i
DEVELOPMENT OF A MICRO-EXTRUDER WITH
VIBRATION MODE FOR MICROENCAPSULATION
OF HUMAN KERATINOCYTES IN CALCIUM
ALGINATE
NURUL HAMIZAH BINTI MD SAI‘AAN
UNIVERSITI TUN HUSSEIN ONN MALAYSIA
ii
DEVELOPMENT OF A MICRO-EXTRUDER WITH VIBRATION MODE FOR
MICROENCAPSULATION OF HUMAN KERATINOCYTES IN CALCIUM
ALGINATE
NURUL HAMIZAH BINTI MD SAI’AAN
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
SEPTEMBER 2017
iii
In the name of Allah, Most Gracious, The Most Merciful
All Praise to Allah
Md Sai’aan bin Jalal & Sabariah binti Hussain
Work hard in silence,
Let your success be your noise.
iv
ACKNOWLEDGEMENT
Alhamdulillah, finally I manage to finish this master project within the time
given. All praise to Allah. Thanks to Almighty for giving me the chances and
strength to complete my master research project. First and foremost, I would like
to dedicate my special appreciation to my supervisor, Associate Professor Dr. Soon
Chin Fhong, for all her support and encouragement towards the completion of this
master project. Her kindness to help me from the beginning until the end of project
will not be forgotten.
Also, I would like to express my acknowledgement to all the staff from other
departments for their time teaching me and lending me a hand to use the laboratory
equipments to complete the experiment for this project. They had given a full
cooperation the time I needed.
Last but not least, my deepest gratitude to my parents and family for their
moral supports especially to me in order to complete this project. Without them,
might probably I might not finish this project. Not to forget to all my colleagues in
the Biosensor and Bioengineering Laboratory for their full support directly or
indirectly to the completion of this project.
v
LIST OF ASSOCIATED PUBLICATIONS
Conference Proceeding:
1. Nurul Hamizah Md Sai’aan, Chin Fhong Soon, and Kian Sek Tee,
“Development of a micro-extruder with vibrational mode for
microencapsulation of cells”, ARPN Journal of Engineering and Applied
Sciences, Vol. 11, No. 14, page 8770-8775, July 2016. (Q3, Scopus
indexed).
2. Nurul Hamizah Md Sai’aan, Chin Fhong Soon, Mohd Khairul Ahmad,
Kian Sek Tee, Mansour Youseffi, and Sayed Ali Khagani,
“Characterisation of encapsulated cells in calcium alginate
microcapsules”, 2016 IEEE EMBS Conference on Biomedical
Engineering and Sciences (IECBES), Malaysia, 2016, pp. 611-616.
doi:10.1109/IECBES.2016.7843522. (Q3, Scopus indexed).
3. Nurul Hamizah Md Sai’aan, Chin Fhong Soon, Mohd Khairul Ahmad,
Kian Sek Tee, Mansour Youseffi, and Sayed Ali Khagani “Growth of
microtissues in microencapsules formed using microextrusion and
vibration”, Proceeding of International Conference on Advances in
Electrical, Electronic and Systems Engineering (ICAEESE), Malaysia,
March 2017, pp. 657-661.10.1109/ICAEES.2016.7888128.
(Q3, Scopus indexed).
vi
ABSTRACT
Microencapsulation is a promising technique to form microtissues. The existing cell
microencapsulation technologies that involved extrusion and vibration are designed
with complex systems and required the use of high energy. A micro-extruder with an
inclusion of simple vibrator that has the commercial value for creating a 3D cell
model has been developed in this work. This system encapsulates human
keratinocytes (HaCaT) in calcium alginate and the size of the microcapsules is
controllable in the range of 500-800 µm by varying the flow rates of the extruded
solution and frequency of the vibrator motor (10-63 Hz). At 0.13 ml/min of flow rate
and vibration rate of 26.4 Hz, approximately 40 ± 10 pieces of the alginate
microcapsules in a size 632.14 ± 10.35 µm were produced. Approximately 100 µm
suspension of cells at different cells densities of 1.55 × 105
cells/ml and 1.37 × 107
cells/ml were encapsulated for investigation of microtissues formation. Fourier
transform infrared spectroscopy (FTIR) analysis showed the different functional
groups and chemistry contents of the calcium alginate with and without the inclusion
of HaCaT cells in comparison to the monolayers of HaCaT cells. From Field
Emission Scanning Electron Microscope (FESEM) imaging, calcium alginate
microcapsules were characterised by spherical shape and homogenous surface
morphology. Via the nuclei staining, the distance between cells was found reduced as
the incubation period increased. This indicated that the cells merged into
microtissues with good cell-cell adhesions. After 15 days of culture, the cells were
still viable as indicated by the fluorescence green expression of calcein-
acetoxymethyl. Replating experiment indicated that the cells from the microtissues
were able to migrate and has the tendency to form monolayer of cells on the culture
flask. The system was successfully developed and applied to encapsulate cells to
produce 3D microtissues.
vii
ABSTRAK
Pengkapsulanmikro adalah teknik yang menjanjikan pembentukan tisumikro.
Teknologi sel pemikrokapsulan sedia ada yang melibatkan penyemperitan dan
getaran telah direka dengan sistem yang kompleks dan memerlukan penggunaan
tenaga yang tinggi. Pengekstrud mikro dengan penambahan penggetar mudah yang
mempunyai nilai komersial untuk mewujudkan satu model sel 3D telah telah
dibangunkan dalam kerja ini. Sistem ini merangkumkan sel kulit manusia (HaCaT)
dengan kalsium alginat dan saiz kapsulmikro adalah dikawal dalam julat 500-800
mikron dengan mengubah kadar aliran pengekstrud dan kekerapan motor penggetar
(10-63 Hz). Pada 0.13 ml/min kadar aliran dan kadar getaran 26.4 Hz, di anggarkan
40±10 biji kapsulmikro alginat bersaiz 632.14 ± 10.35 µm dapat dihasilkan. Kira-
kira 100 µl kumpulan sel-sel dengan ketumpatan yang berbeza pada 1.55×105 sel/ml
dan 1.37×107 sel/ml telah di kapsulkan untuk kajian pembentukan tisumikro.
Analisis FTIR menunjukkan kumpulan berfungsi yang berbeza dan kandungan kimia
alginat kalsium dengan dan tanpa kemasukan sel HaCaT dan juga monosel HaCaT.
Dari pengimejan FESEM, kapsulmikro kalsium alginat telah disifatkan berbentuk
bulat dan permukaan morfologi homogen. Melalui penandaan nukleus, jarak antara
sel telah berkurang dengan peningkatan tempoh pengeraman. Ini menunjukkan
bahawa sel-sel digabungkan menjadi tisumikro yang baik dengan pelekatan sel-sel.
Selepas 15 hari pengeraman, sel masih hidup seperti yang ditunjukkan oleh
ungkapan hijau pendarfluor daripada calcein-acetoxymethyl. Pengulangan
eksperimen menunjukkan bahawa sel-sel dari tisumikro dapat berhijrah dan
mempunyai kecenderungan untuk membentuk lapisan monosel pada kultur kelalang.
Sistem ini telah berjaya di bangunkan dan di gunakan untuk merangkumi sel-sel
untuk menghasilkan tisumikro 3D.
TABLE OF CONTENT
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
LIST OF ASSOCIATED PUBLICATIONS v
ABSTRACT vi
ABSTRAK vii
TABLE OF CONTENT viii
LIST OF FIGURES xii
LIST OF TABLES xvi
LIST OF ABBREVIATIONS xvii
CHAPTER 1 INTRODUCTION 1
1.1 Background of study 1
1.2 Problem statement 3
1.3 Objectives 4
1.4 Scope of project 4
1.5 Organisation of thesis 5
CHAPTER 2 LITERATURE REVIEW 6
2.1 Cells and tissue 6
2.2 Epidermis and human keratinocytes 7
2.3 Rational of growing 3D cells 9
2.4 Methods for growing 3D microtissues 12
2.5 Microencapsulation technology 14
2.6 Polymeric material for microencapsulation of cells 17
2.7 Different microencapsulation techniques 20
viii
2.7.1 Extrusion and dripping technique 20
2.7.2 Extrusion and vibration technology 22
2.7.3 Microfluidic 23
2.7.4 Electrospray 24
2.8 Comparison of different microencapsulation
techniques
25
2.9 Application of cell microencapsulation 26
2.10 Review on electronic system 27
2.10.1 Stepper motor 27
2.10.2 Motor driver L298N 31
2.10.3 Arduino UNO microcontroller 32
2.11 Analytical technique 34
2.11.1 Inverting phase contrast microscopy 34
2.11.2 Fourier transform infrared 35
2.11.3 Field emission scanning electron microscope 36
2.11.4 4’, 6- Diamidino-2- Phenylindole (DAPI)
staining
37
2.11.5 Live and dead assay staining 38
CHAPTER 3 METHODOLOGY 39
3.1 Introduction 39
3.2 Development of a micro-extruder system 42
3.2.1 Hardware part 43
3.2.2 Electronic Part 45
3.2.3 Programming the microcontroller 49
3.3 Verification on the motor speed 51
3.4 Alginate and chloride solutions preparation 52
3.5 Cell preparation 52
3.6 Cell microencapsulation 53
ix
3.7 Characterisation of microencapsulated cell 54
3.7.1 Fourier transform infrared spectroscopy 54
3.7.2 DAPI staining 56
3.7.3 Live and dead assay 56
3.7.4 Alginate lyase 56
3.7.5 Field emission scanning electron microscope 57
CHAPTER 4 RESULTS AND DISCUSSION 58
4.1 Introduction 58
4.2 The functions of micro-extruder system 60
4.3 The effects of motor speed to the size of
microcapsules without vibration mode
63
4.4 The relationship of percentage of PWM to the
vibration frequency
63
4.5 Distribution of microcapsules size 64
4.5.1 Verifying the microcapsules size 66
4.6 Microencapsulation of HaCaT cell at different
densities
68
4.7 Characterisation of microtissues 72
4.7.1 Chemistry contents of microcapsules 72
4.7.2 Nucleus staining using DAPI 75
4.7.3 Live and dead assay 75
4.7.4 FESEM analysis 76
4.8 Degradation of alginate 77
CHAPTER 5 CONCLUSION 80
5.1 Conclusion 80
5.2 Future work 81
5.3 Thesis contributions 81
x
REFERENCES 82
APPENDICES 92
VITA
xi
LIST OF FIGURES
2.1 A schematic representation of cell 7
2.2 A schematic representation of skin 8
2.3 Schematic presentation of cellular distribution in two- and
three-dimensional (2D and 3D) microenvironments
9
2.4 Cell interactions with polymeric material 12
2.5 Milestone for cell microencapsulation since its first
conception
15
2.6 Different structures of microcapsules 16
2.7 Structural characteristics of alginate (a) alginate
monomers (b) chain conformation (c) block distribution
17
2.8 Extrusion technology 21
2.9 Main part of encapsulation device with a concentric
nozzle
22
2.10 Apparatus for producing alginate beads by the vibration
method
23
2.11 Microfluidic channel 24
2.12 Electrospray device setup 25
2.13 Position change in stepper motor 28
2.14 Two types of common stepper motor driver (a) Unipolar
and (b) Bipolar
29
2.15 Basic stepper motor system 30
2.16 L298N Module 31
2.17 L298N block diagram 32
2.18 Arduino UNO board 33
2.19 Image captured using phase contrast microscope (Scale
bar: 100 µm)
34
xii
2.20 Previous FTIR analysis of HaCat cells 35
2.21 FESEM basic operation 36
2.22 FESEM image for alginate beads surface and their cross-
section
37
2.23 DAPI chemical structure 37
3.1 Flow of project methodology 40
3.2 Flow chart of micro-extruder development 42
3.3 Hardware design 44
3.4 Controller board for micro-extruder system 45
3.5 Circuit block diagram 46
3.6 Schematic diagram 48
3.7 Arduino chart flow 49
3.8 Digital tachometer 51
3.9 Experimental setup for micro-extruder system 54
3.10 FTIR Sampling Accessory 55
3.11 Prepared sample for FTIR analysis (a) Calcium alginate
microcapsules (b) Monolayers HaCaT cells (c)
Microencapsulated cells with calcium alginate
55
3.12 Alginate lyase preparation 57
4.1 (a) Prototype micro-extruder system, (b) Linear slider and
(c) Vibrator motor
59
4.2 Controller box 60
4.3 Relationship between flow rate and motor speed 61
4.4 Microcapsules produce size without vibration mode
(Scale bar: 100 µm)
62
4.5 Relationship between size of the beads and programmed
speed for stepper motor without vibration mode
62
4.6 Relationship between frequency and PWM of vibrator
motor
63
xiii
4.7 Photomicrograph of microcapsules generated by different
vibrator frequency (a) 11.7 Hz (b) 19.0 Hz (c) 26.4 Hz (d)
35.2 Hz (e) 42.5 Hz (f) 48.3 Hz (g) 57.1 Hz and (h) 63.0
Hz (Scale bar: 100 µm)
65
4.8 The distribution of microcapsules size with different
frequency at (a) 11.7 Hz, (b) 19.0 Hz, (c) 26.4 Hz, (d) 35.2
Hz, (e) 42.5 Hz, (f) 48.3 Hz, (g) 57.1 Hz and (h) 63.0 Hz
67
4.9 Relationship between size of microcapsules and frequency
of vibrator motor
68
4.10 Microencapsulated cell with low cell densities at day (a) 1
(b) 3 (c) 5 (d) 7 (e) 9 (f) 11 (g) 13 and (h) 15 (Scale bar:
100 µm)
70
4.11 Microencapsulated cell with high cell densities at day (a) 1
(b) 3 (c) 5 (d) 7 (e) 9 (f) 11 (g) 13 and (h) 15 (Scale bar:
100 µm)
71
4.12 Calcium alginate FTIR spectra 73
4.13 HaCaT cell FTIR spectra 73
4.14 FTIR spectra (a) CaAlg and cells (b) HaCaT cell (c) Ca-
Alg
74
4.15 DAPI staining of microencapsulated HaCaT cell at
different incubation period (a) 5 days, (b) 10 days, and (c)
15 days (Scale bar: 100µm)
75
4.16 Image of live and dead assay staining (a) days 5 and (b)
days 15 (Scale bar: 100 µm)
76
4.17 Figure 0.1: FESEM image of 3D cells microcapsules with
different magnification. (a) Overall morphology of
microcapsules (Scale bar: 1 mm) (b) and (c)
Microcapsules structure of encapsulated HaCaT cell
(Scale bar: 100 µm), (d) Surface morphology for the
77
xiv
microcapsules (Scale bar: 10 µm)
4.18 Phase contrast micrographs of microtissues after alginate
lyase
78
4.19 The grow of cell after lyase (a) 24 hours, (b) 36 hours, and
(c) 72 hours (Scale bar: 100 µm)
79
xv
LIST OF TABLES
2.1 Differences in cellular characteristics and processes in
two-dimensional and three-dimensional culture systems
10
2.2 Different method of growing 3D microtissues 13
2.3 Alternative microencapsulation material 17
2.4 Cell encapsulation approaches based on different alginate
matrices
19
2.5 A comparison of microencapsulation techniques 26
3.1 Establishment of experiments 41
xvi
LIST OF ABBREVIATIONS
Symbol Description
2D Two dimensional
3D Three dimensional
AD Alzheimer’s disease
Alg Alginate
ATR Attenuated total reflectance
CFR Code of Federal Regulation
CNTF Ciliary neurotrophic factor
CO2 Carbon dioxide
DAPI 4’,6-diamidino-2-phenylindole
DMEM Dulbecco’s Modified Eagles’s Medium
DNA Deoxyribonucleic acid
dsDNA Double strand deoxyribonucleic acid
ECM Extracellular Matrix
FDA Food and Drug Administration
FESEM Field Emission Scanning Electron Microscope
FTIR Fourier Transform Infra Red
GND Ground
HaCaT Human keratinocytes cell lines
HBSS Hank’s Balanced Salt Solution
HEMA-MMA hydroxyethyl methacrylate-methyl methacrylate
HD Huntington's disease
Hz Hertz
IC Integrated circuit
LCD Liquid Crystal Display
NaCl Sodium Chloride
xvii
NGF Nerve growth factor
NM 1 Long –lived keratinocytes line
PCB Printed circuit board
PDADMAC polydiallyldimethyl ammonium chloride
PDMS Polydimethylsiloxane
PEG poly(ethylene glycol)
PLO Poly-L-ornitine
PMCG Poly(methylene-co-guanidine)
PVA polyvlnyalcohol
PWM Pulse Width Modulation
RNA Ribonucleic acid
RPM Rotation per minute
SEI Secondary electron image
SIK Spontaneously immortalized keratinocytes
USB Universal Serial Bus
VR Variable Reluctance
CHAPTER 1
INTRODUCTION
1.1 Background of study
Microencapsulation is the process of coating a biologically active material to
form a microcapsule inside a membrane [1–3]. This microencapsulation procedure
received increasing interest over the last 20 years [4]. This technology shows
promising potential in agricultural, food industry, cosmetics, and pharmaceutical and
also biomedicine. Although several microencapsulation methods had been
developed, but there are some features that can be improved such as
microencapsulation size to meet the requirements for encapsulating cells.
Recently, new emerging research has been focusing on creating new
microencapsulation techniques. Microencapsulation had become more promising
alternative technique to form the cell spheroids and has the significant potential in
tissue engineering [5]. Before moving further towards the microencapsulation
process itself, there are a few aspects that must be put into consideration. Very basic
knowledge about the need of microencapsulation is required. The need for
microencapsulation thrives for improvements of existing microencapsulation
techniques.
The significant interest on microencapsulation using vibration technology
was gained due to its simplistic approach to produce microcapsules. Based on the
simpler extrusion and dripping method, the microcapsules formed with a very large
diameter of 2-5 mm, which is too large for biotechnology or medical application [6].
Moreover, some of the technologies used the highly viscous polymers in their system
2
[7]. Previous research had developed the vibrational encapsulation device based on
vibrating-nozzle to encapsulate the pancreatic islet. However, this technique suffered
from high complexity and possibly suitable for large number of batches which is not
suitable for laboratory studies [8]. Therefore, a simple technique based on
microextrusion with vibration mode is proposed for the microencapsulation of
human keratinocytes cell lines (HaCaT) in calcium alginate into a size similar to the
thickness of the epidermis.
In cell microencapsulation, there are three main components that are being
focused: the encapsulated cell lines, type of material or polymer used and the
microencapsulation technology itself. In typical cell microencapsulation process,
cells are suspended in a solution, which can become gelled or solidified leading to
the encapsulation of cells in a matrix. Based on both natural and synthetic polymers,
hydrogels continued to be a relevant material for encapsulation of cells. Three
different methods for production of microcapsules include chemical, mechanical and
physiochemical have been described by previous researchers [8 - 9].
Previously, researcher [11] indicated that microencapsulated particles are
usually in the size ranging between 1 and 1000 μm depending on the technique used.
The microencapsulated cells were shown to be able to grow into microtissues or
encouraged formation of cell aggregates. But, different type of cells reacts with
bioactive growth factor based on their biocompatibility properties of the
encapsulation material. The growth factor can provide controlled release into local
micro-environment to yield desirable concentration periods over a day [12].
Alginate is naturally derived polysaccharides from the brown algae forming
the linear binary copolymers consisting β-D- mannuronic acid (M) and α-L-
guluronic acid (G) residues [13]. Sodium alginate has found biomedical and
biotechnology applications mainly as a material for the encapsulation of a variety of
cells for immunoisolatory and biochemical processing applications. Although the
exchange of gases is subjected to diffusion limitations, cells can maintain viability
within the cross linked gel [14]. It has been employed for encapsulating cells to be
transplanted, since it is biocompatible both within the host and with enclosed cells
[15].
Creating a three dimensional (3D) cell culture models instead of two
dimensional (2D) cell model have recently garnered a great attention for
pharmacological study because of their accuracy of the models in representing the
3
tissue structure in-vivo. Multilayer cell model promotes cell differentiation and tissue
organisation [1]. For example, organ on chip as regenerative medicine permits the
study of human physiology in an organ-specific context to create in-vitro organ
culture microenvironment [2]. Therefore, biomedical engineer are provided with new
engineering tools to generate the tissue model in multicellular structure and up to the
extent of creating blood capillary into the tissue model.
1.2 Problem statement
The alginate microcapsules can simply be produced by dripping the sodium alginate
solution into the sodium chloride solution. Simple dripping technique can be used to
generate microcapsules of alginate but this technique produced microcapsules in the
range of 1000 to 3000 µm which are too large for the cells to grow. It was reported
that the optimum size of the microcapsules for the microtissues to grow should be
less than 1000 µm. Furthermore, smaller size of capsules can provide better
transportation of nutrients and oxygen, easier implantation and better mechanical
strength [11, 16]. Extrusion technology with vibration mode for microencapsulation
method had been developed. Some of the technologies used the laminar liquid jet
break up by a superimposed vibration and others used the monocentric or concentric
nozzle system for cell microencapsulation [7]. However, most of the method comes
with some disadvantages such as high complexity, difficult to conduct and high
maintenance [6].
This project is proposed for the development of micro-extruder system that
could generate controllable size of microcapsule that can form 3D cell. The system is
straight forward, cost effective and suitable as laboratory scale device. The
microencapsulation of cells involved calcium alginate that is non-toxic and could be
removed to extract the microtissues formed. The biophysical structures of the
microtissues formed is characterised using Fourier Transform Infrared Spectroscopy
(FTIR), surface morphology using Field Emission Scanning Electron Microscope
(FESEM), nucleus staining, live and dead assay staining.
4
1.3 Objectives
The main objectives of the research are:
a) To design and develop a micro-extruder with vibration mode that generates
microcapsules for encapsulation of cells.
b) To encapsulate human keratinocytes in sodium alginate to form 3D cells.
c) To extract 3D cells from sodium alginate shells using alginate lyase.
d) To characterise the biophysical properties of the 3D cells.
1.4 Scope of project
The design of the micro-extruder system was done using Google Sketch-up. The
micro-extruder system consists of stepper motor, linear slider, Arduino UNO
microcontroller, vibrator motor, syringe holder, switches and power supply. The
stepper motor has different speed which is 5 to 25 rpm referring to the flow rate of
sodium alginate flow at the range of 0.1 to 0.5 ml/min. The developed micro-
extruder used the stepper motor to control the linear slider that clamp the 0.5 ml
insulin syringe (BD U-100) to get approximately 500-800μm of microcapsules.
This study is mainly focus on the extrusion method that can produce 40 ± 10
numbers of microcapsules for 300 µl of alginate solution at a time. Besides that,
vibration mode with frequency of 10-63 Hz will be adjusted to get the best size of
microcapsules. The natural polymer used was calcium alginate which is the
polymerisation of sodium alginate and calcium chloride solution. Keratinocytes
(HaCaT) were used in this research because it can keep undergoing division and
allowed characterisation of several processes. Biophysical characterisation
characterised the size, shape and morphology of the microcapsules. Analysis such as
FTIR, FESEM and staining provided information associated with the properties of
the microcapsules produced.
5
1.5 Organisation of thesis
Chapter 1 consists of an introduction of thesis covering the problem statement,
objective and scopes of project. This chapter presented the reader the proper
understanding about the purpose of this research being carried out.
Chapter 2 covers the microstructure of skin and background of microencapsulation
including the cell line, technique and material used by previous researcher. Since
microencapsulation technology had been widely discovered before, so there will be
many aspects should be taken into consideration to avoid misleading on that fact or
study.
Chapter 3 basically presents the method used to achieve the objectives of this
research project. The selection of stepper motor as the main requirement to control
the extruder is presented. Based on the microcontroller coding, the speed of stepper
motor can be programmed according to required speed value. The adjustable
frequency of the vibrator motor also will affect the production of microcapsules.
Hence, the relationship between speed of motor, frequency of vibrator motor and
microcapsules size produced can be analysed beside the biophysical analysis of the
encapsulated cell.
Chapter 4 totally discusses the finding of the work being conducted. Final result on
experimental study is detailed in this chapter. Figures, graphs and images are
provided to show the finding and discussions of the finding are also in this chapter.
Chapter 5 finally presented the overall conclusion for this project. The
recommendation and future work are also discussed in this chapter.
CHAPTER 2
LITERATURE REVIEW
2.1 Cells and tissue
The human body composed of trillion numbers of cells. Cells function as basic
building blocks of all living cells which have the capability to reproduce themselves.
A cell is the smallest unit of life that has the basic structural, functional and
biological unit known as living organism with its own function. It is consist of
cytoplasm enclosed within a membrane which contains biomolecules include
proteins and nucleic acid such as nucleus, mitochondria, Golgi apparatus,
endoplasmic reticulum, lysosome and some other else as shown in Figure 2.1.
Tissue is a group or layer of specialised cells that bind together to perform the
specific functions. In human body, there are four basics types of tissue include
epithelial, connective, muscular and nervous tissue. Each of the tissues has their
further classification according to their physiological function. The complex
combinations and gradients of extracellular matrix (ECM) component with specific
biological and mechanical influence are having by almost all human tissue. Since the
interest of reproducing the biomaterial environment for tissue and organ are greatly
developed, so does the properties of adaptive materials required to recapitulate the
tissue function [17].
7
Figure 2.1: A schematic representation of cell
2.2 Epidermis and human keratinocytes
Human skin consists of many different types of cells include keratinoctytes,
melanocytes and fibroblasts [17]. Figure 2.2 illustrates the schematic presentation of
a skin that depicts the structure of human skin. The outermost tissue and largest
organ in human body is skin in term of surface area and weight. It has complex
structure consists of many components such as cells, fibers, and several different
layers of skin structure. Resulting from chemical and physical reactions inside these
components, the major function of skin is to act as barrier to the exterior
environment. Other than that, skin can also prevents water lost from the body and
regulates body temperature by evaporation of sweat [18].
Endoplasmic
Reticulum
Microtubules
Microfilament
s
Secretory granules
Lysosomes
Mitochondria
Golgi
Apparatus
Nucleus
Nucleolus
8
Figure 2.2: A schematic representation of skin
The term HaCaT (Ha = human adult, Ca = calcium, T= temperature) was
designed to indicate its origin and the initial culture conditions of normal human
skin. It was developed through a long term culture of normal human adult skin
keratinocytes at reduced calcium concentration and elevated temperature [19]. The
first two lines of skin are NM1 and SIK which are taken from keratinocytes of
neonatal foreskins. HaCaT is the third lines of keratinocytes isolated from adult
epidermis (epithelial outer layer of skin) at the periphery of malignant melanoma.
This cell line is the most extensively characterised of the three spontaneously
immortalised human keratinocytes cell line [20]. In vitro, HaCaT cell lines exhibit
the entire of the major surface marker and functional activities characteristic of
isolated keratinocytes and is able to differentiate, forming stratified epidermal
structures [21]. The different of HaCaT cell with other human cell include suitable
for experiment or scientific research. It is also utilised for their high capacity to
differentiate and proliferate in vitro. Other than that, HaCaT cell allows
characterisation of several processes and keep undergoing division.
Stratum
basale
Stratum
spinosum
Dermis
Stratum
corneum
Melanocytes
Living
keratinocytes
Dead
keratinocytes
9
2.3 Rational of growing 3D cells
Conventional two dimensional (2D) cell culture is extensively used in research study
regenerative medicine and also tissue engineering. This is because of the simplicity
and reproducibility properties. However, since 1970s, the 2D cell cultures had shown
their limitation with the increasingly evident and relevance of appropriate for three
dimensional (3D) cell systems [22].
In 2D cell culture, cell-to- plastic interactions occurred rather than cell-to-cell
and cell-to- extracellular matrix (ECM) interactions that form the normal cell
function [23]. Since the growth and maintenance of normal tissue is depends on a
continuous series of cellular interactions in a microenvironment, the various growth
factors, hormones, adhesion molecules as well as a complex ECM were composed to
perform their functions [24].
Figure 2.3: Schematic presentation of cellular distribution in two- and three-
dimensional (2D and 3D) microenvironments
Cells
Microfibrillar structure of
hydrogel
2D culture 3D culture
10
From the schematic presentation in Figure 2.3, the cellular distribution in 2D and 3D
microenvironment of cell culture shows the different in term of their structural
construction. That is the important of the method for producing the 3D microtissues
because the cell-biomaterial interactions will affect the growth factors of the
encapsulated cell. Then, the different in cellular characteristics and processes in 2D
and 3D culture system were summarised in Table 2.1.
In biological research, cell culture is an important aspect to take as consideration.
The removal of cells from a tissue before growth on the favorable artificial
environment is known as culture cell process [25]. The appropriate models system
can be provided for studying the standard physiology and biochemistry of cells. The
term ‘3D cell culture’ is referred to a suitable micro-environment for optimal cell
growth, differentiation and function and the capability to create tissue-like constructs
in-vitro [26]. 3D cell allowed to increase cell-cell interactions, formation of
intercellular junctions and intercellular communication, providing a more
physiologically relevant microsystem [27]. Many attempts had been proposed to
develop 3D cell culture system mimics the microenvironment to the surrounding cell.
Table 2.1: Differences in cellular characteristics and processes in two-dimensional
and three-dimensional culture systems [28]
Cellular characteristics 2D 3D
Morphology Sheet-like flat and stretched in
monolayer
Mimic natural shape of cell in
aggregate structures
Proliferation Faster proliferation rate than in vivo Proliferation rate depends on the
cell types or type of 3D model
system
Exposure to
medium/drugs
Cells are equally exposed to nutrients
that are distributed in growth medium
Some of the nutrients may not fully
penetrate the spheroid
Stage of cell cycle Same stage of cell cycle because of
equally exposed to the medium
Spheroids contain proliferating,
quiescent, hypoxic and necrotic
cells
Gene/ protein
expression
Displays the differential gene and protein
expression levels
Exhibit gene/protein expression
more similar to those in vivo tissue
origins
Drug sensitivity Very effective to treatment and drug More resistant to treatment
11
Extracellular matrix (ECM) function as scaffold to maintain tissue and organ
structure regulates the aspects of cell behavior including cell proliferation and
growth, survival, change in shape, migration and differentiation. The development of
all multicellular organisms is influenced by the interaction between cells and the
ECM. ECM assembly is regulated by the 3 dimensional (3D) environments and the
cellular tension that is transmitted through integrins [29]. ECM is composed of
collagen, non-collagenous glycoproteins and proteoglycans. These components are
secreted from cells to create an ECM meshwork that surrounds cells and tissues. The
ECM regulates many aspects of cellular function, including the cells dynamic
behavior, cytoskeletal organisation and intercellular communication [30]. Today, the
fabrication of 3D matrices which mimic the better geometry, chemistry and signaling
environment of natural ECM had been increasing. Hence, the intensive research on
the interaction between the matrix and cells had been studied.
Development of material systems in tissue engineering and cell biologists to
culture mammalian cells within 3D ECM had begun over the past few decades. The
ability of 3D ECM mimics to circumvent limitation of 2D cell culture traditionally.
Hence, hydrogel are the most suitable to developing synthetics ECM analogs because
of the highly attractive material which are able to simulate the nature of most soft
tissue [31]. As the encapsulated cell with hydrogel can provide the 3D cells with
tissue-like extracellular environment, they offer several possible applications such as
in vitro model systems for drug screening, diagnostics tools and toxicological assays
[32]. In HaCaT cells, integrins appear as the major receptors, by which cells attach to
the extracellular matrix, and some integrins also mediate important cell-cell adhesion
events. Moreover, integrins make transmembrane connections to the cytoskeleton
and activate many intracellular signaling pathways to mediating cell adhesion [33].
This cell interaction is as shown in Figure 2.4.
12
Figure 2.4: Cell interactions with polymeric material
2.4 Methods for growing 3D microtissues
Productions of microtissues are recently available in many approaches. 3D cell
cultures give advantages on their technical and functional in many applications. The
comparison of 3D spheroids or microtissues formation technique had been discussed
previously [34]. Basically, the method to produce can be dividing into two categories
which are scaffold based and scaffold free method. As shown in Table 2.2, each of
the 3D producing method was discussed briefly. Hydrogels seem to be suitable for
each of application in tissues engineering. They can exhibit low immunogenicity and
low cytotoxicity and allows the exchange of gases and nutrients between cells and
environment [1]. The list of others synthetic and natural polymers for the 3D matrix
building were listed here [25].
Scaffolds technique offers the unique clinical opportunities in tissue
engineering. For scaffolds free method, it consists of microcapsules and spheroid
formation for producing the 3D microtissues. Each of the method offers different
output depends on their application. Microencapsulation offer many advantages over
the conventional method to produce microtissues since it can achieve high densities
and enhanced product recover [35]. Hanging drop method is not standardised and
difficult to upscale even though it is cost effective, gentle method and guaranties
reproducibility [36].
Integrins
crosslinked
alginate network
ECM
ECM to cell
signaling
Cells
13
Basically, for scaffolds based method there are two types of polymers can be used to
be the scaffold which are synthetic and natural polymers. To make the biomaterials
successfully on their application, the biocompatible polymers must be governing
with suitable morphology and properties to allow the creation of desired molecular
architectures [37]. Although synthetic biomaterials are good in physical and
mechanical properties control, but their biocompatibility had an issue during the
attachment of cell and growth on these materials.
Table 2.2: Different method of growing 3D microtissues
Categories
Method for culturing 3D microtissues
Scaffold free Scaffold based
Microcapsules Spheroids Synthetic Natural
Method Electrostatic
droplet generator
Hanging drop Freeze drying 3D printing
Polymers/materials/equipment Calcium alginate
and gelatin
Microtiter
plate
Polylactic
acid (PLLA)
Hyaluronic
acid (HA)
Advantages Simple and easier
to carry out
Can produce
spheroids of a
homogeneous
size without
sieving or
manual
selection
Controllable
degradable
behaviour
Cells
proliferated
well in the
designed
scaffolds
Application Drug research and
regenerative
medicine
The
development
of
bioartificial
tissue
Tissue
engineering
Bone tissue
engineering
Cell type Feline renal
fibroblast cell line
(CRFK)
Human
hepatoma cell
line (HepG2)
and variety of
cell line
Mouse
fibroblast
MC3T3-E1
murine
fibroblasts
14
2.5 Microencapsulation technology
Microencapsulation is a promising technique used to encapsulate cells, drugs and
nanoparticles in polymers. Cell microencapsulation is a technology towards building
of artificial tissues or organs with the use of bioactive materials or polymeric
materials [38]. Many techniques [16] had been proposed by previous researcher and
give a significant advantage to form microcapsules. The idea is to encapsulate the
cell to form 3D cell that mimic the human environment so that it can be used for
regenerative medicine, tissue engineering or drug testing.
In 1999, Stratowa et al. and Taylor et al. in 2001 stated that cell based testing
method had being progressively employed in the early of discovery drug research
process. According to Bhadriraju and Chen in 2002, they believed that cell-based
systems are specifically engineered to mimic in vivo behaviour which can reduce
costs, increase efficiencies and predictive accuracy of the drug discovery process
[39].
Figure 2.5 shows the overall milestone or timeline how the cell
microencapsulation being started by the previous research. Over the year, more
invention on the cell microencapsulation had been proposed until now. Over other
conventional methods for suspension cultures, cell microencapsulation offer many
advantages such as it can achieve very high cell densities and enhanced product
recovery [35].
In 1931, Bungen burg de Jon et al. were the first people discovered the
microencapsulation procedure. They prepared gelatin sphere through coacervation
process. This technique was employed by Bisceglie in 1933 to transplant tumor cells
in polymeric membrane into a pig’s abdominal cavity. In the 1960’s, Chang
proposed the first encapsulation for the entrapment of bioactive materials such as
enzyme, proteins and cells in an immunoprotection semi-permeable membrane. In
1980s, Lim and Sun had successfully encapsulated implantable islets cells in
alginate-poly (L-lysine) to form multilayer microcapsules using natural
polysaccharides based biomaterials which open a new chapter for cell encapsulation.
As time progresses, the adherent cells in encapsulation turn into masses of
microtissues and the value of these microtissues is recognised in pharmacological
assay, toxicity screening and regenerative medicine. Microencapsulation of drugs is
aimed to device method for controllable release of drug in the in-vivo system [9].
15
Figure 2.5: Milestone for cell microencapsulation since its first conception [3, 5, 42]
A process of enclosing micron-sized particles of solids, droplet of liquids or
gases in an inert shell is known as microencapsulation. The material inside the
capsule is referring as core, whereas, the wall is known as shell, coating or
membrane. Usually, the core material contains an active ingredient whereas the shell
materials cover or protect the core material [42]. The products with such a structure
are known as microcapsules, microbeads, microspheres or microparticles. Generally,
particles having a diameter in the range of 1 to 1000 micrometers are termed as
microcapsules. On the other hand, spherical encapsulation with size larger than 1000
micrometers are known as macroparticles (macrospheres) [1, 22].
The microcapsules or microspheres in different structure of encapsulation are shown
in Figure 2.6. The differences of these microcapsules are in the stiffness of materials
and structure of encapsulation. The microcapsules can be fully encapsulated in a
shell or in a mixture of matrix. Encapsulation efficiency may be reduced due to the
irregular shape of microcapsules because of the presence of pores [45]. In cell and
1933 1964 1980 1994 1996 2000 2006 2012
Chang: concept
of artificial cells
First human clinical trial in
encapsulated islet
allotrasplantation performed in
a 38-year-old man with
diabetes
Bachoud-levi: first implant into
ventricles (CNTF for HD)
Wahlberg: first
intraparenchymal brain
implant (NGF and AD)
Bisceglie: first attempt to
encapsulate cells
Lim: xenograft
islets
Intrathecal delivery of CNTF
using encapsulated
genetically modified
xenogeneic cells in patients
with amyotrophic lateral
sclerosis
First intravitreous implantation
of a cell encapsulation device
16
tissue engineering, microencapsulation is applied in capturing cells microcapsules of
hydrogels [46]. Microencapsulation can achieve a certain goals set based on their
application include converting liquids to solids, providing environmental protection,
altering colloidal, changing surface properties and controlled release characteristic
[4,23]. The mutli-core structure of microcapsules were applied in this study because
it mimic the biological cells properties compares to other strucutre.
Figure 2.6: Different structures of microcapsules [24–26]
2.6 Polymeric material for microencapsulation of cells
Finding the best and suitable materials is challenging in order to get the optimum
encapsulation result. Many natural and synthetic polymers include hydrogels are
being tested and reported to be interest for microencapsulation of cells [49]. Table
2.3 shows the alternative polymers that had been designed and used by previous
researchers in microencapsulation of cell. Hydrogels are 3D hydrophilic polymer
networks that absorb water and form swollen material up to thousands times of their
dry weight in water. Properties include chemically stable or may degrade and
eventually disintegrate and dissolve allow for efficient transports of nutrients, growth
factors and drugs to the encapsulated cell [12, 17].
Irregular Multi-shell
Core Shell
Single core Multi-core Matrix
17
Table 2.3: Alternative microencapsulation material [38]
Microcapsule design Advantages
Alg- PLO
Increased biocompatibility
Alg-Cellulose sulfate-PMCG Independent adjustment of capsule parameters
HEMA-MMA
Improved mass transfer, stability and durability
Agarose-polystyrene sulfonate Blocks the activation of complement via enhancing the activity of C1
inhibitors.
Alg-Agarose Increased mechanical stability
Barium-alg Increased mechanical and chemical stability
PDADMAC
Increased mechanical stability
Alg-chitosan Increased biocompatibality
PVA
Improved intracapsular nutrient transport
Photopolymerised PEG-
diacrylate
Increased biocompatibilty
According to Code of Federal Regulation (CFR) in the Food and Drug
Administration (FDA) of the United States of America [41, 42], calcium alginate is
the calcium salt of alginate acid which is a natural polyuronide constituent of various
species brown algae or seaweed [25, 26]. It had been approved as the one of the
important biomaterials in regenerative medicine, nutrition supplements and also
stabilizer. The chemical composition and sequence of alginate may vary widely
because of the different species of algae. Principally, alginate are unbranched
polysaccharides which are binary, linear copolymers consisting of (1→4) linked β-D-
mannuronic acid (M) and α-L- guluronic acid (G) residues as shown in Figure 2.7 [6,
25, 27].
Figure 2.7: Structural characteristics of alginate: (a) alginate monomers, (b) chain
conformation, (c) block distribution [13]
18
By using various techniques for cell encapsulation the proposed polymeric materials
can be explored. Each polymeric material that can be used must be biocompatible in
order to obtain successful cell encapsulation. This may provide immune protection
by isolating encapsulated cells from host tissue but also keeping the cells well
distributed in capsules and maintaining the phenotype of cells. The rough surface of
microcapsules must be avoided to prevent the immunological reactions when
implanted. The feasibility of encapsulation of cells depends on the cell type and
materials for encapsulation. Table 2.4 shows the materials, the type of cells used for
microencapsulation and their clinical applications. From the literature review,
microencapsulation of epithelial cells is still rare and presented opportunity for
research.
A variety of parameters such as different concentration of coating alginate,
variety in exposure time in second gelling solution, different gelling ions (both for
core of alginate beads first and second gelling solution (for coating layer) and
different washing solution (mannitol or saline), to see if this treatment can affect the
binding and distribution of coating alginate in coated capsules [56].
In cell microencapsulation, alginate become the most common polymer employed
because of its features. It is the selective binding of multivalent cations, which is the
basis for hydrogels formation and the transition of alginate does not influenced by
temperature. Alginates also possess a soft nature, making them physically similar to
most native tissue. Transparent condition allow alginate hydrogels the routine
analysis of cell entrapment using standard microscopical techniques and enable cell
recovery without cell damage [54].
For this research study, the selection of alginate as microencapsulation
polymer is promoting because of it properties which is can mimics the extracellular
matrix (ECM) and supports cells functions and metabolism.
19
Table 2.4: Cell encapsulation approaches based on different alginate matrices [57]
Material Cell Implantation site Application
Alginate Bone marrow stromal cells,
murine derived adiposed tissue
stromal cells, islets of Langerhans
Subcutaneous space,
peritoneal cavity and
under the kidney
capsule
Bone and cartilage
engineering,
diabetes and cancer
Alginate
(Atomization)
Monocytes and mesenchymal
stem cells
In vitro study
Alginate (with
RGD)
MC3T3-E1, myoblasts and
satellite cells
Muscle Bone regeneration
and muscle
regeneration
Alginate
(Enzymatic
modification)
In vitro study Increased stability
Alginate
(Chemoenzymatic
modification)
In vitro study Increased stability
Alginate
(Photoreactive
liposomes)
Bone derived cells In vitro study Substrates
containing cells
immobilized in
precise locations
Alginate (Phenol
moieyies)
Crandall-Reese feline kidney
cells
In vitro study Increased stability
Alginate-PLL-
alginate
Embryonic stem cells, bone
marrow mesenchymal stem cells,
islets of Langerhans, chromaffin
cells and myoblasts
Peritoneal cavity,
subarachnoid space
and subcutaneous
space
Bone repair and
regeneration,
chronic neuropathic
pain and anemia
Alginate-PLL-
alginate
(Covalent cross-
links between
membranes)
Islets of Langerhans and EL-4
thymoma
Peritoneal cavity Increased stability
Alginate-PLL-
alginate
(polymerization)
Peritoneal cavity
Alginate-agarose Feline kidney cells and human In vitro study Subsieve-size
capsules
Alginate-chitosan Baby hamster kidney cells and
human mesenchymal stem cells
Subcutaneous space Tissue engineering
Alginate-chitosan
(Lactose modified
chitosan)
Chondrocytes In vitro study Increased
mechanical
properties
Alginate-PLO-
alginate
Choroid plexus and islets of
Langerhans
Brain, peritoneal
cavity and
subcutaneous space
Diabetes and
neuroprotection
20
2.7 Different microencapsulation techniques
2.7.1 Extrusion and dripping technique
Extrusion method or technique is simple devices where the cell and biopolymer
solutions-containing syringe being extruded through a nozzle or needle to be cross-
linked in a hardening solutions [27, 28]. When the way of controlled droplet
formation is there, the method is known as prilling. In this type of system, external
energy is required to reduce the droplet size [23, 27]. Hence, a few extrusion
approaches to the production of microcapsules had been developed and introduced
by previous researcher. This includes electrostatic generator, nozzle resonance
technology, jet cutter, spinning disk and other common techniques [23, 27–30].
Figure 2.8 shows the extrusion technologies in producing microcapsules. Each of the
technologies has different advantages and disadvantages when producing the
microcapsules.
The disadvantage of simple dripping technique is that the microcapsules
formed is very large to be used in biotechnological or medical applications. Dripping
with concentric air jet can produced microcapsules small in size but only in small
batches. This is different from dripping and spraying with electrostatic forces. There
are potentials put in opposite sites between orifice and the gelation bath. With the
slow flow rate, the technology can only produce small batch of microcapsules.
Although rotating disk and jet cutter technologies can produce large batches of bead,
but the size of the beads are too big [6].
Nonetheless, the vibration technology offers the best microcapsules
production with uniform, monodisperse and small in size. The process can be
controlled and easy to scale-up. For vibration technology, it can be applied to
encapsulate living things, liquids and special features based on the end product
requirement.
21
Figure 2.8: Extrusion technology [47]
(a) (b) (c) (d) (e)
(a) (b)
(c) (d)
(e)
Simple dripping Dripping with a concentric air jet
Dripping and spray with electrostatic
forces
Rotating disk and jet cutter
technologies
Vibration technology
22
2.7.2 Extrusion and vibration technology
Different extrusion technologies for encapsulation of cells, microbes or any other
liquids include by applying the vibration on a laminar jet for controlled break-up into
monodisperse microcapsules. A superimposed vibration in vibration technologies is
conducted based on the principle that a liquid jet breaks up into equally sized
microcapsules.
The encapsulation laboratory scale device [6] had been produced to perform
the experiment. In Figure 2.9, the main parts of the device were briefly described.
The device is possibly used to encapsulate cells without any significant loss of cell
viability. It also can produce capsules between 100 up to 2000 μm controlled by
several parameters including the vibration frequency, nozzle size, flow rate and
physical properties of the polymeric material used.
Figure 2.9: Main part of encapsulation device with a concentric nozzle [6]
23
Several methods for producing protein-immobilised calcium alginate beads have
been reported [61, 62]. In Figure 2.10, the study reported by [63], shows the used of
loudspeaker as vibrating device. The sine wave sound generator is connected to the
loudspeaker with the frequency set to 200 Hz. Using this method, the small size
microcapsules below 20 µm that produced were easily arrange with the help of
optical tweezers or laser manipulation. However, another flexible silica capillary
connected to the syringe needle might increase the complexity of this system.
Figure 2.10: Apparatus for producing alginate beads by the vibration method [63]
2.7.3 Microfluidic
Microfluidic has emerged as a powerful platform for the generation of micoparticles
with tailored structure and properties. This technique allows direct integration of
different input fluid into the PDMS microfluidic channel as shown in Figure 2.11.
Flexible silica capillary
24
The system involves with cells suspended in culture media that is dispersed through
the channel continuously flushed with oil. Although microscale techniques has been
applied to biological for nearly two decades, but it has not been widely integrated as
common tools in biological laboratories. This could be due to the tedious
preparation procedures. However, the complexity of the microfluidic design has
increased tremendously with more functionalities and flow channels.
Figure 2.11: Microfluidic channel [64]
2.7.4 Electrospray
Previously, Bugarski et al. first reported the preparation of microcapsule using
electrospray technique. The technique had demonstrated the effective preparation of
size-controlled microcapsules. However this technique comes with high complexity
of operation [65] and come with a risk since it required the used of high voltage
generator. Basically, electrospray system consists of syringe pump, stainless steel
needle and high voltage generator.
The microcapsules were generated by extruding the polymeric material or solution
through the stainless steel needle using a syringe pump. The electric force or voltage
generator was applied between the gelling bath and the needle [11, 20–22]. As shown
in Figure 2.12, it is the example of the electrospray device setup. Although the device
seem to be simple and easy to conduct, but the electric force supply might be the risk
to the user.
82
REFERENCES
[1] J. P. Frampton, M. R. Hynd, M. L. Shuler, and W. Shain, “Fabrication and
optimization of alginate hydrogel constructs for use in 3D neural cell culture.,”
Biomed. Mater., vol. 6, p. 015002, 2011.
[2] D. Huh, G. A. Hamilton, and D. E. Ingber, “From 3D cell culture to organs-
on-chips,” Trends Cell Biol., vol. 21, no. 12, pp. 745–754, 2011.
[3] M. Rimann, B. Angres, I. Patocchi-Tenzer, S. Braum, and U. Graf-Hausner,
“Automation of 3D cell culture using chemically defined hydrogels,” J. Lab.
Autom., vol. 19, no. 2, pp. 191–197, 2013.
[4] D. Lewinska, J. Bukowski, M. Kozuchowski, A. Kinasiewicz, and A.
Werynski, “Electrostatic microencapsulation of living cells,” Biocybern.
Biomed. Eng., vol. 28, no. 2, pp. 69–84, 2008.
[5] Y. W. Chen, C. W. Kuo, D. Y. Chueh, and P. Chen, “Surface modified
alginate microcapsules for 3D cell culture,” Surf. Sci., vol. 648, pp. 47–52,
2016.
[6] C. Heinzen, A. Berger, and I. Marison, “Use of vibration technology for jet-
break for encapsulation of cells, microbes and liquids in monodisperse
microcapsules,” vol. 7, pp. 731–733, 2008.
[7] M. Whelehan and I. W. Marison, “Microencapsulation using vibrating
technology,” J. Microencapsul., vol. 28, no. 8, pp. 669–688, 2011.
[8] S. Sugiura, T. Oda, Y. Izumida, Y. Aoyagi, M. Satake, A. Ochiai, N.
Ohkohchi, and M. Nakajima, “Size control of calcium alginate beads
containing living cells using micro-nozzle array,” Biomaterials, vol. 26, no.
16, pp. 3327–3331, 2005.
[9] M. N. Singh, K. S. Y. Hemant, M. Ram, and H. G. Shivakumar,
“Microencapsulation: A promising technique for controlled drug delivery.,”
Res. Pharm. Sci., vol. 5, no. 2, pp. 65–77, 2010.
83
[10] N. K. Sachan, B. Singh, and K. R. Rao, “Controlled drug delivery through
microencapsulation,” J. Pha, vol. 4, no. 1, pp. 65–81, 2006.
[11] E. P. Herrero, E. M. Martín Del Valle, and M. A. Galán, “Development of a
new technology for the production of microcapsules based in atomization
processes,” Chem. Eng. J., vol. 117, no. 2, pp. 137–142, 2006.
[12] E. Anitua, M. Sánchez, G. Orive, and I. Andia, “Delivering growth factors for
therapeutics,” Trends Pharmacol. Sci., vol. 29, no. 1, pp. 37–41, 2008.
[13] K. I. Draget and C. Taylor, “Chemical, physical and biological properties of
alginates and their biomedical implications,” Food Hydrocoll., vol. 25, no. 2,
pp. 251–256, 2011.
[14] L. Wang, R. M. Shelton, P. R. Cooper, M. Lawson, J. T. Triffitt, and J. E.
Barralet, “Evaluation of sodium alginate for bone marrow cell tissue
engineering,” Biomaterials, vol. 24, no. 20, pp. 3475–3481, Sep. 2003.
[15] I. Ghidoni, T. Chlapanidas, M. Bucco, F. Crovato, M. Marazzi, D. Vigo, M. L.
Torre, and M. Faustini, “Alginate cell encapsulation: new advances in
reproduction and cartilage regenerative medicine.,” Cytotechnology, vol. 58,
no. 1, pp. 49–56, Oct. 2008.
[16] C. Tomaro-Duchesneau, S. Saha, M. Malhotra, I. Kahouli, and S. Prakash,
“Microencapsulation for the therapeutic delivery of drugs, live mammalian
and bacterial cells, and other biopharmaceutics: current status and future
directions,” J. Pharm., vol. 2013, pp. 1–19, 2012.
[17] J. L. García, A. Asadinezhad, J. Pacherník, M. Lehocký, I. Junkar, P.
Humpolíček, P. Sáha, and P. Valášek, “Cell proliferation of HaCaT
keratinocytes on collagen films modified by argon plasma treatment,”
Molecules, vol. 15, no. 4, pp. 2845–2856, 2010.
[18] T. Igarashi, K. Nishino, and S. K. Nayar, “The appearance of human skin,”
2005.
[19] Martin T. Rekus, “Characterization of growth and differentiation of a
spontaneously immortalized keratinocyte cell line (HaCaT) in a defined,
serum-free culture system.,” University of Dusseldorf, 2000.
[20] B. L. Allen-Hoffmann, S. J. Schlosser, C. a Ivarie, C. a Sattler, L. F. Meisner,
and S. L. O’Connor, “Normal growth and differentiation in a spontaneously
immortalized near-diploid human keratinocyte cell line, NIKS.,” J. Invest.
Dermatol., vol. 114, no. 3, pp. 444–455, 2000.
84
[21] L. Micallef, F. Belaubre, A. Pinon, C. Jayat-Vignoles, C. Delage, M.
Charveron, and A. Simon, “Effects of extracellular calcium on the growth-
differentiation switch in immortalized keratinocyte HaCaT cells compared
with normal human keratinocytes,” Exp. Dermatol., vol. 18, no. 2, pp. 143–
151, 2009.
[22] M. Rimann and U. Graf-Hausner, “Synthetic 3D multicellular systems for
drug development,” Curr. Opin. Biotechnol., vol. 23, no. 5, pp. 803–809,
2012.
[23] T.-M. Achilli, J. Meyer, and J. R. Morgan, “Advances in the formation, use
and understanding of multi-cellular spheroids,” Expert Opin. Biol. Ther., vol.
12, no. 10, pp. 1347–1360, 2012.
[24] J. Bin Kim, “Three-dimensional tissue culture models in cancer biology.,”
Semin. Cancer Biol., vol. 15, no. 5, pp. 365–377, 2005.
[25] J. M. Kelm, N. E. Timmins, C. J. Brown, M. Fussenegger, and L. K. Nielsen,
“Method for generation of homogeneous multicellular tumor spheroids
applicable to a wide variety of cell types,” Biotechnol. Bioeng., vol. 83, no. 2,
pp. 173–180, 2003.
[26] E. Knight and S. Przyborski, “Advances in 3D cell culture technologies
enabling tissue-like structures to be created in vitro,” J. Anat., vol. 227, no. 6,
pp. 746–756, 2015.
[27] P. K. Kabadi, M. M. Vantangoli, A. L. Rodd, E. Leary, S. J. Madnick, J. R.
Morgan, A. Kane, and K. Boekelheide, “Into the depths: Techniques for in
vitro three dimensional microtissue visualization,” Biotechniques, vol. 59, no.
5, pp. 279–286, 2015.
[28] R. Edmondson, J. J. Broglie, A. F. Adcock, and L. Yang, “Three-dimensional
cell culture systems and their applications in drug discovery and cell-based
biosensors.,” Assay Drug Dev. Technol., vol. 12, no. 4, pp. 207–18, 2014.
[29] W. P. Daley, S. B. Peters, and M. Larsen, “Extracellular matrix dynamics in
development and regenerative medicine.,” J. Cell Sci., vol. 121, no. Pt 3, pp.
255–64, 2008.
[30] R. P. Mecham, “Overview of extracellular matrix,” Curr. Protoc. Cell Biol.,
no. SUPPL.57, pp. 1–14, 2012.
[31] M. W. Tibbitt and K. S. Anseth, “Hydrogels as extracellular matrix mimics for
3D cell culture,” Biotechnol. Bioeng., vol. 103, no. 4, pp. 655–663, 2009.
85
[32] H. Geckil, F. Xu, X. Zhang, S. Moon, and U. Demirci, “Engineering
hydrogels as extracellular matrix mimics.,” Nanomedicine (Lond)., vol. 5, no.
3, pp. 469–484, 2010.
[33] R. O. Hynes, “Integrins: Bidirectional, allosteric signaling machines,” Cell,
vol. 110, no. 6, pp. 673–687, 2002.
[34] R. Lin and H. Chang, “Recent advances in three-dimensional multicellular
spheroid culture for biomedical research,” pp. 1172–1184, 2008.
[35] V. Breguet, U. Von Stockar, and I. W. Marison, “Characterization of alginate
lyase activity on liquid, gelled, and complexed states of alginate,” Biotechnol.
Prog., vol. 23, no. 5, pp. 1223–1230, 2007.
[36] J. Günter, P. Wolint, A. Bopp, J. Steiger, E. Cambria, S. P. Hoerstrup, and M.
Y. Emmert, “Microtissues in cardiovascular medicine : Regenerative potential
based on a 3D microenvironment,” Stem Cells Int., vol. 2016, pp. 1–20, 2016.
[37] F. Khan, M. Tanaka, and S. R. Ahmad, “Fabrication of polymeric
biomaterials : a strategy for tissue engineering and medical devices,” J. Mater.
Chem. B, vol. 3, pp. 8224–8249, 2015.
[38] G. Orive, R. Maria Hernández, A. Rodr guez Gasc n, R. Calafiore, T. M. Swi
Chang, P. De Vos, G. Hortelano, D. Hunkeler, I. Lac k, and J. Luis Pedraz,
“History, challenges and perspectives of cell microencapsulation,” Trends
Biotechnol., vol. 22, no. 2, pp. 87–92, Feb. 2004.
[39] T. Sun, S. Jackson, J. W. Haycock, and S. MacNeil, “Culture of skin cells in
3D rather than 2D improves their ability to survive exposure to cytotoxic
agents,” J. Biotechnol., vol. 122, no. 3, pp. 372–381, 2006.
[40] W. Wang, X. Liu, Y. Xie, H. Zhang, W. Yu, Y. Xiong, W. Xie, and X. Ma,
“Microencapsulation using natural polysaccharides for drug delivery and cell
implantation,” J. Mater. Chem., vol. 16, no. 32, pp. 3252–3267, 2006.
[41] G. Orive, E. Santos, D. Poncelet, R. M. Hernandez, J. L. Pedraz, L. U.
Wahlberg, P. De Vos, and D. Emerich, “Cell encapsulation: technical and
clinical advances,” Trends Pharmacol. Sci., vol. 36, no. 8, pp. 537–546, 2015.
[42] N. Agnihotri, R. Mishra, C. Goda, and M. Arora, “Microencapsulation – A
novel approach in drug delivery : A review,” J. Pharm. Sci., vol. 2, no. 1, pp.
1–20, 2012.
86
[43] M. Jadupati, D. Tanmay, and G. Souvik, “Microencapsulation: An
indispensable technology for drug delivery system,” J. Pharm., vol. 3, no. 4,
pp. 8–13, 2012.
[44] N. V. N. Jyothi, P. M. Prasanna, S. N. Sakarkar, K. S. Prabha, P. S. Ramaiah,
and G. Y. Srawan, “Microencapsulation techniques, factors influencing
encapsulation efficiency,” J. Microencapsul., vol. 27, no. 3, pp. 187–197,
2010.
[45] H. K. Solanki, D. D. Pawar, D. a. Shah, V. D. Prajapati, G. K. Jani, A. M.
Mulla, and P. M. Thakar, “Development of microencapsulation delivery
system for long-term preservation of probiotics as biotherapeutics agent,”
Biomed Res. Int., vol. 2013, p. 620719, 2013.
[46] J. Wan, “Microfluidic-based synthesis of hydrogel particles for cell
microencapsulation and ell-based drug delivery,” Polymers (Basel)., vol. 4,
no. 4, pp. 1084–1108, 2012.
[47] D. Poncelet, “Microencapsulation: fundamentals, methods and applications,”
Surf. Chem. Biomed. Environ. Sci., pp. 23–34, 2006.
[48] a. Jamekhorshid, S. M. Sadrameli, and M. Farid, “A review of
microencapsulation methods of phase change materials (PCMs) as a thermal
energy storage (TES) medium,” Renew. Sustain. Energy Rev., vol. 31, pp.
531–542, 2014.
[49] A. S. Hoffman, “Hydrogels for biomedical applications,” Adv. Drug Deliv.
Rev., vol. 64, pp. 18–23, 2012.
[50] Š. Selimović, J. Oh, H. Bae, M. Dokmeci, and A. Khademhosseini,
“Microscale strategies for generating cell-encapsulating hydrogels,” Polymers
(Basel)., vol. 4, no. 4, pp. 1554–1579, 2012.
[51] J. Sun and H. Tan, “Alginate-based biomaterials for regenerative medicine
applications,” Materials (Basel)., vol. 6, no. 4, pp. 1285–1309, Mar. 2013.
[52] “Code of Federal Regulations Title 21,” 2015. [Online]. Available:
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=1
84.1187. [Accessed: 11-Jan-2016].
[53] L. Fan, M. Cao, S. Gao, T. Wang, H. Wu, M. Peng, X. Zhou, and M. Nie,
“Preparation and characterization of sodium alginate modified with collagen
peptides,” Carbohydr. Polym., vol. 93, no. 2, pp. 380–385, 2013.
87
[54] S. J. Bidarra, C. C. Barrias, and P. L. Granja, “Injectable alginate hydrogels
for cell delivery in tissue engineering,” Acta Biomater., vol. 10, no. 4, pp.
1646–1662, 2014.
[55] C. H. Goh, P. W. S. Heng, and L. W. Chan, “Alginates as a useful natural
polymer for microencapsulation and therapeutic applications,” Carbohydr.
Polym., vol. 88, no. 1, pp. 1–12, 2012.
[56] S. Hadjialirezaei, “Coating of alginate capsules,” Norwegian University of
Science and Technology, 2013.
[57] A. Murua, A. Portero, G. Orive, R. M. Hernández, M. de Castro, and J. L.
Pedraz, “Cell microencapsulation technology: Towards clinical application,”
J. Control. Release, vol. 132, no. 2, pp. 76–83, 2008.
[58] J. M. Rabanel, X. Banquy, H. Zouaoui, M. Mokhtar, and P. Hildgen,
“Progress technology in microencapsulation methods for cell therapy,”
Biotechnol. Prog., vol. 25, no. 4, pp. 946–963, 2009.
[59] S. Jyothi, A. Seethadevi, K. S. Prabha, and P. Muthuprasanna,
“Microencapsulation: A review,” J. Pharma Bio Sci., vol. 3, no. 1, pp. 509–
531, 2012.
[60] D. Serp, E. Cantana, C. Heinzen, U. Von Stockar, and I. W. Marison,
“Characterization of an encapsulation device for the production of
monodisperse alginate beads for cell immobilization.,” Biotechnol. Bioeng.,
vol. 70, no. 1, pp. 41–53, 2000.
[61] S. Sakai, T. Ono, H. Ijima, and K. Kawakami, “Synthesis and transport
characterization of alginate/aminopropyl-silicate/alginate microcapsule:
Application to bioartificial pancreas,” Biomaterials, vol. 22, no. 21, pp. 2827–
2834, 2001.
[62] R. Srivastava and M. J. McShane, “Application of self-assembled ultra-thin
film coatings to stabilize macromolecule encapsulation in alginate
microspheres,” J. Microencapsul., vol. 22, no. June, pp. 397–411, 2005.
[63] Y. Zhou, S. Kajiyama, H. Masuhara, Y. Hosokawa, T. Kaji, and K. Fukui, “A
new size and shape controlling method for producing calcium alginate beads
with immobilized proteins,” J. Biomed. Sci. Eng., vol. 02, pp. 287–293, 2009.
[64] K.-S. Huang, M.-K. Liu, C.-H. Wu, Y.-T. Yen, and Y.-C. Lin, “Calcium
alginate microcapsule generation on a microfluidic system fabricated using the
optical disk process,” J. Micromechanics Microengineering, vol. 17, no. 8, pp.
88
1428–1434, 2007.
[65] Y. Fukui, T. Maruyama, Y. Iwamatsu, A. Fujii, T. Tanaka, Y. Ohmukai, and
H. Matsuyama, “Preparation of monodispersed polyelectrolyte microcapsules
with high encapsulation efficiency by an electrospray technique,” Colloids
Surfaces A Physicochem. Eng. Asp., vol. 370, no. 1–3, pp. 28–34, 2010.
[66] W. Zhang and X. He, “Encapsulation of living cells in small (~100 μm)
alginate microcapsules by electrostatic spraying: A parametric study,” J.
Biomech. Eng., vol. 131, no. 7, pp. 1–6, 2009.
[67] J. Xie, W. J. Ng, L. Y. Lee, and C.-H. Wang, “Encapsulation of protein drugs
in biodegradable microparticles by co-axial electrospray,” J. Colloid Interface
Sci., vol. 317, no. 2, pp. 469–476, 2008.
[68] J. Xie and C.-H. Wang, “Electrospray in the dripping mode for cell
microencapsulation,” J. Colloid Interface Sci., vol. 312, no. 2, pp. 247–255,
2007.
[69] L. Gasperini, J. F. Mano, and R. L. Reis, “Natural polymers for the
microencapsulation of cells,” J. R. Soc. Interface, vol. 11, pp. 1–19, 2014.
[70] R. M. Hernández, G. Orive, A. Murua, and J. L. Pedraz, “Microcapsules and
microcarriers for in situ cell delivery,” Adv. Drug Deliv. Rev., vol. 62, pp.
711–730, 2010.
[71] A. Leung, Y. Ramaswamy, P. Munro, G. Lawrie, L. Nielsen, and M. Trau,
“Emulsion strategies in the microencapsulation of cells: Pathways to thin
coherent membranes,” Biotechnol. Bioeng., vol. 92, no. 1, pp. 45–53, 2005.
[72] A. Kang, J. Park, J. Ju, G. S. Jeong, and S.-H. Lee, “Cell encapsulation via
microtechnologies,” Biomaterials, vol. 35, no. 9, pp. 2651–2663, 2014.
[73] “Structure of stepper motor,” Oriental Motor USA Corp. [Online]. Available:
http://www.orientalmotor.com/stepper-motors/technology/stepper-motor-
overview.html.
[74] Austin Hughes and A. Hughes, Electric Motors and Drives Fundamentals,
Types and Applications, Second edi. Newnes, 1990.
[75] P. Acarnley, Stepping motor a guide to theory and practice, 4th editio.
Institution of Electrical Engineers,London, 2002.
[76] STMicroelectronics, “L298 Dual Full-bridge Driver,” pp. 1–13, 2000.
[77] I. Rehman and W. Bonfield, “Characterization of hydroxyapatite and
carbonated apatite by photo acoustic FTIR spectroscopy,” J. Mater. Sci.
89
Mater. Med., vol. 8, no. 1, pp. 1–4, 1997.
[78] T. Nicolet and C. All, “Introduction to Fourier Transform Infrared
Spectrometry,” A Thermo Electron Bussines, pp. 1–8, 2001.
[79] A. D. Meade, F. M. Lyng, P. Knief, and H. J. Byrne, “Growth substrate
induced functional changes elucidated by FTIR and Raman spectroscopy in
in-vitro cultured human keratinocytes,” Anal. Bioanal. Chem., vol. 387, no. 5,
pp. 1717–1728, 2007.
[80] S. M. Lim, S. H. Oh, H. H. Lee, S. H. Yuk, G. Il Im, and J. H. Lee, “Dual
growth factor-releasing nanoparticle/hydrogel system for cartilage tissue
engineering,” J. Mater. Sci. Mater. Med., vol. 21, no. 9, pp. 2593–2600, 2010.
[81] S. N. A. Rahim, A. Sulaiman, F. Hamzah, K. H. K. Hamid, M. N. M. Rodhi,
M. Musa, and N. A. Edama, “Enzymes Encapsulation within Calcium
Alginate-clay Beads: Characterization and Application for Cassava Slurry
Saccharification,” Procedia Eng., vol. 68, pp. 411–417, 2013.
[82] B. Chazotte, “Labeling Nuclear DNA Using DAPI,” Cold Spring Harb
Protoc, 2011.
[83] Sigma Aldrich, “DAPI.” [Online]. Available:
http://www.sigmaaldrich.com/content/dam/sigma-
aldrich/docs/Sigma/Product_Information_Sheet/d9542pis.pdf.
[84] Y. Kubota, K. Kubota, and S. Tani, “DNA binding properties of DAPI (4’,6-
diamidino-2-phenylindole) analogs having an imidazoline ring or a
tetrahydropyrimidine ring: Groove-binding and intercalation,” Nucleic Acids
Symp. Ser., no. 44, pp. 53–54, 2000.
[85] B. Inc, “Viability / Cytotoxicity Assay Kit For Animal Live & Dead Cells,”
vol. 30002, no. 510.
[86] S. Sakai, C. Mu, K. Kawabata, I. Hashimoto, and K. Kawakami,
“Biocompatibility of subsieve-size capsules versus conventional-size
microcapsules.,” J. Biomed. Mater. Res. A, vol. 78, no. 2, pp. 394–8, Aug.
2006.
[87] M. Garrod and D. Y. S. Chau, “An Overview of Tissue Engineering as an
Alternative for Toxicity Assessment,” J. Pharm. Sci., vol. 19, no. 1, pp. 31–
71, 2016.
[88] Sigma Aldrich, Fundamental Techniques in Cell Culture. United Kingdom:
European Collection of Authenticated Cell Cultures, 2010.
90
[89] A. K. A. S. Brun-Graeppi, C. Richard, M. Bessodes, D. Scherman, and O. W.
Merten, “Cell microcarriers and microcapsules of stimuli-responsive
polymers,” J. Control. Release, vol. 149, no. 3, pp. 209–224, 2011.
[90] C. Sartori, “The characterisation of alginate systems for biomedical
applications,” Brunel University, 1997.
[91] B. Sarmento, D. Ferreira, F. Veiga, and A. Ribeiro, “Characterization of
insulin-loaded alginate nanoparticles produced by ionotropic pre-gelation
through DSC and FTIR studies,” Carbohydr. Polym., vol. 66, no. 1, pp. 1–7,
2006.
[92] C. G. van Hoogmoed, H. J. Busscher, and P. de Vos, “Fourier transform
infrared spectroscopy studies of alginate-PLL capsules with varying
compositions.,” J. Biomed. Mater. Res. A, vol. 67, no. 1, pp. 172–178, 2003.
[93] K. Y. Lee and D. J. Mooney, “Alginate: properties and biomedical
applications.,” Prog. Polym. Sci., vol. 37, no. 1, pp. 106–126, 2012.
[94] A. Meade, C. Clarke, H. Byrne, and F. Lyng, “Fourier transform infrared
microspectroscopy and multivariate methods for radiobiological dosimetry.,”
Radiat. Res., vol. 173, no. 2, pp. 225–237, 2010.
[95] C. Soon, K. Thong, K. Tee, A. Ismail, M. Denyer, M. Ahmad, Y. Kong, P.
Vyomesh, and S. Cheong, “A scaffoldless technique for self-generation of
three-dimensional keratinospheroids on liquid crystal surfaces,” Biotech.
Histochem., vol. 91, no. 4, pp. 283–295, 2016.
[96] M. Fertah, A. Belfkira, E. montassir Dahmane, M. Taourirte, and F.
Brouillette, “Extraction and characterization of sodium alginate from
Moroccan Laminaria digitata brown seaweed,” Arab. J. Chem., no. MAY,
2014.
[97] M. Jackson and H. H. Mantsch, “The use and misuse of FTIR spectroscopy in
the determination of protein structure.,” Crit. Rev. Biochem. Mol. Biol., vol.
30, no. 2, pp. 95–120, 1995.
[98] J. A. Rowley, G. Madlambayan, and D. J. Mooney, “Alginate hydrogels as
synthetic extracellular matrix materials,” Biomaterials, vol. 20, no. 1, pp. 45–
53, 1999.
[99] J. D. Denstedt, T. A. Wollin, and G. Reíd, “Biomaterials used in urology:
current issues of biocompatibilty, infection and encrustation,” J. Endourol.,
vol. 12, no. 6, 1998.
91
[100] P. Thevenot, W. Hu, and L. Tang, “Surface chemistry influences implant
biocompatibility.,” Curr. Top. Med. Chem., vol. 8, no. 4, pp. 270–280, 2008.
[101] W. Chen, M. Lisowski, G. Khalil, I. R. Sweet, and A. Q. Shen,
“Microencapsulated 3-dimensional sensor for the measurement of oxygen in
single isolated pancreatic islets,” PLoS One, vol. 7, no. 3, pp. 1–10, 2012.
[102] D. P. R. Gaspar, “A novel strategy to produce a drug delivery system for skin
regeneration,” University of Beira Interior, 2012.
[103] H. S. Kim, C. G. Lee, and E. Y. Lee, “Alginate lyase: Structure, property, and
application,” Biotechnol. Bioprocess Eng., vol. 16, no. 5, pp. 843–851, 2011.
[104] U. Remminghorst and B. H. A. Rehm, “Bacterial alginates: From biosynthesis
to applications,” Biotechnol. Lett., vol. 28, no. 21, pp. 1701–1712, 2006.
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