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

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