatocytes attached to the
surface post 3 hrs had a lower OTM as opposed to hepatocytes possesses immediately after
isolation and 1 hr post isolation. There was no statistical difference between T=3 hr, 4 hr, 21 hr
and 24 hr incubation of primary rat hepatocytes, with respect to the OTM, on collagen coated
pre-coated culture dishes. We chose to culture the hepatocytes on the collagen coated surface for
a span of 24 hrs.
Isolated rat hepatocytes were seeded on polystyrene dishes that were incubated with 500gg/ml
(v/v) collagen-I and held for 24 hours after isolation at 370 C in an incubator. The dishes were
trypsinized to lift off the cells and were quenched with C+H medium with serum. The cells were
resuspended in 0.7 % gel at a concentration of 1x10 5 cells/mL in eppendorf tubes. The cell-gel
suspension was pipetted onto regular slides treated by dipping into industrial grade agarose and
dried. This provides adhesion to the cell-gel suspension on solidification. The gels were
solidified by keeping in a cold room environment. The cells embedded in solidified agarose gel
were irradiated with 0 Gy, 1 Gy, 2 Gy, 4 Gy and 10 Gy and were immediately put into lysis after
irradiation to prevent any cellular repair. Each radiation dose group was replicated on three slides
to validate the results. The viability of the cells 24 h post culture was - 90 % (at isolation - 95%)
as determined by trypan blue exclusion.
X-ray Dose Response Comparison (OTM) of Primary Rat
Hepatocytes at 0 h and 24 h
25 -3-9
24
SOh
20 -
0 -
.5
-T1-
__T_
1
2
4
X-ray Radiation Dose (Gy)
Figure 12 X-ray dose response on primary rat hepatocytes comparing the Mean OTMs at 0 h and 24 h.
X-ray Radiation Dose
0 Gy
1 Gy
2 Gy
4 Gy
10 Gy
Mean Olive Tail Moment + S.D
24h: 1.3 + 1.5
Oh: 3.6 + 3.9
24h: 2.2 ± 2.5
Oh: 5.6 ± 5.0
24h: 4.0 + 2.9
Oh: 6.0 4.8
24h: 5.4 + 3.1
Oh: 7.8 + 5.8
24h: 13.6 ± 5.3
Oh: 12.6 ± 6.6
±
Table 4. Dose response values of hepatocytes at Oh, 24h.
Comparison of the mean OTM of hepatocytes processed 24h after incubation and the ones taken
through alkaline comet assay immediately suggests that the cells present greater DNA damage
immediately after isolation (except at 10 Gy). Statistical significance cannot be associated with
the difference in the mean OTM at 0 h and 24 h because of the highly variable damage level
incurred by each cell when exposed to radiation. Further, smaller standard deviation values are
seen for the cells assayed 24 h later. We hypothesize that smaller OTM values and tighter
standard deviations 24 h post isolation are due to the role of repair mechanisms. Culturing the
hepatocytes for 24 h after isolation allows the cells to recover from the physical or chemical
trauma that might be associated with DNA damage and/or enrich for healthier cells that can
attach and spread [83]. Further, the cells that are viable post 24 h culture might repair the damage
associated with the initial damage during isolation and present a smaller OTM. These cells have
the ability to repair the damage and survive through the comet assay protocol indicating true
DNA damage values. The highly damaged cells lose their ability to adhere to the surface and are
possibly washed out during the media washing step before trypsinizing the cells.
Chapter 4: Comet Assay on Adherent Hepatocytes
4.1 Introduction
Comet assay is one of the most sensitive methods to detect DNA damage [12, 82]. In the
past the comet assay has been used to detect genotoxic damage on cells that are
biologically adherent as well as non adherent in nature [41-44, 83]. Procedurally, cells of
either nature are embedded in agarose gel and are run through the single cell
electrophoresis method to determine the amount of DNA damage present in the cells. In
this chapter we propose a modification to the existing comet assay protocol. This new
protocol allows the cells to be in their native adherent state during irradiation as opposed to
the current comet assay protocol where adherent cells are irradiated on a culture dish and
then trypsized. By maintaining the cells in their natural adherent configuration, we exclude
several artifacts that are likely to influence the DNA damage responses. These include
anoikisis induced due to trypsin, mechanical damage, and the variable time from
detachment to analysis. These advantages are not restricted to hepatocytes and can
therefore be extended to all adherent cells for obtaining accurate results and for studying
repair processes in a highly reproducible manner.
4.2 Methods
The comet assay provides an attractive feature to do single cell analysis as opposed to most other
techniques that rely on the averaged signal from the population. It is therefore important that we
address issues pertaining to patterning single cells. Island size for cell capture and the cell
seeding concentration were the two primary factors that were optimized in order to obtain large
areas of single cell capture.
4.2.1 Optimization of the Collagen Island Size for Single Hepatocyte Capture
Regular glass slides (75 cm x 50 cm, pre-cleaned VWR) were cleaned using a sequential
acetone, iso-propanol and methanol wash and were spin dried at 1000 rpm for 1 min before use.
The cleaned slides were coated with positive photoresist S 1813 (Shipley Inc.) using a spin coater
47
and the speed was slowly ramped up to 2000 rpm for 1 min. The photoresist coated slides were
kept on the hot plate for 1 min to evaporate the solvent from the resist. The time for the soft bake
was optimized keeping in view that it had to be lifted off easily using acetone at a later stage. A
transparency containing the pattern to be transferred onto the photoresist coated slide was
designed in Corel Draw 11.0 and printed out at 5080 dpi (Fineline Imaging Inc.). The
transparency was aligned with the photoresist coated slide and exposed to UV radiation for 75
sec (HTG System 3HR 2-3, 300 nm, 100 s, 10 mW/cm 2). The slide was developed for 15-20 sec
(Microposit MF-321) dissolving the resins that were exposed to the UV radiation and was
thoroughly washed with water to remove any photoresist residue on the surface. Due to the need
for a clean glass surface, the patterned photoresist slides were exposed to oxygen plasma for 5
minutes (Technics 500 Asher, 200 mT, 200 W, 5 min).The plasma treatment time was enough to
ensure clean glass surface and not etch away the photoresist completely. The photopatterned
slide was then incubated in collagen for 2.5 hrs at 370 C which physico-chemically adsorbed to
the slide. The slides were then acetoned to "lift-off' the photoresist and leave behind collagen
islands. Collagen islands 15 um, 20 um and 25 um in size were fabricated reliably using the
above mentioned protocol. Collagen island patterned microscope slides were placed in T75
tissue culture dishes and seeded with hepatocytes (1x106 cells in 15 ml) for 1 hr. The dishes
were gently shaken every 10-15 minutes in order to homogenize the cell concentration over the
dish area. The cells were diluted in hepatocyte media without serum, in order to avoid any non
specific adhesion of hepatocytes due to adsorption of proteins in the serum
4.2.2 Optimization of Hepatocyte seeding concentration for Single Cell Capture
Single hepatocyte cell patterning on collagen islands imprinted using photolithography, briefly
described in the above section, and was optimized against the hepatocyte seeding concentration.
Collagen island patterned microscope slides were placed in T75 tissue culture dishes and seeded
with different hepatocyte concentrations (0.5x10 6, 1x10 6, 2x106, 3x10 6, 4x10 6 cells in 15 ml) for
1 hr. The dishes were gently shaken every 10-15 minutes in order to homogenize the cell
concentration over the dish area. The cells were diluted in hepatocyte media without serum, in
order to avoid any non specific adhesion of hepatocytes due to adsorption of proteins in the
serum.
4.2.3 Modified Protocol for Patterned Adherent Comet Assay on Primary Rat Hepatocytes
For adherent cells, like primary hepatocytes, the process of suspending in agarose and running a
comet assay on them poses several problems. Primary hepatocytes show excessive damage if
they are taken through the comet assay immediately after isolation from the source. This is
probably due to the physical and chemical trauma experienced by the cells during the process.
Culturing the primary adherent cells on a suitable surface for a certain length of time (optimized
for that particular cell) after isolation allows the cells to recover from the trauma suffered during
isolation. Trypsinization of the cells from the culture surface before suspension of the cells in
agarose is prolonged after irradiation with ionizing radiation thereby reducing cell viability and
allowing partial repair, with the consequence of reduced DNA damage radiosensitivity after
irradiation. In addition, it has previously been shown that there is additional damage in cells
trypsinized from the surface before suspending in Agarose. This could confound the DNA
damage radiosensitivity of the adherent cells and result in lower signal to noise ratio for studies
evaluating DNA damage at low levels [83].
Consequently, we describe a new protocol for cells adherent in nature. Isolated hepatocytes are
diluted to the required concentration (in medium without serum) and seeded on patterned
collagen slides fitted with Gel Bond Gasket (100 pg/ml) for 1 h. The hepatocytes that attach and
pattern on collagen islands attain a differentiated morphology. The Gel Bond gasket helps pin the
agarose gel on the slide. The adherent hepatocyte slides are then placed in hepatocyte media with
serum and left in the incubator at 370 C for 24 hrs based on the experiment described in section
3.4.3. Agarose gel (500 pl, 0.7% w/v) at 370C was gently poured over each slide. The gel was
gelled at 40C for 10 minutes and the slides were immersed in media with serum at 40C in order to
maintain the viability of the hepatocytes. The tissue culture dishes holding the slides were placed
in the X-ray machine and the cells were exposed to high energy X-rays till the dose requirement
was met. The part of the slide which was used as control (0 Gy) was covered with 1 cm thick
lead sheet in order to block the radiation as opposed to the portion of the slide exposed to
radiation. The lead block was placed 0.5 cm above the slide. The slides were then processed
under the alkaline comet assay protocol described in section 3.3.1.
Pattented Hepatocytes on a
Mlicioscope Slide
X-ray Sou rce
Lead Block
C(ell Cultine DiIh with
Sample
Adjustable Stan(d
Figure 13. X-ray setup for patterned, adherent hepatocytes. To incorporate multiple radiation doses
on the same slide, selective blocking with lead sheets is used.
4.3 Results and Discussion
This section is dedicated to extending the use of comet assay on adherent patterned hepatocytes.
We explore the dose response characteristics on patterned adherent hepatocytes and compare
them with the current standard where the cells are suspended in gel.
4.3.1 Dose Response using Comet Assay on Adherent Primary Rat Hepatocytes
This experiment was designed to attain a set of X-ray radiation doses that will cause DNA
damage in adherent hepatocytes.
The sample preparation involved 5 radiation experiment groups, namely, 0 Gy, 1 Gy, 2 Gy, 4 Gy
and 10 Gy for adherent hepatocyte categories. Each condition for the adherent was replicated on
3 slides in order to validate the results. The protocol for performing the experiment is described
in section 4.2.1.
Dose Response on Adherent
Hepatocytes
S Adhe re nt He patocyte
SII
~I
1
0
2
4
10
X-ray Radiation Dose ( Gy)
Figure 14. X-ray dose response on adherent hepatocytes.
X-ray Radiation Dose (Gy)
0 Gy
1 Gy
2 Gy
4 Gy
10 Gy
Mean Olive Tail Moment + Std. Dev.
3.0 + 1.5
5.1 + 3.2
7.2 + 3.2
8.4 ± 4.6
12.1 ± 3.3
Table 5. Dose response Mean OTM values for adherent hepatocytes.
Exposure to X-ray radiation showed an increase in DNA damage, measured by OTM, with the
increase in radiation dose indicating that adherent hepatocytes are responsive at the given doses.
However, the data was statistically insignificant when comparing OTM's at different X-ray
doses owing to the large standard deviation in the response of each individual cell to the
radiation dose. The DNA damage as quantified by Olive Tail Moment increase with the increase
in X-ray Dose and was found to be maximum at the 10 Gy dose (Mean Olive Tail Moment + Std.
Dev. : 12.1 + 3.3) .
4.3.2 Dose response comparison between adherent primary rat hepatocytes and suspended
primary rat hepatocytes using comet assay
The comet assay has been used to investigate the DNA damage in cells that are either nonadherent in nature or are trypsinized and suspended in an agarose gel after subculture for a
certain amount of time. Here we characterize how primary rat hepatocytes in their adherent state
respond to X-ray radiation with respect to DNA damage compared to suspended hepatocytes
assessed by alkali Single Cell Gel Electrophoresis.
The sample preparation protocol involved 5 radiation experiment groups, namely, 0 Gy, 1 Gy, 2
Gy, 4 Gy and 10 Gy for suspended as well as adherent hepatocyte categories. Each condition for
the adherent and suspended categories was replicated on 3 slides in order to validate the results.
The protocol for the adherent hepatocyte comet assay is as described in section 4.2.1. The
protocol for the suspended hepatocyte comet assay counterpart is described in section 3.3.6.The
processing (post-trypsinizing adherent cells) of the adherent and the suspended Hepatocyte
categories was done simultaneously in order to control for any differences in the damage levels.
The alkaline SCGE protocol post gelling is described in section 3.3.1.
The DNA damage induced in adherent primary rat hepatocytes was lower at low X-ray doses (0
Gy, 1Gy and 2 Gy) and higher at high X-ray doses (4 Gy and 10 Gy) with respect to its agarose
gel suspended counterparts (Figure 15-1 and Figure 15-2). Summary of the mean OTM for the
adherent and suspended categories is presented in Table 6.
Suspended vs. Adherent Hepatocyte Dose
a8
Response
17
16
15
a
Adherent
8
Suspended
Hepatocytes
Hepatocytes
14
*
E
15
S 12
11
9
--
S
r
-I
I
I
7 i
r!
I
> I1
I
3 J,
T
T
2
0
i
i
2
+
x+
o
0
-.1-1
1
2
4
X-rayRadiation Dose (Gy)
10
Figure 15-1. Dose response comparison of OTM of suspended and adherent hepatocytes.
52
Adherent Hepatocytes
Mean Olive Tail Moment ± Std.
Dev.
1.7 ± 0.8
3.5 ± 2.4
4.6 + 3.5
7.2 + 3.8
13.1 ± 3.4
X-ray radiation Dose
(Gy)
0 Gy
1 Gy
2 Gy
4 Gy
10 Gy
Suspended Hepatocytes
Mean Olive Tail Moment + Std.
Dev.
1.9 ± 3.2
3.6 + 4.8
5.9 + 4.4
6.1 ± 5.4
10.0 ± 5.6
Table 6. Mean OTM and standard deviation X-ray dose response values for suspended and adherent
hepatocytes.
Comparison of Standard Deviation of
Dose Response
5
rentHepatocyte
ard Deviation
c2
C-
nded Hepatocyte
ard Deviation
0
0
1
2
4
............................
10
X-ray Radiation Dose (Gy)
Figure 15-2. Compasion of standard deviations in mean OTMs between suspended and adherent
hepatocytes.
The OTM's for the adherent and the suspended categories showed an increasing trend with X-ray
dose. The differences in the damage induced in the two categories were statistically insignificant
indicating no obvious change in the DNA damage incurred by the adherent or suspended
hepatocytes. Further, for each dose it was observed that the standard deviations in the olive tail
moments for the adherent hepatocytes was smaller in comparison to the suspended hepatocytes
(Figure 15-2). We hypothesize that the smaller standard deviation in the olive tail moment for
adherent hepatocytes is due to the enrichment of a more healthy population of cells via
attachment. Contrary to the comet assay protocol for hepatocytes suspended in agarose gel, the
protocol for comet assay on adherent hepatocytes exclude dead cells that detach from the
surface. Further, hepatocytes suspended in gel are dispersed in different planes. Selection of
hepatocytes present in different planes in the gel get exposed to different electric field lines
leading to a differential in the extent of migration of. This might lead to a larger standard
deviation in OTM for the suspended hepatocytes.
In contrast, attached hepatocytes on the
surface of the microscope slide get exposed to the same electric field lines and exhibit a much
tighter standard deviation in the olive tail moment.
4.3.3 Rationale for using Patterned Comet Assay
Hepatocytes are adherent in nature and present a stereotypical polygonal morphology in culture
on attachment to the underlying protein e.g. collagen-I, II, III, IV etc. The polygonal morphology
of the hepatocytes affects the morphology of the underlying DNA nucleoid. This could lead to
unusual comet assay results as the Komet 5.5 software used for analysis requires a circular
symmetric distribution of the DNA nucleoid. In the suspended comet assay protocol the cells
embedded in the solidified Agarose gel occupy a volume equivalent to the cell volume. Upon
lysis, the cellular and well as the nuclear membrane dissolve, the proteins denature and there are
remnants of intracellular debris. Since the size of the DNA nucleoid is smaller than the cellular
volume, the DNA fills up the space originally occupied by the cell. In the setting where cells
assume a polygonal morphology upon attachment the results of the analysis cannot be conclusive
due to asymmetric shape of the DNA nucleoid morphology. This is illustrated in Figure 16.
In order to maintain a spherical shape of adherent Hepatocytes for valid analysis we make use of
the patterning strategy to confine the spreading of the cells on a circular island.
Figure 16. Cell morphology and the corresponding comet morphology.
4.3.4 Collagen Island Fabrication Characterization
It is important to characterize the size of the collagen islands in order to pattern single cells, a
necessity to carry out the comet assay. A large collagen island allows more than one Hepatocyte
cell (10-15 micrometer in diameter) to sediment onto the adhesive collagen island thereby
resulting in poor single cell patterning. Alternatively, a smaller than optimal size of the collagen
island for single cell patterning would result in a 'vacant' spot. A consistent protocol for
fabricating the collagen islands using photolithography was developed whereby the discrepancy
between the starting mask size (Mylar transparency, Fineline Imaging), the intermediate
photoresist mold size for the collagen and the end collagen islands size was insignificant
(p < 0.05). Measurements of the photoresist molds revealed that the photoresist was
overexposed due to an increase in size from the photomasks, and this was consistent across all
sizes. Measurements of the collagen islands revealed that there was no undercutting of the
photoresist when incubated with collagen solution. This maintained the collagen island size
similar to the size of the photoresist mold (Figure 17).
Theoretical vs. Experimental Collagen Island Size
35.00
30.00
i
Mask Hole Size {um)
MPhotoresist Hole Size fum)
I
a
E
1 Collagen antibody island size (um)
25.00
E
...................
..
. .... ,
[] Collagen antibody island size (urn)
.
C
*
20.00
e
*
4W
10.00
C
a
E
*
0.
10.00
S
M
a
VI
5.00
r I110I
---...
'25 micrometer'
'20 micrometer'
'15 micrometer'
island Size (Theoretical Value)
Figure 17. Quantitation of the collagen islands fabricated using photolithography and comparison to the
starting mask size and the intermediate photoresist mold. The difference in the sizes of the mask hole,
photoresist hole size and the collagen antibody island size was insignificant for all categories (p<0.0 5 ).
4.3.5 Optimization of Single Hepatocyte Capture on Photolithographically Microfabricated
Collagen Islands
The comet assay provides an attractive feature to do single cell analysis as opposed to most other
techniques that rely on the averaged signal from the population. It is therefore important that we
address issues pertaining to patterning single cells. Island size for cell capture and the cell
seeding concentration were the two primary factors that were optimized in order to obtain large
areas of single cell capture.
4.3.5.1 Optimization of Hepatocyte seeding concentration for Single Cell Capture
The protocol for carrying out the experiment is described in section 4.2.2.
No substantial change in the single cell patterning was seen with the increasing number of cells
seeded. However, there was an increase in the non specific binding of cells with the total number
of cells seeded. In this sense, the optimal cell seeding concentration was found to be 0.5x106
cells in 15 ml of media in a T75 tissue culture dish.
4.3.5.2 Hepatocyte Single Cell Capture Distribution
Large patterned areas (consistently 60%-70% of the total slide area) of single hepatocytes were
obtained on the microscope slides using the patterning protocol mentioned in the section above
(Figure 18, Figure 19). The protocol describing the fabrication of collagen optimized for single
cell capture is described in section 4.2.1. In order to quantify single cell capture distribution for a
particular island size we considered areas on the slide patterned in an array of 10 x10 cells or
more and visually count the number of collagen islands that captured 0,1,2,3,4 and 5 or more
cells under the microscope.
The optimum island size for single cell capture was found to be 20 pm, whereas the maximum
vacant spots in an array were most prominent in the 15 pm island size. Cells captured on the
order of 3 or more on an island were prominent on the 25 pm island size (Figure 18-3). A right
shift in the number of cells captured per island and the frequency of such islands was seen as the
size of the collagen islands increased from 15 pm to 25 pm. As can be seen from Figure 18-4,
the percentage single cell capture for 15 pm, 20 pm and 25 pm islands were 36 %,46% and 20%
respectively. All the differences were statistically significant (p<0.05).
1000
ýim
Figure 18-1. Large patterned areas of hepatocytes at 2X magnification.
Figure 18-2. Single Cell hepatocyte patterning at 10X magnification elucidating single cell patterning.
Cell Capture Distribution
120
1
80
I15umrnisland
,
100
1L
1 Ibin
0
20 um island
-25umisland
20
0
1
2
3
4
5
Number of cells captured per island
Figure 18-3. Hepatocyte cell capture as a function on island size. Only areas on the slide with an array of
S10x10 patterned cells were considered.
Percentage Single cell capture vs. Island Size
+
50o
T
15 um island
um island
W0
B5um island
30
20
10
15um
20 urn
25 um
IslandSize
Figure 18-4. Percentage single cell hepatpcyte capture as a function of island size. * indicates statistical
significance between 15 pm and 20 pm,t indicates statistical significance between 20 pm and 25 pm
and * indicates statistical significance between 15 pm and 25 pm islands respectively (p < 0.05).
4.3.4 Characterization of X-ray Radiation Setup for Patterned Comet Assay
We are interested in localized radiation exposure so that the cells are exposed to multiple
radiation doses on the same slide. We want to achieve this in order increase the throughput of the
assay and reduce slide to slide variability.
In order to achieve localized exposure we used a lead block to shield the radiation from the
desired regions on the slide. Here we characterize the amount of radiation shielded by the lead
block and the amount of radiation that infiltrated into the blocked region, at the edge of the lead
block.
The adherent patterned hepatocyte slide was placed in the X-ray machine and selective areas of
the slide were dosed or blocked using a lead block, allowing the cells to be exposed to multiple
doses. Complete blocking of X-ray radiation is a function of the lead block thickness (avoiding
penetration) and the distance from the slide (avoiding penumbra effect/leaking of the X-rays into
the blocked region). The choice of the thickness of the lead block and the distance at which it
should be placed from the slide to provide complete blocking of X-rays on a particular section of
the slide was characterized by using an X-ray plate. Based on the observations from Figure 19,
we can see that the region on the X-ray plate exposed to the radiation resulted in the desired (10
Gy) dose, whereas the region blocked by the lead block ( 1 cm thick, placed 0.5 cm from the Xray plate) showed a penumbra effect - 200 pm. The image on the X-ray plate was analyzed using
a simple imaging software (ImageJ, NIH).
10 Gy
Figure 19. Intensity profile of the region exposed to radiation (Left, 10 Gy) and the region shielded by a
Icm thick lead block placed 0.5 cm from the plate (Right).
4.3.5 Comet Assay on Patterned Hepatocytes
Only the patterned cells at a distance of 500 irn or more from the edge of the lead block were
considered due to the confounding penumbra effect - 200 =rn (section 4.3.4). Figure 20-1 shows
a snapshot (Magnification 2X) of the patterned comets on a microscope slide. A comparison of
the mean OTMs of the patterned and the unpatterned comets at 0 Gy and 10 Gy revealed that the
OTM and hence the DNA damage was greater for patterned hepatocytes at 0 Gy as opposed to
the unpatterned hepatocytes. Conversely, OTM for 10 Gy was higher for the unpatterned
category as opposed to the patterned (Figure 20-2).
10 Gv
0 Gv
/
K-
200 pm
Electrophoresis
Figure 20-1. Patterned heptocyte microscope slide with marked regions of comet analysis (0 Gy, 10 Gy).
The analysis region was chosen at a distance greater than 200 pm from the edge of the lead sheet. The
distance between each comet - 250 pm.
Dose Response Comparison of Patterned and Unpattered Adherent
18
Primary Rat Hepatocytes
TT
1
- 14
E 12
Unpatterned Adherent
He patocytes
-
• Patterned Adherent
HPepatocytes
10 ,
U6
4
2
0
10
X-ray Radiation Dose 1(0v
Figure 20-2. Dose response comparison between patterned ( 0 Gy: 1.7 ± 1.5 , 10 Gy: 12.8 + 4.0 ) and
unpatterned ( 0 Gy: 3.6 + 1.9, 10 Gy: 13.9 + 3.0) adherent primary rat hepatocytes.
We hypothesize that the discrepancy in the result is due to the effect of cell shape on DNA
damage. Bretland and coworkers have previously established the role of anoikis in cell death and
how integrin mediated mechanical transduction due to the cell spreading and its effect on
functions of the cell. Confining the cell shape as opposed to unrestrained spreading instigates the
apoptotic pathway due to loss of integrin [84]. Higher levels of OTM for patterned hepatocytes
on 20 pm circular islands might be due to the inclusion of cells damaged due to cellular
confinement. However, at 10 Gy, the anomalous effects of analysis on polygonal morphology
overshadow the DNA damage due to cell patterning. We therefore conclude that even though
patterning of adherent cells might cause DNA damage by confining the cell shape, cells on an
average are still responsive to the X-ray dose response. Further, it equips the assay with the
possibility of exposing cells to multiple doses, thereby reducing the complexity, variability and
cost associated with handling multiple slides.
Chapter 5: Conclusions
The goal of this thesis was to implement an assay that would measure DNA damage in
cells adherent in nature and in a high throughput fashion. Although we chose the X-ray
radiation as the model DNA damaging agent and hepatocytes as the model cell type for the
proof of concept towards the above stated goal, the principals can be extended to any
radiation type (ionizing/non-ionizing) and any adherent cell type.
Singh and coworkers hypothesize that maintaining the cells in their natural adherent
configuration; we exclude several artifacts that are likely to influence the DNA damage
responses. These include anoikisis induced due to trypsin, mechanical damage, and the
variable time from detachment to analysis. Comparing the DNA damaging effects on
hepatocytes when they were irradiated in situ (attached to the surface) as opposed to when
they were trypsinized from the surface and then exposed to radiation (current standard
protocol), showed a non-linear dose dependent increase. However, no statistical difference
in the amount of DNA damage induced between the two categories could be established.
The lack of statistical credence in the DNA damage response between the two categories
could be due to the different radio-sensitivity of each cell. On the other hand, we see a
smaller variation in the DNA damage response for the cells irradiated in situ. We
hypothesize that this is due to enrichment of a healthy population of cells during culture
time, which fosters enhanced DNA damage repair and exclusion of cells that undergo
anoikisis.
Further, we explored the concept of running an in situ irradiated comet assay on
micropatterned array of adherent cells. Micropatteming cells helped us in exposing cells on
the same slide to two radiation doses by making use of spatial encoding. Selective
shielding of radiation from certain regions of the slide allowed us to do a dose response on
the same slide elucidating the proof of concept of possibly running a high throughput
comet assay on adherent cells. The OTM analysis showed that the DNA damage incurred
by the hepatocytes through the in situ irradiated patterned protocol as opposed to the
current standard protocol showed no statistically significant difference indicating that both
the assays will lead to comparable DNA damage assessment.
Even though we successfully demonstrated the use of in situ irradiated patterned comet assay in
increasing the throughput of the comet assay for adherent cells, we came across some challenges
associated with our approach to run a comet assay on adherent cells attached to the surface.
Firstly, adherent hepatocytes present a stereotypical polygonal morphology in culture on
attachment to the underlying protein. The polygonal morphology of the hepatocytes affects the
morphology of the underlying DNA nucleoid and could therefore lead to non representative
analysis of the induced DNA damage by the software, which requires a spherical DNA nucleoid
for correct analysis. Secondly, unexplained surface interactions in the form of cellular DNA
streaking was occasionally observed after the cells were taken through the modified comet assay
for adherent cells. We identified gel micromovement after electrophoresis as one of the potential
causes which could be overcome by careful handling of the sample. These shortcomings must be
addressed in the future generations of this technology by exploring new and improved methods
to micropattern cells.
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