DEVELOPMENT OF AN ELECTRONIC AEROSOL ATOMISATION SYSTEM FOR
GENERATING THREE-DIMENSIONAL (3D) CELLS IN
MICROENCAPSULATIONS AND MICROTISSUES CHARACTERISATION
LEONG WAI YEAN
A thesis submitted in
fulfilment of the requirement for the award of the
Degree of Master of Electrical Engineering
Faculty of Electrical and Electronic Engineering
Universiti Tun Hussien Onn Malaysia
DECEMBER 2016
iii
Special dedication with full gratitude on the guidance and encouragement to families
who loved, especially my beloved father and mother and not forgotten to my supervisor
that contributed ideas and opinions
iv
ACKNOWLEDGEMENT
First, I would like to extend my deepest appreciation and heartfelt gratitude to my
supervisor, Assoc. Prof. Dr. Soon Chin Fhong for her patience, tremendous support, and
excellent guidance in terms of knowledge and continuous encouragement through my
master studies, research, and thesis work, where ideas and supervision from her is very
important in the completion of this project.
My special appreciations to my parents, family members and friends who has
encourage, support, love, patience and understanding me throughout my involvement in
this research project. Thank you for all the encouragement and affection given.
Finally thanks to my friends and any party involved directly or indirectly in this
project which was conducted in Biosensor and Bioengineering Laboratory,
Microelectronics and Nanotechnology – Shamsuddin Research Centre (MiNT-SRC),
Faculty of Electrical and Electronic Engineering, Universiti Tun Hussein Onn Malaysia
(UTHM).
We acknowledge Professor Cheong Sok Cheng from Cancer Research Malaysia
for her kind contribution of oral squamous cell carcinoma (OSCC) cell line (ORL-48).
v
LIST OF ASSOCIATED PUBLICATIONS
Journal
1. Wai Yean Leong, Soon Chuan Wong, Kian Sek Tee, Sok Ching Cheong, Siew
Hua Gan, Mansour Youseffi, Chin Fhong Soon, “In vitro growth of human
keratinocytes and oral cancer cells into microtissues: an aerosol-based
microencapsulation technique”,Biotechnology and Applied Biochemistry. Impact
factor: 1.429 (Q3, JCR, ISI Indexed). [In preparation]
2. Wai Yean Leong, Chin Fhong Soon, Soon Chuan Wong, Kian Sek Tee,
“Development of an electronic aerosol system for generating microcapsules”,
Journal Teknologi, Volume 78, Issue 5-7, Pages: 79-85, May 2016.
http://dx.doi.org/10.11113/jt.v78.8718 (Scopus Indexed).
vi
ABSTRACT
Cell encapsulation is a micro technology widely applied in cell and tissue engineering,
tissue transplantation and regenerative medicine. Various techniques had been
developed for microencapsulation of cells but these techniques presented threat to the
cells due to the harsh or chemical treatment applied. In this research, a simple and
economic electronic aerosol atomisation system was proposed for producing calcium
alginate microcapsules. The system was developed with the incorporation of a
conventional syringe pump, a customised air pump and motor controller circuits. The
microcapsules and 3D microtissues were biophysically characterised. For the output of
the system, the microcapsules size slightly increased with the extrusion rates and
decreased significantly with the airflow rates. At an extrusion rate of 20 µl/min and
airflow rate of 0.3 l/min, microcapsules with a diameter ranging from 220 - 270 µm
were generated. The polymerisation time for the microcapsules was approximately 10
minutes after the immersion in calcium chloride solutions. The microcapsules showed
high porous surface structure in field emission-scanning electron microscopy (FE-SEM)
imaging. Keratinocytes (HaCaT) and Oral Squamous Cell Carcinoma (ORL-48) cells at
cell densities of 3 × 107 and 9 × 10
7 cells/ml, respectively were applied for encapsulation
and successfully grew into microtissues after 16 days of culture. The fourier transform
infrared (FTIR) spectroscopy of the 3D cells showed stretching in phosphate bond of
Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) backbone, lipid and protein.
The cells of HaCaT and ORL-48 microtissues were viable and they were characterised
by different nucleus size. Replating experiment demonstrated that the cells in the
microtissues could spread and proliferate in the culture dish. The electronic aerosol
atomisation system developed in this work has successfully produced microcapsules
with controllable size and applicable for growing microtissues. The microtissues
produced are potentially a useful cell model for the study of cytochemicals.
vii
ABSTRAK
Pengkapsulan sel adalah teknologi mikro digunakan secara meluas dalam bidang
penyelidikan sel dan tisu, pemindahan tisu dan perubatan regeneratif. Pelbagai teknik
telah dibangun untuk menghasilkan kapsul mikro untuk membalut sel tetapi memberi
ancaman kepada sel disebabkan layanan kasar atau kimia semasa proses pengkapsulan.
Dalam kajian ini, sistem pengabusan aerosol elektronik yang mudah dan ekonomi telah
dicadang untuk menghasilkan kapsul mikro kalsium alginat. Sistem ini dibangunkan
dengan penggabungan pam picagari konvensional, pam udara dan litar pengawal motor.
Kapsul mikro dan tisu mikro telah dicirikan. Bagi output sistem, saiz kapsul mikro
menunjukkan sedikit peningkatan dengan kadar penyemperitan dan menurun nyata
sekali dengan kadar aliran udara. Pada 20 µl/min kadar penyemperitan dan 0.3 l/min
kadar aliran udara, kapsul mikro dengan diameter 220 - 270 µm telah dihasilkan. Masa
jangkaan polimerisasi kapsul mikro adalah 10 minit selepas rendam dalam larutan
kalsium klorida. Kapsul mikro menunjukkan struktur permukaan yang berliang tinggi
dalam pengimejan mikroskopi elektron imbasan-emisi medan (FE-SEM). Sel
keratinocytes (HaCaT) dan Oral Squamous Cell Carcinoma (ORL-48) pada kepadatan 3
× 107 dan 9 × 10
7 sel/ml telah digunakan untuk pengkapsulan dan berjaya tumbuh
menjadi tisu mikro selepas 16 hari kultur. Inframerah transformasi Fourier (FTIR) bagi
sel 3D menunjukkan peregangan ikatan fosfat dalam tulang belakang asid
deoksibonukleik (DNA) dan asid ribonukleik (RNA), lipid dan protein. Sel tisu mikro
HaCaT dan ORL-48 hidup tetapi menunjukkan perbezaan dalam saiz nukleus.
Eksperimen pemplatan semula menunjukkan bahawa sel-sel dalam tisu mikro boleh
mengasingkan diri dan proliferat dalam bekas kultur. Sistem pengabusan aerosol
elektronik dihasil dalam kerja ini berjaya menghasilkan saiz kapsul mikro yang boleh
dikawal dan dapat digunakan untuk menumbuh tisu mikro. Tisu mikro yang dihasilkan
adalah berpotensi untuk dijadikan model sel yang berguna untuk kajian sitokimia.
viii
CONTENTS
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
LIST OF ASSOCIATED PUBLICATIONS v
ABSTRACT vi
ABSTRAK vii
CONTENTS viii
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF SYMBOLS AND ABBREVIATIONS xxii
LIST OF APPENDICES xxvi
CHAPTER 1 INTRODUCTION 1
1.1 Research background 1
1.2 Problem statement 3
1.3 Aim 5
1.4 Objectives 5
1.5 Scopes 5
1.6 Thesis contribution 6
1.7 Thesis outline 7
ix
CHAPTER 2 LITERATURE REVIEW 8
2.1 Introduction 8
2.2 Cells and tissue 8
2.2.1 Extracellular matrix (ECM) and cell 10
adhesion
2.3 Epithelial cells and skin 12
2.3.1 Human keratinocyte cell lines (HaCaT) 12
2.3.2 Oral squamous cell carcinoma cell line 13
(ORL-48)
2.4 Rationale of growing 3D cells 14
2.5 Methods for culturing microtissues 15
2.6 Microencapsulation 16
2.6.1 Application of microencapsulation 18
2.6.2 Technologies for microencapsulation of 20
cells
2.6.2.1 Extrusion, Jet break-up methods 21
and spinning disc
2.6.2.2 Micro nozzle array and vibrating 22
nozzle
2.6.2.3 Microfluidic device 23
2.6.2.4 Electrostatic droplet generation 25
2.6.2.5 Atomisation technique 27
2.6.3 Biopolymers used for cell 30
microencapsulation
2.7 Review on microscopy and spectroscopy 33
techniques
2.7.1 Inverted phase contrast microscopy 33
2.7.2 Fluorescence microscopy 34
x
2.7.3 Field emission scanning electron 35
microscopy (FE-SEM)
2.7.4 Fourier transform infrared (FTIR) 37
spectroscopy
2.8 4‟, 6-diamidino-2-phenylindole dihydrochloride 39
(DAPI) staining
2.9 Live/dead viability assay kit 39
2.10 Alginate lyase 40
2.11 Summary 40
CHAPTER 3 METHODOLOGY 41
3.1 Introduction 41
3.2 Development of an electronic aerosol atomisation 45
system
3.2.1 Hardware design of an electronic aerosol 45
atomisation system
3.2.2 Controller circuit design of an electronic 49
aerosol atomisation system
3.2.3 Programming the microcontroller for the 51
air pump
3.2.4 Performance validation of the electronic 56
aerosol atomisation system
3.2.4.1 Verification of the PWM signals 56
generated by the circuit of aerosol
atomisation system
3.2.4.2 Investigate the relationship of 56
potentiometer voltage and PWM
signals
xi
3.2.4.3 Investigate the relationship of PWM 57
signals and output voltage to air pump
3.2.4.4 Investigate the effect of PWM signals 57
to airflow rate
3.2.4.5 Extrusion rate calibration of the 58
commercial syringe pump
3.2.4.6 Airflow rate calibration of the 59
aerosol atomisation system
3.3 Experimental setup of aerosol atomisation system 59
for producing microcapsules
3.3.1 Validation of microcapsules drop distance 61
3.3.2 Determining the size of calcium alginate 63
microcapsules
3.3.3 Spectroscopy analysis of the calcium
alginate microcapsules
3.4 Microencapsulation of cells 64
3.4.1 Cell culture and preparation 64
3.4.2 Preparation of cells-alginate suspension 65
3.4.3 Microencapsulation of cells using the 66
developed aerosol atomisation system
3.4.4 3D cell culture and monitoring 67
3.5 Biophysical properties characterisation of the 67
microcapsules and microtissues
3.5.1 Fourier transform infrared (FTIR) 67
spectroscopy measurement
3.5.2 FE-SEM imaging of the calcium alginate 68
microcapsules and 3D microtissues
xii
3.5.3 DAPI (4‟, 6-diamidino-2-phenylindole 70
dihydrochloride) staining
3.6 Live and dead cell stainings 70
3.7 Degradation of calcium alginate microcapsules 71
membranes using alginate lyase
3.8 Replating of microtissues 71
3.9 Summary 71
CHAPTER 4 RESULTS AND DISCUSSION 73
4.1 Introduction 73
4.2 The electronic aerosol atomisation system 73
4.2.1 The mechanism and operation of an 74
electronic aerosol atomisation system
4.2.2 System verification 78
4.2.2.1 Duty cycle of the pulse width 78
modulation (PWM)
4.2.2.2 The relationship of potentiometer 79
voltage and pulse width modulation
4.2.2.3 The relationship of pulse width 80
modulation and output voltage to
air pump
4.2.2.4 Airflow rate measurement 81
4.2.2.5 Calibration of the extrusion rate of 82
the syringe pump
4.2.2.6 Airflow rate calibration of the 83
aerosol atomisation system
4.3 The effect of drop distance to the structure of 84
the microcapsules
4.4 The effects of extrusion and airflow rates to the size 87
of microcapsules
xiii
4.4.1 The effects of different extrusion rates to 87
the size of microcapsules
4.4.2 The effects of different airflow rates to 90
the size of microcapsules
4.5 Polymerisation time of calcium alginate 95
microcapsules based on spectroscopy analysis
4.6 In vitro growth of encapsulated cells (3D cells) 97
into microtissues
4.7 The biophysical properties of the microcapsules 101
and microtissues
4.7.1 FTIR spectrum of calcium alginate 101
encapsulated cells
4.7.2 FE-SEM physical and surface structure 105
scanning
4.7.2.1 Physical structure of calcium 105
alginate microcapsules
4.7.2.2 Physical structure of 3D 106
microtissues
4.7.3 Nucleus distribution of the cells in the 108
microtissues
4.8 Viability of the cells in microtissues 109
4.9 3D microtissues extracted from degraded 110
calcium alginate microcapsules
4.10 The effect of replating the 3D microtissues 111
4.11 Summary 113
CHAPTER 5 CONCLUSION AND FUTURE WORK 114
5.1 Conclusion 114
5.2 Recommendations for future works 115
xiv
REFERENCES 117
APPENDIX A 144
APPENDIX B 147
APPENDIX C 154
VITA 156
xiv
LIST OF TABLES
2.1 A summary of 3D cell culture methods for culturing
3D microtissues
15
2.2 Comparison of different microencapsulation
technologies for encapsulation of cells
20
2.3 Summary of materials and cell types involved with
microfluidic technologies used for cell encapsulation
25
2.4 An overview on biopolymers used for cells
encapsulation, the encapsulated cells type and their
applications
30
3.1 The specification of electronic aerosol atomisation
system and the parameters used to generate 3D cells
43
3.2 Establishment of experiments 44
4.1 The standard operating procedures of the electronic
aerosol atomisation system
77
xv
LIST OF FIGURES
2.1 The anatomy of human cell 9
2.2 The four basic types of tissue 10
2.3 Cell adhesion to the ECM. (a) Suspended cells
adhere to the surface of ECM via integrins (b) The
structures of actin cytoskeleton, focal adhesion
complexes, integrin receptors, and adhesion proteins
to form cross-linked platforms
11
2.4 Phase contrast micrographs of HaCaT cells cultured
for 3 days at 1 : 6 dilutions (Scale bar: 100 µm)
13
2.5 Phase contrast micrographs of ORL-48 cells cultured
for 3 days at 1 : 6 dilutions (Scale bar: 100 µm)
14
2.6 Different morphology of microcapsules (a) Mono-
core, Single-core or reservoir type, (b) Poly-core or
Multiple-core, (c) and (d) Matrix type
17
2.7 Principle of immunoisolation by a microcapsule 18
2.8 Schematic diagram of different microencapsulation
processes in forming microcapsules: (a) Extrusion,
(b) Jet cutter and (c) Spinning disc
22
2.9 Schematic diagram of microencapsulation processes
in alginate: (a) Micro nozzle array and (b) Vibrating
nozzle
23
2.10 Illustrations of microfluidics system mechanism for
microencapsulation
24
2.11 Illustration of microcapsules fabrication methods
based on microfluidics device. (a) Flow-focusing and
24
xvi
(b) T-junction beads formation
2.12 A schematic view of electrostatic droplet generation
system
26
2.13 Electric charges distribution when the charged
droplet is hanging on the needle tip. Capillary,
electrostatic and gravitational forces are exerted on
the charged droplet
27
2.14 The coaxial air-flow experiment setup 28
2.15 The illustration of the airflow based on the pressure
at two different point
29
2.16 The working principle of aerosol atomisation system 29
2.17 The monomers of alginate 31
2.18 The molecular structure of calcium alginate 32
2.19 (a) Calcium binding site in G-blocks and (b) “Egg-
box” model for alginate gel formation
33
2.20 The working principal of phase contrast microscope 34
2.21 The working principal of fluorescence microscope 35
2.22 The working process of field emission-scanning
electron microscopy
36
2.23 A FE-SEM available in Microelectronic and
Nanotechnology-Shamsuddin Research Centre,
Universiti Tun Hussein Onn Malaysia
37
2.24 Working principle of FTIR spectroscopy 38
2.25 Fourier transform infrared spectroscope, Perkin
Elmer Spectrum 100
38
3.1 Flow chart for the development of electronic aerosol
atomisation system and techniques used to
characterise the microcapsules and 3D microtissues
42
3.2 Three major parts of the electronic aerosol
atomisation system
45
3.3 The conceptual design of a customised electronic 46
xvii
aerosol atomisation system
3.4 The control panel of the electronic aerosol
atomisation system
47
3.5 The direct current air pump of the electronic aerosol
atomisation system
47
3.6 The block diagram of an electronic aerosol
atomisation system
48
3.7 The schematic circuit design diagram of the
electronic aerosol atomisation system
50
3.8 The PCB layout for the circuit connection 51
3.9 The programming flow of the aerosol atomisation
system
52
3.10 Source code for controlling the PWM signal 53
3.11 Source code of start or stop button 53
3.12 Source code of airflow rate function control 55
3.13 The setup for airflow rate measurement of the air
pump
58
3.14 A schematic illustration of the experimental setup of
an electronic aerosol atomisation system for
generating calcium alginate microcapsules
60
3.15 An illustration of the aerosol nozzle. (a) The
schematic diagram of the insulin syringe needle head
area and (b) the picture of the insulin syringe needle
head
61
3.16 (a) The microcapsules drop distance validation setup
and (b) the schematic diagram of the dispersed
coverage (C), angle (θ) and drop distance (D)
62
3.17 The 96 wells plate containing calcium alginate
microcapsules for spectroscopy analysis
64
3.18 Preparation of 1.5 % wt/v cell-alginate suspension 66
3.19 Insulin needle with 100 µl cell-alginate suspension 66
(b) Airflow
xviii
3.20 The samples on FTIR spectroscopy stage. (a) Sodium
alginate powder, (b) calcium alginate microcapsules
and (c) calcium alginate encapsulated cells
68
3.21 The mounting stub with microcapsules and 3D
microtissues
69
4.1 The overall experiment of an electronic aerosol
atomisation system
74
4.2 (a) The electronic circuit boards in the casing and (b)
the front panel of the electronic aerosol atomisation
system
75
4.3 The airflow rate knob used to select the airflow rate
and the LCD displays the selected airflow rate of the
electronic aerosol atomisation system
76
4.4 The output signals of PWM: (a) 0 %, (b) 20 %, (c) 40
%, (d) 60 %, (e) 80 % and (f) 100 % duty cycle. T
denotes the period for a cycle of pulse
79
4.5 The corresponding value of PWM to the
potentiometer voltages manipulation for controlling
the air pump
80
4.6 The corresponding value of output voltage to the
duty cycle of PWM signals manipulation
81
4.7 The correspond value PWM to the airflow rate of the
aerosol atomisation circuit developed
82
4.8 Calibration results for the extrusion rate of the
commercial syringe pump
83
4.9 The airflow rates calibration result of the aerosol
atomisation system
84
4.10 The drop distance between needle tip and CaCl2 bath
surface at (a) 3, (c) 6 and (e) 9 cm and the
photomicrographs of polymerised calcium alginate
formed at drop distance of (b) 3, (d) 6 and (f) 9 cm
86
xix
(Scale bar: 200 µm)
4.11 The drop distance between the aerosol nozzle and the
CaCl2 solution surface determine the microdroplets
coverage region
86
4.12 Morphological and size distribution of calcium
alginate microcapsules with the extrusion rate of (a)
5, (b) 10, (c) 15 and (d) 20 µl/min and a fixed airflow
rate of 0.3 l/min (Scale bar: 200 µm)
88
4.13 The effect of different extrusion rates generated by
the aerosol atomisation system on average diameter
distribution of microcapsules (airflow rate = 0.3
l/min)
89
4.14 The size distribution of calcium alginate
microcapsules prepared by aerosol atomisation
system with (a) 5, (b) 10, (c) 15 and (d) 20 µl/min
extrusion rate and a fixed airflow rate of 0.3 l/min
90
4.15 Morphological and size distribution of calcium
alginate microcapsules with the airflow rate of (a)
0.2, (b) 0.3, (c) 0.4 and (d) 0.5 l/min and a fixed
extrusion rate of 20µl/min (Scale bar: 200 µm)
92
4.16 The effect of different airflow rates generated by the
aerosol atomisation system on average diameter
distribution of microcapsules (extrusion rate = 20
µl/min)
93
4.17 The size distribution of calcium alginate
microcapsules prepared by aerosol atomisation
system with (a) 0.2, (b) 0.3, (c) 0.4 and (d) 0.5 l/min
airflow rate and a fixed extrusion rate of 20 µl/min
94
4.18 The effects of extrusion rate and airflow rate to the
size of microcapsules
95
4.19 The polymerisation absorbance of the microcapsules 96
xx
upon irradiation at wavelength of 330 nm light
4.20 The microcapsules of calcium alginate (a) before and
(b) after polymerisation in calcium chloride bath
97
4.21 Phase contrast microscopic images of calcium
alginate encapsulated 3D HaCaT cells in growth
transition for 16 days of culture. (a) Day 0, (b) 2, (c)
4, (d) 6, (e) 8, (f) 10, (g) 12, (h) 14 and (i) 16 (Scale
bar: 100 µm)
99
4.22 Phase contrast microscopic images of calcium
alginate encapsulated 3D ORL-48 cells in growth
transition for 16 days of culture. (a) Day 0, (b) 2, (c)
4, (d) 6, (e) 8, (f) 10, (g) 12, (h) 14 and (i) 16 (Scale
bar: 100 µm)
100
4.23 Protrusion of cells starting from Day 8 of cells
culture, the dissolved calcium alginate over time and
2D monolayer cells (Scale bar: 100 µm)
101
4.24 FTIR spectra of (a) sodium alginate, (b) calcium
alginate microcapsules, calcium alginate
encapsulated with (c) HaCaT and (d) ORL-48 cells
104
4.25 The size, shape and surface structure of the calcium
alginate microcapsules at (a) 150 ×, (b) 300 × and (c)
10,000 × magnification
106
4.26 Field emission-scanning electron micrographs of 3D
HaCaT microtissue under FE-SEM at (a) 150 ×, (b)
300 × and (c) 1,500 × magnification, respectively
107
4.27 Field emission-scanning electron micrographs of 3D
ORL-48 microtissue under FE-SEM at (a) 150 ×, (b)
300 × and (c) 1,500 × magnification, respectively
108
4.28 DAPI staining of cells in the 3D microtissues of (a)
HaCaT and (b) ORL-48 after 16 days of culture
(Scale bar: 100 µm)
109
xxi
4.29 Live and dead staining fluorescence microscopic
micrographs of calcium alginate encapsulated (a)
HaCaT and (b) ORL-48 3D microtissues after 16
days of culture (Scale bar: 100 µm)
110
4.30 Phase contrast microscopic images of the calcium
alginate encapsulated (a) HaCaT and (b) ORL-48
microtissues before degradation process, and the
extracted (c) HaCaT and (d) ORL-48 microtissues
after degradation process at 100 × magnification
(Scale bar: 100 µm)
111
4.31 Phase contrast microscopic image of replating the 3D
HaCaT microtissues (a) Day 0, (b) Day 1, (c) Day 2
and (d) Day 3 (Scale bar: 100 µm)
112
4.32 Phase contrast microscopy image of replating the 3D
ORL-48 microtissues (a) Day 0, (b) Day 1, (c) Day 2
and (d) Day 3 (Scale bar: 100 µm)
113
xxii
LIST OF SYMBOLS AND ABBREVIATIONS
2D - Two-Dimensional
3D - Three-Dimensional
- Alpha
- Beta
oC - Degree Celsius
< - Lower Than
% - Percent
cells/ml - Cells per Milli Litre
cm - Centimeter
cm2 - Centimeter Square
f - Frequency
F - Force
cm-1
- Reciprocal Centimeter
kg/m3 - Kilo Gram per Cubic Meter
kV - Kilo Volt
l/min - Litre per Minute
µg/ml - Micro Gram per Milli Litre
µl - Micro Litre
µl/min - Micro Litre per Minute
µm - Micro Meter
µM - Micro Molar
mA - Milli Ampere
mg/l - Milli Gram per Litre
mg/ml - Milli Gram per Milli Litre
min - Minute
xxiii
ml - Milli Litre
mm - Milli Meter
mM - Milli Molar
ms - Milli Second
ms-1
- Milli per Second
nm - Nano Meter
nM - Nano Molar
R2 - Coefficient of Determination
s - Second
units/ml - Units per Milli Litre
v - Velocity
V - Volume
A - Ampere
ARES - Advanced Routing and Editing Software
A-T - Adenine−Thymine
ATR - Attenuated Total Reflection
A-U - Adenine−Uracil
BD - Becton Dickinson
CaCl2 - Calcium Chloride
CLS - Cell Line Services
CO2 - Carbon Dioxide
DAPI - 4‟, 6-Diamidino-2-Phenylindole Dihydrochloride
dc - Direct Current
DI - Deionised
DMEM - Dulbecco‟s Modified Eagle Medium
DNA - Deoxyribonucleic Acid
ECM - Extracellular Matrix
ER - Endoplasmic Reticulum
EthD-1 - Ethidium Homodimer
ex/em - Excitation/Emission
FA - Focal Adhesion
xxiv
FBS - Fetal Bovine Serum
FDA - Food and Drug Administration
FE-SEM - Field Emission-Scanning Electron Microscope
FTIR - Fourier Transform Infrared
G - Guluronate
HaCaT - Human Keratinocyte Cell Line
HBSS - Hank‟s Balanced Salt Solution
HTS - High-throughput Screening
Hz - Hertz
I-C - Hypoxanthine−Cytosine
ICF - Inertial Confinement Fusion
IL - Illinois
ISIS - Intelligent Schematic Input System
LABE - Low Angle Backscatter Imaging
LCD - Liquid Crystal Display
LED - Light Emitting Diode
LEI - Lower Secondary Electron Imaging
M - Mannuronate
MiNT-SRC - Microelectronics and Nanotechnology-Shamsuddin Research
Centre
MO - Missouri
N - Newton
Na+ - Sodium
NaCl - Sodium Chloride
NIH - National Institutes of Health
ORL-48 - Oral squamous cell carcinoma (OSCC) cell line
OSCC - Oral Squamous Cell Carcinoma
Pa - Pascal
PCB - Printed Circuit Board
Pd - Dynamic Pressure
PDMS - Polydimethylsiloxane
xxv
PEG - Polyethylene Glycol
PFPE-PEG - Perfluoropolyether - Polyethylene Glycol
PVC - Polyvinyl Chloride
PWM - Pulse Width Modulation
Q - Airflow Rate
RNA - Ribonucleic acid
RPM - Revolutions Per Minute
SD - Standard Deviation
SEI - Upper Secondary Electron Imaging
UK - United Kingdom
US - United States
USA - United States of America
UTHM - Universiti Tun Hussein Onn Malaysia
V - Volt
Vdc - Volt Direct Current
wt/v - Weight per Volume
xxvi
LIST OF APPENDICES
APPENDIX TITLE PAGE
A List of hardware used and specification 144
B The Arduino source code of electronic aerosol
atomisation system
147
C The mechanism and specification of PVC airflow
meter
154
CHAPTER 1
INTRODUCTION
1.1 Research background
Monolayer cultures in plastic vessels are routinely used in biological studies. However,
the use of two-dimensional (2D) cell models for cell biological studies has its limitations
[1, 2]. In 2D culture, the proliferation, differentiation, gene and protein expression,
functionality and morphology of cells is considerably different from their physiological
origin in vivo [3]. By contrast, the three-dimensional (3D) cell culture creates an
artificial environment where cells are permitted to grow or interact with its surroundings.
3D cell culture is believed to have a better approximation to the tissue model for cell and
tissue research because it restores specific biochemical and morphological features
similar to the corresponding tissue in vivo [4]. In 3D cell culture, the connections
between cells are more native-like and the behaviour of cells is more reflective of in vivo
cellular responses [3, 5].
Regenerative medicine or biotechnology for creating living functional tissues in
vitro is urgently needed for repair or replacement of damaged organs [6], application in
cell culture and tissue engineering [7], pharmacological testing and bioengineering fields
[8]. Microencapsulation is an intensive research area to create cell and tissue model for
rehabilitation of functional tissues [9] and therapeutics purpose [10, 11]. It is a technique
which encloses cells within a membrane or shell. It has been widely studied since 1960s
[12]. A microcapsule is a hollow chamber with diameters in the range of a few
micrometers to several thousands of micrometers [13, 14]. The semipermeable
2
membrane of the microcapsule can facilitate the transportation of proteins,
deoxyribonucleic acid (DNA), and drug and allows the diffusion of oxygen, nutrients,
therapeutic products and wastes, while blocking the entry of antibodies and
immunocytes [15]. In tissue transplantation, microcapsules segregate cells from the
surrounding tissue to protect the implanted cells from the recipient‟s immune system
[16]. Therefore, cell encapsulation in biocompatible and semipermeable biopolymeric
membranes is an effective method to overcome rejection of the implanted organ [17].
There are various types of biopolymer such as agarose, collagen, alginate,
chitosan and gelatin that are widely applied for encapsulation of cells [14, 18]. These
materials are different in polymerisation process and hence this consideration greatly
influences the design of the microencapsulation system. Among them, alginate is the
most commonly used biopolymer for encapsulation of living cells because of many
advantages it offers [19, 20]. Alginate is a naturally derived polymer, biocompatible in
vitro and in vivo, with excellent biodegradability and provide rapid gelation process in
the presence of divalent cations at room temperature [21]. Indeed, alginate has been
employed for encapsulating cells and tissues to be transplanted into human body, as it is
biocompatible to both the host and the enclosed cells [22]. Furthermore, alginate has
been studied extensively and it is currently recognised as a clinically ready application
material by the United States Food and Drug Administration (US FDA) [15, 23].
A few methods had been developed for the microencapsulation of cells such as
simple dripping [24, 25], micromolding [26, 27], extrusion [28], microfluidic device [29,
30], electrostatic droplet generation [31-33], coaxial air-flow [34-36], vibration [37] and
jet cutting techniques [38-40]. For simple dripping technique, the diameter of the
capsules produced is usually ranging between 600 and 1000 µm [41]. This technique is
used to produce microcapsules that does not involve with chemical or mechanical
treatment. Other techniques such as microfluidic, micromolding and electrostatic
dropping could produce smaller size of microcapsules ranging from 200 to 600 µm [34]
but these techniques are considered harsh because involvement of the organic solvent,
oil phase, high voltage and ultra-violet treatment to produce the microcapsules. The
requirement for post-processing treatment due to the harsh generation techniques may be
3
threatening the survival rate of the living organism encapsulated in the microcapsules [8,
28, 42].
Amongst previous methods discussed [26-28, 30-33, 37-39, 43], aerosol
atomisation technique is a simple and efficient method to generate microcapsules with
well-controlled size and shape without the use of harsh chemicals [19, 36, 44]. In this
study, an electronic aerosol atomisation system had been developed for the generation of
3D human keratinocytes (HaCaT) and oral squamous cell carcinoma (OSCC) cells
(ORL-48) in microencapsulations of calcium alginate that leads to the growth of 3D
microtissues in vitro.
1.2 Problem statement
Alginate based capsules can be generated by simply extruding droplets of sodium
alginate solution from a syringe needle and the droplets are immediately allowed to
polymerise in the calcium chloride bath. However, the simple dripping technique usually
produced large diameter capsules of alginate in millimeter, that were recognised as
unsuitable for medical and biotechnological applications [37, 45]. The size of the droplet
is mainly dependent on the orifice diameter [46] and dripping can only be achieved
when the extruded droplet‟s of alginate continue to grow until its mass overcomes the
surface tension at the tip of the needle [47]. Because of this limitation, other approaches
have been developed to create microcapsules with smaller diameter [43]. Smaller
alginate capsules in micron size are desirable because this range of microcapsules can
equilibrate rapidly across the ultrathin membrane with larger surface to volume
relationship, and hence provide better transport of gases and nutrients for the
encapsulated cells [48]. Generation of alginate droplets by the electrostatic and JetCutter
technique were shown to decrease the size of the droplets or capsules compared to
normal dripping [24]. However, these techniques required sophisticated high voltages or
strong electric fields, complex and bulky design of devices, respectively, that might be
of high demand of energy and time during the fabrication of microencapsulation system
[39, 49-51]. Microcapsules formed based on microfluidic emulsion technique are
covered with oil and hence post-processing treatment is required to remove the oil film
4
for application in cell microencapsulation [52]. This is because the oil layer could block
the exchange of gas and nutrient to the cells in the microcapsules. Involvement of harsh
treatment to remove the oil film causing the cells in the microcapsules exposed more to
the divalent ions or solvents which may present threats to the survival rate of the cells [8,
28]. Hence, the simpler the production process(without harsh and post-processing
treatment), the less threat to the cells whilst ensuring cells to proliferate in the
encapsulations.
In this thesis, an electronic aerosol atomisation system is proposed to generate
the desired size of calcium alginate microcapsules for the microencapsulation of cells.
Although the aerosol atomisation method has been developed previously [53, 54], but
the microcapsules size was ranging from 10 to 40 µm which is too small and not suitable
for cells encapsulation. Current applications (air jets, fuel injection and spray coating)
based on aerosol atomisation technique required high air flow rate (50 - 600 l/min) and
large volume (millilitre) of solution to create small beads size (approximately 1 - 3.5
µm) [55]. Thus, an adjustable electronic aerosol atomisation system employed for this
research was designed to produce different airflow rates (0.2 - 0.5 l/min), in which it can
be used to disperse small volume of cells-alginate suspension (microlitre) and to
generate larger size of microcapsules with controllable size (range in 80 - 360 µm)
which is suitable for cells encapsulation. Instead of using compressed air from a gas
cylinder [19, 36] which is costly, the electronic aerosol atomisation system presented a
different approach in the generation of airflow by using a direct current (dc) air pump.
Moreover, this research applied OSCC and HaCaT cell lines for the microencapsulation
that had not been reported previously. Oral cancer is the most common disease and it is a
silent killer in the developing world, particularly in Southeast Asia country [56, 57].
Although the etiological factors of oral cancer are well established, the mechanism
developed has rarely been studied and it is not well understood [56, 57]. In vitro
microtissues models of OSCC could support the cancer research. Therefore,
development techniques for generation of OSCC and HaCaT microtissues are essential.
The OSSC (ORL-48) and HaCaT cells encapsulated are expected to grow into
microtissues models that have applications for pharmacology study and preliminary
prediction performance of their efficacy in therapeutic strategies.
5
1.3 Aim
The aim of the research is to develop an electronic aerosol atomisation system to
generate calcium alginate microcapsules that are size controllable and able to
encapsulate cells that leads to the growth of 3D microtissues.
1.4 Objectives
The following research objectives were established to achieve the aim. The objectives
for this research are:
a) To develop an electronic aerosol atomisation system for generating calcium
alginate based microcapsules of cells.
b) To encapsulate human Keratinocytes (HaCaT) and OSCC cell lines (ORL-48)
using microcapsules of calcium alginate to form 3D cells.
c) To characterise the biophysical properties of calcium alginate microcapsules
and the 3D microtissues produced.
1.5 Scopes
The four scopes of the research work are as follows:
a) Development and characterisation of an electronic aerosol atomisation
system to generate microcapsules.
b) Synthesis of calcium alginate microcapsules with a diameter ranging from
200 to 300 µm, as the thickness of human epidermis by using an aerosol
atomisation system.
c) Determine the extrusion rates and airflow rates of the aerosol atomisation
system to generate appropriate size of microcapsules for cell encapsulation.
6
d) Encapsulation of HaCaT and OSCC (ORL-48) cells using calcium alginate to
form 3D cells, monitor their growth in the encapsulation and investigate their
biophysical properties.
1.6 Thesis contribution
The main contributions of this thesis are:
a) Electronic aerosol atomisation system with controllable airflow rate
The aerosol atomisation system has revived previous cell encapsulation techniques with
no post-treatment process, no complex fabrication design of nozzle or high voltage
requirement that would affect the cell survival rate in the alginate microcapsules [8, 28,
29, 42, 58].
b) Round shape and suitable size of 3D cells generated for the application
The findings obtained from the aerosol atomisation system have contributed to the
understanding of how alginate is involved in driving the growth of both HaCaT and
ORL-48 microtissues whereby the microcapsules and 3D cells generated were round
shape and in consistent size [8, 30].
c) Encapsulate new cell lines of HaCaT and ORL-48
This is the first demonstration of microencapsulation of HaCaT and ORL-48 using
calcium alginate microcapsules to be applied as a cell model for cancer research.
7
1.7 Thesis outline
Chapter 1 introduces the overview of this project with technology and technique of
microencapsulation. The problems of the current 3D cell encapsulation technique were
discussed, followed by the problem statement, aims, objectives, scopes, thesis
contribution and thesis outline.
Chapter 2 consists of the review of the essential background study information in
understanding the current body knowledge of microencapsulation and the latest
development or technique developed in the field associated with the research topic.
Chapter 3 presents the methodology used to develop the electronic aerosol
atomisation system and technique to produce 3D cells based on the calcium alginate
microencapsulation. Calcium alginate microencapsulation to generate 3D cell technique,
the development of an electronic aerosol atomisation system, the programming of
microcontroller of the air pump, the circuit design and simulation of the aerosol
atomisation system, the procedure in preparing the cells and calcium alginate for
microencapsulation and the biophysical properties characterisation of the microcapsules
and microtissues were discussed.
Chapter 4 unveils the performance of the aerosol atomisation system based on
the pulse width modulation (PWM), potentiometer voltage and the effects of airflow rate
and extrusion rates to the size of microcapsules. The biophysical properties of
microcapsules generated were assessed using the optical microscopy, fourier transform
infrared spectroscopy (FTIR) and field emission-scanning electron microscopy (FE-
SEM). The growth of 3D HaCaT and ORL-48 cells into microtissues were monitored
using inverted phase contrast and fluorescence microscopy. Nonetheless, the results of
the biophysical properties of the microtissues formed were reported and discussed.
Chapter 5 summarises the problem statement that have been solved, the
objectives that have been achieved and the future works to enhance this research.
CHAPTER 2
LITERATURE REVIEW
2.1 Introduction
This chapter discussed and explained the background knowledge and information of
cells and tissues, types of epithelial cells applied for microencapsulation, rationale of
growing 3D cells, microencapsulations, applications of microcapsules,
microencapsulation technics, biopolymers used for fabrication of microcapsules for cell
microencapsulation and the review on microscopy and spectroscopy techniques applied
in this research.
2.2 Cells and tissue
The basic building blocks of all living things is cell [59]. Cells provide structure for
human body, take in nutrients that are consumed, convert it into energy, and use them to
carry out specialised functions. Organelles are specialised structures that perform
important cellular functions within the cell. Human cells contain nine major organelles
such as the cytoplasm, cytoskeleton, endoplasmic reticulum (ER), golgi apparatus,
lysosomes, mitochondria, nucleus, plasma membrane and ribosomes as shown in Figure
2.1.
9
Figure 2.1: The anatomy of human cell [60]
Tissue is structural organisation of cells with similar or identical specialised
characteristics, contributing to the performance of a specific function. Tissues are parts
of organs that provide numerous functions of organs necessary to maintain biological
life. In humans, there are four basic types of tissue, which are epithelial, connective,
muscular, and nervous tissues (Figure 2.2). Epithelial tissue covers the body surface and
forms the lining for most internal cavities. The major function of epithelial tissue
includes protection, secretion, absorption, and filtration. The skin is an organ made up of
epithelial tissue which protects the body from harmful microbes [61]. Cells of the
epithelial tissue have different shapes. Connective tissue is tissue that supports and binds
other tissues. It consists of connective tissue cells embedded in a large amount of
extracellular matrix.
Mitochondria
Lysosome
Centrioles
Microtubules
Golgi
apparatus
Vesicle
Cytoplasm
Plasma
membrane
Microfilaments
Smooth ER
Ribosome
Rough ER
Nucleus
Nuclear pore
Free ribosome
Peroxisome
10
Figure 2.2: The four basic types of tissue [62]
2.2.1 Extracellular matrix (ECM) and cell adhesion
All cells in solid tissue are surrounded by extracellular matrix (ECM). ECM is
composed of proteins and polysaccharides. In animal cells, the ECM surrounds cells as
fibrils that contact the cells. Cells are linked directly to each other by cell adhesion
molecules at the cell surface. ECM provides mechanical support [63], a biochemical
barrier [64], a medium for extracellular communication [65], cell matrix adhesion [66],
and adhesion matrix for cell migration [67-69] during cell development.
Adhesion of cells to the ECM is key to the regulation of cellular morphology,
migration, proliferation, survival, and differentiation [70]. These functions are essential
during development, maintenance of tissue architecture and the induction of tissue
repair. Integrin are the predominant receptors that mediate cell adhesion to the ECM
proteins [71, 72].
Attachment of cells to ECM components induces clustering of integrin on the
cell surface [73]. The cytoplasmic portions of the clustered integrin then function as a
11
platform for the recruitment of cellular proteins and signaling proteins to the inner
surface of the plasma membrane, where they form structures called focal adhesions (FA)
(Figure 2.3 (a)) [74]. The FA provide strong linkages to the actin cytoskeleton mediated
by integrins to connect cells firmly to the ECM [75].
Cells adhere to the ECM via integrins that function as a heterodimer that
composed of subunits alpha (α) and beta (β) transmembrane linked to cell cytoskeleton
actin microfilaments via talin and vinculin [76]. Talin is a main regulator of the initial
process of FA assembly [77]. During the initial step of FA formation, the binding of
talin to integrin stabilises the ligand-induced clustering by mediating crosslinking of
integrins with vinculin and α-actinin (Figure 2.3(b)) [78].
(a)
(b)
Figure 2.3: Cell adhesion to the ECM. (a) Suspended cells adhere to the surface of ECM
via integrins (b) The structures of actin cytoskeleton, focal adhesion complexes, integrin
receptors, and adhesion proteins to form cross-linked platforms
ECM
Cell Focal adhesion (FA) Nucleus
Talin
Integrin
Bilayer
membrane
Src Paxillin
Actin filament
Tensin
Laminin protein
ECM
12
2.3 Epithelial cells and skin
HaCaT and OSCC (ORL-48) are non-cancer and cancer epithelial cells, respectively.
The microtissue models for epidermis and oral cancer cell study are scarce. Hence, the
growth of both cell types into biomimetic microtissues would provide value in tissue
implant [79], pharmacology [80] or even cancer therapeutic drugs study [11].
2.3.1 Human keratinocyte cell lines (HaCaT)
The epidermis is a squamous epithelium that forms the protective layer of the skin. It
consists of renewing tissue with the main cell type (keratinocytes), superpositioned and
organised into four histologically distinct cellular layers: stratum corneum, stratum
granulosum, stratum spinosum and stratum basale [81]. The HaCaTs have a close
similarity in functional competence to normal keratinocytes [82]. HaCaT is a
spontaneously immortalised and transformed aneuploidy immortal keratinocyte cell line
from adult human skin [83]. The naturally immortalised human HaCaT cell line can be
grown in culture vessel (Figure 2.4) for long periods of time [84]. This cell line has been
widely used for studies of skin biology, differentiation and scientific research as a
paradigm for epidermal cells [84-86]. HaCaT grew in the form of monolayer and
adherent to the culture dish easily. Under typical culture conditions, HaCaT cells have a
partially to fully differentiated phenotype due to the high calcium content of both
standard media and fetal bovine serum. HaCaT cells are used for high differentiate and
proliferate capacity in vitro [87]. HaCaT cells drastically reduced tissue regeneration
compared to normal epidermal keratinocytes [88, 89]. The deficiency in HaCaT cells
were not due to the permanent loss of differential functions and can be solved by the
addition of growth factors [84].
13
Figure 2.4: Phase contrast micrographs of HaCaT cells cultured for 3 days at 1 : 6
dilutions (Scale bar: 100 µm)
2.3.2 Oral squamous cell carcinoma cell line (ORL-48)
Oral cancer is defined as malignant lesion within oral cavity. Most cancerous oral cells
originate from the oral squamous epithelium cell which is the primary surface structure
of the lips and mucous membrane of the oral cavity [90]. OSCC has been histologically
characterised as irregular nests, columns or malignant epithelial cells [91].
Abnormalities of oral cancerous cells are believed to be associated with several
consecutive genetic mutations [92]. By clonal selection of viable cells which have
accumulated genetic damages, normal mucosa cells ultimately evolve into malignant
mucosa cells over an indefinite period [93].
ORL-48 is one of the OSCC cell lines derived in Cancer Research Malaysia.
ORL-48 was surgically explanted specimens obtained from untreated primary human
oral squamous cell carcinomas of the oral cavity [56]. It was derived from a female
donor patient at the age of 79 years old having cancer tumour in the mouth and gum
[56]. ORL-48 cell lines grew in the form of monolayers (Figure 2.5) with the population
doubling times ranging between 26.4 and 40.8 hours and they are immortal [56].
14
Figure 2.5: Phase contrast micrographs of ORL-48 cells cultured for 3 days at 1 : 6
dilutions (Scale bar: 100 µm)
2.4 Rationale of growing 3D cells
Sub-culturing monolayer of cells in plastic vessels is a routine procedure for cell biology
study. However, the validity of using such a 2D cell model for cell biology or
pharmacological study is controversial [1, 2]. Cells grown in monolayer proliferate
involuntarily due to the contactless spreading of cells and it has been shown to produce
limited amount of extracellular matrix proteins [94]. The cell behaviour such as
proliferation, differentiation, gene and protein expression, general cell function and
morphology is considerably different from their physiological origin in vivo [3]. In
contrast, 3D cell culture creates an artificial environment in which biological cells are
allowed to grow and interact with its surrounding environment in three dimensional. 3D
cell culture is also proven to have better approximation to the tissue model for cell and
tissue research because it reconstructs specific biochemical and morphological features
similar to the tissue in vivo [4]. The connections between cells are more native-like and
the cellular behaviour is more realistic.
15
2.5 Methods for culturing microtissues
3D cell culture methods are commonly accepted as more physiologically relevant
methods and are believed to improve prediction of drug development process [95, 96].
There are several methods for culturing cells into 3D microtissues, which involved
scaffolds, matrices (scaffold-free), gels or hydrogels and bioreactor as listed in Table
2.1. Scaffold based method is available in variety of materials with different porosities,
permeabilities and mechanical characteristics designed to mimic the in vivo ECM of the
specific tissues [1]. Whereas, microtissue culture using scaffold-free platforms do not
contain added biomaterials or ECM. Cells grown and organised with their own
generated ECM [96]. Gels or hydrogels culturing method aim to mimic the ECM and it
has a soft tissue-like stiffness [97]. Cells can be cultured directly on the hydrogels
(agarose, collagen and alginate) to form microtissues [98-100]. This method can be
combined with other methods, such as scaffolds and microchips. The most ideal 3D cell
culture method for high volume cell production and in vitro tissue engineering
applications are the bioreactors method [101]. Microtissues cultured by using bioreactor
method allows circulation of nutrients and removal of wastes within the reactor.
Table 2.1: A summary of 3D cell culture methods for culturing 3D microtissues
Methods Advantages Disadvantages Applications Reference
a) Scaffolds
• Polymeric Hard Scaffolds
• Biologic Scaffolds
• Micropatterned Surface
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 (HTS)
options limited
• Basic research
• Drug discovery
• Cell expansion
[102-105]
16
Table 2.1 (continued): A summary of 3D cell culture methods for culturing 3D
microtissues
Methods Advantages Disadvantages Applications Reference
b) Matrices
(Scaffold-free)
• Hanging Drop Microplates
• Microfluidic
• Microarray
No added
materials
Consistent
spheroid
formation
(control over
size)
Co-cultures
possible
Transparent
HTS capable
Compatible with
liquid handling
tools
Inexpensive
No support or
porosity
Limited
flexibility
Size of spheroid
limiting
Basic research
Drug discovery
Personalised
medicine
[96, 106,
107]
c) Gels / Hydrogels
• Micromold
• Microencapsulation
• Large variety of
natural or
synthetic
materials
• Customisable
• Co-cultures
possible
• Inexpensive
• Gel-to-gel
variation and
structural changes
over time
• Undefined
constituents in
natural gels
• May not be
transparent
• HTS options
limited
• Basic research
• Drug discovery
[103, 104,
108]
d) Bioreactor
• Several options
available
• High volume cell
production
• Customisable
• Cost
• HTS options
limited
• Basic research
• Tissue
engineering
• Cell expansion
[101]
2.6 Microencapsulation
Microencapsulation is a technology of packaging solids, liquids or gases to be
encapsulated inside a tiny sphere, called microcapsule. A microcapsule is a small sphere
with hollow chamber, micro-porous and semi-permeable wall around it [109].
Microencapsulation of active compounds is defined as a series of techniques whereby a
17
compound is coated or masked, to present it in the form of multiparticulate system.
Microencapsulation process can be classified in terms of the microparticles or
microspheres, based on their external morphology and internal structure (homogeneous
or solid spheres) in micrometer range diameters [13, 34]. The material inside the
microcapsule is referred to as the core, internal phase, or fill, whereas the wall is
sometimes called a shell, coating, or membrane. Microcapsules can be classified into
three basic categories as mono-core (also called single-core or reservoir type), poly-core
(also called multiple-core) and matrix types (Figure 2.6). Mono-core is microcapsule
which has a single hollow chamber within the capsule [110]. Poly-core is microcapsule
which has a number of different size chambers within the shell [110]. Matrix type is of
microparticle that has the active compounds integrated within the matrix of the shell
material [110]. However, the morphology of the internal structure of a microparticle
depends mainly on the shell materials and the microencapsulation methods that are
employed [110].
(a) (b) (c) (d)
(a) (b) (c) (d)
Figure 2.6: Different morphology of microcapsules. (a) Mono-core, Single-core or
reservoir type, (b) Poly-core, Multiple-core, (c) and (d) Matrix type [110]
The main functions of microencapsulation are to isolate, immobilise, stabilise
and protect the core from its surroundings. Capsular membrane is to shield the material
within and control the flow of materials across the membrane (Figure 2.7). It allows
manipulating the diffusion rate of molecules leaving the microcapsule under specific
conditions and protecting against degradation agents (humidity, light, pH and gases)
[111]. Microcapsules are also performing as carriers for drugs delivery, removal of
fragrances and other compounds to facilitate product handling and improve material
18
ability [34]. For microencapsulation of cells, the selection of a suitable encapsulating
material is critical. The material is required to have appropriate porosity, which can
facilitate the transport of nutrients, proteins, DNA, and drug while blocking attack of
antibodies and immune cells [6]. The capsules must be mechanically stable and easy to
handle. These requirements may be fulfilled by controlling the pore size and the
thickness of encapsulating polymer membrane at microscale. Smaller pore size and
thicker capsules membrane showed higher mechanical stability [112, 113]. The cell
viability and metabolic status must be optimal if the encapsulated cells are in the order
of hundreds micron in size [15].
Figure 2.7: Principle of immunoisolation by a microcapsule [15]
2.6.1 Application of microencapsulation
Microencapsulation offers the possibility to microencapsulate any substances in
polymeric materials [6]. It is well known as the promising technique to fabricate novel
19
micro and nanostructured materials applied to a wide variety of applications. There are
various application of microcapsules that already been introduced in the market.
One of the most important applications of microencapsulated products is in the
area of crop protection [114, 115]. Polymer microcapsules, such as gelatin, serve as
efficient delivery vehicles to deliver pheromone by spraying the capsule dispersion and
protect the pheromone from oxidation and light during storage and release [116].
The major applications area of encapsulation technique is pharmaceutical or
biomedical for controlled drug delivery [117-120]. Several drug delivery systems are
replacement of therapeutic agents, gene therapy and vaccines use. The capsules are
engineered to stick tightly to and even penetrate linings in the gastrointestinal track
before transferring the drug contents over time into circulatory system or the targeted
spot [119, 121]. Other than that, one of the most important medical applications of
microencapsulation technology is to serve as a cushion or implant, such as breast
implant [110].
Microencapsulation is used to overcome all the challenges in food industry by
providing technology to incorporate minerals, vitamins, flavours [122] and essential oils
in food [123]. Microencapsulation simplify the food manufacturing process by
converting liquids to solid powder, decreasing production cost, help fragile and sensitive
materials survive processing and packaging conditions and stabilise the shelf life of the
active ingredient [124-126].
Microencapsulation also plays a crucial role in energy generation field. Hollow
and multilayered plastic microspheres loaded with gaseous, deuterium, a fusion fuel, are
used to harness nuclear fusion for producing electrical energy [127]. This fusion
experiment process has been named as inertial confinement fusion (ICF) and it has been
in use since 1980s [128].
Design and development of nanofiber-based microencapsulation as a novel
materials by the inclusion of carbonaceous materials such as graphene, in aeronautics-
grade matrixes (thermoplastics and thermoset resins) through the application of
microcoatings and intermediate microlayers in sandwich panels and reinforcement of
matrixes have been widely used in aeronautics application.
20
2.6.2 Technologies for microencapsulation of cells
The encapsulation of various materials and living cells inside capsules for different
purposes in the pharmaceutical, chemical, food industry, agriculture, tissue engineering,
biotechnology and medicine is of great importance. Microencapsulation of cells in
hydrocolloid gel matrices is the technique that the cells are entrapped during gel
formation, leading to spherical droplets containing cells. Some of the popular
microencapsulation technologies generally produce the capsules of micron to millimeter
size for microencapsulation of cells were listed in Table 2.2.
Table 2.2: Comparison of different microencapsulation technologies for encapsulation of
cells
Micro-
encapsulation
process
Cost Complex
design
High
voltage
Material
volume
Uniform Size
(mm)
Post-
processing
/harsh
treatment
Reference
Extrusion Low No No Large No 2 - 10 No [129-131]
JetCutter
break-up
High Yes Yes Large Yes < 1 No [39, 49,
132-134]
Spinning disc High Yes No Large Yes 0.2 - 5 No [135]
Micro nozzle
array
High Yes No Large Yes > 0.5 Yes [8, 19]
Vibration
nozzle
Low Yes No Medium No 0.1 - 3 No [37, 136,
137]
Coacervation/
emulsion
method
High Yes No Large Yes 0.02 - 2 Yes [130, 131,
138, 139]
Electrostatic
droplet
generation
High Yes Yes Medium Yes > 0.1 No [31-33,
133]
Flicking High No No Medium Yes 0.2 - 0.4 No [97]
Air
atomisation
Low No No Medium No 0.08 -
0.6
No [35, 140,
141]
21
2.6.2.1 Extrusion, Jet break-up methods and spinning disc
Extrusion (Figure 2.8 (a)) is the most common methods widely used to produce
microcapsules due to its ease, simplicity, low cost, gentle condition and high quantity of
encapsulated cells. Jet break-up and spinning disc techniques are also originated from
the extrusion method. In a basic extrusion technique, the alginate containing cells are
extruded through a syringe needle as droplets into calcium chloride (CaCl2) solution to
be polymerised. The size and shape of the capsules were influenced by the aperture size
of the needle, concentration of the CaCl2 solution and the surface tension of the CaCl2
solution. The basic extrusion technique produced capsules size ranging from 2 - 10 mm
[129-131].
Jet cutter method is suitable to be used with high viscosity polymer solutions
such as poly(vinyl alcohol) solutions [134]. In this technique, the mixture of cells-
alginate suspension was forced through a nozzle to form liquid jet and then cut by a
rotating cutting wire (Figure 2.8 (b)). The number of cutting wires, rotations speed of
cutting tool and the infusion rate manipulates the size of the capsules.
For the spinning disc technique, the capsules are formed by infusing the cells-
alginate suspension onto the high velocity spinning disc (Figure 2.8 (c)) due to the
centrifugal force at the edge of the spinning disc, the droplets are formed and dropped
into the CaCl2 solution to be polymerised. The size of the capsules is controlled by the
rotating disc speed [142]. This method produces capsules with the size ranged from a
few hundreds of micrometers up to several millimeters. In contrast to the jet cutting
method in which, this method is suitable for fluid at low viscosity and it has a very high
productivity.
22
(a) (b) (c)
Figure 2.8: Schematic diagram of different microencapsulation processes in forming
microcapsules: (a) Extrusion, (b) Jet cutter and (c) Spinning disc [143]
2.6.2.2 Micro nozzle array and vibrating nozzle
Micro nozzle is a developed technique for microencapsulation in year 2000. In this
technique, the cell-alginate suspension is flew through silicon micro nozzle array and
then cut off by the high stream of oil to form droplets [8]. The gel droplets drop into the
oil stream that directs the flow of the droplets to a solution of positive ions (Figure 2.9
(a)). Due to the high flow pressure conditions, micro nozzle array are suitable to be used
with high viscosity solution [144]. If this method is to be scaled up for large production,
the cost of the oil and its disposability could be the limitations of this technique [145].
For vibration nozzle technique (Figure 2.9 (b)), the microcapsules are formed by
oscillating and purging the mixture cells suspension through a nozzle into the hardening
bath, resulting in size distribution of capsules as 0.1 - 3.0 mm in diameter [137].
Collecting
bath
Liquid jet
Cutting
tool with
wires
Spinning
disc
23
(a) (b)
Figure 2.9: Schematic diagram of microencapsulation processes in alginate: (a) Micro
nozzle array [8] and (b) Vibrating nozzle [146]
2.6.2.3 Microfluidic device
Microfluidics device has emerged as a powerful platform for the generation of
microparticles with tailored structure and properties [147-150]. This technique allows
direct integration of different input fluids into the polydimethylsiloxane (PDMS)
microfluidic channel as shown in Figure 2.10. The working principle of microfluidic to
generate microcapsules is based on the emulsification of alginate solution.
Microcapsule fabrication methods based on microfluidics device may be
classified into two major approaches, that are flow-focusing and T-junction capsule
formation. The flow-focusing microfluidic approach, as shown in Figure 2.11, forms
microcapsules by allowing a core fluid (cell-alginate suspension) to be surrounded by
sheath stream (oil) flowing. In contrast, T-junctions microfluidic is designed to form
microcapsules by permitting the core fluid to be swept away by one sheath stream in
only one direction. A summary of the microfluidic emulsification technologies based on
PDMS microfluidic chip design for both flow-focusing and T-junction capsules
formation methods, used for the application of cell encapsulation were listed in Table
2.3 [6].
Alginate CaCl2
Microcapsules Vegetable
oil
Vibration
of nozzle
Vibration
of liquid
24
Figure 2.10: Illustrations of microfluidics system mechanism for microencapsulation
[151]
(a) (b)
Figure 2.11: Illustration of microcapsules fabrication methods based on microfluidics
device. (a) Flow-focusing and (b) T-junction capsules formation [6]
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