TITLE

DEVELOPMENT OF A FLICKING SYSTEM FOR PRODUCING 3-DIMENSIONAL

CELLS IN MICROBEADS

WONG SOON CHUAN

A thesis submitted in

fulfilment of the requirement for the award of the

Degree of Master in Electrical Engineering

Faculty of Electrical and Electronic Engineering

Universiti Tun Hussein Onn Malaysia

OCTOBER 2016

iii

DEDICATION

For my beloved family

iv

ACKNOWLEDGEMENT

Thanks to God for all the blessings given to me, both in wisdom and health that I am

able to finish this project successfully. Special thanks to my supervisor, Assoc. Prof. Dr.

Soon Chin Fhong for her supports, patience and guidance given throughout the duration

for this research. We acknowledge the help from Prof. Dr. Cheong Sok Ching from

Cancer Research Malaysia for donating oral squamous cell carcinoma (ORL-48) cells to

this research. Continuous research discussion and supports from the colleagues from

Biosensor and Bioelectronics Laboratory, MiNT-SRC and FKMP, UTHM is another

important factor that helps in the completion of this project. Appreciation is also

extended to the lecturers or panels from FKEE who has provided constructive criticism

in improving the research.

Not forgetting all my friends whom always being there for me when I needed

them most. Appreciation also goes to everyone involved directly and indirectly towards

the compilation of this thesis. Thanks also to my family members for giving me the all

the support that I need in completing this project and thesis especially in spiritual and

financial support.

v

LIST OF ASSOCIATED PUBLICATION

Journal

1. Soon Chuan Wong, Chin Fhong Soon, Wai Yean Leong, Kian Sek Tee,

“Flicking technique for microencapsulation of cells in calcium alginate leading

to the microtissue formation”, Journal of Microencapsulation, Volume 33, Issue

2, Pages: 162-171, 11 February 2016. http://dx.doi.org/10.3109/02652048.2016.

1142017. Impact factor: 1.631 (Q2, JCR, ISI Indexed).

2. Soon Chuan Wong, Chin Fhong Soon, Wai Yean Leong, Kian Sek Tee,

“Development of a flicking system for producing calcium alginate microbeads”,

Jurnal Teknologi, Volume 78, Issue 6-2, Pages: 103-109, June 2016.

http://dx.doi.org/10.11113/jt.v78.8909 (Scopus Indexed).

3. Wai Yean Leong, Chin Fhong Soon, Soon Chuan Wong, Kian Sek Tee,

“Development of an electronic aerosol system for generating microcapsules”,

Jurnal Teknologi, Volume 78, Issue 5-7, Pages: 79-85, May 2016.

http://dx.doi.org/10.11113/jt.v78.8718 (Scopus Indexed).

vi

Conference proceeding

1. Chin Fhong Soon, Soon Chuan Wong, Wai Yean Leong, Mohd Khairul Ahmad,

Kian Sek Tee, “A flicking method for generation of polymer microbeads”,

Japanese Journal of Applied Physics (JJAP) Conference Proceedings, Volume 4,

011110 (2016) doi:10.7567/JJAPCP.4.011110, 14th

International Conference on

Global Research and Education, Inter-Academia 2015, September 28-30, 2015,

Hamamatsu, Japan.

vii

LIST OF AWARD

1. Gold Medal in Research & Innovation Festival 2015 (UTHM),

“A novel flicking system for producing high throughput 3D cells”,

16 – 17th

November 2015, Universiti Tun Hussein Onn Malaysia.

2. Bronze Medal in Malaysia Technology Expo (MTE) 2016,

“A novel flicking system for producing high throughput 3D cells”,

18 – 20th

February 2016, Putra World Trade Centre, Kuala Lumpur, Malaysia.

PATENT FILING

1. In the process of patent filing. (Patent pending)

“Novel flicking system for producing high throughput 3D cells”

viii

ABSTRACT

Culturing cells on planar compliant substrates produces low yield of microtissues. A

variety of microtechnologies was introduced and developed to encapsulate cells for

producing microtissues. However, the size control of the microcapsules is challenging to

the current technologies for cells encapsulation. In this research, we proposed a simple

yet efficient technique to encapsulate cells leading to the growth of microtissues. The

technique involved with the development of a flicking system which can be applied to

encapsulate cells in calcium alginate microbeads at different flow rates and flicking

speed. The microbeads size increased with the flow rate but decreased with the flicking

speed. Flicking speed of 80 rpm and flow rate of 4 µl/min was chosen to use in the cells

encapsulation. The cells encapsulated in the microbeads at cell density of 31.9 × 106

cells/ml (HaCaT) and 94.2 × 106 cells/ml (ORL-48) were cultured and observed for

proliferation over a period of time. The microbeads had a smooth surface with spongy

and porous surface texture in FE-SEM imaging. In nucleus (DAPI) staining, the

encapsulated HaCaT and ORL-48 cells in microbeads grew from scatter individual cells

to form cells aggregate and then microtissues. In live and dead cells stainings, majority

(≈99 %) of the microtissues cultured in the calcium alginate microbeads were stained in

green which indicated the cells were alive. Alginate lyase was used to dissolve the

alginate shells in which, the intact microtissues was released. The microtissues obtained

were characterised by a rough and uneven surface could be due to the extracellular

matrix proteins adjoining the cells. The cells of microtissues were viable and able to

proliferate and spread into 2D monolayer in replating experiment. The flicking system

has produced microbeads with controllable size and allows the growth of microtissues.

HaCaT and ORL-48 cells encapsulated in calcium alginate microbeads can integrate into

microtissues after two weeks of culture.

ix

ABSTRAK

Pengkulturan sel pada substrat yang satah menghasilkan mikrotisu yang rendah.

Pelbagai mikroteknologi diperkenalkan dan dibangunkan untuk merangkumi sel untuk

menghasil mikrotisu. Namun, pada masa ini teknologi kawalan saiz mikrokapsul untuk

sel enkapsulasi adalah mencabar. Dalam kajian ini, kami mencadangkan satu teknik

yang mudah lagi berkesan untuk merangkumi sel untuk pertumbuhan mikrotisu yang

mempunyai potensi dalam kegunaan penilaian ubat. Teknik yang terlibat dengan

pembangunan sistem jentik boleh digunakan untuk merangkumi sel dalam mikromanik

kalsium alginat pada kadar aliran dan kelajuan jentik yang berbeza. Saiz mikromanik

meningkat dengan kadar aliran tetapi menurun dengan kelajuan jentik. 80 rpm kelajuan

jentik dan 4 µl/min kadar aliran telah digunakan dalam pengkapsulan sel. Sel yang

terkandung dalam mikromanik pada kepadatan sel 31.9 × 106

sel/ml (HaCaT) dan 94.2 ×

106

sel/ml (ORL-48) dikulturkan dan diperhatikan untuk percambahan dalam satu

tempoh masa. Mikromanik itu mempunyai permukaan yang licin dengan tekstur yang

lembut dan berliang dalam pengimejan FE-SEM. Dalam pewarnaan nukleus (DAPI),

HaCaT dan ORL-48 sel yang terkandung dalam mikromanik membesar daripada

berselerak sel individu jadi sel agregat dan kemudian membentuk mikrotisu. Dalam

pewarnaan sel hidup dan mati, majoriti (≈99 %) mikrotisu diternak dalam mikromanik

diwarnakan hijau yang menunjukkan sel itu masih hidup. Alginat lyase digunakan untuk

melarutkan shell alginat untuk membebaskan mikrotisu. Mikrotisu itu mempunyai sifat

permukaan yang kasar dan tidak rata mungkin disebabkan protein matriks ekstraselular

bersebelahan sel. Sel mikrotisu itu masih hidup dan mampu berkembang dan merebak

jadi ekalapis 2D dalam eksperimen pemplatan semula. Sistem jentik telah menghasilkan

mikromanik dalam saiz dikawal untuk pertumbuhan mikrotisu. Sel HaCaT dan ORL-48

boleh membentuk mikrotisu selepas dua minggu diternak dalam mikromanik alginat.

x

CONTENTS

TITLE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

LIST OF ASSOCIATED PUBLICATION v

LIST OF AWARD vii

ABSTRACT viii

CONTENTS x

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF SYMBOLS AND ABBREVIATIONS xxii

LIST OF APPENDICES xxv

CHAPTER 1 INTRODUCTION 1

1.1 Overview 1

1.2 Research background 1

1.3 Problem statement 4

1.4 Objectives of the research 5

1.5 Scope of research 5

1.6 Thesis outline 5

CHAPTER 2 LITERATURE REVIEW 7

2.1 Introduction 7

2.2 Cell and tissue 7

2.3 Human keratinocytes 10

2.4 Oral squamous cell carcinoma cell line (ORL-48) 12

xi

2.5 Extracellular matrix (ECM) and cell adhesion 13

2.6 Phases of cell growth 15

2.7 Rationale to generate 3D cells 16

2.8 Previous methods for growing 3D cells 17

2.9 Microencapsulation 19

2.10 Different methods of microencapsulation 22

2.10.1 Simple dripping 22

2.10.2 Microfluidic 22

2.10.3 Electrostatic 23

2.10.4 Vibration technique 24

2.10.5 Jet cutter 25

2.10.6 Rotating disc and rotating nozzle atomiser 27

2.10.7 The summary of different microencapsulation 29

methods

2.11 Biopolymers used for microencapsulation of cells 30

2.12 Characterisation methods 32

2.12.1 Phase contrast microscopy 32

2.12.2 Field emission scanning electron microscope 33

(FE-SEM)

2.12.3 Fourier transform infrared spectroscopy (FTIR) 35

2.12.4 4′,6-Diamidino-2-phenylindole dihydrochloride 36

(DAPI) staining

2.12.5 Live/dead viability assay 37

CHAPTER 3 METHODOLOGY 38

3.1 Introduction 38

3.2 Overview of the research methodology 38

3.3 Overall architecture of the flicking system 42

3.3.1 Design of the flicking device 46

3.3.1.1 Circuit design of the flicking device 48

3.3.1.2 Program flow for the flicking device 51

3.3.2 Design the infusion pump of the flicking 57

system

xii

3.3.2.1 Circuit design of the infusion pump 59

3.3.2.2 Program flow for the infusion pump 63

3.4 Performance assessment of the flicking system 67

3.4.1 Verify the PWM signals generated by the 67

circuit of flicking device

3.4.2 Investigate the relationship of the 67

potentiometer voltage to the PWM signals

3.4.3 Investigate the effect of PWM signals to 68

motor speed

3.4.4 Flow rate calibration of the commercial syringe 68

pump and the customised infusion pump

3.5 The setup of the flicking experiment 69

3.6 Investigating the effects of flow rate and flicking 70

speed to the size of the microbeads

3.7 Microencapsulation of cells using the developed 71

flicking system

3.7.1 Cell culture of HaCaT and ORL-48 71

3.7.2 Microencapsulation of cells and monitoring 72

3.8 Characterising the biochemistry and surface 73

structure of the microbeads

3.8.1 FTIR scanning of the calcium alginate 73

microbeads with and without cells

3.8.2 FE-SEM scanning of the calcium alginate 73

microbeads

3.9 Characterising the biochemistry properties of the 3D

microtissues 73

3.9.1 DAPI staining 73

3.9.2 Live and dead cells staining 74

3.9.3 Extraction of 3D microtissues from 74

calcium alginate

3.9.4 FE-SEM scanning of the extracted 3D 75

microtissues

3.9.5 3D microtissues replating 75

xiii

CHAPTER 4 RESULTS AND DISCUSSION 76

4.1 Introduction 76

4.2 The functions of flicking system 76

4.3 Assessment of the flicking system performance 79

4.3.1 Verification of PWM signals 79

4.3.2 The relationship of potentiometer voltage 81

to the PWM signals

4.3.3 Effect of PWM signals on motor speed 82

4.3.4 Battery level indication of the flicking 83

device

4.3.5 Flow rate calibration of the commercial 84

syringe pump (New Era NE-4002X)

4.3.6 Flow rate calibration of the customised 85

infusion pump

4.4 The effects of flow rate and flicking speed to 86

the size of microbeads

4.4.1 The effects of different flow rates to the 86

size of microbeads

4.4.2 The effects of different flicking speeds to 93

the size of microbeads

4.5 Microencapsulation of HaCaT and ORL-48 cells 98

at different cell densities

4.5.1 HaCaT cells encapsulation at a cell density 98

of 1.75 × 106 cells/ml

4.5.2 HaCaT cells encapsulation at cell density 99

of 31.9 × 106 cells/ml

4.5.3 ORL-48 cells encapsulation at cell density 101

of 3.97 × 106 cells/ml

4.5.4 ORL-48 cells encapsulation at cell density 102

of 94.2 × 106

cells/ml

4.6 The biochemistry and surface structure of 104

the microbeads

4.6.1 The functional groups of the microbeads 104

with and without cells

xiv

4.6.2 The surface morphology of the microbeads 107

4.7 The biophysical properties of the 3D microtissues 108

4.7.1 Nucleus staining of the encapsulated 108

HaCaT and ORL-48 cells

4.7.2 The viability of the encapsulated 111

HaCaT and ORL-48 cells

4.7.3 3D microtissue extracted from calcium 112

alginate microbeads

4.7.4 The surface morphology of the 3D 113

microtissues

4.7.5 Spread of cells in microtissue replating 115

experiment

CHAPTER 5 CONCLUSION 118

5.1 Conclusion 118

5.2 Thesis contribution 119

5.3 Recommendations for future work 119

REFERENCES 121

APPENDIX 134

VITA 151

xv

LIST OF TABLES

2.1 Summary of 3D cell culture systems 18

2.2 Application of encapsulation and target of encapsulation 21

2.3 Comparison of six microencapsulation techniques on their

capabilities

30

2.4 Common biopolymers used for microencapsulation of cells

and their applications

32

3.1 Establishment of experiments 41

3.2 The relationship between “mode” and communication lines

data

65

4.1 The steps for operating the flicking system 79

xvi

LIST OF FIGURES

2.1 The anatomy of human cell 8

2.2 Human body tissues 10

2.3 Layers of the epidermis 11

2.4 Monolayer of HaCaT cells cultured in a petri dish

(Scale bar: 100 µm)

12

2.5 Monolayer of ORL-48 cells cultured in a petri dish

(Scale bar: 100 µm)

13

2.6 Integrin-mediated cell adhesion to the ECM. (a) Suspension

cell adhere to the surface of ECM. (b) The structures of

ECM

14

2.7 Different phases of cell growth 15

2.8 The five main structural forms of microcapsules 20

2.9 Calcium alginate beads formation by simple dripping 22

2.10 Calcium alginate microbeads formation by microfluidic

device

23

2.11 Calcium alginate microbeads formation by electrostatic 23

2.12 Apparatus for producing calcium alginate beads by the

vibration method, comprising a syringe pump for forcing

sodium alginate solution from a syringe, a loudspeaker, and

a sine wave sound generator

24

2.13 Arrangement of nozzle and cutting tool, with 48 stainless

steel wires

25

2.14 Scheme of the cutting process 26

2.15 The setup of JetCutter technique 26

xvii

2.16 Scheme of the rotating disc and nozzle technology for bead

production

27

2.17 Bead formation by a rotating disc device 28

2.18 Bead formation by a rotating nozzle device 28

2.19 The molecular structure of calcium alginate 31

2.20 The working principal of Phase Contrast Microscope 33

2.21 FE-SEM (Jeol, JSM – 7600F) in Microelectronic and

Nanotechnology - Shamsuddin Research Center, Universiti

Tun Hussein Onn Malaysia

34

2.22 A FTIR spectrometer (Perkin Elmer Spectrum 100) 35

2.23 Chemical structure of 4'-6-diamidine-2-phenylindole

(DAPI) dichloride

36

3.1 Flow chart of research methodology 40

3.2 Two major parts of the flicking system 42

3.3 The architecture sketchup of the flicking system (front side) 42

3.4 The architecture sketchup of the flicking system (back side) 43

3.5 Control panel of the flicking system 44

3.6 Infusion pump of the flicking system 44

3.7 Flicking device of the flicking system 45

3.8 The block diagram of flicking device circuit 46

3.9 The layout of the flicking device and controller circuit 47

3.10 The flicking device was used with a conventional syringe

pump (New Era NE-4002X) to generate Ca-Alg microbeads

during the preliminary work

47

3.11 The overall schematic diagram of the flicking device 50

3.12 Programming flow chart of the flicking device 52

3.13 Coding of custom character 1, head of battery bar (filled) 53

3.14 Coding of custom character 2, head of battery bar (hollow) 53

3.15 Coding of custom character 3, middle of battery bar (filled) 53

3.16 Coding of custom character 4, middle of battery bar

(hollow)

54

xviii

3.17 Coding of custom character 5, end of battery bar (filled) 54

3.18 Coding of custom character 6, end of battery bar (hollow) 54

3.19 Coding to read the remaining battery level 55

3.20 Coding to read the communication data from the Arduino of

infusion pump

56

3.21 Coding to start or stop the flicking device 57

3.22 The block diagram of infusion pump circuit 58

3.23 The preliminary design of the infusion pump with a

temporary casing

59

3.24 The overall schematic diagram of the infusion pump 60

3.25 The communication connections between Arduino ARD1

(flicking device) and Arduino ARD2 (infusion pump)

62

3.26 Programming flow chart of the infusion pump 64

3.27 Coding used to select the speed of infusion pump 65

3.28 Coding used to create the low, middle and high pulse

frequency and output the speed of infusion pump to the

flicking device for display on the LCD

66

3.29 The experimental setup to generate calcium alginate

microbeads

70

4.1 Front view of the flicking system prototype 77

4.2 Top view of the flicking system prototype 77

4.3 Flicking motor of the flicking system prototype 78

4.4 LCD display of the flicking system prototype 78

4.5 The output signals of PWM at different duty cycles: (a) 0 %

duty cycle, (b) 25 % duty cycle, (c) 50 % duty cycle, (d) 75

% duty cycle and (d) 100 % duty cycle

80

4.6 The relationship between the potentiometer input voltage

and duty cycle of PWM signal

81

4.7 Graph of motor speed versus duty cycle of PWM 82

4.8 (a) High, (b) middle, (c) low and (d) extremely low battery

levels

83

xix

4.9 Syringe pump (New Era NE-4002X) flow rates verification

result

84

4.10 Customised infusion pump flow rates verification 85

4.11 One of the microbeads sample generated by flow rates: (a)

5, (b) 10 and (c) 15 µl/min (Scale bar: 100 µm)

87

4.12 The size distribution graph of microbeads produced at

different flow rates of (a) 5, (b) 10 and (c) 15 µl/min

88

4.13 Size distribution of calcium alginate microbeads in different

flow rates (5, 10, 15 µl/min)

89

4.14 Some of the microbeads sample generated by flow rates: (a)

2 µl/min, (b) 4 µl/min, (c) 6 µl/min, (d) 8 µl/min and (e) 10

µl/min (Scale bar: 200 µm)

91

4.15 The size distribution graph of microbeads produced at

different flow rates of (a) 2, (b) 4, (c) 6, (d) 8 and (e) 10

µl/min

92

4.16 Size distribution of calcium alginate microbeads in different

flow rates (2, 4, 6, 8, 10 µl/min)

93

4.17 Some of the microbeads sample generated by flicking

speeds: (a) 60, (b) 70, (c) 80, (d) 90 and (e) 100 rpm (Scale

bar: 200 µm)

95

4.18 The size distribution graph of microbeads produced at

different flicking speed of (a) 60, (b) 70, (c) 80, (d) 90 and

(e) 100 rpm

96

4.19 Size distribution of calcium alginate microbeads in different

flicking speeds

97

4.20 HaCaT cells in calcium alginate with cell density of 1.75 ×

106

cells/ml after: (a) day 1, (b) day 3 and (c) day 6 of

culture (Scale bar: 100 µm)

98

4.21 HaCaT cells protrusion from the microbead (Scale bar: 100

µm)

99

xx

4.22 HaCaT cells in calcium alginate with cell density of 31.9 ×

106 cells/ml after: (a) day 1, (b) day 3, (c) day 5, (d) day 7,

(e) day 9, (f) day 11, (g) day 13 and (h) day 15 of culture

(Scale bar: 100 µm)

100

4.23 ORL-48 cells in calcium alginate with cell density of 3.97 ×

106 cells/ml after: (a) day 1, (b) day 3 and (c) day 6 and (d)

day 9 of culture (Scale bar: 100 µm)

101

4.24 ORL-48 cells in calcium alginate with cell density of 94.2 ×

106 cells/ml after: (a) day 1, (b) day 3, (c) day 5, (d) day 7,

(e) day 9, (f) day 11, (g) day 13 and (h) day 15 of culture

(Scale bar: 100 µm)

103

4.25 FTIR spectra of (a) calcium alginate, (b) HaCaT cells and

(c) calcium alginate encapsulated with HaCaT cells

106

4.26 Morphology image of calcium alginate microbead with

different magnification: (a) ×150 (Scale bar: 100 µm), (b)

×300 (Scale bar: 10 µm) and (c) ×10000 (Scale bar: 1 µm)

107

4.27 DAPI staining on HaCaT cells in calcium alginate: (a) after

3 days cultured (white light), (b) after 3 days cultured

(fluorescence), (c) after 9 days cultured and (d) after 15

days cultured. (Scale bar: 100 µm)

109

4.28 DAPI staining on ORL-48 cells in calcium alginate: (a)

after 3 days cultured, (b) after 9 days cultured and (c) after

15 days cultured. (Scale bar: 100 µm)

110

4.29 Live and dead staining on 3D HaCaT cells encapsulated in

calcium alginate microbead: (a) sample 1 and (b) sample 2.

(Scale bar: 100 µm)

112

4.30 The images of microtissues after extraction: (a) HaCaT

microtissue before and (b) after alginate lyased, (c) ORL-48

microtissue before and (d) after alginate lyased (Scale bar:

100 µm)

113

4.31 FE-SEM image of 3D HaCaT microtissue with different 114

xxi

magnifications: (a) ×150 (Scale bar: 100 µm), (b) ×300

(Scale bar: 10 µm) and (c) ×1500 (Scale bar: 10 µm)

4.32 FE-SEM image of 3D ORL-48 microtissue with different

magnifications: (a) ×150 (Scale bar: 100 µm), (b) ×300

(Scale bar: 10 µm) and (c) ×1500 (Scale bar: 10 µm)

115

4.33 Monitoring of replated 3D HaCaT microtissue on: (a) day 1,

(b) day 2, (c) day 3 and (d) day 4 (Scale bar: 100 µm)

116

4.34 Monitoring of replated 3D ORL-48 microtissue on: (a) day

1, (b) day 2, (c) day 3 and (d) day 4 (Scale bar: 100 µm)

117

xxii

LIST OF SYMBOLS AND ABBREVIATIONS

% - Percent sign

- Pulse duration

oC - Degree Celsius

2D - Two dimensional

3D - Three dimensional

BSC - Biological safety cabinet

Ca-Alg - Calcium alginate

cm - Centimeter

CO2 - Carbon dioxide

DAPI - 4′,6-Diamidino-2-phenylindole dihydrochloride

DC - Direct current

DMEM - Dulbecco‟s modified eagle medium

DNA - Deoxyribonucleic acid

ECM - Extracellular matrix

ER - Endoplasmic reticulum

ETFE - Ethylene tetrafluoroethylene

EthD-1 - Ethidium homodimer-1

FDA - Food and Drug Administration

FE-SEM - Field emission scanning electron microscope

FITC - Fluorescein isothiocyanate

FTIR - Fourier transform infrared spectroscopy

H & E - Hematoxylin and eosin

HA - Hyaluronic acid

HaCaT - Human keratinocyte

xxiii

HBSS - Hank‟s balanced salt solution

Hz - Hertz

kg-cm - Kilogram per centimeter

LABE - Low Angle Backscatter Imaging

LCD - Liquid crystal display

LEI - Lower Secondary Electron Image

MiNT-SRC - Microelectronic and Nanotechnology –

Shamsuddin Research Center

mg/ml - Miligram per mililiter

min - Minute

ml - Mililiter

mm - Milimeter

nm - Nanometer

ORL-48 - Oral squamous cell carcinoma cell line

OSCC - Oral squamous cell carcinoma

PAA - Poly(acrylic acid)

PAam - Polyacrylamide

PCB - Printed circuit board

PCL - Poly(ε-caprolactone)

PDMAEM - Poly(dimethylaminoethylmethacrylate) hydrochloride

PEG - Poly(ethylene glycol)

PGA - Polyglycolide

PLA - Polylactide

PLAGA - Poly(lactic acid-glycolic acid)

PLGA - Poly(l-lactide-co-glycolide)

PLLA - Poly(L-lactic acid)

PMMA - Polymethylmethacrylate

PNIPAAm - Poly(N-isopropylacrylamide)

PPF - Poly(propylene fumarate)

xxiv

PWM - Pulse width modulation

RNA - Ribonucleic acid

rpm - Revolutions per minute

SD - Standard deviation

SEI - Secondary Electron Image

SEM - Scanning electron microscope

µl - Microliter

µl/min - Microliter per minute

µm - Micrometer

µM - Micromolar

UK - United Kingdom

US - United States

USA - United States of America

UTHM - Universiti Tun Hussein Onn Malaysia

V - Volt

w/v - Weight per volume

xxv

LIST OF APPENDICES

APPENDIX TITLE PAGE

A Arduino source code of the flicking device 134

B Arduino source code of the infusion pump 145

C List of hardware used and specification 149

1. CHAPTER 1

INTRODUCTION

1.1 Overview

Microbeads or microcapsules have wide applications in biomedical engineering field

that include drug delivery, encapsulation of biomolecules, tissue padding and tissue

regeneration. General information concerning microbeads or microcapsules is briefly

illustrated in the research background. The problem statement highlights the weakness

of current methods to fabricate microbeads. Consequently, a method which is very

simple, reliable and uses cheap materials available in the lab was introduced to generate

microbeads. Nonetheless, this chapter also covers the objectives of the research, scope of

research and thesis outline.

1.2 Research background

In the development of tissue engineering, technologies for creating living functional

tissues in the laboratory are being developed and discovered. Tissue engineering is an

alternative to organ transplantation, drug therapy and gene therapy [1]. Animal models

used for drug testing and drug discovery studies are not encouraged due to ethical and

feasibility issues. Thus, in vitro tissue models (self cultured microtissues) are actively

used in research as an alternative for quantitative, repetitive and systematic investigation

of drugs in order to reduce the usage of animal models. These tissue models can be used

2

for high-throughput screening of drugs and for pharmacokinetic analyses of drugs [2].

For example, one man was brain dead and another five people were in hospital after an

experimental drug was administered to 90 people in a French clinical trial, as reported

by British Broadcasting Corporation (BBC) on 15 January 2016. This incident revealed

the vital of using alternative tissue models with more availability for experimental drug

trial might avoid sacrifice of life.

Various in vitro methods had been developed to culture microtissues. The most

common method to culture microtissue is culturing cells on scaffolds of biopolymers

that can be produced using electrospinning, salt leaching and ice particle leaching

method [3]. With this method, the cells are scattered in the pores of the scaffolds.

Culturing cells on a planar compliant substrate such as a hydrogel produces low yield

and inconsistent size of microtissues. This technique is arguably because it is different

from the actual biological microenvironment in the tissue [4]. Alternatively, microtissue

can be cultured by using microencapsulation technique that allows a compound (cells) to

be encapsulated inside a tiny spheroid known as microsphere or microbead, having an

average diameter as small as one micro meter to several hundred micro meters [5].

Microencapsulation of cells in the calcium alginate microbeads is leading to the

formation of microtissues (multicellular spheroids) [6, 7]. The unique advantage of this

technique is that cell culture is performed within a 3D environment that completely

surrounds cells and enabling the delivery of intense signals to cells from all directions

[8]. Compared to the 2D monolayer cell culture, 3D microtissue models mimic the cell

structural organisation in the in vivo system and the cellular microenvironment

established in the 3D models often plays a more significant role in cellular responses to

drugs and disease progression [2].

Among the previous methods, microencapsulation of cells is a promising method

because microencapsulation leads to the formation of living microtissues which is

suitable to be used for therapeutic tests. Microbeads or microcapsules have wide

applications in biomedical engineering field that include drug delivery, encapsulation of

biomolecules, tissue padding and tissue regeneration. The selection of a suitable

encapsulating material with appropriate porosity, which can facilitate the transport of

nutrients, proteins and drugs while blocking attack of antibodies and bacteria is critical

3

[9]. The capsule must be mechanically stable and easy to handle. These requirements

may be fulfilled by controlling the size and thickness of the encapsulating polymer

membrane at microscale range.

Microcapsules are spherical particles containing a core substance with the size

varying between 50 nm to 2 mm [10]. During the early days in the 80‟s,

microencapsulation technologies were used for the microencapsulation of drugs and

liquidified food [11, 12]. Later in the 90‟s, this technology was discovered with new

application in tissue engineering [13]. In tissue engineering, microcapsules of sodium

alginate for microencapsulation of cells have the potential for the application in

immunoisolatory and biochemical assays [14]. Biomaterials for microencapsulation of

cells play a major roll to determine the fate of the cells encapsulated and also the

proliferation of cells. There are various types of materials used to produce microcapsules

such as agarose, collagen, alginate, chitosan and gelatin [15]. Sodium alginate has been

found suitable for biotechnology and biomedical applications mainly as a material for

the encapsulation of different cells for biochemical processing and immunoisolatory

while the cells can still maintain viability within the hydrogel [14]. It has been employed

for encapsulating cells to be transplanted, since it is biocompatible both to the host and

enclosed cells [8].

Some of the popular methods developed particularly for the microencapsulation

of cells are the microfluidic device [16], emulsification [17], extrusion, electrospray

[18], electrostatic [19-21] and airflow [22]. However, these methods are complex in

design [23], required high voltage supply [20], post cleaning process [24] and large

volume of reagents. The purpose of this research is to develop a novel flicking system

that could be used to produce uniform and controllable sized of calcium alginate

microbeads for 3D cells encapsulation. Calcium alginate was proposed to be used for

encapsulation of cells is because this biomaterial is non-toxic, degradable and it is a

biocompatible material approved by US Food and Drug Administration (FDA) [9, 25].

Under the FDA approval, calcium alginate is permitted to be an implantable material in

the human body [9, 25]. The contribution of this study is expected to be useful for

generating 3D cells model for studying therapeutic cytochemicals. The biochemistry

properties of the microbeads and biophysical properties of the microtissues formed will

4

be characterised using microscopy observation, FTIR scanning, Field Emission

Scanning Electron Microscope (FE-SEM), DAPI staining, live and dead stainings and

3D microtissues replating. The backbone of this study is to be useful for generating the

3D microtissues, which is identified to be relevant to drive major innovations in tissue

engineering.

1.3 Problem statement

The microbeads of calcium alginate can be easily generated by extruding droplets of

sodium alginate solution in air using a syringe and allowed to polymerised in the

calcium chloride solution. However, the extrusion technique provided limited control

over the size of the microbeads and usually large beads of calcium alginate in millimeter

size were produced. Small calcium alginate beads in a few hundreds of micron are

desirable because this range of microbeads provide higher mechanical strength, easier

implantation, better transport of oxygen and nutrients for the cells [26]. Although

alginate beads can be fabricated by using microfluidic approach in micron size, the

alginate microbeads formed based on microemulsion technique were covered with oil

film and hence, post cleaning process is required to remove the oil film before

incubating the cells. The oil film can be sealing the gas from the cells in encapsulation.

Strong mechanical and chemical treatments are required to remove the immiscible fluid,

which prolongs exposure of the cells to divalent ions or solvents which may harm and

kill the cells [27]. Other methods such as microextrusion techniques assisted by

electrostatic field and electrospraying were shown to decrease the alginate drop size.

However, these methods required sophisticated high power circuit to create atomization

of alginate droplets which is dangerous with electric shock hazard [20, 28].

Based on the literature review, a simple yet efficient technique could be

designed and developed to address the several issues raised for current techniques of

microencapsulation. Hence, the purpose of this study is to develop a novel flicking

system that could be used to produce calcium alginate (Ca-Alg) microbeads for the

microencapsulation of cells. The flicking method used by the flicking system to

encapsulate cells is simple and highly reproducible.

5

1.4 Objectives of the research

The aim of the research is the development of a flicking system for producing 3D cells

in microbeads leading to the growth of microtissues. Four objectives were outlined to

achieve the aim:

a) To develop a flicking system to produce calcium alginate microbeads size in

200 to 300 micron.

b) To investigate the effects of infusion pump flow rates and flicking motor

speed to the size of the microbeads produced.

c) To microencapsulate cells using the developed flicking system.

d) To characterise the biophysical properties of the 3D microtissues formed in

calcium alginate microbeads.

1.5 Scope of research

This research was divided into four scopes which are:

a) Design and development of a mechatronic system of microextrusion system

with a flicking device (flicking system).

b) Determining the syringe flow rates and flicking speed of the system to

encapsulate cells in the appropriate microbeads size.

c) Encapsulating human keratinocyte cell lines (HaCaT) and oral squamous cell

carcinoma (ORL-48) cells in the calcium alginate microbeads.

d) Monitoring the growth of the microencapsulated cells into microtissues.

1.6 Thesis outline

This thesis was divided into five chapters. Chapter 1 introduced an overview of the

research background, problem statement, objectives of the research, scope of research

6

and thesis outline. Chapter 2 revealed the cells type, the method used in

microencapsulation, biopolymers used for microencapsulation of cells, rationale to

generate 3D cells, instrument utilised in the research and stainings used to characterise

the biophysical of 3D microtissues. Chapter 3 outlined the experiment procedure of this

research including the materials and methods used in the development of flicking

system, experimental setup, investigating the effects of flow rates and flicking speed to

the size of microbeads, characterising the biochemistry and surface structure of the

microbeads, preparation for microencapsulation of cells using the developed flicking

system and characterising the biophysical properties of the 3D microtissues formed from

the 3D cells. Chapter 4 presented the results and discussion of the research project which

included the functions of flicking system, assessment of the system performance, the

effects of flow rates and flicking speed to the size of microbeads, the biochemistry and

surface structure of the microbeads, microencapsulation of HaCaT and ORL-48 cells at

different cell densities and the biophysical properties of the 3D microtissues. At last but

not least, Chapter 5 delivered the conclusion, thesis contribution and recommendations

for future work.

2. CHAPTER 2

LITERATURE REVIEWS

2.1 Introduction

This chapter has included the summarised information of the relevant studies on cells

microencapsulation leading to microtissues formation in the calcium alginate

microbeads. The literature review covered the fundamental knowledge of cell and tissue,

microencapsulation techniques and techniques used for biophysical characterisation of

microtissues are discussed in the following subchapters which are cell and tissue,

extracellular matrix (ECM), cell adhesion, phases of cell growth, microencapsulation,

different technology of microencapsulation, biopolymer used for microencapsulation of

cells, rationale to generate 3D cells, inverted phase contrast microscopy, field emission

scanning microscope (FE-SEM), fourier transform infrared spectroscopy (FTIR), DAPI

staining and live/dead viability assay.

The sources of the literatures reviewed are journals, articles, and information

from books and internet, which are important on identifying problems as well as give

ideas in this research.

2.2 Cell and tissue

The cell is the fundamental structural, functional, and biological unit of all known living

organisms. Cells are the smallest unit of life that can replicate freely. Cells consist

8

of cytoplasm enclosed within a membrane, which contains many biomolecules such

as nucleic acids and proteins [29]. Organisms can be classified as unicellular (cell and

bacteria) or multicellular (plants and animals). Human has about 100 trillion (1014

) cells

while the number of cells in plants and animals varies from species to species [30]. Most

animal and plant cells are in dimensions between 1 and 100 micrometres and only can be

visible under the microscope [31]. The cells build the structure of body, consume

nutrients from food, convert those nutrients into energy, and carry out specialist

functions. Organelles are structures that perform specialised tasks within the cell. These

organelles containing in the cytoplasm are such as pinocytotic vesicle, golgi vesicles,

cytoskeleton, endoplasmic reticulum (ER), golgi apparatus, lysosomes, mitochondria,

centrioles, microtubules, nucleus, nucleolus, plasma membrane and ribosomes as shown

in Figure 2.1.

Figure 2.1: The anatomy of human cell [32]

Centriole

(within

centrosome)

Lysosome

Golgi

apparatus

(Golgi body)

Nucleus

Nucleus membrane

Genetic material

(DNA)

Nucleolus

Endoplasmic

reticulum

Ribosomes

Plasma

membrane

(cell

membrane)

Cytoplasm

Mitochondrion

9

Cytoplasm is a jelly-like fluid which surrounds the cell‟s organelles and nucleus.

The cytoskeleton is a chain of long fibers that build up the cell‟s structural framework

that has several critical functions, allowing cells to move, cell division and determining

cell shape. In addition, cytoskeleton provides a track-like system that directs the

movement of organelles and other substances within cells. The endoplasmic reticulum is

responsible in molecule processing and transport to their specific destinations either

outside or inside the cell. Nonetheless, the golgi apparatus is responsible in packaging

molecules for export from the cell. Lysosomes destroy toxic substances and recycle

exhausted cell parts. In addition, mitochondria provide the energy for cells to replicate

themselves with their own genetic material. Ribosomes use the cell‟s genetic

instructions to make proteins at the same time. Besides that, the nucleus contains most of

the cell‟s genetic material such as RNA (ribonucleic acid) and DNA (deoxyribonucleic

acid). The plasma membrane is the outer most layer which enclosed the cell and allows

the transport of materials to leave and enter the cell.

Tissue is an assembly of cells with similar characteristics which performs a

specific function. Different types of tissues can be found in different organs as shown in

Figure 2.2. Organs are made of tissues that provide the various functions of organs

required to maintain biological life. There are four basic types of tissue in human, which

are connective, epithelial, nervous and muscular tissue. The function of connective

tissue supports and binds with other tissues. Connective tissue cells can be found in a

large amount of extracellular matrix (ECM). Epithelial tissue covers the body surface

and forms the lining for most internal cavities of organs. Epithelial tissues have different

cells shapes which are squamous, cuboidal and columnar epithelium. The main functions

of epithelial tissue are filtration, protection secretion, and absorption of glucose from the

lumen of the intestine. Skin is one of the organs that consist of most epithelial tissue

which protects the body from bacteria, dirt and microbes that may be harmful.

10

Figure 2.2: Human body tissues [33]

2.3 Human keratinocytes

The epidermis is made of squamous epithelium that forms the protective layer of the

skin [34]. Keratinocytes are the main cell type in the epidermis layer. Epidermis consists

of five histologically distinct cellular layers such as stratum corneum, stratum lucidum,

stratum granulosum, stratum spinosum and stratum basale as shown in Figure 2.3.

Stratum spinosum is the layer with the most human keratinocytes in the epidermis.

Human keratinocytes cell line (HaCaT) is transformed immortal keratinocyte cell line

with abnormal chromosomes number from adult human skin that has been widely used

for scientific research, studies of cell biology and differentiation. HaCaT cells are

Connective

tissue

Skeletal muscle

Epithelial tissue Smooth muscle

Nervous

tissue

Cardiac muscle

11

utilised for their high capacity to proliferate and differentiate in vitro [35]. HaCaT cells

have reduced tissue proliferation and regeneration compared to normal epidermal

keratinocytes [36, 37]. By the addition of growth factors can stimulate the proliferation

rate of HaCaT cells [38], the characterisation of human keratinocyte using this model

becomes reproducible and overcome the short culture lifespan issue of normal human

keratinocyte. Figure 2.4 shows a phase contrast photomicrograph of HaCaT cells in

monolayer of culture. In monolayer or 2D structure, HaCaT cells are displayed with

polygonal shape and tightly coupled to the neighbouring cells when grown into

confluency.

Figure 2.3: Layers of the epidermis [39]

Stratum corneum

Stratum lucidum

Stratum granulosum

Stratum spinosum

Stratum basale

Melanocyte

Dermis

Dead cells filled

with keratin

Lamellar granules

Keratinocyte

Merkel cell

Sensory neuron

12

Figure 2.4: Monolayer of HaCaT cells cultured in a petri dish (Scale bar: 100 µm)

2.4 Oral squamous cell carcinoma cell line (ORL-48)

Oral cancer is a common disease and silent killer in the developing country, especially in

Southeast Asia country. Microtissue models of cancer cells are necessary in drug

discovery and the cancer research. Oral squamous cell carcinoma (OSCC) cell line

(ORL-48) was established by Cancer Research Malaysia. They are surgically explanted

epithelial specimens obtained from untreated primary human oral carcinoma cell in the

squamous layer of the oral cavity. These epithelial cells were genetically modified to be

immortal and grown into monolayer with doubling times ranging between 26.4 and 40.8

hours [40]. Figure 2.5 shows a phase contrast photomicrograph of ORL-48 cells in

monolayer culture. In 2D structure or monolayer, ORL-48 cells are typically irregular

elongated polygonal shape and coupled randomly with the neighbouring cells.

13

Figure 2.5: Monolayer of ORL-48 cells cultured in a petri dish

(Scale bar: 100 µm)

2.5 Extracellular matrix (ECM) and cell adhesion

Extracellular matrix (ECM) is made up of polysaccharides and proteins, including

laminins, collagens, elastins, proteoglycans and fibronectins, all secreted by the cell

[41]. In animal cells, the ECM enclosed cells as fibrils that contact the cells [42]. Cells

in animals are also joined directly to each other by cell adhesion proteins at the cell

surface. In addition, ECM provides a biochemical barrier, mechanical support and a

medium for extracellular communication that is assisted by cell adhesion proteins,

moreover ECM provides tensile strength for tendons, compressive strength for cartilage,

hydraulic protection for cells and elasticity to the walls of blood vessels [42].

Adhesion of cells to the extracellular matrix (ECM) is key to the regulation of

cellular morphology, differentiation, proliferation, migration and survival [43]. These

functions are necessary during tissue development, maintenance of tissue structure and

the initiation of tissue repair. Integrins are the main receptors that moderate cell

adhesion to the components of ECM [44, 45]. Figure 2.6(a) shows a suspension cell

adhesion to ECM proteins via integrins and Figure 2.6(b) shows the structures of ECM

which consist actin cytoskeleton, integrin receptors, adhesion proteins and focal

adhesion complexes. Integrins are categorised as the heterodimers which consisted of

14

two different chains, the α (alpha) and β (beta) subunits [46]. During cell-ECM

adhesion, the specific ligands in the ECM decide the type of α and β subunits of the

integrins being attached by the cell [47]. There are numerous integrins exist on the cell

membrane and they work collaboratively with the cell adhesion proteins in cell-ECM

adhesion [48].

Figure 2.6: Integrin-mediated cell adhesion to the ECM. (a) Suspension cell adhere to

the surface of ECM. (b) The structures of ECM [49]

(a)

(b)

15

2.6 Phases of cell growth

A regular growth curve for cultured cells displayed a sigmoid pattern of proliferation.

The growth phases correlative with normal cells are divided into lag phase, logarithmic

growth phase, stationary (or plateau) phase and decline phase as shown in Figure 2.7. It

was vital to understand the growth characteristics of the cell line because any change in

cellular growth can represent a significant problem of the cell line and affect the

experimental results [50]. At lag phase, the cells do not divide but adapting to the culture

conditions and the length of this phase will depend upon the growth phase of the cell

line at the seeding density and time of subculture. During log growth phase, the cells

actively proliferate and exponentially increase in cell density. The cell population is

considered to be the most viable at this phase, therefore it was recommended to assess

cellular function at this stage. Each cell line will show different cell proliferation

kinetics during the log phase, therefore it is the optimal phase for determining the

population doubling time [50]. After the log phase, the cellular proliferation slows down

due to the cell population becoming confluent at stationary phase. Ultimately, the cell

death predominates in this death phase and there will be a reduction in the number of

viable cells. Cell death was not due to the reduction in nutrient supplements but the

natural path of the cellular cycle.

Figure 2.7: Different phases of cell growth [50]

Log N

um

ber

of

Cel

ls

Lag Log Stationary

/ Plateau Death

Live

Total

1 2 3 4 5 6 7 8 9 10 Days

16

2.7 Rationale to generate 3D cells

In the in vitro or two-dimensional (2D) culture, cells are tightly spread and coupled to

the bottom surface of culture flask which is contrarily to cells that retain in-vivo [4]. The

extracellular matrix components, cell-to-cell and cell-to-matrix interaction that are

important for proliferation, differentiation and cellular functions in vivo are lost in the

conventional 2D culture [51].

Cell to cell interaction in 2D culture is mainly concentrated at the boundary of

the flattened cells [52]. Cells grown in 2D spread involuntarily by producing limited

amount of extracellular matrix proteins due to the contactless spreading of cells [53].

The spatial organisation of the cells in 2D is distorted compared with the three-

dimensional (3D) tissue in-vivo [54].

3D cells are good models as “near-to-in vivo” systems and reveal useful insights

from a variety of ways [54]. 3D cell model is a cost effective screening platform for drug

development and testing, simultaneously provides a better and more realistic predictive

value for risk and safety assessment [54].

A major benefit of the 3D over the 2D culture is the decrease in the gap between

cell cultures system and the cellular physiology [54]. Several researches show that 3D

organisation of cells reveals more novel and surprising visions into the tumorigenesis

mechanism which could represent an integral missing component in the in vitro cancer

studies [55].

Thus, 3D cell culture is believed to have better similarity to the actual tissue

model for cancer cell research because 3D culture restore specific morphological and

biochemical features similar to the corresponding tissue in-vivo [56].

Besides that, modelling the complexity of cancer utilising these cell lines on

standard plastic substrate does not accurately represent the tumour microenvironment

[57]. However, tumour cells cultured in 2D monolayer conditions do not respond to

cancer therapeutics [57]. Thus, 3D cells culture is useful for drug testing on cancer

tumour.

Many researchers want to mimic the actual cancer tumour in human body by

culturing cancer cells into 3D cells because the 3D cells reveal a more realistic drug

17

response, precisely characterises the disease and mimics the tumour microenvironment

in human body [58]. Other than that, the applications of 3D cell are differentiation

studies, drug discovery and pharmacological applications, cancer research, gene and

protein expression studies [59].

2.8 Previous methods for growing 3D cells

3D cells culture exhibits features that are closer to the complex in vivo conditions and

the 3D culture models have proven to be more realistic for translating the study findings

for in vivo applications [59].

Various in vitro methods had been developed for growing 3D cells as shown in

Table 2.1. While each method presents its unique factors, the appropriate method must

be selected to meet the requirements for the specific type of cells in different

applications.

The most common method to culture microtissue is culturing cells in suspension

such as hanging drop method. With this method, the cells are scattered in the suspension

media with limited flexibility. It is not a safe to use when one is dealing with extremely

pathogenic organisms as the user can easily get an infection.

Matrigel, Extracel and AlgiMatrix are the common methods used to culture 3D

microtissues on substrate. Culturing cells on a planar compliant substrate produces low

yield and inconsistent size of microtissues.

Other than that, microencapsulation techniques are also applied in microtissue

culturing that allows 3D cells growing within tiny spheroid matrix. The unique

advantage of this technique is that cell culture is performed within a 3D environment

that completely surrounds cells, enabling the delivery of intense signals to cells from all

directions [8].

18

Table 2.1: Summary of 3D cell culture systems [60]

3D cell culture systems

Pros

Cons Methods

3D spheroids grown in

suspension.

No added materials

Consistent spheroid

formation; control

over size

Co-cultures possible

Transparent

High-throughput

screening capable;

compatible with

liquid handling tools

Inexpensive

No support or

porosity

Limited flexibility

Size of spheroid

limiting

Magnetic cell

levitation [4] and

hanging drop [61]

3D spheroids grown on

matrix.

Large variety of

natural or synthetic

materials

Customisable

Co-cultures possible

Inexpensive

Gelling mechanism

Gel-to-gel variation

and structural

changes over time

Undefined

constituents in

natural gels

May not be

transparent

High-throughput

screening options

limited

Hydrogel [3],

Matrigel, Extracel

and AlgiMatrix [62]

3D spheroid grown

within matrix.

Large variety of

materials possible

for desired

properties

Customisable

Co-cultures possible

Medium cost

Possible scaffold-to-

scaffold variation

May not be

transparent

Cell removal may be

difficult

High-throughput

screening options

limited

Microencapsulations

[5, 7]

Culture media

3D spheroid

Culture media

3D spheroid

Matrix

Matrix

3D spheroid

Culture media

19

2.9 Microencapsulation

Vincenzo Bisceglie was the first to encapsulate cells using polymer membranes in 1933

[63]. Based on Bisceglie‟s work, Thomas Chang proposed the idea of encapsulating

cells within ultra thin polymer membrane microcapsules to provide immunoprotection to

the cells and introduced the term "artificial cells" to define this concept of

bioencapsulation in 1964 [64]. Microcapsule, microsphere or microbead are similar

terms that is defined as a spherical particle containing a core substance with a size

between 50 nm to 2 mm [10]. However, microcapsules may not be spherical structurally

but are referred to empty particles made of hydrogel (Figure 2.8). Many researches were

worked on microspheres and microcapsules for the applications of controlled drug

release formulations. They have been proven suitable for many applications which can

be produced from natural or synthetic materials, but they are usually found in nature, for

example, microencapsulation is also a natural phenomenon as what we can observe in

the bacterial spores, plants seeds and egg shells [65].

Microcapsules are divided into three categories that are determined by the

structure and layers of encapsulation. Mononuclear or single-core (Type A) is the

simplest form of microcapsules. The core material of mononuclear microcapsules

usually is a liquid, thus they can be called as liquid-core microcapsules also which is

surrounded by a layer of membrane [66]. The diameter of the core material and the

membrane thickness can vary in size. Multi-shells (Type B) microcapsules have multiple

shell layers which modify the original permeability and stability characteristics of the

microcapsules [67]. Polynuclear or multi-core (Type C) microcapsules containing two or

more individual cores [68]. Microcapsules of Type A-C are normally looked like

spherically shaped particles with a well-defined shell and core structure but other non-

spherical forms of microcapsules are categorised as Type D and Type E of

microcapsules. Type D microcapsule is the most common type of microcapsules without

multilayer of membrane applied for encapsulation of cells, particles and does not possess

multiple layer of membrane. We always referred these microcapsules as microbeads or

microspheres [69]. In addition, Type E are non-spherical or irregular shaped

20

microcapsules that can be polynuclear, mononuclear or solid particle encapsulation and

they are commonly used in the pharmaceutical and food industry [70].

Figure 2.8: The five main structural forms of microcapsules [65]

The microencapsulation technology has been applied to biotechnological and

medical disciplines recently such as the encapsulation of mammalian cells [71],

implantation of cells [72] and encapsulation of recombinant therapeutic proteins [73].

There are six main applications of microencapsulation within a membrane compared to

its non-encapsulated form as shown in Table 2.2 with the examples of where the process

was implemented.

A. Single-core B. Multi-shell C. Multi-core

D. Entrapment E. Irregular

21

Table 2.2: Application of encapsulation and target of encapsulation [65] [65]

Application of encapsulation Target of encapsulation

Protection or stabilization of the encapsulant from

interactions with reactive environments or future

surroundings

Cells (Prevention)

Mammalian (immuno-response and cell damage

due to agitation and aeration)

Yeast (ethanol toxicity)

Enzymes

Improved stability and reactivity

Prevent denaturing

Food additives and bioactives

Off-setting loss and deterioration by:

High temperature food processing

passage through the GI tract

hygroscopicity

evaporation (aroma compounds)

oxidation

Recombinant proteins

Improved stability and protection

Sustained, controlled or timed release of the core

material Agrochemicals (delivery of fertilizers, repellents,

herbicides, fungicides, etc.)

Folic acid (controlled release)

Pharmaceuticals (controlled delivery)

Antibody (sustained release)

Adhesive (dispensed upon pressure)

Butan-1-ol flavour (delayed release)

Fragrances (dispensed upon pressure)

Targeting of encapsulated compounds to specific

sites DNA to phagocytic cells

Vitamin C (oral route)

Probiotic bacteria (intestine)

Doxycycline for localized treatment of septic

arthritis

Enabling core material to be used as an extraction

device for product removal including in situ

product recovery

Environmental pollutants

Extraction of pharmaceuticals,

herbicides/pesticides and heavy metals from water

In situ product recovery

Culture environments

Recovery of primary and secondary metabolites

Bioconversion processes

Removal of products from hydrolysis of Penicillin

G and a lipase-catalysed reaction

Improved flow properties of the encapsulant for

enhanced handling, usage and storage including

safety

Pesticides (enhanced safety handlng and usage)

Biosorbents (improved usage and storage)

Enzyme (better storage)

Bioactives (enhanced handling and storage)

Hydrophobic liquids (new applicability)

Improved organoleptic properties of the

encapsulation product Shark liver oil (preventing undesirable taste)

Tea bags (improved appearance)

Antibiotic colistin (hiding taste)

Ultra Rice (preventing undesirable appearance

and taste)

22

2.10 Different methods of microencapsulation

2.10.1 Simple dripping

Calcium alginate beads can be easily generated by extruding drops [74] of sodium

alginate solution in the air using a syringe and collecting the droplets of sodium alginate

into calcium chloride solution, it offers little control over drop formation and large

calcium alginate beads in millimeter size were produced as shown in Figure 2.9.

Figure 2.9: Calcium alginate beads formation by simple dripping

2.10.2 Microfluidic

Microfluidic device can be used to produce small calcium alginate microbeads in a few

hundred micron size (nanometric size is also possible) with the aid of dispersion oil to

disperse the alginate solution into small droplets as shown in Figure 2.10. Although this

method produced smaller alginate drops, the precise control over the gelation process is

complicated and the removal of the immiscible fluid (oil) requires strong chemical and

mechanical treatments, which prolongs exposure of the cells to solvents which will be a

threat to the cells [27].

Alginate droplet

Calcium chloride

solution

Calcium alginate

bead

Syringe

Alginate solution

Syringe needle

23

Figure 2.10: Calcium alginate microbeads formation by microfluidic device

2.10.3 Electrostatic

Alternatively, microbeads generation techniques assisted by electrostatic field and

electrospray could decrease the size of the alginate droplets as shown in Figure 2.18. In

this method, the high voltage (up to 25 kV) of electricity in either static or pulse is used

to create the electrostatic potential between the needle and the hardening bath (calcium

chloride) in order to disperse the alginate solution into small droplets [20, 28]. This

method is able to produce smaller microbeads (≥ 50 μm in diameter) under sterile

conditions with uniform size and shape under reproducible conditions compared to

normal dripping method [75].

Figure 2.11: Calcium alginate microbeads formation by electrostatic

Oil

Oil

Oil Alginate

Alginate droplets

Syringe

Alginate solution

Syringe needle

High voltage

Alginate droplet

Calcium chloride

solution

Calcium alginate

bead

24

2.10.4 Vibration technique

The vibration technique is the most sophisticated dropping technique for

microencapsulation. Vibration nozzle was used to produce alginate beads with

immobilised proteins [76]. The vibration technique used mechanical vibrations

generated by speaker to break up a jet of cell-alginate mixture into uniform droplets as

shown in Figure 2.12. The calcium alginate bead size is controlled by varying the jet

velocity, vibration frequency and jet diameter [76]. The main disadvantage of vibrating

technique is low production yields of small microbeads, as it only produce a single

droplet, one after another at once. The production flow rate is mainly dependent on the

nozzle diameter with increasing diameter resulting in higher production rate, however

the production rate is still low for the largest nozzle diameter [75].

Figure 2.12: Apparatus for producing calcium alginate beads by the vibration method,

comprising a syringe pump for forcing sodium alginate solution from a syringe, a

loudspeaker, and a sine wave sound generator [76]

Wood

board

Syringe pump

Syringe

Sodium alginate solution

with protein

100% Isoamyl

alcohol

50% CaCl2 aq and 50%

Isopropyl alcohol

mixture

Speaker

Sine wave sound

generator

REFERENCES

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Replacement Organs and Tissues. Oxford: Oxford University Press. 2004.

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