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A New Dimension in
Modeling Infectious Disease
Three-dimensional cell cultures closely mimic in vivo tissues, offering a
powerful new means for studying infectious diseases
Cheryl A. Nickerson and C. Mark Ott
ultured mammalian cells, including
primary cultures, organ cultures,
and continuous cell lines, have long
been used to investigate host-pathogen interactions that lead to infections. While these models continue to contribute
to our understanding of infectious diseases, each
has inherent limitations, raising questions about
their relevance or practicality.
Hence, we and other researchers are moving
toward developing novel physiologically relevant, three-dimensional (3-D) tissue culture
models and away from the decades-old standard
that consisted of monolayer cultures of mammalian cells grown in two dimensions on plastic or
glass. As compared to standard monolayers,
these newer, 3-D cell cultures contain structural
and functional properties that more closely approximate conditions that pathogenic microorganisms encounter in the host tissues they infect.
One popular option for generating 3-D cells
that retain a differentiated phenotype is to embed them in an extracellular matrix of structural proteins such as collagen and laminin. In
another approach, we use rotating-wall vessel
(RWV) bioreactors, designed by investigators at
the National Aeronautics and Space Administration (NASA), to grow 3-D cell cultures that
faithfully model structures and behaviors that
occur in tissues in vivo. In addition to providing physiologically relevant models of human
tissues, this novel approach confers other important advantages when studying tissue-level
mechanisms of infectious disease. In particular,
these 3-D cell cultures represent a highly controlled and reproducible model that can create
extremely high n values per experiment, and
C
enormous numbers of cells which can be studied
by techniques not possible in matrix culture and
somewhat limited in monolayer culture.
Problems in Using Traditional 2-D
Cultures in Infectious Disease Studies
Researchers who use primary cell and organ
cultures to study infectious diseases have recognized several important limitations associated
with these systems. For instance, the mitotic
potential of cells growing in such culture systems is limited, thereby restricting experiments
to short-term studies, causing reproducibility
problems, and significantly increasing costs.
Continuous cell lines solve many of the challenges associated with using primary cell and
organ cultures to study infectious diseases.
However, problems are also associated with using continuous cell lines, including the fact that
they are not truly representative of the in vivo
tissues from which they derive. In particular,
propagating such cell lines as standard, 2-D
monolayers on impermeable surfaces prevents
cells from differentiating and acquiring polarity.
These steps are blocked because cells grown as
monolayers on flat plastic or glass surfaces cannot form the cell-cell and cell-matrix interactions as they would in their parental tissues.
In the body, tissues are found as well-organized 3-D structures that are critical for establishing and maintaining differentiated form and
functions. Accordingly, flat layers of cells being
cultured under standard 2-D conditions differ
physiologically from their in vivo counterparts.
Many of these differences are believed to result when cells dissociate from their native 3-D
tissue structure in vivo as they are made to
Cheryl A. Nickerson
is Associate Professor in the Department of Microbiology and
Immunology at the
Tulane University
Medical Center,
New Orleans, La.
C. Mark Ott is the
Laboratory Manager
at the Microbiology
Laboratory at the
NASA Johnson
Space Center,
Houston, Tex. This
article is based on a
presentation during
the 103rd ASM
General Meeting,
Washington, D.C.,
May, 2003.
Volume 70, Number 4, 2004 / ASM News Y 169
optimized technology for growing
3-D cells under conditions that
maintain many of the specialized features of in vivo tissues (Fig.1A).
The primary advantage of the
RWV over either dynamic or static
tissue culture systems is that its lowshear environment allows cells to aggregate, grow three-dimensionally,
and differentiate. Within RWV bioreactors, cells are maintained in a
gentle fluid orbit, enabling them to
attach to one another and to form
the fragile connections that are required for complex 3-D structures
and to attain a more tissue-like phenotype. Thus, unlike cell and tissue
cultures grown in standard 2-D
monolayers, cells cultured in the
RWV are structurally and functionally similar to in vivo tissues.
Moreover, because of their lowshear, low-turbulence operation,
RWV bioreactors minimize mechanical cell damage and thus largely
solve many challenges associated
with suspension culture. That is,
(A) The operation of the RWV. The cylindrical culture vessel is filled with growth medium and the
cells and microcarriers can be suscells are added. All air bubbles are removed from the culture vessel. The vessel is attached to
the rotator base and rotated about the horizontal axis (power supply not shown). Cell aggregate
pended without turbulence, while
particles establish a fluid orbit within the culture medium in the rotating vessel. As 3-D tissues
providing adequate nutrition and
grow in size, the rotation speed is adjusted to compensate for the increased settling rates of the
oxygenation. The low-shear condilarger particles. Oxygen supply and carbon dioxide removal is achieved through a gas-permeable
silicone rubber membrane at the back of the bioreactor. (B) Cells cultured in the RWV are
tions in RWV bioreactors are
maintained in a gentle fluid orbit. Cell aggregates establish a fluid orbit within the culture medium
thought to be similar to particular
in the RWV. The sedimentation of the cells within the vessel is offset by the rotating fluid,
environments in the body, such as becreating a constant free fall of the cells through the culture medium. (C) Depiction of a single
collagen-coated microcarrier bead to which cells have attached on the surface. The microcarrier
tween the brush border microvilli of
beads are porous, which allows for the establishment of an internal compartment (i.e., the lumenal
epithelial cells, that are encountered
space) in the 3-D cells, which is essential for the function and architecture of epithelial cells in vivo.
by numerous bacterial pathogens during infections of the gastrointestinal,
respiratory, and urogenital tracts.
propagate as 2-D monolayers on impermeable surfaces.
The principal design feature of the RWV bioTreating vessel surfaces with matrix products such as collagen or
reactor is based on a horizontally rotating vessel
growing cells on collagen-coated inserts can enhance cell-cell and
that is bubble-free when filled with culture mecell-matrix interactions and preserve some aspects of the differentidium. As it rotates, both the wall and fluid rotate
ated phenotype. However, these systems do not fully model the
at the same angular rate, as if they formed a solid
complex 3-D cell architecture that is important for the function of in
body. The sedimentation of the cells within the
vivo tissues. Moreover, researchers observe several important differvessel is offset by the rotating fluid, creating a
ences between the pathogenesis of microorganisms in human infecconstant free fall of cells through the culture
tions and in commonly used cell culture models.
medium (Fig. 1B). This constant free fall
through the medium facilitates nutrient exchange and localized mixing. Oxygen is proRWV Apparatus To Generate 3-D Cell Cultures
vided to the aggregates through a hydrophobic
NASA engineers designed the RWV bioreactor in the early 1990s to
membrane on the back of the bioreactor.
model effects of weightlessness on cells. That bioreactor led to an
Ample oxygen and nutrient exchange allow
FIGURE 1
170 Y ASM News / Volume 70, Number 4, 2004
these aggregates to grow to a substantial size.
Limited shear in the vessel not only reduces cell
damage but also facilitates 3-D aggregation
based on natural cellular affinities. By manipulating the rotational velocity, the cell aggregates
can be maintained in suspension with minimal
contact against the walls of the reactor. The
result is assembly of 3-D tissue-like aggregates—
sometimes called “organoids”—that closely resemble natural tissues.
These 3-D tissue cultures are being used in a
wide variety of fundamental biomedical applications besides infectious disease research, including studying immune-cell interactions,
growing tissues for transplantation, cancer biology, developing and testing novel therapeutic
drugs, angiogenesis, toxicology, and producing
a variety of important products, such as antibodies, hormones, and vitamins. Researchers
have published many peer-reviewed articles about
3-D cell culture in RWV bioreactors and, to date,
more than 50 normal and transformed cell types
have been grown under such conditions.
Applying 3-D Cell Cultures to the Study of
Infectious Diseases
One advantage of cell culture in the RWV is its
simplicity. Cells that are to be cultured in RWV
bioreactors are first grown as monolayer cultures. When cells reach an appropriate density,
they are removed from their flasks, resuspended
in fresh medium, and incubated with dextran
microcarrier beads that are coated with collagen
or another suitable extracellular matrix protein
to allow for cell attachment (Fig. 1C).
Cells that are cultured on microcarrier beads
represent a dual-chamber system where the porous bead matrix constitutes a second compartment that is absent in standard flat monolayer
cultures. This second chamber (i.e., the internal
compartment within the porous microcarrier) is
important because it is highly likely that ion and
solute gradients established by the 3-D cells
between these two compartments contribute to
the establishment of differentiated phenotypes.
(This concept is depicted in an animated supplement to the online version of this article.) Once
cell-bead complexes are introduced into the RWV
vessel, it is made to rotate. The medium subsequently is changed as necessary, and the rotation
speed is increased as aggregates develop to maintain the cells in free-falling suspension.
When used in infection studies, the 3-D cells
are removed from the bioreactor and distributed
evenly in wells of tissue culture plates. In this
regard, the 3-D cells are treated in the same
manner as monolayers, except care should be
taken when removing media and washing the
cells because they do not adhere to the wells.
The 3-D cells can also be treated to remove them
from beads for studies that require homogeneous cell suspensions, such as when flow cytometry analysis is being used. If warranted,
studies also may be conducted on cells while
they are in the bioreactor.
RWV bioreactor-grown cells are being used
to study microbial pathogenesis, providing
physiologically relevant model systems. For example, we have developed a variety of different
3-D cell culture models of human cells and tissues that recapitulate many aspects of cellular
structure, differentiation, and function that occur in vivo, and which are currently being used
in infection studies by us and our colleagues.
The key component of each of these models is a
novel 3-D architecture generated in the RWV
bioreactor. In each of these models, the 3-D cells
display a phenotype that more closely approximates the parental in vivo tissue than do the
same cells grown as standard monolayers.
Modeling Salmonella Pathogenesis
Our laboratory reported the first use of 3-D cell
cultures of human intestinal epithelial cells
generated in RWV bioreactors for studying the
enteric bacterial pathogen Salmonella enterica
serovar Typhimurium. S. enterica serovar
Typhimurium is among the most common
causes of foodborne infections resulting in gastroenteritis and diarrheal disease in humans
worldwide, making it a major public health
problem. An essential feature of the pathogenicity of Salmonella is its interaction with host
intestinal epithelial cells. However, investigators
studying such interactions have relied primarily
on animal models and conventional tissue cultures of human cell lines, neither of which accurately duplicates human intestinal mucosa.
Thus, we developed 3-D cell culture models of
human small intestinal epithelium that approximate the behavior of in vivo tissues, bringing
these models closer to the environment encountered by Salmonella during the natural course of
infections. Specifically, we used the human in-
Volume 70, Number 4, 2004 / ASM News Y 171
FIGURE 2
Transmission electron micrographs of 3-D Int-407 aggregates as compared to Int-407
monolayers. This figure shows TEM images of Int-407 monolayers (A) and 3-D Int-407
cells (B). The monolayers demonstrated inferior tight junction formation as compared to
the 3-D aggregates (concave arrows). Note the presence of numerous, well-formed
vacuolar-like structures in the 3-D aggregates as compared to their poorly developed
counterparts in the monolayers (arrow heads). (C-F) Scanning electron micrographs of
uninfected and Salmonella-infected Int-407 monolayers and 3-D Int-407 cell aggregates
(2,000X). Uninfected monolayer control (C). At 2 hours postinfection, Int-407 cell
monolayers exhibit major loss of structural integrity with numerous membrane alterations including blebs and the formation of pathological lesions, as well as some surface
bound bacteria (D). Uninfected 3-D Int-407 control showing dense masses of cells with
extracellular secretions (E). At 2 hours postinfection, the surface of the 3-D Int-407 cells
is quite irregular and associated with what appear to be numerous surface lesions. (F).
Note that the 3-D Int-407 cell aggregates infected with Salmonella do not display as
extensive loss of structural integrity as observed for infected monolayers following the
same time-course of infection. Few surface-bound bacteria are observed. (Reprinted
with permission from ASM, Nickerson et al., Infect. Immun. 69:7106 –7120).
testinal epithelial cell line Int-407, which is one of the most extensively studied for this purpose. However, when grown in monolayers,
Int-407 cells are poorly differentiated, lack polarity, do not produce
mucus, and lack other physiological features associated with their
172 Y ASM News / Volume 70, Number 4, 2004
in vivo counterparts, including tight
junctions, desmosomes, and secretory
granules.
Thus, the Int-407 cell line provided
us an excellent opportunity for determining whether the RWV bioreactors
would enable such cells to differentiate
into 3-D tissue-like masses that more
accurately model in vivo human intestinal epithelial tissues. On the basis of immunohistochemical, histological, and
morphological analyses, the Int-407
cells that we grew in the RWV bioreactors indeed formed 3-D aggregates that
retain many in vivo features.
For example, unlike monolayers,
these 3-D cells exhibit inherent apical
and basolateral polarity, and also form
extensive tight junctions and desmosomes, which are important for organizing and maintaining normal epithelial structure (Fig. 2A-B). Moreover,
according to immunofluorescence and
light microscopy studies, the 3-D cells
express numerous markers of epithelial
differentiation, including type IV collagen, laminin, cadherin, desmoplakin
(Fig. 3A), ZO-1, epithelial-specific antigen, villin, vimentin, fibronectin, the
M-cell glycoconjugate sialyl Lewis A
antigen, and a variety of cytokeratins.
In addition, these 3-D Int-407 cells exhibit well-formed and numerous microvilli at apical cell surfaces, abundant
and well-developed vacuoles (Fig. 2AB), and produce mucus.
All of these are important physiological features of in vivo tissues which
were either absent or not expressed or
distributed at physiologically relevant
levels in monolayer cultures of the same
cells. Moreover, cytokeratins 18 and
19, which are markers for tumor and
undifferentiated cells, are down-regulated in 3-D Int-407 aggregates compared to monolayers. This finding is
important because the Int-407 small intestinal epithelial cell line also contains
HeLa cells that derive from a human
cervical adenocarcinoma cell line.
3-D Cultured Cells Outperform 2-D Cells
as Infection Models
When we infected 3-D Int-407 cells with S.
enterica serovar Typhimurium, we noticed im-
portant differences and a closer resemFIGURE 3
blance to typical in vivo infections compared to the same infections in
monolayer cells, including differences
in tissue pathology, adherence, invasion, apoptosis, and expression of cytokines. A representative confocal image
of the 3-D Int-407 cells infected with
serovar Typhimurium shows the complex, tissue-like arrangement of these
cells (Fig. 4D).
We also used scanning electron microscopy (SEM) to examine surface interactions and membrane structural
alterations during the course of Salmonella infections of 3-D and monolayer
Int-407 cells. Following the same time
course, the 3-D Int-407 cells display
minimal loss of structural integrity and
more rapid recovery of cell structure,
whereas their monolayer counterparts
show a major loss of structural integrity
(Fig. 2C-F). The response of the 3-D
Int-407 cells following infection is relevant to that observed in vivo, where
serovar Typhimurium-induced damage
(A) Immunofluorescent image of 3-D Int-407 intestinal cell aggregates stained with a
to the small intestinal epithelium is
monoclonal antibody against desmoplakin. Note the enrichment of the intercellular
junction marker, desmoplakin, a protein component of desmosomes, at cell-cell borders
short-lived, followed by rapid repair of
(see arrows), especially where multiple cell aggregates express desmoplakins. Magnifidamaged cells along with restoration of
cation, x400. Arrowheads point to desmoplakin associated with cell-cell interfaces. The
the mucosal surface.
150␮m carrier beads are delineated by a dotted outline. (B) Immunofluorescent image of
3-D HT-29 colon cell aggregates stained with a monoclonal antibody against villin. HT-29
Additional results from adherence,
3-D aggregates show well-organized staining, and clear junction association for the
invasion, and apoptosis studies support
cytoskeletal marker villin (an abundant protein in the brush border of intestinal epithelial
the tissue pathology observations recells). Arrowheads point to villin associated with cell-cell interfaces. (C) Immunofluorescent image of 3-D A549 lung cell aggregates stained with a monoclonal antibody against
garding epithelial damage. Specifically,
occludin. The distribution of the tight junction-associated protein occludin in the 3-DSalmonella are less able to adhere to
cultured A549 cells shows a concentration of this protein at epithelial junctions
and invade 3-D Int-407 cells, compared
(arrowheads), which is reflective of stabilization of tight junctions. (D) Immunofluorescent
image of 3-D SGHPL-4 placental extravillous cytotrophoblast cell aggregates stained with
to infected monolayers. Moreover,
an antibody against collagen IV. SGHPL-4 cells were derived from first trimester chorionic
there was a rapid onset of apoptosis
villous explants from a normal, healthy human placenta. The 3-D aggregates express
following Salmonella infection of Intcollagen type IV (see arrowheads), an important extracellular matrix protein involved in
cytotrophoblast differentiation.
407 monolayers, with 70 –90% of cells
undergoing apoptosis within 90 minutes. In contrast, there was no difference in apoptosis between infected and uningrowth factor-␤1 (TGF-␤1) mRNA and prostafected 3-D Int-407 cells during this same
glandin E2 (PGE2) as compared to uninfected
monolayers. Both of these molecules have the
postinfection period. Considering that fewer
potential to limit damage to the intestinal epithan 5% of cases of Salmonella-induced gastrothelium following infection with invasive enteric
enteritis are reported, it seems unlikely that bebacteria such as Salmonella. TGF-␤1 frequently
tween 70 –90% of human intestinal epithelial
serves in an immunosuppressive role. The encells would undergo apoptosis after infection
hanced basal level of expression of TGF-␤1 in
with this pathogen.
the uninfected 3-D Int-407 cells as compared
In further support of these tissue pathology
to monolayers resembles conditions found in
studies, uninfected 3-D Int-407 cells constituvivo, where the intestinal mucosa constitutively
tively express higher levels of transforming
Volume 70, Number 4, 2004 / ASM News Y 173
FIGURE 4
(A, B) Monolayers and 3-D-aggregates, respectively, of the human colon cell line HT-29, infected with S. enterica serovar Typhimurium.
HT-29 cells were infected with S. typhimurium and incubated for 1hr at 37°C. The actin filaments are stained with phalloidin (red), the cell
nucleus with DAPI (blue), and serovar typhimurium with a FITC-conjugated LPS antibody (green). The arrowheads point to actin accumulation
near the site of Salmonella invasion. Note the complex tissue-like architecture which is clearly evident in the 3-D HT-29 cells as compared
to monolayers of the same cells. (C) Immunofluorescent image of SGHPL-4 human placental 3-D-aggregates infected with human
cytomegalovirus (HCMV). SGHPL-4 3-D cells were infected with HCMV and incubated for 48 hours at 37°C. The 3-D cells stain positive for
the nuclear HCMV immediate early proteins (IE1/2, red), indicating that the cells are permissive for in vitro HCMV infection. Cell nuclei are
stained with DAPI (blue). Magnification, x100. (D) Confocal image of Int-407 3-D cells following infection with serovar Typhimurium.
Phalloidin labeling of the actin cytoskeleton of 3-D Int-407 cells (red) at 90 minutes after infection with GFP-labeled serovar Typhimurium
(green). Reprinted with permission from Synthecon, Inc. (E-F) SEM analysis of 3-D A549 human lung aggregates before and after infection
with Pseudomonas aeruginosa. Panels E–F correspond to uninfected 3-D aggregates (E), and 3-D aggregates 6 hours postinfection with P.
aeruginosa (F). Magnification, x2,000. Note the complex tissue-like architecture in these cells as well as the clear difference in tissue
morphology between the uninfected 3-D control (E) and 6 hour infected 3-D cells (F). The arrowhead points to a Pseudomonas bacterium
on the surface of the 3-D cells.
produces high levels of this cytokine. Prostaglandins may protect the mucosa by downregulating proinflammatory cytokines such as
interleukin-1 (IL-1). Indeed, we observed that
IL-1␣ and IL-1␤ mRNA expression were upregulated in Int-407 monolayers compared to
3-D aggregates following Salmonella infection.
The results from these studies are in agreement
with a role for prostaglandins in mediating a
protective response against Salmonella-induced
damage to the epithelial mucosa.
Versatility of 3-D Cells for Modeling Other
Infectious Diseases
Based on our studies of Salmonella infection of
3-D intestinal epithelial cells, we believe that
174 Y ASM News / Volume 70, Number 4, 2004
RWV bioreactors could be used to generate 3-D
aggregates from a variety of other cell types to
model many other infectious diseases. Hence,
we have been generating other 3-D cell cultures
from different human tissues, including the colon, lung, placenta, bladder, and periodontal
ligaments that are currently being used in collaborative infectious disease studies.
Specifically, the 3-D colon cells are being used
to study infections with Salmonella sp., Vibrio
sp., and other enteric pathogens. Meanwhile the
3-D lung cells are being used to study infections caused by Pseudomonas aeruginosa and
Klebsiella pneumoniae, while the 3-D placental
tissues are serving as models of human cytomegalovirus infections during pregnancy. Additionally, the 3-D bladder cells are being used to
investigate host-pathogen interactions with uropathogenic Escherichia coli, and the 3-D periodontal ligament cells are serving as useful models to study Porphymonas gingivalis and other
pathogens of the oral cavity.
On the basis of immunohistochemical profiling, each of these newer 3-D cell cultures is
already proving to be representative of their
respective in vivo parental tissues (Fig. 3B–D).
Indeed, numerous markers appear to be distributed in a more physiologically relevant manner
in 3-D cells compared to those grown in monolayer cultures on collagen-coated vessels. We
have used fluorescent staining and SEM to study
several pathogens infecting these 3-D cell cultures, including serovar Typhimurium in the
colon, cytomegalovirus in the placenta, and P.
aeruginosa in lung cells (Fig. 4A-C, E-F).
We can further modulate 3-D cell culture
systems—for instance, by introducing biological
signals that mimic what such tissues encounter
in vivo. Many different relevant materials could
be introduced into the culture system, including
extracellular matrix components of the interstitial and basement membranes, bioscaffolding
materials, growth factors, hormones, nutritional
supplements, cytokines, chemokines, and other
bioactive molecules. In addition, such studies also
could include the use of several relevant cell types
simultaneously in a “co-culture” model, including established cell lines and primary cells from
particular tissues, as part of a broader effort to
understand particular host-pathogen interactions.
The RWV bioreactor technology represents a
versatile new approach for investigating infectious diseases— one that is just beginning to
be tapped. Because cells generated in the RWV
so closely approximate cells in native tissues,
3-D cultures offer a powerful approach for understanding host-pathogen interactions at a
detailed level and will surely provide insights
helpful for developing novel products and procedures for diagnosing, preventing, and treating
infectious diseases.
ACKNOWLEDGMENTS
We thank Jim Wilson and Kerstin Höner zu Bentrup for critical reading of this manuscript; Kerstin Höner zu Bentrup for
generating fluorescent images and figure panels; and Rajee Ramamurthy, Carly LeBlanc, Duane Pierson, Neal Pellis, Cindy
Morris, Heather LaMarca, Steve Alexander, Kamal Emami, Tom Goodwin, Mayra Nelman-Gonzalez, Michael Schurr, Alex
Carterson, Allen Honeyman, Virginia Miller, and Scott Hultgren for helpful discussions and/or sharing aspects of their work
for this manuscript. We thank Synthecon Inc. for their permission to use the confocal image of the 3-D Int-407 cells that
Kerstin Höner zu Bentrup generated with the assistance of Luis Marrero and Chassidy Johnson. Work in Cheryl Nickerson’s
lab is supported, in part, by a grant from the National Aeronautics and Space Administration (NASA-Ames grant NAG
2–1378), and by a generous grant from the W. M. William Keck Foundation of Los Angeles, Calif.
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