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.

iv

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.

v

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

vii

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

viii

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

x

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

xiv

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)

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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

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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)

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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).

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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

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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).

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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

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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.

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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

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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).

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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.

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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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