In Vitro Models for Airway Epithelial
I4"1
Cell Culture
by
Vivek Sivathanu
I
Bachelor of Technology in Mechanical Engineering
Indian Institute of Technology, Madras, 2010
Submitted to the Department of Mechanical Engineering in partial fulfillment of
the requirements for the degree of
Master of Science in Mechanical Engineering
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2013
@ Massachusetts Institute of Technology,,2013. All rights reserved.
Author..........................................
.........................
Depar
C ertified by .................................
.
ent of Mechanical Engineering
May 20, 2013
........................
Roger Dale Kamm
Professor of Mechanical and Biological Engineering
Thesis Supervisor
A ccepted by .....................
................
David Hardt
Chairman, Committee on Graduate Students
Department of Mechanical Engineering
In Vitro Models for Airway Epithelial Cell
Culture
by
Vivek Sivathanu
Submitted to the Department of Mechanical Engineering on May 20, 2013 in
partial fulfillment of the requirements for the degree of
Master of Science in Mechanical Engineering
Abstract
This work is about the development of a physiologically relevant model of the human airway.
Various factors such as the cell model, physiochemical factors such as the cell substrate
properties including its stiffness, shear stress, stretch, the air-liquid interface and the biochemical
factors in the medium influence the biology of the cells. The aim of this work is to closely
approximate conditions in an in vivo situation by engineering the above conditions in to the in
vitro platform. An assay to introduce the cell substrate properties was developed in a glass
bottomed petri dish type culture as well as a microfluidic device culture. The influence of the cell
substrate on airway epithelial cell monolayer formation was investigated in detail by changing
the stiffness of the substrate independently by changing the gel concentration, the gel formation
pH and the height of the gel from a hard substrate. Further, we found that biochemical growth
factors have a huge role in cell monolayer formation. A real-time measurement of monolayer
integrity using electrical resistance measurements was developed. A shear stress application
platform was developed and a stretch application platform was designed. The applications of
such a platform with the inclusion of various physiologically relevant factors include the study of
physiologic evolution of microbes such as the influenza virus.
Thesis supervisor: Roger Dale Kamm
Title: Professor of Mechanical and Biological Engineering
Acknowledgements
I would like to acknowledge the great support and encouragement from Prof. Roger Kamm,
my thesis adviser, without whom this work would not have been possible. He has been
instrumental in shaping my experience at MIT. The flexibility he offered me to explore and
develop my skills and ideas, both inside and outside the lab, is unprecedented.
I thank members of the Kamm lab who have been great team members in research and
beyond. I'd especially like to thank past and current Kamm lab members Ioannis
Zervantonakis, Vernella Vickerman, William Polacheck, Tharathorn Rimchala, Jessie Jeon,
Sebastien Uzel, Choong Kim, Andrea Pavesi, Jordan Whisler and Ran Li for the numerous
times they helped me in my research and in creating a very supportive environment in the
lab.
I would like to thank members of the Charles Stark Draper laboratory for financial support
and collaboration, especially Jeffrey Borenstein, James Comolli, Gayatri Perlin, Rachelle
Prantil-Baun, Amanda Lever and Ernest Kim. I'd also like to thank Jorge Valdez of the
Griffith lab at MIT for support with PEG-DA hydrogels.
My friends, both old and new whom I've made in Boston have made my life here a lot of fun.
The many people I have met here at MIT have inspired me to become a better person, both
professionally and otherwise.
Last but not the least I'm very grateful to my parents and the rest of my family for their
unconditional love and support during my studies.
2
Table of Contents
Chapter 1: Introduction......................................................................................................................6
The im portance of physiologically relevant culture m odels..................................................................
6
Influen za evolution ...................................................................................................................................
7
Chapter 2: Objective ........................................................................................................................
10
Viral evolution platforms ........................................................................................................................
10
10
State of the art in viral culture system s ..............................................................................................
12
---..
.............
--..
.........
Our m odel.......................................................................................................-
Chapter 3: The System and Technologies..........................................................................................14
Com ponents of the cell culture system ..............................................................................................
Cell M odel................................................................................................................................---..---
14
14
Glass bottom ed petri dish cultures...................................................................................................15
16
M icrofluidic system culture ................................................................................................................
18
Air liquid interface culture ..................................................................................................................
Scaffolds....................................................................................................................
.....
--. 19
-..........
Biological hydrogel scaffold ..........................................................................................................
19
PEG-DA-RGD scaffold .....................................................................................................------.........
19
Quantification .........................................................................................................................................
imaging ...............................................................................................................................................
20
20
Autom ated TEE R platform ..................................................................................................................
Two and four probe m easurement in a transwell cham ber .......................................................
21
Assays for more physiologic factors........................................................................................................
24
23
Shear stress application platform .....................................................................................................
24
Stretch application platform ...............................................................................................................
28
Chapter 4: Results and Discussion.....................................................................................................29
Petri dish culture parametric experiments .........................................................................................
30
Effect of gel thickness ........................................................................................................................
31
Effect of gel form ation pH ..................................................................................................................
Effect of basem ent mem brane ligand ...........................................................................................
33
Influence of biochem ical factors in the cell culture medium .......................................................
34
M icrofluid ic device culture .....................................................................................................................
M onolayer form ation in a m icrofluidic device (with ATCC m edium ) ..............................................
35
M onolayer form ation in a m icrofluidic device (with optim ized m edium ) .....................................
Chapter 5: Sum m ary and Future w ork ..........................................................................................
36
38
Sum m ary .......................................................................--Future work
..
. ----.......
-----------...............................
.....................................................................................................................
Bibliography ...............................................................................................--------------....................
3
34
35
38
38
40
List of figures
Fig 1: Creation of a novel viral strain in the upper airway cell acting as a mixing chamber.........8
Fig 2: State of the art in vitro models for airway culture..............................................................12
Fig 3: Final objective: Using airway epithelial cell cultures as physiologic mixing chambers for
influenza viruses......................................................................................................................................13
Fig 4: Cell models for airway epithelial cell cultures.....................................................................
15
Fig 5: Glass bottom ed petri dish cultures........................................................................................
16
Fig 6: M icrofluidic device schem atic...............................................................................................
17
Fig 7: Phase contrast image of Calu-3 cells growing on a transwell membrane in an air-liquid
interface culture ALI (left) and in a liquid covered culture LC (right) .........................................
18
Fig 8: Schematic of microfluidic device cell culture.......................................................................19
Fig 9: Phase contrast images of Calu-3 cells growing on polystyrene as a control (left), on a PEGDA gel not functionalized with RGD (middle) and on a PEG-DA gel functionalized with RGD
(rig ht)........................................................................................................................................................20
Fig 10: Fluorescence image of 1pam beads on top of the Calu-3 cells in an ALI culture: 2X image
(left), 4X(middle) and time lapse image (right) showing the movement of the beads possibly due
to beating cilia..........................................................................................................................................2
1
Fig 11: Schematic of impedance measurement setup(left) and the reference electrode(right)..22
Fig 12: Schematic of equivalent electrical circuit of the airway epithelial monolayer..............22
Fig 13: Pictures of impedance measurement setup in a transwell chamber: Two probe(left),Four
probe measurement (middle), Arduino/custom circuit to increase throughput (right) .......... 23
Fig 14: Variation of TEER (barrier function measure) over 17 days around a Calu-3 monolayer in
an A LI and LC culture...............................................................................................................................24
Fig 15: Shear stress calculations from a FEM of airflow over a transwell membrane...............25
Fig 16: Shear stress calculations from a FEM of airflow over a transwell membrane in a modified
transwell setup with a plug to focus the airflow............................................................................ 26
Fig 17: Summary of shear stresses on the cell culture membrane calculated using FEM in a
m icrochannel setup.................................................................................................................................27
Fig 18: Example of shear stress profile on the cell culture membrane along with the associated
flow rate and pressure head calculated using FEM in the microchannel setup....................... 27
Fig 19: Design of a stretch application platform with a PDMS based setup for epithelial and
endothelial cells with a matrix to visualize transport...................................................................
28
Fig 20: Calu-3 cells seeded uniformly in a microfluidic device on a soft collagen gel 2.5mg/ml in
ATCC recommended medium forms clumps instead of a monolayer........................................ 29
Fig 21: Calu-3 cells growing in a glass bottomed petri dish on top of a collagen 6mg/ml gel
formed at pH 11 with fibronectin coating at various thicknesses...............................................30
4
Fig 22: The effect of substrate collagen gel thickness on Calu-3 monolayer formation on four
separate gel com positions.....................................................................................................................32
Fig 23: The effect of substrate collagen gel formation pH on Calu-3 monolayer formation in a
collagen gel with concentrations 2.5mg/ml and 6mg/ml...........................................................
Fig 24: The effect of basement
membrane
33
ligand coating on Calu-3 cell monolayer
fo rm atio n ...................................................................................................................................................
34
Fig 25: The effect of biochemical growth factors in the cell culture medium on Calu-3 cell
m onolayer form ation..............................................................................................................................35
Fig 26: Clump formation in Calu-3 cell cultures in a microfluidic device with ATCC recommended
m ed iu m .....................................................................................................................................................
36
Fig 27: Formation of a Calu-3 cell monolayer in a microfluidic device with the optimized
medium containing the added growth factors..............................................................................
37
Fig 28: Calu-3 cells do not grow well on a stiffness gradient......................................................
38
Fig 29: Schematic of a viral evolution platform using fitness gradients......................................39
5
Chapter 1
Introduction
The importance of physiologically relevant culture models
The drug development pipeline is fundamentally broken'. In the last half century, there
have been tremendous improvements in efficiency of various parts of the drug development
process. In preclinical discovery, there has been often Moore's law like improvements in many
steps including the number of available compound libraries and in high throughput screening
systems. Animal models have become much better transgenic mouse models used to validate
these compounds. The efficiency of running human clinical trials has also increased through the
use of outsourcing and more advanced project management methodologies.
In spite of all
these improvements to parts of the drug development pipeline, the overall efficiency of drug
development defined as the number of new drugs brought to market per billion US dollars of
R&D has been steadily coming down. This trend has been often called Eroom's law (Moore's
law backwards).1
There are several reasons for this'. Primarily, it is due to the higher bar in terms of
clinical outcome required to approve new drugs over existing drugs, increased regulatory
standards of safety by the FDA and because of the brute force methods used in basic research.
The first two have led to increased number of preclinical and clinical tests required per patient
which leads to higher costs per drug. The last reason, namely the effect of brute force methods
is one of the motivations of this work.
What are these brute force methods? Much of pharmaceutical research in drug
discovery is based on the hypothesis that a small molecule drug candidate binding to a specific
target would lead to a benefit in terms of reducing the disease progression or a potential cure'.
As the number of compounds available to screen has increased tremendously, we screen for
more compounds. This follows finding the ones that have the highest binding affinity. Medicinal
chemists then alter and optimize these molecules shapes to further increase their binding
affinity. This hypothesis rarely translates to success in human clinical trials in reality due to
unpredictable off target effects and other system's effects that go beyond a single receptorligand binding. Therefore we need better methods to predict how preclinical discoveries may
translate in human clinical trials.
There are approaches actively being researched to achieve this goal include systems
biological modeling techniques 2 to predict the effect of single molecule drugs on entire systems
6
as opposed to a single target, humanized animal models which are used to study the in vivo
behavior of these drugs and most recently physiologic in vitro systems3-6
Physiologic in vitro systems aim to recapitulate the in vivo microenvironment of the
modeled organ system. Eg. Interstitial flow in cancer7 , shear stress in endothelial cells8 , soft
matrix substrate in neuronal cultures in a 3D tissue culture platform, chemical gradients across
monolayers, multiple cell types interacting with each other are all biophysical parameters which
when controlled would enable controlled studies of cellular processes in a manner that closely
simulates the in various complex processed occurring in the in vivo situation. It has been shown
by many groups that the behavior of cells and tissues in a physiologic microenvironment is
quite different from its behavior in simplistic in vitro platforms such as cells growing on petri
dishes 3-5 . Hence, the idea of physiologic in vitro systems is to be an in vivo like platform which
can also be controlled well unlike in vivo models so that high throughput screening of potential
drug candidates and study of biological systems would more closely mimic in vivo behavior in
humans than non-physiologic systems. Further unlike in vivo animal models in which animal
(mice, pig etc.) cells are tested, one can use human cells in physiologic in vitro systems to more
closely mimic human biology. Hence, such a model is expected to be in the best case scenario
much better than our best animal models if all the complexities if in vivo models are
incorporated. However, if more we increase the complexity of these systems, the more difficult
it is to control the microenvironment. However, this is an engineering challenge and the classic
tradeoff between complexity, control and physiologic relevance has to be made. We will discuss
the development of one such in vitro model in this work.
Influenza evolution
"Influenza-associated mortality in the next five decades is likely to exceed that of any
other global catastrophe" 9 . The Spanish Flu pandemic of 1918 killed over 50 million people,
more flu mortalities in 24 weeks than AIDS mortalities in 24 years'0 . Currently, almost a century
since then, we have a much more dangerous virus H5N1 circulating in birds. About 60% of the
humans infected by this virus die. Fortuitously, this virus has not yet been able to spread easily
between humans and hence we have avoided a pandemic. However, recently Ron Fouchier and
colleagues" engineered mutations in this virus that enabled it to attach to human airways and
also allowed the virus to evolve over 10 generations of ferrets, resulting in a highly pathogenic
and airborne virus. This virus is considered one of the most dangerous of all time; even the
anthrax virus pales in comparison
12
This is an imminent threat and we must develop scientific solutions to prevent a
disaster. Influenza vaccines that are being developed are often ineffective as they are usually
based off of vaccines for the previous seasonal flu strains. This does not always work because
7
the flu virus mutates rapidly crossing over between birds, pigs and humans. The unexpected
H1N1 strain that caused the swine flu outbreak in 2009 is testament to this.
Genetic drift and shift occur to create mutated strains of influenza. Genetic drift is the
slow and gradual pointwise mutation of the virus in pigs, birds and humans. Genetic shift is the
sudden appearance of a new strain in a species (say humans) by cross over from another
species (say swine), by novel mixes strains or both.
H1N1
HSN1
Human infection and pandemic
Co-infection of host
(acts as miixing tbarnber)
Mbdng of viral genes
New Infuenza strain
Novel viral strain
Fig 1: Creation of a novel viral strain in the upper airway cell acting as a mixing chamber
Influenza is an RNA virus with 8 strands of RNA. When an influenza virus enters a host
cell (say a human cell), all the 8 RNA strands mix inside this cell. This is an instance in which
novel virus particles are created. This isshown in Fig 1. In 1961, the extremely pathogenic avian
influenza strain H5N1 was found to lethally infect wild aquatic birds. Eventually, after 36 years
of genetic drift and shift, in 1997 the H5N1 virus crossed the chicken- human barrier and many
cases of lethal human infections have been reported ever since. However to date, the H5N1 is
not easily transmissible between humans. A H5N1 virus normally infects mainly the lower
airways deep in our respiratory tract. The lower airways hold most of the H5N1 virus receptors
although the upper airways also have some H5N1 receptors. It is a deadly strain but it does not
typically spread between humans because it mostly infects only the deep airways. So coughing
or sneezing is not likely to eject particles from deep in the lungs to the surroundings. But in case
the H5N1 mutates by shifting and drifting to acquire the right proteins on its surface to attach
8
and enter upper human airway cells more than it does currently, it might end up mixing with
other less transmissible viruses. Strains of influenza such as HINI are not very virulent but are
highly transmissible between humans as they infect the upper airways. If such a mixing happens
between a lethal strain and a transmissible strain, it could create a novel strain that could
potentially be as virulent as H5N1 and as easily transmissible as HiN1 (Fig 1). These are the
kinds of viruses that could lead to a pandemic.
9
Chapter 2
Objective
Viral evolution platforms
As shown in Fig. 1 and as discussed above, human upper airway cells could be a
dangerous mixing chamber for transmissible viruses such as HiN1 and more virulent yet less
transmissible viruses such as H5N1. Hence, studying the biology of such a mixing process would
be an important step in preventing such a mixing.
We are pursuing many directions to solve this problem. Bioinformatics collaborators at
the University of Massachusetts, Medical School evolve flu viruses by infecting MDCK cells
(canine kidney epithelial cells) with these viruses, subject them to various selective pressures
(anti-viral drugs) and use these data to train an algorithm that can predict viral evolution
trajectories. Their viral culture model is of widespread use because growing MDCK cells on
extremely stiff petri dishes and infecting them with flu viruses is well established and relatively
robust protocol. However, infecting canine kidney cells growing on non-physiologic substrates
is a poor model for infection and viral mixing in the human lungs. The influenza virus receptor
distribution (between H5N1 and HINI at least) is quite different in MDCK cells than what it is in
human upper airway cells.
State of the art in viral culture systems
State of the art systems today have many limitations. Most of them have been
optimized for vaccine production (viral reproduction). They are not suitable for physiologic
influenza evolution. MDCK cultures are cultures of canine kidney cells in polystyrene flasks. This
is the most widely used method because MDCK cells give a high yield of influenza cells. They
can be used to culture both H5N1 and HIN1 viruses with high yield as they have receptors for
both these kinds of viruses. Further, culturing MDCK cells themselves is a very robust process as
the culture conditions are well defined and widely used. This would be a great system if the
purpose of the culture was to get a high viral yield in applications such as cell culture derived
vaccines. But we want a physiologic system that can help replicate mixing in the upper airways.
Further, this is not even a human cell line. Vero and Per.C6 cells are also similarly used in
commercial vaccine production. So the use of any of these cells in petri dish cultures may not
model viral evolution in human airways very well.
10
Don Ingber's lung on a chip platform 3 has a micro engineered platform designed to capture the
oscillatory strain experienced by alveolar epithelial cells and the underlying endothelial cells.
This system has incorporated many of the physiologic conditions in the alveoli and would be
great to culture H5N1 cells. But alveolar epithelium is not typically the mixing chamber in which
highly transmissible novel virus strains are created. The human airway epithelium is the correct
model for this.
NHBE cultures on transwell membranes at an air-liquid interface are a good model system for
evolution of viruses. Firstly, it is a human airway model, which is exactly what we need.
Secondly, it is a pretty physiologic system because this involves creation of a differentiated
pseudostratified airway epithelium. It is a little short of being a perfectly physiologic system
because the cells grow on a hard polycarbonate/polyester membrane as opposed to actual
airway epithelial cells which grow on a soft substrate with other cells encapsulated in the
substrate tissue. Further there is a strain in the substrate and a shear stress due to the
breathing motion. These physical parameters are expected to change the way that cells react to
their surroundings.
Illustration
Cell
Drawback
MDCK
x
Not a human lung cell
Canine
x
Can reproduce both H1N1
H5N1
Kidney Cell
and
Vero
physiologically
Monkey
Kidney Cell
Per.C6
Human
retinal Cell
11
non-
Alveolar
Epithelial
Cells
Don Ingber
vacum
Aleolsvaum
Lung
chip
EnOtheium
on
x It is an alveolar model, not
an airway model
x
airway tissues well.
a
3
The system can't hold a
thick matrix to represent
x
H5N1/H1N1
Reproduces
but
W
non-physiologic
for
H1N1
and
of
evolution
H5N1 for the creation of
highly transmissible stains.
Transwel
nse
Apical
chamber
Cep
monolayer
Permeable
membrane
Basolateral
chamber
NHBE
/
The correct cell line
normal
x
But
in
a
non-physiologic
microenvironment
human
bronchial
epithelial
cells
in
transwells
Fig 2: State of the art in vitro models for airway culture
Our model
To overcome the drawback of using a non-physiologic system for the viral culture, we
are designing and prototyping a bio-mimetic airway infection system that is better than all the
current systems. There are many characteristics which influence the airway microenvironment.
We will grow airway cells on a substrate with in vivo like stiffness while also mimicking the
shear stress felt due to breathing. In spite of experiencing a relatively low shear stress
compared to the levels that endothelial cells feel, this level of shear stress has been shown to
increase mucus production' 3 and acts as an additional selective barrier to influenza infection.
We have already investigated the effect of stiffness of substrates such as collagen, fibronectin,
vitronectin, laminin and matrigel. We are also developing a technique to incorporate an RGDconjugated Poly Ethylene Glycol (PEG) hydrogel of tunable stiffness in to our systems. Further,
instead of growing canine kidney cells, we grow human airway cells (NHBE and Calu-3). NHBE:
Normal Human Bronchial Epithelial cells and Calu-3: Human Lung Carcinoma cell line. Influenza
viral infection in this system is expected to simulate evolution in the real airways much better
than in over-simplistic MDCK cultures on petri dishes because substrate stiffness, cell
phenotype, shear stress on the surface and many other factors influence infection dynamics.
12
Moreover, the infection is also much more controllable than in extremely complex animal
models more commonly used. In our microfluidic device, we also have the ability to co-culture
several different cell types, which gives our system the potential to mimic complex heterotypic
cellular interactions such as those with smooth muscle cells, cartilaginous structures,
fibroblasts, endothelial cells etc.
What constitutes a physiologic micro-environment
for airway host cells?
*
Shear Stress (0.05 Pa due to normal breathing)
*
Multiple Cell types (ciliated, goblet, basal, fibroblasts)
*
Airway stromal tissue stiffness, basal lamina
*
Stretch
_____
Humns
Few HsN1reeptr
HSN1 ecepors
(evisn fu)
Virtual lung group,
UNC, 2009
Fig 3: Final objective: Using airway epithelial cell cultures as physiologic mixing chambers for
influenza viruses
13
Chapter 3
The System and Technologies
In this chapter, the various components that make up our cell culture system and associated
technologies that were developed in the process will be discussed. In the next chapter, we will
discuss their application to this study.
Components of the culture system
Cell Model
There are many different cell types we could use for in vitro lung epithelial cell culture. We use
Calu-3 cells for lung airway epithelial cultures because of its many advantages over other
systems.
It forms tight monolayers
. Hence it is good for transport experiments in which
microbes might disrupt the epithelial cell-cell junctions. By being tight at baseline, one could
use dextran transport or electrical impedance based methods to monitor the cell-cell junction
of Calu-3 monolayers. These cells also express mucins15, which contain many anti-microbial
factors. Influenza viruses can be cultured on them 16. In addition, these mucins present a
transport barrier to viruses. This is because viral entry in to the airway cells is key to their
replication. Certain species of viruses may have a selective advantage over others in terms of
their ability to penetrate mucins. Hence having a good representation of mucins is essential if
one were to model viral evolution in the airways. Calu-3 cells are also known to possess cilia
and microvilli in patches. Cilia are key cellular structures which beat and help clear the mucins.
This is part of the airway cells' mucociliary escalator by which it clears away particles trapped in
the mucus. Hence, it is also a critical part of the infection model that we are developing. Cilia
might also prove important in adhesion to receptors on the cell surface, hence in the invasive
ability of bacteria17 . Calu-3 cells have the ability to be cultured at an air-liquid interface, which is
key to having a physiologic model of the airway.
Eventually we will move on to primary cell cultures of NHBE (Normal human bronchial epithelial
cells), which also has all the above properties (tight junctions, mucins, cilia) and are known to
differentiate in to pseudostratified epithelia at an air-liquid interface. But for now Calu-3 cells
are the optimal choice to develop our system as cell lines are easier and less expensive to
culture compared to primary cells.
14
Cell type
Origin
Barrier
Mucus
function
Calu-1
Bronc~al Ep.
Cancer
Good
16HBE14o
Good
-
Immortalized
Bronch. Ep.
NHBE15
Human
46 primary C'4
Go401od
A549
Adenocarcinoma
Poor
Yes
MDCK
Canine kidney
GoodjBest
N/A
NCI-H292
Pulmonary
mucoepidermold
carcinoma
Poor
Yes
1IHAo-
Immortalized Arr.Ep
Goba
Yes
s
Lysozyme
Lactoferrin
(anti-
(nti-
bacterial)
bactiffungi)
yes
No
Yes
No
Yes
Ye
I
(High)
Yes
Fig 4: Cell models for airway epithelial cell cultures
Glass bottomed petri dish cultures
Cell culture has been shown for many different cell types to depend on the stiffness of the
substrate and the basement membrane ligand that the cells attach to in the substrate1 -25 . So
we would like to culture cells on soft physiologically relevant substrate like in actual airways. In
order to do a mass screening of the properties of the stiffness and the basement membrane,
we used a glass bottomed petri dish based culture.
1.
As shown in the figure, we use a collagen gel of various heights.
2. We then coat this substrate with a basement membrane protein cocktail such as matrigel,
vitronectin or fibronectin.
3. The varying heights of collagen change the stiffness felt by the cells,
4. At "0" height of collagen, the cells grow on glass and feel an effectively infinite stiffness
(~100GPa). However, at a height of 0.3mm of collagen, the cells feel mainly the collagen
stiffness (~1kPa). So by varying the collagen gel height over this range, we effectively vary
the stiffness felt by the cells over a wide range.
5. We also change the stiffness of the gel independent of the collagen height change by
changing the gel composition
and polymerization
itself by changing the collagen
concentration and the pH at which the gel forms.
6. Once the gel forms, Calu-3 cells, which were previously grown on polystyrene flasks up to
about 70% confluence, are trypsinized, centrifuged down, resuspended to the appropriate
concentration and added to the top of the gel.
7. Cells are cultured in this chamber and the cell culture medium is replaced every day.
15
Cells
ECM
Coating
Collagen gel
Cells
Collagen
gel
Estimated Gel thickness (without accounting for the meniscus)
332pm
38pm
76pLm
168pm
12pm
Fig 5: Glass bottomed petri dish cultures
Microfluidic system cultures
In order to perform controlled heterotypic cell cultures, to measure changes in cell-cell junction
strength with a higher accuracy and to image the cell with high resolution in a very controlled
environment, we culture the cells in a PDMS based microfluidic device. As described in many
articles before 7 , we manufacture the PDMS microfluidic devices pictured here by the following
protocol.
1.
Make an SU-8 mold of the inverse of the final PDMS device.
2. Pouring a well-mixed and degased mixture uncured PDMS prepolymer and curing agent at a
10:1 ratio on the SU-8 mold.
3.
Allowing the PDMS to polymerize at 80 0 C overnight to create a solid block of PDMS with the
features from the SU-8 mold in to it.
4. Peel the PDMS device from the SU-8 mold using a knife and peeling it off.
5. Wet autoclave the PDMS device in water for 20 minutes to remove any uncured PDMS
monomers.
6. Dry autoclave the device for 20 minutes to sterilize and dry it. Also bake the device in the
80*C oven overnight to remove most of the water in the device.
7. Plasma-treat the surface of the device in an Oxygen plasma chamber at ~650 Torr for 2
minutes and coat the central region of the device with the posts(the orange region in which
collagen is held) with a solution of Poly-D-Lysine (PDL) in order to covalently bond the PDL
to the PDMS. Let it incubate in a cell culture incubator for 4 hours. PDL is important for
collagen to bond well to the PDMS via the PDL.
16
8. Aspirate the PDL on top of the devices and wash well with cell culture water as un-bonded
PDL iscytotoxic to cells. Dry the device in the 800Coven for four hours.
9. Prepare collagen gel with final concentrations between 2mg/ml to 6mg/ml by mixing rat tail
collagen I, 10XPBS, NaOH and water at the appropriate ratios to obtain the desired collagen
concentration, pH and osmolarity. Store the collagen gel in an ice bucket.
10. Plasma treat the surface of the device again for 2 minutes. Leave the device in room
temperature in a petri dish for 10 minutes so that it recovers some of its hydrophobicity and
then pipette about 10tL of collagen gel on top the posts in the device. Immediately bond a
cleaned class coverslip on the device to form an irreversible seal and encapsulate the
collagen gel in the microfluidic device.
11. Allow the collagen gel to polymerize in the 370C incubator in a humidified chamber for
about 30 minutes.
12. Fill both the channels with cell culture medium so that the gel does not dry up. And make
sure to avoid any bubbles by triturating gently.
13. Calu-3 cells, which were previously grown on polystyrene flasks up to about 70%
confluence, are trypsinized, centrifuged down, resuspended to the appropriate
concentration and added to one of the channels of the device.
14. The device is then tilted 90 degrees for about 2.5 hours so that the cells settle down by
gravity on top of the gel (green) as shown.
15. The device is left in the incubator with fresh medium replaced each day and the cells are
allowed to grow.
Hydrogel
(orange)
Cell monolayer
(green)
Air bubble
controlled by
a syringe
pump (blue)
Fig 6: Microfluidic device schematic
17
Air liquid interface culture
The process described above for both the petri dish and microfluidic device cultures is used to
culture airway cells in a liquid covered culture. But physiologically airway epithelial cells have
nourishment from the basal side where they are attached to the substrate and air exposed to
the apical side. Such a condition is necessary for the cells to differentiate in to a
pseudostratified monolayer that is really physiologic28 . In order to create an air-liquid interface,
we first let the cells grow to confluence and create a tight monolayer in a liquid-covered
culture.
In order to create an air-liquid interface in a petri dish type environment, we use transwells.
Cells are cultured on clear polyester membranes in a transwell setup. The basal chamber is
always filled with medium. Medium in the apical chamber is present in a liquid-covered culture.
For an air-liquid interface culture, the medium on the top is removed once the cells reach
confluence. As seen from the phase contrast image of the cell culture in transwell after 6 days
(below), there are some clustered structures present in the air-liquid interface (ALI) culture that
are not present in the liquid covered culture (LC).
ALl
LC
6 days after
seeding
Fig 7: Phase contrast image of Calu-3 cells growing on a transwell membrane in an air-liquid
interface culture ALI (left) and in a liquid covered culture LC (right)
In the microfluidic device culture, once the cells grow to confluence, we remove the liquid in
the channel by introducing a long air bubble. In order to create an air liquid interface, we plug
in a syringe pump filled with air in to the channel with the airway cells (shown). The syringe in
the pump is fitted with a sterile filter and pumps long air bubbles in to the channel to create a
physiologic air-liquid interface environment for the cells to grow on.
18
Epithelfal monolayerat en
air liquid Interface
To Syringe Pump
substrate has a tunable
Stiffness
M
Matrix composition
Dynamics
(other cell types)
Height of each channel
Width of get region
=180 um
1mm
Fig 8: Schematic of microfluidic device cell culture
Scaffolds
Biological hydrogel scaffold
Collagen type I is used as the major protein of the substrate as it is the main structural protein
present in the tissues. Matrigel, Laminin, Fibronectin and Vitronectin. The collagen gel
concentration used is between 2mg/ml and 6mg/ml. The advantage of using a collagen scaffold
is its physiologic nature.
PEG-DA-RGD scaffold
We are also developing a Polyethylene glycol (PEG) gel based system in order to vary the
stiffness of the substrate much more controllably than in collagen gels. While the PEG-DA gel
system is not as physiologic as the collagen based system, it is much easier to control than the
collagen based system. In a collagen gel, many different factors affect collagen polymerization
and hence the collagen microstructure. So it is difficult to modify the stiffness of collagen alone
without changing the ligand distribution, the fiber size etc. To make a PEG-DA gel, we inject a
mixture of Polyethylene Glycol Diacrylate (PEG-DA) and photo-initiator in the desired cavity
(either between two glass plates or between a microfluidic device and a glass coverslip). UV
radiation from a 10mw/cm 2 UV lamp with a 365 nm wavelength output is applied on the gel for
30 seconds. This would polymerize the gel in about 20 minutes depending on the size of the
sample. We chose to use 3.4kDa PEG-DA monomer as it gives a high stiffness of 20kPa-100kPa
depending on the PEG-DA concentration. At 20% concentration it is known to have a stiffness of
19
about lOOkPa29 . We also used 10% CGRGDSPY fragments to help the cells attach to the PEG gel
as cells do not attach to pure PEG as shown in the figure.
Fig 9: Phase contrast images of Calu-3 cells growing on polystyrene as a control (left), on a
PEG-DA gel not functionalized with RGD (middle) and on a PEG-DA gel functionalized with
RGD (right)
Quantification
Imaging
In order to quantify the monolayer and its developing, we primarily use imaging methodologies.
Mucus is one of the parameters important to creating a physiologic viral evolution platform.
Hence, while characterizing this system, imaging the level of mucus secreted by the cells is
important. MUC5AC is secreted by goblet cells and is representative of the level of mucus
secreted3 0 . Immunostaining could be used to quantify this. Imaging the cilia is another
parameter which determines the level of differentiation of these cells in to a physiologic
phenotype. Imaging R-Tubulin IV would give us a measure of the cilia present30. Lectin
expression staining would help quantify the ratio of influenza receptors on the surface of the
Calu-3 cells that would allow either H1N1 or H5N1 to attach on to it 31 .
Sambucus niger
agglutinin and Maackia amurensis lectins could be used to identify specific Sialic acid residues
SAa2,6Gal and SAa2,3Gal residues which help preferential binding of HIN1 and H5N1
respectively3 2 . Mucocilliary clearance tests could be used to test the ability of beating cilia to
clear the mucus layer on top. We performed an initial test with fluorescent lum polystyrene
beads and tracked the motion of these beads on the surface of the Calu-3 cells. As seen in the
figure, there are local patches of swirly motion but also a long range movement of the beads,
indicating synchronously beating cilia. This test was done after two weeks of culturing Calu-3
cells at an air-liquid interface on a transwell membrane, indicating a good level of
differentiation. Further optimization could be done using this protocol by quantifying the height
of mucus and the corresponding mucus flow rate induced by the beating cilia.
20
Fig 10: Fluorescence image of 1pm beads on top of the Calu-3 cells in an ALI culture: 2X image
(left), 4X(middle) and time lapse image (right) showing the movement of the beads possibly
due to beating cilia
Automated TEER platform
A less invasive measurement of the monolayer could be an electrical impedance based method.
We developed a method to measure the electrical method of the cell monolayer using a 2 and 4
probe type electrical resistance setup. We have two electrodes far from the gel which are
connected to a stable power source and used to pass a current through the cell monolayer.
These electrodes are placed far from the actual monolayer to create a uniform current density
near the location of the cell monolayer. We also have two voltage sensitive reference
electrodes that are used to measure the potential across the monolayer and placed right across
the monolayer. By applying a current and measuring the voltage across the monolayer, we
obtain the complex impedance of the monolayer itself. As shown in the equivalent electrical
circuit figure of a cell monolayer, this impedance measurement could be used to measure a
number of parameters such as the membrane capacitances, the barrier function and the
intracellular (or transcellular resistance). One simple example is the application of a DC current
which will all pass through the intercellular space, thereby giving us the barrier function (or
equivalently TEER - trans epithelial electrical resistance). If we use high frequency currents it
would help us measure the combined membrane capacitance because a lot of the current will
also pass through the membranes which act as capacitors.
We make our electrodes ourselves using some commercially available parts. We buy a 0.2mm
diameter Silver (Ag) wire which we use for the current passing electrodes. In order to make the
valtage sensitive electrical reference electrodes, we dip one of these silver wires in to a 100%
bleach solution (Sodium hypochlorite) for 5-10 minutes to coat the surface of the silver wire by
Silver Chloride (AgCl). Silver gets oxidized by bleach in to silver chloride. Ag/AgCI could be used
as a reference electrode with a stable junction potential. The junction potential depends on the
local concentration of chloride ions around the Ag/AgCI interface. To maintain this, we
21
encapsulate the Ag/AgCI wire in IM KCI gel in a 10Ip pipette tip. The KCI gel is prepared by
heating a mixture of IM KCI in 5% Agar at an 80"C with constant mixing just until the Agar starts
to dissolve well and form a gel. When the gel is still in its liquid state, we pipette the gel in to
the 10lpL pipette tip with the Ag/AgCI wire held inside. Then we allow to gel to cool down and it
forms a solid block with a stable reference electrode. It is then stored in a KCI solution at 4*C
until use.
11
_
Ag
AgCl coating
Conductive
gel 5% Agar in
IM KCI
Fig 11: Schematic of impedance measurement setup (left) and the reference electrode (right)
Membrane
capac ance
Barrier function (related to TEER)
I
I
I
IA_
I
I
II
T Current
I
Equivalent circuit
Apical membrane
capacitance
Transcellular resistanc
TEER
Basal membrane
capacitance
Fig 12: Schematic of equivalent electrical circuit of the airway epithelial monolayer
22
Two and four probe measurement in a transwell chamber
Fig 13: Pictures of impedance measurement setup ina transwell chamber: Two probe (left),
Four probe measurement (middle), Arduino/custom circuit to increase throughput(right)
In a two probe measurement, we just apply a known voltage across the transwell membrane
using the Silver wires and measure the current that passes through using an ammeter (as
shown). This results in a baseline resistance of 600 Ohms when nothing but 3ml of medium is
filled in the two chambers. But this includes the error (additional resistance in series with the
transwell resistance) contact resistance of the probes. However using a four probe
measurement limits this error and the baseline resistance drops to 150 Ohms. Clearly a four
probe measurement is a better choice. Using this four probe measurement, we first take a
baseline resistance measurement across the transwell membrane. Then we plate cells on it and
monitor the resistance across the monolayer as often as we would like. Typically a resistance
measurement once a day is sufficient to capture the normal development of the monolayer.
However, in the presence of infectious agents or anything that might affect the normal
development of the monolayer, one should monitor the resistance even every few minutes to
capture the dynamics. It would be non-invasive because we would apply a short pulse and
record the current in that short time.
We monitored TEER development in a Calu-3 monolayer for about 3 weeks. As shown in the
figure, the cells become confluent at about day 10 at which point, the resistance starts
increasing. This suggests that the cell-cell junctions are slowly developing and getting stronger
(barrier function increases). But there isa point about the 17 day point around when the barrier
function is the highest. It is at this point that we desire to do infection and microbial invasion
based studies. We also note how the ALI and LC differ in the way they develop as denoted by
the TEER. In general liquid covered cultures (LC) have a higher barrier function than Air-liquid
interfaced cultures (ALI). This suggests that although some microbes may not disrupt the
monolayer in a LC culture, they may actually disrupt the monolayer in an All culture because
the ALI monolayers are less tight. ALI cultures are also more physiologic and so might be more
representative of the in vivo situation. We also note how sensitive the monolayers are because
the monolayers were disrupted when we introduced microspheres to monitor mucociliary flow
23
at day 17 in the ALI culture. Within a few hours of introduction of the microspheres, the TEER
dropped massively to the baseline TEER. But the phase image at day 17 after the microsphere
introduction looks to have a healthy monolayer visually. However, TEER is able to detect
monolayer disruption. Hence TEER measurements are clearly a good measure of monolayer
integrity above and beyond a visual inspection.
1400
1200
TEER (Ohm cm2)
-+-LCC1
-0- LCC2
-r-LCC3
TEER
1000
-Au1
-i--AU2
800
-e-Au3
600
400
e Sudden drop is due to
the disruption of the
monolayer due to
added fluorescent
200
o
-microscpehres.
1 202
3
4
5
6
7
8
9
10 11
12 13 14 15
16
17
-200
Fig 14: Variation of TEER (barrier function measure) over 17 days around a Calu-3 monolayer
in an ALI and LC culture
Assays for more physiologic factors
Shear stress application platform
To test the effect of shear stress on the monolayer, we are developing a platform with
machined channels to apply a shear stress in a transwell based setup and in a more controlled
microchannel type setup. The main design requirement was to have uniform shear stress on
the cells at a level of experienced by the airways is in the range of 0.04-0.2Pa as this is the shear
stress level experienced by cells in the airways33
As shown in the figure, we have an air inlet and outlet on top of the transwell chamber which
allows for a flow of air. This air is humidified by passage through a water column. We
performed a finite element simulation of the air flow using COMSOL Multiphysics to figure out
the shear stress profile that the cells feel and the appropriate flow rate that would give a shear
24
stress in the range felt by cells physiologically (0.04-0.2Pa). As shown, the shear stress is
actually quite non-uniform whenever the shear stress approaches physiologic levels (happens
only at high air flow rates). So we designed a plug that fits in to the transwell chamber to focus
the flow a bit more to get a more uniform shear stress profile. This gives a reasonably uniform
shear stress profile at physiologic levels that would be appropriate for the cells.
20b
Air IN
Air OUT
-'040
Streamlines
Q0
Cells
20
20
-20
x10"
;i
Fig 15: Shear stress calculations from a FEM of airflow over a transwell membrane
25
Air IN
Air OUT
0_
Iii
4
- We can get shear stresses of 10-2 Pa
with this configuration
- But in all these transwell setups, the
shear stress is quite non-uniform and
control is limited.
Fig 16: Shear stress calculations from a FEM of airflow over a transwell membrane in a
modified transwell setup with a plug to focus the airflow
In order to make the shear stress application system much more controlled and uniform, we
are designing and prototyping a microchannel version of the same. Essentially, rectangular
channels are machined in to a polystyrene block and humidified air is flown in to the channels
to apply the appropriate level of shear stress over a wide range of channel length. Our design
criteria were chosen for the purpose of easy manufacturing. In the figure shown, only h2 is
varied. Thereby with the same flow rate, the cells with experience lower shear stresses as h2 is
increased. We simulated the shear stress on the cells assuming a laminar flow of air using finite
element method (COMSOL). The results are summarized here. A representative plot of the
shear stress on the cell culture area is also shown. It has a symmetric shear stress profile and so
only half the plane is shown.
26
Air flow
h
(rate QarWi1
w1
h1
w2
2 mm
0.5 mm
2.5 mm
enannei
I
Only h2 is varied. The rest of the
Cells are cultured on a membrane that attaches geometry is fixed.
to the bottom of this face (area = w1 x L)
Length of the channel is 1 cm or longer
h2
x-shear (mean) at
Qair = 10 ml/min
t-shear (mean) at
Qair = 120 ml/min
1 mm
0.003 Pa
0.04 Pa
0.5 mm
0.008 Pa
0.1 Pa
0.25 mm
0.015 Pa
0.18 Pa
0 mm
0.034 Pa
0.41 Pa
Fig 17: Summary of shear stresses on the cell culture membrane calculated using FEM in a
microchannel setup
SWWOc
Vnaan ofw.-*Con",sWt I#AI11
I
.4
Q = 60 mI/mn for ~ 0.OSPa average
shear stress
Pressure head = 3.68 Pa
MV -
-
Peak Shear = 0.0779Pa
U
4
4
7
*
~
*~*
YA*~A.1~~
Fig 18: Example of shear stress profile on the cell culture membrane along with the associated
flow rate and pressure head calculated using FEM in the microchannel setup
27
Stretch application platform
We are also designing a new device which helps apply a stretch to the cells in order to also
capture alveolar epithelia. This device was inspired by Don Ingber's lung on a chip model 3 . But
our design has the additional ability to include tertiary cells types (besides alveolar epithelial
cells and endothelial cells) in the gel encapsulated in the device.
Alveolar epithelial
monolayer
Alveolar
layer stretch
control
ctann"
vacuum
Ar
m
Endothelial
layer stretch
control
cM
vacuuS
.
=
m
Endothelial monolayer
-
Thin perforated PDMS
membrane
E
WITHOUT STRETCH
Vacuum
Air
Vacuum
Meim
Vacuum
vacuum
WITH STRETCH
Fig 19: Design of a stretch application platform with a PDMS based setup for epithelial and
endothelial cells with a matrix to visualize transport
28
Chapter 4
Results and Discussion
As discussed earlier, the goal is to create a monolayer of Calu-3 cells in a microfluidic device
under physiologic conditions. So we fabricated microfluidic devices as described above and
introduced a 2.5mg/ml collagen I gel in it. Calu-3 cells were added to the device at a range of
seeding densities. Calu-3 cells were prepared in a 2M cells/ml solution of ATCC recommended
Calu-3 cells culture medium. Between 5-60pL of this solution was injected in the cell culture
channel and the device was tilted for 2.5 hours so that the cells rest on the gel by gravity. As
shown in the figure, the cells are uniformly distributed on top of the gel. However, after 15
hours, the cells form huge clusters instead of being a continuous monolayer. This is not a
sudden process. As shown in the figure, it is a dynamic process in which cells slowly migrate
towards one another. The homotypic interaction between the cells is much stronger than the
cell-matrix interaction. This kind of clump formation was independent of the cell seeding
density. We used a range of seeding densities as mentioned above ranging from cells being a
few diameters away from each other to cells piling up on top of each other. And in all the cases,
in a microfluidic device, cells do not form a monolayer on a 2.5mg/mI collagen I gel.
Time = 15hrs.
Time = 0
Fig 20: Calu-3 cells seeded uniformly ina microfluidic device on a soft collagen gel 2.5mg/ml
inATCC recommended medium forms clumps instead of a monolayer
29
Petri dish culture parametric experiments
Cell behavior is heavily influenced by the stiffness of the substrate and the ligand that the cells
5
attach tois-2
. In order to investigate if the substrate properties could be tuned to enable the
Calu-3 cells to attach to the gel, we conducted a series of experiments in a petri dish type
culture.
Cells cultured on a soft substrate such as collagen feel the stiffness of the underlying hard
substrate (polystyrene/glass) if the thickness of the soft substrate is less than a few tens of
microns. So we pipetted gels in to glass bottomed petri dishes and grew Calu-3 cells on top of
them. Based on the volume of gel pipetted and the cross section area of the dishes, we
estimated the height of the gel in each plate (ignoring the meniscus at the walls). Collagen I was
used as the main hydrogel. Once the collagen polymerized, the surface was further coated with
a 5 pg/ml solution of fibronectin, vitronectin or matrigel. Cells were then plated on top of the
coated gel and ATCC recommended medium was used and replaced every day. Besides
changing the height of the gel, we also changed the composition of the collagen gel itself to
change the stiffness. This gave us a wide range of parameters to modify to find the optimum
substrate that would allow cell monolayer growth while also being soft and physiologic.
Cells
Collagen
gel
Estimated Gel thickness (without accountingfor the meniscus)
12pm
38pm
76pm
168pm
332im
Gel thickness
12pm
38pm
168pm
76pm
332pm
Fig 21: Calu-3 cells growing in a glass bottomed petri dish on top of a collagen 6mg/ml gel
formed at pH 11 with fibronectin coating at various thicknesses
30
Effect of gel thickness
We performed experiments with Collagen gels 2.5mg/ml and 6mg/ml formed both at pH 7.4
and pH11. The Calu-3 cells were seeded on to them and allowed to grow out for a week. The %
confluence of these cells was then calculated from images such as the one shown in the figure.
These percentages were normalized with respect to the sample of highest confluence within
each (gel concentration, pH). These airway epithelial cells in general preferred growing on
stiffer substrates. They grew faster to confluence a stiffer substrate. As shown in the figure with
collagen gel concentration 2.5mg/ml formed at pH 7.4, cells that grew on gels with lower
thicknesses became confluent faster. Cells that grew on the 12pm gel can sense the stiffness of
the glass surface underneath easily and hence grow very well to form a confluent monolayer on
this substrate. As the thickness increases, we see that the cells get to confluence slower than
otherwise. In fact, there seems to be a sharp cutoff a little beyond a 38Im gel at which point
the cells probably don't feel the stiffness of the underlying glass surface anymore and they
grow much slower on the soft substrate. Comparing the effects of the collagen gel with collagen
concentrations 2.5mg/ml and 6mg/mI, both formed at pH 7.4, it is expected that collagen gel
with concentration 6mg/ml is stiffer. From the figure, we see that the effect of decrease in
speed to confluence with increase in gel thickness is reduced in the stiffer collagen 6mg/ml gel.
A similar effect if observed when comparing collagen 2.5mg/ml gels formed at pH 7.4 and pH 11
because the collagen gel formed at pH 11 is expected to be multi-fold stiffer than a collagen gel
formed at a neutral pH.
Effect of gel thickness on monolayer formation
100
80
U12pm
U
40
M76pm
2 168pm
E 20
Z
38pm
60
0
I 332.Lm
0
Collagen 2.5mg/ml formation pH 7.4
31
Effect of gel thickness on monolayer formation
100
80
U 12pm
60
11 38pm
S40
76pm
I
J 168pm
20
z
0 332pim
0
0
Collagen 6mg/ml formation pH 7.4
Effect of gel thickness on monolayer formation
100
80
0 12[im
60
I
a 38pm
0E76ptm
40
a 168pm
E 20
E0
0
*M332pm
Collagen 2.5mg/ml formation pH 11
Effect of gel thickness on monolayer formation
100
80 -0
U 12pm
8 60 -
N 38pm
M76pm
40 -
E 20 -
N 168pm
~M 332p.m
0
0
Collagen 6mg/ml formation pH 11
Fig 22: The effect of substrate collagen gel thickness on Calu-3 monolayer formation on four
separate gel compositions
32
Effect of gel formation pH
If a collagen gel is formed at a higher pH the negatively charged ions make the backbone of the
collagen stiffer due to a higher repulsion between the collagen fibers. So a collagen gel formed
at pH 11 is stiffer than a collagen gel formed at pH 7.4 assuming that all the other factors such
as collagen nucleation time, conditions during polymerization (temperature, humidity) and the
collagen concentration are the same. As shown in the figure, keeping all those factors constant
besides pH, we see that an increase in % confluence with an increase in pH over a neutral pH.
The relative increase in % confluence gives an indication also of the level to which stiffness
changes are perceived by cells. In the collagen gel 2.5mg/ml, as shown, the increase in
%confluence with pH when the cells are on a soft substrate (higher gel thicknesses) is much
more than the corresponding increase in the collagen 6mg/mI gel. This suggests that the cells
react more strongly to an increase in stiffness via an increase in pH in the softer 2.5mg/ml gel
than in the much stiffer 6mg/mi gel.
Effect of gel formation pH on monolayer formation
a week after cell seeding
Gel formation pH 7.4
* Gel formation pH 11
100 90
80
70
60
40
30
20
10
0
12pnm
38pm
76pm
168pm
332pm
Collagen gel 2.5mg/mI thickness
Effect of gel formation pH on monolayer formation
a week after cell seeding
m Gel formation pH 7.4
g Gel formation pH 11
100
80
70
60
50
30
20 -A
10
0
12Am
38Wn
76pm
168pm
332pm
Collagen gel 6mg/ml thickness
Fig 23: The effect of substrate collagen gel formation pH on Calu-3 monolayer formation in a
collagen gel with concentrations 2.5mg/ml and 6mg/ml
33
Effect of basement membrane ligand
The use of any basement membrane coating (5pg/ml) such as fibronectin, matrigel or
vitronectin increases % confluence in general.
Cells
ECM Coating
Collagen gel
Collagen 2.5mg/ml with
Collagen 6mg/mi with an ECM
an ECM coating
coating
100
100
80
80
~40
40
20
Culture time
1 week
20~ ~
20 -
pH 7.4
pH 11
Fig 24: The effect of basement membrane ligand coating on Calu-3 cell monolayer formation
Influence of biochemical factors in the cell culture medium
It was shown 3 4 that the use of retinoic acid or retinol (one of the forms of Vitamin-D) is
essential to create tracheobronchial cell monolayers without clumps. However, retinoic acid
was not used in the ATCC recommended medium (MEM/F12/FBS). Further a number of other
growth factors such as Insulin, Transferrin, EGF, Dexamethasone, Cholera Toxin and Bovine
Hypothalamus extract (BHE) were not explicitly added to the MEM in the ATCC medium
although some cocktail of growth factors in the form of FBS were added to it. It was shown 35
that these growth factors are essential to the growth of airway epithelial cells and that these
factors have a significant effect of mucus production.
So we grew cells in a petri dish culture again. Significant differences were noted between cell
growth in the ATCC recommended medium and the optimized medium which includes all the
above factors in addition to minimum essential medium (MEM).
34
Control on ATCC medium
In the optimized medium
-
-ATCC
recommended medium
MEM + Fetal Bovine Serum
(FBS).
> Growth factors in FBS are
not fully characterized
> Resulted in cyst like clumps
of cells
1 day
after
seeding
Optimized medium
MEM/F12/EGF/Vitamin
AlTransferrin/Insulin/Cholera
Toxin/Dexamethasone/BHE
2 days
after
seeding
Scalebar 200p1m
Fig 25: The effect of biochemical growth factors in the cell culture medium on Calu-3 cell
monolayer formation
Microfluidic device culture
Having investigated the effect that the substrate and cell culture medium properties have on
the growth of a cell monolayer, we tried to grow a monolayer again in a microfluidic device. We
tried parametric experiments in the microfluidic device with collagen gels 2.5mg/ml and
6mg/ml formed at pH 7.4 and pH 11 with various coating solutions - matrigel, vitronectin and
fibronectin.
Monolayer formation in a microfluidic device (with ATCC medium)
When we used the ATCC recommended medium with any of these gels, we were never able to
get a monolayer. In the figure shown, the monolayer seen is the growth of cells on the glass
coverslip. However, on the gel (the square posts hold the gel in place), the cells form clumped
structures.
35
200pm scale bar
Fig 26: Clump formation in Calu-3 cell cultures in a microfluidic device with ATCC
recommended medium
Monolayer formation in a microfluidic device (with optimized medium)
When the optimized medium was used, containing all the growth factors listed above, we
found that on stiffer substrates such as Collagen gel 2.5mg/ml formed at pH 11 and coated with
vitronectin, the cell monolayer formed and no clumps were found. As seen in the figure below,
in which different Z sections of the cells are focused on, an almost continuous monolayer is
present. The cells have been stained with Texas Red conjugated Phalloidin after being fixed
using 4% paraformaldehyde. Zi is the bottom-most section which shows the cell monolayer
growing on the glass coverslip. Every other section shows a continuity of cells from the glass
coverslip on to cells growing on the collagen gel. The black space at the bottom is where the gel
is present and the concentration of red is fluorescence from cells in many different planes being
captured.
36
Z5 (just at the PDMS top)
Z3
Z6(just above the top surface of PDMS)
Fig 27: Formation of a Calu-3 cell monolayer in a microfluidic device with the optimized
medium containing the added growth factors. The actin was stained with RhodaminePhalloidin
37
Chapter 5
Summary and Future work
Summary
We have developed a microfluidic system in which we can grow a monolayer of airway
epithelial cells on a physiologically relevant substrate. Specialization does not correlate with
robustness. The Calu-3 airway epithelial cell line optimized to grow on hard surfaces in
DMEM/F12/FBS medium does not form a monolayer at all on a soft substrate with the same
medium. The use of the optimized medium containing many growth factors essential for the
formation of a monolayer on a soft substrate solves this problem. One of the future directions
to form a specialized cell line that could grow on soft substrates is the use of an evolutionary
methodology. Cells could be grown on a soft substrate and a subpopulation that grows well on
soft substrates could be sub-cultured to create a new cell line for growth on soft substrates. In
general, it was found that cells grow to confluence faster as the stiffness of the substrate is
increased by many independent methods (gel height change, gel concentration change and PH
of gel formation which changes the gel microstructure). In the future, we plan to also use PEGDA gels/PA gels/other hydrogels much stiffer than collagen gels in our microfluidic device as the
stiffness of these substrates can be tuned much more controllably.
Future work
It was found that Calu-3 cells do not grow well on a stiffness gradient as shown in the figure.
This is an interesting phenomenon that could be investigated in the future with more controlled
PEG-DA enabled stiffness gradient experiments.
GEL
CELLS
CELLS
CELLS
GEL
On top o psasic
Fig 28: Calu-3 cells do not grow well on a stiffness gradient
38
We also need to test the effect of shear stress, stretch on relevant parameters such as mucus,
lectin expression and mucociliary flow. Once characterized well, we would test the effect of
viruses on the system.
One of the future applications of such as system is the accelerated evolution of microbes in a
chip in which we gradient a gradient of fitness. It has been observed in the viral culture
community that getting sufficient viral diversity in in vitro cultures is a challenge. One possible
solution could be the use of the following viral evolution platform. The gradient of fitness would
accelerate the evolution of microbes and could lead to potential studies of drug resistance and
predicting future vaccines.
Problem:
In vitro viral propagation does not lead to sufficient diversity
Hypothesis: Absence of a gradient of fitness
fe
Figl A
F
I j~
0
00
0 0mutin
outnumbered
e
'0.0
i
I
0 00i
Mutantis
-
I
i
Fig1B
Random
%
[ ]]
No selective
g
Wild type
With selective
pressure
+
Mutant
pressure
0090-
Zhang et al (2011)
Acceleration of Emergence
of Bacterial Antibiotic
Resistance in Connected
Microenvironments,
Science, 1764-1767
Experiment: Viral propagation with a gradient of anti-viral agent
Beating cilia propel mucus towards the right
I
Gradient of Oseltamivir
ycell
with cilia
*
Influenza virion
Mucus layer
Fig 2 A(side view)
Periciliary fluid
layer
Oseltmivir(n
Mucus flow
Semi-permeable
membrane
Fig 2 8 (to pview)
Fig 29: Schematic of a viral evolution platform using fitness gradients
In a microfluidic system such as the one we described earlier, studying viral transport would be
possible due to the great imaging capabilities. Our microfluidic system is designed such that the
entire cell culture occurs within 180.im of the glass cover slip. Hence the study of the ability of
viruses to invade the airway cells and cross the barrier in our platform would be interesting and
might lead to insights in to the mechanisms of viral invasion. Other untested hypotheses such
as bacterial-viral co-infection and their role in respiratory diseases might also be investigated
here.
39
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