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CELL CULTURE AT THE LIQUID-LIQUID INTERFACE:
EFFECTS OF FLUOROCARBON LAYER THICKNESS ON
CELL GROWTH

presented by
Victoria A. Sanocki

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CELL CULTURE AT THE LIQUID-LIQUID INTERFACE: EFFECTS OF
FLUOROCARBON LAYER THICKNESS ON CELL GROWTH

By

Victoria A. Sanocki

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Chemical Engineering

2004

ABSTRACT

CELL CULTURE AT THE LIQUID-LIQUID INTERFACE: EFFECTS OF
FLUOROCARBON LAYER THICKNESS ON CELL GROWTH

By

Victoria A. Sanocki

This study assessed the effect of fluorocarbon layer thickness on the adsorption of
proteins at the liquid-liquid interface, and on subsequent attachment, growth and
differentiation of 3T3 fibroblasts cultured at that interface. Cells were grown at the
interface between culture medium and fluorinert liquid FC-7O layers of 5 mm, 1 mm, and
0.5 mm. The interfaces were treated with either fibronectin (FN) or human serum
albumin (HSA). Growth and proliferation improved as FC-7O layer thickness decreased,
with the best growth on the 0.5 mm layer. Cell growth on the FN-treated interfaces was
better than on the HSA-treated ones. The occurrence and size of cell-free gaps (or
“lakes”) decreased with thickness, with the smallest cell-free areas occurring on the 0.5
mm FN-treated fluorocarbon layer. The adsorption of HSA and FN was also studied at
the liquid-liquid interface, using total internal reflection fluorescence microscopy and
fluorescence photobleaching recovery (TIRFM/FPR) on oil layers ranging in thickness
from 4 pm to 50 pm. The fraction of irreversibly adsorbed proteins, go, did not vary with
thickness for either FN or HSA; however, FN had higher g0 values than HSA in all cases.
Taken together, the cell culture and TIRFM/FPR data suggest a sigmoidal behavior for
the rate of cell growth as a function of fluorocarbon layer thickness, with cell density

approaching some maximum value as the fluorocarbon layer becomes infinitely thin.

DEDICATION

To my parents, for all of their love and support.

iii

ACKNOWLEDGMENTS

I wish to thank my major advisor, Dr. R. Y. Ofoli, for his guidance and support
throughout my studies. I would also like to thank Drs. C. Chan and R. M. Worden for

serving on my advisory committee, and for their suggestions.

I would especially like to thank Dr. Chan for generously donating cells and the use of her
laboratory facilities for this project. Special thanks go to Srivatsan Kidambi for his
invaluable help with cell culture and for answering all my questions about it. I would
also like to thank Sachin Vaidya and Lavanya Parthasarathy for lending me their expertise
on total internal reflection fluorescence microscopy and for being there to commiserate

and offer advice when things did not go as expected.

I would like to thank my fiance, Kevin Brown, for his love and for being there to

encourage me and keep me going when I was feeling down. I would also like to thank

my sisters, Ellen and Abigail Sanocki, for being there.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES .......................................................................................................... viii
NOMENCLATURE ............................................................................................................ x
1. INTRODUCTION ....................................................................................................... 1
1.1 Objective ............................................................................................................. 5

2. LITERATURE REVIEW ............................................................................................ 6
2.1 Cell Culture at the Liquid-Liquid Interface ......................................................... 6
2.2 Total Internal Reflection Fluorescence ............................................................. lO

3. THEORETICAL CONSIDERATIONS .................................................................... 17
3.1 Total Internal Reflection ................................................................................... 17
3.2 Fluorescence Photobleaching Recovery ............................................................ 18

4. EXPERIMENTAL MATERIALS AND TECHNIQUES ......................................... 22
4.1 Materials ............................................................................................................ 22
4.2 Experimental Methods ...................................................................................... 23
4.2.1 Cell Culture ............................................................................................... 23
4.2.1.1 Preparation of liquid-liquid interface for cell culture ............................ 23
4.2.1.2 Growth Curves ...................................................................................... 23

4.2.2 TIRFM/FPR .............................................................................................. 24
4.2.2.1 TIRFM Apparatus ................................................................................ 24
4.2.2.2 Procedure for Labeling Proteins ............................................................ 27

4.2.2.3 Beam Alignment and Microscope Focusing Procedure ........................ 28
4.2.2.4 Photobleaching ...................................................................................... 29

4.2.2.5 Data Analysis ........................................................................................ 29

5. RESULTS AND DISCUSSION ................................................................................ 30
5.1 Cell Culture ....................................................................................................... 30
5.2 TIRFM/FPR ...................................................................................................... 43

6. SUMMARY AND CONCLUSIONS ........................................................................ 51
7. SUGGESTIONS FOR FUTURE WORK ................................................................. 55

8. APPENDICES ........................................................................................................... 57

8.1 Appendix A: Physical Properties of FC-70 ....................................................... 57
Properties ................................................................................................................... 57

8.2 Appendix B: Sigma Plot Output ....................................................................... 58

9. BIBLIOGRAPHY ..................................................................................................... 60
9.1 General References ............................................................................................ 64

vi

LIST OF TABLES

Table 1: TIRFM/PPR results for HSA. Data are presented i the standard error from

regression. ................................................................................................................. 47
Table 2: TIRFM/PPR results for FN. Data are presented i the standard error from
regression. ................................................................................................................. 47
Table 3: Physical properties of FC-70. Values are determined at 25 °C. ........................ 57

vii

LIST OF FIGURES

Figure 1.1: Schematics of cell culture at the liquid-liquid interface. We hypothesize that,
as the fluorocarbon layer gets thinner, the liquid-liquid interface would become more
stable on the solid substrate and promote better protein adhesion and subsequent cell
attachment, growth and proliferation. ......................................................................... 4

Figure 3.1: Total internal reflection at an interface, induced by laser beam entering the
interface at an angle exceeding the critical angle. The resulting evanescent wave
decays exponentially with distance into the optically rarer medium ......................... 18

Figure 4.1: The experimental set up for TIRFM. The double flat system (F) splits the
beam into an intense photobleaching beam (pb) and a much weaker monitoring
beam (m), and then recombines them to ensure that both beams are incident at the

same spot on the interface. Mirrors (M) enable focusing of the beam into the sample
can”. ......................................................................................................................... 25

Figure 4.2: The experimental flow cell. Spin coating controls the thickness of the oil
layer on the top slide. The entire oil-water assembly is approximately 800 pm thick.
................................................................................................................................... 27

Figure 5.1: Growth curves for FN-treated FC-70-culture media interfaces. 'Untreated'
refers to a 5 mm FC-7O layer on which FN was not adsorbed prior to the cell culture
experiment. Lines do not represent models, but have been added to aid in
visualizing trends. All cell count data were normalized against the initial seeding
density. ...................................................................................................................... 32

Figure 5.2: Cell behavior on 0.5 mm (A) and 1 mm (B) FN-treated FC-7O layers, after 3
days of incubation. There are only a few small cell-free gaps on the 0.5 mm layer
(A). However, there is evidence of more protein layer rupture and aggregate
formation (dark spot) on the 1 mm layer (B). The two images are of identical
magnification ............................................................................................................. 33

Figure 5.3: Growth curves for HSA—culture media interfaces. 'Untreated' refers to a 5
mm thick FC-7O layer on which HSA was not adsorbed prior to the cell culture
experiment. Lines have been added to aid in visualizing trends. All cell count data
were normalized against the initial seeding density. ................................................. 35

viii

Figure 5.4: Cell behavior on 0.5 mm (A) and 1 mm (B) HSA-treated FC-70 layers, after
3 days of incubation. Cell growth in both cases is sparse. There are more and larger
aggregates (dark spots) on the 1 mm layer (B). ......................................................... 36

Figure 5.5: Cell count as a function of fluorocarbon layer thickness for FN and HSA on
Days 2 (top), 3 (middle), and 4 (bottom). Lines have been added to aid in
visualizing trends. All cell count data were normalized against the initial seeding
density. ...................................................................................................................... 37

Figure 5.6: Cell growth on 0.5 mm FC-70-culture media interfaces after 3 days of
incubation for FN (A) and HSA (B). Cell growth on the FN-treated interface is
clearly much more robust than on the HSA-treated interface. .................................. 38

Figure 5.7: Cells before (A) and after (B) 20 minutes of trypsin-EDTA treatment on 1
mm of FN-treated FC-70-medium interface. The cells have not rounded up after
being treated with trypsin-EDTA, so they cannot be completely removed by trypsin-
EDTA treatment. ....................................................................................................... 40

Figure 5.8: Cells before (A) and after (B) 20 minutes of trypsin-EDTA treatment on 1
mm of HSA-treated FC-70-medium interface. Most of the cells have rounded up
and are ready to be detached after trypsin-EDTA treatment. .................................... 41

Figure 5.9: Cells before (A) and after (B) 20 minutes of trypsin-EDTA treatment on
polystyrene culture plate. All of the cells have rounded up and are ready to be
removed from the plastic surface (B). ....................................................................... 42

Figure 5.10: Sample fluorescence recovery curve for HSA adsorbed at an interface
featuring a 6 pm oil layer. The fluorescence units are arbitrary (A.U.). The slow
recovery of fluorescence after photobleaching is an indication that a large fraction of
protein is bound irreversibly to the interface ............................................................. 44

Figure 5.11: Sample g(t) data and regression fit for HSA on 6 pm oil layer. g(t) is
normalized from the fluorescence recovery curve (Figure 5.10). The single
exponential model fits the data well, even at later times. ......................................... 45

Figure 5.12: Most likely cell growth curve as a function of fluorocarbon layer thickness
for FN on Day 3. The dashed line represents the most plausible cell growth profile,
based on the combination of results from TIRFM/FPR and cell culture experiments.
................................................................................................................................... 50

ix

NOMENCLATURE

6 angle of incidence of laser beam

BC critical angle

10 wavelength of incident laser beam

A(r, z, t) bulk concentration of molecules

[A] equilibrium bulk concentration of molecules

B(r, I) surface concentration of free binding sites

[B] equilibrium surface concentration of free binding sites
C(r, t) surface concentration of bound molecules

[ C I equilibrium surface concentration of bound molecules

C(r, t) surface concentration of unbleached fluorescent molecules

(1 penetration depth of the evanescent wave

0;, bulk diffusion coefficient

EDTA Ethylenediaminetetraacetic acid

F equilibrium pre-bleach fluorescence emission intensity

F ( 0) fluorescence emission intensity immediately after photobleaching
F ( I) measured fluorescence emission intensity as a function of time
FITC fluorescein-5-isothiocyanate (fluorescent dye)

FN fibronectin

FPR fluorescence photobleaching recovery

g0 fraction of molecules that are irreversibly bound at the interface

g.-
HSA

10

fir)

1(z)

k1

k2“)

PMT

TIRFM

2

fraction of molecules belonging to binding class 1'
human serum albumin

laser intensity at the interface (2:0)

dimensionless profile function

intensity of evanescent wave at any depth 2
surface adsorption rate
surface desorption rate of molecule in class i
refractive index of media j
photomultiplier tube
system constant
radial position vector
time
total internal reflection fluorescence

depth coordinate into rarer media

xi

1. INTRODUCTION

The purpose of cell culture is to produce, nurture and maintain viable cells outside of
their original multicellular host organism. The behavior of the cells and how they are
influenced by certain conditions can then be observed without interference from other
systems or functions in the animal they were taken from. To keep it viable, the
environment around the cultured cell must mimic the cell’s original surroundings. Cells
must be kept clean and fed in a setting that will promote growth, and potentially harmful
byproducts of cellular activity must be removed from the environment. Culture media
must be carefully formulated to provide the nutrients required for cell viability. For
example, oxygen must diffuse through the culture media to reach cells. Too much culture
medium may retard diffusion, and result in oxygen deprivation and eventually cell death.
To this end, researchers have been perfecting culture media and techniques over the past

century.

In 1907, Ross Harrison grew frog neural tissue in a sealed container filled with frog
lymph'. His experiments fostered more interest in maintaining cells and organs outside
of animals for research purposes. Due to the desire to examine systems that better
approximated humans, mammalian cells became the primary focus for many tissue
culturists. Virology and cancer research further improved tissue culture by standardizing
techniques and media, along with establishing many continuous cell lines for future

research.

The standardization of procedures and media, along with the increased availability of
cells for culture opened tissue culture up to many other applications and areas of research
beyond cell biology. Cultured cells provide a source of DNA sequences and a means of
determining the effects of transfected genes for gene therapy. Tissue culture decreases
the need for animal experimentation in drug, cosmetic, and pollutant toxicity tests.
Large-scale cell cultures, whether genetically altered or not, can lead to products such as
insulin for the treatment of diseases. Tissue grafts and scaffolds seeded with cultured
cells can be used to replace damaged tissue. Therefore, tissue culture is not just

academic; it leads to products that can enhance the quality of life.

Tissue culture of adherent or anchorage—dependent cells has been traditionally carried out
on solids, primarily glass or plastic. These solid substrates provide the support adherent
cells need to grow and proliferate. The most commonly used plastic is polystyrene,
which must be treated with y irradiation, chemically, or with gas-plasma discharge to
support cell growth]. These treatments do not always produce uniform surfaces and the
quality varies from manufacturer to manufacturer. In addition, some organic solvents
erode polystyrene. Plastic or glass surfaces can also be treated with proteins such as
fibrinogen or flbronectin to enhance cell growth. To be removed from the interface, cells
must be treated with digestive enzymes like trypsin-ethylenediaminetetraacetic acid
(EDTA) or be scraped off. These treatments can damage the cell. For example, scraping
may cause cells to burst if too much force is applied. Alternatives to cell culture on a

solid substrate have been used to avoid some of these problems.

An interesting but little used alternative for growing anchorage-dependent cells is to use a
liquid-liquid interface, a technique Rosenberg2 introduced in 1964. The lower phase is
dense immiscible oil, typically a fluorocarbon, while the upper phase is a culture medium.
The resulting interface provides support for cell growth, with cells growing on a layer of
adsorbed interfacial proteins. However, since cells do not adhere to a liquid-liquid
interface as strongly as they would to a solid-liquid interface, pipetting or centrifugation

can be used to remove cells from the surface, resulting in little or no damage“.

Fluorocarbons are usually the preferred hydrophobic substrate for cell culture at the
liquid-liquid interface, because their properties make them ideal for that purpose. The
medical field already employs them as blood substitutes and for short-term organ
preservations. They are chemically and biologically inert and are thermally stable. With
a high solubility to respiratory gases, fluorocarbons can easily provide oxygen to cellss.
Therefore, the supply of oxygen is not just limited to diffusion through the culture

medium. Fluorocarbons can also be re-sterilized and reused.

Despite the advantages of fluorocarbons, there are some drawbacks to cell culture at the
liquid-liquid interface. Purified fluorocarbons are often poor substrates for cell growth,
because trace impurities generally improve protein adsorption at the interface and
improve protein film quality, both of which are essential for ensuring the growth of
anchorage-dependent cellsé. However, some of these impurities may be toxic at high
concentration. Much more care is required when handling cultures. For example,

sloshing the oil may cause cells to detach and form aggregates. If the protein layer is

ruptured, large lake-like Openings will form in the cell sheet7. The forces cells exert on
the protein layer as they grow and proliferate may induce protein desorption from the

interface or cause the protein layer to rupture, which leads to clump formation.

In this study, we hypothesized that decreasing the thickness of the fluorocarbon layer may
provide a solution to the problem of protein layer rupture. When the thickness of the
fluorocarbon layer is decreased, the liquid-liquid interface is brought closer to the
underlying solid substrate (Figure 1.1), promoting stability of the liquid-liquid interface
and anchoring it more securely to the solid substrate. This increased stability should lead
to better anchoring of proteins upon adsorption at the interface, promote better cell
growth and proliferation, and reduce the possibility of protein layer rupture and formation
of lake-like openings in the cell sheet. The result is that cell growth characteristics will be
similar to those on a solid surface, while still maintaining many of the benefits of cell

culture at a liquid-liquid interface.

 

 

 

 

 

 

 

Media
Media
Fhorocarbon Fhorocarbon
/ / / / / / / / / /v/ / / / / / / / /
Solid Interface Solid

 

 

 

Figure 1.1: Schematics of cell culture at the liquid-liquid interface. We hypothesize that, as the
fluorocarbon layer gets thinner, the liquid-liquid interface would become more stable on the solid
substrate and promote better protein adhesion and subsequent cell attachment, growth and

proliferation.

1.1 Objective

The objective of this study was to assess the effect of fluorocarbon layer thickness on the
adsorption of proteins at the liquid-liquid interface, and on the attachment, growth and
differentiation of cells cultured at the interface. The study focused on the following
specific activities:

0 Use of total internal reflection fluorescence microscopy and fluorescence
photobleaching recovery (TIRFM/FPR) to study the irreversibility of protein
adsorption at the oil-water interface as a function of oil layer thickness; and

0 Use of growth curves to assess the effects of fluorocarbon layer thickness on cell

attachment, growth, and proliferation at the interface.

Cells were cultured at fluorocarbon layers of varying thickness treated with either
fibronectin or human serum albumin. Fibronectin is important for cellular adhesion,
migration, and proliferationg. Human serum albumin, on the other hand, transports other
substances in the blood, such as bile acids, drugs, toxins, and excess hormones and
vitaminsg. Human serum albumin is also known to decrease the adhesion of cells on

solid substratalo" '.

The adsorption of these proteins at the liquid-liquid interface was
studied directly with total internal reflection fluorescence microscopy, along with

fluorescence photobleaching recovery, to determine the effects of oil layer thickness.

2. LITERATURE REVIEW
2.1 Cell Culture at the Liquid-Liquid Interface

Rosenberg2 introduced the technique of culturing cells at a liquid-liquid interface. He
wanted to clarify the mechanisms that underlie cell-surface interactions, and believed that
a) a liquid-liquid interface would be ideally suited to such studies, b) a liquid substrate
would avoid the molecular inhomogeneities of a solid substrate, and c) proteins and
phosphatide monolayers could be adsorbed to the interface to simulate half of a cell
membrane. Rosenberg found that, for the most part, cells grew well on the liquid
substrates he had chosen; however, their behavior was quite different on different oils.
On FC-43 and FC—75, cells were not in contact with each other, and their growth was less
sustained. On the other hand, cells were epithelial with uniform spreading on DC 710,
‘Kel-F’-10 and ‘Kel-F’-4O oils. He hypothesized that the differences were the results of
variations in oil composition and impurities at the interface. He also studied the effects
of different surfactants on cell growth. For example, cell spreading was reduced upon the

injection of lecithin at the interface.

Cells grow on the layer of proteins adsorbed at the liquid-liquid interface. If proteins are
not purposely pre-adsorbed, they are adsorbed from the culture medium. Due to the
greater molecular homogeneity of liquids, the specific interactions of cells with a
particular protein may be examined more directly than on a solid surface”. Ando et al.‘2
pretreated fluorocarbon-culture medium interfaces with extracellular matrix proteins

(ECM) to determine their effects on cell attachment and growth. The density of human

endothelial cells was very low on untreated and bovine serum albumin (BSA)-treated
interfaces, while it was significantly higher on interfaces treated with collagen type IV
(COL), laminin (LN), fibronectin (FN), and fibrinogen (FG). Improving the strength of
the adsorbed protein layer improves cell growth. Crosslinking the adsorbed protein layer
with gluteraldehyde or inducing the formation of a double protein layer with polylysine
lead to enhanced cell growth with reductions in the formation of aggregates and openings

in the cell sheet7.

Different cell types behave differently at the liquid-liquid interface. Bovine endothelial
cells grew on untreated interfaces, albeit with lower attachment and proliferation rates
than for treated surfaces. Human endothelial cells,'however, required treatment with
ECM in order to show any growth”. 3T3-L1 cells attached and spread to form a nearly
confluent monolayer on the FC-7O interface, while SV-T2 cells formed aggregates7.
Different cell types may exert different forces on the interface, causing the protein layer to

12.13

break up and form aggregates . An alternative explanation may be that cells exert the
same force but the surface area of their attachment plaques to the substrate varies. The

force is delocalized for cells with larger plaques, leading to less damage and strain”.

Greater attractive forces between cells may also lead to the formation of aggregates”.

Surface-active compounds or surfactants, often present as trace impurities in
fluorocarbons, facilitate cell growth at the liquid-liquid interface. Shiba et al.3 found little
difference in cell growth and morphology among the fluorocarbons they studied, despite

differences in their physical properties, leading them to conclude that cell growth may not

depend on the physical properties of the liquid-liquid interface. Two of the fluorocarbons
studied by Keese and Giaever6, FC-72 and L-2345, exhibited very different cellular
behavior even though they had very similar compositions. They attributed this difference
to trace impurities in the compounds. Several studies have found that a fluorocarbon’s
ability to support cell growth is lost or decreased upon alumina column purification6’12’l4.
Treatment of FC-43 with fibrinogen after purification showed decreased cell attachment
and proliferation compared to unpurified, fibrinogen treated FC-43'2. While protein does
adsorb at a purified interface, the interactions between the protein molecules are weaker
and succumb more readily to forces exerted by cells6. Purified fluorocarbons regain their
ability to support cell growth upon addition of surfactants. For example, addition of
pentafluorobenzoyl chloride (F5BzCl) to purified fluorocarbons restored the ability to
support cell growth up to a concentration of 2 ug/mlé. Greater concentrations resulted in

a loss of cell growth and spreading as a result of toxicity. Impurities containing hydrogen

also restored cell growth”.

While growth is mainly dependent on surface-active impurities, the underlying liquid
substrata have an effect on both growth and morphology. Sparrow et al.'4 found
differences in growth between different alumina treated fluorocarbons. The high stability
of C-F bonds makes saturated perfluorocarbons practically inert while heteroatoms such
as nitrogen and oxygen increase surface reactivity, leading to better cell growth even with

alumina purification.

Cells do not adhere as strongly to a liquid substrate as to a solid substrate. The low
degree of adhesiveness enhances the attractiveness of the liquid-liquid interface to certain
studies, since strong adhesion is thought to interfere with rapid cell division. Shiba et 31.3
noticed an accelerated growth rate for L-929 cells on a liquid substrate as opposed to a
solid one, which they attributed to weaker cell adhesion to the liquid-liquid interface.
Kwon et al.15 reported enhanced gene transduction on FC-40 interfaces treated with poly-
L-lysine (PLL), gelatin (ON), and fixed gelatin (fGN) as opposed to polystyrene surfaces.
They attributed this improved transduction to the flexibility of the FC-40 interface, also
noting that the transduction efficiency was less for the fGN-treated FC—40, which
exhibited the lowest transduction efficiency of the three protein-precoated interfaces

before contact inhibition took place.

The low degree of adhesiveness lends itself to the study of membrane proteins produced
by adherent cells. Cells can be removed without the use of trypsin-EDTA or scraping,
both of which tend to damage the cell and alter the membrane proteins recovered. Terada
et al.16 found that there was a difference in the amount of protein recovered between cells
grown on plastic removed with either scraping or trypsin-EDTA and cells grown at a
liquid-liquid interface and removed by pipetting. Trypsin-EDTA treatment had the most
diminished protein recovery while scraping only differed a little from pipetting cells from
the liquid interface. Sobel and Rosenbergl7 reported similar findings. They noted that
there was no evidence of cell damage when cells were pipetted from a liquid-liquid
interface, as opposed to the harsher treatments of scraping or trypsinization used in

studies of cell membrane proteins on a solid substrate.

Rappaport et al. reported multilayer growth for HeI.a18 and Hep G24 cells at the liquid-
liquid interface. They found that multilayer growth depended on providing higher and
more uniform oxygen concentrations to cells, a substratum with a lesser degree of
adhesiveness, and a nutrient rich culture medium“. Perfluorocarbons have a high degree
of 02 solubility as well as being more flexible with a lower degree of adhesiveness. They
determined that, in this environment, cells were able to produce albumin in greater

quantities per cell than those growing on a solid substrate.

2.2 Total Internal Reflection Fluorescence

Total internal reflection fluorescence (TIRF) is a non-invasive technique used to study
molecular interactions at interfaces. In TIRF, an evanescent wave is generated when a
light beam, traveling in a medium of high refractive index, is totally internally reflected at
the interface between the medium and one of a lower refractive index. The evanescent
wave can be configured for shallow penetration (_<_100 nm) into the medium of lower
refractive index, which then selectively excites significantly more fluorophores at the
interface than in the bulk. The fluorescence emission intensity curves provide
information on the degree of molecular binding at the interface. For example, the
concentration of a species can be determined as a function of distance from the interface,

allowing the adsorption and desorption kinetic rates to be determined").

TIRF has been used for a wide variety of studies, foremost among which are those that

target biological phenomena. For example, TIRF has been used for visualization of cell-

10

substrate contact regions and the long term observation of cells, which is made possible
because the short penetration depth of the evanescent wave limits damage to
photosensitive cell organelles”. TIRF may be used to measure kinetics of binding of
proteins at interfaces, or to characterize receptor-ligand interactions. For example, in a
recent study, Conboy et al.21 examined the interaction of HIV-1 surface glycoprotein gp
120 (rgp 120) with lactosylceramide (LacCer), glucosylceramide (GlcCer), and
galactocylceramide (GalCer) in planar supported lipid bilayers. Binding to glycoproteins
in the cell membrane is one of the first steps for HIV entry into a cell. They found that, at
rgp 120 concentrations between 0.2 and 15 nM, rgp 120 has a high affinity for GalCer

only.

The effect of surface prOperties on protein adsorption and desorption in the presence of
other proteins are often studied with TIRF as well. Hlady and Ho22 studied the effect of
human serum albumin (HSA) on the adsorption of low density lipoprotein (LDL) in
relation to the hydrophobicity of the adsorbent surface. They reported that the kinetics of
LDL adsorption and LDL apparent binding affinity decrease with increasing
hydrophobicity. They found that the kinetics of LDL adsorption on hydrophilic silica was
not affected by the co-adsorption of HSA, and pre-adsorption of HSA slowed the
adsorption of LDL on silica with an even more significant decrease on the more
hydrophobic end of the sample. TIRF spectroscopy has also been used for the detection

of individual molecules at an interface20‘23.

ll

The total internal reflection geometry can be combined with a variety of other
spectroscopic techniques. When combined with fluorescence photobleaching recovery
(FPR), the kinetics of desorption of molecules from an interface can be deterrrrined24'25.
Similarly, TIR can be combined with fluorescence correlation spectroscopy (FCS), which
monitors the random fluctuations in the number of fluorophores in a very small
observation volume, to determine the kinetic rate and bulk diffusion near the surface“.
TIR and resonance light scattering (RLS) have also been used to characterize interfacial
species without the use of fluorescent dyes, while limiting interference from other
molecules that is commonly observed in bulk solutions”. TIR-fluorescence lifetime
measurements provide information on the molecular state and structure of fluorophores at

an interface”.

Various studies using total internal reflection have been performed at the liquid-liquid
interface. Gajraj and Ofoli27 developed a total internal reflection fluorescence
microscopy (TIRFM) apparatus to examine the liquid-liquid interface, using a geometry
in which the entire oil-water layer was about 1.0 mm thick. They studied the adsorption
of bovine serum albumin (BSA) and lysozyme at the interface, to determine if the thin
oil-water interface was thermodynamically true. They were able to accurately estimate
the critical micelle concentration of BSA, demonstrating that the thin oil-water layer
accurately mimics a standard interface. The adsorption dynamics of a protein to a liquid-
liquid interface can be affected by conditions at the interface, as well as conditions of the
molecule itself. Using the same apparatus, Gajraj and Ofoli28 examined the effects of

fluorescent labels on the adsorption kinetics and diffusion of BSA at the oil-water

12

interface using TIR/FPR. They reported that the apparent diffusion coefficient of BSA
labeled with two FITC (BSA-ZFIT C) molecules was about 40% greater than that of BSA
labeled with only one FIT C molecule. BSA-ZFITC also had a slower rate of desorption
from the interface. These differences were attributed to increased hydrophobicity with
increased labeling ratio.

Jones and Bohnzg’30

used TIRF in conjunction with electrocapillary measurements to
monitor the potential dependent adsorption of dye di—Nbutylaminonaphthylethenyl-
pyridiniumpropylsulfonate (dye I), a zwitterionic amphiphile, at bare and dilauroyl-
phosphatidylcholine (DLPC)-modified 1,2-dichloroethane (DCE) -water interfaces. The
fluorescence behavior of dye I changed in the presence of DLPC. Without DLPC, the
probe fluorescence decreased with increasingly positive potentials over 0.25 V but, with
it, fluorescence emission increased with increasingly positive potentials over 0.29 V.
This behavior was attributed to the formation of dye I aggregates, which do not fluoresce,
on the bare interface. On the other hand, the surface excess of dye I was reduced and
aggregates could not be readily formed on the DLPC modified interface. This was

because, as more dye I replaced DLPC, the affinity for the interface drops with

increasingly positive potential.

Hashimoto et al.23 developed a method to detect single molecules adsorbed at the
dodecane-water interface. The lateral diffusion coefficient of single cyanine dye
molecules (DiI) was estimated from the maximum duration of photon bundles detected in

the observation area. The effects of two surfactants, sodium dodecyl sulfate (SDS) and

13

dimyristoyl phosphatidylcholine (DMPC), on the lateral diffusion dynamics of DH were
also investigated. The lateral diffusivity of DH was only slightly affected by the presence

of SDS, while DMPC drastically reduced it.

The physical properties of the liquid-liquid interface can also be examined with TIRF.
Ishizaka et al.31 studied the polarity of several oil-water interfaces with sulforhodamine B
(SRB), a polarity sensitive probe. The roughness/thickness of the oil-water interface was
determined by fluorescence dynamic anisotropy and excitation energy transfer dynamics
and related to the interfacial polarity determined by TIRF spectroscopy. For thin, sharp
oil-water interfaces, the interfacial polarity was well predicted by the arithmetic average
of the polarities of the water and oil phases. Cyclohexane-water, CCl4-water, and
toluene-water interfaces were considered thin and sharp. On the other hand, when the oil-
water interface was determined to be rough, the interfacial polarity deviated from the
average as in the case of dichlorobenzene-water and DCE-water interfaces. Oils with
higher polarity tended to form rough interfaces. Hashimoto et al.23 estimated the
interfacial viscosity, 1],, from the lateral diffusion dynamics determined by single
molecule fluorescence at the dodecane-water interface. The interfacial viscosity was as
high as that for dodecane (1.4 mPa s) and higher than the value for water (0.89 mPa s). In
the presence of SDS, the interfacial viscosity was slightly higher than that of dodecane.

In the presence of DMPC it was two orders of magnitude greater.

Yamashita et al.32 used time-resolved TIRF spectroscopy to study the solvation dynamics

of 12-(9-anthroyloxy) steric acid (12-AS) and 4-(9-anthroyloxy) butanoic acid (4—ABA) at

14

the heptane-water interface. These probes are sensitive to changes in their solvated
environments due to strong charge—transfer in the excited state. 12-AS is soluble in
heptane while 4—ABA is water-soluble. From observation of the time-dependent
fluorescence spectral shifts, it was determined that 4-ABA was surrounded by more water
molecules and was located closer to the water phase than 12-AS. The solvent relaxation

of 12-AS occurred more slowly at the interface than in the bulk solution.

Feng et al. used TIRF at the CCl4—water interface to determine the amounts of
chlortetracycline (CTC)33 and berberine”, and TIR/KLS to determine the amount of
HSA35 present in a sample. CTC is an antibiotic drug often used in medicine and food
science. Berberine is used to cure digestive system ailments. In the case of CT C, a
fluorescent complex was formed between CT C, europium (Ill), and trioctylphosphine
(TOPO) at the CCl4-water interface. TOPO was dissolved in the CCl4 phase while CT C
and Eu(Il]) were dissolved in the water phase. The fluorescence of berberine was
enhanced when it was adsorbed at the CCl4-water interface in the presence of sodium
dodecyl benzene sulfonate (SDBS). A complex that was formed between HSA, thorium
(IV), and SDBS resulted in enhanced TIR/RLS signals at the CCl4-water interface. To
determine the sensitivity and selectivity of the TIRF or TIR/RLS signal, model samples
were made by adding other substances such as metal ions, amino acids, and
carbohydrates. y-globulin was added to model solutions for HSA as it interferes with
most common HSA assays. Body fluid samples were also used to detect CTC and HSA.
The TIRF and TIR/RLS detection methods were accurate in all three cases and there was

limited interference from the other substances. There was no interference from y-globulin

15

in the case of HSA. The TIR assay methods were simpler and less time consuming than

many of the assays currently in use for the quantification of these compounds in a sample.

16

3. THEORETICAL CONSIDERATIONS
3.1 Total Internal Reflection

The theory of total internal reflection is well developed elsewhere”. Briefly, a light beam
traveling from an optically dense medium, with refractive index m, to an optically rarer
medium, with refractive index n2, will be totally internally reflected when the angle of

incidence, measured from the normal to the interface, exceeds a critical angle, 6,, where

6C = sin”l (fl). (3.1)
n]

Although the beam is totally internally reflected at the interface, it produces an
electromagnetic field called the evanescent wave, which penetrates into the rarer medium
(Figure 3.1). The intensity of the evanescent wave, I(z), decays exponentially with

distance, 2, from the interface according to the relationship
1(z) = 1(0) exp[—§) (3.2)

where [(0) is the intensity of the incident light at the interface and d, the penetration

depth, is given by

 

d = 10 . (3.3)
471'sz sin2 (Si-n:

10 is the wavelength of incident light at the interface. The penetration depth, d, is

independent of the polarization of the light beam. It is on the order of 10 or less and

decreases with increasing (9, except as 67—96,, in which case d -900.

17

 

Laser Beam

 

Evanescent Wave

 

 

 

Figure 3.1: Total internal reflection at an interface, induced by laser beam entering the interface at
an angle exceeding the critical angle. The resulting evanescent wave decays exponentially with

distance into the optically rarer medium.

3.2 Fluorescence Photobleaching Recovery

The theory of total internal reflection/fluorescence photobleaching recovery (TIR/FPR) is

24,25

well treated elsewhere . In its essence, the binding of molecules to the interface may

be represented by the chemical equation:

k
___L._\
A(r,z,t)+B(r,t)TC(r,t) (3.4)

where Ah: 2. t) is the bulk concentration of molecules, B(r,!) is the surface concentration
of free binding sites, and C(r, t) is the surface concentration of bound molecules. The
constants k, and k2 are the surface adsorption and desorption rate constants respectively.

The equilibrium constant, K, is then

18

[C] __/_<._

= [AHB] _ k2 ’ (35)

where [A], [B], and [ C ] are the equilibrium concentrations.

Assuming that molecules within the evanescent wave are bound to the surface, the

measured fluorescence, F ( t), is then
F(t) = Q10 [1m C(r,t)d2r, (3.6)
where Q is a product of the efficiencies of excitation light absorption and fluorescence

emission and detection, 10 is the maximum intensity and I( r) is a dimensionless profile

function with a maximum amplitude of unity.

For FPR

G(t) = 1-5—F(t) (3.7)
where F(t) is the fluorescence emission after photobleaching at time t = 0 and IT" is the
equilibrium pre-bleach fluorescence emission intensity. The surface concentration of

unbleached fluorescent molecules, 6’ (r, t), is

C(r,t) = [C]— C(r,t) (3.8)
Combining Equations (3.6), (3.7) and (3.8), we obtain
G(t) = Q1O jz(r)C(r,t)d2r (3.9)

To solve for C(t), the differential flux equations for A(r, z, t) and C(r, t) are solved by

linear transformation theory, with initial conditions derived from the intensity profile of

19

the monitoring beam and boundary conditions based on the fact that only surface bound

molecules within a finite surface area are bleached.

After integration and application of the boundary conditions, G(t) can be simplified based
on the conditions of the system. The simplest form of G(t), and the one for which the
theory is best defined, is that of the “reaction limited” regime, where bulk diffusion is
rapid relative to the adsorption and desorption of molecules form the interface. In this

C886

G(t) z G(0)exp(-k2t). (3.10)

To determine if the system is in the reaction limited regime, the characteristic time for

bulk diffusion, (1/ RBND) , is calculated

1 :(lclllAl) (3.“)
RBND DA

 

where DA is the bulk diffusion coefficient. If the characteristic time of recovery in a

TIR/FPR experiment is much larger than l/RBND , then the experiment is reaction limited.

Once it is determined that the experiment is reaction limited, the fluorescence recovery
curve, F(t), is normalized to give g(t):

_ F(t)— F(O)

_ 3.12
F-F(0) ( )

g(t)=1

where F is the equilibrium pre-bleach fluorescence intensity, F(O) is the intensity

immediately following photobleaching at time t = 0, and F(t) is the subsequent

20

fluorescence recovery. The variable g(t) is G(t)/G(0) as defined by Equation (3.7), and

can be written as the sum of exponentials:

g(t) = g0 +2 g, exp(—k,(i)t) (3.13)

i=1
where go is the fraction of molecules which are irreversibly bound to the interface, the g,-
are different binding classes of molecules (for example, rapidly versus slowly desorbing),
and the k2( i) are the desorption rates of those classes. The parameters are determined by

curve fitting.

2]

4. EXPERIMENTAL MATERIALS AND TECHNIQUES
4.1 Materials

Fluorinert liquid FC-70 was purchased from 3M (St. Paul, MN). FC-70 was sonicated for
30 minutes to improve stability at the interface4 and sterilized by exposure to UV-light for
up to 24 hours and/or by passage through a 0.22-um filter (Millipore Corporation,
Bedford, MA). Human plasma fibronectin (FN) (FC010, Chemicon International,
Temecula, CA) and human serum albumin (HSA) (A8763, Sigma Chemical Co., St.
Louis, M0) were dissolved in PBS buffer solutions (pH 7.4) to give the concentrations
required for each experiment. Both protein solutions were sterilized by passage through a

0.22-um filter. Fluorescein-S-isothiocyanate (FITC) was purchased from Molecular

Probes (F1907, Eugene, OR).

NIH/3T3 (ATCC) fibroblasts were generously provided by Dr. C. Chan, Michigan State
University. Culturing was done in a humid 37°C, 5% C02 incubator (Class [1 Water
Jacketed C02 Incubator, Therrno Forrna, Marlotta, OH) in a pH 7.4 culture medium
consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO, Carlsbad, CA)
supplemented with 10% fetal bovine serum (GIBCO, Carlsbad, CA). Industrial grade

CO; was purchased from AGA Gases (Lansing, M1) at a purity of 99%.

22

4.2 Experimental Methods
4.2.1 Cell Culture
4.2.1.1 Preparation of liquid-liquid interface for cell culture

The liquid-liquid interface was prepared following the method of Ando et al.12. In a
sterile biological safety cabinet (Class H A/B3 Biological Safety Cabinet, Thermo Forrna,
Marlotta, OH) FC-70 was added to the wells of a 48—well tissue culture plate (Corning
Incorporated, Corning, NY) to give oil layer thicknesses of 5 mm, 1 mm, and 0.5 mm.
After this, 0.7 ml of either 4 jig/ml FN solution or 1 mg/ml HSA solution was carefully
pipetted on top of the oil. The plate was then incubated at 37°C for approximately three
hours. After incubation, the protein solution was removed and the interfaces gently
washed three times with culture medium. After that, 0.5 ml of cell suspension was

pipetted on top of the interface.

4.2. I .2 Growth Curves

Growth curve experiments were run over a period of up to four days for each of the oil
layer thicknesses and at least one of two controls: an untreated 5 mm thick fluorocarbon
layer, and plastic. Cells were removed from the interface with trypsin-EDTA (GIBCO,
Carlsbad, CA) and scraping and counted using a hemocytometer. Duplicates of each

sample were counted.

23

4.2.2 TIRFM/F PR
4.2.2.1 TIRFM Apparatus

The experimental setup (Figure 4.1) is described in detail elsewhere”. The system uses
an inverted microscope (Zeiss Axiovert 135 M, Carl Zeiss, Inc., Thomwood, NY), which
houses a motorized stage and flow cell. The 488 nm line of a 5W continuous wave argon
ion laser (Lexel Lasers Model 95, Fremont, CA) was used in all experiments. Intensity
measurements were made with a side-on photomultiplier tube (PMT) (Hamamatsu
R4632, Bridgewater, NJ) jacketed in a therrnoelectrically cooled housing (TE177TSRF,
Products for Research, Danvers, MA). A modular automation controller with an RS232
interface (MAC 2000, Ludl Electronic Products, Hawthorne, NY) provides the high
voltage required for PMT operation. A double-syringe pump system (Model 551382,
Harvard Apparatus, South Natick, MA) simultaneously infuses and withdraws samples
from the flow cell, to prevent damage to the interface. The entire apparatus, with the
exception of the automation controller, is mounted on a vibration isolation table (RS

4000, Newport Corporation, Irvine, CA) to ensure interfacial stability.

The laser beam is attenuated using neutral density filters (O3FNQ, Melles Griot, Boulder,
CO). An optical chopper (SR540, Stanford Research Systems, Sunnyvale, CA) prevents
unintended photobleaching when long experiments are being conducted. A system of two
optical flats and a programmable shutter (D122, Uniblitz, Vincent Associates, Rochester,
NY) are used to split the laser beam into a photobleaching and a much weaker monitoring

beam such that the ratio of the two intensities is approximately 10,0002]. The first flat

24

splits the beam while the second flat recombines the two so that they are accurately
superimposed at the interface. Between the two flats, the shutter blocks only the
photobleaching beam. When activated, the shutter allows for photobleaching with a sub-
millisecond laser flash. After passing through the flats, the laser beam is conveyed by
mirrors and finally focused onto the interface using a plano-convex lens (f = 200 nm,

Oriel Corporation, Stratford, CT).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

    

 

 

 

 

 

 

controller ‘ computer 1
a
s
e
r
P
M
l NDF --
NDF .,
M1 V I C
filter F2 Sh F1
inverted microscope

 

 

 

Figure 4.1: The experimental set up for TIRF M. The double flat system (F) splits the beam into an
intense photobleaching beam (pb) and a much weaker monitoring beam (m), and then recombines
them to ensure that both beams are incident at the same spot on the interface. Mirrors (M) enable

focusing of the beam into the sample cell”.

25

A Zeiss LD achroplan microscope objective (440651, 32x, 0.4, Carl Zeiss Inc.,
Thomwood, NY) is focused at the interface and captures the fluorescence emission from
the sample. An interference filter (500EFLP-EM, Omega Optical, Brattleboro, VT)
inside the microscope allows the fluorescence emission wavelengths to be separated from
the excitation wavelength. The PMT is operated in digital (photon counting) mode. A
fast preamplifier (SR 445, Stanford Research Systems, Sunnyvale, CA) amplifies the
output signal from the PMT, which is then fed to a photon counter (SR 400, Stanford
Research Systems, Sunnyvale, CA) for measurements. Fluorescence intensities are
recorded with programs written in Labview 6.0 (National Instruments, Austin, TX). An
output reference voltage from the chopper triggers the photon counter so that data

collection only occurs when the laser beam illuminates the sample cell.

The flow cell consists of two microscope slides separated by a 800 um thin aluminum
spacer with an oval cut into it to form the sample cell (Figure 4.2). The underside of the
top slide is coated with Cargille immersion oil (n = 1.46, Fisher Scientific, Pittsburgh,
PA). Oil layer thickness, ranging from 4 pm to 50 um, was controlled by spin coating
microscope slides with oil at different speeds. Higher speeds produced thinner oil layers.
Two holes are cut into the bottom slide to allow for infusion and withdrawal of protein
solutions by the double-syringe pump. The top slide is optically coupled to a 70° dovetail
prism (n = 1.46) and the entire assembly is secured onto an anodized aluminum shell that
sits on top of the microscope stage. The entire oil-water assembly for TIRFM

experiments was approximately 800 pm thick.

26

 

Aluminum Spacer
1 Prism

 

 

 

Inlet/Outlet

 

 

ii ii

T To Syringe Pump 1' Oil-Water Interface

 

 

“i”

O-ring

 

 

 

 

 

 

Figure 4.2: The experimental flow cell. Spin coating controls the thickness of the oil layer on the top
slide. The entire oil-water assembly is approximately 800 pm thick.

4.2.2.2 Procedure for Labeling Proteins

Proteins were labeled following the procedure provided in the Molecular Probes product
information for amine reactive probes“. Approximately 10 mg of protein was dissolved
in 5 ml of sodium bicarbonate buffer (0.1 M, pH 9.0). A labeling ratio of 1.3:1 was used
to determine the appropriate concentration of FITC, dissolved in sodium bicarbonate
buffer. The FITC solution was slowly added drop wise (about 50 pl) to the continuously
stirred protein solution. The reaction was incubated at room temperature for 4 to 6 hours
with continuous stirring. The stop reaction was performed using 0.1 ml of a 1.5 M
hydroxylamine solution (pH 8.5) and incubated for an hour. Hydroxylamine removes dye
6

from unstable conjugates with tyrosine, serine, threonine, and histidine3 . A dialysis

process, performed over a period of 48 hours, was used to remove the unreacted dye. The

27

final protein concentration and labeling ratio were determined by measuring absorbance
at 280 nm and 496 nm with a diode array spectrophotometer (8452 A, Hewlett Packard,

Brielle, NJ).

4.2.2.3 Beam Alignment and Microscope Focusing Procedure

For the TIRFM system to be properly aligned, normal incidence of the laser beam on the
prism must be achieved while the point of total internal reflection (TIR) is exactly above
the microscope objective. First, the flow cell was filled with PBS buffer using the syringe
pump. Normal incidence was accomplished by adjusting the mirrors until the back
reflection from the prism is aligned along the original path of the laser beam. This
indicated that normal incidence had been achieved. The position of TIR in the flow cell
was then checked. If it was not over the microscope objective, the mirrors were adjusted

in the opposite direction and the procedure for normal incidence was repeated.

To focus the microscope, the piano-convex lens was put into place and alignment was
first done by eye to place the point of TIR over the objective. The interference filter was
removed from the microscope. Then, looking through the PMT cavity on the microscope,
the lens was adjusted to position the evanescent wave over the microscope objective. The
microscope was then focused until the evanescent wave filled the objective. The shutter
was then used to block the photobleaching beam, and the PMT was put into place on the
microsc0pe. Small adjustments were made to the microscope to maximize the PMT
signal. The interference filter was then replaced in the microscope and the protein

solution was injected into the flow cell.

28

4.2.2.4 Photobleaching

The flow cell was filled with protein solution for an hour, during which time readings
were taken every 2 to 5 minutes until pseudo-equilibrium adsorption has been achieved.
At this point, the chopper was switched off, the shutter was triggered, and the sample was
subjected to a brief flash of the photobleaching beam for approximately 100 ms. During
the photobleaching flash, a shutter in the microscope, which is triggered along with the
other shutter, protects the PMT. After the photobleaching pulse, the fluorescence

recovery was recorded.

4.2.2.5 Data Analysis

The data were analyzed with a user defined regression equation in Sigma Plot 2001
(SPSS Inc., Chicago, IL). An iterative method was used to determine the values of the
coefficients in Equation (3.13). The regression program was provided with initial guesses
of the coefficients; the resulting numbers were then input into the program as new initial
guesses until the percent variance was minimized. An example of the output is given in

Appendix B.

29

5. RESULTS AND DISCUSSION

5.1 Cell Culture

The attachment and growth of 3T3 fibroblasts at the interface between culture medium
and FC-70 layers of 5 mm, 1 mm, and 0.5 mm was studied, with 0.5 mm being the
minimum thickness attainable in our system. When a smaller layer of FC-70 was placed
in the well, it completely covered the bottom of the well but, upon addition of culture
medium, it would pool to the side of the well. Two other fluorocarbons, HEB—7100 and
HFE-7500, were also investigated. These are primarily composed of hydrofluoroethers,
and both exhibited the same tendencies observed with FC-70. The polystyrene culture
plates used in this study were treated to make them hydrophilic and negatively charged, in
order to promote cell attachment. The interaction between the hydrophobic fluorocarbons
and the hydrophilic polystyrene plate is likely one of high energy. It is possible that, by
pushing the medium off the interface so that the medium can directly interact with the

plate is a mechanism to lower the total free energy of the system.

Results show that cell growth was influenced by the thickness of the FC-70 layer, most
likely the result of better initial cell adhesion to the adsorbed protein layer. Figure 5.1
shows the growth curves (cell count versus time) for interfaces on which fibronectin (FN)
had been adsorbed, and for one interface that was not pretreated with the protein. All cell
counts data were normalized against the initial seeding density. Clearly, both growth and
proliferation improved as the fluorocarbon layers became thinner. As was discussed in
Chapter 1, this is most likely because thinner fluorocarbon layers promote interfacial

stability of the adsorbed protein layer, which reduces protein layer rupture and subsequent

30

formation of lake-like openings in the protein film. The stronger protein film enables

better cell attachment, growth and proliferation.

The cell morphology is shown in Figure 5.2 for the 0.5 mm and 1 mm layer thicknesses.
The cell sheets were nearly confluent in both cases, with slightly bigger openings in the 1
mm case. The cells were more densely packed on the 0.5 mm thick fluorocarbon layer,
even though there are still some areas in the sheet with only a sparse covering of cells.
The cells on the 1 mm thick layer have densely packed areas, but there are larger cell-free
gaps. The cells appear to be forming an aggregate (see the dark spot in Figure 5.2 B),
which is probably the result of breakage of the protein monolayer as the cells grow and
proliferate. Phase contrast images could not be taken of the 5 mm thick layer on the
available microscope because it could not be precisely focused on the interface. Cell
growth on the 5 mm interface was observed on another microscope in the lab, to ensure

that the growth was as expected.

Cell growth and proliferation studies were also conducted with human serum albumin
(HSA) adsorbed at the liquid-liquid interface, to provide a comparison for the data
obtained for FN. Results were similar to those for FN with regards to the effect of
fluorocarbon layer thickness. Figure 5.3 presents the growth curves for HSA. The cell
morphology on 1 mm and 0.5 mm HSA-treated FC-70 layers is shown in Figure 5.4.
There are large cell-free gaps on the HSA-treated FC-70-media interfaces, and aggregates
appear to be forming on the 1 mm thick fluorocarbon layer. In comparison to FN, cells

did not grow as well on adsorbed HSA, and the cell sheets were not as confluent.

31

 

 

 

 

 

 

 

 

 

 

 

 

'0.
V
"U
B
8
A b—v
1::
D
l
r
I
Ewe.
('1
V5
9
EM“
o—c
Ema.
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‘9. g
0 V
l E
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O
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O O O O O O O O O O O
O O O O O O O O O O O
O 0\ w h \0 V3 V m N F‘ O
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1111100 “90 DOZITBUUON

 

 

 

Figure 5.1: Growth curves for FN-treated FC-70-culture media interfaces. 'Untreated' refers to a 5
mm FC-70 layer on which FN was not adsorbed prior to the cell culture experiment. Lines do not
represent models, but have been added to aid in visualizing trends. All cell count data were
normalized against the initial seeding density.

32

 

Figure 5.2: Cell behavior on 0.5 mm (A) and 1 mm (B) FN-treated FC-70 layers, after 3 days of
incubation. There are only a few small cell-free gaps on the 0.5 mm layer (A). However, there is
evidence of more protein layer rupture and aggregate formation (dark spot) on the 1 mm layer (B).

The two images are of identical magnification.

33

Figure 5.5 shows the cell counts as a function of FC-70 layer thickness for adsorbed HSA
and FN, for 2, 3, and 4 days. For the entire culture period, growth was better on the 0.5
mm and 1 mm FN-treated interfaces than those treated with HSA. In the 5 mm case, the
only major difference in cell numbers was on the final day of culture, where the} FN-
treated interface had more cells. Cell layers were more densely packed on FN-treated
interfaces than on those treated with HSA (Figure 5.6). In comparison to FN, there are
large areas on the HSA-treated interface where cells have not grown at all. In light of
data recently acquired in our laboratory”, it would be expected that FN-treated interfaces
would promote greater initial cell attachment and subsequent growth than those treated
with HSA, since FN adsorbs more strongly at the interface than HSA. Therefore, the

trend observed in this study is as expected.

As a control, experiments were also conducted on an untreated surface with a 5 mm layer
of FC-70. As can be seen in Figures 5.1 and 5.3, there was not much difference between
protein-treated and untreated 5 mm layers with respect to the density of cells growing on
them. The probable reason is that, at this FC-70 layer thickness, the interactions between
the adsorbed proteins and the underlying substrate are not as strong as they are in the
cases where the interface is much closer to the underlying solid. Therefore, the cells can
tear the protein monolayer apart as they proliferate and move around on the surface,

leading to reduced cell growth.

34

 

 

4.5

 

3
8
5 - .
\' l
l I". i
l '1': E _ 2
l 3: ;
u a =
" E - n
’5 ...

l 3‘} S _ W. .-.
l 'l\ V} N 5‘
l 2: ° 7;

E. l -.. a

 

 

 

/
15

 

 

 

— v!
o
\ i
Q
r r r r ‘ 1 ' O
8 O o O o O 8
O. O O. O O
\O V) V n N H o
IIIIIOO [[90 DOMINION

 

 

 

Figure 5.3: Growth curves for HSA-culture media interfaces. 'Untreated' refers to a 5 mm thick FC-
70 layer on which HSA was not adsorbed prior to the cell culture experiment. Lines have been added

to aid in visualizing trends. All cell count data were normalized against the initial seeding density.

35

 

Figure 5.4: Cell behavior on 0.5 mm (A) and 1 mm (B) HSA-treated FC-70 layers, after 3 days of
incubation. Cell growth in both cases is sparse. There are more and larger aggregates (dark spots)
on the 1 mm layer (B).

36

 

 

 

 

6

554

8

=4“

0

U

'03-

3

.a2_

E
1..

Z +FN-I-HSA
0 I l l l l

 

0 1 2 3 4 5 6
Thickness (mm)

Day 3

 

1 1

l

1

1

Normalized cell count
0 '-‘ N w A LII ON \I W

i

 

 

 

 

Day 4

 

Normalized cell count
A
l-l-l
I

 

 

 

Thickness (mm)

Figure 5.5: Cell count as a function of fluorocarbon layer thickness for FN and HSA on Days 2 (top),
3 (middle), and 4 (bottom). Lines have been added to aid in visualizing trends. All cell count data
were normalized against the initial seeding density.

37

 

bation for FN

(A) and HSA (B). Cell growth on the FN-treated interface is clearly much more robust than on the

HSA-treated interface.

men

a interfaces after 3 days of '

Cell growth on 0.5 mm F C-70-culture

Figure 5.6

38

It is reasonable to expect that cells could be more easily removed from the liquid-liquid
interface than from the solid-liquid interface. As a result, it was originally hypothesized
that trypsin-EDTA treatment would be enough to ensure that all the cells were removed
from the liquid-liquid interfaces. However, it turned out that trypsin-EDTA treatment
alone was not enough to remove cells, especially in the case of FN. Figures 5.7, 5.8, and
5.9 show images before and after trypsin-EDTA treatment for FN, HSA, and bare plastic,
respectively. Cells are generally ready for removal when they have detached from the
interface and “rounded up’“. In the cases of plastic and HSA, the cells had rounded up
and were ready to be pipetted off. For FN, however, there were still quite a few cells that
had not rounded up even after 20 minutes of treatment with trypsin-EDTA. A possible
explanation for this is that serum proteins, such as fibrinogen in the culture medium,
interact with FN adsorbed at the liquid-liquid interface, thus strengthening cellular

8

adhesion . It is likely that FN is interacting with the cells as well as the interface; this

may be over-riding the normal route of detaching cells with trypsin-EDTA.

39

 

Figure 5.7: Cells before (A) and after (B) 20 minutes of trypsin-EDTA treatment on 1 mm of FN-
treated FC-70-medium interface. The cells have not rounded up after being treated with trypfin-
EDTA, so they cannot be completely removed by trypsin-EDTA treatment.

40

 

Figure 5.8: Cells before (A) and after (B) 20 minutes of trypsin-EDTA treatment on 1 mm of HSA-
treated FC-70-medium interface. Most of the cells have rounded up and are ready to be detached
after trypsin-EDTA treatment.

41

 

Figure 5.9: Cells before (A) and after (B) 20 minutes of trypsin-EDTA treatment on polystyrene
culture plate. All of the cells have rounded up and are ready to be removed from the plastic surface
(B).

42

5.2 TIRFM/FPR

The adsorption of protein was studied at the liquid-liquid interface with TIRFM/FPR at
oil-water interfaces featuring oil layers ranging in thickness from 4 pm to 50 pm. A
typical fluorescence recovery curve for HSA is shown in Figure 5.10. After equilibrium
adsorption, the interface was exposed a brief flash of the bleaching beam. The
subsequent fluorescence emission intensity is approximately 60% of its pre-bleach level.
The recovery is slow, indicating a slow exchange of interface-bound bleached proteins
with unbleached protein from the bulk. This implies that a large fraction of protein is

irreversibly bound to the interface.

The gaps in the curve result from the fact that, to prevent photobleaching over the long
duration of the experiment, the monitoring beam was periodically blocked for a few
seconds as the data were acquired. The fluorescence recovery data from Figure 5.10 were
normalized with Equation (3.12) and then fitted to Equation (3.13), using Sigma Plot.
Data were fitted with i = 1 (see Eqn. (3.13)). The purpose of the second exponential term
in the equation was to help model the kinetics of recovery of the slowly desorbing class of
molecules. However, the timescale over which the recovery data were collected
(approximately 95 seconds) was most likely too short to see the dynamics of the slowly
desorbing class of molecules. As a result, the double exponential form of the equation (i
= 2) was not used to fit the experimental data. However, since the irreversibly adsorbed,

rapidly desorbing and slowly desorbing fractions must add up to unity, the slowly

43

desorbing fraction can be determined from the single exponential fit, even though the

kinetic rate of that fraction is not captured by this procedure.

Sample g(t) data are shown in Figure 5.11, along with a plot of the model obtained from
the Sigma Plot of g(t) from the HSA recovery data in Figure 5.10. Normalizing the data
with Equation (3.12) causes the signal at time t = 0 to become unity, and the increase in
fluorescence is manifested as a decrease in g(t). Despite the omission of the second

exponential term, the equation fits the data well, especially at later times.

 

 

 

 

 

 

 

 

 

 

30000
A. 25000 t
:1
3 20000
0.)
a 15000 —W
8
§ 10000
O
:1 e
E: 5000
0 ’. r . . .
-20 0 20 40 60 80 100
time (s)

 

 

 

Figure 5.10: Sample fluorescence recovery curve for HSA adsorbed at an interface featuring a 6 pm
oil layer. The fluorescence units are arbitrary (A.U.). The slow recovery of fluorescence after

photobleaching is an indication that a large fraction of protein is bound irreversibly to the interface.

 

 

 

 

 

 

 

 

 

1?
‘53 0.6
0.4 a
0 g(t) data
0.2 - — g(t) fit
0.0 r 1 r r
0 20 4O 6O 80 100

time (s)

Figure 5.11: Sample g(t) data and regression fit for HSA on 6 pm oil layer. g(t) is normalized from
the fluorescence recovery curve (Figure 5.10). The single exponential model fits the data well, even at
later times.

The results of the TIRFM/FPR experiments with HSA at a concentration of 0.2 mg/ml are
shown in Table 1. It was determined that this concentration was in the reaction limited
regime, by experiment and by comparison with published reaction limited data of 0.1
mg/ml for BSA, a structurally and functionally similar protein”. Experimental
confirmation was obtained by comparing the results of a 0.7 mg/ml experiment to those
of a 0.2 mg/ml experiment at the same oil layer thickness, and showing that they are
statistically similar. This indicates that 0.2 mg/ml is within the reaction limit since an
increase in protein concentration did not result in a change in the values determined for

the constants in Equation (3.13).

45

In these experiments, oil layer thickness did not appear to influence the irreversibly bound
fraction, go, as would be expected. This would seem to indicate that there is a limit at
which the thickness of the oil layer no longer plays a role in strengthening the interaction
between the solid substrate and proteins bound at the liquid-liquid interface. That is, the
stability conferred to the liquid-liquid interface by its proximity to the solid substrate has
reached its maximum, and there is no further improvement in the interactions between the
bound proteins and the substrate. Due to the viscosity of the oil, there is a limit to the
thickness of the oil layer that can be attained in our system. In fact, 50 um was the

maximum thickness that could be reproducibly spin-coated on the microscope glass.

The oil layer thickness did not significantly affect g1, the fraction of proteins that
constitutes the rapidly desorbing class, or k2(1), the desorption rate of the rapidly
desorbing class of proteins (Table 1). There appears to be a difference in the k2(1) for the
50 um oil layer. However, we believe this difference is more likely the result of
photobleaching by the monitoring beam as the experiment progressed. The exponential
parameters are greatly affected by data at longer adsorption timescales; therefore,

photobleaching by the monitoring beam will have an impact on them.

The results of TIRFM/FPR experiments with fibronectin (FN) at a concentration of 0.05
mg/ml are shown in Table 2. As with HSA, g0 did not depend on oil layer thickness.
However, go is higher for FN than for HSA. This is to be expected, since FN has been
shown to be more aggressive at the oil-water interface than HSA”. Vaidya and Ofoli37

showed that FN has the ability to displace HSA at the liquid-liquid interface, even when

46

HSA has been pre-adsorbed at the interface for as long as six hours; on the other hand,

HSA is unable to displace much FN from the interface.

Table 1: TIRFM/FPR results for HSA. Data are presented :1: the standard error from regression.

 

 

 

 

 

 

Thickness (um) g0 g1 _ k2(1)
6 0.83 i 0.001 0.10 i 0.003 0.13 i 0.009
12 0.84:0.001 0.11 $0.006 0.14i0.014
38 0.84 i- 0.001 0.09 i 0.002 0.12 :1: 0.006
50 0.85 i 0.002 0.09 i- 0.012 0.31 :1: 0.064

 

 

 

 

There is more variation in g, and k2(1) with thickness in the case of FN than was seen
with HSA, with both g, and k2(1) increasing with increasing oil thickness. To
understand the reasons for this, we should note that g, and k2(1) represent the fraction and
desorption rate, respectively, of proteins in the rapidly desorbing class. Burghardt and
Axelrod25 discussed the possibility that the binding of proteins in this class may result
from the formation of a monolayer of protein on a rough interface. It is possible that the
interface may become rougher as the thickness of the oil layer increases, which could
account for the increase in g, and k2(1). It is also possible that the concentration of FN in
these experiments was on the edge of the reaction limited regime, and that the variations

in g) and k2(I) are a result of this.

Table 2: TIRFM/FPR results for FN. Data are presented :1: the standard error from regression.

 

 

 

 

 

Thickness (pm) g0 g I k2(1)
4 0.92 i 0.000] 0.06 i 0.0004 0.03 i 0.0004
16 0.93 i 0.0002 0.04 i 0.0005 0.05 i 0.0011
33 0.90 i 0.0002 0.08 i 0.0010 0.08 i 0.0016

 

 

 

 

47

 

 

The results of the TIRFM/FPR experiments suggest that there is a point beyond which
further decreases in the thickness of the oil layer do not affect protein adsorption at the
liquid-liquid interface. On the other hand, the cell culture results show that cell growth
and proliferation strongly depend on the fluorocarbon layer thickness. If improved cell
growth is a function of better protein attachment, then the sigmoidal behavior presented
in Figure 5.12 would be suggested by the TIRFM/FPR data, which show that there is no

further improvement in protein layer stability at very thin oil layers.

Taken together, the two sets of data would strongly suggest the dashed line in Figure 5.12
as the most likely profile for cell growth with respect to fluorocarbon layer thickness. If,
as the TIRFM/FPR data suggest, there is a point where making thinner FC-70 layers does
not improve protein attachment at the liquid-liquid interface, and if cell growth at the
liquid-liquid interface is affected by the strength of attachment of the underlying protein
layer, then cell growth must indeed level off to some maximum asymptotic value at very

thin fluorocarbon layers, as depicted in Figure 5.12.

Normal cell growth should not exceed the maximum number of cells that could be
accommodated by the surface area of the vessel, because contact inhibition causes cells to
cease proliferating once they are in contact with one another. This is true at the liquid-
liquid interface as well. If the plastic surface is considered to have the maximum cell
density available for the surface area of the culture vessel, then as the fluorocarbon layer
gets thinner, cell growth should approach this maximum number of cells. As the

thickness of the fluorocarbon layer approaches zero, cell growth should level off towards

48

the maximum allowed by the surface area of the culture plate, since 3T3 cells are contact-

inhibited”.

49

 

3
Thickness (mm)

 

 

 

— N
r- H
//

'l/ l l l l l I T o
O O O O O O O O O O
'O. O O O O O O O O O
Ox 00 I“ \O V) V M N H O

mnoo 119:) P92119W0N

Figure 5.12: Most likely cell growth curve as a function of fluorocarbon layer thickness for FN on
Day 3. The dashed line represents the most plausible cell growth profile, based on the combination of
results from TIRFM/FPR and cell culture experiments.

50

6. SUMMARY AND CONCLUSIONS

Cell culture at a liquid-liquid interface provides an alternative to cell culture on a solid
substrate, and eliminates some of the problems commonly associated with a solid
substrate. It is generally believed that a liquid-liquid interface is more uniform than a
solid substrate”. Cells can be removed from a liquid-liquid interface without the use of
damaging treatments such as application of trypsin-EDTA or scraping. There are,
however, some drawbacks to cell culture at the liquid-liquid interface. Chief among them
is the breakage of the layer of protein that adsorbs at the interface and supports cell
growth. When this happens, it causes cells to form aggregates and large cell-free open
areas commonly referred to as “lakes.” In this work, it was hypothesized that decreasing
the thickness of the underlying fluorocarbon layer would provide a solution to this
problem, because it would impart more interfacial stability and improve the anchorage of
proteins at the interface. This should decrease the incidence of protein layer rupture and

lead to improved cell growth and proliferation.

The objective of this work, therefore, was to study the effect of fluorocarbon layer
thickness on the adsorption of human serum albumin (HSA) and fibronectin (FN) at the
liquid-liquid interface, and assess the influence of fluorocarbon layer thickness on the
attachment and growth of cells cultured at the interface. Cell attachment and growth at
the interface between culture medium and FC-70 layers of 5 mm, 1 mm, and 0.5 mm
were measured, with either FN or HSA preadsorbed at the interface. The adsorption and
reversibility of HSA and FN was examined directly by total internal reflection

fluorescence microscopy (TIRFM) on oil layers ranging in thickness from 4 pm to 50 pm.

51

Cell attachment and growth were clearly influenced by the thickness of the FC—70 layer.
Growth and proliferation improved as the fluorocarbon layers became thinner, with the
best growth on the 0.5 mm thick layer. Cells were also more densely packed on the 0.5
mm layer than on the 1 mm and 5 mm ones. There were larger cell free gaps on the 1 mm
and 5 mm thick layers and more aggregates formed, indicating that the layers of adsorbed
protein had ruptured more on the thicker fluorocarbon layers than on the thinnest one.
This is most likely because the thinner fluorocarbon layers promote interfacial stability of
the adsorbed protein layer, facilitating better cell attachment, growth and proliferation,

and diminishing protein layer breakage, as was hypothesized in this study.

The type of protein adsorbed at the liquid-liquid interface also influenced the growth of
cells. While cell growth improved as the thickness of the fluorocarbon layer decreased
for both proteins, cells grew much better on FN-treated interfaces than on HSA-treated
ones. In all cases, cell numbers were typically higher for FN-treated interfaces over the
culture period. FN-treated interfaces had larger areas of densely packed cells on both the
0.5 mm and 1 mm thick layers. More aggregates formed on HSA-treated interfaces and

there were larger areas with no cell growth.

An interesting problem was encountered when cells were removed from the interface.
Based on the assumption that cells do not adhere as strongly to the liquid-liquid interface,
it was hypothesized that trypsin-EDTA treatment would lead to total removal of cells

from the interface. However, trypsin-EDTA alone was not enough to remove all of the

52

cells. In some cases, no cells were removed at all following treatment with trypsin-
EDTA, especially from FN-treated interfaces. Normally, cells should be rounded up and
ready to be detached after about 20 minutes of treatment by trypsin-EDTA. Results in
this study showed that, after treatment, cells on HSA-treated FC-70 and on bare plastic
had rounded up and were apparently ready to be removed. However, on FN-treated
interfaces, the cells were still spread out and were clearly not ready for removal.

Therefore, it was necessary to scrape the interfaces to detach the cells.

Protein adsorption at the liquid—liquid interface was studied by TIRFM/FPR, with oil
layers ranging from 4 pm to 50 pm thick. In these experiments, oil layer thickness did
not significantly influence go, the fraction of protein irreversibly bound at the interface,
which was taken to be an indicator of the strength of the adsorbed protein layer, for both
HSA and FN. In all cases, g0 was greater for FN than for HSA. For HSA, the rapidly
desorbing fraction of protein, g1, and its desorption rate, k2(1), did not vary with thickness

either. On the other hand, both g. and k2(l) for FN did vary with oil layer thickness.

The TIRFM/FPR results for both proteins show that there is no difference in the fraction
of proteins irreversibly adsorbed at the interface for oil layers ranging from 4 to 50 pm.
This would suggest that, within 50 pm of the interface, oil layer thickness has no effects
on protein adsorption at the liquid-liquid interface. Therefore, cell attachment and
proliferation could not be further improved, except for the responses of the cells to
different proteins adsorbed at the interface. On the other hand, the cell culture results

show that, down to a thickness of 0.5 mm, cell growth and proliferation strongly depend

53

on the thickness of the fluorocarbon layer for a given protein. If, as hypothesized,
improved cell growth is a function of the strength or quality of the adsorbed protein layer,
then these data together would suggest a sigmoidal behavior for cell growth at the liquid-
liquid interface, with cell growth leveling off asymptotically to some maximum value as

the thickness of the fluorocarbon layer decreases.

54

7. SUGGESTIONS FOR FUTURE WORK

Among the studies total internal reflection fluorescence has been used for is
visualization of cell-substrate contact regions”. The degree of cell adhesion to a
substrate can be studied and the strength of cellular adhesion can be inferred
from the number of focal contacts“). The current TIRFM apparatus can be
adapted to study cells cultured at the liquid-liquid interface. Such studies would

provide further confirmation of the hypothesis.

To further verify our results, it would be useful to examine cell growth on even
thinner fluorocarbon layers. This may be achieved by looking into the use of
other fluorocarbons, which may not show the same effects as FC-70, HFE-7100,
and FIFE-7500. Alternatively, the cell culture plates may be treated with
something, like photoresist, to make them more hydrophobic. There are
drawbacks to treating the culture plates with other chemicals, as they may be
toxic to the cells and are not necessarily sterile. Another option would be to try

bacteriological culture plates, which are treated to by hydrophobic.

Different cell types behave differently when cultured at the liquid-liquid
interface. Repeating these experiments with a different cell line, such as HeLa

cells, may provide further verification of the thickness results.

Atomic force microscopy (AFM) is a valuable tool for studying protein

configuration and attachment and cell attachment on surfaces. AFM is being

55

adapted to the study of liquid-liquid interfaces4'43. In the future, AFM may be
useful for studying protein adsorption and cells cultured at the liquid-liquid
interface, providing information on the conformation of protein adsorbed at the

interface as well as the strength of cellular attachment at the interface.

56

8. APPENDICES
8.1 Appendix A: Physical Properties of FC-70

FC-70 is a clear, colorless liquid comprised of a mixture of fully-fluorinated compounds

that are blended to achieve specific physical properties.

Table 3: Physical properties of F C-70. Values are determined at 25 °C.

 

 

 

 

 

 

 

Properties

Average Molecular Weight 820

Boiling Point (1 atm) 215 °C
Liquid Density 1940 kg/m3
Absolute Viscosity 24 cp
Surface Tension 18 dynes/cm

 

 

57

 

8.2 Appendix B: Sigma Plot Output

A sample of the output from Sigma Plot for the determination of the parameters in
Equation (3.13) from fluorescence recovery data is given below. Initial guesses for the
coefficients were provided to the regression program, and then the resulting solutions
were input back into the program as new initial guesses until the percent variance was

minimized.

[Variables]

t=col(l)

y=col(2)

[Parameters]

g0 = 0.75 ' {{previous: 0.829841 }}
g1 = 0.25 ' {{ previous: 0.0883611 }}
kl = 12' {{previous: 0.0968088”
[Equation]

f=g0+gl *exp(-kl *0

fit f to y

[Constraints]

kl>0

g1>0

g0>0

[Options]

tolerance=0.000100

stepsize=100

iterations=100

R = 0.5081 qur = 0.2582 Adj qur = 0.2549

58

Standard Error of Estimate = 0.0419

Coefficient
g0 0.8298
g1 0.0884
k1 0.0968

Analysis of Variance:

DF
Regression 2
Residual 447
Total 449

PRESS = 0.7951

Durbin-Watson Statistic = 1.8886

Normality Test:

Constant Variance Test:

Std. Error t P
0.0049 169.8753 <0.0001
0.0073 12.0816 <0.0001
0.0238 4.0674 <0.0001

SS MS F P
0.2729 0.1364 77.7896 <0.0001
0.7840 0.001 8
1 .0569 0.0024

K-S Statistic = 0.0349

Significance Level = 0.6351

Passed (P = 0.5840)

Power of performed test with alpha = 0.0500: 1.0000

59

10.

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9.1 General References

1.

Basic Cell Culture: A Practical Approach (ed. Davis, J. M.) (Oxford University
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Harrison, M. A. & Rae, I. F. General Techniques of Cell Culture (Cambridge
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64

ill";

fl

‘1

. ‘1 .11.‘ O‘. ‘1‘. ‘1. ‘0

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