An-Najah National University 

Faculty of Graduate Studies 

 

 

 

 

 

Development of Three-Dimensional 

Neuronal Model Systems for 

Mechanical Stress Application 

 

 

By 

Mahmoud Ardah 

 
Supervisors 

Dr. Ashraf Sawaftah 

Dr. Feras Al-Battah 

 

This Thesis is Submitted in Partial Fulfillment of the Requirements for 

the Degree of Master of Biology, Faculty of Graduate Studies, An-Najah 

National University, Nablus, Palestine. 

2021  





iii 
 

Dedication 

I humbly dedicate this piece of work to my loving parents 

(Basim and Jasmin), for their endless g u i d a n c e  a n d  s u p p o r t .  

To  my brothers  and  s is ters  who shared their words 

of advice and encouragement to finish this study. 

 

 

 

  



iv 
 

Acknowledgment 

First of all, I would like to express my sincere appreciation to my German 

Supervisor, PD. Dr. Bernd Hoffmann and his crew in IBI-2 Jülich research 

center/ Germany and in addition to my Palestinian supervisors Dr. Ashraf 

Sawaftah (An-Najah National University) and Dr. Feras Al-Battah (Arab 

American University) for their guidance and encouragement during my 

master research work and writing. I do not forget the appropriate guidance 

of Dr. Jella Abraham during my research and thesis writing in Jülich 

research center Germany. 

I would like to thank PALAST and the PGSB for allowing me to do my 

master thesis at Jülich research center Germany and giving me the 

opportunity for this fellowship. 

My acknowledgment for the IBI-2 institute and colleagues in Jülich research 

center Germany. 

Finally, special thanks to my appreciative and beloved parents ( Basem and 

Jasmin). 

To my brothers and sisters (Mohamed, Anas, and Bilal), and sisters (Amira, 

Hiba, and Tasnim), and my nephews (Malak and Khaled) for their support 

during my study and thesis preparation.  





vi 
 

Table of Contents 

Acknowledgment ......................................................................................... iv 

Declaration .................................................................................................... v 

Table of Contents ......................................................................................... vi 

List of Tables .............................................................................................. viii 

List of Figures .............................................................................................. ix 

List of Appendices ..................................................................................... xiv 

Abstract ....................................................................................................... xv 

Chapter 1 Introduction .................................................................................. 1 

1.1 The human brain ................................................................................... 1 

1.2 The Neuronal cytoskeleton ..................................................................... 6 

1.3 Traumatic injury in the central nervous system (CNS) .......................... 8 

1.4 Mechanical strain .................................................................................... 9 

1.5 Stretch experiments ............................................................................... 10 

1.6 Traditional two-dimensional (2D) cell culture vs. three-dimensional 

(3D) cell culture .................................................................................. 12 

1.7 Aim of the study .................................................................................... 15 

Chapter 2 Materials and Methods ............................................................... 17 

2.1 Cell culture ............................................................................................ 17 

2.1.1 Elastomeric chamber fabrication ....................................................... 17 

2.1.2 Primary cortical neuronal cell isolation ............................................. 19 

2.1.3 Matrigel as a natural product for a 3D scaffold ................................. 21 

2.1.3 A Sandwich method ........................................................................... 22 

2.1.3 B Three-dimension (3D) Suspension method .................................... 24 

2.1.4 Synthetic scaffold for three-dimension (3D) cell cultivation in 

Polyvinylpyrrolidone (PVP) copolymerized with Glycidyl 

methacrylate (GMA) ........................................................................... 27 

2.1.5 Synthetic scaffold for three-dimension (3D) cell cultivation in 

VitroGel 3D-RGD ............................................................................... 29 

2.1.6 Cell Stretching of cortical neuronal cells ........................................... 30 

2.1.7 Calcein Staining ................................................................................. 32 

2.1.8 Immunocytochemistry stain ............................................................... 34 

2.1.9 Sample fixation and preparation for LSM ......................................... 35 

2.1.10 Microscopy ....................................................................................... 36 

2.1.11 Imaris imaging processing software ................................................ 37 



vii 
 

Chapter 3  Results ....................................................................................... 38 

3.1 Three dimensional (3D) cell culture ..................................................... 38 

3.2 Neuronal cell culture in a 3D system and stretch experiments using 

Matrigel as a natural scaffold. ............................................................. 39 

3.2A The 3D Suspension method ............................................................... 40 

3.2B Sandwich method ............................................................................... 42 

3.3 Matrigel is a naturally suitable 3D culture system but a non-stretchable 

scaffold. ............................................................................................... 44 

3.4 Classical PDMS chamber reconstruction and development ................. 46 

3.5 VitroGel 3D-RGD as a synthesized polymer. ....................................... 47 

3.6 PVP co-GMA a synthesized stretchable 3D scaffold ........................... 49 

Chapter 4  Discussion and Conclusion ....................................................... 55 

4.1 Matrigel non-suitable three-dimensional (3D) system stretch scaffold 56 

4.2 VitroGel 3D-RGD cultivation scaffold ................................................. 58 

4.3 PVP copolymerized with GMA (PVP co-GMA) the 3D artificially 

synthesized stretchable scaffold .......................................................... 59 

Chapter 5  Future perspectives .................................................................... 61 

References ................................................................................................... 63 

Appendices .................................................................................................. 70 

 ج ............................................................................................................ الملخص

 

  



viii 
 

List of Tables 

Table 2.1: The 3D suspension method Matrigel stock and final concentration 

for 13 mm pre-drilled Petri dish glass and PDMS traditional 

elastomeric chamber. ................................................................ 26 

Table 2.2: Matrigel volumes for stock and final concentration for the newly 

reconstructed PDMS elastomeric chamber. .............................. 26 

Table 2.3: VitroGel mixing protocol from producer company was adjusted 

to fit our newly reconstructed PDMS chamber (The 

wellbioscience). ........................................................................ 30 

Table 2.4: Stretch parameters on natural and synthetic 3D polymers with 

different frequencies. ................................................................ 31 

Table 2.5: Each type of  microscope was used for special purposes as 

mentioned in the table above. ................................................... 36 

 



ix 
 

List of Figures 

Figure 1.1: The three main human brain parts. The cerebrum, cerebellum, 

and brain stem. The cerebrum is the major part of the brain, 

which is composed of two brain hemispheres (left and right). . 2 

Figure 1.2: The human central nervous system vs. peripheral nervous 

system. Adapted from lumen learning anatomy of the nervous 

system book [2]. ........................................................................ 3 

Figure 1.3: Neuronal cell body structure and cell-cell connection. Signal 

transfer from one cell to the other cell through the synaptic cleft. 

Adapt form journal of athletic trainer (Craig, 2003). ............... 4 

Figure 1.4: Neuronal cytoskeleton components. The neuronal cytoskeleton 

consists of three types of bio-polymers actin filament 

(microfilament) (green), microtubules (red), and intermediate 

filaments (grey) (Malacrida et al., 2019). ................................ 7 

Figure 1.5: The figure represents a comparison between 2D and 3D cell 

culture. A represents a 2D culture that is coated with protein, 

and B is a 3D culture on a scaffold. As shown in the figure, the 

cell shape is flat in the 2D culture, while in the 3D culture it is 

taking a spherical shape due to the ECM protein or peptide 

interaction. The 3D culture is closer to the real environment as 

shown in C (Stukel & Willits, 2016). ..................................... 13 

Figure 1.6: Comparison between 2D cell culture on flat culture vial vs. 3D 

cell culture on a scaffold from the growth and adhesion 

direction. The 3D culture shows the growth in an XYZ direction 

while the 2D culture shows growth only in an XY direction 

(Baker & Chen, 2012). ............................................................ 15 

Figure 2.1: PDMS crosslinking reaction by mixing them and curing it for 16 

h at 60 °C (Wisser et al., 2015). 18 

Figure 2.2: The 50 kPa PDMS fabrication mold chamber steps (Traditional 

chamber). The chamber cultivation area dimension is 2 cm * 2 



x 
 

cm. Images by Jens Konrad Forschungszentrum Jülich/ ICS-

7. .............................................................................................. 18 

Figure 2.3: Classical PDMS elastomeric chamber used for stretch 

experiments in the ICS-7. Images by Jens Konrad FZJ ICS-

7. .............................................................................................. 19 

Figure 2.4: Rat E18 – E19 brain dissection steps and cortex isolation. Brain 

images under a dissecting microscope in HBSS solution. 

Images by Mahmoud Ardah/ ICS-7. (A) Rat embryo head. (B) 

Top view of the rat E18 – E19 brain. The two hemispheres can 

be seen and the cerebellum. (C) The brain hemispheres are 

separated and meninges were removed. (D) Brain hemisphere 

without meninges. (E) Whole hemisphere vs. cortex. (F) Single 

rat embryo cortex. ................................................................... 20 

Figure 2.5:  Sandwich method system explanation figure. The pink color 

indicates the gel layer, and the blue color indicates the neuronal 

cell layer suspension. The two cell layers were separated by a 

gel layer, through this gel layer neuronal cells built connections 

between both cell layers. ......................................................... 23 

Figure 2.6: The 3D suspension method explanation. The neuronal cells 

(Blue) are randomly distributed inside the Matrigel in purple 

color. ....................................................................................... 25 

Figure 2.7: The chemical formula of Polyvinylpyrrolidone (PVP) 

copolymerized with Glycidyl methacrylate (GMA) PVP co-

GMA. This chemically synthesized polymer produced by DWI 

Aachen. ................................................................................... 28 

Figure 2.8:  Uniaxial stretcher device set developed by Wolfgang Rubner, 

ICS-7. A) PDMS chambers 1:40 where the cells were cultivated 

and stretched. B) Chamber holder where the chamber is 

screwed/ fixed in. C)Stretch apparatus where the holder with 

chamber was built-in to be stretched. Images by Jens Konrad 

FZJ ICS-7. ............................................................................... 31 



xi 
 

Figure 2.9: Cell Esterase converts Calcein-AM to Calcein, pathway inside 

alive cell. Image adapted from Dojindo. Living and dead cell 

staining, 2016 [6]. ................................................................... 33 

Figure 2.10: Immunocytochemistry mechanism. Primary and secondary 

antibodies bind together to the target and the fluorophore. .... 34 

Figure 3.1: Four different optimized Matrigel concentrations (12, 10, 6, 5 

mg/ml) respectively were used in the 3D suspension method as 

shown. The above-shown figure represents an un-stretched 

classical PDMS chamber. The higher the Matrigel 

concentration, the more the cells are clustered together and 

harder to distribute within the Matrigel. The PDMS chamber 

was stained with calcein and observed under the upright 

microscope with a 40X objective lens. The figure scale bar was 

20 µm. 41 

Figure 3.2:  Sandwich method cultivated in Matrigel with two different 

optimized concentrations (10, 20 mg/ml). This method was 

plated on the classical PDMS elastomeric chamber in an 

unstretched position. Neuronal cells were observed only on the 

upper cell layer. Cells were stained with calcein and observed 

under the upright microscope with a 40X objective lens. The 

figure scale bar is 20 µm. ........................................................ 43 

Figure 3.3: Traditional (classical) 50 kPa PDMS chamber, empty chamber 

vs. cultured chamber. Image by Jens Konrad (ICS-7). ........... 44 

Figure 3.4: Neuronal cells cultivated on Matrigel in the newly developed 

and reconstructed PDMS elastomeric chamber. Matrigel 

detachment from the inserted grooves wall. Chamber stained 

with MAP2 in green, anti-tubulin in red, and NFH in blue. A 

40x objective LSM 880 image. ............................................... 45 

Figure 3.5: Classical elastomeric chamber vs. newly developed and 

reconstructed elastomeric chamber. The external chamber 

dimensions were constant as the classical and the inserted wells 

dimensions were 5 mm* 5 mm*0.7 mm. reconstruction and 



xii 
 

modifications were performed by Mahmoud Ardah. The 

reconstruction were of the PDMS elastomeric chamber was 

performed and designed to fit 3D experiments. Images by Jens 

Konrad FZJ ICS-7. .................................................................. 47 

Figure 3.6: An un-stretched sample of neuronal cells cultivated in VitroGel 

3D-RGD, neuronal cells, and some cell branching can be 

observed after 24h of incubation at 37°C. Cells were stained 

with calcein and observed under the upright microscope with a 

40x objective lens with transmission light (TL). Figure Scale 

bar 30µm. ................................................................................ 48 

Figure 3.7: PVP co-GMA polymer plated without neuronal cells in a newly 

developed PDMS elastomeric chamber. The chamber was 

stretched for 1hr at a frequency of 300 mHz and an amplitude 

of 15%. The white arrow shows the stretch direction which was 

applied to the newly reconstructed PDMS chamber. No 

detachment was observed during stretch and chamber medium 

change. .................................................................................... 49 

Figure 3.8: Primary isolated neuronal cells cultivated in PVP co-GMA with 

high fibrin concentration (16.3 mg/ml). Slow neuronal cell 

growth due to high fibrinogen concentration. Cells were stained 

with calcein and observed under the upright microscope with a 

40x objective lens. The figure scale bar is 20 µm. A) Represents 

primary neuronal cells after 24h of cultivation and incubated in 

37°C. B) Neuronal cells after 1 week of cultivation. ............. 50 

Figure 3.9: Primary neuronal cells cultivated in PVP high fibrinogen 

concentration (16.3 mg/ml). Neuronal cells were stained and 

treated with MAP2 (green), NFH (blue), and Tubulin (red). The 

sample was observed under the LSM 880 with an air objective 

20x. The figure scale bar is 50 µm. ........................................ 51 

Figure 3.10: Different fibrinogen concentrations were tested for primary 

neuronal cell cultivation in PVP co-GMA gel.  Neuronal cells 

were stained with MAP2 in green and tubulin in red staining. 

A) High fibrinogen concentration 16.3 mg/ml low and slow 



xiii 
 

neuronal cell growth. B) Moderate fibrinogen concentration of 

8.16 mg/ml was acceptable for neuronal cell growth. C) Low 

fibrinogen concentration of 4.03 mg/ml favorable for neuronal 

cell growth. The figure scale bar is 50 µm. ............................ 52 

Figure 3.11. 1: Primary neuronal cells cultivated in PVP co-GMA with a 

moderate amount of fibrinogen concentration 8.16 mg/ml. 

Neuronal cells were stained with MAP2 in green and tubulin in 

red. Neuronal cells under 15% strain for 24 h with a frequency 

of 300 mHz The figure scale bar is 50 µm. 53 

Figure 3.11. 2: Primary neuronal cells cultivated in PVP co-GMA gel with 

a low amount of fibrinogen 4.03 mg/ml. Neuronal cells were 

stained with MAP2 in green and tubulin in red. Neuronal cells 

under 15% strain for 24 h with a frequency of 300 mHz The 

figure scale bar is 50 µm. ........................................................ 54 

 

  



xiv 
 

List of Appendices 

 

Consumable Materials ................................................................................. 71 

Instruments .................................................................................................. 71 

Software ...................................................................................................... 72 

Chemicals .................................................................................................... 73 

Media and Buffers ....................................................................................... 74 

Neurobasal medium +++ ............................................................................. 74 

3D culture media ......................................................................................... 75 

Imaging medium ......................................................................................... 75 

Antibodies ................................................................................................... 76 

 

  



xv 
 

Development of Three-Dimensional Neuronal Model Systems for 

Mechanical Stress Application 

By 

Mahmoud Ardah 

Supervisors 

Dr. Ashraf Sawaftah 

Dr. Feras Al-Battah 

Abstract 

Most of the known biological processes were discovered in two-dimensional 

(2D) cell culture. An increasing need for an in vitro model that mimics the 

brain's physiological environment to understand neurodegenerative 

diseases. The human brain is in continuous stretching-release movement due 

to the normal and repeated heartbeats and blood circulation in our body. This 

regular stretching-release brain movement changed the size and stretched 

the neuronal cells. To mimic this size change in the brain, the neuronal cells 

were cultivated on different scaffolds and stretched on an elastomeric 

Polydimethylsiloxane (PDMS) silicon chamber in vitro with different 

amplitudes. Previous stretch experiments on cortical neurons were applied 

to two-dimensional (2D) cell cultures testing the neuronal cells' behavior 

and cytoskeleton due to the mechanical strain applied by the stretcher 

device. The 2D culture results were attractive to go a step further to come 

closer to the real brain movement by creating and developing the three-

dimensional (3D) cell culture system. The brain movement was mimicked 

by a stretch apparatus and a mechanical strain was applied to the cortical 

neurons. 



xvi 
 

To create a 3D culture stretch experiment, a stretchable 50 kPa PDMS 

elastomer was needed. The elastomeric chamber was fixed in a specific 

holder, and primarily isolated rat embryo neuronal cells were cultivated in a 

3D scaffold on the PDMS chamber. Lastly, the complete 3D culture was 

screwed to the stretcher device. In the development process; of a 3D stretch 

culture, many cultivation scaffolds were studied: three different gel types 

were used and tested as a scaffold for the study. Naturally synthesized 

Matrigel, isolated from EngelbrethHolm-Swarm (EHS) mouse tumor was 

the first scaffold used for our research, in addition to two artificial 

synthesized gel scaffolds. Matrigel's experiment showed neuronal growth in 

the used two methods, but it was not stretchable. Hydrogel (VitroGel 3D-

RGD) with a modified functional group, and finally a fibrin gels the PVP 

co-GMA. In this study, three different scaffolds (Matrigel, VitroGel, and 

PVP co-GMA) had been studied and tested; in addition, the classical ICS-7 

PDMS stretch chamber was modified and reconstructed to fit the 3D stretch 

experiment; and finally, a 3D neuronal stretch method with a stretchable 

artificial scaffold (PPVP co-GMA) was developed and analyzed with special 

software and equipment in the lab. 



1 
 

Chapter 1 

Introduction 

1.1 The human brain 

The human brain is responsible for controlling and coordinating the 

information collected from other body organs and liable for making 

decisions. The brain is the least understood organ in the human body. It is 

difficult to access, highly susceptible to damage, and complex in structure 

and function (Forstmann, Keuken, & Alkemade, 2015). The poor 

understanding of the human brain is reflected in the lack of effective 

treatments for various neurological disorders such as Parkinson’s, 

Alzheimer’s disease, and motor neuron disorders. To address this research 

gap, new methods for the culture of human neural (neuronal and glial) 

lineage cells, particularly in vitro three-dimensional (3D) culture, are being 

developed to more accurately reconstruct the complex in vivo structure and 

function of the human brain (Murphy, Laslett, O'Brien, & Cameron, 2017).  

In a highly simplified view, three main parts are forming the brain, i.e. the 

cerebrum, cerebellum, and brain stem, as shown in Figure 1.1. The major 

part of the human brain is the cerebrum, which is divided into two 

hemispheres (right and left). These two hemispheres are surrounded by the 

brain's outer layer, which is called the cortex. The cortex is composed of a 

neuronal network, which is primarily responsible for thinking and body 

movement. The second part is the cerebellum, which plays a role in balance 



2 
 

and body coordination. The main body functions are controlled in the third 

part of the brain, which is the brain stem (Nowinski, 2019). 

 

Figure 1. 1: The three main human brain parts. The cerebrum, cerebellum, and 

brain stem. The cerebrum is the major part of the brain, which is composed of two 

brain hemispheres (left and right). 

The human brain, the spinal cord, and the nerve cells are forming the nervous 

system. The nervous system is composed of a complex and complicated 

network of neuronal cells. They are responsible for transferring and 

receiving messages from the brain and spinal cord to other body parts. This 

system is composed of two main parts: the first part is the Central Nervous 

System (CNS), which is called the control center of the body and the largest 

part of the nervous system. This part consists of the brain and spinal cord 

that are responsible to coordinate voluntary and involuntary reactions in our 

bodies. The second part of the nervous system is the Peripheral Nervous 

System (PNS). This part is capable of sensation and connection of the CNS 

to other body parts [1] [Figure 1.2; (Gibson & Fambrough, 2018)]. 



3 
 

The central nervous system is protected within the brain in the cranial cavity, 

while the spinal cord is inside the vertebral column (Edition, 2014). The 

major component of these three brain parts is the neuronal cells. Neurons 

are the structural and functional building units of the nervous system and 

brain (Beniaguev, Segev, & London, 2020), which are distributed all over 

the body and are responsible for human body management and control. The 

neuronal cells are the basic subunit of the nervous system and are specialized 

for the response to chemical and physical activity by receiving and 

transferring cell signals to and from neighborhood cells and tissues (Chung 

& Chung, 2008). The neuronal cell shape plays a major role in its function 

that connects to other cells and/or targets with connectors called synapses 

(Craig, 2003). Cell signals are transferred through these branches and 

connections (synapses) from one to another cell (Figure 1.3). 

 

Figure 1. 2: The human central nervous system vs. peripheral nervous system. 

Adapted from lumen learning anatomy of the nervous system book [2].  



4 
 

Through these synapses, the signal (action potential) is carried from one cell 

to another releasing a neurotransmitter. The sending cell is called 

presynaptic, where the signal departures the neuron through the axon 

terminal to the postsynaptic signal receptor, and enters through the dendrites 

in the neighbor cell. Between every two neuronal cells, there is a small gap 

that is separating the axon terminal and the dendrite or tissue signal receptor 

called the synaptic cleft. Through this space (cleft) the neurotransmitters are 

transferred from sender to receiver. Along the neuronal axons, there are 

some shielding’s covering the axon arm, these shielding’s are called the 

myelin sheaths they serve as signal speeders. Myeline sheaths are not found 

in all brain neurons (Micheva et al., 2016). 

 

Figure 1. 3: Neuronal cell body structure and cell-cell connection. Signal transfer 

from one cell to the other cell through the synaptic cleft. Adapt form journal of 

athletic trainer (Craig, 2003). 



5 
 

Electrochemical signals cross along the neurons to transfer information to 

and from the brain. The process of signal sending through the neurons has 

two steps along with the cell, it is called an action potential, and between the 

cells, it is called the neurotransmitter. The chemical signal 

(neurotransmitter) is the release of the electrical or chemical signals from 

the axon into the synaptic cleft, these neurotransmitters bind to their specific 

receptors in the postsynaptic region (Stufflebeam, 2008) (Carter & Bean, 

2009).  

The action potential is an electrical impulse that circulates the signal 

between the neuronal cells in the body, which occurs due to the change in 

the membrane voltage along the cell axon, which happens by the ion 

exchange through the cell (Carter & Bean, 2009). All sodium and potassium 

channels are closed during the rest state. These channels are closed with 

gates, and these gates are voltage-gated because of the response only to 

voltage change. Two types of ion exchange channels responsible for charge 

change are found in neurons the sodium and potassium ion channels. 

Recent studies on neuronal biomechanics highlight the considerable role of 

mechanical forces in neuronal development and synaptic formation. The 

need for a well-understanding brain development process is related to 

understanding and treatment of many associated diseases like traumatic 

brain injury (TBI), Alzheimer's, Parkinson’s disease, and how this complex 

tissue is regulated. 

 



6 
 

1.2 The Neuronal cytoskeleton 

A neuronal cell structure is composed of a cell body, axon, and dendrites 

and it is a highly polarized cell (Xiao, Hu, Wei, & Tam, 2016). Cells' shape, 

polarization, and motility of all types of cells are guided by a filamentous 

structure that creates the cytoskeleton. The filamentous structure includes 

the filamentous actin that is composed of G-actin and intermediate filaments 

(neurofilaments). Three types of protein-polymer are forming the neuronal 

cytoskeleton: the actin filament (microfilament), microtubules, and 

intermediate filaments are composed by protein subunit self-association 

(Figure 1.4)(Malacrida, Meregalli, Rodriguez-Menendez, & Nicolini, 

2019). They are nanometers in diameter and are micrometers in length, 

which allows them to integrate into the intracellular space. These polymers 

can overcome the mechanical tension, and the compression allows them to 

give the shape, and the cellular architecture is stabilized, and cell movement 

occurs. Their long length approves them to move the intracellular 

components. 

 In the nervous system, neurofilaments are present and composed of 

intermediate filament proteins and the cytoskeletal microtubule contains α 

and β-tubulin heterodimer (Menon & Gupton, 2016). The neuronal 

cytoskeleton is essential to maintain the cell function since the development 

and the entire existence of the nervous system (Muñoz-Lasso, Romá-Mateo, 

Pallardó, & Gonzalez-Cabo, 2020). Many proteins are connected with 

microfilaments and microtubules, and they are not responsible just for the 



7 
 

structure; they are related also to the dynamic and function (Muñoz-Lasso 

et al., 2020).  

They can connect one filament type with another and form a network to the 

plasmic membrane. The neuronal cytoskeleton is not a fixed structure, it has 

the role of maintaining the structural integrity of the cell and promotes 

axonal growth. It plays an essential role in axonal transport. 

Specific functions are related to cytoskeletal proteins to ensure the electrical 

and chemical transmission between neuronal cells. The neuronal cytoskeleton 

has to be flexible and dynamic to maintain the functioning circuits of the 

neurons throughout the organism's life (Muñoz-Lasso et al., 2020). 

 

Figure 1. 4: Neuronal cytoskeleton components. The neuronal cytoskeleton consists 

of three types of bio-polymers actin filament (microfilament) (green), microtubules 

(red), and intermediate filaments (grey) (Malacrida et al., 2019). 



8 
 

The neuronal cytoskeleton is essential for the cell shape and physiology. 

Furthermore, it is responsible for the formation of specialized structures like 

the growth cones, which play a role in the axon elongation and the 

development of synaptic boutons and dendritic spines [4]. The structural 

difference in the cytoskeleton affects the cell shape, due to this change the 

cell signaling and behavior are affected (Stukel & Willits, 2016). 

1.3 Traumatic injury in the central nervous system (CNS) 

The nervous system is a complex and highly specialized network in our 

bodies. The central nervous system is composed of the brain and the spinal 

cord, the brain has a high level of function coordination and the spinal cord 

serves for the pathway communication between the CNS and PNS. Central 

nervous system traumatic injury refares to traumatic brain injury (TBI) and 

spinal cord injury (SCI) (Walter & Zweckberger, 2018). The traumatic brain 

injury is caused by external physical damage, the result of this damage 

severity depends on the anatomical location of the injury. Traumatic brain 

injuries, in the CNS have their unique challenge for clinical and preclinical 

treatment because of their complex pathophysiology. For traumatic injury 

patients, specialized centers are responsible for their treatment.  

Mechanical loading of the brain is caused by head trauma (Hemphill, Dauth, 

Yu, Dabiri, & Parker, 2015). TBI occurs due to an external force that affects 

the brain, which affects brain function. The pathophysiology of this injury 

is not clear it can be caused by open or closed head injuries. The head injury 



9 
 

can vary from small to large structural changes in the brain (Valente & 

Fisher, 2011). Neuronal cell damage in traumatic brain injury leads to 

cytoskeletal rearrangement or damage, microtubule bundle confusion, and 

protein degradation cascade. Irregular calcium levels lead to the severity of 

the CNS injury. 

1.4 Mechanical strain 

To regulate cell and tissue physiology mechanical cues are essential 

(Boulter, Tissot, Dilly, Pisano, & Féral, 2020). The human tissue's 

mechanical properties differ depending on the composition, structure, and 

role (Kim & Choi, 2019). A vital role is played by mechanical stress in the 

development and maintenance of tissue functionality. The mechanical 

stimulations regulate different cell functions like proliferation, 

differentiation, and survival. Cytoskeleton undergoes remodeling in 

response to mechanical stimuli  (Masuda et al., 2007). Environmental, 

mechanical, and biochemical cues were sensed and integrated by all cells to 

coordinate tissue development and homeostasis maintenance. A specialized 

sensor located on the cell membrane receives the stress and force applied on 

the cell that works as a converter of this signal into a biochemical signal that 

affects cells' behavior in surviving ECM remodeling, and differentiation 

(Boccafoschi, Mosca, Ramella, Valente, & Cannas, 2013). All cell types in 

our body including neurons do forces on their surroundings. To measure and 

calculate the substrate strain and exerted cell stress, newly developed 



10 
 

procedures and protocols were used for this purpose. A linearly elastic 

substrate is chosen (Franze, 2013).  

Mechanical forces are translated into biochemical signals that affect cell and 

tissue behavior. The human brain has its appropriate mechanical properties 

that play an essential role in neural stem cell behavior and tissue 

development (Barnes, Przybyla, & Weaver, 2017). Cells adapt mechanically 

to the surrounding forces applied to increase the ECM rigidity and to 

transmembrane receptor integrins by producing stress-induced response 

strengthening. By increasing the applied stress level in cells mechanical 

stiffness increase linearly (Matthews, Overby, Mannix, & Ingber, 2006).   

1.5 Stretch experiments 

There is a high interest in understanding how cells and tissues respond to 

mechanical stimuli, but the approaches are limited until now. Many tools 

and devices were fabricated and invented to mimic the actual cell response 

to stimuli. Newly some 2D stretch experiments are today arising and 

applicable in cell culture for measuring cell response to stimuli. Till today 

the stretch experiments are applied to 2D cultures on different cell and tissue 

types. Stretching experiments are tools that employ mechanical forces on 

cells in vitro, which mimics the real force applied on cells in vivo. These 

techniques and tools bring us to understand the progression of different 

diseases that are altered by mechanical cues  (Tremblay, Cuerrier, 

Andrzejewski, O'Brien, & Pelling, 2014). 



11 
 

In our lab, the stretcher device was constructed and developed by Wolfgang 

Rubner in the ICS-7 Jülich research center for 2D stretch experiments (Faust 

et al., 2011). Cells respond to cyclic substrate stretch that mimics the body's 

vital physiological conditions. The adherent cell response to the external 

forces starting from a random orientation to reorient into a particular angle 

depends on the applied stretch. At the subcellular level, the cytoskeleton and 

the stress fibers generate internal contractile forces to reorient into the same 

angle (Livne, Bouchbinder, & Geiger, 2014).   

The first 3D stretch experiments on primary isolated neuronal cells were 

tested in our study on our house-made stretcher device using an elastomeric 

PDMS (silicone rubber) chamber. During the study, we developed and 

reconstructed the PDMS elastomeric chamber to fit the 3D stretch 

experiment, the chamber modifications and development were needed to 

build up our 3D system stretcher system. Cyclic stretch experiments were 

performed on cortical neuronal cells after 24 h of cell cultivation and 

adherence in the 3D scaffold. The stretch experiment was performed under 

sterile conditions. A uniaxial stretcher device was utilized to provide strain 

with different amplitudes and frequencies (Abraham et al., 2018). 

Stretch experiments are generally performed with different types of cells and 

tissues to study different cell issues. At the beginning mechanical stimulus 

was used and performed in bone remodeling and now it is used and 

investigated for different and several type of cells (Seriani et al., 2016). 



12 
 

1.6 Traditional two-dimensional (2D) cell culture vs. three-dimensional 

(3D) cell culture 

Two-dimensional (2D) culture in mechanotransduction studies provides 

basic information on the cellular-material interaction, whereas three-

dimensional (3D) culture mimics the actual in vivo environment (Stukel & 

Willits, 2016). The 3D culture is the building of cell culture in the XYZ 

direction.  The three-dimensional (3D) cell culture is not only that the culture 

is growing in three dimensions, concerning the normal tissue, but the 

function is also determined by cellular and non-cellular components. The 

more the cell culture comes closer to the actual conditions, the more the cell 

culture can mimic the original cell response and behavior. The cell culture 

showed that cell aggregation in a 3D shape provides a high physiological 

cell response compared to 2D cell culture is plated on plastic or glassware. 

The 3D spherical shape of cell aggregation, as a result of cell adherents 

together and not to cell culture tubes, or due to 3D scaffolds (natural or 

synthetic gels).  



13 
 

  

Figure 1. 5: The figure represents a comparison between 2D and 3D cell culture. A 

represents a 2D culture that is coated with protein, and B is a 3D culture on a 

scaffold. As shown in the figure, the cell shape is flat in the 2D culture, while in the 

3D culture it is taking a spherical shape due to the ECM protein or peptide 

interaction. The 3D culture is closer to the real environment as shown in C (Stukel 

& Willits, 2016). 

In the two-dimensional culture (2D), cells grow flat only in two dimensions 

and attach to the culture flask surface (Caliari & Burdick, 2016). Two-

dimensional cultures are simple and cheap for experiment replication. Some 

disadvantages of 2D cultures; it does not mimic the real tissue structure and 

behavior and the absence of cell-cell and cell-matrix interaction (Meeßen, 

Graeve, & Offenhäusser). In a three-dimensional (3D) cell culture in vitro 

the cell-cell and cell-ECM interactions mimic’s the natural in vivo 

interactions, on the cellular morphology level the shape looks close to the 

real cell shape (Langhans, 2018) (Edmondson, Broglie, Adcock, & Yang, 



14 
 

2014). The interactions are required for proliferation, cell differentiation, 

and cell vitality. Cell division and morphology are affected when moving 

the cell from the tissue to 2D cultures. Cell morphology changes affect cell 

function, structure, and secretion (Breslin & O’Driscoll, 2013). The lake of 

the external environment interaction leads to cell response change. Owing 

to 2D culture disadvantages, an alternative system is needed to mimic the 

real tissue environment as three-dimensional cell culture (3D) 

(Kapałczyńska et al., 2018). In addition, neuronal cell culture on 2D cultures 

receives their information only from the single layer, while neurons in our 

bodies receive their information from 3D. 

Among the first 3D cell cultures was made in soft agar solution by Hamburg 

and Salmon in the 1970s (Hamburger & Salmon, 1977). Three-dimensional 

culturing models can be divided into 1) culture in gel-like substances, 2) 

culture on a scaffold and, 3) suspension cultures on non-adherent plates. The 

3D sphere concept is based on building or creating a spherical structure 

composed of many layers. The sphere structure mimics the physical and 

biochemical properties of the tissue. Cultivated cells in a 3D structured 

environment mimic the real cell environment and architecture more 

accurately than 2D cultures and systems. Three-dimensional (3D) cell 

cultures keep the cell density constant as in the real tissue. The 3D model 

system is a perfect intermediate model between 2D cultures and animal 

models (Kapałczyńska et al., 2018). 



15 
 

 

Figure 1. 6: Comparison between 2D cell culture on flat culture vial vs. 3D cell 

culture on a scaffold from the growth and adhesion direction. The 3D culture shows 

the growth in an XYZ direction while the 2D culture shows growth only in an XY 

direction (Baker & Chen, 2012). 

1.7 Aim of the study 

To come closer to the real brain-like environment, researchers were 

developing and creating systems to understand how this organ is working 

and how the connections are formed. The present awareness about biological 

development is not completely dissolved by two dimensional (2D) culture 



16 
 

systems. The cell-cell and cell-matrix interactions are not represented as 

they are in the real and three-dimension (3D) cell culture. 

The 2D cultures (single layer) lack cell-cell and cell-matrix interactions. The 

main aim of this study was to establish and develop a 3D system that is 

mimics better the in vivo environment of the real brain. The newly 

established 3D system should be comparable with previously obtained 2D 

culture results. Besides, the 3D in vivo like model should be tested for the 

ability to withstand mechanical deformation. A deformable 3D model would 

allow us to investigate the physiological occurring mechanical strain in 

neuronal development and disease in more detail. Several in vitro models 

and scaffolds should be tested and investigated for their ability to perform 

3D stretch experiments. Finally, one suitable 3D model should be optimized 

and chosen for stretch experiments. To perform further 3D stretch 

experiments, an additional aim was to evaluate the model according to the 

ability of data analysis and optimizing a protocol for data retrieval with the 

Imaris software (for more details refer to 2.1.11 page 46). Setting up a 

reproducible and analyzable stretch 3D model was the main aim of the 

research to study the brain environment closely.  



17 
 

Chapter 2 

Materials and Methods 

List of materials and equipment’s are available in the appendix page 82. 

2.1 Cell culture 

2.1.1 Elastomeric chamber fabrication 

Creating a stretchable three-dimensional (3D) system requires an 

elastomeric chamber to build in the 3D scaffold. This elastomeric chamber 

is composed of a mixture of two synthetic components, 

the Polydimethylsiloxane (PDMS) and its crosslinker methyl-hydro 

siloxane-dimethyl siloxane. PDMS was mixed with its crosslinker (methyl-

hydro siloxane-dimethyl siloxane) in a ratio of 1:40 weight to weight (w/w). 

The previously mentioned mixing ratio needs to be mixed thoroughly in a 

plastic mixing cup. The homogenized mixture was degassed in a 5L 

desiccator to remove the air bubbles, which could disrupt the transparency 

of the elastomeric chamber. Finally, the degassed mixture is poured into 

special fabrication molds, and incubated for crosslinking for 16h at 60°C 

(Figure 2.2). After crosslinking, this mixture proportion gives a deformable 

substrate with an elastic modulus of 50 kPa (Wisser, Schumm, Mondin, 

Grothe, & Kaskel, 2015). 

 



18 
 

Figure 2. 1: PDMS crosslinking reaction by mixing them and curing it for 16 h at 

60 °C (Wisser et al., 2015). 

Treated and cured PDMS chambers were screwed out of the mold and 

sterilized with isopropanol alcohol to prevent contamination during 

neuronal three-dimensional (3D) cell culture. Sterilized PDMS silicone 

chambers were fixed in their special metal chamber holders. Then they were 

placed in the incubator at 37 °C for around 1h to get rid of isopropanol by 

evaporation. Finally, the sterilized PDMS chambers were used for three-

dimensional (3D) system cell cultivation (Figure 2.2). 

 

Figure 2.2: The 50 kPa PDMS fabrication mold chamber steps (Traditional 

chamber). The chamber cultivation area dimension is 2 cm * 2 cm. Images by Jens 

Konrad Forschungszentrum Jülich/ ICS-7.   

  



19 
 

The traditional chamber mold shown in Figure 2.2 is used for two-

dimensional (2D) experiments in our institute (ICS-7) to cultivate neuronal 

cells. In the traditional elastomeric PDMS chamber, 2D stretch experiments 

were performed. The traditional elastomeric chamber was used and tested 

for 3D stretch experiments as they have been used in 2D cultures.  

 

 

Figure 2. 3: Classical PDMS elastomeric chamber used for stretch experiments in 

the ICS-7. Images by Jens Konrad FZJ ICS-7. 

2.1.2 Primary cortical neuronal cell isolation 

Cortical neurons were obtained from pregnant rats (Wistar, Charles River) 

at (E18- E19) of gestation. Pregnant rats were anesthetized with CO2 in a 

large beaker then the embryos were removed surgically from the pregnant 

rat. The embryos heads were dissected in HBSS Petri dishes under a 

dissecting microscope, and the cortices were transferred to ice-cold trypsin 

and incubated for 15 min at 37 °C in the incubator. Then the cortices were 

transferred to a fresh Neurobasal medium (NB+++) for washing. Neurobasal 

medium NB is supplemented with B27, GlutaMAX, and Gentamicin. The 

cortices were washed three times in the NB+++ medium. The dissociated 

cells were counted using the hematocytometer under the inverted 



20 
 

microscope, up to 7 million neuronal cells were obtained per one embryonic 

rat head. After that, the cells were seeded within the 3D different scaffolds 

on glass dishes or/and PDMS elastomeric chambers according to each 

protocol method. The cortical cell isolation follows the steps, as shown in 

Figure 2.4.  

 

Figure 2. 4: Rat E18 – E19 brain dissection steps and cortex isolation. Brain images 

under a dissecting microscope in HBSS solution. Images by Mahmoud Ardah/ ICS-

7. (A) Rat embryo head. (B) Top view of the rat E18 – E19 brain. The two 

hemispheres can be seen and the cerebellum. (C) The brain hemispheres are 

separated and meninges were removed. (D) Brain hemisphere without meninges. 

(E) Whole hemisphere vs. cortex. (F) Single rat embryo cortex. 

The complete dissection process and cell isolation were performed under 

sterile conditions, using the laminar flow and 80% alcohol to minimize and 

prevent the contamination possibility. The embryonic head was placed in 

the frontal direction, to see the brain's hemispheres from the upper side of 

the head. The embryo's head was dissected in an HBSS solution petri dish 



21 
 

under the stereomicroscope inside the laminar flow. The dissection process 

was performed using fine-pointed forceps due to the softness of the embryo's 

brain tissue. Considering the head skin removal and thin skull bones, the 

brain hemispheres were visible. The brain is covered by a membrane called 

the dura mater, which was removed smoothly and slowly using fine forceps. 

After that, the brain was removed from the skull using fine curved forceps. 

The cerebellum and meninges were separated from the hemispheres. The 

two hemispheres were separated from each other, and the hippocampus was 

removed. Lastly, the cortices were transferred to ice-cold trypsin and 

incubated for 15 min at 37°C for cell dissociation. After the incubation 

period, the cortices were washed smoothly with neurobasal medium 

(NB+++) three times. Then the cells were counted using trypan blue and the 

hematocytometer. Finally, they were seeded in the 3D polymer for each 

experiment according to the experiment's protocol as described in the 

coming sections. 

2.1.3 Matrigel as a natural product for a 3D scaffold 

Matrigel is a gelatinous protein mixture used as a 3D cultivation scaffold 

that is secreted and isolated from Engelbreth-Holm-Swarm (EHS) mouse 

tumor. This soluble basement membrane contains several extracellular 

matrix molecules (Hughes, Postovit, & Lajoie, 2010) (Lee et al., 2015). The 

isolated natural polymer is rich with ECM proteins (Laminin, Collagen IV, 

and Entactin), in addition to several growth factors in this product. Matrigel 

polymerizes at a temperature of 22 °C to 35 °C, which makes it proper for 



22 
 

primary neuronal cell culturing (Corning company). Three-dimensional 

(3D) cell culture requires an appropriate cultivation scaffold, but the used 

scaffold should consider the same original conditions but in vitro 

environment. Matrigel was a suitable option that contains most of the 

features needed from protein contents, brain plasticity and was supposed to 

mimic the in vivo environment. Matrigel was used to build the 3D neuronal 

cell culture system, it was plated on glass dishes, and a 50 kPa PDMS 

elastomeric stretch chamber. With this type of gel (scaffold), two different 

cell cultivation methods were used: the sandwich method and the 3D 

suspension method. In the following parts, the cultivation methods were 

described in detail for each manner, presenting the differences and 

cultivation methods. 

2.1.3 A Sandwich method 

As its name indicates, it is a multi-layer method that is composed of two gel 

and two cell layers (Figure 2.5 for method clarification). The two cell layers 

were separated with a Matrigel layer in between. Through the second gel 

layer, which separates the two cell layers, we have a cell-cell connection 

between them. Different Matrigel concentrations were tested and used to 

apply the sandwich method. Two stock Matrigel concentrations of 10 and 

20 mg/ml were studied and used in the research. The sandwich method was 

applied on glass Petri dishes and 50 kPa elastomeric PDMS chambers. By 

applying this system on both PDMS chambers or glass, they were kept on 



23 
 

ice to delay the gelatinization and polymerization time during gel layer 

preparation; an ice block was used for this step for each gel layer addition. 

 

Figure 2. 5:  Sandwich method system explanation figure. The pink color indicates 

the gel layer, and the blue color indicates the neuronal cell layer suspension. The 

two cell layers were separated by a gel layer, through this gel layer neuronal cells 

built connections between both cell layers. 

Previous to neuronal cell isolation the first Matrigel layer was applied to the 

culturing chambers a volume of 76 µl of the stock concentration 10 mg/ml 

or 20 mg/ml were plated on the glass or 50 kPa PDMS elastomeric 

chambers. The applied layer was incubated for 30 min at 37 °C for 

polymerization. The neuronal cells were plated and cultured on the 

polymerized gel layer. Each cell layer is composed of 350.000 cells 

suspended in 45 µl of pre-warmed NB+++ medium, which gives a cell 

density of 7.777 cells per 1 mm3. Then they were incubated for 4h at 37 °C 

for cell adherence. After the incubation period, the second Matrigel layer 

was added, a 53.2 µl of Matrigel was plated over the first cell layer, and 

incubated for 30 min at 37 °C for gelatinization (polymerization). The 

second cell layer is prepared fresh from a stored cortex in the HBSS medium 

on ice, and freshly isolated cells were seeded for the second layer to avoid 



24 
 

cell death. The same cell number was seeded, 350.000 cells were suspended 

in 45 µl pre-warmed NB+++ medium, and the culture was incubated for 1-

2 h at 37 °C for cell adherence on Matrigel. Finally, after the incubation 

period and cell adhesion additional 1.5 ml or 500 µl, warm NB+++ medium 

is added for the glass dish or chamber, respectively.  

In the newly developed and reconstructed PDMS chamber, the number of 

cells and the Matrigel volumes were calculated to fit in the inserted grooves 

to the PDMS chamber. In the newly developed chamber, 62187 cells were 

suspended in an 8.48 µl warm NB+++ medium, and this amount of cells was 

used for each cell layer added in the newly developed and reconstructed 

elastomeric PDMS chamber having a cell density of 7.333 cells per 1 mm3. 

For each gel layer, 10.5 µl and 7 µl of Matrigel were used for the first and 

second layers, respectively. Finally, an additional 500 µl warm NB+++ 

medium is added. The PDMS elastomeric chambers were incubated for 24h 

at 37 °C then the experimental protocol was applied to the PDMS chambers 

like stretching and analyzing.  The method was applied to glass and 50 kPa 

PDMS chambers. The cultivation processes for the newly developed 

chamber were the same as the traditional chamber. 

2.1.3 B Three-dimension (3D) Suspension method 

The 3D Suspension method differs from the sandwich method in the cell 

location. The three-dimension (3D) suspension is supposed to fill the gap 

between two-dimensional (2D) cultures and animal systems (Febles, Ferrie, 



25 
 

& Fang, 2014). In the first method, the cells were on the gel surface, while 

in the second method, the cells are embedded in the Matrigel itself. In this 

system, the cells are distributed randomly within the gel components, as 

illustrated in figure 2.6. The cells were suspended in NB+++ medium and 

mixed with Matrigel. The number of cells is constant for this system at all; 

3 Million cells were suspended in different NB+++ volumes depending on 

the final Matrigel concentration. Different Matrigel concentrations were 

tested and used for this study (5,6,10, and 12 mg/ml of Matrigel). The values 

in table 2.1 are for the 13 mm pre-drilled Petri dish and the traditional PDMS 

chamber. Due to the gelatinization and polymerization temperature of the 

Matrigel, the elastomeric PDMS chamber and glass are prepared on ice to 

delay the gelatinization time. 

 

Figure 2. 6: The 3D suspension method explanation. The neuronal cells (Blue) are 

randomly distributed inside the Matrigel in purple color. 

 

 

 



26 
 

Table 2. 1: The 3D suspension method Matrigel stock and final 

concentration for 13 mm pre-drilled Petri dish glass and PDMS 

traditional elastomeric chamber. 

Matrigel 

stock conc. 

Cell suspension 

volume [µl] NB+++ 

Matrigel 

volume  [µl] 

Matrigel final 

conc. 

20 mg/ml 60 90 12 mg/ml 

 75 75 10 mg/ml 

10 mg/ml 60 90 6 mg/ml 

 75 75 5 mg/ml 

To keep the experimental conditions constant in both PDMS chambers 

(traditional and reconstructed) all component volumes were recalculated to 

fit in the inserted wells. The total volume of the inserted wells was 17.5 µl 

each. Only 350.000 neuronal cells were needed instead of three Million cells 

for the 3D suspension method. The cells were suspended in a warm NB+++ 

medium with different volumes regarding the final concentration of 

matrigel, as shown below in Table 2.2. In this method, we have a cell density 

of 20.000 cells per 1 mm3 in both chambers. These cells were distributed 

randomly within the gel scaffold. 

Table 2. 2: Matrigel volumes for stock and final concentration for the 

newly reconstructed PDMS elastomeric chamber. 

Matrigel 

stock conc. 

Cell suspension 

volume  [µl] NB+++ 

Matrigel 

volume [µl] 

Matrigel final 

conc. 

20 mg/ml 7 10.5 12 mg/ml 

 8.75 8.75 10 mg/ml 

10 mg/ml 7 10.5 6 mg/ml 

 8.75 8.75 5 mg/ml 
 

 



27 
 

2.1.4 Synthetic scaffold for three-dimension (3D) cell cultivation in 

Polyvinylpyrrolidone (PVP) copolymerized with Glycidyl methacrylate 

(GMA) 

An artificial synthesized water-soluble polymer for three-dimension (3D) 

cell cultivation was produced by Deutsches Wollforschungs institute 

Aachen (DWI Aachen) [5]. Due to its content of fibrinogen and thrombin, it 

is classified as fibrin gel. Fibrin gels are a mixture of fibrinogen and 

thrombin, which is formed from peripheral blood that is found in wound 

regions for repairing the injured regions. Fibrin formation occurs during the 

coagulation process of fibrinogen cleavage by thrombin to fibrin monomers, 

and they polymerize to form a three-dimensional (3D) polymer (Ju, Janmey, 

McCormick, Sawyer, & Flanagan, 2007). It is not only used for therapeutical 

uses but is also investigated as a biodegradable material in biomedical 

applications like tissue engineering and drug delivery. Hydrophilic cross-

linked fibrils contain fibrin gels that are suitable for three-dimensional (3D) 

cell culture. The PVP chemical structure is shown in figure 2.7 below. 

 

 



28 
 

 

Figure 2. 7: The chemical formula of Polyvinylpyrrolidone (PVP) copolymerized 

with Glycidyl methacrylate (GMA) PVP co-GMA. This chemically synthesized 

polymer produced by DWI Aachen. 

Fibrin gels components assemble rapidly by a modified polycondensation 

reaction, unlike the extracellular matrices and basement membrane 

components which assemble in a slow ordered manner directed by the cell 

that secret them (Janmey, Winer, & Weisel, 2009). 

In the study, the primary isolated cell suspension was mixed with the 

artificially synthesized PVP polymer. Additionally, fibrinogen and 

thrombin were added to the cell suspension. The complete mixture was 

plated and cultivated in the newly reconstructed PDMS elastomeric 

chamber. After that, the PDMS chamber was incubated for 10 to 15 min at 

37°C for polymerization. Then, an additional 500 µl of pre-warmed 

Neurobasal medium (NB+++) supplemented with Tranexamic acid, 

GlutaMAX, N2 supplement, and fetal bovine serum (FBS) was added to the 

chambers and incubated at 37°C for 24h. Culturing medium was 

supplemented with tranexamic acid to avoid fibrinolysis (Shakur et al., 

2016). The NB+++ medium was supplemented with N2 supplements to 



29 
 

provide optimal growth conditions for neuronal cells (Dhara & Stice, 2008). 

Finally, after 24 h of incubation, the chamber was ready for stretching 

periods at different amplitudes and analysis. With an LSM 880 microscope, 

the results were analyzed and observed. 

2.1.5 Synthetic scaffold for three-dimension (3D) cell cultivation in 

VitroGel 3D-RGD 

This gel is an artificial polymer synthesized by a company, which mimics 

the natural extracellular matrix (ECM) [3]. VitroGel 3D-RGD type is a 

modified polymer with integrin-binding ligand (RGD), the modified 

polymer is responsible for cell adhesion and cell-matrix interaction (Haruna 

& Huang). VitroGel polymer was tested with four different concentrations 

regarding the producer's protocol on the 3D suspension method. The gel was 

diluted with 0.5x PBS and mixed gently to be homogenous (The 

wellbioscience). Finally, (200.000 or 350.000) cells were suspended in 

NB+++ medium and mixed with the gel mixture, then they were plated on 

glass dishes or PDMS elastomeric chambers. The producer's protocol is 

suitable for large cultivation areas. To keep the gel concentrations constant 

in the newly reconstructed PDMS elastomeric chamber, the producer's 

protocol volumes were adapted (Table 2.3). 

  



30 
 

Table 2.3: VitroGel mixing protocol from producer company was 

adjusted to fit our newly reconstructed PDMS chamber (The 

wellbioscience). 

Dilution Vitrogel [µl] 0.5X PBS [µl] NB+++ [µl] 

1:0 18 0 4.5 

1:1 9 9 4.5 

1:2 10.8 21.64 8.13 

1:3 4.5 13.5 4.5 
 

2.1.6 Cell Stretching of cortical neuronal cells 

For this purpose, a special uniaxial mechanical stretcher device was 

developed in the ICS-7 by Wolfgang Rubner to mimic brain movement. The 

stretcher device is composed of the elastomeric PDMS chamber fixed in a 

special metal chamber holder, and the stretcher device is connected to the 

PC for parameter control. The elastomeric chamber was equipped in a 

horizontal state in a pre-stretch position (1.5 mm stretched) to prevent the 

drop of the bottom of the chamber. The cells were cultivated in their 3D 

scaffold (gel) on the elastomeric PDMS chamber. After cultivating the 3D 

culturing system in the chambers, they were incubated for 24h at 37 °C. By 

the end of the incubation time, the complete holder (holder and elastomeric 

chamber) was fixed and screwed into the uniaxial stretch apparatus. The 

stretcher device is driven by a motor that moves uniaxially, as shown in 

figure 2.8. This device is connected to a PC where the stretching parameters 

were controlled manually in the software. To apply strain on the sample, the 

holder's frame should be removed by starting the device, the mechanical 

strain was applied with different amplitudes and parameters (for optimizing 



31 
 

the system). Table 2.4 shows the different parameters used in the elastomeric 

chambers. 

 

Figure 2. 8:  Uniaxial stretcher device set developed by Wolfgang Rubner, ICS-7. A) 

PDMS chambers 1:40 where the cells were cultivated and stretched. B) Chamber holder 

where the chamber is screwed/ fixed in. C)Stretch apparatus where the holder with 

chamber was built-in to be stretched. Images by Jens Konrad FZJ ICS-7. 

Table 2. 4: Stretch parameters on natural and synthetic 3D polymers 

with different frequencies.  

Stretch 

percent 

Amplitude 

[mm] 

Frequency 

[mHz] 
Speed  [mm/s] Pause [s] 

7% 1.505 100 0.401 1.250 

15% 3.225 100 0.860 1.250 

30% 6.45 100 1.720 1.250 

7% 1.505 300 1.204 0.417 

15% 3.225 300 2.580 0.417 

30% 6.45 300 5.160 0.417 

The stretcher parameters were forming a sinusoidal-like curve by repeating 

the wave speed and amplitude continuously regarding the entered 

parameters, as shown in the above table. The sine curve in the experiment 



32 
 

represents the heartbeat in our bodies, which causes stretching and release 

in the brain cells. 

Depending on experiment conditions, stretch periods of 1h, 4h, 24h, and one 

week were used and applied. After the stretch cycles were finished, the 

stretcher device was stopped in the release position. To visualize the 

stretched and un-stretched cortical neurons within a 3D culture scaffold, the 

PDMS chambers were stained using: Calcein staining or 

immunocytochemistry stain. These two staining methods were used to 

observe the results of the samples before and after the stretch. The following 

sections describe each staining method in detail. 

2.1.7 Calcein Staining 

For neuronal cell analysis, cell staining was obtained for this step. Calcein 

stain is used for the analysis with a fluorescent dye with a specific 

wavelength of 495/515 nm. Calcein stain can distinguish live cells from dead 

cells. Calcein stain is found in two forms Calcein-AM (Calcein-AM, AM: 

acetoxymethyl), the non-fluorescent form of the stain before passing the cell 

membrane (figure 2.9), and Calcein is the fluorescent form after passing the 

cells membrane. The calcium affine binding site in the chemical structure is 

covered with the acetoxymethyl group that allows the dye to penetrate 

through the cell membrane without being damaged. When the stain 

penetrates the cell membrane, the non-fluorescent calcein-AM is converted 

to fluorescent calcein by cell esterase. Regarding the dissociation of the 



33 
 

acetoxymethyl group, calcein binds to calcium ions in the alive cell and 

appears in a fluorescent green color upon adequate illumination. 

To stain the neuronal cells, the calcein stock (Calcein-AM) was diluted in a 

normal NB+++ medium, and then the stain was added to the sample and 

incubated for 15 min at 37°C inside the incubator. After the stain incubation 

period, the sample was washed for 5 min with a normal NB+++ medium to 

remove the excess of calcein from the sample. Finally, 500 µl of the imaging 

medium was added to the sample, and the medium was supplemented with 

10 mM of HEPES. Diluted HEPES was added to the imaging medium for 

sample observation under the upright microscope with a 40x objective lens. 

 

Figure 2. 9: Cell Esterase converts Calcein-AM to Calcein, pathway inside alive cell. 

Image adapted from Dojindo. Living and dead cell staining, 2016 [6]. 



34 
 

2.1.8 Immunocytochemistry stain 

A staining method is used for cell screening. It uses special antibodies to 

determine specific proteins or antigens in cells based on antigen-antibody 

interaction. This technique uses two types of antibodies primary and 

secondary antibodies. Primary antibodies are unconjugated antibodies, 

while the secondary antibodies are conjugated to a fluorophore, which 

absorbs light in a certain wavelength and emits it in different ranges. Primary 

antibodies bind directly to the target protein. Primary antibodies were 

incubated and washed with skim milk powder. Secondary labeled antibodies 

were added to the sample, which detects the primary antibody attached to 

the target. The secondary antibodies are directed against the 

immunoglobulin class or subclass of the primary antibody’s species (Figure 

2.10). Finally, the sample is observed under the confocal microscope (LSM 

880). 

 

Figure 2. 10: Immunocytochemistry mechanism. Primary and secondary antibodies 

bind together to the target and the fluorophore. 



35 
 

2.1.9 Sample fixation and preparation for LSM 

To observe a stretched sample in PDMS chambers, the stretcher device is 

stopped in a relaxation (release) position. The sample was fixed with 3.7% 

paraformaldehyde (PFA) solution diluted in 1X CB buffer and incubated for 

10 min at 37 °C. The sample was washed with 30 mM glycine 1X CB 

solution, and the chamber was washed 3 times with 1X CB to remove the 

excess of PFA. The chamber was treated with 0.05% Triton X 100 diluted 

in 1X CB for 10 min for cell membrane permeabilization. After the treatment 

with Triton X 100, the chamber was washed 3 times with 1X CB buffer and 

blocked with 5% skim milk powder diluted in 1X CB buffer for 1h at room 

temperature to block unspecific antibody binding. Anti-MAP2 antibody and 

Anti-Tubulin Antibody clone YL 1/2 primary antibodies are prepared in 1% 

to skim milk powder in a dilution ratio of 1:500 and incubated for 2h while 

shaking. Primary antibodies excess is removed, then the chamber was 

washed 3 times with 1% milk powder. The chambers were treated with Anti-

Rat IgG (H&L) Alexa Fluro 647 and Anti-Rabbit IgG (H&L) Alexa Fluro 

488 as a secondary antibody. They were diluted in 1% skim milk powder in 

the ratio of 1:500 and incubated for 1h in dark (covered) with shaking. After 

the shaking period, secondary antibodies excess is removed, and the 

chamber was washed 3 times with 1X CB, and finally with H2O. Before 

covering the chamber, it was mounted on an object slide, which serves as a 

sample holder for the confocal microscope. Both wells are covered with 40 

µl of the mixture of Fluoromount mounting media and 15 mM DABCO (1,4-



36 
 

diazabicyclo [2.2.2] octane), then covered with a coverslip Ø = 15 mm. The 

sample is settled for drying for the next day. After the fixation and mounting 

process, the chamber walls are removed, and the sample was examined 

upside down on confocal LSM 710 or LSM 880. Secondary antibodies 

Alexa Fluro 647 and Alexa Fluro 488 were excited with a helium-neon laser 

at 633 nm and an argon-ion laser, respectively. In one microscope scan, two 

fluorophores were detected by the mean beam filter MBS 488/543/633.  

2.1.10 Microscopy 

Different types of microscopes were used for different purposes as follow: 

Table 2. 5: Each type of  microscope was used for special purposes as 

mentioned in the table above.  

Microscope 

type 
Company Lenses Camera Purpose 

Dissecting / 

stereo 

microscope 

ZEISS stemi 

508 

microscope 

10x ocular 

lens 
 

Neuronal cell 

isolation 

Inverted 

microscope 

ZEISS Axio 

Vrt.A1 

5x, 10x,20x 

objective. 

10x ocular 

lens. 

AxioCam 

ICm1 

Cell counting 

and 

observation 

during 

cultivation 

time 

Upright 

microscope 

ZEISS Axio 

Imager.M2 
40x objective  

Axio 503 

mono 

Live cell 

imaging and 

stained 

samples 

LSM 880 

confocal 

microscope 

ZEISS LSM 

880 

20x air 

objective 
 

Observing 

fixed sample 

stained with 

antibodies 

 

  



37 
 

2.1.11 Imaris imaging processing software 

A computer software used for 3D and large microscopic data analysis. This 

software provides many functions for the segmentation and interpretation of 

the 3D data. The data were imported to the program, and different program 

tools were used and applied. Advanced visualization and analysis methods 

were supported with this software. This program allows the data vision in a 

3D perspective. With the obtained results from Imaris software, we can 

compare both data from the original and derived from the software together. 

Different tools from distance measurement and neurite length can be applied 

to the data using this software.  

  



38 
 

Chapter 3 

 Results 

To understand one of the most complex organs in our body, how does it 

work, and how to overcome and deal with its diseases, it is essential to 

develop an in vitro brain-like model (Frimat et al., 2015). Researchers 

started to study the brain neuronal network in vitro in typical 2D cultures, 

but 2D cultures do not mimic the actual brain environment. To come closer 

to the original brain environment and brain-behavior 2D cultures were not 

enough to fill the study gap, so there should be an alternative culturing 

system. 

We, therefore, aimed to create and develop a three-dimensional (3D) cell 

culture system. Several materials and natural or synthesized gels were used 

for this purpose. The used gels and scaffolds will be discussed in detail in 

the coming sections. 

3.1 Three dimensional (3D) cell culture 

The commonly used cell culture is the two-dimensional (2D) cultures. 

Recent research on three-dimensional (3D) cultures gained popularity 

(Kapałczyńska et al., 2018). Creating a three-dimensional (3D) culture is 

extremely sensitive and requires appropriate culturing matrixes (scaffolds) 

for building the systems used in the study. The step after chamber fabrication 

and cell isolation is the cultivation of primary isolated cortical neurons in a 

3D environment. To mimic the brain-like environment, the used scaffolds 



39 
 

should include suitable and essential growth conditions close to the real 

brain environment. Two-dimensional (2D) cell cultures do not mimic and 

do not provide the physiology and morphology of the real brain 

environment, whereas the 3D cell culture provides the required and needed 

environment. The cell-cell and cell-matrix interaction in 2D cultures are not 

represented as they were in the reality. These mentioned interactions mimic 

the natural environment in vivo (Edmondson et al., 2014; Neto, Levkin, & 

Mano, 2018).  

Two types of gels are used in the study a natural and artificial synthesized 

to build the 3D culture. According to the experiment's protocols, the cells 

were seeded and mixed with the gels in different concentrations and ratios 

used for each method. Depending on the culturing system, as illustrated in 

the following sections. 

3.2 Neuronal cell culture in a 3D system and stretch experiments using 

Matrigel as a natural scaffold. 

Two different cultivation systems were tested using the naturally secreted 

and isolated gel, the 3D suspension, and the sandwich method. Both 

neuronal cell cultivation methods were plated on the classical PDMS 

elastomeric chamber. Both cultivation methods used different Matrigel 

concentrations as described and shown in the following sections. 

 



40 
 

3.2A The 3D Suspension method 

In this system, the cells were distributed randomly within the Matrigel in all 

directions, a droplet-like system (sphere-like) (Figure 2.6 for illustration). 

The difference in Matrigel concentrations affected cell growth and 

distribution in the gel. Clearly visible is cell aggregation with increasing 

Matrigel concentrations (12 mg/ml). In contrast, the homogeneous 

distribution was seen for lower Matrigel concentrations, (5 and 6 mg /ml). 

Different Matrigel concentrations were tested for the 3D suspension method. 

Only the four optimized concentrations were suitable and stable on PDMS 

elastomeric chamber and glass dishes, but in an un-stretched position 

(5,6,10, and 12 mg/ml) as shown in figure 3.1. 

 



41 
 

 

Figure 3. 1: Four different optimized Matrigel concentrations (12, 10, 6, 5 mg/ml) 

respectively were used in the 3D suspension method as shown. The above-shown 

figure represents an un-stretched classical PDMS chamber. The higher the Matrigel 

concentration, the more the cells are clustered together and harder to distribute 

within the Matrigel. The PDMS chamber was stained with calcein and observed 

under the upright microscope with a 40X objective lens. The figure scale bar was 

20 µm. 

In the above-shown figures, cortical neurons were plated and cultivated in 

the classical PDMS chamber. Cortical neurons grow in a 3D sphere-like 

environment and form neurites in all Matrigel concentrations and all 

directions. Neuronal cells clump and cluster together in high Matrigel 

concentrations (12 mg/ml) and well cell distribution in low Matrigel 

concentrations (5 and 6 mg/ml). High protein concentrations are found in 

high Matrigel concentrations. This high protein content prevents the neurons 



42 
 

from homogeneous distribution and cell growth. In low matrigel 

concentrations (5 and 6 mg/ml), cortical neurons are well distributed equally 

in the 3D suspension method and form neurites in all directions because of 

low protein concentrations and amount in low gel concentration. 

3.2B Sandwich method  

In addition to the 3D suspension method, we had another cultivation and 

culturing method for the 3D system the Sandwich method is studied. This 

method differs from the 3D Suspension method as it is composed of two gel 

layers and two neuronal cell layers, as shown in Figure 2.5. Thus, neuronal 

cells were distributed in a horizontal direction on each matrigel layer in 

contrast to the 3D Suspension method. This method is not close to reality 

compared to the 3D Suspension method from the culture shape. Two gel 

concentrations were studied (10 mg/ml and 20 mg/ml) and plated on the 

classical elastomeric PDMS chamber. 



43 
 

 

Figure 3. 2:  Sandwich method cultivated in Matrigel with two different optimized 

concentrations (10, 20 mg/ml). This method was plated on the classical PDMS 

elastomeric chamber in an unstretched position. Neuronal cells were observed only 

on the upper cell layer. Cells were stained with calcein and observed under the 

upright microscope with a 40X objective lens. The figure scale bar is 20 µm. 

Two cell layers were observed by staining the culture with calcein, but the 

second Matrigel layer in between cannot be recognized clearly. The top cell 

layer cells were clear to identify, and the beneath neuronal cell layer is 

blurry. As shown in the figures above, the first cortical neuronal cell layer 

cannot be identified clearly because of the gel and cell layers above. 

In both systems, the 3D suspension and sandwich method, the neuronal cells 

were cultivated in Matrigel on 50 kPa classical PDMS chambers (Figure 

3.3). The cultivation of neuronal cells in Matrigel was optimized at different 

gel concentrations, and the neuronal cells survived within these different 

concentrations in both methods. 

   



44 
 

 

Figure 3. 3: Traditional (classical) 50 kPa PDMS chamber, empty chamber vs. 

cultured chamber. Image by Jens Konrad (ICS-7).  

3.3 Matrigel is a naturally suitable 3D culture system but a non-

stretchable scaffold. 

Matrigel provides a suitable neuronal cell growth and cell culture 

environment. Neuronal cell growth and branch formation occurred during 

the first 24h of cell cultivation in the scaffold. Both cultivation methods the 

sandwich and the 3D suspension were used and tested on Matrigel. These 

two cultivation methods were applied on glass dishes and classical PDMS 

elastomeric chamber. The scaffold (Matrigel) detached from the classical 

PDMS chamber during medium change, and even without any stretch force 

on the elastomeric classical PDMS chamber. Due to the shear force that 

occurred in the chamber gel detaches from the classical elastomeric PDMS 

chamber. The classical PDMS elastomeric chamber was cross-linked with 

different substances like glutarate and different Matrigel concentrations to 

prevent the detachment of Matrigel culture from the elastomeric PDMS 

chamber. With or without cross-linking the PDMS chambers Matrigel 



45 
 

detached from the elastomer even without applying any force on the 

chamber. 

Newly developed and reconstructed elastomeric PDMS chambers 

(developed and reconstructed by Mahmoud Ardah) were designed to reduce 

the shear force applied by the medium change and stretch movement in the 

classical PDMS chambers. The new PDMS chambers walls work as Z-

direction supporters for the complete cell culture. 

Neuronal cells were cultivated in the newly developed PDMS elastomeric 

chambers, so matrigel detachment during medium change was observed 

again. By applying even small amplitudes on the culture Matrigel 

detachment was observed beginning from the small, inserted grooves wall 

as shown in (Figure 3.4). 

 

Figure 3. 4: Neuronal cells cultivated on Matrigel in the newly developed and 

reconstructed PDMS elastomeric chamber. Matrigel detachment from the inserted 

grooves wall. Chamber stained with MAP2 in green, anti-tubulin in red, and NFH 

in blue. A 40x objective LSM 880 image. 



46 
 

3.4 Classical PDMS chamber reconstruction and development 

The classical PDMS elastomeric chamber design fits 2D stretch and 

cultivation experiments, the chamber was tested and used for 3D neuronal 

cultivation and stretch experiments. The 3D cultivation and stretch 

experiments in the classical PDMS elastomeric chamber were not successful 

at all. The different types of scaffolds detached from the classical 

elastomeric PDMS chamber. There was a need for an alternative and new 

elastomeric chamber design that fits the 3D stretch and cultivation 

experiment. The newly reconstructed chamber had the same external 

dimension as the classical chamber with small, inserted wells. The 

reconstruction idea came from that the 3D culture should have Z-direction 

supporters, and the newly reconstructed elastomeric chamber was designed 

to have these supporters. The elastomeric chamber modification and 

reconstruction were performed by Mahmoud Ardah. The newly developed 

and reconstructed elastomeric chamber had small, inserted wells with 5 mm 

*5 mm * 0.7 mm dimensions. The wells measurement was obtained using a 

40x and 63x objective lens of an upright microscope to determine the actual 

working distance in the chamber. The figure 3.5 below shows the 

reconstruction and modification carried out to the classical elastomeric 

chamber. 

 



47 
 

 

Figure 3. 5: Classical elastomeric chamber vs. newly developed and reconstructed 

elastomeric chamber. The external chamber dimensions were constant as the 

classical and the inserted wells dimensions were 5 mm* 5 mm*0.7 mm. 

reconstruction and modifications were performed by Mahmoud Ardah. The 

reconstruction were of the PDMS elastomeric chamber was performed and 

designed to fit 3D experiments. Images by Jens Konrad FZJ ICS-7. 

3.5 VitroGel 3D-RGD as a synthesized polymer. 

An artificial chemically synthesized polymer classified as hydrogels that 

closely mimics the endogenous physiological microenvironment for in 

vitro cell cultivation. Different concentrations of VitroGel were tested for 

cell growth, cultivation, and stability on both PDMS elastomeric chambers 

(classical and newly developed). Cells were cultivated in the VitroGel 3D-

RGD model and were tested in the 3D suspension method in different gel 

concentrations. This type of hydrogel was not suitable for neuronal primary 

cell cultivation in addition to its instability in PDMS elastomeric chambers. 

Polymer detachment from the PDMS chambers was observed during the first 



48 
 

24h of cultivation, even in the newly reconstructed PDMS chambers. The 

cell suspension was mixed with 0.5x PBS and enough VitroGel, which gave 

different concentrations regarding the company’s cultivation protocol 

(Figure 3.6). 

 

Figure 3. 6: An un-stretched sample of neuronal cells cultivated in VitroGel 3D-RGD, 

neuronal cells, and some cell branching can be observed after 24h of incubation at 37°C. 

Cells were stained with calcein and observed under the upright microscope with a 40x 

objective lens with transmission light (TL). Figure Scale bar 30µm. 

Figure 3.6 illustrated above shows the primary neuronal cell growth in 

VitroGel 3D- RGD forming branches in an unstretched position. Primary 

neurons grow slowly on VitroGel 3D- RGD, but the polymer detaches from 

the 50 kPa PDMS elastomeric chambers by the medium change even 

without stretching like the Matrigel. 

  



49 
 

3.6 PVP co-GMA a synthesized stretchable 3D scaffold 

A preliminary test was performed on the third type of cultivation polymers. 

This type of fibrin gel the PVP co-GMA was designed by the DWI Aachen 

Institute to fit the three-dimensional (3D) cultures. According to its sticky 

“glue-like” property, it was tested separately without neuronal cells on a 

PDMS elastomeric chamber for stretch experiments with different 

parameters. After 24h of incubation, a stretch experiment was applied with 

different parameters on the chamber to test the PVP co-GMA gel stability 

on the PDMS elastomeric chamber during stretch experiments (Figure 3.6).  

 

Figure 3. 7: PVP co-GMA polymer plated without neuronal cells in a newly 

developed PDMS elastomeric chamber. The chamber was stretched for 1hr at a 

frequency of 300 mHz and an amplitude of 15%. The white arrow shows the stretch 

direction which was applied to the newly reconstructed PDMS chamber. No 

detachment was observed during stretch and chamber medium change. 

  



50 
 

The figure above 3.7 represents the preliminary test experiment of PVP co-

GMA gel stability on PDMS elastomeric chambers without neuronal cells. 

The experiments showed that the synthesized polymer was stable on PDMS 

chambers during stretch experiments and chamber medium change. 

Fibrin concentration plays a major role in neuronal cell growth in this type 

of cultivation polymers. High Fibrin concentrations lead to low neuronal cell 

growth rate due to crowded contents of fibrin and thrombin mixture in PVP 

polymer (Figure 3.7). 

 

Figure 3. 8: Primary isolated neuronal cells cultivated in PVP co-GMA with high 

fibrin concentration (16.3 mg/ml). Slow neuronal cell growth due to high fibrinogen 

concentration. Cells were stained with calcein and observed under the upright 

microscope with a 40x objective lens. The figure scale bar is 20 µm. A) Represents 

primary neuronal cells after 24h of cultivation and incubated in 37°C. B) Neuronal 

cells after 1 week of cultivation. 

  



51 
 

Different fibrinogen concentrations were tested and changed to get the 

optimal concentration for neuronal cell growth and cultivation. Neuronal 

cells do not grow in any fibrinogen concentration, because of the dense 

fibrinogen thrombin interaction which prevents smooth cell growth. As 

tested before the neuronal cell grows in PVP co-GMA gel the sample shows 

to be suitable for 3D cell cultivation and stretch experiment model as shown 

in the figure below (Figure 3.8). 

 

Figure 3. 9: Primary neuronal cells cultivated in PVP high fibrinogen concentration 

(16.3 mg/ml). Neuronal cells were stained and treated with MAP2 (green), NFH 

(blue), and Tubulin (red). The sample was observed under the LSM 880 with an air 

objective 20x. The figure scale bar is 50 µm.  



52 
 

The different fibrinogen concentrations were tested varied from high to low 

concentrations. Low fibrinogen concentration was more suitable than high 

fibrinogen concentration for primary neuronal cell cultivation in PVP 

modified gel. Slow neuronal cell growth in high fibrinogen concentration 

16.3 mg/ml compared to lower fibrinogen concentration faster neuronal cell 

growth and branch formation (Figure 3.10). 

 

Figure 3. 10: Different fibrinogen concentrations were tested for primary neuronal 

cell cultivation in PVP co-GMA gel.  Neuronal cells were stained with MAP2 in 

green and tubulin in red staining. A) High fibrinogen concentration 16.3 mg/ml low 

and slow neuronal cell growth. B) Moderate fibrinogen concentration of 8.16 mg/ml 

was acceptable for neuronal cell growth. C) Low fibrinogen concentration of 4.03 

mg/ml favorable for neuronal cell growth. The figure scale bar is 50 µm. 

 

  



53 
 

PVP co-GMA was mixed with primary isolated neuronal cells and plated on 

the 50 kPa PDMS elastomeric chambers. Neither detachment nor tear-off 

from the PDMS chambers was observed during the medium change, even 

by applying stretch on the elastomeric chambers. PVP co-GMA gel was a 

stretchable and analyzable polymer. Due to its stability on the PDMS 

elastomeric chambers stretch experiments were applied to them with an 

amplitude of 300 mHz and 15% of stretch for 24 h. Two different fibrinogen 

concentrations were used with these conditions, after the stretch period the 

chambers were stained with MAP2 in green and tubulin stain in red (Figure 

3.10 a&b). 

 

Figure 3.11. 1: Primary neuronal cells cultivated in PVP co-GMA with a moderate 

amount of fibrinogen concentration 8.16 mg/ml. Neuronal cells were stained with 

MAP2 in green and tubulin in red. Neuronal cells under 15% strain for 24 h with a 

frequency of 300 mHz The figure scale bar is 50 µm.  

 



54 
 

 

Figure 3.11. 2: Primary neuronal cells cultivated in PVP co-GMA gel with a low 

amount of fibrinogen 4.03 mg/ml. Neuronal cells were stained with MAP2 in green 

and tubulin in red. Neuronal cells under 15% strain for 24 h with a frequency of 

300 mHz The figure scale bar is 50 µm. 

As seen from the previous images the neuronal cell grows in 3D culture in 

an opposite direction to the strain direction to reduce the force applied on 

the cells from the stretch. Unstretched neuronal cells remain randomly 

growing and distributed in all directions of the polymer as shown in the 

above images.  



55 
 

Chapter 4  

Discussion and Conclusion 

The movement from 2D cultures in vitro to 3D cultures in vitro is a quantum 

leap in the research world. In the 3D research, we are coming closer to the 

actual tissue environment, especially the brain. The 3D cell cultures 

demonstrate the practical and real application for the in vivo studies (Ravi, 

Paramesh, Kaviya, Anuradha, & Solomon, 2015). The brain is among the 

tissues that are in continuous movement regarding the heartbeats. This brain 

movement is simulated in in vitro environment by using a house-made 

stretcher device (Faust et al., 2011). Cortical isolated neuronal cells were 

stretched on PDMS elastomeric chambers cultivated in their 3D scaffold. 

The used 3D scaffold was either naturally isolated or chemically 

synthesized. The 3D cell cultures represent the actual cell-cell and cell-

matrix interactions; these interactions are required for normal cell behavior 

to feel or behave as in the brain, unlike 2D cultures. Two-dimension cell 

cultures are low in cost, unlike the 3D culture that requires many 

possibilities to build the culture (Kapałczyńska et al., 2018) (Neto et al., 

2018). Owing to the 2D reality shortness and disadvantages, there was a 

necessary aim and need to create and develop a system that touches the 

reality in the research field. The three-dimension cell culture mimics the 

actual brain environment. The 3D culture is an intermediate model between 

2D cultures and animal models, this intermediate model (3D) can be used 

for neurological disorders including traumatic brain injury, Alzheimer, and 



56 
 

Parkinson's diseases (Febles et al., 2014). The 3D cell culture's importance 

lies in the treatment of brain trauma, injuries, and brain stroke and aided in 

pre-clinical therapies (Benton, George, Kleinman, & Arnaoutova, 2009). 

Different types of 3D scaffolds were tested and stretched in our house-made 

stretcher, among these gels (scaffolds) naturally, and chemically synthesized 

polymers were studied.  

Naturally synthesized scaffold contains the proteins and molecules found in 

the brain tissue that is responsible for cell growth, unlike the chemically 

synthesized polymers growth factors. Moreover, proteins must be added to 

the culture (Benton et al., 2009). Each of the polymers has its advantages 

and disadvantages among each other as described in the paragraphs below. 

4.1 Matrigel non-suitable three-dimensional (3D) system stretch 

scaffold 

Matrigel provided an applicable neuronal cell growth due to the richness of 

essential proteins and ECM molecules. The cultivation environment was 

close to the actual brain environment. Matrigel allowed the neuronal cells to 

grow and form neurites, as shown in the results part in the previous sections. 

Both cultivation systems, 3D suspension, and sandwich methods were 

suitable for cell growth, but they were not appropriate for stretch 

experiments.  

Matrigel advantages provide applicable 3D neuronal cell growth conditions 

and environment. Otherwise, Matrigel had a considerable disadvantage in 



57 
 

our study; it detaches from our 50 kPa elastomeric PDMS chamber. High 

Matrigel concentrations prevent equal neuronal cell distribution in the 

cultivation scaffold, it avoids neurite formation and smooth cell growth. 

High gel concentrations are rich and dense with the internal components that 

determine cell growth and neurite formation. Low Matrigel concentrations 

allow equal or homogeneous neuronal cell distribution that allows the cells 

to form neurites smoothly. Cell growth and neurite formations depend on 

the internal components of the scaffold (Matrigel) from proteins and other 

molecules. Matrigel is suitable for neuronal cell culture without applying 

stretch experiments. 

  



58 
 

4.2 VitroGel 3D-RGD cultivation scaffold 

VitroGel 3D-RGD is a chemically synthesized polymer that had a longer 

polymerization time than Matrigel and PVP co-GMA but was not 

appropriate for primary isolated neuronal cell cultivation. As with Matrigel, 

VitroGel detaches from PDMS elastomeric chamber by cell cultivation and 

the medium change. This type of polymer; was not applicable for primary 

neuronal cell cultivation and was even not suited for 3D neuronal cell culture 

in our research. The chemically synthesized polymer was not suitable for 

cortical neuron cultivation, but this type is applicable for other cell-type 

cultivation. A considerable disadvantage of this type of gel is the detachment 

of the PDMS elastomeric chamber during stretch cycles and medium 

change. Due to this disadvantage of VitroGel, it was not more tested in our 

study for neuronal cultivation and stretch experiments.  



59 
 

4.3 PVP copolymerized with GMA (PVP co-GMA) the 3D artificially 

synthesized stretchable scaffold 

This type of polymer is an artificially synthesized one it has an advantage 

among the other gels previously mentioned so that it does not detach from 

our 50 kPa PDMS elastomeric chambers like the other natural and 

synthesized polymers.  This polymer is classified as fibrin gel due to its 

content of fibrinogen and thrombin. Due to its fibrinogen content, the 

neuronal cell growth is controlled by the component concentration, high 

fibrinogen concentrations lead to slow neuronal cell growth due to the small 

space inside the gel between the contents of the polymer in contrast to low 

fibrinogen concentration where the polymer is less crowded and high cell 

growth and neurite formation. The development of a 3D stretch experiment 

with this type of polymer was successful as the cultivation of primary 

neuronal cells in the suspension method on our newly developed 50 kPa 

PDMS elastomeric chambers was stretched without polymer detachments. 

The result of the experiment was stained with MAP2 and tubulin stain and 

observed under the LSM 880 microscope. The results showed neuronal cell 

reorientation perpendicular (opposite direction) to the stretch direction. 

Primary isolated neuronal cells grow in a perpendicular (opposite) to stretch 

direction even under low amplitudes. The obtained results were analyzed 

using a computational software called IMARIS. 

Additional experiments are required on the stretchable optimized fibrin gel 

(scaffold) concentrations and system. Two-dimension experiments and 



60 
 

studies regarding structural and functional aspects can be performed in the 

optimized three-dimension (3D) stretchable cell culture. 

  



61 
 

Chapter 5  

Future perspectives 

To overcome the organ environment difficulties, the experiments should be 

close enough to the actual organ environment. In our study, to investigate a 

suitable 3D stretch model, the neuronal cells were seeded on a suitable 

scaffold on a stretchable silicone PDMS elastomer. This elastomer is fixed 

on a house-made stretcher device to apply force and strain to the culture 

sample. More stretch experiments will be needed to study the synthesized 

artificial polymer PVP co-GMA, in addition to short and long-term cell 

cultivation. Three-dimensional (3D) cell stretch experiments with different 

amplitudes will be studied in more and specific details. The synthesized 

polymer was optimized on two suitable fibrinogen concentrations, and these 

concentrations were examined in our research and study. The tested 

fibrinogen concentrations were advisable for neuronal cell growth, as shown 

in the results part. Further experiments are needed on the optimized 

fibrinogen concentrations. The applied researches on two-dimensional (2D) 

neuronal cell cultures could be practiced with the newly developed three-

dimensional (3D) systems and the optimized fibrinogen concentrations. The 

achieved results from the 3D system culture could be compared with the 

previous 2D experiment results. Cytoskeletal and functionality stretch 

experiments of neuronal cells will be studied in more detail. Neuronal cell 

morphology and growth behavior can be studied in detail in a 3D 

environment. In the newly developed three-dimensional (3D) system life 



62 
 

cell imaging can be performed to study neuronal synchronization. 

Additionally, extreme amplitudes can be performed on the newly 

reconstructed PDMS elastomeric chambers using the optimized PVP co-

GMA polymer. 

Finally, with the newly developed 3D system we come to a step closer to the 

actual brain environment. This step leads us to overcome some brain 

illnesses like Alzheimer's and Parkinson's diseases and go a step further with 

brain trauma researches. 

  



63 
 

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