Monolayer and Spheroid Culture of Human Liver
Hepatocellular Carcinoma Cell Line Cells Demonstrate
Distinct Global Gene Expression Patterns
and Functional Phenotypes
Tammy T. Chang, M.D., Ph.D.,1,2and Millie Hughes-Fulford, Ph.D.2,3
Understanding cell biology of three-dimensional (3D) biological structures is important for more complete ap-
preciation ofin vivotissue functionand advancingex vivoorganengineering efforts.Toelucidatehow3Dstructure
may affect hepatocyte cellular responses, we compared global gene expression of human liver hepatocellular
carcinoma cell line (HepG2) cells cultured as monolayers on tissue culture dishes (TCDs) or as spheroids within
rotating wall vessel (RWV) bioreactors. HepG2 cells grown in RWVs form spheroids up to 100mm in diameter
within 72h and up to 1mm with long-term culture. The actin cytoskeleton in monolayer cells show stress fiber
formation while spheroids have cortical actin organization. Global gene expression analysis demonstrates upre-
gulation of structural genes such as extracellular matrix, cytoskeletal, and adhesion molecules in monolayers,
whereas RWV spheroids show upregulation of metabolic and synthetic genes, suggesting functional differences.
Indeed, liver-specific functions of cytochrome P450 activity and albumin production are higher in the spheroids.
Enhanced liver functions require maintenance of 3D structure and environment, because transfer of spheroids to a
TCD results in spheroid disintegration and subsequent loss of function. These findings illustrate the importance of
physical environment on cellular organization and its effects on hepatocyte processes.
donor organs is a major limitation.1Tissue engineering is a
burgeoning field of investigation and explores the possibility
of ‘‘building’’ a liver ex vivo for therapeutic replacement.2
A number of challenges exist for constructing a functioning
complex solid organ such as the liver. One of the first re-
quirements is to provide a three-dimensional (3D) environ-
ment for the cells to form tissues. A variety of strategies have
been used to provide a 3D structure for culturing primary
hepatocytes and hepatic cell lines. A major approach is to use
biodegradable scaffolds.3–14However, hepatocytes can also
self-assemble into spheroids without scaffolding. This can be
achieved by culturing in spinner flasks15or on specially trea-
from 50 to 100mm in size. Yoffe et al.18,19first described one
efficient culture technique for generating larger hepatic
RWV is a disc-like vessel completely filled with medium that
iver transplantation is currently the only cure for
patients with end-stage liver disease, but availability of
a low-turbulence, low-shear-force environment with 3D spa-
tial freedom for the cells to aggregate and grow.20,21In our
research, we use spheroids formed in the RWV to study the
biology of hepatocyte 3D culture without confounding inter-
actions with scaffolding or substratum materials.
hepatocyte 3D cultures than in conventional two-dimensional
(2D) monolayers,3,17,22however, the mechanisms of the func-
tional improvement remain unclear. There is a growing body
of evidence that mechanical stress mediated by adhesion to
extracellular matrix (ECM) or other cells modulates signal
transduction and gene transcription in a variety of cell
types.23,24In this study, we demonstrate that human liver
hepatocellular carcinoma cell line (HepG2) cells respond to
differing physical environments of 2D and 3D culture with
altered actin cytoskeleton structure and cell shape. Through
global gene expression analysis, we find that distinct genetic
programs are initiated depending on the physical structure of
the cells. Monolayers express high levels of ECM, cytoskele-
ton, and adhesion molecules. These transcripts are down-
regulated in the spheroids while metabolic and synthetic
functional genes are upregulated. The differences in gene
1Department of Surgery, University of California, San Francisco, San Francisco, California.
2Laboratory of Cell Growth, Veterans Affairs Medical Center, San Francisco, San Francisco, California.
3Department of Medicine, University of California, San Francisco, San Francisco, California.
TISSUE ENGINEERING: Part A
Volume 15, Number 3, 2009
ª Mary Ann Liebert, Inc.
expression reflect the greater cytochrome P450 activity and
albumin production in spheroids. Enhanced liver-specific
functions are dependent on maintenance of 3D structure
because they are lost after transfer of spheroids to a tissue
culture dish (TCD). Together, these results illustrate the im-
portance of the physical environment on hepatocyte cellular
function and inform future efforts in liver tissue engineering.
Material and Methods
HepG2 Cells (ATCC, Manassas, VA) were maintained in
T75 culture flasks in 10% fetal calf serum (Hyclone, Logan,
UT) in Eagle’s minimum essential medium supplemented
with glutamine, antibiotics and pyruvate (Fisher, Phila-
delphia, PA). For experiments, cells were placed in 6cm
TCDs or 10mL RWVs with a diameter of 6cm (high-
aspect-ratio vessels, HARVs; Synthecon, Houston, TX). Cells
were cultured in 10mL of medium at a cell density of 5?104
cells=mL for TCDs and RWVs. For short-term cultures up to
7 days, no medium was exchanged in TCD or RWV cultures
and 10mL HARVs were rotated at 16rpm with the RCCS-4
culture system (Synthecon). Cell densities in TCDs and
RWVs were approximately 3?105=mL at day 3 of culture
and 4?105to 5?105=mL at day 7 of culture. For long-term
culture (6–10 weeks), 50mL HARVs were rotated at 16rpm
for the first 7 days and then at 20rpm thereafter to keep
spheroids in the center of the rotational axis. Medium was
changed weekly in long-term cultures.
Light and fluorescence microscopy
Phase contrast photos were taken using a Canon PowerShot
A540 (Canon USA, Lake Success, NY) adapted to the micro-
scope eyepiece. Cell sizes were measured using a stage mi-
crometer. For fluorescence microscopy, cells were stained with
and Hoechstdye (2mg=mL; CalBiochem,San Diego, CA)in1%
bovineserum albumin=phosphate buffered salinefor 30min at
room temperature. Cells were then washed and mounted on
slides with Flouromount (SouthernBiotech, Birmingham, AL).
Spheroids were mounted as a wet preparation without a cov-
erslip to preserve the natural 3D architecture. Fluorescent
images were taken using a Zeiss Axioscope fluorescent mi-
croscope (Carl Zeiss, Oberkochen, Germany) and Orca ER
CCD digital camera (Hamamatsu, Bridgewater, NJ)
Microarray sample preparation and analysis
RNA from day 3 cultures of monolayers and spheroids
were amplified and biotinylated using the MessageAmp II-
Biotin Enhanced Kit per the manufacturer’s instructions
(Ambion, Austin, TX). Initial RNA integrity was verified and
final fragmented amplified RNA (aRNA) analyzed using the
Agilent 2100 Bioanalyzer (Santa Clara, CA). Fifteen mg of
biotinylated aRNA were hybridized onto the Human U133
Plus 2.0 Array (Affymetrix, Santa Clara, CA). Microarray
data were analyzed using GeneSpring 7.3 software (Silicon
Genetics, Redwood City, CA). Expression was normalized to
median and raw expression data cut off at 50 to eliminate
non-specific background. Significant genes were flagged
with present calls and demonstrated at least 2-fold changes
in expression between monolayers and spheroids.
Quantitative real-time reverse transcription
polymerase chain reaction
Two-step quantitative real-time reverse transcription poly-
day 3 monolayer and spheroid cultures as previously de-
scribed.25Selected genes were also tested on day 7 cultures (3-
hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR),
low-density lipoprotein receptor (LDLR), albumin (ALB), and
cytochrome P450 1A1 (CYP1A1)). The thermal profile was
508C for 2min, then 958C for 10min, followed by 40 amplifi-
cation cycles consisting of 958C for 30s, 608C for 30s, and 728C
for 1min.Relativequantification ofgenes was calculatedusing
the 2-(Ct gene 1 – Ct CPHI 1)-(Ct gene 2 – Ct CPHI 2)equation, where ‘‘Ct
gene 1’’ represented the calculated threshold cycle (Ct) of the
target gene in culture condition 1 (2D or 3D). ‘‘Ctgene 2’’ was
in each of the respective conditions. Cyclophilin expression
levels remained constant between TCD and RWV conditions
and between all time points tested.
Primers were designed using Oligo 6.0 software to span
introns and custom made by Operon (Huntsville, AL). See
Table 1 for primer sequences.
Cell aliquots were taken for Cyquant Assay following the
manufacturer’s instructions (Invitrogen).
Albumin enzyme-linked immunosorbent assay
Cell culture supernatants were collected and albumin
concentration determined by a human albumin enzyme-
linked immunosorbent assay (ELISA) kit per the manufac-
turer’s instructions (Bethyl, Montgomery, TX).
assay for CYP1A1activity
7-ethoxyresorufin-o-dealkylase (EROD) assays were per-
formed as previously described.7,26
Statistical analyses were performed with InStat 3.0 soft-
ware using the two-tailed Student t-test. Significance is
considered when p<0.05.
HepG2 cells cultured in the 3D environment of RWVs
form cellular spheroids with cortical actin organization
up to 500mm to 1mm in diameter
HepG2 cells cultured on TCDs proliferated as 2D clusters
and eventually became confluent as a monolayer (Fig. 1A,
C). In contrast, within the 3D environment of the RWV,
HepG2 cells formed 3D aggregates and spheroids. By day 3
of culture, visible spheroids up to 100mm in diameter were
formed within the RWV (Fig. 1B). Whereas cells grown in
TCDs were spread and flat, cells in the spheroids were round
560CHANG AND HUGHES-FULFORD
and had more compact cytoplasm. The spheroids increased
in size up to 500mm by day 7 (Fig. 1D), and long-term culture
created large spheroids up to 1mm in diameter (Fig. 2B).
Hoechst staining of nuclei demonstrated that cells were
spread out two-dimensionally on the TCD, but clustered
tightly three-dimensionally in the RWV spheroids (Fig. 1E, F).
Moreover, the actin cytoskeleton of cells in monolayers and
spheroids were remarkably different. Cells in monolayers
showed mainly F-actin stress fibers for attachment to the
surface. In contrast, cells in spheroids demonstrated cortical
actin organization outlining the cells (Fig. 1G, H).
Global gene expression analysis reveals differential
upregulation of structural genes in monolayers
and metabolic functional genes in spheroids
To investigate the underlying cellular and molecular differ-
ences between monolayers and spheroids, we performed a
global gene expression analysis using the whole human
genome microarray from Affymetrix. HepG2 cells were sub-
cultured on TCDs or in RWVs. Three days later, when 100-mm
spheroids were first being formed in the 3D cultures, RNA
from each condition was purified for microarray analysis.
Significant genes were filtered according to background
expression (raw expression greater than 50), presence of flags,
and at least 2-fold expression differences between the culture
conditions. Using these criteria, 250 genes were identified to
be upregulated in the monolayers. A distinct set of 210 genes
were upregulated in the spheroids. Close examination of the
biological functions of these differentially expressed genes
Table 1.Primers for qRT-PCR
Primer NamePrimer Sequence (50to 30)
CYP1A1 F- ggA gCT AgA CAC AgT gAT Tgg C
R- ggT gAA ggg gAC gAA ggA
F- AgA CAT TgT TCT ggT TgC CTA T
R- AAg ggT CAA ATA TCg CAC AT
F- CCA gAA gCA TgA gCg gAT gA
R- CgA CAg gAA CTT gCg gAT gT
F- ACC TTg AAg AAA gCg TCT CC
R- TCC TTg CCA gCg gTA gA
F- CAA TgT CTC ACC AAG CTC T
R- TCT gTC TCg Agg ggT AgC T
F- TAC CAT gTC Agg ggT ACg T
R- CAA gCC Tag AgA CAT AAT CAT
F- AgA gCC CTg ATT gAT ATg TA
R- gTT gCC AAC AAg gTA gTC TT
F- ATC AgT ggg CAC Agg TAA AA
R- TgA CCg AAT ACC gCA gTA g
F- CgT TCC CAA AgA gTT TAA TgC
R- AAg CTg CgA AAT CAT CCA TAA C
F- CAg gTC TCT CCg CTC ATC
R- TTC TCT ggC AAT CCg TTT CA
F- Tgg TCg TgT CCT TCg TCg TC
R- ggg CAC Tgg gTA gTT gTA g
F- gTC CCC CTg gCT CTg CTg gTT
R- Tgg gTA gAA ggA gAg TTT ggT A
F- gTg TgC CAg gAT ACA gC
R- TTg TgC CAg CCA TAg TCA
F- gCC CTg CCA ATC CCg ATg AA
R- CgC CgC CTC CgT ACA T
F- CAT Cgg CAA CAg CAT CgT
R- CgC CAg CAg TgA gTC gT
F- ggT ggg gAA CAA gTg TgA CAT
R- ggg TgT CCg AgA gAC gC
F- TTT CCT gAg TgA AgC ggT CT
R- gCA TCT gAg Tgg gCA ggT ACA C
F- TgA CTT CAC ACg CCA TAA Tg
R- CAC ATg CTT gCC ATC CAA CCA C
F, forward primer; R, reverse primer.
(HepG2) cells form cellular spheroids with cortical actin or-
ganization when cultured in the three-dimensional environ-
ment of rotating wall vessels (RWVs). HepG2 cells were
cultured on tissue culture dishes (TCDs) (A, C, E, G) or in
RWVs (B, D, F, H). Light-field microscopy was performed
after 3 days (A, B) or 7 days (C, D). Cells grown on TCDs had
spread cytoplasm and formed a monolayer by day 7. Spher-
oids in RWVs were up to 100mm in diameter by 3 days and
500mm by 7 days. Cells were stained with Hoechst dye for
of culture and visualized using fluorescence microscopy.
Whereas cells in monolayers formed F-actin stress fibers,
cells in spheroids had cortical actin organization. Magnifica-
Human liver hepatocellular carcinoma cell line
MONOLAYER AND SPHEROID GLOBAL GENE ANALYSIS561
revealed that significantly more genes related to ECM, cyto-
skeleton, and cell adhesion were expressed in monolayer cells
(Table 2). On the other hand, genes involved in liver-specific
in spheroids. In addition, more genes involved in cell cycle
and regulation of growth and proliferation were upregulated
in monolayers. There were also functional categories such as
apoptosis, signal transduction, and transcription in which the
number of genes differentially regulated were not significantly
different between the two conditions.
Representative genes identified using the microarray
analysis were verified with qRT-PCR. Genes upregulated
in spheroids (Fig. 3A) could be broadly categorized into
those involved in metabolic or synthetic pathways. In partic-
ular, expression of genes involved in xenobiotic metabolism
was markedly upregulated compared to monolayers, in-
cluding cytochrome P450 1A1 (CYP1A1) (10.6?2.8 fold),
aldo-keto reductase 1C1 (5.2?2.3 fold), and epoxide
hydrolase 1 (2.8?0.7 fold). Expression of leukotriene B412-
hydroxydehydrogenase, important for metabolic inactivation
pathways, two critical genes for glutathione synthesis, gluta-
ligase (2.7?0.5 fold), were highly induced in spheroids.
Transcription of albumin, an important liver-specific protein
product, was greater (1.4?0.3 fold). There was also a modest
but significant increase in the expression of genes necessary
for adenosine triphosphate (ATP) production (ATP synthase
(1.4?0.2 fold) and nicotinamide adenine dinucleotide dehy-
drogenase (1.3?0.2 fold)).
The expression of metabolic and synthetic functional
genes within spheroids changed differently with time.
Expression of key genes for cholesterol metabolism, low-
density lipoprotein receptor (LDLR) and HMG-CoA reduc-
tase (HMGCR), was comparable to that of controls in day 3
spheroids (Fig. 3) and significantly greater by day 7 (LDLR,
1.4?0.2 fold, and HMGCR, 2.0?0.4 fold, p<0.001). Ex-
pression of albumin transcripts, significantly higher in day 3
spheroids, was not different from that of controls by day 7
(0.9?0.3 fold, not statistically significant). Finally, the
marked 10-fold increase in CYP1A1 expression in spheroids
on day 3 was maintained in day 7 spheroids (10.1?1.0 fold,
There were also genes that were significantly up regulated
in monolayers. Specifically, levels of ECM genes were dra-
matically higher compared to spheroids (Fig. 3B). Expression
2 (versican) was higher in monolayers by 69.2?7.5 fold and
11.4?1.6 fold, respectively, compared to spheroids. Genes
important for cell-to-cell adhesion, such as E-cadherin (CDH1,
3.2?0.4 fold) and claudin 6 (component of tight junctions;
CLDN6, 3.5?0.8 fold), were also expressed significantly
higher in monolayers. In addition to structural genes, expres-
sion of RAB3B (7.5?1.2 fold) and AXL (6.0?1.7 fold), two
genes likely important in regulating cell differentiation and
proliferation, were also increased in monolayers.
Using qRT-PCR, we compared the expression of several
liver-enriched transcription factors, including forkhead box
protein A1 (FOXA1), FOXA2, FOXA3, FOXM1, hepatic nu-
clear factor 1 alpha (HNF1a), HNF4a, HNF6, and CCAAT-
enhancer-binding protein beta. None of these transcription
factors showed significant differential expression between
monolayers and spheroids (data not shown).
Overall proliferation is comparable between monolayer
and spheroid cultures, but liver metabolic and synthetic
functions are enhanced in spheroids
We tested whether differential gene expression between
2D and 3D cultures was reflected in the functional pheno-
types of HepG2 cells and compared their proliferation cul-
tured as monolayers or as spheroids. At 6h, 24h, 72h, and 6
days after subculture in the two conditions, cell numbers
were determined using the Cyquant cell proliferation assay
(Fig. 4). More cell cycle and proliferation genes were upre-
gulated in monolayers (e.g., AXL confirmed using qRT-PCR)
at 72h, correlating with the greater cell proliferation in
monolayers at this time point. However, there were not
consistent significant differences in overall proliferation
between 2D and 3D cultures. Cell numbers were higher in
the spheroid cultures at early time points but became lower
and equivalent to those in monolayers by 72h and 6 days,
tinued culture in rotating wall vessels (RWVs). Human liver
weeks in the three-dimensional (3D) environment of theRWV
bioreactor. Formed cellular spheroids were subcultured in
tissue culture dishes (TCDs) or RWVs for 7 days. Light mi-
croscopy showed disintegration of the spheroids in TCDs (A),
compared with intact spheroids that remained in the 3D en-
vironment of RWVs (B). Magnification 20?.
Spheroid cellular architecture is dependent on con-
2 Fold in Monolayers or Spheroids as Determined
by Microarray Analysis
Number of Genes Upregulated by at Least
Number of Genes
562CHANG AND HUGHES-FULFORD
Liver-specific metabolic and synthetic functions were
significantly higher in spheroids compared to monolayers.
7-ethoxyresorufin-o-dealkylase assays were performed in
7-day monolayers and spheroids to compare CYP1A1 activity
between 2D and 3D cultures. Cell numbers were determined
using the Cyquant assay to calculate the level of CYP1A1 ac-
tivity (i.e. amount of resorufin product formed) per cell.
HepG2 cells within the spheroids exhibited significantly
greater CYP1A1 metabolic activity than in monolayers (Fig.
5A). In addition, culture supernatants were collected and al-
bumin content measured using ELISA assay. Amount of al-
bumin was divided by cell number to determine the
significantly more albumin than in monolayers (Fig. 5B).
Spheroid morphology and enhanced liver functions
are dependent upon continuous culture within
the RWV bioreactor
To determine whether continuous culture within the RWV
was necessary to maintain spheroid architecture and en-
hanced liver-specific functions, we created long-term spher-
oid cultures for 6 to 10 weeks. Spheroids up to 1mm in size
were formed and then subcultured in TCDs or continued in
the 3D environment of RWVs. After 7 days, spheroids in
TCDs were completely dissociated without external disrup-
tion, and some cells appeared to be attaching to the surface
(Fig. 2A). In contrast, spheroids continued in the RWV
maintained their spheroid morphology (Fig. 2B).
Not only was continued culture within the RWV necessary
to maintain spheroid morphology, it was also important for
maintaining the enhanced liver-specific functions. After a 7-
day subculture of the spheroids in TCDs or RWVs, CYP1A1
activity and albumin production were measured. Spheroids
maintained a high level of cytochrome P450 metabolism and
albumin synthesis when cultured continuously in the RWV.
These functions were significantly lower in the cells of
spheroids transferred into TCDs (Fig. 6).
Understanding cell biology in 3D culture is an important
step toward tissue engineering solid organs. We compared
the morphology, global gene expression, and function of
verse transcription polymerase
chain reaction (qRT-PCR) of selected
genes shows that metabolic and
synthetic genes are upregulated in
spheroids, whereas structural genes
and certain oncogenes are upregu-
lated in monolayers. qRT-PCR was
performed on day 3 cultures of
monolayers and spheroids. Gene
expression was normalized to the
housekeeping gene cyclophilin, and
fold increase in one culture condi-
tion was calculated relative to the
other condition. Figure represents
pooled data from three independent
experiments with three independent
biological samples for each condi-
tion (n¼9). Gene symbols:
CYP1A1, cytochrome P450 1A1;
AKR1C1, aldo-keto reductase 1C1;
EPHX1, epoxide hydrolase
1; LTB4DH, leukotriene B412-
low-density lipoprotein receptor;
Quantitative real-time re-
glutaryl coenzyme Areductase; GSTA1, glutathioneS-transferase A1;GCLM,glutamate-cysteine ligase;ALB, albumin;ATP5I,
adenosine triphosphate synthase; NDUFA3, nicotinamide adenine dinucleotide dehydrogenase; COL1A1 – collagen type I,
alpha 1; CSPG2, chondroitin sulfate proteoglycan 2 (versican); CDH1, E-cadherin; CLDN6, claudin 6. *p<0.001; **p<0.005.
liver hepatocellular carcinoma cell line (HepG2) cells cul-
tured as monolayers and spheroids. HepG2 cells were grown
in tissue culture dishes or rotating wall vessels for 6h, 24h,
72h, or 6 days. At each time point, Cyquant DNA quantifi-
cation assays were performed to determine cell number.
Data are representative of three independent experiments.
Overall proliferation is comparable between human
MONOLAYER AND SPHEROID GLOBAL GENE ANALYSIS563
HepG2 monolayers and spheroids to determine the distinct
cellular and molecular responses to 2D and 3D environments.
We showed that culturing within RWV bioreactors is an
efficient method for generating large functioning hepatic
spheroids. Structural genes encoding ECM, cytoskeletal, and
adhesion proteins are upregulated in monolayers, whereas
metabolic and synthetic genes are upregulated in spheroids.
Hepatic spheroids formed in the RWV are larger than self-
assembled spheroids formed using other methods and are
not confined within the lattices of scaffolds. In RWV biore-
actors, spheroids are 100mm in diameter by day 3 of culture
and grow in size up to 1mm with long-term culture, 10-fold
larger than the spheroids formed using other culture tech-
niques.5,15–17RWV bioreactors can form larger spheroids
because cells are free to aggregate and grow three-dimen-
sionally without the constraints of surfaces and scaffolds.
Only diffusion of nutrients to the center and the increasing
shear force experienced by larger spheroids limit spheroid
size.20,21Previous studies showed that cell aggregates in
RWVs may reach 1 to 3mm in length, and there are no ap-
optotic centers in spheroids smaller than 1mm in diameter.19
The lack of necrosis in our spheroids was verified when they
were transferred into tissue culture dishes, because all cells
were viable when the spheroids self-disassembled.
In this study, we examined 3D cell biology of the spher-
oids formed in RWV bioreactors without confounding in-
teractions of scaffolding or surface materials. Through global
gene expression analysis and subsequent verification with
qRT-PCR, we identified several metabolic pathways that
are upregulated in spheroids. Genes involved in xenobiotic
metabolism are most markedly upregulated and expression
of genes involved in leukotriene metabolism, cholesterol
metabolism, glutathione synthesis, albumin synthesis, and
ATP synthesis are all significantly greater. This suggests that
spheroids are overall more metabolically active than mono-
layers. On the other hand, monolayers express significantly
higher levels of ECM genes, specifically collagen type I and
versican. Other cytoskeletal and adhesion genes, including
E-cadherin and claudin 6 (tight junction), are also signifi-
cantly upregulated in monolayers.
lular carcinoma cell line spheroids
cultured in rotating wall vessels
demonstrate better liver-specific
metabolic and synthetic functions
than in monolayers. (A) Cyto-
chrome P450 activity is greater in
spheroids. After 7 days of culture
as monolayers or spheroids, 7-
says were performed to measure
cytochrome P450 1A1 activity.
Cyquant assays determined cell
numbers in order to present data
as amount of resorufin product per
million cells. (B) Albumin produc-
tion was greater in spheroids.
After 7 days of culture, culture
supernatants were collected and
albumin quantified according to
assay. Cyquant assays were performed to determine cell numbers. Data represent concentration of albumin in culture
supernatant per million cells. Data are representative of three independent experiments. *p<0.001.
Human liver hepatocel-
specific functions when placed in
tissue culture dishes (TCDs). Hu-
man hepatocellular liver carcinoma
cell line cells were cultured for 6 to
10 weeks in the rotating wall vessels
(RWVs). Formed cellular spheroids
were subcultured in TCDs or RWVs
for 7 days. Cytochrome P450 activ-
ity as measured according to resor-
ufin formation (A) and albumin
production (B) was determined.
Data were adjusted per million cells
as determined using the Cyquant
assay. *p<0.005, **p<0.001.
Spheroids lose liver-
564CHANG AND HUGHES-FULFORD
We found that expression of various functional genes in
spheroids changed with time. Expression of LDLR and
HMG-CoA mRNA increased in spheroids from day 3 to 7.
CYP1A1 remained 10-fold higher in spheroids than in
monolayers during this period. Albumin gene expression
was higher in spheroids on day 3 and was the same as in
controls by day 7. The accumulated albumin protein in cul-
ture supernatants over 7 days was significantly (2.5 fold)
higher in spheroid cultures than in monolayers. Albumin is a
stable protein and has a serum half life of 20 days.27Albumin
protein synthesis is mostly regulated on the transcriptional
level,28–30and some post-transcriptional events also play a
role.31The accumulated greater albumin protein detected
in day 7 spheroid cultures reflected the continual expression
of albumin messenger RNA throughout the 7-day culture
period. Additional post-transcriptional regulation of albumin
synthesis may be involved and may be investigated in future
studies. Although metabolic and synthetic functions were
higher in RWVs for short-term (7 days) and long-term cul-
ture (6–10 weeks), P450 metabolic activity per cell within
spheroids increased with culture time length, and albumin
production per cell decreased (compare Fig. 5 and 6). This is
consistent with the observation that albumin transcript lev-
els in spheroids decreased with time while cytochrome
P450 1A1 transcripts remained markedly higher compared
to monolayers. It suggests that culture conditions that sup-
port long-term metabolic functions may be different from
those that support synthetic functions. RWV culture may be
more efficient at sustaining P450 activity than albumin syn-
It is interesting to compare our findings in RWV spheroids
with other models of 3D hepatocyte culture. HepG2 cells en-
capsulated within alginate demonstrated greater synthesis of
enhances synthetic and metabolic functions. In contrast, algi-
nate-encapsulated HepG2 showed greater proliferation when
cultured in a rotary culture system4and greater surrounding
ECM as measured using immunohistochemistry than when
cultured in monolayers.32In our system, proliferation of cells
cultured as spheroids was greater at early time points, less by
72h, and was comparable to that of monolayers by 6 days. In
addition, RWV spheroids had significantly lower expression
PCR than monolayers. It is likely that these differences be-
tween RWV spheroids and alginate encapsulation beads
reflect the differing physical culture environments of the two
systems. Alginate-encapsulated HepG2 cells have signifi-
cant interactions with the surrounding alginate. This cell–
scaffold interaction may have characteristics analogous to
2D culture in which cells adhere to an underlying sur-
face. Spheroids in RWVs, on the other hand, are cell aggre-
gates that have no exogenous scaffolding. RWV spheroids
have only cell–cell interactions, while alginate encapsulation
beads have cell–scaffold interactions that most certainly have
additional effects on cell shape and stretch that influence cell
A number of studies have shown that tension forces
and cell shape can determine cell fate in a variety of cell
types.33–36Markers of terminal differentiation are induced in
keratinocytes forced into a spherical shape, as opposed to
cells spread out on a substratum.35Human mesenchymal
stem cells differentiate into osteocytes when flat and spread
out, whereas round cells with limited surface contact un-
dergo adipogenesis.33Cell stretch, in particular, appears to
be an important signal for proliferation in endothelial and
smooth muscle cells.37,38The cytoskeleton and cell shape of
HepG2 cells may play a role in determining their distinct
global gene expression patterns as monolayers or spheroids.
Cells adherent to a surface may be stimulated to express
structural genes such as ECM and adhesion molecules,
whereas RWV spheroids switch off expression of these
structural genes and acquire more metabolic functions.
In addition to greater metabolic functions, 3D organization
of hepatocytes also induced development of more-differen-
tiated cell morphologies. HepG2 cells within alginate3or
porous polystyrene scaffolds39expressed large numbers of
microvilli and formed structures resembling canaliculi. Si-
milarly, previous studies of HepG2 cells and primary hepa-
tocytes cultured in RWVs showed that cells within spheroids
formed bile canaliculi with multiple tight junctions.19These
results demonstrate that more-complex cellular organization
can be achieved using various 3D culture strategies.
Through microarray analysis, we identified potentially
novel pathways that may be important in determining cell
function in 2D and 3D culture. RAB3B is a Ras-family
member small GTPase that is highly expressed in monolay-
ers and downregulated in spheroids. It was recently identi-
fied as one of the key genes regulating mesenchymal stem
cell differentiation40and a specific marker of liver tissue–
based stem cells (oval cells).41,42In addition, AXL, a tyrosine
kinase receptor and oncogene that mediates cell proliferation
and survival signals,43is also differentially upregulated in
monolayers compared to spheroids. The absence of consis-
tent significant differences in proliferation between mono-
layers and spheroids through the various time points tested,
however, may reflect the redundancy of proliferative signals
in HepG2 cells.
RWV spheroids demonstrated enhanced cytochrome P450
6 to 10 weeks. Moreover, the functional phenotype of spher-
oids requires continued culture within a 3D environment.
Enhanced metabolic functions of spheroids are lost when
to their physical environment and modify their gene expres-
sion accordingly. This has important implications for future
tissue engineering efforts of 3D solid organs. In addition to
biochemical signals, physical signals can be manipulated to
achieve thedesired cellular functions andresponses. Our data
suggest that hepatocytes stretched against bioscaffolds may
produce ECM, whereas unstretched cells in the interior are
likely to be more metabolically active. Within a 3D structure,
cells may demonstrate different functional phenotypes de-
important for tissue engineering and for understanding nor-
mal cellular processes in vivo.
This work was funded by the American College of Sur-
geons Research Scholarship and supported by the Veterans
Affairs Medical Center. We thank Dr. Lygia Stewart and
Dr. Carlos U. Corvera for their kind support of this effort.
MONOLAYER AND SPHEROID GLOBAL GENE ANALYSIS565
1. Port, F.K., Dykstra, D.M., Merion, R.M., and Wolfe, R. 2004
Annual Report of the U.S. Organ Procurement and Trans-
plantation Network and the Scientific Registry of Transplant
Recipients: Trasplant Data 1994-2003. Department of Health
and Human Services, Division of Transplantation, Rockville,
MD; United Network for Organ Sharing, Richmond, VA;
University Renal Research and Education Association, Ann
Arbor, MI, 2004.
2. Kulig, K.M., and Vacanti, J.P. Hepatic tissue engineering.
Transpl Immunol 12, 303, 2004.
3. Khalil, M., Shariat-Panahi, A., Tootle, R., Ryder, T.,
McCloskey, P., Roberts, E., Hodgson, H., and Selden, C.
Human hepatocyte cell lines proliferating as cohesive
spheroid colonies in alginate markedly upregulate both
synthetic and detoxificatory liver function. J Hepatol 34, 68,
4. Coward, S.M., Selden, C., Mantalaris, A., and Hodgson, H.J.
Proliferation rates of HepG2 cells encapsulated in alginate
are increased in a microgravity environment compared with
static cultures. Artif Organs 29, 152, 2005.
5. Dvir-Ginzberg, M., Gamlieli-Bonshtein, I., Agbaria, R., and
Cohen, S. Liver tissue engineering within alginate scaffolds:
effects of cell-seeding density on hepatocyte viability, mor-
phology, and function. Tissue Eng 9, 757, 2003.
6. Dvir-Ginzberg, M., Elkayam, T., Aflalo, E.D., Agbaria, R.,
and Cohen, S. Ultrastructural and functional investigations
of adult hepatocyte spheroids during in vitro cultivation.
Tissue Eng 10, 1806, 2004.
7. Seo, S.J., Choi, Y.J., Akaike, T., Higuchi, A., and Cho, C.S.
Alginate=galactosylated chitosan=heparin scaffold as a new
synthetic extracellular matrix for hepatocytes. Tissue Eng 12,
8. Verma, P., Verma, V., Ray, P., and Ray, A.R. Formation and
characterization of three dimensional human hepatocyte cell
line spheroids on chitosan matrix for in vitro tissue engi-
neering applications. In Vitro Cell Dev Biol Anim 2007.
9. Kim, S.S., Sundback, C.A., Kaihara, S., Benvenuto, M.S.,
Kim, B.S., Mooney, D.J., and Vacanti, J.P. Dynamic seeding
and in vitro culture of hepatocytes in a flow perfusion sys-
tem. Tissue Eng 6, 39, 2000.
10. Huang, H., Hanada, S., Kojima, N., and Sakai, Y. Enhanced
functional maturation of fetal porcine hepatocytes in three-
dimensional poly-L-lactic acid scaffolds: a culture condition
suitable for engineered liver tissues in large-scale animal
studies. Cell Transplant 15, 799, 2006.
11. Torok, E., Vogel, C., Lutgehetmann, M., Ma, P.X., Dandri, M.,
Petersen, J., Burda, M.R., Siebert, K., Dullmann, J., Rogiers, X.,
and Pollok, J.M. Morphological and functional analysis of rat
hepatocyte spheroids generated on poly(L-lactic acid) poly-
mer in a pulsatile flow bioreactor. Tissue Eng 12, 1881, 2006.
12. Mooney, D.J., Sano, K., Kaufmann, P.M., Majahod, K.,
Schloo, B., Vacanti, J.P., and Langer, R. Long-term engraft-
ment of hepatocytes transplanted on biodegradable polymer
sponges. J Biomed Mater Res 37, 413, 1997.
13. Fiegel, H.C., Havers, J., Kneser, U., Smith, M.K., Moeller, T.,
Kluth, D., Mooney, D.J., Rogiers, X., and Kaufmann, P.M.
Influence of flow conditions and matrix coatings on growth
and differentiation of three-dimensionally cultured rat he-
patocytes. Tissue Eng 10, 165, 2004.
14. Velema, J., and Kaplan, D. Biopolymer-based biomaterials as
scaffolds for tissue engineering. Adv Biochem Eng Bio-
technol 102, 187, 2006.
15. Abu-Absi, S.F., Hu, W.S., and Hansen, L.K. Dexamethasone
effects on rat hepatocyte spheroid formation and function.
Tissue Eng 11, 415, 2005.
16. Koide, N., Sakaguchi, K., Koide, Y., Asano, K., Kawaguchi,
M., Matsushima, H., Takenami, T., Shinji, T., Mori, M., and
Tsuji, T. Formation of multicellular spheroids composed of
adult rat hepatocytes in dishes with positively charged
surfaces and under other nonadherent environments. Exp
Cell Res 186, 227, 1990.
17. Du, Y.,Han,R.,Ng,S.,Ni,J.,Sun,W.,Wohland,T.,Ong,S.H.,
Kuleshova, L., and Yu, H. Identification and characterization
of a novel prespheroid 3-dimensional hepatocyte monolayer
on galactosylated substratum. Tissue Eng 13, 1455, 2007.
18. Yoffe, B., Darlington, G.J., Soriano, H.E., Krishnan, B., Risin,
D., Pellis, N.R., and Khaoustov, V.I. Cultures of human liver
cells in simulated microgravity environment. Adv Space Res
24, 829, 1999.
19. Khaoustov, V.I., Darlington, G.J., Soriano, H.E., Krishnan, B.,
Risin, D., Pellis, N.R., and Yoffe, B. Induction of three-
dimensional assembly of human liver cells by simulated
microgravity. In Vitro Cell Dev Biol Anim 35, 501, 1999.
20. Unsworth, B.R., and Lelkes, P.I. Growing tissues in micro-
gravity. Nat Med 4, 901, 1998.
21. Hammond, T.G., and Hammond, J.M. Optimized suspen-
sion culture: the rotating-wall vessel. Am J Physiol Renal
Physiol 281, F12, 2001.
22. Elkayam, T., Amitay-Shaprut, S., Dvir-Ginzberg, M., Harel,
T., and Cohen, S. Enhancing the drug metabolism activities
of C3A—a human hepatocyte cell line—by tissue engineer-
ing within alginate scaffolds. Tissue Eng 12, 1357, 2006.
23. Hughes-Fulford, M. Signal transduction and mechanical
stress. Sci STKE 2004, RE12, 2004.
24. Ingber, D.E. Cellular mechanotransduction: putting all the
pieces together again. Faseb J 20, 811, 2006.
25. Li, C.F., and Hughes-Fulford, M. Fibroblast growth factor-2
is an immediate-early gene induced by mechanical stress in
osteogenic cells. J Bone Miner Res 21, 946, 2006.
26. Donato, M.T., Gomez-Lechon, M.J., and Castell, J.V. A mi-
croassay for measuring cytochrome P450IA1 and P450IIB1
activities in intact human and rat hepatocytes cultured on
96-well plates. Anal Biochem 213, 29, 1993.
27. Peters, T.J. All About Albumin: Biochemistry, Genetics, and
Medical Applications. San Diego, CA: Academic Press, 1996.
28. Kimball, S.R., Horetsky, R.L., and Jefferson, L.S. Hormonal
regulation of albumin gene expression in primary cultures of
rat hepatocytes. Am J Physiol 268, E6, 1995.
29. Lloyd, C.E., Kalinyak, J.E., Hutson, S.M., and Jefferson, L.S.
Stimulation of albumin gene transcription by insulin in pri-
mary cultures of rat hepatocytes. Am J Physiol 252, C205,
30. Plant, P.W., Deeley, R.G., and Grieninger, G. Selective block
of albumin gene expression in chick embryo hepatocytes
cultured without hormones and its partial reversal by in-
sulin. J Biol Chem 258, 15355, 1983.
transcriptional regulation of albumin gene expression in Xe-
nopus liver. Embo J 4, 1261, 1985.
32. Selden, C., Khalil, M., and Hodgson, H. Three dimensional
culture upregulates extracellular matrix protein expression
in human liver cell lines—a step towards mimicking the
liver in vivo? Int J Artif Organs 23, 774, 2000.
33. McBeath, R., Pirone, D.M., Nelson, C.M., Bhadriraju, K., and
Chen, C.S. Cell shape, cytoskeletal tension, and RhoA reg-
ulate stem cell lineage commitment. Dev Cell 6, 483, 2004.
566CHANG AND HUGHES-FULFORD
34. Roskelley, C.D., Desprez, P.Y., and Bissell, M.J. Extracellular
matrix-dependent tissue-specific gene expression in mam-
mary epithelial cells requires both physical and biochemical
signal transduction. Proc Natl Acad Sci U S A 91, 12378,
35. Watt, F.M., Jordan, P.W., and O’Neill, C.H. Cell shape con-
trols terminal differentiation of human epidermal keratino-
cytes. Proc Natl Acad Sci U S A 85, 5576, 1988.
36. Dike, L.E., Chen, C.S., Mrksich, M., Tien, J., Whitesides,
G.M., and Ingber, D.E. Geometric control of switching be-
tween growth, apoptosis, and differentiation during angio-
genesis using micropatterned substrates. In Vitro Cell Dev
Biol Anim 35, 441, 1999.
37. Chen, C.S., Mrksich, M., Huang, S., Whitesides, G.M., and
Ingber, D.E. Geometric control of cell life and death. Science
276, 1425, 1997.
38. Liu, W.F., Nelson, C.M., Tan, J.L., and Chen, C.S. Cadherins,
RhoA, and Rac1 are differentially required for stretch-medi-
ated proliferation in endothelial versus smooth muscle cells.
Circ Res 101, e44, 2007.
39. Bokhari,M., Carnachan,R.J., Cameron,N.R.,andPrzyborski,
S.A. Culture of HepG2 liver cells on three dimensional poly-
styrene scaffolds enhances cell structure and function during
toxicological challenge. J Anat 211, 567, 2007.
40. Song, L., Webb, N.E., Song, Y., and Tuan, R.S. Identification
and functional analysis of candidate genes regulating
mesenchymal stem cell self-renewal and multipotency.
Stem Cells 24, 1707, 2006.
41. Batusic, D.S., Cimica, V., Chen, Y., Tron, K., Hollemann, T.,
Pieler, T., and Ramadori, G. Identification of genes specific
to ‘‘oval cells’’ in the rat 2-acetylaminofluorene=partial
hepatectomy model. Histochem Cell Biol 124, 245, 2005.
42. Corcelle, V., Stieger, B., Gjinovci, A., Wollheim, C.B., and
Gauthier, B.R. Characterization of two distinct liver pro-
genitor cell subpopulations of hematopoietic and hepatic
origins. Exp Cell Res 312, 2826, 2006.
43. Hafizi, S., and Dahlback, B. Signalling and functional di-
versity within the Axl subfamily of receptor tyrosine
kinases. Cytokine Growth Factor Rev 17, 295, 2006.
Address reprint requests to:
Tammy T. Chang, M.D., Ph.D.
Veterans Affairs Medical Center, San Francisco
4150 Clement Street
Building 1, Room 110
San Francisco, CA 94121
Received: December 12, 2007
Accepted: June 2, 2008
Online Publication Date: August 22, 2008
MONOLAYER AND SPHEROID GLOBAL GENE ANALYSIS567
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