INVESTIGATION ON HELA CELLS BEHAVIOUR
INDUCED WITH PULSE ELECTRIC FIELD FOR
WOUND HEALING APPLICATION
MOHAMED AHMED MILAD ZALTUM
UNIVERSITI TUN HUSSEIN ONN MALAYSIA
INVESTIGATION ON HELA CELLS BEHAVIOUR INDUCED WITH PULSE
ELECTRIC FIELD FOR WOUND HEALING APPLICATION
MOHAMED AHMED MILAD ZALTUM
A thesis submitted in
fulfillment of the requirement for the award of the
Degree of Doctor of Philosophy in Electrical Engineering
Faculty of Electrical and Electronic Engineering
Universiti Tun Hussein Onn Malaysia
APRIL, 2017
iii
ACKNOWLEDGEMENT
I would like to take this opportunity to express my greatest gratitude to Almighty
Allah, for His help and support during the course of life and the moment of truth.
I would like to express my appreciation and sincere gratitude to my supervisor,
Associate Professor Dr. Muhammad Mahadi bin Abdul Jamil, for his continuous
support, encouragement and endless patience towards completing the course of this
PhD research. I am thankful for his aspiring guidance, invaluable constructive
criticism and friendly advice during the project work. I am sincerely grateful to him
for sharing his truthful and illuminating views on a number of issues related to the
project.
I would like to express my warm thanks to Associate Professor Ir. Dr. Babul
Salam B. Ksm Kader Ibrahimand for his support and guidance. I would also like to
thank Engr. Buhari Hassam Mamman, Engr. Abubaker Sadiq Abdulkadir, Engr. Hadi
Abdullah, Engr. Ijaz Khan, Engr. Yoosuf Nizam, Dr. Mohamad Nazib Bin Adon and
Late Mr. Rashid bin Mahzan (May his gentle soul rest in perfect peace) from Medical
Instrumentation Laboratory, Faculty of Electrical and Electronic Engineering, who
provided me with the required facilities for my PhD research project. Financial support
for material of research was provided by the Fundamental Research Grant Scheme
award (FRGS) phase 2/2014, Vot 1488, from Ministry of Education Malaysia.
I would like to express my appreciation to my beloved father, my beloved
mother, my beloved wife, my children “AHMED & HAWA”, my brothers and my
sisters for their tremendous patient, effort, support and encouragement during my PhD
research. Last but not the least I would sincerely like to thank all my friends who have
contributed directly or indirectly to the accomplishment of this PhD research.
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ABSTRACT
This study focuses on the investigation of pulsed electric field (PEF) exposure effect on
HeLa cells (cervical cancer cells) for in-vitro experiments. The study focused mainly on
real time experimental setup for cell morphological properties imaging. In the
experimental setup, a modified EC magnetic chamber with incubator system is used to
maintain the real time in-vitro environment for exposing the HeLa cells to high electric
field. A Nikon inverted microscope (Ti-series) with Metamorph® time lapse application
is utlized for image capturing and video recording. The first investigation is to look at the
proliferation rate of HeLa cells within 72 hours inside the modified chamber. This first
investigation is utmost important to see if there is any effect from the PEF exposure. From
this, it was found that the HeLa cell growth rate increased up to 50% faster when applied
Electroporation (EP) in comparison to the cell without EP treatment. The investigation
was continued to look at the best PEF parameter that assisted in the growth rate of HeLa
cells. These investegations motivates the need of finding the best parameters for PEF
exposure that are suitable for HeLa cell reversible condition. This study continues to look
at the relation between amplitude and duration of PEF effect on the HeLa cell growth rate.
The cells were subjected to single pulse, constant field strength of 1kV/cm and pulse
durations ranging from 30μs to 600μs. It was found that at 100μs pulse duration the HeLa
cell growth rate increased dramatically and achieving confluency faster in comparison to
the cells exposed with other pulse durations. After obtaining the best parameter for HeLa
cell (1kV/cm, 100μs, & single pulse) the potential application of the EP technique for
wound healing was explored. The result of the exposed cells to PEF revealed a five times
faster healing rate than control group. Additionally, we have used Microcontact printing
(MCP) technique for cell guidance and assembling. The results indicates that the cells
aligned and elongated more on fibronectin pattern substrate under PEF than without PEF.
Thus, PEF usage on biological cells would enable a novel method for assisting drug free
wound repair systems and many other potential biomedical engineering applications.
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ABSTRAK
Kajian ini memberi tumpuan kepada penyiasatan kesan pendedahan denyutan medan
elektrik (PEF) pada sel HeLa (kanser pangkal rahim) dalam ujian eksperimen secara in-
vitro. Kajian ini memberi tumpuan terutamanya kepada persediaan eksperimen pada masa
sebenar untuk pengimejan sifat morfologi sel. Dalam persediaan eksperimen, kami
menggunakan ruang magnet EC yang diubahsuai dengan sistem inkubator untuk
mengekalkan persekitaran in-vitro pada masa yang sebenar untuk mendedahkan sel HeLa
dengan medan elektrik yang tinggi. Mikroskop Nikon inverted (Ti-siri) dengan Aplikasi
time lapse Metamorph® digunakan untuk memantau, tangkapan imej dan rakaman video.
Siasatan pertama adalah untuk melihat kadar percambahan sel HeLa dalam masa 72 jam
di dalam ruang medan EC yang diubah suai. Kajian pertama ini adalah penting untuk
melihat jika terdapat sebarang kesan masa nyata daripada pendedahan PEF itu. Hasil
didapati bahawa kadar pertumbuhan sel HeLa meningkat sehingga 50% lebih cepat apabila
mengunakan elektroporasi (EP) berbanding dengan sel tanpa rawatan EP. Siasatan
diteruskan dengan melihat parameter optimum PEF dalam membantu kadar pertumbuhan
sel HeLa. Penemuan ini mendorong kami untuk melihat parameter optimum untuk
pendedahan PEF yang sesuai untuk sel HeLa keadaan berbalik. Kajian diteruskan dengan
melihat hubungan antara amplitud dan tempoh kesan PEF pada kadar pertumbuhan sel
HeLa. Sel tertakluk kepada denyut tunggal, pada kekuatan medan berterusan 1kV/cm, pada
jangka masa nadi antara 30μs hingga 600μs. Ia juga mendapati bahawa pada kadar tempoh
denyutan 100μs pertumbuhan sel HeLa meningkat secara mendadak dan mencapai kadar
pertumbuhan lebih cepat berbanding dengan sel yang terdedah dengan jangka masa nadi
yang lain. Selepas mendapat parameter optimum untuk sel HeLa (1kV/cm, 100μs, &
denyut tunggal) potensi aplikasi teknik EP untuk penyembuhan luka telah dikaji. Hasil
daripada pendedahan sel kepada PEF menunjukan lima kali lebih cepat dalam kadar
penyembuhan daripada kumpulan kawalan. Selain itu, kami telah menggunakan teknik
Microcontact Printing (µCP), untuk memberi panduan pada sel dan juga pengumpulan.
Keputusan menunjukkan bahawa sel sejajar dan memanjang mengikut corak fibronectin
denagn PEF berbanding tanpa PEF. Oleh itu, penggunaan PEF pada luka sel biologi akan
mewujudkan kaedah baru untuk membantu sistem pembaikan luka tanpa bantuan ubat dan
lain-lain yang berpotensi dalam aplikasi kejuruteraan bioperubatan.
vi
CONTENTS
TITTLE i
ACKNOWLEDGEMENT iii
ABSTRACT iv
ABSTRAK v
CONTENTS vi
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF SYMBOLS AND ABBREVIATIONS xv
LIST OF PUBLICATIONS, RESEARCH GRANT
AWARD xvi
CHAPTER 1 INTRODUCTION 1
1.1 Basic information of electroporation 1
1.2 Problem statement 3
1.3 Objective of research 4
1.4 Scope of research 4
1.5 Thesis outline 5
1.6 Thesis contribution 6
CHAPTER 2 LITERATURE REVIEW 7
2.1 Introduction 7
2.2 Introduction of living cells 7
2.2.1 Human cervical cancer cells (HeLa cells) 9
2.2.2 Membrane of living cells 10
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2.3 Electroporation (EP) 11
2.3.1 Overview of electroporation 12
2.3.1.1 Basic circuit for electroporation 16
2.3.1.2 Method for enhanced electroporation 17
2.3.1.3 Electroporation of suspension 18
2.3.2 Electroporation types 19
2.3.3 Electroporation parameters 19
2.4 Experimental studies 21
2.5 Electroporation applications 22
2.5.1 Electrochemotherapy (ECT) 22
2.5.2 Electrogenetransfection (EGT) 23
2.5.3 Electrofusion (EF) 23
2.5.4 Transdermal drug delivery (TDD) 24
2.5.5 Electroinsertion (EI) 25
2.5.6 Bacterial decontamination 25
2.6 Summary 25
CHAPTER 3 EXPERIMENTAL SETUP FOR ELECTROPORATION
SYSTEM AND LIVE IMAGING 26
3.1 Introduction 26
3.2 Electroporation system setup 26
3.3 Material and methods 29
3.3.1 Electroporation system equipment 29
3.3.1.1 Nikon inverted microscope (Ti-series) 29
3.3.1.2 Nikon eclipse TS100 inverted
microscope 31
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3.3.1.3 Pulse generator 32
3.3.1.4 Chamlide CMB chamber 32
3.3.1.5 Chamlide EC chamber 33
3.3.2 Live visualization 35
3.3.2.1 MetaMorph software 35
3.3.2.2 DinoCapture 2.0 software 36
3.3.3 Cell counting using a hemocytometer 38
3.3.4 Estimation of percentage confluency of cells 39
3.4 Summary 40
CHAPTER 4 CELL CULTURE PROCEDURE AND
INVESTIGATION OF ELECTROPORATION
EFFECT ON HELA CELLS GROWTH RATE 41
4.1 Introduction 41
4.2 Experimental method 42
4.2.1 HeLa cells reagents 42
4.2.2 HeLa cells splitting protocol 42
4.2.2.1 HeLa cells subculture protocol 43
4.2.2.2 HeLa cells subculture process 44
4.2.3 Visualization and monitoring setup for
inducement of pulse electric field 46
4.3 Result and discussion 47
4.4 Summary 52
CHAPTER 5 OPTIMIZATION OF ELECTROPORATION
PARAMETER FOR HELA CELLS GROWTH RATE 53
5.1 Introduction 53
5.2 Electroporation parameters 53
ix
5.3 Material and methods 56
5.3.1 Cell line and culture protocol 56
5.3.2 Imaging system 56
5.3.3 Optimization of electroporation parameter 57
5.3.3.1 Electric field strength 57
5.3.3.2 Electric field pulse number and duration 57
5.4 Result and discussion 58
5.4.1 Investigation of pulse electric field parameter
for growth rate of HeLa cells 58
5.4.2 Verification of the PEF parameters effect on
growth rate of HeLa cells 65
5.5 Summary 71
CHAPTER 6 INVESTIGATION OF PEF PARAMETER EFFECT
ON HELA CELLS WOUND CLOSURE PROPERTIES 72
6.1 Introduction 72
6.2 Material and methods 73
6.2.1 Preparation of HeLa cells 73
6.2.2 Plating cell on the coverslip 73
6.2.3 Cell imaging 74
6.3 Result and discussion 74
6.4 Summary 81
CHAPTER 7 HELA CELLS INTERACTIONS WITH
MICROPATTERNED SURFACE FOR CELL GUIDANCE 82
7.1 Introduction 82
7.2 Material and methods 84
7.2.1 Cell culture 84
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7.2.2 Fabrication of stamp 84
7.2.3 Micro contact printing (MCP) procedure 85
7.2.4 Cell plating 86
7.3 Result and discussion 87
7.3.1 Micro contact printing (MCP) technique 87
7.3.2 HeLa cells interactions with micro-patterned
surface 88
7.4 Summary 91
CHAPTER 8 CONCLUSION AND FUTURE WORK 92
8.1 Conclusion 92
8.2 Recommended further future work 93
REFERENCES 94
xi
LIST OF TABLES
2.1 Summary of the prokaryotic and eukaryotic cells type 9
4.1 Quantitative result of HeLa cell growth rate 51
5.1 HeLa cell growth rate over time 63
5.2 Quantitative result of HeLa cell growth rate 70
6.1 Average wound width over time for PEFexposed and
non- exposed HeLa cells 78
xii
LIST OF FIGURES
2.1 Prokaryotic cell 8
2.2 Eukaryotic cell 8
2.3 Cervical Cancer cells (HeLa Cells) 10
2.4 Cell membrane 11
2.5 Schematic diagram of electroporation process 12
2.6 Basic circuit diagram of the electroporation setup 17
2.7 The basic relationship between the parameters of major
important field Strength and pulse length 18
2.8 Pulse electric field parameters range for electroporation
applications 20
2.9 Cell electrotransfection process 23
2.10 Cell electrofusion process 24
3.1 Electroporation system for PEF exposure 27
3.2 PEF stimulator with cuvette 27
3.3 Integrated electroporation system 28
3.4 Nikon inverted microscope (Ti-series) 30
3.5 (a) Chamlide stage, (b) Magnetic chamber 30
3.6 Nikon eclipse TS100 phase contrast inverted microscope 31
3.7 Pulse generator ECM®830 32
3.8 Chamlide CMB for 25mm coverslip 33
3.9 EC magnetic chamber with 10mm gap platinum
electrodes 34
3.10 Chamlide TC chamber 34
3.11 (a) Automatic CO2 gas mixing; (b) Dynamic temperature
control 35
3.12 Metamorph® time lapse application 36
3.13 DinoLite – DinoCapture 2.0 camera 37
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3.14 DinoLite camera on microscope conected with computer 37
3.15 Images stored into a computer as digital images in JPEG
format 38
3.16 Haemocytometer 39
3.17 Grid lines to estimate the percentage of cells confluence 40
4.1 HeLa cells at 80 – 90% confluence 44
4.2 HeLa cells trypsinization image 44
4.3 Proliferation rate of HeLa cells 46
4.4 HeLa cells attached on coverslip surface 47
4.5 HeLa cells after 6 hours incubated 48
4.6 HeLa cells growth rate in percentage per time (hour) on
round glass coverslip (25 mm diameter) without
electroporation process 49
4.7 HeLa cells growth rate in percentage per time (hour) on
round glass coverslip (25 mm diameter) with
electroporation process 50
4.8 Analysis of HeLa cells growth rate 51
5.1 Range of electric field and pulse width for biological
applications 54
5.2 Formation of pores (a) Initial cell membrane condition,
(b) a cell exposed to pulse electric field. This results in
non-uniform molecular structure (c) Carving of cell
membrane (d) Short-term hydrophobic pore on cell (e)
Restructuring cell membrane 55
5.3 Cervical Cancer Cells (HeLa cells) 56
5.4 HeLa cell growth rate at different pulse duration
parameter over different time 62
5.5 Percentage of HeLa Cells growth rate 63
5.6 HeLa cells growth rate after 60 Hour EP 1kV/cm pulse
with 70μs and 200μs pulse duration time 64
5.7 HeLa cells growth rate after 72 Hour EP 1kV/cm pulse
with 30μs and 300μs pulse duration time 64
5.8 HeLa cells after 6 hours incubated 66
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5.9 HeLa cells growth rate in percentage per time (hour)
without electroporation process (control) 67
5.10 HeLa cells growth rate in percentage per time (hour)
with electroporation process (1kV/cm, 100μs & single
pulse) 68
5.11 HeLa cells growth rate in percentage per time (hour)
with electroporation process (2.7kV/cm, 30μs & single
pulse) 69
5.12 Analysis of HeLa cells growth rate 70
6.1 Wound closure response with PEF treatment 75
6.2 Wound closure rate response without PEF 77
6.3 Time evolution of wound gap distance for HeLa cells
line 79
6.4 Diagram showing wound healing process under different
PEF parameter 80
7.1 PDMS stamps with (a) 25µm, (b) 50µm and (c) 100µm
width 85
7.2 Illustration of the stamping process 86
7.3 Fibronectin patterned surface on glass coverslip 87
7.4 Photomicrographs of HeLa cells after 18hrs of seeding
on coverslip glass 88
7.5 Photomicrographs of HeLa cells after 18hrs of seeding
on Fibronectin patterned surface on glass coverslip 89
7.6 Photomicrographs of HeLa cells after 18hrs of seeding
50µm Fibronectin patterned surface on glass coverslip 90
xv
LIST OF SYMBOLS AND ABBREVIATIONS
ECM - Extra Cellular Matrix
EP - Electroporation
PDMS - Polydimethylsiloxane
PEF - Pulse Electric Field
HT29 - Colon Cell line
HeLa - Cervical Cancer Cell
J3T - Brain Tumors Cells
CHO-K1 - Chinese Hamster Ovary
ECT - Electrochemotherapy
EGT - Electrogenetransfection
EF - Electrofusion
EI - Electroinsertion
TDD - Transdermal Drug Delivery
MCP - Micro-Contact Printing
FBS - Fetal Bovine Serum
NEP - Non-Electroporattion
xvi
LIST OF PUBLICATIONS, RESEARCH GRANT AWARD
The followings are the list of publications associated with this thesis:
Journals:
1. Mohamed A. Milad Zaltum, Muhammad Mahadi Abdul Jamil and Morgan C. T.
Denyer. (2016). “Pulsed Electric Field Exposed HeLa cell Alignment on
Extracellular Matrix Protein Patterned Surface”. Asia Pacific Journal of Molecular
Biology and Biotechnology. (In review).
2. Mohamed A. Milad Zaltum, Muhammad Mahadi Abdul Jamil, Morgan C. T.
Denyer, Mansour Youseffi and Farideh Javid. (2016). “Study on Pulse Electric Field
Exposure Effect on HeLa cell for wound healing application”. Journal of Science
and Technology. (In review).
Book chapter:
3. Mohamed A. Milad Zaltum, Nur Adilah Abd Rahman, & Muhammad Mahadi
Abdul Jamil. (2016). “Pulse Electric Field Effect on the Growth of HeLa Cells”. In
Biomedical Engineering Applications: Cell Engineering (pp. 37-46). UTHM, Johor.
4. Mohamed A. Milad Zaltum, & M. Mahadi Abdul Jamil. (2016). “Pulse Duration
Effect on Growth Rate of HeLa Cells”. In Biomedical Engineering Applications:
Cell Engineering (pp. 47-55). UTHM, Johor.
xvii
Conference proceedings:
5. Mohamed A. Milad Zaltum, Adon, M.N., & M. M. A. Jamil. "Electroporation
effect on growth of HeLa cells." In Biomedical Engineering International
Conference (BMEiCON), 2013 6th: IEEE. 1-4.
6. Mohamed A. Milad Zaltum, M. N. Adon, Sallehuddin Hamdan, & M. Mahadi
Abdul Jamil. “Feasibility Study on MCF-7 Cell Membrane Swelling Properties
Induced by Microsecond Pulsed Electric Field” Proceedings of the 2nd International
Conference on Biomedical Engineering – (ICoBE 2015), 30th -31th march 2015,
Georgetown, Penang, Malaysia.
7. Mohamed A. Milad Zaltum, M. N. Adon, Sallehuddin Hamdan, M. Noh Dalimin
& M. Mahadi Abdul Jamil. “Investigation a Critical Selection of Pulse Duration
Effect on Growth Rate of HeLa cells” Proceedings of the International Conference
on BioSignal Analysis, Processing and System (ICBAPS 2015), 26th -28th May
2015, Kuala Lampur, Malaysia.
8. A. A. Sadiq, Mohamed A. Milad Zaltum, H. B. Mamman, M. Nazib Adon, N. B.
Othman, M. Noh Dalimin, & M. Mahadi Abdul Jamil. An Overview: Investigation
of Electroporation and Sonoporation Techniques. International Conference on
Biomedical Engineering (ICoBE 2015), School of Mechatronic Engineering,
Universiti Malaysia Perlis (UniMAP).
9. Mohamed A. Milad Zaltum & Muhammad Mahadi Abdul Jamil. Optimization of
Pulse Duration Parameter for HeLa cells Growh Rate. International Conference on
Biomedical Engineering (ICoBE 2017), School of Mechatronic Engineering,
Universiti Malaysia Perlis (UniMAP). (In review).
xviii
Research grant award:
Grant Type: Fundamental Research Grant Scheme award (FRGS) phase 2/2014,
Vot 1488, Title: Fundamental Study On Hela Cells Morphological Properties
Induced Via Microsecond Pulse. Funded by: Ministry of Education Malaysia, 16
November 2014.
CHAPTER 1
INTRODUCTION
The focus of this thesis will be on the use of electroporation system to investigate the
HeLa cell morphological properties. Recent developments in electroporation have
enabled a broad range of biological applications. For example, the use of
electroporation systems in biotechnology and medicine has led to new methods of
cancer treatment, gene therapy and drug delivery. Electroporation is an alternative
technique that enables the delivery of foreign materials into the cell by applying an
electric field applied across a cell opens pores in the cell membrane. The effects of
electric field on human body and cells that are recently discovered are discussed this
chapter.
1.1 Basic information of electroporation
Living cells exhibits various electrical properties that make them to react and create
electric field and current (Hondroulis et al., 2013). Therefore, controlling the electrical
characteristic of the cell can provide a significant direct healing choice for wound
repair application and cancer treatment (Hondroulis et al., 2013). A technique that uses
high voltage electrical pulse to tissue in-vivo or cells in-vitro is knows as
Electroporation. This technique was found to improve the cell’s uptake of molecules
by producing transient pores in the cell membrane (Titushkin et al., 2009).
The effect of electroporation is not only extended to opening pores in the cell
membrane, but it can also control the variations in the cytoskeletal restructuring. Thus,
it has a noticeable impact on the cell adhesion and migration (Titushkin et al.,
2009).Though, the physiology of cells can be altered with the help of electric field by
involving intracellular signalling pathology yet the entire process is not fully
2
understood and need further research. Mamman and Abdul Jamil (2015) have stated
that the electric field can significantly affect the attachment speed and spreading
characteristic of colon cell line (HT29) by exposing them to an electric field of
0.6kV/cm for 500µs duration (Mamman et al., 2015).
In-vitro methods for human cell culturing are adopted by many recent
researchers for applying electroporation. Melanoma cells (Daud et al., 2008), J3T
(brain tumours) cells (Andre & Mir, 2004), and HeLa (cervical cancer) cells (Nazib et
al., 2013) are the cell types used in electroporation by different researchers. Different
types of cells can be classified and used based on their structure, shape and content.
However, different types of cells exhibits different characteristics towards electric
field intensities and duration, hence the selection of cells is very important in the
process of electroporation.
In recent years, Yong Hung and Boris Rubinsky (2003) have designed a new
micro fabricated electroporation chip for single cell membrane permeabilization
(Yong et al., 2003). They incorporate a live biological cell in the electrical circuit of a
micro electroporation chip. Moreover, they investigated the fundamental biophysics
of membrane permeabilization on a single cell level. It can control introduction of
macromolecules into individual cells. The experimental results shows that the chip has
a good ability to manipulate and induce electroporation in specific cells.
Furthermore, Rivera et al., (2004) have designed a fluidic microchip to inject
therapeutic molecules in the whole targeted tissue (Rivera et al., 2004). The great
advantage of this device is that it is a stand-alone device. The device uses gold
electrodes and leads are passivized with silicon oxide. A stand-alone device is 500μm
square sections, so that it is small enough to be inserted deep into a target tissue. It can
apply high voltage electric impulse into therapeutic molecules, genes or drugs which
are injected into targeted tissue. Moreover, the device is designed to allow electro
transfection in-vivo because of its invasiveness.
The electroporation microsystem has been developed and combined with a
logic circuit for gene transfection. Min et al., (2005) have designed an electroporation
microchip device for gene transfection and system optimization (Min et al., 2005). The
device combines micro fabrication techniques, logic circuit and electrophoresis design
to create a multi-function gene transfection device. This device can be used in wide
areas of medical science research applications. In their study, they subjected 104 NIH
3T3 cells to an electroporation process with a 50μm electrode gap, 6 volts and two
3
pulses applied. They used a fluorescence microscope to observe the experimental
results. They have reported the efficiency of gene transfection with an electric field
becomes higher than without electric field. The delivery rate was increased to 35.89%
when putting GFP gene into NIH 3T3 cells. This device has been applied in cancer
research, protein transfection, and drug delivery.
Many studies have been conducted in order to explore the mechanism of
electroporation and its applications to cell fusion (Nazib et al., 2013), drug delivery
(Prausnitz et al., 2004), gene therapy (Heller & Heller, 2006). From literature it can
be observed that cells exhibits different kind of characteristics to pulsed electric field
with different intensity and duration such as alteration in morphology of the cells. This
leads to the suggestion of different applications of electroporation.
The implanted electrodes in cuvette are polished to achieve sterility. For real
time visualization experimental setup cuvette alone cannot be used. Therefore, a
customized setup is required to allow high resolution visualization of electroporation.
Once designed with high resolution microscope, it can provide real time information
of electric field introduction to cells. This customized setup also allows the researchers
to investigate its effects on cell behaviour and introduction of foreign materials in cells.
1.2 Problem statement
Although, many research studies focus on pulse electric field effect, the main
phenomenon of the effects of external pulse electric field on cells is still in process of
discovery.
Different applications of electroporation require different EP parameters for
different cells type. An intense external electric field could damage the cell membrane
and lead to cell lysis. Therefore, examining of EP parameter for different applications
is very significant and extended experimental knowledge with theoretical models is
needed.
Normal electroporation setup in biology uses cuvettes which do not support
real time observation. Therefore to observe the morphological characteristics of cells,
an experimental system is required which uses live imaging for observation.
Even though electric field can alter the cell physiology by interacting with the
cell signalling pathways, only little is known about the whole process. Similarly, there
4
is evidence that electric field can considerably influence adhesion and spreading
properties of many cells. Since cell adhesion and migration are strongly associated,
there is potential for investigating electric field in cell migration for drug free wound
healing treatment.
The alignments of cells play a key role in wound healing application. Many
researchers have investigated cell guidance and alignment via micro-contact printing
with different protein. However, the inducement processes of micro-patterned in
combination with electric field excitation towards cell guidance have not been
investigated.
1.3 Objective of research
The main aim of this research is to observe the effects of PEF on growth rate of HeLa
cells for wound healing application. To achieve this, the research objectives are
divided into four parts:
1- To develop an experimental system setup that introduces electric field for
electroporation of cells so that the process can be observed and improved in real
time.
2- To optimize the best electroporation parameters for growth rate of HeLa cells in
the experimental work.
3- To investigate the electroporation process on HeLa cells for applications in wound
closure based on optimized electroporation parameters.
4- To investigate the HeLa cells interactions with micro-patterned surface assisted
by pulse electric field excitation for cell guidance.
1.4 Scope of research
The following scope of research has been identified to achieve the objectives of this
research:
1. Achieving the process of HeLa cell subculture in laboratory using the required
equipment for experimentation process (Plating, Growing and Splitting of
cells).
5
2. Acquisition of custom made of 1mm gap electrode chamber (EC Chamlide
Magnetic Chamber).
3. Investigation of pulse duration exposure rate for electroporation process to get
the best parameter for growth rate of HeLa cells.
4. Time-lapse live imaging for Investigation of drug free wound closure of HeLa
in the presence of pulse electric field.
5. Printing of protein of various patterns on glass coverslip substrates and
subsequent plating of the cells on the patterned surface for cell alignment under
pulse electric field.
1.5 Thesis outline
The thesis is divided into eight chapters. Following are the brief content of each
chapter:
Chapter 1: The structure of this thesis reflects and flow of information identified in
overview of the research is focused in this chapter.
Chapter 2: An overview of the cell and electroporation application methods are
presented in this chapter. The cell structure, electroporation and the parameters that
affect their performance are also discussed.
Chapter 3: This chapter demonstrates HeLa cells culture protocol and the development
process of the experimental setup for electroporation of HeLa cells.
Chapter 4: This chapter investigates the pulse electric field exposure effect on growth
rate of HeLa cell reversible condition.
Chapter 5: The selection of best parameters of pulse electric field for growth rate of
HeLa cells are shown in this chapter.
Chapter 6: This chapter gives the analysis of the wound closure process assisted by
pulse electric field.
Chapter 7: In this chapter, the assembling and guidance of HeLa cells on micro
patterned surface coated fibronectin with and without pulse electric field are
investigated.
Chapter 8: The last chapter will conclude the overall findings and provide
recommendation for future work.
6
1.6 Thesis contribution
In this research, the influence of pulse electric field on the cellular behaviour such as
proliferation, migration and cell guidance of HeLa cell have been investigated. The
following points demonstrate the main contributions of this research:
1. In the experimental setup, a custom made EC magnetic chamber is used to ensure
that the electroporation process is performed under fully controlled enviroment. It
maintains the real time in-vitro experiments for exposing HeLa cells to high
electric field. It also allows the investigation of multicellular behaviour of HeLa
cells during PEF exposure for qualitative and quantitative analysis.
2. Pulse Electric Field parameter is investigated to examine and to get the best value
for PEF parameters (pulse strength 1kV/cm, pulse duration 100µs & single pulse)
towards faster growth rate of HeLa cells. This can be used in clinical applications.
3. This research applies pulse electric field along with micro-contact printing
technique on HeLa cells which facilitates cellular processes like migration, cell
guidance and cell alignment. These cellular processes are vital for wound healing
process, tissue regeneration and cancer treatment.
CHAPTER 2
LITERATURE REVIEW
2.1 Introduction
This chapter presents the background and literature review of the state of the art work
in the field of electroporation. Additionally, biological cells are discussed in this
chapter providing more detail of the type of cells used in this research.
2.2 Introduction of living cells
Cells are the structural and functional unit of all living organisms and are called the
“building blocks of life” (Mariana, 2007). They are divided into two types. Bacteria
cells are prokaryotic; all other cells are eukaryotic of which there are four kinds:
animal, plant, fungi and Protista. Prokaryotic and eukaryotic are compared in table 2.1.
Prokaryotic cells (shown in figure 2.1) are smaller than eukaryotic cells (shown in
figure 2.2). Prokaryotic cells have no nucleus and a typical cell size of less than 5μm.
Typical cell mass is around 1 nano gram. This research used HeLa cell which is
described in detail in section 2.2.1.
8
Figure 2.1: Prokaryotic cell (Source:
http://www.shmoop.com/biology-cells/prokaryotic-cells.html)
Figure 2.2: Eukaryotic cell (Source:
https://openoregonstate.pressbooks.pub/microbiology/chapter/introduction-to-cell-
structure)
9
Eukaryotic cells typically contain additional components. Lysosomes are used to break
down unwanted chemicals, toxins, and organelles. The cell membrane is a very
flexible and thin layer surrounding the cells. The cell wall is a tough and thick layer
outside the cell membrane. The cell wall is used to give a physical rigidity and allows
chemical and cellular material to pass through the cells. These cells are found in plants,
animals and fungi. The Undulipodium is a flexible tail whose function is to provide
motility.
Table 2.1: Summary of the prokaryotic and eukaryotic cells type (Mariana, 2007)
Prokaryotic cells
Eukaryotic cells
Size of cells < 5μm Size of cells > 10μm
Exist in unicellular form Exist in multicellular form
Do not contain nucleus or any
membrane-bound organelles
Have nucleus and other
membrane-bound organelles
DNA is round in shape which do not
contain proteins
DNA is linear and associated with
proteins to form chromatin
Do not contain cytoskeleton Always has a cytoskeleton
Use binary fission for cell division Use mitosis or meiosis
for cell division
Reproduction is always asexual Reproduction is either sexual or
asexual
2.2.1 Human cervical cancer cells (HeLa cells)
HeLa cells are derived from cervical cells which belong to eukaryotic cells type. They
are the main focus in exploring cancer related studies and were first known in 1951.
They were taken from a cancer patient, named Henrietta Lacks, who passed away from
cancer. She passed away due to cervical cancer in eight months; however samples of
her cells were preserved in various laboratories for further research. The first human
cells known to be continuously grown in culture are HeLa cells. HeLa cells have the
capability of growing and division continuously and indefinitely, as long as suitable
10
environment is provided. This made it easy to literally immortalize the cells of
Henrietta Lacks.
In early decades of cancer research, many researchers studied and explored the
characteristics of HeLa cells, because the cells were easily available due to its
continuous culture. A distinctive feature of HeLa cells and many other cultured cancer
cells is that they are very adaptable and they can survive in circumstances where other
cells would die. HeLa cells are so potent that they have the ability to occasionally
contaminate other cell lines as well. HeLa cells are shown in figure 2.3.
2.2.2 Membrane of living cells
The cell membrane or plasma membrane is composed of a lipid bilayer. The cell
membrane contains the cell cytoplasm and are found on all living cells. The cell
membrane is shown in figure 2.4. The function of the cell membrane is to be selectively
permeable to particular chemicals that can pass in and out of cells. It is also an
interlocking surface that binds cells together. The cell membrane is only about 10nm
thick. There are two part molecules called phospholipids which compose the layers.
Hydrophilic phosphate heads facing outwards, and their non-polar, hydrophobic fatty
acid tails facing each other in the middle of the bilayer. The lipids (fatty acids) are a
hydrophobic layer that acts as a barrier to all but the smallest molecules, effectively
isolating the two sides of the membrane.
Figure 2.3: Cervical cancer cells (HeLa cells) (scale
bar 50μm)
11
Figure 2.4: Cell membrane (Source: http://biofoundations.org)
2.3 Electroporation (EP)
Electroporation also called Electropermeabilization is the use of high pulse electric
field to modify the permeability of a cell membrane. This modification in permeability
is achieved by using an electric field pulse to induce microscopic `pores' in the cell
membrane. These pores are commonly called `electropores' that is why the process is
commonly referred to as electroporation.
Many biotechnological applications and research requires transport of
macromolecules such as genes, antibodies, and chemical drugs, into a host cell. For
any particular application, a given transfer process selection is based on its efficacy,
ease of use and side effects. A characteristic shared by most of the chemical and
biological techniques is that they are usually cell-type dependent and have relatively
poor efficiencies. Therefore, both versatile and efficient methods are being
investigated. Electroporation was first reported in 1982 (Neumann et al., 1982) and is
one of the methods reported to be effective for such delivery. Since its inception, this
method has been a valuable tool for in-vitro delivery of small and large molecules into
a large variety of cells. During this time, electroporation has been performed on living
plants, animals, and humans (in-vivo electroporation), with an increasing focus on
therapeutic uses (Dev et al., 2000; Smith et al., 2000; Muramatsu et al., 1998).
12
2.3.1. Overview of electroporation
Electroporation is a technique in which strong, rapid electric pulses are used to make
the membrane of the living cell permeable to chemical species, which otherwise cannot
cross the membrane (Weaver et al., 1996). Figure 2.5 demonstrates the process of
electroporation. It’s a malleable and nontoxic physical technique which normally
requires 1-4kV/cm electric pulse strength for 1µs till few ms in order to make the
transient permeation possible through the cell membrane (Isambert, 1998). Despite the
fact that the mechanism of electroporation is not fully explored, this technique has
been used in biotechnology to incorporate different molecules into many different
types of cells. Few examples of which are the incorporation of DNA and RNA
fragments, proteins, antibodies and drugs into bacteria, yeasts, plant and mammalian
cells (Chang et al., 1992; Neumann et al., 1989).
Figure 2.5: Schematic diagram of electroporation process (Valero, 2006).
The technique of the electric alteration of cell membrane conductivity is widely
known since 1940 (Cole, 1972). Sale and Hamilton (Sale et al., 1967, 1968) stated that
the subjection of cells to high electric field pulses leads to cell lysis. They described
that the cell membranes were impaired by the transmembrane potential induced by the
applied electric field.
The transmembrane potential Vm was estimated from the equation, often
referred to as the (steady-state) Schwan's equation (Schwan, 1957).
𝑉𝑚 = 3
2𝐸𝑎𝐶𝑜𝑠 𝜃 (2.1)
Where E is the applied external electric field, 𝑎 is the radius of the cell, and 𝜃
is the angle between the direction of the field and the normal to the cell surface. The
13
critical transmembrane potential built up for electroporation to occur was found to be
about ±1V. The phenomenon was called `electric breakdown' by Sale and Hamilton
(Sale & Hamilton 1968).
Introduction of high electric fields to cells for long time compared to the
membranes charging time is approximately one microsecond for the plasma membrane
of mammalian cells (Weaver, 2000). This yields in charge redistribution, and making
of a transmembrane voltage addition to the resting voltage. When the transmembrane
voltage increases a few hundred millivolts, structural reconstruction starts. This makes
the membrane to be instable. The shape of membrane then changes and makes aqueous
paths. These are nano-scalable pathways which can provide entrance to foreign
materials (Chang et al., 1992) and the membrane quickly attains some type of new
electrical conduction pathways. Mass transfer can now happen using these pores with
electrical control systems.
There are a number of early reports on the effects of electric fields on the living
cell membrane. In 1958, the nodes of Ranvier of nerves were reported to be involved
in some type of ‘breakdown’ (Stampfli, 1958). A decade later, in the late 1960s, Sale
and Hamilton reported the damaging effects of strong electric fields on
microorganisms and erythrocytes, suggesting non thermal membrane interactions
(Hamilton et al., 1967; Sale et al., 1968). It was first found in the early 1970s that an
induced electric field causes dielectric breakdown of the cell membrane and release
intracellular components (Crowley, 1973; Zimmermann et al., 1974).
In 1970s reconstruction of membrane was discovered which would seal itself
after creating pores (Kinosita et al., 1977; Gauger et al., 1979). By varying pulse
parameters it was discovered that the introduced electric field could either have no
effect on the cell membrane permeability, which causes reversible permeabilization of
the cell membrane. On the other hand it can cause irreversible cell membrane porosity
which damages the cells (Benz et al., 1979; Ho et al., 1996). Further evidence for
chemical transport through membranes was observed in experiments with red blood
cells (Zimmermann et al., 1976; Sukhorukov et al., 1998). Erythrocytes were also used
to show DNA transfer into a cell induced with dielectric opening of the cell membrane
pores (Auer et al., 1976). The cell transfection in-vitro through DNA electro-transfer
were first experimentally shown by Neumann et al. and Wong and Neumann in 1982
(Neumann et al., 1982; Wong et al., 1982). From the start of nineties application of
14
electric field is frequently used technique in biotechnology and genetic engineering
(Chang et al., 1992).
Crowley demonstrated membrane breakdown using electromechanical
instability theory (Crowley, 1973). Crowley proposed that the electrostatic
compressing force causes reduction in membrane thickness. The membrane area
increase because the bilayer volume cannot be compressed, which forms lipids of
increased wedge shaped. Thus, it can be concluded that phase equilibrium shift toward
non-lamellar phase’s leads to destabilization of bilayer which results in membrane
breakdown (Crowley, 1973; Rubinsky, 2004). However experimental evidence
showed that Crowley’s theory failed to differentiate between reversible membrane
breakdowns and irreversible membrane rupture. Bryant, Wolfe and Wilhelm et al.
introduced theories that stated that mechanical stress causes membrane breakdown.
Bryant and Wolfe stated that cell lysis do not occur due to electric field produced in
the membrane but it occurs due to the isotropic mechanical surface tension formed in
distorting the cell (Bryant et al., 1987). Wilhelm et al. demonstrated membrane
breakdown by relating models of pore formation with those of mechanical stress
(Wilhelm et al., 1993).
The knowledge of fusion cells using high-voltage electric pulses gained the
attraction of more scholars among cell biologists and biophysicists towards
electroporation (EP). Demange et al., (2011) introduced Giant cells viability by simply
electro-pulsing a suspension of cells (Demange et al., 2011). Later, it was found that
dielectrophoresis can be used to get interaction among cells (Manaresi et al., 2003).
Dielectrophoresis can be known as the movement of somewhat non conducting
particles or charged cells in non-uniform alternating electric fields (Manaresi et al.,
2003). The cells can aggregate into chain lie structures when many particles are present
in an alternating electric field. To achieve this particle size, particle density, electric
field magnitude and frequency need to be optimized.
Alien genes are transfected into eukaryotic cells by introducing electric fields
(Tsong & SU, 1999). This transfection process opens the pores of the plasma
membrane of cells to intake the proteins which include antibodies or genetic material.
From experimentation it was also seen that transfected genes can be obtained from the
host cells (Tsong & SU, 1999). Similarly, electroporation was recognized as an
efficient method for the introduction of foreign molecules into cells of any basis
(Lavitrano, 1989).
15
Different experiments were performed in 1998 on human skin fibroblast and it was
observed that most resourceful transient chloramphenicol acetyltransferase expression
was detected after transfection with plasmid (Brown, 1998). The point that these cells
show transfected exogenous DNA properties could conclude in many important
applications in the study of human genetic diseases and cancer. Others researches
observed that the electroporation of the skin could be used to enhance transdermal drug
delivery (Henry et al., 1998).
In 1996, it was observed that embedding of the protein in cell plasma
membrane was potentially available by introducing electric fields pulses on a
suspension of cells in the presence of a specific membrane protein (Zimmermann,
1996). This process is called as electroinsertion. In further research, electroporation of
excitable membranes was studied (Chang & Reese, 1990). The morphological
indication showed that electrically induced membrane breakdown of remote
cardiomyocytes cells has signs of the presence of protein (Weaver & Chizmadzhev,
1996).
The electroporation technique is explained in many studies by combining the
theories of pre-existing pores, defects or fluctuations in the cell membrane integrity.
Neumann and Boldt stated that electroporation technique is a transition phase from
hydrophobic pathways to hydrophilic pathways in the lipid layers (Neumann et al.,
1990). Similarly, Chernomordik reported that it is small hydrophobic pores or defects,
which later becomes large in size and hydrophilic when subjected to electric field
(Chernomordik, 1992). The models of denaturation shows that the membrane can be
permeated at the protein channels, where it experiences denaturation after subjected to
electrical modification or Joule heating (Tsong, 1992).
Weaver and Powell reported that electropores are dynamic and transient
structures in the lipid bilayer membrane (Weaver et al., 1989). In Weaver and Powell’s
model either small pores already exist in the membrane or created by electric field in
the membrane. The electrical pulse generates electric potential which expands these
tiny pores. In order to investigate the pore population rate in a membrane and the
number of electropores, Weaver and Barnett established pore population models
(Weaver et al., 1992). If the energy is expended for edge formation and is taken by
increasing pore areas the electropores will remain stable. According to Weaver’s
model, these stable pores acts as passageways for external substances to pass into the
16
cells. Conversely, the existence of these pores has never been reported in the
membrane of mammalian cells.
Various methods for electroporation have been established in different areas
including genetics, immunology, microbiology, biochemistry, medicine,
pharmacology, and toxicology. While in case of in-vivo introduction of pule electric
field to allow molecular entrance is gaining attention for multiple applications
(Somiari, 2000).
Electrochemotherapy is an application of electroporation targets cancer cells.
Short duration high electric field is introduced just enough to target tumour cells. Once
the membrane is open anticancer materials can be introduced in the cells to treat
cancer. For now, these methods are used in in-vivo environments (Somiari, 2000).
Keeping importance of this application, this study focuses on the difficulties faced in
the experiments. This research also presents better understanding of the process by
presenting optimization of parameters for electroporation process.
2.3.1.1 Basic circuit for electroporation
Figure 2.6 presents the basic circuit setup of the electroporation apparatus. The typical
process of electroporation circuit is controlled by switch 1 and switch 2. The capacitor
is charged by the closing the switch 1 and the capacitor stores a high voltage of around
10-100kV/cm. Once the second switch is closed, this voltage discharges through the
liquid of the cell suspension. The pulse DC is thought both to disrupt temporarily the
cell membrane and allow DNA into cells.
17
2.3.1.2 Method for enhanced electroporation
A method for enhancing electroporation involves the application of a short electric
pulse that transiently disrupts cellular membranes (Chang et al., 1992). The electric
field is typically applied as one or more short (μs to ms) pulse with a rectangular or
exponential decay waveform. The increase of cell permeability is thought to be due to
the formation of short lived aqueous pathways “pores” in the plasma membrane. It
depends upon the field strength, length, and number of pulses (Weaver, 2000). The
most important parameters for effective electroporation are electric field strength and
the pulse length (Dower et al., 1988).
Figure 2.6: Basic circuit diagram of the electroporation
setup
Voltage
12V
Resistor
68kΩ
Capacitor
1.1µF
S1
Key = A Swtch 1
S2
Key = A
Switch 1
Switch 2
Cuvette of cells exposed to PEF
18
Figure 2.7: The basic relationship between the parameters of major important field
Strength and pulse length (Dower et al., 1988).
In order to electroporate the cell membrane a transmembrane potential difference of
between 0.7V- 1V needs to achieved. The required field strength E is inversely
proportional to the cell diameter.
High field strength and generator pulse length enhance the permeability of the
cell membrane. Applying too high a field strength can result in the cell being unable
to repair itself. On the other hand, if the field strength is too low, the breakdown of the
cell membrane may not be achieved. If the pulse length is too short, the pore of the
cell membrane will not be opened.
2.3.1.3 Electroporation of suspension
Most researchers have demonstrated that electroporation can be successfully applied
to different types of mammalian cells (Chakrabarti et al., 1989; Gilbert et al., 1987;
Jordan et al., 2004), yeast (Wall et al., 2004), bacteria (Lee et al., 2002) and red blood
cells (Mouneimne et al., 1990). However, the electroporation is not meaningful for
cells e.g. epithelial cells, because it poorly mimics in-vivo cell function and geometry
found in tissues (Gharter, 2004).
19
2.3.2. Electroporation types
A process in which high electric field is applied to cells to open the membrane of cells
of a small time is known as reversible. Reversible electroporation allows introduction
of foreign molecules or genes into cells and change cells properties which can not the
achieved in natural conditions. In biomedical and medical sciences, alien molecules
and other agents to cells are extensively used specially in case of reversible
electroporation. Cells fusion is also one of the applications of reversible
electroporation (Nazib et al., 2013). This method has been used to induce external
substance, for example the introduction of exogenous DNA into cells. For the
eradication of cancer cells this method can also be used and referred as
electrochemotheraphy (Jaroszeski et al., 1997). Irreversible electroporation on the
other hand is mostly used to kill cells. In this process and electric field is introduced
which is higher than what cell’s structure can bear. This leads to the death of cells.
Microbial deactivation is a process in which this technique was used to ill bacterial
cells (Schenk & Laddaga, 1992).
2.3.3. Electroporation parameters
Two of the main parameters for electroporation process are applied electric field
strength and pulse duration of applied electric field. Other type of parameters also
plays their role in efficient electroporation. Other environmental and chemical
conditions can also increase or decrease the intake foreign material. Figure 2.8 shows
the correlation of pulse duration and electric field strength in electroporation.
20
Figure 2.8: Pulse electric field parameters for electroporation applications (Puc et
al., 2004)
Figure 2.8 shows that, the poration do not occur if the strength of electric field and
range of pulse duration is low. When the intensity of electric field or the exposure of
pulse duration increases, it reaches to a certain range where much clear effects are
expected, even though if the variation of temperature is acceptable. If the pulse electric
filed strength and pulse duration increases to a certain value, the phenomenon of cell
lysis can occur, this means that the cells under exposure could be killed.
The preferred range of operation for medical applications is long time duration
pulses and low-electric voltage as shown in the right side of Figure 2.8. The Figure
shows that gene transfection occurs in the range of pulse durations and electric field
amplitudes. Drug delivery needs shorter pulses and higher electric fields. One
microsecond range of pulse duration is required for bacterial decontamination
subjected to electric fields from 10kV/cm to 100kV/cm. A completely different level
of applications can be reported when the values of graph travels to short pulse duration
with very high-electric voltage. With increasing pulse duration the membrane charging
time, the subcellular effects contribute to the intracellular electromanipulation instead
of plasma membrane electroporation.
21
2.4 Experimental studies
Considering theoretical techniques, different experimental setups can be used to carry
out studies for these theories. Electroporation parameters play vital role which can be
observed with experimental setup for best results.
In experimental work, it is shown that higher magnitude of external force
which is electric field increases the permeability of cells five to six times when
compared to moderate electric field application (Esser et al., 2007; Jiang et al., 2015).
The electroporation effect causes pores formation on the membrane by making
liquefied passages from which external substance can travel inside the cells. However,
if electric field strength increases the bearing properties of membrane it causes
irreversible electroporation causing ruptures which eventually kills the cells (Esser et
al., 2007; Jiang et al., 2015). Moreover, the pores reseal and conductivity of the
membrane increases (Jiang et al., 2015).
Considering potential applications of these techniques, extensive experimental
work has been conducted to understand electroporation in detail. Some researchers
conducted the study of electric current through membrane while electroporation and
its relation to porosity while others focused on time duration of voltage applied (Chung
et al., 1998; Sinton & Cuevas, 1996). Fluorescent molecules have also been focused
by many researches.
Since the size of the pores is extremely small usually in nanometres and also
their formation and growth rate is high. Therefore, it is difficult to observe the process
of electroporation directly. Even though many researchers succeeded in providing
substantial amount of progress, still there are various basic aspects of electroporation
that needs further exploration and understanding (Rolong et al., 2014).
So far researchers have provided many techniques in order to find out the
effects of electroporation on biological cells. This research focuses to explain
experimentation technique. The development of this experimental setup will discuss
and explain the best applications of electroporation.
The study of cells and biological materials outside their naturally occurring
environment is called In-vitro. This is the most commonly used techniques in
electroporation for analysis of cell and biological material behaviour (Hatanaka &
Murakami 2002; El-Ali et al., 2006). To make cells survive outside their naturally
22
occurring environment, an artificial environment is created to make the cells survive
and act naturally.
The artificial environment includes the control of temperature, CO2 level and
humidity. It makes the setup flexible to study the effect of not only externally applied
electric field but also artificially maintained environment for electroporation (Davis &
Warren, 1994). The parameters are important factor for quantitative researches. On the
other hand, it is vital to understand that these experiments are in-vivo which has
artificial environments outside the actual biological body. These techniques cannot be
applied on complicated body systems. Hence, it is crucial to understand the outcome
from these experiments and cannot be directly applied on complex biological systems.
2.5 Electroporation applications
Electroporation can be beneficial in many ways in molecular biology, biochemistry,
medicine and other biological research.
2.5.1 Electrochemotherapy (ECT)
In cancer chemotherapy, some drugs do not exhibit anti-tumour effects because of
insufficient transport through the cell membrane (Miklavcic et al., 2004). A combined
use of chemotherapeutic drugs and application of electric pulses is known as
Electrochemotherapy and is useful for local tumour control. Especially, Bleomycin
has been reported to have shown a 700-fold increased cytotoxicity when used in ECT
(Cemazar et al., 2010; Sersa et al., 2008). This helps to achieve a substantial anti-
tumour effect with a small amount of drug that limits its side effects. Bleomycin and
cisplatin have proven to be much more effective in electrochemotheraphy than in
standard chemotherapy when applied to tumour cell lines in-vitro, as well as in-vivo
on tumours in mice (Mir et al., 1991, 1995). Clinical trials have been carried out with
encouraging results (Gothelf et al., 2003; Tozon et al., 2005; Snoj et al., 2005).
23
2.5.2 Electrogenetransfection (EGT)
Electroporation transfer DNA into cells to alter some form of gene therapy, usually
known as Electrogenetransfection. It is currently being used in various pre-clinical
trials (Mir, 2001). As a non-viral technique, Electrogenetransfection has large potential
to transfer genetic material into cells. This process focuses on treating genetic diseases
(Budak et al., 2005; Bertino, 2008).
Figure 2.9 shows the process of DNA transfection using electroporation. In the
first part DNA is introduced which binds with the border of cell as shown in Figure
2.9 (a). Once DNA is introduced, electroporation is performed which opens cell pores
and DNA entrance into the cell as shows in Figure 2.9 (b). Figure 2.9 (c) shows DNA
inside the cell after electroporation.
Figure 2.9: Cell electrotransfection process
2.5.3 Electrofusion (EF)
Application of electric pulses can lead to fusion of membrane in close-contact adjacent
cells. Electrofusion can be used to create genetic hybrids and in order to encapsulate
both original cells intracellular material within a single enclosed membrane
(Zimmermann, 1982). The fusion of tumour cells with an antibody secreting
stimulated B-lymphocytes forms a hybrid cell known as Hybridoma. Later on, this
hybridoma has the ability to continue their growth in a culture environment, and an
enormous amount of desired antibodies can be obtained. Electrofusion is a beneficial
24
technique for the generation of vaccines (Scott et al., 2000; Orentas et al., 2001),
antibodies (Schmidt et al., 2001), and reconstruction of embryos in mammalian
cloning (Gaynor et al., 2005).
Fusion of cells by electrofusion is multiple stage process which involves fusion
of lipid and mixing of cytoplasmic content. Figure 2.10 shows the process of
electrofusion of two cells. When two cells are introduced to electroporation the cells
start to porate. In first stage, since the cell membrane are porous they tend to join
making the fusion of outer layer but the cytoplasmic content is separate. At this stage,
the internal structure of cells is independent and only the outer membrane is merged
to make a common enveloping lipid. In second stage the fusion occurs in both
monolayers of lipid bilayer. This takes the fusion process of the two cells further in
their structures. Once both fusion of monolayers occurs the cytoskeletal networks of
two cells will gradually merge. Once all the content is merged the two cells with appear
to be one cell will complete and shared cytoplasm.
2.5.4 Transdermal drug delivery (TDD)
The transdermal drug delivery is potentially valuable because the technology can be
site-specific, good control of the dose and located outside the body. In order to deliver
drugs through the skin by means of electrical pulses, the stratum corneum with the
thickness of 20μm must be electroporate. Because its thickness corresponds to
approximately 200 times of the lipid bilayer membranes thickness, the required
potential difference across the stratum corneum is in the order of 200V. Introduction
Figure 2.10: Cell electrofusion process
94
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