Optimization of Cell Adhesion
Environments for a Liver Cell
Dn
'W__&
DIUI=ClU LU[
-i
AASSACHUSETTS iNSMTUTE
OF TECHNOLOGY
by
JAN 2 5 2006
Michael B. Wongchaowart
Q (
Ui.s.
LIBRARIES
irrsi^1rv
LjuIVIVjJ y
Massachusetts Institute of Technology, 2004
Submitted to the Division of Bioengineering in Partial
Fulfillment of the Requirements for the Degree of
Master of Engineering in Biomedical Engineering
At the
MassachusettsInstitute of Technology
September 2005 F.r.bv "a:'5
© 2005 Massachusetts Institute of Technology.
All Rights Reserved.
Signature of Author:
` :ivision
of Biological Engineering
August X, 2005
Certified by:
Linda G. Griffith
Professor of Biological and Mechanical Engineering
Thesis Supervisor
Accepted by
,."
/·/Lj
n,vu A Ia39 ..X
A,,..
Lq-UI lI I
llrr....
UIu YCI
Professor Of Biological
an4'Cheical Engineering
MEBEProgram Director
Chair of BE Graduate Committee
1
.CHIVES
Table of Contents
ABSTRACT
4
INTRODUCTION
5
1.1
Overview of the Liver .....................................................................................................................
1.1.1
1.1.2
1.2
B asic Liver Functions .................................................................................................................. 5
Liver Structure and Histology ..................................................................................................... 5
Bioartificial Liver ...........................................................................................................................
1.2.1
1.2.2
5
7
Applications ................................................................................................................................. 7
MilliF Bioreactor ......................................................................................................................... 8
1.3
Cell Adhesion in the Liver ............................................................................................................. 9
1.3.1
Liver Extracellular Matrix ........................................................................................................... 9
10
Integrins ................................................................................................
1.3.2
12
1.3.3
Adhesion Signaling Pathways ...........................................................
2
HEPATOCYTE SPHEROID CULTURE
12
2.1
Rationale for Spheroid Use..............................................................
2.2
Primary Hepatocyte Isolation and Spinning Suspension Culture....
12
............................
13
2.3
Spheroid Characterization ...................................
..........................
14
2.3.1
Toluidine Blue Viability Assay ........................................
......................
14
2.3.2
Liver-Specific Gene Expression .............................................................
15
2.3.3
RNA Isolation and Real-Time PCR........................................................................................... 17
2.4
Polymer Microspheres ..............................................................
18
2.4.1
Objective for Microsphere-Spheroid
2.4.2
2.4.3
19
Microsphere Synthesis and Preparation..............................................................
Hepatocyte Culture with Microspheres on poly-HEMA-Treated Surfaces ............................... 20
2.5
Results and Discussion ....
2.5.1
2.5.2
2.5.3
3
Culture ..............................................................
18
..........................................................
21
Spheroids from Spinning Suspension Culture ........................................................................... 21
26
...........
Microsphere Spheroids .....................................................................................
28
Comparison of Spheroid Formation Methods..............................................................
COMB POLYMER ADHESION SURFACES
30
3.1
Control of Surface Cell Adhesion Properties Using Comb Polymers ............................
3.2
Comb Polymer and Adhesion Peptide Synthesis ...........
2
........................
30......
30
.................... 32
3.3
Cell Spreading Analysis ...................................................
33
3.4
Adhesion Signaling on Comb Polymer Surface ...................................................
3.4.1
Focal Adhesion Kinase Activation States...................................................
3.4.2
Analysis of FAK Activation by Immunoblotting ........................................
...........
3.5
Surface Selectivity and Nonparenchymal Cell Attachment...................................................
34
34
35
36
3.5.1
Kupffer and Stellate Cell Antibodies ...................................................
36
3.5.2
Immunostaining Protocol ...................................................
36
3.6
Results and Discussion ...................................................
3.6.1
3.6.2
3.6.3
Hepatocyte Morphology and Function ........................................
FAK activation ...................................................
Surface Selectivity ...................................................
4
CONCLUSIONS
5
REFERENCES
AND FUTURE WORK
37
...........
37
39
39
39
39
3
Abstract
4
Abstract
The MilliF bioreactor offers great potential for the formation of i vivo-like liver
tissue outside the body, making it a valuable tool for applications such as drug toxicity
models and biosensors. Cell adhesion is an important factor in the maintenance of
differentiated hepatocyte functions. Hepatocyte adhesion environments were examined
in two settings: spheroid culture prior to seeding in the bioreactor and 2D surface culture
methods that could be applied to the bioreactor scaffold.
Spheroids were formed either by culturing in spinning suspension or on a static,
non-adherent surface. In spheroid culture, the addition of extracellular matrix (ECM)
signaling through the use of soluble Matrigel or adhesion protein-coated microspheres
did not improve hepatocyte viability or function as assessed by liver-specific gene
expression. These results suggest the importance of cell-cell rather than cell-surface
interactions in maintaining hepatocytes. Optimal culturing of spheroids in spinning
suspension without the ECM addition was found to be 3 days without media changes.
2D surfaces were treated with an adhesion peptide-conjugated
comb polymer,
preventing nonspecific cell adhesion and allowing attachment through the as31 integrin.
Varying the proportion of adhesion peptide presented to cells was found to regulate
hepatocyte morphology and function; a surface with decreased hepatocyte spreading and
liver-specific gene expression closer to in vivo was characterized. Immunoblotting for
activated focal adhesion kinase (FAK) revealed that FAK signaling was not induced by
attachment to the comb polymer surfaces.
Immunostaining
for other liver cell types
demonstrated that the surface allowed hepatic stellate cell and Kupffer cell adhesion.
Acknowledgements
f would like to thank my advisors, Professor Linda Griffith and Doctor Tommy
Wong.
Their support,
encouragement,
and inspiration
were greatly
appreciated
throughout my graduate experience. It was an honor to work under both of them, and
their guidance was essential for the completion of my thesis.
I would also like to recognize all of the members of the Griffith lab for their
generosity and willingness to help me with my research. I thank Ben Cosgrove, Albert
Hwa, Artemis Kalezi, Ricardo Llamas Vidales, Corey Moore, Joe Moritz, Joe Shuga, and
Anand Sivaraman for their advice and aid. In particular, I am grateful to Alexandria
Sams, Nate Tedford, and Megan Whittemore for their mentorship and the time they
dedicated to helping me with my experiments.
Jim Serdy and Eileen Dimalanta
generously prepared and provided me with essential materials for my research.
Finally, I am deeply indebted to my family and friends, whose love and support
helped me through the struggle.
E 4c<,
Introduction
1.1 Overview of the Liver
1.1.1 Basic Liver Functions
The liver is a vital organ with numerous functions. The liver is important for
carbohydrate metabolism, regulating blood glucose levels and serving as a major site of
glycogen storage. In lipid metabolism, the liver provides fatty acids and cholesterol to
the rest of the body and uptakes and excretes cholesterol. In protein metabolism, the liver
converts ammonia to urea, synthesizes all nonessential amino acids, and secretes all
major plasma proteins. In addition, the liver stores iron and vitamins and serves as a
major blood reservoir. For digestion, the liver synthesizes and secretes bile to emulsify
lipids in the intestines. A large population of phagocytic cells makes the liver a major
line of defense against foreign particles such as bacteria. The liver is also important for
clearing hormones from the blood and processing and excreting drugs and toxins (Berne
et al., 2004; Desmet, 1994).
1.1.2 Liver Structure and Histology
The liver receives blood from two sources: the hepatic artery and the portal vein.
Blood exits the liver in hepatic veins that empty into the inferior vena cava. The liver is
organized into lobules, polyhedral structures with a central vein surrounded by portal
triads consisting of a bile duct, hepatic artery, and portal vein (Figure 1). Blood enters
from arterioles and portal venules at the periphery of the lobule and flows radially inward
through sinusoids to the central vein. Bile flows radially outward through bile canaliculi
and empties into bile ducts.
5
Liver Lobule
Detail of Lobule
patic artery
branch
Hepatocyte~
Sinusoic
Bile
canalit
Bile
di
rtal vein branch
Figure 1. Liver lobule structure. Blood flows from portal triads through sinusoids to the
central vein. Bile secreted by hepatocytes flow through bile canaliculi into bile ducts
(Cunningham and Van Horn, 2003).
Hepatocytes are the main functional cell of the liver, comprising approximately
60% of the total liver cell number. These parenchymal cells are polarized, containing a
canilular domain for bile secretion (13% of the cell surface), a sinusoidal domain in
contact with the space of Disse and bloodstream (37% of the cell surface), and a lateral
domain where cell-cell contact (Weibel et al., 1969). Hepatocytes form cell plates that
line the extensive vasculature of the liver and are responsible for most liver functions
such as carbohydrate, protein, lipid, and drug metabolism.
6
Sinusoidal
endothelial
cells (SEC) line the sinusoidal
vessels and comprise
approximately 20% of the total liver cell number. SEC membranes contain pores, or
fenestrations, that increase hepatocyte exposure to blood and act as filters (Braet and
Wisse, 2002). Unlike most endothelial cells, SEC lack a regular basement membrane;
this also serves to increases hepatocyte-blood contact.
Kupffer cells, comprising about 15% of the total liver cell number, are liver tissue
macrophages. Found attached to the sinusoidal wall, their main function is to clear waste
and foreign materials such as bacteria and endotoxins from hepatic circulation through
endocytosis (Naito et al., 2004). Kupffer cells also secrete factors such as proteases and
cytokines that influence hepatocytes and other sinusoidal cells.
Hepatic stellate cells, also known as perisinusoidal, fat-storing, or Ito cells, are
located between hepatocytes and sinusoidal endothelial cells. Comprising approximately
6% of the total liver cell number, star-shaped stellate cells have long processes that wrap
around the sinusoidal endothelium. Stellate cells functions include production of liver
extracellular matrix proteins, retinoid storage, and the regulation of sinusoidal blood flow
(Burt, 1999). In response to injury, stellate cells secrete growth hormones and increase
matrix protein synthesis.
Pit cells are large granular lymphocytes found attached to sinusoidal endothelial
cells and Kupffer cells. Comprising less than 2% of the total liver cell number, pit cells
are associated with natural killer activity (Nakatani et al., 2004).
1.2 Bioartificial Liver
1.2.1 Applications
7
Liver failure remains
a significant
health problem
and is associated
with
approximately 30,000 deaths per year (1993). The condition of patients with liver
disease might be improved by the use of extracorporeal liver tissue bioreactors, or
bioartificial livers. These bioreactors have potential to support liver functions better than
mechanical devices focusing on processes such as hemodialysis,
hemofiltration,
and
plasma exchange. Bioartificial liver systems will most likely benefit patients with acute
liver failure rather than chronic liver failure (Tzanakakis et al., 2000).
Bioartificial
livers also represent
a valuable
tool for the development
of
pharmaceuticals. The liver is responsible for processing foreign molecules entering the
body, and new drugs must be tested in vitro to predict their toxicity and efficacy.
Bioartificial livers that better mimic the in vivo organ have potential to be superior in
vitro systems for assessing important drug characteristics such as hepatotoxicity,
cholestasis, and drug uptake and metabolism. Finally, the liver's role in drug metabolism
and its exposure to pathogens through its extensive vasculature makes bioartificial livers
especially relevant as a biosensor designed to detect toxins and pathogenic infection.
1.2.2 MilliF Bioreactor
The Griffith lab at MIT has developed a microfabricated array bioreactor for 3D
perfused culture of liver cells (Powers et al., 2002a; Powers et al., 2002b), (Figure 2).
Liver cells are seeded into an array of channels etched through a thin silicon scaffold.
The scaffold rests upon a microporous
filter that is supported by a second, empty
scaffold. After allowing cells to attach to the scaffold (-1 day after seeding), higher fluid
pressure is applied to the upper, cell-filled scaffold. This pressure drives perfusion of
culture medium through the channels and back into the media reservoir for recirculation
8
(Figure 2C). Fluid flow in the bioreactor was designed to provide adequate oxygen levels
to cells while applying physiologic levels of sheer stress. Liver cell morphogenesis is
directed by channel geometry and adhesion properties of the scaffold surface, which is
coated with type I collagen.
The presence
of an optical window allows in situ
observation of the tissue by microscopy.
I
I
Figure 2. MilliF bioreactor system. (A) MilliF bioreactor. (B) Tissue formation in scaffold
channels. (C) Schematic of fluid flow through the bioreactor.
The MilliF bioreactor has shown promise as an improved liver cell culture system
when compared to 2D collagen sandwich culturing of hepatocytes (in press).
The
expression of a number of liver-specific genes including liver-enriched transcription
factors and drug metabolizing enzymes was significantly higher in the bioreactor. In
addition, albumin and urea synthesis were increased relative to collagen sandwich
cultures. While current protocols utilize a type I collagen coating to promote hepatocyte
attachment to the scaffold, other surface adhesion preparations might be explored to
further improve the MilliF bioreactor system. In addition, cell culture steps prior to
seeding into the MilliF bioreactor can be further characterized and optimized.
1.3 Cell Adhesion in the Liver
1.3.1 Liver Extracellular Matrix
While extracellular matrix (ECM) occupies only 3% of the area in sections of
normal liver tissue, it is an important regulator of liver tissue morphology and function
9
(Lin et al., 1998). While ECM is mainly present around vascular structures (portal triads
and central vein branches) and Glisson's capsule (the connective tissue encasing the liver,
hepatic artery, portal vein, and bile ducts), it is also found in the space of Disse (Rojkind
and Greenwel,
1994).
The space of Disse between hepatocytes
and the sinusoidal
endothelial lining contains typical basement membrane components such as sheetforming type IV collagen, fibronectin, laminin, and proteoglycans, as well as some type I
collagen (Stamatoglou
and Hughes,
1994).
However, it lacks a defined basement
membrane; the diffuse ECM facilitates the transfer of molecules between blood and
hepatocytes.
Portal tract and central vein areas are rich in fibrillar collagens (types I, III,
and V) and also include fibronectin (Bedossa and Paradis, 2003). In addition to serving
as a mechanical scaffold for liver tissue, the ECM is an important regulator of cell
processes including survival, proliferation, differentiation, and migration. ECM often
mediates such processes through cell adhesion molecules called integrins, though it is
also capable of binding and releasing signaling molecules such as growth factors or
cytokines.
1.3.2 Integrins
Integrins are a family of glycosylated heterodimeric cell surface receptor proteins
consisting of a a- and 3-subunit. Each subunit spans the plasma membrane once and has
a large extracellular domain and generally short cytoplasmic tail. There are currently 18
known a-subunits and 8 known P-subunits; different combinations of alpha and beta
subunits associate noncovalently
to form distinct integrins, of which 24 have been
presently identified. Most integrins bind to ECM components, and the ligand-binding
process involves divalent cations (Plow et al., 2000). Integrins are thought to have two
10
possible conformations: a bent, inactive state and a straight, active state (Xiong et al.,
2003), (Figure 3A).
The tripeptide sequence arginine-glycine-aspartate (RGD) is often a recognition
site for ECM-binding integrins and is found in ECM proteins such as fibronectin,
vitronectin, and fibrinogen (Koivunen et al., 1993; Ruoslahti, 1996). Integrin binding
specificity is influenced by sequences on both subunits, and the crystal structure of
extracellular integrin heterodimer bound to an RGD peptide reveals that the RGD binding
site lies at the interface between the a- and 3-subunits (Xiong et al., 2002). As integrins
bind to ECM, they cluster and form complexes with numerous other proteins involved in
intracellular signaling and cytoskeletal organization; these complexes are termed focal
adhesions. The following integrins and corresponding ECM ligands are expressed by
hepatocytes: atll (collagen I, collagen IV, and laminin), a231 (collagen I, collagen IV,
laminin, and fibronectin), a 3 31,(fibronectin), and asB3
1 (fibronectin) (Gullber et al., 1992;
Ruoslahti et al., 1994).
Figure 3. Integrin structure and signaling. (A) Crystal structure of the integrin
heterodimer in bent conformation. Integrin conformation corresponds to activity state
11
(Lodish et al., 2004).
(B) Complex integrin signaling pathways lead to cell proliferation,
survival, migration, and differentiation (Guo and Giancotti, 2004).(Guo and Giancotti,
2004; Lodish et al., 2004)
1.3.3 Adhesion Signaling Pathways
When cells adhere to ECM through integrins, the assembly of focal adhesions
structurally connects the cytoskeleton
with the ECM and initiates a complex set of
intracellular signaling events (Figure 3B). Integrin signaling influences a variety of
cellular processes including proliferation, survival, migration, and differentiation
(Lafrenie and Yamada, 1996; van der Flier and Sonnenberg, 2001).
For example,
activation of p3 integrins by treatment with RGD peptides or anti-integrin antibodies has
been shown to prevent apoptosis in cultured hepatocytes (Pinkse et al., 2004).
When
clustered, most integrins recruit focal adhesion kinase (FAK) to the focal adhesion. FAK
recruitment in turn leads to the MAP kinase, JNK, and AKT signaling pathways through
Src-family kinases (SFKs) and phosphatidylinositol
3-kinase (PI3K) (Guan, 1997; Guo
and Giancotti, 2004). In addition to these complex pathways, integrins can also interact
with and behaviorally influence other integrins, recteptor tyrosine kinases, and cadherins
(van der Flier and Sonnenberg, 2001).
2 Hepatocyte Spheroid Culture
2.1 Rationale for Spheroid Use
Isolated hepatocytes can aggregate to form compact multicellular structures called
spheroids when they are cultured using a variety of conditions including positively
charged surfaces (Koide et al., 1990), nonadherent surfaces (Landry et al., 1985), and
spinner vessels (Wu et al., 1996). Hepatocyte spheroids have been shown to maintain
liver functions such as albumin secretion better than traditional monolayer culture
12
techniques, suggesting the importance of 3D cell-cell interactions (Koide et al., 1990;
Tong et al., 1990). Spheroids continue to offer potential as a tissue-like system for
modeling the liver, and have shown liver function such as cytochrom P450 induction and
UDP-glucuronyltransferase activity after long-term culture (Tong et al., 1992).
Experiments with the MilliF bioreactor have made use of spheroids cultured in
spinner vessels because spheroids show superior structural stability and liver function
maintenance compared to seeding single cells (Powers et al., 2002a), (Powers et al.,
2002b). However, the formation and maintenance of hepatocyte spheroids in spinner
vessels has not been thoroughly investigated.
Spheroid health was characterized after 2,
3, and 7 days of culture with and without daily half media changes to ascertain optimal
culture conditions. In addition, soluble Matrigel was added to some to determine if
extracellular matrix signaling affects spheroid formation and maintenance. Matrigel, a
complex, soluble mixture of basement membrane proteins extracted from EngelbrethHolm-Swarm mouse sarcoma cells, has previously been shown to better maintain
hepatocyte function and morphology than collagen alone in 2D cultures (Moghe et al.,
1996). Matrigel is mainly composed of laminin, followed by collagen IV, heparan sulfate
proteoglycans, entactin and nidogen (Kleinman et al., 1982).
2.2 Primary Hepatocyte Isolation and Spinning Suspension
Culture.
Liver cells were isolated from 150 to 230g male Fischer rats using a modified
two-step collagenase perfusion (Seglen, 1976). The liver cell suspension was centrifuged
three times for 2 min at 50 x g. The cell pellet was then resuspended in hepatocyte
growth medium (HGM) similar to that used by Block et al. (1996), but without
13
hepatocyte growth factor. Cell viability, assessed by trypan blue exclusion, was between
88% and 93% following resuspension. The liver cell suspension typically included 95%
hepatocytes and 5% nonparenchymal cells.
Isolated hepatocytes were generously
provided by Emily Larson, Megan Whittemore, and Laura Vineyard.
Hepatocyte spheroids were formed in spinner vessels using a protocol similar to
that used by Wu et al. (1996).
Spinner vessels were coated with Sigmacote (Sigma
Chemical Co.) to prevent cell adhesion, rinsed with deionized water, and sterilized. 20 x
106 freshly isolated hepatocytes were added to cold HGM to a total volume of 100 ml.
While being cultured in a 37°C, 8.5% CO2, humidified incubator, cells were stirred at 85
rpm with a suspended magnetic stirbar. For flasks with daily media changes, 50ml media
were removed after allowing cells to settle. The media was centrifuged for 3 min at 50 x
g to collect cells unintentionally removed, the supernatant was discarded, and the small
cell pellet was resuspended in 50ml fresh 37°C HGM and returned to the spinner flask.
For flasks testing the effects of soluble matrix components, ml of growth factor reduced
Matrigel (BD Biosciences #356230) was added to the media prior to cell seeding.
Spheroids with diameters between 100 and 300ptm were selected using open mesh nylon
filters (SEFAR #03-100/49 and #03-300/54). This spheroid size range was used for
seeding into the MilliF bioreactor, in which scaffold channels have 300 Im x 300 jim
rounded square geometry (Powers et al., 2002a; Powers et al., 2002b).
2.3 Spheroid Characterization
2.3.1 Toluidine Blue Viability Assay
Spheroids were fixed in 2.5% glutaraldehyde for 6 hours at 40 C and washed in
phosphate buffered saline (PBS) overnight at 4C.
14
Cells were dehydrated with 100%
acetone for 1 hour at 4°C and embedded in glycol methacrylate using a Technovit 8100
embedding kit. 3-4 tm thick sections were prepared from embedded spheroid samples
using an Ultracut E microtome (Reichert). Sections were floated on water droplets and
incubated at to affix to slides.
Spheroid sections were stained with 0.5% Toluidine Blue O (Electron Microscopy
Sciences #22050) for 1.5 min and washed for 2 min in distilled water. Toluidine blue can
be used to preferentially stains live cells a deep blue while only lightly staining dead cells
(Li et al., 1998). Photos were taken using a AxioCam HRc camera (Carl Zeiss #BLANK)
attached to an Axiovert 200 inverted microscope (Carl Zeiss #M202662), and AxioVision
3.1 software (Carl Zeiss). Adobe Photoshop 7.0 was used for live/dead image analysis to
determine the proportion of live cell area to dead cell area.
Threshold luminescence
values were selected for live-dead and dead-background transitions. The numbers of
image pixels falling within the live and dead cell luminescence
ranges were counted
using a pixel histogram function. Average spheroid section diameters were calculated by
adding live and dead cell areas and assuming circular geometry.
2.3.2 Liver-Specific Gene Expression
In order to characterize how well liver functions are being maintained in vitro, the
expression levels of a number of liver-specific genes were determined using real-time
PCR.
These genes included transcription factors, drug metabolizing enzymes, and
albumin.
Hepatocyte nuclear factor 1 alpha (HNFla) is a liver enriched transcription factor
that influences the expression of a large number of liver specific genes. HNF1 binding
15
sites are found in the promoters or enhancers of many genes involved in liver functions
such as carbohydrate metabolism, detoxification, lipid metabolism, and protein secretion
(Odom et al., 2004). HNF1 binds to a palindromic DNA sequence as a dimer (Chouard
et al., 1990).
CCAAT/enhancer binding protein beta (C/EBPP3)is a liver-enriched transcription
factor involved in inducible gene regulation in response to inflammatory stress
(Lekstrom-Himes and Xanthopoulos, 1998). C/EBPP is activated by interleukin-6 in
hepatocytes and regulates acute-phase reactive genes, including C-reactive protein and
complement C3. C/EBPP has also been found to be function in controlling noninducible
liver-specific genes such as CYP2D5, PEPCK, and transferrin (Tronche and Yaniv,
1994).
Albumin, synthesized by the liver, is the most abundant plasma protein and is
important for maintaining osmotic pressure through its high concentration in the blood.
Albumin is responsible for transporting a variety of substances including metals,
hormones, free fatty acids, unconjugated bilirubin, and many drugs. Albumin expression
is controlled both by several liver-enriched transcription factors (HNF1, HNF3, and
C/EBP) and other transcription factors (Cereghini et al., 1987; Lichsteiner et al., 1987).
Albumin synthesis is often used to assess hepatic function in liver cell cultures.
Cytochrome P450s (CYPs) are heme-containing enzymes involved in endogenous
metabolism and phase I biotransformation of xenobiotics (Parkinson, 1996). Located in
hepatocyte
microsomes
or
endoplasmic
reticulum,
CYPs
often
catalyze
monooxygenation reactions that add a functional group to their substrates to which other
molecules can be conjugated. CYP2B1 is the major phenobarbital-inducible
16
CYP in rats;
while it is constitutively expressed, phenobarbital treatment has been shown to greatly
increase CYP2B1 mRNA levels (Morris and Davila, 1996). CYP2Cll is constitutively
expressed at high levels in adult male rat hepatocytes and is involved in testosterone
metabolism (Morishima et al., 1987).
CYP3A1 and CYP3A2 are both constitutively
expressed in the liver, and a variety of drugs have been found that can induce increased
mRNA levels (Morris and Davila, 1996).
2.3.3 RNA Isolation and Real-Time PCR
Spheroids were resuspended in
ml TRIzol
Reagent (Invitrogen Life
Technologies #15596-026) to stabilize RNA. The suspension was homogenized using a
syringe and 20 gauge needle, pumping 5 times.
2001il chloroform was added to the
homogenate, and the mixture was vortexed briefly and centrifuged for 15 min at 12,000 x
g and 4°C. The upper aqueous phase was mixed with an equal volume of 70% ethanol,
and then RNA was isolated with a Qiagen RNEasy Mini Kit (Qiagen #74104). Isolated
RNA was treated with DNase I (Invitrogen #18068-015) to degrade any contaminating
DNA. Total RNA was converted to cDNA using an Ominscript Reverse Transcription
Kit (Qiagen #205111), RNase inhibitor (Ambion #2682), and random hexamer primer
(Qiagen #SP200-0).
Liver specific mRNA expression levels were evaluated by real-time polymerase
chain reaction. Real-Time PCR experiments were carried out on a DNA Engine Peltier
Thermal Cycler with Chromo 4 Four-Color Real-Time Detector (MJ Research) using
Opticon Monitor 2.0 software (MJ Research).
cDNA samples with a final reaction
volume of 20pl were prepared using a QuantiTect SYBR Green PCR Kit (Qiagen
#204143).
After an initial denaturing step at 95°C for 15min, the following steps were
17
cycled 41 times: 94°C for l5sec (melting), 55°C to 620 C for 1 min (annealing), 72°C for
30sec (extension), and plate read. The annealing temperature varied for different primer
sets. PCR product purity was assessed by melting curve analysis. Liver specific mRNA
levels were normalized using 18S ribosomal RNA, a house-keeping
gene that has a
constant expression level across different conditions. In order to compare to in vivo gene
expression, RNA was isolated from rat liver tissue sections that were immediately placed
in TRIzol Reagent and homogenized after surgical removal. After homogenization, RNA
isolation from in vivo samples was carried out as described above.
2.4 Polymer Microspheres
2.4.1 Objective for Microsphere-Spheroid Culture
Hepatocyte spheroid formation is necessary for seeding into the MilliF bioreactor
(Powers et al., 2002a). However, spheroid formation is often an inefficient process in
which only a fraction of the total cells forms spheroids in the desired size range; many
hepatocytes either fail to aggregate at all or form large aggregates inappropriate for use in
the bioreactor. Polymer microspheres coated with extracellular matrix proteins were
utilized to test whether uniform hepatocyte spheroids consisting of a monolayer of cells
surrounding a central microsphere could be generated. A degradable biocompatible
polymer was selected so that microspheres would degrade after long-term culture in the
MilliF bioreactor.
In addition,
hepatocyte
spheroids
often include a significant
proportion of dead cells. The microspheres were used to test whether the addition of cellmatrix interactions could improve viability and liver function maintenance in spheroids,
which are presumably dominated by cell-cell interactions.
18
2.4.2 Microsphere Synthesis and Preparation
Poly(co-lactic-glycolic acid) (PLGA) microspheres were synthesized from
polylactic acid (Sigma-Aldritch) (PLA) and polyglycolic acid (Sigma-Aldritch) (PGA).
15.0g PLA in 3kg water and 42.11g PGA in 800g methylene chloride. The PGA solution
was added to the PLA solution stirring at 400rpm in a Lightnin Labmaster Mixer (VWR
#52339-958). Copolymer beads formed during slow evaporation of methylene chloride
while stirring over 24 to 36 hours.
Microspheres were sieved to select for diameters
between 24 and 38 plm. PLGA microspheres were generously provided by James Serdy.
Microspheres were sterilized for 2 hours in 70% ethanol.
They were then
incubated with either bovine dermal type I collagen (Angiotech #FXP-019) or human
plasma fibronectin (Invitrogen #33016015) overnight at room temperature with gentle
agitation. Collagen-coated microspheres were incubated with 0.31mg/ml collagen in
PBS with a total collagen content of 100 ptg/cm2 microsphere surface area (assuming a
smooth surface).
Fibronectin-coated
microspheres
were incubated
with 10 ptg/ml
fibronectin with a total fibronectin content of 1.77Ltg/cm2 microsphere surface area.
Microsphere surfaces are porous, so effective adhesion protein surface densities are most
likely significantly lower. Microspheres sometime aggregated during incubation with
adhesion adhesion proteins; these samples were vortexed to break apart aggregates.
Microspheres
were centrifuged for 1 min at 1 x 103 rpm and the supernatant was
removed.
19
O
arr
OCH
OCH3
O
C-CH-O-C-CH-O
0O
polylactide
(PLA)
}C-CH 2 - -
O-C-CH2-0
O
0oo 0O m
polyglycolic acid
(PGA)
A
0
0
B0m
50
O
B
Figure 4. (A) Chemical structure of PLA and PGA. The PLGA copolymer degrades slowly
by backbone hydrolysis to form lactic acid and glycolic acid monomers. A greater
proportion of PLA leads to slower degradation.
(B) 24 to 381pm PLGA microspheres.
2.4.3 Hepatocyte Culture with Microspheres on poly-HEMATreated Surfaces
Initial attempts to add the microspheres to the spinning suspension culture failed.
The vast majority of microspheres failed to incorporate into cell aggregates, most likely
due to their inefficient dispersion in the media. To address this issue, spheroids were
instead generated by static culturing on a non-adherent plastic surface (Landry et al.,
1985). This alternative method did allow efficient incorporation of microspheres in the
spheroids.
60mm tissue culture dishes were coated with poly(2-hydroxyethyl methacrylate)
(poly-HEMA)
by evaporating
2 ml of 2.5% poly-HEMA
in 95% ethanol.
Isolated
hepatocytes were mixed with microspheres at a ratio of 6 cells to each microsphere.
Microsphere numbers were estimated by assuming random close packing of 31.5plm
20
diameter spheres.
1 x 106 primary hepatocytes with microspheres in 4.5ml HGM were
seeded into each dish. Cells were cultured in a 37°C, 5% C0 2, humidified incubator.
2.5 Results and Discussion
2.5.1 Spheroids from Spinning Suspension Culture
Spheroid viability was examined by toluidine blue staining of sections and image
analysis (Figure 5).
Daily media changes and soluble adhesion factors did not
significantly affect viability after 2 or 3 days of culture; viability remained around 70%
(Figure 6A). Spheroids showed similar viabilities from day 2 to day 3, and either period
might be an appropriate length of culture time before proceeding to the bioreactor.
Viability declined more quickly from day 3 to 7 in spheroids without daily media
changes, suggesting that nutrient availability or buildup of cell waste do not affect
viability until after 3 days in culture. However, daily media changes only slightly
increased spheroid viability at day 7. This result implies that other factors most likely
contribute to cell death. Such factors could include programmed cell death due to a lack
of cell-surface signaling.
Day 7 spheroids were often characterized by a central dead region, with live cells
located on spheroid surfaces (Figure 6B). This pattern was observed in spheroids with
daily media changes, indicating that cell death in the spheroid core was not due to
unavailability of nutrients or growth factor from the media.
The live/dead cell
configuration suggests the presence of pro-survival signaling at the surface, perhaps
through deposition of ECM proteins on the spheroid exterior. In future studies, the
presence of ECM components on or throughout hepatocyte spheroids might be
characterized through immunostaining. Alternatively, the survival of cells at the spheroid
21
surface might be promoted through fluid shear. Shear stress-induced integrin signaling
through the FAK pathway has been noted in vascular endothelial cells (Li et al., 1997).
Soluble Matrigel was found to modulate spheroid size in spinning suspension
culture. While control and media change spheroids were of similar size after 3 days of
culture (93.0 and 114.6[tm respectively), Matrigel spheroids were approximately twice as
large (213.5[pm). Spheroid aggregate size is thought to increase in the spinner vessels
over time through the fusion of smaller spheroids (Wu et al., 1996). The significant size
difference observed between Matrigel and non-Matrigel
spheroids might be due to
increased fusion. Such fusion could be promoted by the deposition of Matrigel adhesion
proteins on spheroid surfaces, increasing adhesiveness between individual spheroids.
The ability of Matrigel to influence spheroid formation might be better characterized
through particle size analysis.
Real-time PCR results were difficult to analyze due to high variability between
samples (Figure 7).
While the spheroid-forming
process was difficult to reproduce
consistently, some trends were discernable. For control spheroids, day 3 spheroids
generally showed gene expression levels closer to in vivo than day 2 spheroids. Since
viabilities are similar at both days, 3 days is most likely the optimal culture time for
hepatocytes in spinner flasks. Overall, daily media changes did not significantly improve
gene expression relative to control spheroids. Since viability reaches unacceptably low
levels by day 7 with or without changing media, this data suggests that daily media
changes are unnecessary and would only increase the risk of contaminating the cultures.
Addition of Matrigel did not improve overall liver-specific gene expression. While there
was no clear-cut trend, gene expression was often down-regulated in Matrigel spheroids.
22
Matrigel still might be useful for regulating spheroid size and kinetics of formation, but it
does not appear to significantly promote the maintenance of hepatocyte differentiation in
spinning suspension culture.
%P-945
4 0
%ft
Figure 5. Spheroid Viability. (Left) Day 3 hepatocyte spheroids fixed, embedded,
sectioned, and stained to distinguish live and dead cells. Live cells stain a deeper blue than
dead cells. (Right) Live cell images. Pixels with intensities in the range of dead cells have
23
been removed. (A) Control spheroids.
(B) Daily media change spheroids.
(C) Matrigel
Figure 6. (A) Spheroid viability over time in culture. Daily media changes only slightly
mitigate the decline in viability over time in culture. (B) Hepatocyte spheroids after 7 days
of culture with daily media changes. Media changes do not prevent the development of a
central dead core.
24
2
-
X-
-------HNF1a
1-
237
2 3 7
23
7
2
2
7
37
2 3 7
23
7
1 ff _l
0B -1 -
~-3
-4
Albumin
C/EBPb
r L I-el
"%'
I8
-
~-5-6a)
ct00 - -
O Control
E Media Change
-8 -
* Matrigel
u
-J
3
CYP2C11
1
CYP3A1
CYP3A2
2
1
237
237
237
237
237
0-
o
237
237
237
237
237
4
-1-
-2 -3 'E
._
-4
C
0
-5
-6 w
"
I
I
-8 -
-j0
-9 -10
-11
-12
I
I I
I
II
-7 -
LC
I. REW
BIE
I
UI I
1
I.
I
I
.I .
. ....
...
1
nn
frr..............................
I
(2ntral
O Media Change
IL'
1
U
!
I
1lII
I
I
* Matrigel
Figure 7. Liver-specific gene expression in control, media change, and Matrigel hepatocyte
spheroids after 2, 3, and 7 days of culture. Spheroid formation in spinning suspension is
difficult to reproduce, and there is a high degree of variability between experiments.
25
2.5.2 Microsphere Spheroids
Microsphere incorporation into hepatocyte aggregates was achieved for both
collagen- and fibronectin-coated
microspheres, cell attachment to the microspheres was
observed within 15min of seeding (Figure 8A). However, the spheroids generated were
extremely heterogeneous in size and shape (Figure 8C). Complex aggregates containing
many microspheres were often formed rather than cell monolayers on individual
microspheres, and some spheroids contained no microspheres
at all (Figure 8B).
In
addition to generating spheroids more irregular than controls, extremely large macro-
scale aggregates would sometimes form (Figure 8D).
This heterogeneity makes
microsphere spheroids inappropriate for use in the MilliF bioreactor.
Microsphere incorporation led to a decrease in cell viability in spheroids after 3
days of culture; viability was 54.8% for collagen-coated microsphere spheroids and
50.2% for fibronectin-coated microsphere spheroids. In addition, the expression levels of
a number of liver-specific genes tested using real-time PCR were not closer to in vivo in
microsphere spheroids (Figure 9). These results suggest that cell-cell interactions might
be more important than cell-substratum interactions for the maintenance of differentiated
hepatocytes. An ideal surface for a hepatocyte bioreactor might be one that allows
maximal cell-cell contact by reducing cell-matrix contact to the minimum required for
stable attachment and pro-survival signaling. Discuss cadherin signaling.
26
I
Figure 8. Hepatocyte spheroid formation with PLGA microspheres. (A) In static culture,
isolated hepatocytes begin adhering to microspheres within 15 min of seeding. (B) A
heterogeneous population of spheroids develops, with spheroids containing zero, one, and
multiple microspheres coexisting in culture. (C) Microsphere spheroids are often less
regular in shape than control spheroids. (D) Extremely large spheroid-microsphere
aggregates formed over time in culture.
27
.,
r~
/
HNF-1 a
C/EBP-b
Albumin
CYP2B1
1
CYP2C11
CYP3A1
CYP3A2
_T
--
0-
_
-1
,·
4 -3
'.2
0
-4
_
* -6
WU-7
I
'a
i
I
I
LL-8
-9 9
.II
.......-
__--.-- ---.....-
-10 - _-......... ---
1 ...
_
I
I
O Control Spheroids
I
-4
O Spheroids + Collagen-coated
Microspheres
-11-
-I
* Spheroids + Fibronectin-coated
Microspheres
-19 -
.~~~~~~~~
Figure 9.
Liver-specific gene expression in microsphere spheroids. Microsphere
incorporation did not improve maintenance of hepatocyte gene relative to control
spheroids.
2.5.3 Comparison of Spheroid Formation Methods
After forming spheroids on polyHEMA and in spinning suspension culture, there
appeared to be significant differences between the two processes.
Liver-specific gene
expression was down-regulated in control spheroids formed on polyHEMA compared to
control spheroids formed in spinner flasks (Figures 7 and 9).
This difference in
expression might be attributed to unfavorable oxygen levels in static culture or to
signaling induced by fluid shear stress in the spinner flask. Functionally, hepatocytes in
spinner flask spheroids appear to be superior to those formed on polyHEMA. However,
static culture was better with respect to efficiency of incorporation into appropriately
sized aggregates.
Standard curves have been previously generated to correlate the
28
amount of total RNA isolated to hepatocyte cell number (- 0.063ng RNA / cell). This
correlation was used to estimate spheroid formation efficiency, the ratio cells in spheroids
to total cells input at the time of seeding (Figure 10). 50 to 300rtm diameter day 3
spheroids were selected after formation on polyHEMA and in spinner flasks with varying
culture conditions.
Lower efficiencies with coated microspheres on polyHEMA and
Matrigel in spinner flasks were most likely due to an increased proportion of cell
aggregates larger than 300pm. Efficiencies were significantly lower when 100 to 300pm
diameter spheroids were selected. While spinning suspension culture offers better liver
function as shown by gene expression analysis, more efficient spheroid generation
methods such as polyHEMA might be necessary if only limited numbers of primary cells
are available.
II UUo
-1,
80%
o
C
o
LU
60%
.o
0
LL
a)
0.
20%
0%
polyHEMA Culture
Spinning Suspension Culture
Figure 10. Spheroid formation efficiency by polyHEMA and spinning suspension culture
methods. Efficiency estimated by total RNA isolated from 50 to 300pm day 3 spheroids.
29
3
Comb Polymer Adhesion Surfaces
3.1 Control of Surface Cell Adhesion Properties Using
Comb Polymers
Cells in culture can secrete a variety of ECM proteins that adsorb nonspecifically
to surfaces, allowing cell adhesion. Attachment to this variable and undefined surface
can influence cell phenotype and behavior through adhesion-induced signaling. This
effect is potentially detrimental for the MilliF bioreactor system because culture surface
has been shown to affect the maintenance of differentiation in hepatocytes. The ECM
density in experiments with hepatocytes cultured on Matrigel-coated surfaces has been
shown to regulate cell morphology and function (Mooney et al., 1992; Powers et al.,
1997). Hepatocytes spread on dense ECM and take on a dedifferentiated, proliferative
phenotype. On more diffuse ECM they fail to spread or proliferate, but retain higher
levels of differentiated function such as albumin secretion.
Uncontrolled protein
adsorption and nonspecific cell adhesion in the MilliF bioreactor might and promote cell
proliferation and adversely affect hepatocyte function. In order to prevent nonspecific
attachment and more rigorously control cell adhesion, a comb polymer was utilized.
The comb polymer consists of a hydrophobic backbone adsorbed to the surface
with hydrophilic side chains extending into the aqueous environment (Figure 11). While
the comb polymer alone is resistant to cell adhesion, the side chains can be modified with
adhesion molecules that allow cells with the appropriate receptors to adhere. The as5
1
integrin receptor, which binds the ECM component fibronectin, was chosen because this
integrin has been shown to be important for hepatocyte adhesion to liver ECM. Unlike
most epithelial cells, hepatocytes are exposed to fibronectin at both their canilicular and
30
sinusoidal domains (Enrich et al., 1988).
The asp5 integrin has correspondingly
been
shown to be distributed evenly over the sinusoidal, canilicular, and lateral domains of
hepatocytes (Stamatoglou et al., 1990). Adhesion to fibronectin has shown promote
differentiated hepatocyte function over other ECM molecules such as collagen or laminin
(Sanchez et al., 2000).
A branched adhesion peptide specific for the a5s3 1 integrin, referred to as the
synKRGD peptide, was attached to the comb polymer (Fig. 2B). This peptide consists of
the linear chain PHSRNGGGKGGRGDSPY
containiing
an RGD sequence and a
synergy. The central lysine serves as the branching point with the peptide GGC linked to
its sidechain.
While the RGD sequence is recognized by many integrins, the synergy
sequence PHSRN has been shown to preferentially enhance a 5 ,31 integrin-binding (Mould
et al., 2000). The branched structure of the synKRGD peptide gives both a5p1 integrin-
binding motifs the conformational freedom to optimally bind the receptor. Previous
experiments established that the synKRGD peptide comb polymer system facilitates
hepatocyte attachment and showed that epidermal growth factor can mediate cell
spreading (Yin, 2004).
31
Figure 11. Comb polymer adhesion system. (A) Comb polymer structure. Cell adhesion
peptides (orange) are linked to the comb polymer backbone (gray) through hydrophilic side
chains (green). The hydrophilic side chains move freely and prevent protein adsorption to
the surface. (B) Chemical structure of the as5P adhesion peptide. The branched structure
allows the RGD (red) and synergy (blue) binding motifs to find their optimal binding
positions. (C) Fibronectin structure in which the RGD sequence and synergy region are
presented to integrins (Lodish et al., 2004).
3.2 Comb Polymer and Adhesion Peptide Synthesis
The comb polymer comprised a poly(methyl methacrylate) (PMMA) backbone
with hydroxy-poly(ethylene oxide) (HPOEM) side chains.
The comb polymer was
synthesized as previously described (Banerjee et al., 2000). The copolymer contained
approximated BLANK% HPOEM, within the range to enable both comb polymer
insolubility and cell adhesion resistance (Irvine et al., 2001). The SynKRGD peptide was
generateded by linking the main linear peptide to the cysteine-containing branch peptide
(both made using an automated peptide synthesizer) as previously described (Yin, 2004).
32
The SynKRGD peptide was conjugated to the comb polymer using p-maleimidophenyl
isocyanate (PMPI).
linkage to PMPI.
Varying proportions of HPOEM sidechains were activated by
Side chains were then reacted with various SynKRGD peptide
concentrations, in which the cysteine residue in the peptide bonds with the maleimide in
PMPI.
After preliminary experiments, two surfaces were chosen for further
characterization:
(10% 25ltM) and (20% 25lpM) referring respectively
activation by PMPI and SynKRGD concentration during conjugation.
to side chain
SynKRGD-
conjugated comb polymer surfaces were generously provided by Eileen Dimalanta.
3.3 Cell Spreading Analysis
Comb polymer-treated coverslips were placed in 24-well tissue culture plates and
sterilized under UV light for 10min. 1.5cm sections of silicone tubing (Nalgene #80600140) were sterilized in 100% ethanol for 1 hour, rinsed twice in PBS, and placed firmly
in wells to hold down the coverslips.
This setup left 0.775cm 2 of surface area per well
for cell attachment. Hepatocytes were isolated as described in Section 2.2. After rinsing
wells with 300[1l HGM, cells were seeded at either low density (6.4 x 103 cell/cm 2 ) or
high density (3 x 104 cells/ cm2 ) to observe behavior in single cells and large aggregates
respectively. Cells were cultured in a 370 C, 5% C0 2, humidified incubator.
After 48 hours, cell membranes were fluorescently stained with 5l/ml Vybrant
CM-DiI cell-labeling solution (Molecular Probes #V-22888) for 20min at room
temperature.
Cell nuclei were fluorescently stained with
then fixed in 2% paraformaldehyde for
ll/ml Hoechst dye. Cells were
hour at 4C and rinsed twice in PBS. Cells
were mounted on slides in Fluoromount-G (Electron Microscopy Sciences #17984-25).
Microscopy and image capture were carried out as described in Section 2.3.1. Fields at
33
20x magnification were photographed for bright field, cell membrane, and nuclei images.
Adobe Photoshop 7 was used for the cell spreading analysis (Figure 12). The cell area in
each field was determined using a pixel luminescence threshold function and cell pixels
were counted using a pixel histogram function. Cell area was normalized to the number
of nuclei in the field. For low cell density analysis, aggregates containing more than 5
nuclei were excluded. For high cell density analysis, only aggregates containing 6 or
more nuclei were included.
Figure 12. Cell spreading image analysis of cells cultured at low (top) and high (bottom)
densities. (A) Cell area stain. (B) Nuclei stain. (C) Cell area quantification.
3.4 Adhesion Signaling on Comb Polymer Surface
3.4.1 Focal Adhesion Kinase Activation States
FAK is a 125kDa nonreceptor protein tyrosine kinase involved in cell adhesion
signaling (Schlaepfer et al., 1999). FAK is dephosphorylated and inactive in nonadherent
cells. When cells attach to extracellular matrix, FAK is recruited to focal adhesions and
34
becomes phosphorylated and activated. Autophosphorylation of tyrosine 397 (Tyr397), a
major site of FAK phosphorylation, is an early and critical step in FAK signaling
(Schaller et al., 1994). For example, phosphorylation at Tyr397 creates a binding site
signaling proteins with an SH2 domain such as Src and phosphatidylinositol
3-kinase
(Chen et al., 1996; Xing et al., 1994). Phosphorylation of FAK at other tyrosine sites is
then mediated by Src or other Src-family kinases (Calalb et al., 1995).
In order to
characterize the extent of FAK-mediated cell adhesion signaling on the comb polymer
adhesion surfaces, immunoblotting experiments to detect FAK phosphorylated at Tyr397
were carried out.
3.4.2 Analysis of FAKActivation by Immunoblotting
Isolated hepatocytes were cultured on comb polymer surfaces as described in
Section 3.2.1. After aspirating culture media, 100p1of cold lysis buffer containing 89[pl
1% NP40 detergent (VWR #PI28324), 1pt10.1M phenylmethanesulfonyl fluoride (SigmaAldrich #P7626), and 10pl Protease Inhibitor Cocktail (Sigma-Aldrich #P8340) was
added to each well. Cells were disrupted by pipetting up and down repeatedly and then
incubated at 4°C for 15min.
The lysate was mixed again by pipette, collected, and
centrifuged at 12,000 rpm for 15min at 4°C.
The supernatant was collected in a fresh
tube and protein concentration was determined using a Micro BCA Protein Assay Kit
(Pierce Biotechnologies #23235) and a NanoDrop ND-1000 Spectrophotometer
(NanoDrop Technologies).
Proteins from freshly isolated hepatocytes were also
extracted by a similar method.
25pg
samples of total protein from each surface were separated by
polyacrylamide gel electrophoresis on a 7.5% gel (Bio-Rad #161-1100) and transferred to
35
a polyvinylidene difluoride membrane (Bio-Rad #162-0174).
After blocking with a
solution of 3% bovine serum albumin (Blank ) in PBST for 2 hours at room temperature,
activated FAK was detected by incubating with a 1:1,000 dilution of rabbit anti-phosphoFAK (Tyr397) polyclonal primary antibody (Upstate Biotechnology #07-012) overnight
at 4°C. After washing for 15 min in PBST four times, the membrane was incubated for
3hr at room temperature with a 1:10,000 dilution of horse radish peroxidase-conjugated
goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch Labs # 111-035-144).
The membrane was then washed ffor 15 min in PBST five times and developed by
incubating for 1.5min in Western Blot Chemiluminescence Reagent Plus (Perkin Elmer
Life Sciences #NEL104).
Immunoblot images were captured using a Kodak Image
Station 1000 and Kodak ID v3.6 software.
3.5 Surface Selectivity and Nonparenchymal Cell
Attachment
3.5.1 Kupffer and Stellate Cell Antibodies
NPC surface attachment or integration into cell aggregates was examined by
immunostaining. Kupffer cells were detected using a mouse anti-rat ED2 antibody
conjugated to FITC. ED2 binds to an antigen expressed in resident tissue macrophages
and is a known marker for Kupffer cells (Dijkstra et al., 1994; Meijer et al., 2000).
Hepatic stellate cells were detected using a mouse anti-GFAP, a known marker
(Neubauer et al., 1996; Niki et al., 1996).
3.5.2 Immunostaining Protocol
Liver cells were isolated as described in Section 2.2. Since NPCs are rare in the
95% hepatocyte fraction, total liver isolate was used prior to hepatocyte enrichment
36
through centrifugation. Comb polymer adhesive surfaces were setup as described in
section 3.2.1. After culturing 48 hours, cells were fixed in 2% paraformaldehyde for 1
hour at 4C and rinsed twice in PBS. All subsequent incubations were carried out at
room temperature.
Cells were permeabilized with 0.1% Triton-X-100 in PBS for 15min
and washed for 5min in PBS three times. Cells were then washed with PBG three times
and blocked with 5% goat serum in PBG for 30min. Cells were incubated with a 1:100
dilution of mouse anti-rat GFAP primary antibody (BLANK) for lhr at room
temperature. After washing for 5min in PBG four times, cells were incubated with a goat
anti-mouse IgG secondary antibody conjugated to Cy3 (BLANK). Cells were washed
three times with PBG and three times with PBS. Nuclei were stained with 2% Hoechst
dye (BLANK) in PBS for 3min. Cells were rinsed once with PBS and mounted on slides
in Fluoromount-G.
3.6 Results and Discussion
3.6.1 Hepatocyte Morphology and Function
Varying comb polymer surfaces were found to distinctly regulate cell spreading
and maintenance of liver-specific functions. Average cell area per nuclei was greater on
the 10% 25[tM surface than on the 20% 25ltM (Figure 13A). This finding suggests that
the 10% 25gM surface is more adhesive for hepatocytes despite containing a smaller
proportion of adhesion peptide for presentation to the cells.
On both surfaces, cells
seeded at low density spread more than cells seeded at high density.
Culturing hepatocytes cultured on the two comb polymer surfaces revealed
functionally distinct phenotypes. For the set of liver specific genes examined by realtime PCR, expression was significantly closer to in vivo in hepatocytes cultured on the
37
20% 25pM surface (Figure 13B). This improved gene expression correlates with less cell
spreading, linking hepatocyte morphology and function.
A lesser degree of cell
spreading entails a rounder shape reminiscent of in vivo hepatocytes.
1200
.
..
.
_
_
..
[ 10% 25uM
*20% 25uM
T
1000-
N
cm
E
U)
_
U) 800U
0
z
0
a
0Q)
600 -
.
__----
X
0
0)
0
O
I)
400-
__ ....
0
:
200- _~__
0-
A
l
Low Cell Density
High Cell Density
38
1
HNFla
C/EBPb
Albumin
CYP2B1
CYP2C11
CYP3A1
CYP3A2
L
0
''
R
,"
-1
0
0 -2
o -3
w
0
0
*0
C -4
cc'a
U.
-5
[
I
I
I
0)
-j0 -6
I
-7
_,10% 25uM
B
-8
L
20% 25uM
Figure 13. Morphological and functional differences between hepatocytes cultured on two
RGD comb-polymer surfaces. (A) Cell spreading on the surfaces characterized by average
area per nucleus. Hepatocytes spread more on the 10%, 25ptM surface. (B) Liver-specific
gene is significantly closer to in vivo in cells cultured on the 20%, 25F1Msurface.
3.6.2 FAK activation
3.6.3 SurfaceSelectivity
4 Conclusions and Future Work
5 References
1993. Vital Statistics of the U.S. National Centor for Health Statistics.
39
Banerjee, P., D.J. Irvine, A.M. Mayes, and L.G. Griffith. 2000. Polymer latexes for
cell-resistant and
cell-interactivesurfaces. Journal of BiomedicalMaterialsResearch.50:331-339.
Bedossa, P., and V. Paradis. 2003. Liver extracellular matrix in health and disease.
Journal of Pathology.200:504-515.
Berne, R.M., M.N. Levy, B.M. Koeppen, and B.A. Stanton. 2004. Physiology.
Mosby, St. Louis.
Braet, F., and E. Wisse. 2002. Structural and functional aspects of liver sinusoidal
endothelial cell fenestrae: a review. Comparative Hepatology. 1:1-17.
Burt, A.D. 1999. Pathobiology of hepatic stellate cells. Journal of Gastroenterology.
34:299-304.
Calalb, M.B., T.R. Polte, and S.K. Hanks. 1995. Tyrosine phosphorylation of focal
adhesion kinase at sites in the catalytic domain regulates kinase activity: a
role for Src family kinases. Molecular and Cellular Biology. 15:954-963.
Cereghini, S., M. Raymonjdjean, A.G. Carranca, P. Herbomel, and M. Yaniv. 1987.
Factors involved in control of tissue-specific expression of albumin gene. Cell.
50:627.
Chen, H.C., P.A. Appeddu, H. Isoda, and J.L. Guan. 1996. Phosphorylation of
tyrosine 397 in focal adhesion kinase is required for binding
phosphatidylinositol 3-kinase. Journal of Biological Chemistry.271:2632926334.
Chouard, T., M. Blumenfeld, I. Bach, J. Vandekerkhove, S. Cereghini, and M.
Yaniv. 1990. A distal dimerization domain is essential for DNA-binding by
the atypical HNF1 homeodomain. Nucleic Acid Research. 18:5853.
Cunningham, C.C., and C.G. Van Horn. 2003. Energy availability and alcohol-
related liver pathology. Alcohol Research & Health. 27:281-299.
Desmet, V.J. 1994. Organizational Principles. In The Liver: Biology and
Pahtobiology. I.M. Arias, J.L. Boyer, N. Fausto, W.B. Jakoby, D. Schachter,
and D.A. Shafritz, editors. Raven Press, Ltd., New York. 3-11.
Dijkstra, C.D., E.A. Dopp, T.K. van den Berg, and J.G.M.C. Damoiseaux. 1994.
Monoclonal antibodies against rat macrophages. Journal of Immunological
Methods. 174:21-23.
Enrich, C., W.H. Evans, and C.G. Gahmberg. 1988. Fibronectin isoforms in plasma
membrane domains of normal and regenerating rat liver. FEBS Letters.
28:135-138.
Guan, J.-L. 1997. Focal Adhesion Kinase in Integrin Signaling. Matrix Biology.
16:195-200.
Gullber, D., K.R. Gehlson, D.C. Tuner, K. Ahlen, L.S. Zijenah, M.J. Barnes, and K.
Rubin. 1992. Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1
integrins in cell--collagen interactions: identification of conformation
dependent alpha 1 beta 1 binding sites in collagen type I. EMBO Journal.
11:3865-3873.
Guo, W., and F.G. Giancotti. 2004. Integrin Signalling During Tumour Progression.
Nature Reviews Molecular Cell Biology. 5:816-826.
40
Irvine, D.J., A.M. Mayes, and L.G. Griffith. 2001. Nanoscale Clustering of RGD
Peptides at Surfaces Using Comb Polymers. 1. Synthesis and
Characterization of Comb Thin Films. Biomacromolecules.2:85-94.
Kleinman, H.K., M.L. McGarvey, L.A. Liotta, P.G. Robey, K. Tryggvason, and G.R.
Martin. 1982. Isolation and characterization of type IV procollagen, laminin,
and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry.
21:6188-6193.
Koide, N., K. Sakaguchi, Y. Koide, K. Asano, M. Kawaguchi, H. Matsushima, T.
Takenami, T. Shinji, M. Mori, and T. Tsuji. 1990. Formation of Multicellular
Spheroids Composed of Adult Rat Hepatocytes in Dishes with Positively
Charged Surfaces and under Other Nonadherent Environments.
Experimental Cell Research. 186:227-235.
Koivunen, E., D.A. Gay, and E. Ruoslahti. 1993. Selection of Peptides Binding to the
alpha5 betal Integrin from Phage Display Library. Journal of Biological
Chemistry. 268:20205-20210.
Lafrenie, R.M., and K.M. Yamada. 1996. Integrin-Dependent Signal Transduction.
Journal of CellularBiochemistry.61:543-553.
Landry, J., D. Bernier, C. Oullet, R. Goyette, and N. Marceau. 1985. Spheroidal
Aggregate Culture of Rat Liver Cells: Histotypic Reorganization, Biomatrix
Deposition, and Maintenance of Functional Activities.Journal of Cell
Biology. 101:914-923.
Lekstrom-Himes, J., and K.G. Xanthopoulos. 1998. Biological Role of the
CCAAT/Enhancer-binding Protein Family of Transcription Factors. Journal
of Biological Chemistry.273:28545-28548.
Li, C.K.F., R. Seth, T. Gray, R. Bayston, Y.R. Mahida, and D. Wakelin. 1998.
Production of Proinflammatory Cytokines and Inflammatory Mediators in
Human Intestinal Epithelial Cells after Invasion by Trichinella spiralis.
Infection and Immunity. 66:2200-2206.
Li, S., M. Kim, Y.-L. Hu, S. Jalali, D.D. Schlaepfer, T. Hunter, S. Chien, and J.Y.-J.
Shyy. 1997. Fluid Shear Stress Activation of Focal Adhesion Kinase. Journal
of BiologicalChemistry.272:30455-30462.
Lichsteiner, S., J. Wuarin, and U. Schibler. 1987. The interplay of DNA-binding
proteins on the promoter of the mouse albumin gene. Cell. 51:963.
Lin, X.Z., M.H. Horng, Y.N. Sun, S.C. Shiesh, N.H. Chow, and X.Z. Guo. 1998.
Computer morphometry for quantitative measurement of liver fibrosis:
comparison with Knodell's score, colorimetry and conventional description
reports. Journal of Gastroenterologyand Hepatology.13.
Lodish, H., A. Berk, P. Matsudaira, C.A. Kaiser, M. Krieger, M.P. Scott, S.L.
Zipursky, and J. Darnell. 2004. Molecular Cell Biology.W. H. Freeman and
Company, New York.
Meijer, C., M.J. Wiezer, A.M. Diehl, S.-Q. Yang, H.J. Schouten, S. Meijer, N.v.
Rooijen, A.A.v. Lambalgen, C.D. Dijkstra, and P.A.M.v. Leeuwen. 2000.
Kupffer cell depletion by CI2MDP-liposomes alters hepatic cytokine
expression and delays liver regeneration after partial hepatectomy. Liver.
20:66-77.
41
Moghe, P.V., F. Berthiaume, R.M. Ezzell, M. Toner, R.G. Tompkins, and M.L.
Yarmush. 1996. Culture matrix configuration and compositionin the
maintenance of hepatocyte polarity and function. Biomaterials.17:373-385.
Mooney, D., L. Hansen, J. Vacanti, R. Langer, S. Farmer, and D. Ingber. 1992.
Switching from differentiation to growth in hepatocytes: Control by
extracellular matrix. Journal of CellularPhysiology.151:497-505.
Morishima, N., H. Yoshioka, Y. Higashi, K. Sogawa, and Y. Fujii-Kuriyama. 1987.
Gene Structure of Cytochrome P-450(M-1) Specifically Expressed in Male
Rat Liver. Biochemistry.26:8279-8285.
Morris, D.L., and J.C. Davila. 1996. Analysis of Rat Cytochrome P450 Isoenzyme
Expression Using Semi-Quantitative Reverse Transcriptase-Polymerase
Chain Reaction (RT-PCR). Biochemical Pharmacology. 52:781-792.
Mould, A.P., J.A. Askari, and M.J. Humphries. 2000. Molecular Basis of Ligand
Recognition by Integrin alpha5 betal. Journal of BiologicalChemistry.
275:20324-20336.
Naito, M., G. Hasegawa, Y. Ebe, and T. Yamamoto. 2004. Differentiation and
function of Kupffer cells. Medical Electron Microscopy. 37:16-28.
Nakatani, K., K. Kaneda, S. Seki, and Y. Nakajima. 2004. Pit cells as liver-
associated natural killer cells: morphology and function. Medical Electron
Microscopy. 37:29-36.
Neubauer, K., T. Knittel, S. Aurisch, P. Fellmer, and G. Ramadori. 1996. Glial
fibrillary acidic protein--a cell type specific marker for Ito cells in vivo and in
vitro. Journal of Heptology.24:719-730.
Niki, T., P.J. de Bleser, G. Xu, T.K. van den Berg, E. Wisse, and A. Geerts. 1996.
Comparison of glial fibrillary acidic protein and desmin staining in normal
and CC14-inducedfibrotic rat livers. Hepatology.23:1538-1545.
Odom, D.T., N. Zizlsperger, D.B. Gordon, G.W. Bell, N.J. Rnaldi, H.L. Murray,
T.L. Volkert, J. Schreiber, P.A. Rolfe, D.K. Giffor, E. Fraenkel, G.I. Bell, and
R.A. Young. 2004. Control of Pancreas and Liver Gene Expression by HNF
Transcription Factors. Science. 303:1378-1381.
Parkinson, A. 1996.Biotransformation of Xenobiotics.In Casarett and Doull's
Toxicology: The Basic Science of Poisons. C.D. Klassen, editor. McGrawHill.
Pinkse, G.G.M., M.P. Voorhoeve, M. Noteborn, O.T. Terpstra, J.A. Bruijn, and E.
de Heer. 2004. Hepatocyte survival depends on betal-integrin-mediated
attachment of hepatocytes to hepatic extracellular matrix. Liver
International. 24:218-226.
Plow, E.F., T.A. Haas, L. Zhang, J. Loftus, and J.W. Smith. 2000. Ligand Binding to
Integrins. Journal of BiologicalChemistry.275:21785-21788.
Powers, M.J., K. Domansky, M.R. Kaazempur-Mofrad, A. Kalezi, A. Capitano, A.
Upadhyaya, P. Kurzawski, K.E. Wack, D.B. Stolz, R. Kamm, and L.G.
Griffith. 2002a. A Microfabricated Array Bioreactor for Perfused 3D Liver
Culture. Biotechnologyand Bioengineering.78:257-269.
Powers, M.J., D.M. Janigian, K.E. Wack, C.S. Baker, D.B. Stolz, and L.G. Griffith.
2002b. Functional Behavior of Primary Rat Liver Cells in a Three-
Dimensional Perfused Microarray Bioreactor. Tissue Engineering. 8:499-513.
42
Powers, M.J., R.E. Rodriguez, and L.G. Griffith. 1997. Cell-Substratum Adhesion
Strength as a Determinant of Hepatocyte Aggregate Morphology.
Biotechnologyand Bioengineering.53:415-426.
Rojkind, M., and P. Greenwel. 1994.The Extracellular Matrix of the Liver. In The
Liver: Biology and Pathobiology. I.M. Arias, J.L. Boyer, N. Fausto, W.B.
Jakoby, D. Schachter, and D.A. Shafritz, editors. Raven Press, Ltd., New
York. 843-868.
Ruoslahti, E. 1996. RGD and Other Recognition Sequences for Integrins. Annual
Review of Cell and DevelopmentalBiology. 12.
Ruoslahti, E., M.D. Pierschbacher, and W.A. Border. 1994. Cell-Extracellular
Matrix Interactions. In The Liver: Biologyand Pathobiology. I.M. Arias, J.L.
Boyer, N. Fausto, W.B. Jakoby, D. Schachter, and D.A. Shafritz, editors.
Raven Press, Ltd., New York. 899-906.
Sanchez, A., A.M. Alvarez, R. Pagan, C. Roncero, S. Vilaro, M. Benito, and I.
Fabregat. 2000. Fibronectin regulates morphology, cell organization and
gene expression of rat fetal hepatocytes in primary culture. Journal of
Heptology. 32:242-250.
Schaller, M.D., J.D. Hildebrand, J.D. Shannon, J.W. Fox, R.R. Vines, and J.T.
Parsons. 1994.Autophosphorylation of the focal adhesion kinase, pp125FAK,
directs SH2- dependent binding of pp60src. Molecularand Cellular Biology.
14:1680-1688.
Schlaepfer, D.D., C.R. Hauck, and D.J. Sieg. 1999. Signaling through focal adhesion
kinase. Progressin Biophysics& Molecular Biology.71:435-478.
Seglen, P.O. 1976. Preparation of Isolate Rat Liver Cells. Methods in Cell Biology.
13.
Stamatoglou, S.C., and R.C. Hughes. 1994. Cell adhesion molecules in liver function
and pattern formation. FASEB Journal. 8:420-427.
Stamatoglou, S.C., K.H. Sullivan, S. Johansson, P.M. Bayley, I.D. Burdett, and R.C.
Hughes. 1990. Localization of two fibronectin-binding glycoproteins in rat
liver and primary hepatocytes. Co-distribution in vitro of integrin (alpha 5
beta 1) and non-integrin (AGpll10)receptors in cell-substratum adhesion
sites. Journal of Cell Science. 97:595-606.
Tong, J.Z., O. Bernard, and F. Alvarez. 1990. Long-Term Culture of Rat Liver Cell
Spheroids in Hormonally Defined Media. ExperimentalCell Research.
189:87-92.
Tong, J.Z., P. de Lagausie, V. Furlan, T. Cresteil, O. Bernard, and F. Alvarez. 1992.
Long-Term Culture of Adult Rat Hepatocyte Spheroids. Experimental Cell
Research. 200:326-332.
Tronche, F., and M. Yaniv. 1994. Liver Gene Expression. R.G. Landes Company,
Paris, France.
Tzanakakis, E.S., D.J. Hess, T.D. Sielaff, and W.-S. Hu. 2000. Extracorporeal Tissue
Engineered Liver-Assist Devices.Annual Review of BiomedicalEngineering.
02:607-632.
van der Flier, A., and A. Sonnenberg. 2001. Function and interactions of integrins.
Cell and Tissue Research.305:285-298.
43
Weibel, E.R., W. Staubli, H.R. Gnagi, and F.A. Hess. 1969. Correlated
morphometric and biochemical studies on the liver cell. I. Morphometric
model, stereologicmethods, and normal morphometric data for rat liver.
Journal of Cell Biology.42:68-91.
Williams, E., G. Williams, B.J. Gour, O.W. Blaschuk, and P. Doherty. 2000. A Novel
Family of Cyclic Peptide Antagonists Suggests That N-cadherin Specificity Is
Determined by Amino Acids That Flank the HAV Motif.Journal of
BiologicalChemistry.275:4007-4012.
Wu, F.J., J.R. Friend, C.C. Hsiao, M.J. Zilliox, W.-J. FKo, F.B. Cerra, and W.-S.
Hu. 1996. Efficient Assembly of Rat Hepatocyte Spheroids for Tissue
Engineering Applications.Biotechnologyand Bioengineering.50:404-415.
Xing, Z.H., C. Chen, J.K. Nowlen, S.J. Taylor, D. Shalloway, and J.L. Guan. 1994.
Direct interaction of v-Src with the focal adhesion kinase mediated by the Src
SH2 domain. Molecularand CellularBiology.5:413-421.
Xiong, J.-P., T. Stehle, S.L. Goodman, and M.A. Arnaout. 2003. New insights into
the structural basis of integrin activation. Blood. 102:1155-1159.
Xiong, J.-P., T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S.L. Goodman, and
M.A. Arnaout. 2002. Crystal Structure of the Extracellular Segment of
Integrin alpha Vbeta 3 in Complex with an Arg-Gly-Asp Ligand. Science.
296:151-155.
Yin, D. 2004. The Applications of Comb Polymer to the Study of Liver Cell
Adhesion and Signaling. In Division of Bioengineering. Massachussetts
Institute of Technology, Cambridge, MA. 48.
44