Critical Reviews in Food Science and Nutrition

ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: http://www.tandfonline.com/loi/bfsn20

Intestinal in vitro cell culture models and their

potential to study the effect of food components
on intestinal inflammation

Maria del Carmen Ponce de León-Rodríguez, Jean-Pierre Guyot & Caroline

Laurent-Babot

To cite this article: Maria del Carmen Ponce de León-Rodríguez, Jean-Pierre Guyot & Caroline
Laurent-Babot (2018): Intestinal in�vitro cell culture models and their potential to study the effect of
food components on intestinal inflammation, Critical Reviews in Food Science and Nutrition, DOI:
10.1080/10408398.2018.1506734

To link to this article: https://doi.org/10.1080/10408398.2018.1506734

Published online: 02 Oct 2018.

Submit your article to this journal

View Crossmark data

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=bfsn20
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
https://doi.org/10.1080/10408398.2018.1506734

REVIEW

Intestinal in vitro cell culture models and their potential to study

the effect of food components on intestinal inflammation

n-Rodr
ıguez, Jean-Pierre Guyot, and Caroline Laurent-Babot
Maria del Carmen Ponce de Leo
NUTRIPASS—University of Montpellier, IRD, Montpellier SupAgro, Montpellier, France

ABSTRACT KEYWORDS
Cell cultures are widely used in pharmaceutical, medical, food/nutrition and biological sciences. In Bioactive compounds; gut;
food and nutrition science, intestinal cell culture models of human origin are attracting increasing intestinal epithelial cells;
interest but are still rarely used in investigations of the effects of bioactive food compounds on immune cells
intestinal inflammation. However, such in vitro models would, among other benefits, limit the use
of in vivo models and could provide new molecular data.
This review is an overview of two-dimensional (2D) and three-dimensional (3D) intestinal cell cul-
ture models and their potential use in gut inflammation studies. After describing the features of
healthy and inflamed intestinal barriers, we describe the main intestinal cell lines (Caco2, HT29,
T84) and their use in investigations of the transport and antioxidant/anti-inflammatory potential of
some bioactive food compounds. Finally, different co-culture models of gut inflammation, in asso-
ciation with immune cells (PBMC, THP1 and RAW 264.7 cell lines) in both 2D and 3D models are
presented. 3D models called organs-on-chips or biochips are the most recent and very promising
approach made possible by bioengineering and biotechnological improvements and more accur-
ately mimic the gut microenvironment.

Introduction components, including bioactive compounds with potential

The intestinal epithelium is a permeable barrier that plays a
major role in the regulation of solute and fluid exchange
and has direct consequences for the absorption and trans-
port of nutrients. Different types of cells (enterocytes, goblet
cells, M cells, Paneth cells and others such as endocrine and
immune cells) form the intestinal epithelium. Beneath the
epithelial monolayer, immune cells (e.g. macrophages, lym-
phocytes) are spread into the lamina propria. Good cooper-
ation between all these cells maintains mucosal integrity,
which is challenged daily by external factors in the luminal
environment. The loss of the intestinal barrier function con-
sequently has clinical and nutritional consequences including
chronic disorders, inflammation and malnutrition. The eti-
ology of gut inflammation, whose incidence and prevalence
is increasing worldwide, is still not fully understood,
although multiple causes are known to be involved
(Molodecky et al. 2012; Loftus 2004). Low or high grade gut
inflammation is often associated with other pathological
conditions (e.g. infectious diseases, inflammatory bowel dis-
eases) but also with metabolic syndromes and with some
noncommunicable diseases (NCDs) (e.g. obesity) (Ding and
Lund 2011; Esser et al. 2014; Walters, Xu, and Knight 2014).
Moreover, malnutrition is a common feature related to the
inflammatory status of the intestine (Benjamin et al. 2008;
Forbes et al. 2017; Massironi et al. 2013; Mijac et al. 2010).
The intestinal epithelium is intensely exposed to food
risks or benefits for health. Among these compounds, phyto-
chemicals such as polyphenols or carotenoids have been
shown to have anti-inflammatory and antioxidant effects
and benefits for chronic disorders.
In recent decades, in vitro, ex vivo and in vivo models
have been developed to study different intestinal epithelium
functions and metabolism, in particular in the inflamed
state. Although animal models can simulate the physiology
of an entire organism, the main disadvantages of in vivo
models are the variation in responses due to differences
between species and the difficulty in extrapolating results to
humans (Fitzgerald et al. 2015). In addition, the use of cell
culture in vitro models has increased because the use of ani-
mal models involves ethical issues and is quite expensive.
Cell culture models have been widely used in bioavailability
and toxicological studies in both the food and pharmaceut-
ical fields. Improved in vitro models using cells of a human
origin have the advantage of reducing animal experimenta-
tion and could enable the study of molecular mechanisms in
a simple and reproducible way. For instance, the molecular
study of bioactive food compounds and their interactions
with the intestinal epithelial barrier and microbiota has
become possible.
Originally, cell lines were used in cancer research but
later some of these models were further used for in-depth
investigations of physiological mechanisms thanks to their
ability to differentiate into cells expressing normal

CONTACT Caroline Laurent-Babot caroline.laurent@umontpellier.fr NUTRIPASS—University of Montpellier, IRD, Montpellier SupAgro, Montpellier, France
ß 2018 Taylor & Francis Group, LLC
2

M. C. PONCE DE LEON-RODR
IGUEZ ET AL.
characteristics. In addition, co-culture models have been
developed to better mimic the complex composition of the
intestinal barrier as described above. Most currently
employed cell culture models are performed in two-dimen-
sional (2D) conditions with one or more cell lines in (co-
)culture, and frequently use insert systems composed of rigid
porous membranes. The intrinsic limitations of those models
drove the development of three-dimensional (3D) culture
systems. As a consequence of technological improvements,
the use of 3D in vitro models to reconstitute the in vivo cel-
lular microenvironment has increased significantly.
In the context of the extensive use of cell cultures in
many different fields, this review provides an overview of
2D and 3D intestinal cell culture models and their potential
use in gut inflammation studies. These models have great
potential for studies of the effect of bioactive food com-
pounds on intestinal inflammation.
Features of healthy and inflamed intestinal barrier
Characterized by a single-cell layer, the intestinal epithelium
is mainly composed of polarized absorptive cells called
enterocytes. However, other specialized epithelial cells are
also present including goblet cells, microfold cells (M cells)
and Paneth cells, forming the whole gut wall. Goblet cells
are responsible for the production of mucins that form a
protective mucus layer and Paneth cells secrete antimicrobial
peptides (defensins) and growth factors. The intestinal epi-
thelium is an important barrier between the lumen and the
intestinal lamina propria and functions as a dynamic inter-
face between the external environment and intestinal tissues
(Viladomiu et al. 2013; Wehkamp et al. 2005). These cells
all cooperate to prevent the entrance of microorganisms and
other particles into the host and are also involved in epithe-
lial growth and repair maintaining the mucosal integrity.
Beneath the intestinal epithelial monolayer, immune cells
(macrophages, dendritic cells, lymphocytes) are spread into
the lamina propria. These defense cells are more concen-
trated in Peyer’s patches (secondary lymphoid organ) and
interact with epithelial cells via soluble factors like growth
factors, cytokines and enzymes (Abraham and Cho 2009).
The intestinal epithelium is a permeable barrier that also
plays a major role in the regulation of solute and fluid
exchange with direct consequences for the absorption and
transport of nutrients. This necessary exchange occurs via
intercellular junctions present between the cells, which allow
paracellular transport. Accordingly, direct cell damage
observed for example in inflammatory bowel diseases (IBD),
provokes loss of barrier function. Clinical evidence has shown
that malnutrition is a common feature in IBD patients
(Benjamin et al. 2008; Forbes et al. 2017; Massironi et al.
2013; Mijac et al. 2010). Diverse micronutrient deficiencies
e.g. minerals (Fe, Zn), vitamins (D, A, B9, B12) and proteins
have been reported in patients with chronic intestinal disor-
ders (Conklin and Oliva-Hemker 2010; Mallon and Suskind
2010; Vagianos et al. 2007). Additionally, changes in perme-
ability can cause closer interaction between immune cells of
the lamina propria and the lumen content (Hering and
Schulzke 2009). Hence, food molecules and diverse microor-
ganisms, including microbiota promote sustained activation
of the mucosal immune system (Geremia et al. 2014;
Shanahan and Quigley 2014; Vindigni et al. 2016).
The epithelial intercellular junctions that form the apical
junction complex (AJC) are crucial to preserve the intestinal
barrier function. The AJC formed by transmembrane proteins
is represented by the tight junctions (TJs, e.g. occludin and
claudins) and the adherens junctions (AJs, e.g. E-cadherin)
that either interact with the cytoskeleton via cytoplasmic pro-
teins like the zonula occludens proteins (ZO) or the catenins
family. Paracellular transport is regulated by the TJs, which
seals the space between adjacent epithelial cells and control
the passage of molecules based on size and charge selectivity.
Moreover, the active nutrient transport pathway (e.g., Naþ-
co-transporters), depends on the transepithelial concentration
gradient generated by the flux of ions through the TJs (Liang
and Weber 2014; Turner 2009). Alterations in permeability
will result in loss of water in addition to malabsorption of
nutrients, therefore, homeostasis depends to a great extent on
a thoroughly functional TJ system.
In addition to behaving as a physical barrier, the intestinal
epithelium also plays a major role in the host defense system.
As described above, immune cells are present in the lamina
propria (Peyer’s patches), making the gut the main immune
organ of the human body. In healthy circumstances, the intes-
tinal barrier is in a necessary and continuous state of
“balanced” inflammation with a fragile equilibrium that can
be altered by several factors. Homeostasis can be disrupted by
both acute and chronic processes. For example, during intes-
tinal infections, pathogens such as Salmonella spp,
Escherichia coli, Clostridium difficile or Giardia often destabil-
ize the host TJ system and subsequently cause episodes of
diarrhea (Nusrat et al. 2001; O’Hara and Buret 2008;
Simonovic et al. 2000). The etiology of intestinal chronic dis-
orders such as IBD is however, not yet clear, although it is
certain that multiple causes are involved. Ulcerative colitis
(UC) and Crohn’s disease (CD) are the main inflammatory
bowel diseases studied, as they have the highest prevalence
worldwide (Loftus Jr 2004; Molodecky et al. 2012).
Many studies suggest that alterations in TJs and subse-
quent changes in permeability in IBD exacerbate the inflam-
matory process as the result of an imbalance between pro-
inflammatory and anti-inflammatory cytokines produced by
immune cells (Neurath 2014; Pandolfi et al. 2009; Suzuki
2013). It has been demonstrated that cytokines like TNF-a,
INF-c, IL-1b and IL-13 induce intestinal epithelial barrier
dysfunction which increases the paracellular infiltration of
luminal potentially antigenic molecules (Al-Sadi et al. 2011;
Heller et al. 2005; Turner 2006; Utech et al. 2005; Wang
et al. 2005; Watson et al. 2005). This suggests that the intes-
tinal barrier function plays a crucial role in IBD pathophysi-
ology, but it remains unclear whether these changes are part
of the cause or the consequence of chronic inflammation.
Likewise, different studies have shown the effects of diet on
TJ integrity (Kosi
nska and Andlauer 2013; Suzuki 2013).
According to in vitro studies, dietary components such as
polyphenols or casein peptides may improve the intestinal

barrier function, whereas others like ethanol and acetalde-
hyde its metabolite, are thought to have a negative impact
(Ferguson 2014; Samak, Aggarwal, and Rao 2011; Shimizu
2017; Suzuki, Tanabe, and Hara 2011; Yasumatsu and
Tanabe 2010).
Recent reports strongly suggest that intestinal microbiota
play an important role in preserving gut health. A disturb-
ance in the equilibrium of microbiota composition may rep-
resent a risk of infections developing (Khosravi and
Mazmanian 2013). This imbalance can also modify the
response of the intestinal immune system and the epithelial
barrier function that subserve the development of IBD and
could even have extraintestinal consequences (Brown,
Sadarangani, and Finlay 2013; Cammarota et al. 2015;
Fyderek et al. 2009; Kamada et al. 2013; Kostic, Xavier, and
Gevers 2014; Manichanh et al. 2012; Reiff and Kelly 2010).
Changes in microbiota composition were also linked to
reduced short-chain-fatty acids (SCFAs) metabolism which are
known to have anti-inflammatory and immune-regulatory
activity (Arpaia et al. 2013; Maslowski et al. 2009; Smith et al.
2013; Vieira et al. 2012). Despite limited available information,
there are reports concerning dietary impact on intestinal
microbiota and the link that those changes might have with
IBD (Sheehan, Moran, and Shanahan 2015; Wu et al. 2011;
Zimmer et al. 2012). As mentioned above, the intestinal epithe-
lium plays an important role in immunity. Macrophages and
dendritic cells present in the lamina propria are responsible for
sensing luminal antigens while regulatory T lymphocytes
(Treg) maintain a balance by suppressing abnormal immune
response against the microbiota or dietary antigens (Geremia
et al. 2014; Strober, Fuss, and Blumberg 2002). It is thus clear
that altered interactions between intestinal microbiota and the
immune response also play an important role in intestinal
inflammation (Kamada and N
~ez 2014). However, further
un
studies are needed to clearly understand this relationship.
Based on epidemiological studies, changes in food con-
sumption with increased intake of animal proteins, n-6 fatty
acids and ‘fast food’ among other dietary components, are
thought to be linked to changes in microbiota and to the
risk of developing IBD. In contrast, a Mediterranean diet,
rich in vegetables, fruit, cereals and fish, but also in olive oil
and red wine has been reported to reduce the risk of IBD.
However, no causal food item has been identified in studies
of the association between diet and IBD due to interpret-
ation difficulties (Ng et al. 2013). In addition to dietary
factors, TJ and immune dysregulation and a potential micro-
biota disequilibrium, genetic, environmental and socioeco-
nomic factors may also influence the development of IBD
and are reviewed elsewhere (Cho 2008; Khor, Gardet, and
Xavier 2011; Liu and Stappenbeck 2016; Ng et al. 2013).
In vitro cell culture models of gut inflammation
Technological advances in cell culture in the last 40 years
have increased research into intestinal disorders and physio-
logical epithelial barrier mechanisms with the aim of mini-
mizing in vivo experiments prior to clinical trials. Cell
culture has become an essential tool in toxicology and
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3
nutrition since the establishment of in vitro cell lines from
both animal and human origin. The use of primary cell cul-
tures is limited by their availability, repeatability and their
use in long term studies because of their short life span, but
transformed cell lines are largely used instead (Langerholc
et al. 2011; Zucco et al. 2005). The development of in vitro
models to mimic the in vivo environment required the char-
acterization of cell lines as single or co-culture models
before they could be used successfully. In this context,
human cell lines have been favored.
Main intestinal cell lines
Caco-2 cell line
The human epithelial cell line Caco-2 has been the cell line
most widely used as an intestinal model for absorption,
transport and bioavailability studies in recent decades
(Cheng, Li, and Uss 2008; Hidalgo, Raub, and Borchardt
1989; Hillgren, Kato, and Borchardt 1995). This cell line was
isolated from a human colorectal adenocarcinoma and
established by Fogh & Trempe in 1974 (Fogh and Trempe
1975). It is well characterized as an enterocyte model thanks
to its morphological and functional features expressed after
differentiation. It takes these cells about five days to reach
confluence and spontaneously start to differentiate during
30 days of culture. Once differentiated, they form a polarized
cell monolayer with apical and basolateral membranes, a
junction complex and a brush border with microvilli on the
apical side typical of human enterocytes (Chantret et al.
1988; Hidalgo, Raub, and Borchardt 1989; Pinto et al. 1983).
The expression of enzymes such as hydrolases (sucrose-iso-
maltase, lactase, etc.) present in intestinal microvilli
(Chantret et al. 1994; Howell, Kenny, and Turner 1992;
Vachon and Beaulieu 1992) and different membrane trans-
porters are also traits of the Caco-2 cell line (Lea 2015).
However, the Caco-2 parental cell line has certain limita-
tions including the formation of a heterogeneous monolayer
linked to culture time and to the number of passages
(Artursson, Palm, and Luthman 2001) and the presence of
multilayer zones (Briske-Anderson, Finley, and Newman
1997). The cultivation conditions may also select subpopula-
tions of cells (Lea 2015). In addition, this cell line cannot
differentiate into goblet cells, so one important limitation is
their inability to produce mucus.
Diverse Caco-2 clones have been established to reduce
heterogeneity (e.g. Caco-2/TC7, Caco-2/15, Caco-2/AQ)
(Sambuy et al. 2005). The TC7 clone was derived from a
late passage (198) of the Caco-2 line and differentiates faster
than the parental line (20 vs. 30 days) by retaining its mor-
phological and functional characteristics and the monolayer
formed is more homogeneous and stable (Caro et al. 1995;
I. Chantret et al. 1994; Ranaldi et al. 2003; Turco et al.
2011). Moreover, the TC7 clone has been shown to be a
suitable model of the small intestinal barrier in terms of
reproducibility and performance (Zucco et al. 2005).
Caco-2 cells have also been shown to be able to produce
inflammatory markers (e. g., cytokines) in response to other
cytokines such as IL-1b, TNF-a and/or IFNc (Hollebeeck
4

M. C. PONCE DE LEON-RODR
IGUEZ ET AL.
et al. 2012; Rodr
ıguez-Ramiro et al. 2013; WeR glarz et al.
2007) and/or in some cases, in response to external stimuli
like lipopolysaccharides (LPS) that are present on the mem-
branes of microorganisms (Barrera and S
anchez 2015;
Maccaferri et al. 2012; WeR glarz et al. 2007; Xu et al. 2012).
It should be pointed out that reports also showed Caco-2
cells were hypo-responsive to LPS stimulation due to the
limited expression of the Toll-like receptor involved in LPS
recognition (TLR-4), even though these cells express the co-
receptor MD-2, which is essential for NF-jB activation and
the subsequent secretion of pro-inflammatory cytokines
(Suzuki, Hisamatsu, and Podolsky 2003; Vamadevan
et al. 2010).
HT-29 cell line
The HT-29 is another intestinal cell line, established by
Fogh and Trempe. This cell line was also derived from a
colon adenocarcinoma (Fogh and Trempe 1975) and has
been used as an enterocyte model in bioavailability and cell
mechanism studies (Andoh et al. 2001; Hagesaether 2011; Yi
et al. 2016). The HT-29 line is considered as a pluripotent
intestinal cell line because changes in the culture media can
lead to different enterocytic differentiation paths. Unlike in
the Caco-2 cell line, in HT-29, differentiation is not spon-
taneous but rather depends on nutritional and culture con-
ditions (Huet et al. 1987; Viallard et al. 1986; Zweibaum
et al. 2011). In a glucose free medium, in a similar way to
the Caco-2 line, HT-29 cells differentiate into enterocyte-
type cells expressing hydrolases like sucrose-isomaltase,
which are restricted to the intestinal brush border, whereas
when grown in presence of glucose, they remain undifferen-
tiated. Furthermore, the differentiation in HT-29 cells can
be inhibited and reversed by the addition of glucose to the
medium (Zweibaum et al. 1985). Other differences between
Caco-2 and HT-29 lines are a longer differentiation period
and the absence of lactase expression in the HT-29 line.
Nevertheless, the main difference between HT-29 and Caco-
2 cell lines is that under certain culture conditions, HT-29
can differentiate into goblet-like cells and thus has the abil-
ity to produce mucus (Zweibaum et al. 2011). The mucus
produced by goblet cells is a water insoluble gel mainly
composed of glycoprotein oligomers and related monomers
that forms the protective layer in the intestine (B
eduneau
et al. 2014).
When treated with sodium-butyrate, for example, HT-29
cells differentiate into distinct phenotypes, as demonstrated
by Augeron and Laboisse (1984), who isolated different
HT-29 subpopulations, some of which were mucus secreting
clones morphologically analogous to intestinal goblet cells
(Augeron and Laboisse 1984). Later, when the focus shifted
to the ability of HT-29 cells to adapt to metabolic stress
conditions, various populations of mucus-secreting cells
were isolated including HT29-FU, HT29-18N2 and HT29-
MTX (Huet et al. 1987; Lesuffleur et al. 1993). The stable
clone HT29-MTX was isolated from the HT-29 cell resist-
ance (irreversible) to high concentrations of Methatrexate
(MTX), after a gradual adaptation to this anticancer drug.
This clone is capable of spontaneously differentiating into
goblet-like mucus-producing cells (Lesuffleur et al. 1990),
which have been employed as a model to study intestinal
cell adhesion of lactic acid bacteria (Turpin et al. 2012).
The HT29-MTX has also been widely used in co-culture
systems along with Caco-2 cell line to mimic the human
intestinal barrier in absorption and permeability studies
(Barnett et al. 2016; B
eduneau et al. 2014; Behrens et al.
2001; Kaulmann, Andr
e, et al. 2016; Laparra, Glahn, and
Miller 2009; Mahler, Shuler, and Glahn 2009; Nollevaux
et al. 2006; Poquet, Clifford, and Williamson 2008; Selby-
Pham et al. 2017). Other mucus secreting cell lines have
been characterized including LS174T and LS180 (Tom et al.
1976) and clones from these lines have been isolated to
study mucins (Kuan et al. 1987).
T84 cell line
A third cell line frequently used in intestinal studies is T84,
which was established in 1980 by Murakami et Masui to
study the hormonal control of human colon carcinoma cell
growth (Murakami and Masui 1980). T84 cells grow in
monolayers of polarized cells that show the presence of TJs
and small microvilli on the surface (Dharmsathaphorn et al.
1984). Unlike spontaneous differentiation of Caco-2, a par-
ticularity of the T84 cell line is that differentiation into
crypt-like cells (Madara and Dharmsathaphorn 1985) com-
mences only after induction with human recombinant trans-
forming growth factor (TGFb1) or other soluble factors
from mesenchymal cells. This feature makes it possible to
study the differentiation process (Juuti-Uusitalo et al. 2006).
T84 cells have also been used to study electrolyte trans-
port mechanisms (Alzamora et al. 2011; Beltr
an et al. 2015;
Nichols et al. 2015; Tang et al. 2010) and intestinal perme-
ability (Ewaschuk et al. 2008; Huang et al. 2016; Watson
et al. 2005). Besides, this cell line has also been employed in
studies of signaling pathways related to the inflammatory
response of intestinal epithelium (Cario et al. 2000; Cho and
Park 2015; de Kivit et al. 2011; Juuti-Uusitalo et al. 2011;
Ou et al. 2009; Vamadevan et al. 2010) and bacteria-entero-
cyte interaction (Otte and Podolsky 2004).
Despite the wide use of Caco-2, HT29 and T84, other
human gut cell lines (e.g. SW480, H4) and animal cell mod-
els are available; some of them are reviewed elsewhere
(Langerholc et al. 2011; Leibovitz et al. 1976). A gene
expression profile of some of these commonly exploited
intestinal cell lines was published in 2012 to help choose the
appropriate in vitro model for specific studies (Bourgine
et al. 2012).
These in vitro models enable the simple and reproducible
study of cell mechanisms at molecular level that may be dif-
ficult to perform in vivo. Nowadays, semipermeable plastic
filter inserts used as culture systems facilitate the observation
of molecules moving across the cell monolayer (compart-
mentalized systems). Accordingly, in recent years, many
studies have been performed to characterize the absorption,
transport and bioavailability of food components or mole-
cules of therapeutic interest. Even if caution should be exer-
cised when comparing in vitro and in vivo results, these
research works provide interesting information.

Bioactive compounds: transport and
antioxidant/anti-inflammatory potential in
intestinal cell culture models
Bioactive compounds like phytochemicals such as polyphe-
nols and carotenoids, present interesting benefits for health
particularly due to their anti-inflammatory and anti-oxidant
potential. Intestinal cell culture models have been extensively
used to study these properties and their absorption and
transport to assess their bioavailability and regulation of
intestinal permeability.
For example, a Cocoa flavan-3-ols absorption/secretion
study was conducted by Kosi
nska and Andlauer (2012)
using Caco-2 cell monolayers. Their results suggest a para-
cellular pathway of transport for the cathechin, epicatechin
and procyanidin B2. In addition, these authors concluded
that compounds of a purified extract of cocoa powder might
be able to increase permeability with no harmful effects on
cells, thereby increasing the bioavailability of flavan-3-ols
(Kosi
nska and Andlauer 2012). Recently, Denis et al. (2015)
provided evidence for the capacity of cranberry phenolic
fractions to reduce intestinal oxidative stress and inflamma-
tion while improving mitochondrial dysfunction using the
cell clone Caco-2/15 as a model (Denis et al. 2015).
Similarly, the results obtained by Bitzer et al. (2015) in a
study of cocoa procyanidin fractions in Caco-2 and HT29
cell lines suggest that high-molecular-weight polymeric pro-
cyanidins are highly effective at preserving membrane integ-
rity (in Caco-2 cells) and reducing inflammation markers
such as IL-8 (in HT29 cells) (Bitzer et al. 2015). In 2017,
Martins et al. published a study on the antioxidant and anti-
inflammatory effects of grape pomace (GP), which is rich in
polymeric and glycosidic polyphenols. These authors showed
a reduction in inflammatory markers (IL-8, PGE2) on IL-1b
stimulated Caco-2 monolayers after the addition of tannase
(tannin acyl hydrolase) to the cultures, suggesting an
improvement in the antioxidant and anti-inflammatory
activities of GP polyphenol extracts (Martins et al. 2017).
The potential health benefits of polyphenol have been also
studied by other researchers. The positive effect of kaempferol
on the integrity of the TJ barrier was investigated using Caco-
2/HTB-37 cells and showed improvement in TJ protein assem-
bly after flavonoid administration (Suzuki, Tanabe, and Hara
2011). Likewise, Rodr
ıguez-Ramiro et al. (2013) studied the
molecular mechanism underlying the anti-inflammatory activ-
ity of polyphenols and concluded that cocoa polyphenols
effectively down-regulate the levels of inflammatory markers
in vitro (Caco-2 cells) and in vivo (rat model) (Rodr
ıguez-
Ramiro et al. 2013). A flavonoid-rich concentrate from
Opuntia ficus indica juice, a species of cactus, was evaluated by
Matias et al. (2014). Their results showed that co-incubation
of the concentrated with a stress-inductor attenuates the pro-
duction of radicals significantly more than pre-incubation.
This suggests that some of the main compounds present in the
concentrate, e.g. isorhamnetins derivatives that present low
bioavailability, inhibit the formation of reactive oxygen species
(ROS) in the environment of epithelial intestinal cells. A simi-
lar positive response to inflammatory activity was observed
during co-incubation (Matias et al. 2014). All these studies
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 5
demonstrate the antioxidant and anti-inflammatory potential
of the polyphenols usually present in fruit and vegetables. The
transport (Dhuique-Mayer et al. 2007; During et al. 2002) and
anti-inflammatory effect (Kaulmann et al. 2012) of carotenoids
have also been studied using cell culture models.
As already mentioned, cell monocultures have been
extensively used to assess immune response modulation,
antioxidant and anti-inflammatory effects of different com-
ponents of fruits, vegetables and other natural products such
as human breast milk lipids or phytic acid (Barrera and
S
anchez 2015; Hollebeeck et al. 2012; Kang et al. 2009;
Sergent et al. 2010; WeR glarz et al. 2007; Wojtal et al. 2013;
Xu et al. 2012). Shimizu (2010) reviewed the interaction of
food components with the intestinal epithelium using the
Caco-2 line as cell model, particularly in studies of transport
pathways, modulation of transporter functions, TJ perme-
ability and immune system regulation (Shimizu 2010).
It should be also pointed out that food additives, adju-
vants or food matrices may influence the intestinal transport
and bioavailability of these bioactive compounds (Nerurkar,
Burton, and Borchardt 1996) or have a direct influence on
intestinal function (Jiang et al. 2013). Recently, the inter-
action of nanomaterials employed in the food industry with
intestinal models, including cell cultures, was reviewed by
Lefebvre (Lefebvre et al. 2015). The migration of food con-
tact materials like silver nanoparticles (AgNPs) from utensils
and storage containers was studied using the T84 cell line,
as well as its impact on intestinal permeability and barrier
function. These authors concluded that small-size AgNPs
may increase intestinal permeability even in the absence of
any toxic effects on cells (Williams et al. 2016).
Cell models have been useful not only for the study of food
components but also for pharmaceutical research. Studies in
this field aim to evaluate potential toxic effects of drug candi-
dates and to improve the absorption and bioavailability of
drugs, nanoparticles or other components used in drug deliv-
ery. For instance, in the case of the improvement of oral deliv-
ery of poorly permeable drugs, Maher et al. (2016) reviewed
more than 200 intestinal permeation enhancers (PEs) that have
been tested in preclinical intestinal delivery models, including
Caco-2 cells. These permeation enhancers alter intestinal per-
meability in two different ways. First generation paracellular
PEs target cell signaling pathways involved in disbandment of
TJs and the second generation can directly target the physical
disruption of TJs by interfering in intercellular hemophilic
interactions. Transcellular PEs, act via modification of the
integrity of the cell plasma membrane or via hydrophobisation
of the target therapeutic peptide. Maher et al. (2016) concluded
that models like epithelial monolayer cultures enable the detec-
tion of PEs that modify barrier integrity and improve the bio-
availability of peptides even though such systems do not
consider drug formulation (Maher, Mrsny, and Brayden 2016).
Intestinal and immunological cells Mono- and
co-cultures as gut inflammation models
To improve on simple cellular models, researchers developed
2D co-cultures as gut inflammation models (summarized in
6

M. C. PONCE DE LEON-RODR
IGUEZ ET AL.
Table 1). These models were established using the associ-
ation of intestinal and immune cells, the second most
numerous type of cells present in intestinal epithelium, and
compartmentalized systems largely employed to grow intes-
tinal cell lines, as mentioned above. Since the late 1990’s,
researchers have shown increasing interest in improving
intestinal mucosal barrier models by including immune cells
in the cultures (Kern
eis et al. 2000). The main immune cells
used to establish these co-culture systems with intestinal cell
lines include primary cells like human peripheral blood
mononuclear cells (PBMC) and macrophage cell lines of
either human (THP-1) or murine origin (RAW 264.7).
Pbmc
PBMC are obtained from the buffy coats of healthy donors
frequently through Ficoll gradient centrifugation and include
lymphocytes, monocytes and dendritic cells. The presence of
cell markers for macrophages and dendritic cells has been
described in co-cultures using PBMC (Leonard, Collnot, and
Lehr 2010). However, given the differences in their pheno-
type, cells isolated from blood might have different
responses from those present in the intestinal lamina propria
as they present (Kleiveland 2015).
THP1 cell line
The THP-1 cell line was isolated from the peripheral blood
of a 1-year old male patient suffering from acute monocytic
leukemia and characterized as a monocytic cell line
(Tsuchiya et al. 1980). These cells can be cultured in vitro
for about 3 months (passage 25) with no modification in cell
sensitivity or activity. THP-1 cells can differentiate into mac-
rophages after phorbol-12-myristate-13-acetate (PMA) treat-
ment or into dendritic cells by transferring them into
serum-free medium, and subsequently treating them with a
mixture of IL-4, GM-CSF, TNF-a and ionomycin (Chanput,
Peters, and Wichers 2015). This cell line has been character-
ized as a model for immune modulation (Chanput et al.
2013; Chanput, Mes, and Wichers 2014) and has also been
widely used to study ROS production and inflammation. For
instance, researchers have employed THP-1 cell line as a
model to analyze the anti-inflammatory effects of polyphe-
nols, (Chanput et al. 2010; Huang, Wu, and Yen 2006; Wu
et al. 2011) or fatty acids (Huang et al. 2012).
The use of THP-1 could have certain advantages over
PBMC for example, due to its homogeneous genetic back-
ground that reduces the variability of the cell phenotype,
thereby improving the reproducibility of the experiments.
RAW 264.7 cell line
Finally, a monocytic murine cell line (RAW 264.7) has also
been frequently used in co-cultures with Caco-2 cells. The
RAW 264.7 cell line was established from a murine tumor
induced by the Abelson leukemia virus and presents macro-
phage properties (Raschke et al. 1978). It has been shown
that these cells mimic murine bone marrow-derived macro-
phages in terms of cell surface receptors and response to
microbial ligands involving Toll-like receptors (TLRs)
(Berghaus et al. 2010). However, these authors concluded
that caution is required when interpreting and extrapolating
results due to possible changes in RAW cells in continuous
culture (Berghaus et al. 2010). Moreover, possible inter-spe-
cies cross-reactions should also be taken into account in co-
culture models that combine murine (e.g. RAW cells) and
human cell lines as Caco-2 cells to study anti-inflamma-
tory effects.
2D co-culture models. Establishing co-cultures of different
cell types enables the study of cell-cell (intercellular) com-
munication and the investigation of the influence of these
interactions on growth, differentiation and immune
response. This also makes it possible to explore the effect of
food components and the response of cells to exposure to
the components in either healthy or pathological conditions.
Even though in vitro models do not fully mimic the com-
plexity of in vivo physiology, these models facilitate the
study of cell contact mechanisms and responses to soluble
factors such as cytokines. Nowadays, the majority of the co-
culture models involving intestinal and immune cells use
standard insert systems in which intestinal cells (upper com-
partment) are not in direct contact with the immunological
ones (lower compartment). However, there have been a few
studies in which both intestinal and immune cells are seeded
in the same compartment (Susewind et al. 2016) or using
inverted models (Ara
ujo et al. 2016; des Rieux et al. 2007;
Mori, Satsu, and Shimizu 2003; Tyrer et al. 2011). This
allows direct contact between the two cell types and hence
the study of migration of the immune cells into the intes-
tinal monolayer (see particular features in Table-1).
However, 2D cell culture models have some limitations
including reduced cell-cell interactions, lack of cell-matrix
interactions, and the tissue architecture is not completely
recreated; Fitzgerald et al. (2015) reviewed these disadvan-
tages in terms of evaluating new drug delivery strategies
(Fitzgerald et al. 2015). Despite their limitations, 2D in vitro
models are a useful tool to study the interactions between
food components and intestinal cells during intestinal
inflammation. To date, only a few studies have linked the
effects of food components to gut inflammation using 2D
co-culture models. In this context, Caco-2 cells have been
co-cultivated with different immune cells in an attempt to
mimic the presence of the latter in the lamina propria
beneath the intestinal epithelium. For example, the Caco-2
cell line has been associated with THP-1 cells to study
effects of lycopene, a carotenoid present in red fruits and
vegetables, on pro-inflammatory targets. The authors of that
study found that a low concentration (2 mM) of lycopene
reinforced pro-inflammatory gene expression in THP-1 cells,
whereas higher concentrations (5–20 mM) had an anti-
inflammatory effect with a decrease in ROS (Makon-
S
ebastien et al. 2014). Other authors associated Caco-2 cells
with the RAW 264.7 cell line to study the anti-inflammatory
effects of natural extracts. In one study, researchers observed
anti-inflammatory properties of a fruit juice enriched with
pine bark extract which decreased after digestion (Frontela-
Table 1. Gut inflammation 2D co-culture models.
Model Cell type(s) & source
Caco-2/THP1 Human colon adenocarcinoma /
Human peripheral blood (acute
monocytic leukemia) cell lines
Caco-2/Raw 264.7 Human colon adenocarcinoma /
Murine macrophage cell lines
Caco-2/ PBMC Human colon adenocarcinoma
cell line / Human peripheral
blood mononuclear cells
Human colon adenocarcinoma
cell line / Murine peripheral
blood mononuclear cells
Caco-2/HT29-MTX/PBMC Human colon adenocarcinoma
cell lines / Human peripheral
blood mononuclear cells
Caco-2/HT29-MTX/THP-1 Human colon adenocarcinoma /
Human peripheral blood
(acute monocytic leukemia)
cell lines
Objective(s)
Effect of lycopene on pro-inflammatory targets of intes-
tinal and immune cell lines
Microparticle uptake
Effect of macrophages on intestinal epithelial cells
Evaluation of the cross talk between intestinal epithe-
lium and macrophages
Migration of immune cells into intestinal epithelial
cell monolayers
Effect of chickpea protein hydrolysates on cell
proliferation
Bioaccessibility, bioavailability and anti-inflammatory
effects of polyphenols
Anti-inflammatory effects of hit or lead drugs from the
MicroSource Pure Natural Products Collection and
Sigma-Aldrich database
Effect of fucoidan (from brown algae) on gut
inflammation
Anti-inflammatory effects of Smabucus nigra fruit extract
on LPS-stimulated macrophage-type cells
Anti-inflammatory effects of Sasa quelpaertensis leaf
extract (SQE) on LPS-stimulated co-culture
Anti-inflammatory capacity of fruit juices enriched with
pine bark extract (PBE)
Effect of arsenical species on the release of cytokines
Lactobacillus casei subsp. rhamnosus (Lcr35) anti-inflam-
matory effect on Caco-2 cells.
Effect of bacterial products (LPS, MDP) on enterocyte-
macrophage interaction
Interaction between PBMC and enterocytes during chal-
lenge with bacteria (Nonpathogenic E. coli)
Validation of an in vitro M-cell model
Salmonella enterica and intestinal cells interactions
under different Fe concentrations using an in vitro
colonic fermentation system inoculated with immobi-
lized child microbiota
Anti-inflammatory and antioxidant capability of carote-
noids/polyphenols from plum and cabbage digesta
on a stimulated model (LPS, TNF-a & IL-1b)
Particular features Reference
/ (Makon-S
ebastien et al. 2014)
/ (Moyes, Morris, and Carr 2010)
/ (Satsu et al. 2006)
/ (Kanzato, Manabe, and
Shimizu 2001)
Caco-2 cells were cultured on inverted (Mori, Satsu, and Shimizu 2003)
Millicell inserts (12 mm pore size) and
then re-orientated before adding THP-1
to allow contact with the porous mem-
brane at the basolateral side of the cells.
/ on-Calle, Alaiz, and
(Gir

Vioque 2010)
/ (Zhang et al. 2017)
Compounds were chosen according to a (Galvez-Llompart et al. 2013)
quantitative structure-activity relationship
model based on molecular topology to
predict the IL-6 mediated anti-UC activity.
/ (Tanoue et al. 2008)
/ (Olejnik et al. 2015)
/ (Kim et al. 2015)
/ (Frontela-Saseta et al. 2013)
/ (Calatayud et al. 2015)
/ (Fang et al. 2010)
Caco-2 cells were cultured on inverted (Tyrer et al. 2011)
Transwells and then re-orientated before
adding PBMC to allow cell-cell contact.
/ (Parlesak et al. 2004)
Caco-2 cells were cultured on inverted (Tyrer et al. 2002)
Transwells (0.3 mm pore size) and then
re-orientated before adding isolated
Peyer’s patch lymphocytes to allow cell-
cell contact.
Intestinal cell lines were seeded on the (Dostal et al. 2014)
apical side of the inserts to which S.
enterica N-15 in DMEM or fermentation
effluents, both with different Fe concen-
trations, was added. Freshly collected
PBMC were added to the basolateral com-
partment with different concentrations
of Fe.
Intestinal cell lines were seeded on inserts (Kaulmann, Legay, et al. 2016)
apical side and immune cells on the baso-
lateral compartment.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
(Continued)
7
Table 1. Continued.
Model Cell type(s) & source
Caco-2/Raji B Human colon adenocarcinoma /
Human Burkitt’s lymphoma
cell lines
Caco-2/HT29-MTX/Raji B Human colon adenocarcinoma /
Human Burkitt’s lymphoma
cell lines
Caco-2/ISO-HAS-1 Human colon adenocarcinoma/
Human microvascular endo-
thelial cell lines
SW480/THP-1 Human colorectal adenocarcin-
oma / Human peripheral
blood (acute monocytic leu-
kemia) cell lines
m-ICd2 / DCs Murine intestinal epithelial cells/
dendritic cells / murine bone
marrow-DCs cells
Objective(s)
Improvement of an in vitro model of human follicle-
associated epithelium (FAE) to study nanoparticle
transport mechanisms by M cells
Establishment of a tissue-engineered model for oral
drug delivery studies
Development of a triple culture intestinal model to
study nanoparticle mobility
Evaluation of co-culture interactions with nanosized
drug vehicles or contrast agents during an inflamma-
tory induced state
Investigation of the effects of HMGB1 inhibitors in a
LPS stimulated model
Probiotic bacteria strain-specific immune-modulatory
capacities: impact on dendritic cells maturation
8
Particular features Reference
Two models were set up, one with Raji B (des Rieux et al. 2007)
cells in the basolateral compartment of a
standard compartmentalized model and
the other where inserts containing Caco-2
cells were inverted in order to seed Raji B
cells in contact with the porous mem-
brane at the basolateral side of the intes-


tinal cells.
Two models were set up: one in which (Antunes et al. 2013; Ara
ujo
Intestinal cell lines were seeded on et al. 2016; Ara

ujo and
M. C. PONCE DE LEON-RODR
Transwell inserts and Raji-B cells were Sarmento 2013)
added to the basolateral compartment
and an inverted model in which intestinal
cells were seeded on the basal side of
the membrane and then Raji B cells were

IGUEZ ET AL.
seeded on the apical side of the insert.
Intestinal cell lines were seeded on the (Schimpel et al. 2014)
apical side of Transwell inserts and Raji-B
cells were seeded on the basolateral
compartment.
Transwell inserts were coated with collagen I (Kasper et al. 2016)
and inverted, ISO-HAS-1 cells were seeded
on the lower side and after 2 h of adhe-
sion, the inserts were re-inverted and
Caco-2 cells seeded in the upper
compartment.
SW480 cells were prepared in the lower (Wang 2015)
chamber of Transwell inserts and THP-1
cells were loaded into the
upper chamber.
/ (Zoumpopoulou et al. 2009)

Saseta et al. 2013). Furthermore, it has been shown in other
studies that fruit (e.g. eldelberry) and leaf (e.g. bamboo)
extracts down-regulated the expression of pro-inflammatory
cytokine (IL-1b, IL-6, TNF-a) and antioxidant enzymes
(iNOS, COX) genes and/or suppressed their secretion (Kim
et al. 2015; Olejnik et al. 2015). Recently, Kaulmann et al.
(2016) associated two intestinal cell lines (Caco-2/HT29
MTX) with THP-1 cells to study the anti-inflammatory and
antioxidant capability of plum and cabbage digesta. Their
results revealed a reduction in certain markers of inflamma-
tion (IL-6, IL-8 and NF-jB) and oxidative stress (Nrf2),
although the effects were moderate and could not be aligned
to either cabbages or plums, nor to carotenoids or polyphe-
nols (Kaulmann, Legay, et al. 2016).
3D (co)-culture models. In vitro models are constantly
evolving from simple monocultures, through 2D (co-) cul-
tures using plastic supports, to 3D cultures in which cells
are grown within an extracellular matrix (ECM). Kim et al.
(2014) observed differences in barrier function (permeabil-
ity) between a 2D and 3D villi model using Caco-2 cells (S.
H. Kim et al. 2014). Similarly, Yu et al. (2012) reported var-
iations during cell differentiation; according to their experi-
ments, 3D models could be more time efficient with a more
and realistic cell differentiation (Yu et al. 2012). Another of
the limitations of 2D models, particularly in tumor research,
is the absence of a true ECM with changes in structure,
adhesive potential, mechanotransduction, and results of
exposure to soluble factors (Ryan et al. 2016). In fact, the
ECM gives tissues mechanical properties and favors cell
communication. The anchorage of membrane receptors to
the ECM regulates the cells’ interpretation of signals from
their environment (Abbott 2003).
Thanks to advances bioengineering, 3D co-culture sys-
tems have been designed using different platforms like scaf-
fold constructions, hydrogels (e.g. collagens, laminins),
spheroids and microfluidic devices with channels that enable
controlled diffusion of nutrients (Fitzgerald et al. 2015). The
aim of 3D models and the frequently termed organs-
on-chips or biochips (microfluidic devices), is to provide a
cellular microenvironment that brings in vitro conditions
closer to the physiological reality of the organization and
dynamic properties of tissues (summarized in Table 2). For
example, Juuti-Uusitalo et al. (2011) employed 3D models
(T84 and Caco-2 cells) to study the effect of TNF (TNFSF2)
and INFc on intestinal epithelial morphogenesis and perme-
ability. Their work revealed differences in the response to
stimuli using a 3D model, and showed that apoptosis is an
important mechanism in increased permeability after TNF
stimulation. They also demonstrated that this pro-inflamma-
tory cytokine inhibits normal intestinal epithelial morpho-
genesis resulting in the formation of ‘leaky’ monolayers in
the case of T84 3D model (Juuti-Uusitalo et al. 2011).
To our knowledge, most of these 3D and microfluidic
models have been applied in the pharmaceutical field with a
focus on drug toxicology and screening. For instance,
Leonard et al. (2010, 2012), established an inflamed 3D
model using the intestinal cell line Caco-2 (C2Bbe1) and
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 9
PBMC as immune cells embedded in a collagen layer. The
model was used to evaluate the efficacy of different formula-
tions of an anti-inflammatory drug (budesonide) used in the
treatment of IBD. Their results show the ability of the model
to differentiate therapeutic efficacy among different formula-
tions while adequately representing the complex patho-
physiological changes observed in vivo (Leonard et al. 2012;
Leonard, Collnot, and Lehr 2010). Li et al. (2013) also devel-
oped a 3D intestinal model involving intestinal cells (Caco-2,
HT29MTX), fibroblast-type cells and macrophage-type cells
to study drug absorption. The authors concluded that their
model mimics the native intestinal epithelial layer more
closely. They also demonstrated an improved correlation
between the absorptive permeability across the 3D co-culture
model and the human absorption fraction in the case of
moderate and highly permeable drugs (Li et al. 2013).
More recently, Wu et al. (2017) assessed the toxicity and
inflammatory effects of ZnO nanoparticles (ZnO NPs) using
2D and 3D Caco-2 cell culture models. ZnO NPs have been
used in dietary supplements and in food additives and food
packaging materials because of their antimicrobial activity.
Wu et al. results showed that ZnO NPs induced the toxicity
of 2D and 3D cultures in different size-dependent manners.
Their results also indicated that ZnO NPs induced a chronic
inflammatory response in 2D cell culture and an acute
inflammatory response in 3D cell culture, indicating that
cell space organization have an important impact on cell
responses (Wu et al. 2017). Even if today, most research
using 3D models has pharmaceutical goals, in the food field,
such models could be used to study absorption and the anti-
oxidant and/or anti-inflammatory properties of bioactive
food compounds during intestinal inflammation.
Several biochips have been established that are thought to
be more representative of the in vivo microenvironment try-
ing to mimic intestinal functions. The development of these
complex devices, passing from static to fluidic cultures,
opens new opportunities to understand organ-level interac-
tions. In the food field, they could be employed to study
nutrient absorption and the impact of food components on
immune modulation. For instance, a new integrated micro-
fluidic platform, called Nutrichip, has been developed to
investigate the potential of the immune modulatory function
of dairy foods (Ramadan et al. 2013; Vergeres et al. 2012).
This system consists of a monolayer of epithelial cells inter-
acting with immune cells mounted on a chip holder device.
Different perfusion microchannels allow a uniform radial
fluid flow of the culture medium and an inflammatory
stimulus or a digesta; cytokine production is quantified
using an on-chip immunomagnetic assay. More recently, a
similar miniaturized Caco-2/U937 co-culture model was
used to characterize TJ disruption and immune response
modulation at intestinal level (Ramadan and Jing 2016).
More research is still needed to completely control all the
model features (Pampaloni, Reynaud, and Stelzer 2007; Ravi
et al. 2015). For example, ECM composition and stiffness
(Langhans 2018; Wallace and Rosenblatt 2003), gas
exchange, chemical and mechanical signal control and inter-
stitial flows have a crucial impact on cell behavior and
Table 2. Gut 3D culture models.
Model Cell type(s) & source
HEALTHY GUT D human small intes- Caco-2 (human colon
tinal villous adenocarcinoma)
Multicellular organotypic HCT-8 (human ilieocecal adenocarcin-
model of human intes- oma), CCD-18Co (human colon
tinal mucosa fibroblast), HUVEC (human umbil-
ical vein endothelial cells)
3D setup of intes- Caco-2 (human colon adenocarcin-
tinal mucosa oma), THP-1 (human macro-
phages), MUTZ-3 (human
dendritic cells)
3D co-culture (intestinal Caco-2 (human colon adenocarcin-
epithelial cells-fibro- oma) / NHDF (normal human der-
blast model) mal fibroblasts)
3D intestinal culture T84 (human colon carcinoma, lung
metastasis), Caco-2 (human colon
adenocarcinoma)
Caco-2 (human colon
adenocarcinoma)
Caco-2 (human colon
adenocarcinoma)
Caco-2 (human colon
adenocarcinoma)
Human gut-on-a-chip Caco-2 (human colon
adenocarcinoma)
Intestinal microflui- Caco-2 (human colon
dic system adenocarcinoma)
Intestinal microscale cell Caco-2 and HT29-MTX (human
culture analog (mCCA) adenocarcinoma), HepG2/C3A
(human hepatocellular carcinoma)
Objective(s)
Development of a 3D hydrogel scaffold
model to accurately replicate the shape
and size of human small intestinal villi to
test drug permeability
Development of an organotypic model with
close structural and functional resem-
blance to the human intestinal mucosa
Development of a 3D intestinal model for
drug delivery studies and to assess nano-
material toxicity in healthy or dis-
ease conditions
To study TJ expression and cell differenti-
ation in a 3D model
Effect of TNFa and IFNc on paracellular per-
meability and morphogenesis in 3D T84
and Caco-2 luminal spheres
Development of microporous, polymeric
membranes that are either flat or contain
controllable 3-dimensional shapes that
can be integrated in microfluidic, multi-
organ cell culture systems
Study of epithelial morphogenesis
Toxicity and inflammatory effects of differ-
ent-sized ZnO nanoparticles (NPs) at vari-
ous concentrations seeded in 3D cultures
Development of a biomimetic ‘human gut-
on-a-chip’
Development of a microchip-based system
to mimic the intestine
Development of an in vitro microscale cell
culture analog (mCCA) to the gastrointes-
tinal tract
10
Particular features Reference
Cells cultured on a soft-hydrogel structure. (Yu et al. 2012)
Model includes fibroblasts, lymphocytes, epi- (Salerno-Goncalves,
thelial and endothelial cells embedded in Fasano, and
a protein enriched collagen I matrix. Cells Sztein 2011)
can differentiate into several lineages


(goblet cells, M cells and differentiated
enterocytes).
Immune cells were embedded in a collagen (Collnot, Susewind, and
M. C. PONCE DE LEON-RODR
scaffold, seeded on the apical side of Lehr 2013; Susewind
inserts and Caco-2 cells, on top of et al. 2015)
this layer.
Co-cultures of intestinal and endothelial (Matsusaki et al. 2015)

IGUEZ ET AL.
cells; 1L-Caco-2/1L-NHDF and 1L-Caco-2/
8L-NHDF were constructed using a cell-
coat (nano-ECM of fibronectin and gel-
atin) technology.
Cells were seeded in MatrigelTM in order to (Juuti-Uusitalo et al. 2011)
form spheres.
Membranes can be integrated with micro- (Esch et al. 2012)
fluidic, multi-organ cell culture systems,
providing access to both apical and baso-
lateral sides.
To produce cysts, Caco-2 cells were plated (Jaffe et al. 2008)
either on top of the ECM (MatrigelTM), for
time lapse or embedded in the ECM (col-
lagen I and MatrigelTM), for
immunofluorescence.
The cells were seeded and immobilized in (Wu et al. 2017)
agarose gel.
Micro-device composed of two microfluidic (Kim et al. 2012; Kim and
channels separated by a porous flexible Ingber 2013)
membrane coated with ECM (collagen
and MatrigelTM) and lined with human
intestinal epithelial (Caco-2) cells.
Microchip composed of a glass slide, a per- (Imura et al. 2009)
meable membrane, and polydimethylsi-
loxane sheets, containing microchannels
and Caco-2 cells seeded on
the membrane.
Intestinal cells seeded on polycarbonate (0.4 (Mahler, Shuler, and
mm) membranes, mCCA coated with poly- Glahn 2009)
D-lysine and plasma fibronectin and
HepG2/C3A cells seeded into the liver
chamber. A peristaltic pump was used to
simulate medium re-circulation.
(Continued)
Table 2. Continued.
Model Cell type(s) & source
Intestinal Caco-2 (human colon Development of an integrated micro-
microfluidic adenocarcinoma) fluidic system for long-term perfu-
device sion culture and on-line
monitoring of intestinal tis-
sue models
Microfluidic Caco-2 (human colon Reproduce the 3D villi structure and
gut-on- adenocarcinoma) the fluidic shear in a microflui-
a-chip dic chip
Intestinal Caco-2 (human colon Evaluation of Ca2þ transport
microfluidic adenocarcinoma)
chip
Intestinal Caco-2 (human colon Development of a microfluidic device
microfluidic adenocarcinoma that mimics human intes-
device tinal properties
INFLAMED GUT 3D in vitro intestinal Caco-2 and HT29-MTX (human
mucosa model adenocarcinoma), THP-1 (human
peripheral blood -acute monocytic
leukemia), MEFs, P0 (primary
mouse embryonic fibroblast)
Intestinal inflamed 3D Caco-2, HT29, T84 (human colon
cell-culture model adenocarcinoma) / PBMC (human
peripheral blood mono-
nuclear cells)
Caco-2 (human adenocarcinoma),
THP-1 (human peripheral blood
-acute monocytic leukemia)
3D co-culture (intestinal HT29 (human colon adenocarcin-
epithelial cell-macro- oma), U937 (human monocyte-
phage) model histiocytic lymphoma)
NutriChip Caco-2 (human colon adenocarcin-
oma), U937 (human monocyte-
histiocytic lymphoma)
Microfluidic chip Caco-2 (human adenocarcinoma),
U937 (human monocyte- histio-
cytic lymphoma)
Objective(s)
The microfluidic structure is divided into two
independent channels separated by a
semipermeable membrane enabling the
detection of polarized Caco-2 trans-
port activity.
Cells were seeded on a collagen scaffold
(villi) incorporated into a microfluidic
device consisting of three layers of polydi-
methyl-siloxane (PDMS), a slide glass and
a polyester (PET) membrane
The chip was made of two PDMS layers, two
polymethylmethacrylate (PMMA) layers, a
PET membrane (0.4mm) and a glass slide.
Ag/AgCl electrodes were included in
the device.
Caco-2 cells were seeded on a porous mem-
brane coated with fibronectin between
two layers of polydimethylsilox-
ane (PDMS).
Development of an improved 3D in vitro
intestinal mucosa model to evaluate
drug absorption.
Development of a 3D co-culture of human
intestinal and immune cells stimulated
model for anti-inflammatory drug screen-
ing and their formulations
Evaluation of the delivery efficacy of
nanoparticles
Study of Salmonella enterica coloniza-
tion patterns
Development of an integrated microfluidic
platform to investigate the potential
immuno-modulatory function of
dairy food
Development of a microfluidic-based
dynamic in vitro model of human intes-
tinal barrier
Particular features Reference
(Kimura et al. 2008)
(Shim et al. 2017)
(Huang et al. 2014)
(Chi et al. 2015)
MEFs were dispersed in a type rat tail colla- (Li et al. 2013)
gen solution. Intestinal cells were seeded
on the top of the formed collagen gel.
The co-culture was then transferred to a
receiver plate pre-seeded with THP-1
derived macrophages.
Macrophages and dendritic cells derived (Leonard et al. 2012;
from PBMC were embedded in a collagen Leonard, Collnot, and
layer on a Transwell insert and Caco-2 Lehr 2010)
cells were seeded on top.
THP-1 and Caco-2 cells were embedded in a (Huang et al. 2014)
Matrigel TM solution.
U937 cells were activated upon collagen- (Barrila et al. 2017)
coated scaffolds. HT-29 epithelial cells
were then added and the 3D model was
cultured in the NASA Rotating Wall Vessel
bioreactor until optimal differentiation
was reached.
The core component of the NutriChip is a (Ramadan et al. 2013;
miniaturized artificial human gastrointes- Vergeres et al. 2012)
tinal tract (GIT), which consists of a con-
fluent layer of epithelial cells separated
from a co-culture of immune cells by a
permeable membrane.
Continuous perfusion of culture media from (Ramadan and Jing 2016)
the apical and basolateral side of the por-
ous membrane mimics physiological flow.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
11
12

M. C. PONCE DE LEON-RODR
IGUEZ ET AL.
responses (Griffith and Swartz 2006). Other challenges asso-
ciated with the use of these kinds of models concern the
impact of seeding density on cell responses and develop-
ment, as it has been shown for other type of tissues (Dvir-
Ginzberg et al. 2003; Holy, Shoichet, and Davies 2000;
Ohnuma, Arkin, and Holland 1986; Watt 1988).
Optimization of these models is thus required to be able to
correlate 3D model and in vivo results.
Finally, it should be pointed out that recent advances in
human intestinal epithelial stem cell culture methods, such
as the discovery of the growth factors required for stem cells
homeostasis, have allowed the generation of long-term cul-
ture systems: human intestinal organoids (HIOs) and enter-
oids/colonoids (HIEs). These ex vivo and in vitro models
reproduce physiological characteristics of the human intes-
tine in vivo (Zachos et al. 2015). HIOs are established in
vitro using induced pluripotent stem cells (iPSCs) or embry-
onic stem cells (ESCs) (Spence et al. 2011; McCracken et al.
2011). HIEs are developed from dissociated crypts/stem cells
from intestinal tissue (Sato et al. 2011; Saxena et al. 2015).
In contrast to other culture techniques that contain cells of
mesenchymal origin, HIEs allow the study of isolated epithe-
lial cells (VanDussen et al. 2014). HIEs models are cultivated
in monolayers or in 3D forms (spheroids) using Matrigel as
ECM (VanDussen et al. 2014; Costantini et al. 2018). These
models include several differentiated cell types (enterocytes,
goblet, enteroendocrine and Paneth cells) and could be
established from different segments of the intestine which
could help to better understand the specific-region functions
(Middendorp et al. 2014). HIEs models are derived of non-
transformed small intestine and colonic tissues established
from individuals biopsies (VanDussen et al. 2014) or from
surgically resected intestinal tissues (Sato et al. 2011). This
could be considered as an inconvenient due to ethical issues,
however it can also be seen as an advantage because it could
simplify the study of individual variations in human epithe-
lial function. Especially host-pathogen interactions with
potential use in patient-specific assays with repeatability
already observed by VanDussen et al. (2014). These models
also bring new opportunities, for instance, for the develop-
ment of personalized treatments for chronic diseases.
However, disadvantages of this type of cell culture systems
include the high cost (growth factors) and the fact that they
are labor intensive (Costantini et al. 2018).
Recently, researchers used HIEs models to study norovi-
rus and rotavirus (food/water spread). The authors con-
cluded that this type of models are suitable to study human
enteric virus pathogenesis. Previous attempts to study virus
replication using other in vitro models have been unsuccess-
ful after many years because of the lack of a robust and
reproducible intestinal adapted cell culture system (Ettayebi
et al. 2016; Saxena et al. 2015, 2017; Costantini et al. 2018).
Thus, organoids and enteroids/colonoids may be helpful in
other areas of food research and beyond. For instance, the
possibility to co-cultivate them with immune cells or micro-
organisms would allow the generation of a more complex
intestinal model in order to study intestinal inflammation.
Technical difficulties to extract and maintain viable primary
human epithelial cells may be in the way to be overcome
even if further improvements are required to optimize
these models.
Conclusion and perspectives
Functional in vitro models of the gut have an essential role
in the assessment of the risks or benefits of food as an alter-
native to animal studies. Despite the advances in cell culture
models, much remains to be achieved in the development of
complex gut inflammation models. From simple intestinal
monocultures, through co-cultures to 3D cultures within an
ECM, in vitro models of the intestine have gain considerable
importance as a tool to create more physiologically appro-
priate conditions. Most inflamed gut models are currently
run in 2D conditions to study interactions between the gut
and bioactive food compounds or drugs. In addition, 3D
systems including microfluidic devices, HIOs and HIEs are a
promising way to understand complex whole-organ human
physiology, and are a good alternative to in vivo studies.
Major challenges such as the characterization and validation
of such models as gut inflammation models will concern
how to integrate these technologies in the drug and food
fields. Although caution should be exercised when compar-
ing in vitro and in vivo results, such research should provide
interesting information.
References
Abbott, A. 2003. Biology’s new dimension. Nature 424(6951):870.
doi:10.1038/424870a.
Abraham, C., and J. H. Cho. 2009. Inflammatory bowel disease. New
England Journal of Medicine 361(21):2066–78. doi:10.1056/
NEJMra0804647.
Al-Sadi, R., D. Ye, H. M. Said, and T. Y. Ma. 2011. Cellular and molecu-
lar mechanism of interleukin-1b modulation of caco-2 intestinal epi-
thelial tight junction barrier. Journal of Cellular and Molecular
Medicine 15(4):970–82. doi:10.1111/j.1582-4934.2010.01065.x.
Alzamora, R., F. O’Mahony, W.-H. Ko, T. W.-N. Yip, D. Carter, M.
Irnaten, and B. J. Harvey. 2011. Berberine reduces cAMP-Induced
Chloride secretion in T84 human colonic carcinoma cells through
inhibition of basolateral KCNQ1 channels. Frontiers in Physiology 2:
33. doi:10.3389/fphys.2011.00033.
Andoh, A.,. K. Kinoshita, I. Rosenberg, and D. K. Podolsky. 2001.
Intestinal trefoil factor induces decay-accelerating factor expression
and enhances the protective activities against complement activation
in intestinal epithelial cells. Journal of Immunology 167(7):3887–93.
doi:10.4049/jimmunol.167.7.3887.
Antunes, F., F. Andrade, F. Ara
ujo, D. Ferreira, and B. Sarmento. 2013.
Establishment of a triple co-culture in vitro cell models to study
intestinal absorption of peptide drugs. European Journal of
Pharmaceutics and Biopharmaceutics 83(3):427–35. doi:10.1016/
j.ejpb.2012.10.003.
Ara
ujo, F., C. Pereira, J. Costa, C. Barrias, P. L. Granja, and B.
Sarmento. 2016. In vitro M-like cells genesis through a tissue-engi-
neered triple-culture intestinal model. Journal of Biomedical
Materials Research Part B: Applied Biomaterials 104(4):782–8.
doi:10.1002/jbm.b.33508.
Ara
ujo, F., and B. Sarmento. 2013. Towards the characterization of an
in vitro triple co-culture intestine cell model for permeability stud-
ies. International Journal of Pharmaceutics 458(1):128–34.
doi:10.1016/j.ijpharm.2013.10.003.
Arpaia, N., C. Campbell, X. Fan, S. Dikiy, J. van der Veeken, P.
deRoos, H. Liu, J. R. Cross, K. Pfeffer, P. J. Coffer, and A. Y.

Rudensky. 2013. Metabolites produced by commensal bacteria pro-
mote peripheral regulatory T-cell generation. Nature
504(7480):451–5. doi:10.1038/nature12726.
Artursson, P., K. Palm, and K. Luthman. 2001. Caco-2 monolayers in
experimental and theoretical predictions of drug transport1.
Advanced Drug Delivery Reviews 46(1-3):27–43. doi:10.1016/S0169-
409X(00)00128-9.
Augeron, C., and C. L. Laboisse. 1984. Emergence of permanently dif-
ferentiated cell clones in a human colonic cancer cell line in culture
after treatment with sodium butyrate. Cancer Research 44(9):3961–9.
Barnett, A. M., N. C. Roy, W. C. McNabb, and A. L. Cookson. 2016.
Effect of a semi-purified oligosaccharide-enriched fraction from cap-
rine milk on barrier integrity and mucin production of co-culture
models of the small and large intestinal epithelium. Nutrients
8(5):267. doi:10.3390/nu8050267.
Barrera, G. J., and G. S
anchez. 2015. Cytokine modulation (IL-6, IL-8,
IL-10) by human breast milk lipids on intestinal epithelial cells
(caco-2). Journal of Maternal-Fetal and Neonatal Medicine 0(0):1–8.
doi:10.3109/14767058.2015.1091879.
Barrila, J., Yang, J. Crabb
e, A. Sarker, S. F. Liu, Y. Ott, C. M. Nelman-
Gonzalez, M. A. Clemett, S. J. Nydam, S. D. Forsyth. R. J. et al.
2017. Three-dimensional organotypic co-culture model of intestinal
epithelial cells and macrophages to study Salmonella enterica colon-
ization patterns. Npj Microgravity 3(1):10. doi:10.1038/s41526-017-
0011-2.
B
eduneau, A., C. Tempesta, S. Fimbel, Y. Pellequer, V. Jannin, F.
Demarne, and A. Lamprecht. 2014. A tunable caco-2/HT29-MTX
co-culture model mimicking variable permeabilities of the human
intestine obtained by an original seeding procedure. European
Journal of Pharmaceutics and Biopharmaceutics 87(2):290–8.
doi:10.1016/j.ejpb.2014.03.017.
Behrens, I., P. Stenberg, P. Artursson, and T. Kissel. 2001. Transport of
lipophilic drug molecules in a new mucus-secreting cell culture
model based on HT29-MTX cells. Pharmaceutical Research
18(8):1138–45. doi:10.1023/A:1010974909998.
Beltr
an, A. R., Carraro-Lacroix, L. R. Bezerra, C. N. A. Cornejo, M.
Norambuena, K. Toledo, F. Araos, J. Pardo, F. Leiva, A. Sanhueza.
C. et al. 2015. Escherichia coli heat-stable enterotoxin mediates naþ/
H þ exchanger 4 inhibition involving cAMP in T84 human intestinal
epithelial cells. PLoS ONE 10(12):e0146042. doi:10.1371/
journal.pone.0146042.
Benjamin, J., G. K. Makharia, M. Kalaivani, and Y. K. Joshi. 2008.
Nutritional status of patients with Crohn’s disease. Indian Journal of
Gastroenterology: Official Journal of the Indian Society of
Gastroenterology 27(5):195–200.
Berghaus, L. J., J. N. Moore, D. J. Hurley, M. L. Vandenplas, B. P.
Fortes, M. A. Wolfert, and G.-J. Boons. 2010. Innate immune
responses of primary murine macrophage-lineage cells and RAW
264.7 cells to ligands of toll-like receptors 2, 3, and 4. Comparative
Immunology, Microbiology & Infectious Diseases 33(5):443–54.
doi:10.1016/j.cimid.2009.07.001.
Bitzer, Z. T., S. L. Glisan, M. R. Dorenkott, K. M. Goodrich, L. Ye,
S. F. O’Keefe, J. D. Lambert, and A. P. Neilson. 2015. Cocoa procya-
nidins with different degrees of polymerization possess distinct
activities in models of colonic inflammation. The Journal of
Nutritional Biochemistry 26(8):827–31. doi:10.1016/
j.jnutbio.2015.02.007.
Bourgine, J., I. Billaut-Laden, M. Happillon, J.-M. Lo-Guidice, V.
Maunoury, M. Imbenotte, and F. Broly. 2012. Gene expression
profiling of systems involved in the metabolism and the disposition
of xenobiotics: Comparison between human intestinal biopsy sam-
ples and Colon cell lines. Drug Metabolism and Disposition
40(4):694–705. doi:10.1124/dmd.111.042465.
Briske-Anderson, M. J., J. W. Finley, and S. M. Newman. 1997. The
influence of culture time and passage number on the morphological
and physiological development of caco-2 cells. Experimental Biology
and Medicine 214(3):248–57. doi:10.3181/00379727-214-44093.
Brown, E. M., M. Sadarangani, and B. B. Finlay. 2013. The role of the
immune system in governing host-microbe interactions in the intes-
tine. Nature Immunology 14(7):660–7. doi:10.1038/ni.2611.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 13
Calatayud, M., J. V. Gimeno-Alca~ niz, V. Devesa, and D. V
elez. 2015.
Proinflammatory effect of trivalent arsenical species in a co-culture
of caco-2 cells and peripheral blood mononuclear cells. Archives of
Toxicology 89(4):555–64. doi:10.1007/s00204-014-1271-1.
Cammarota, G., G. Ianiro, R. Cianci, S. Bibb o, A. Gasbarrini, and D.
Curr o. 2015. The involvement of gut microbiota in inflammatory
bowel disease pathogenesis: Potential for therapy. Pharmacology &
Therapeutics 149 :191–212. doi:10.1016/j.pharmthera.2014.12.006.
Cario, E., I. M. Rosenberg, S. L. Brandwein, P. L. Beck, H.-C.
Reinecker, and D. K. Podolsky. 2000. Lipopolysaccharide activates
distinct signaling pathways in intestinal epithelial cell lines express-
ing toll-like receptors. Journal of Immunology 164(2):966–72.
doi:10.4049/jimmunol.164.2.966.
Caro, I., X. Boulenc, M. Rousset, V. Meunier, M. Bourri
e, B. Julian, H.
Joyeux, C. Roques, Y. Berger, A. Zweibaum, and G. Fabre. 1995.
Characterisation of a newly isolated caco-2 clone (TC-7), as a model
of transport processes and biotransformation of drugs. International
Journal of Pharmaceutics 116(2):147–58. doi:10.1016/0378-
5173(94)00280-I.
Chanput, W., J. Mes, R. A. M. Vreeburg, H. F. J. Savelkoul, and H. J.
Wichers. 2010. Transcription profiles of LPS-stimulated THP-1
monocytes and macrophages: A tool to study inflammation modu-
lating effects of food-derived compounds. Food & Function
1(3):254–61. doi:10.1039/C0FO00113A.
Chanput, W., J. J. Mes, H. F. J. Savelkoul, and H. J. Wichers. 2013.
Characterization of polarized THP-1 macrophages and polarizing
ability of LPS and food compounds. Food & Function 4(2):266–76.
doi:10.1039/C2FO30156C.
Chanput, W., J. J. Mes, and H. J. Wichers. 2014. THP-1 cell line: An in
vitro cell model for immune modulation approach. International
Immunopharmacology 23(1):37–45. doi:10.1016/j.intimp.2014.08.002.
Chanput, W., V. Peters, and H. Wichers. 2015. THP-1 and U937 Cells.
In The impact of food bioactives on health, eds. K. Verhoeckx, P.
Cotter, I. L
opez-Exp
osito, C. Kleiveland, T. Lea, A. Mackie, T.
Requena, D. Swiatecka, and H. Wichers, 147–159. New York, NY:
Springer International Publishing.
Chantret, I., A. Barbat, E. Dussaulx, M. G. Brattain, and A. Zweibaum.
1988. Epithelial polarity, villin expression, and enterocytic differenti-
ation of cultured human colon carcinoma cells: A survey of twenty
cell lines. Cancer Res 48(7):1936–42.
Chantret, I., A. Rodolosse, A. Barbat, E. Dussaulx, E. Brot-Laroche, A.
Zweibaum, and M. Rousset. 1994. Differential expression of sucrase-
isomaltase in clones isolated from early and late passages of the cell
line caco-2: Evidence for glucose-dependent negative regulation.
Journal of Cell Science 107(1):213–25.
Cheng, K.-C., C. Li, and A. S. Uss. 2008. Prediction of oral drug
absorption in humans – from cultured cell lines and experimental
animals. Expert Opinion on Drug Metabolism & Toxicology
4(5):581–90. doi:10.1517/17425255.4.5.581.
Chi, M., B. Yi, S. Oh, D.-J. Park, J. H. Sung, and S. Park. 2015. A
microfluidic cell culture device (lFCCD) to culture epithelial cells
with physiological and morphological properties that mimic those of
the human intestine. Biomedical Microdevices 17(3):58. doi:10.1007/
s10544-015-9966-5.
Cho, J. A., and E. Park. 2015. Curcumin utilizes the anti-inflammatory
response pathway to protect the intestine against bacterial invasion.
Nutrition Research and Practice 9(2):117–22. doi:10.4162/
nrp.2015.9.2.117.
Cho, J. H. 2008. The genetics and immunopathogenesis of inflamma-
tory bowel disease. Nature Reviews Immunology 8(6):458–66.
doi:10.1038/nri2340.
Collnot, E.-M., J. Susewind, and C.-M. Lehr. 2013. Advanced in vitro
Models of the Intestinal Mucosa for Drug Delivery Studies. In
Cellular in vitro testing: Methods and protocols, ed. J.W. Haycock, A.
Ahluwalia, and J.M. Wilkinson, 123–133. Boca Raton: CRC Press-
Taylor & Francis Group.
Conklin, L. S., and M. Oliva-Hemker. 2010. Nutritional considerations
in pediatric inflammatory bowel disease. Expert Review of
Gastroenterology & Hepatology 4(3):305–17. doi:10.1586/egh.10.23.
14

M. C. PONCE DE LEON-RODR
IGUEZ ET AL.
Costantini, V., E. K. Morantz, H. Browne, K. Ettayebi, X. L. Zeng,
R. L. Atmar, M. K. Estes, and J. Vinj
e. 2018. Human norovirus rep-
lication in human intestinal enteroids as model to evaluate virus
inactivation. Emerging Infectious Diseases 24 (8):1453–64. doi:
10.3201/eid2408.180126.
Denis, M.-C., Y. Desjardins, A. Furtos, V. Marcil, S. Dudonn
e, A.
Montoudis, C. Garofalo, E. Delvin, A. Marette, and E. Levy. 2015.
Prevention of oxidative stress, inflammation and mitochondrial dys-
function in the intestine by different cranberry phenolic fractions.
Clinical Science 128(3):197–212. doi:10.1042/CS20140210.
Dharmsathaphorn, K., J. A. McRoberts, K. G. Mandel, L. D. Tisdale,
and H. Masui. 1984. A human colonic tumor cell line that maintains
vectorial electrolyte transport. American Journal of Physiology-
Gastrointestinal and Liver Physiology 246(2):G204–8. doi:10.1152/
ajpgi.1984.246.2.G204.
Dhuique-Mayer, C., P. Borel, E. Reboul, B. Caporiccio, P. Besancon,
and M.-J. Amiot. 2007. b-Cryptoxanthin From citrus juices:
Assessment of bioaccessibility using an in vitro digestion/caco-2 cell
culture model. British Journal of Nutrition 97(05):883–90.
doi:10.1017/s0007114507670822.
Ding, S., and P. K. Lund. 2011. Role of intestinal inflammation as an
early event in obesity and insulin resistance. Current Opinion in
Clinical Nutrition & Metabolic Care 14(4):328–33. doi:10.1097/
MCO.0b013e3283478727.
Dostal, A., M. Gagnon, C. Chassard, M. B. Zimmermann, L.
O’Mahony, and C. Lacroix. 2014. Salmonella adhesion, invasion and
cellular immune responses are differentially affected by iron concen-
trations in a combined in vitro gut fermentation-Cell Model. PLoS
ONE 9(3):e93549. doi:10.1371/journal.pone.0093549.
During, A., M. M. Hussain, D. W. Morel, and E. H. Harrison. 2002.
Carotenoid uptake and secretion by CaCo-2 cells b-carotene isomer
selectivity and carotenoid interactions. Journal of Lipid Research
43(7):1086–95. doi:10.1194/jlr.M200068-JLR200.
Dvir-Ginzberg, M., I. Gamlieli-Bonshtein, R. Agbaria, and S. Cohen.
2003. Liver tissue engineering within alginate scaffolds: Effects of cell-
seeding density on hepatocyte viability, morphology, and function.
Tissue Engineering 9(4):757–66. doi:10.1089/107632703768247430.
Esch, M. B., J. H. Sung, J. Yang, C. Yu, J. Yu, J. C. March, and M. L.
Shuler. 2012. On chip porous polymer membranes for integration of
gastrointestinal tract epithelium with microfluidic ‘body-on-a-chip’
devices. Biomedical Microdevices 14(5):895–906. doi:10.1007/s10544-
012-9669-0.
Esser, N., S. Legrand-Poels, J. Piette, A. J. Scheen, and N. Paquot. 2014.
Inflammation as a link between obesity, metabolic syndrome and
type 2 diabetes. Diabetes Research and Clinical Practice
105(2):141–50. doi:10.1016/j.diabres.2014.04.006.
Ettayebi, K., S. E. Crawford, K. Murakami, J. R. Broughman, U.
Karandikar, V. R. Tenge, and F. H. Neill. 2016. Replication of
human noroviruses in stem cell–derived human enteroids. Science
353(6306):1387–93. doi:10.1126/science.aaf5211.
Ewaschuk, J. B., H. Diaz, L. Meddings, B. Diederichs, A. Dmytrash, J.
Backer, M. L. Langen, and K. L. Madsen. 2008. Secreted bioactive
factors from Bifidobacterium infantis enhance epithelial cell barrier
function. American Journal of Physiology-Gastrointestinal and Liver
Physiology 295(5):G1025–34. doi:10.1152/ajpgi.90227.2008.
Fang, H.-W., S.-B. Fang, J.-S. Chiang Chiau, C.-Y. Yeung, W.-T. Chan, C.-
B. Jiang, M.-L. Cheng, and H.-C. Lee. 2010. Inhibitory effects of
Lactobacillus casei subsp. rhamnosus on salmonella lipopolysaccharide-
induced inflammation and epithelial barrier dysfunction in a co-culture
model using caco-2/peripheral blood mononuclear cells. Journal of
Medical Microbiology 59(5):573–9. doi:10.1099/jmm.0.009662-0.
Fitzgerald, K. A., M. Malhotra, C. M. Curtin, F. J. O’ Brien, and C. M.
O’ Driscoll. 2015. Life in 3D is never flat: 3D models to optimise
drug delivery. Journal of Controlled Release 215 :39–54. doi:10.1016/
j.jconrel.2015.07.020.
Fogh, J., and G. Trempe. 1975. New human tumor cell lines. In
Human tumor cells in vitro. ed. J. Fogh, 115–159. New York:
Springer.
Forbes, A., J. Escher, X. H
ebuterne, S. KłeR k, Z. Krznaric, S. Schneider,
R. Shamir, K. Stardelova, N. Wierdsma, A. E. Wiskin, and S. C.
Bischoff. 2017. ESPEN guideline: Clinical nutrition in inflammatory
bowel disease. Clinical Nutrition 36(2):321–47. doi:10.1016/
j.clnu.2016.12.027.
Frontela-Saseta, C., R. L
opez-Nicol
as, C. A. Gonz
alez-Berm
udez, C.
Mart
ınez-Graci
a, and G. Ros-Berruezo. 2013. Anti-inflammatory
properties of fruit juices enriched with pine bark extract in an in
vitro model of inflamed human intestinal epithelium: The effect of
gastrointestinal digestion. Food and Chemical Toxicology 53 :94–9.
doi:10.1016/j.fct.2012.11.024.
Fyderek, K., M. Strus, K. Kowalska-Duplaga, T. Gosiewski, A.
WeR drychowicz, U. Jedynak-Wa˛sowicz, M. Sładek, S. Pieczarkowski,
P. Adamski, P. Kochan, and P. B. Heczko. 2009. Mucosal bacterial
microflora and mucus layer thickness in adolescents with inflamma-
tory bowel disease. World Journal of Gastroenterology 15(42):5287.
doi:10.3748/wjg.15.5287.
Galvez-Llompart, M., M. Iglesias, C. R. del, J. G
alvez, and R. Garc
ıa-
Domenech. 2013. Novel potential agents for ulcerative colitis by
molecular topology: Suppression of IL-6 production in caco-2 and
RAW 264.7 cell lines. Molecular Diversity 17(3):573–93. doi:10.1007/
s11030-013-9458-6.
Geremia, A., P. Biancheri, P. Allan, G. R. Corazza, and A. Di Sabatino.
2014. Innate and adaptive immunity in inflammatory bowel disease.
Autoimmunity Reviews 13(1):3–10. doi:10.1016/j.autrev.2013.06.004.
Gir
on-Calle, J., M. Alaiz, and J. Vioque. 2010. Effect of chickpea pro-
tein hydrolysates on cell proliferation and in vitro bioavailability.
Food Research International 43(5):1365–70. doi:10.1016/
j.foodres.2010.03.020.
Griffith, L. G., and M. A. Swartz. 2006. Capturing complex 3D tissue
physiology in vitro. Nature Reviews Molecular Cell Biology
7(3):211–24. doi:10.1038/nrm1858.
Hagesaether, E. 2011. Permeation modulating properties of natural pol-
ymers – effect of molecular weight and mucus. International Journal
of Pharmaceutics 409(1-2):150–5. doi:10.1016/j.ijpharm.2011.02.046.
Heller, F., Florian, P. Bojarski, C. Richter, J. Christ, M. Hillenbrand, B.
Mankertz, J. Gitter, A. H. B€ urgel, N. Fromm. M. et al. 2005.
Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis
that affects epithelial tight junctions, apoptosis, and cell restitution.
Gastroenterology 129(2):550–64. doi:10.1053/j.gastro.2005.05.002.
Hering, N. A., and J.-D. Schulzke. 2009. Therapeutic options to modu-
late barrier defects in inflammatory bowel disease. Digestive Diseases
27(4):450–4. doi:10.1159/000233283.
Hidalgo, I., T. Raub, and R. Borchardt. 1989. Characterization of the
human-Colon carcinoma cell-Line (caco-2) as a model system for
intestinal epithelial permeability. Gastroenterology 96(3):736–49.
doi:10.1016/0016-5085(89)90897-4.
Hillgren, K. M., A. Kato, and R. T. Borchardt. 1995. In vitro systems
for studying intestinal drug absorption. Medicinal Research Reviews
15(2):83–109. doi:10.1002/med.2610150202.
Hollebeeck, S., J. Winand, M.-F. H
erent, A. During, J. Leclercq, Y.
Larondelle, and Y.-J. Schneider. 2012. Anti-inflammatory effects of
pomegranate (Punica granatum L.) husk ellagitannins in caco-2
cells, an in vitro model of human intestine. Food & Function
3(8):875–85. doi:10.1039/C2FO10258G.
Holy, C. E., M. S. Shoichet, and J. E. Davies. 2000. Engineering three-
dimensional bone tissue in vitro using biodegradable scaffolds:
Investigating initial cell-seeding density and culture period. Journal
of Biomedical Materials Research 51(3):376–82. doi:10.1002/1097-
4636(20000905)51:3 < 376::aid-jbm11 > 3.0.co;2-g.
Howell, S., A. Kenny, and A. Turner. 1992. A survey of membrane
peptidases in 2 human colonic cell-Lines, caco-2 and ht-29.
Biochemical Journal 284 :595–601. doi:10.1042/bj2840595.
Huang, C., Q. Ramadan, J. B. Wacker, H. C. Tekin, C. Ruffert, G.
Vergeres, P. Silacci, and M. A M. Gijs. 2014. Microfluidic chip for
monitoring Ca2þ transport through a confluent layer of intestinal
cells. RSC Advances 4(95):52887–91. doi:10.1039/C4RA09370D.
Huang, H.,. J.-Q. Liu, Y. Yu, L.-H. Mo, R.-T. Ge, H.-P. Zhang, Z.-G.
Liu, P.-Y. Zheng, and P.-C. Yang. 2016. Regulation of TWIK-related
potassium channel-1 (Trek1) restitutes intestinal epithelial barrier
function. Cellular & Molecular Immunology 13(1):110–8.
doi:10.1038/cmi.2014.137.

Huang, S., J. M. Rutkowsky, R. G. Snodgrass, K. D. Ono-Moore, D. A.
Schneider, J. W. Newman, S. H. Adams, and D. H. Hwang. 2012.
Saturated fatty acids activate TLR-mediated proinflammatory signal-
ing pathways. Journal of Lipid Research 53(9):2002–13. doi:10.1194/
jlr.D029546.
Huang, S.-M., C.-H. Wu, and G.-C. Yen. 2006. Effects of flavonoids on
the expression of the pro-inflammatory response in human mono-
cytes induced by ligation of the receptor for AGEs. Molecular
Nutrition & Food Research 50(12):1129–39. doi:10.1002/
mnfr.200600075.
Huang, Z., Z. Wang, S. Long, H. Jiang, J. Chen, J. Zhang, and L. Dong.
2014. A 3-D artificial Colon tissue mimic for the evaluation of
nanoparticle–based drug delivery system. Molecular Pharmaceutics
11(7):2051–61. doi:10.1021/mp400723j.
Huet, C., C. Sahuquillo-Merino, E. Coudrier, and D. Louvard. 1987.
Absorptive and mucus-secreting subclones isolated from a multipo-
tent intestinal cell line (HT-29) provide new models for cell polarity
and terminal differentiation. Journal of Cell Biology 105(1):345–57.
doi:10.1083/jcb.105.1.345.
Imura, Y., Y. Asano, K. Sato, and E. Yoshimura. 2009. A microfluidic
system to evaluate intestinal absorption. Analytical Sciences
25(12):1403–7. doi:10.2116/analsci.25.1403.
Jaffe, A. B., N. Kaji, J. Durgan, and A. Hall. 2008. Cdc42 controls spin-
dle orientation to position the apical surface during epithelial mor-
phogenesis. The Journal of Cell Biology 183(4):625–33. doi:10.1083/
jcb.200807121.
Jiang, H. A. I.-Y. U. E., F. E. N. G. Wang, H. A. I.-M. I. N. Chen, and
X. I. A. O.-J. U. N. Yan. 2013. j-carrageenan induces the disruption
of intestinal epithelial caco-2 monolayers by promoting the inter-
action between intestinal epithelial cells and immune cells.
Molecular Medicine Reports 8(6):1635–42. doi:10.3892/
mmr.2013.1726.
Juuti-Uusitalo, K., L. J. Klunder, K. A. Sjollema, K. Mackovicova, R.
Ohgaki, D. Hoekstra, J. Dekker, and S. C. D. van IJzendoorn. 2011.
Differential effects of TNF (TNFSF2) and IFN-c on intestinal epithe-
lial cell morphogenesis and barrier function in three-Dimensional
Culture. PLoS ONE 6(8):e22967. doi:10.1371/journal.pone.0022967.
Juuti-Uusitalo, K. M., K. Kaukinen, M. M€aki, J. Tuimala, and H.
Kainulainen. 2006. Gene expression in TGFbeta-induced epithelial
cell differentiation in a three-dimensional intestinal epithelial cell
differentiation model. BMC Genomics 7 :279. doi:10.1186/1471-2164-
7-279.
Kamada, N., and G. N
~ez. 2014. Regulation of the immune system by
un
the resident intestinal bacteria. Gastroenterology 146(6):1477–88.
doi:10.1053/j.gastro.2014.01.060.
Kamada, N., S.-U. Seo, G. Y. Chen, and G. N
~ez. 2013. Role of the
un
gut microbiota in immunity and inflammatory disease. Nature
Reviews Immunology 13(5):321–35. doi:10.1038/nri3430.
Kang, K.-H., C.-S. Kong, Y. Seo, M.-M. Kim, and S.-K. Kim. 2009.
Anti-inflammatory effect of coumarins isolated from corydalis heter-
ocarpa in HT-29 human Colon carcinoma cells. Food and Chemical
Toxicology 47(8):2129–34. doi:10.1016/j.fct.2009.05.036.
Kanzato, H., M. Manabe, and M. Shimizu. 2001. An in vitro approach
to the evaluation of the cross talk between intestinal epithelium and
macrophages. Bioscience, Biotechnology, and Biochemistry
65(2):449–51. doi:10.1271/bbb.65.449.
Kasper, J. Y., M. I. Hermanns, C. Cavelius, A. Kraegeloh, T. Jung, R.
Danzebrink, R. E. Unger, and C. J. Kirkpatrick. 2016. The role of
the intestinal microvasculature in inflammatory bowel disease:
Studies with a modified caco-2 model including endothelial cells
resembling the intestinal barrier in vitro. International Journal of
Nanomedicine Volume 11 :6353–64. doi:10.2147/IJN.S92608.
Kaulmann, A., C. M. Andr
e, Y.-J. Schneider, L. Hoffmann, and T.
Bohn. 2016. Carotenoid and polyphenol bioaccessibility and cellular
uptake from plum and cabbage varieties. Food Chemistry em 197
Part A: :325–32. doi:10.1016/j.foodchem.2015.10.049.
Kaulmann, A., S. Legay, Y.-J. Schneider, L. Hoffmann, and T. Bohn.
2016. Inflammation related responses of intestinal cells to plum and
cabbage digesta with differential carotenoid and polyphenol profiles
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 15
following simulated gastrointestinal digestion. Molecular Nutrition &
Food Research 60(5):992–1005. doi:10.1002/mnfr.201500947.
Kaulmann, A., T. Serchi, J. Renaut, L. Hoffmann, and T. Bohn. 2012.
Carotenoid exposure of caco-2 intestinal epithelial cells did not
affect selected inflammatory markers but altered their proteomic
response. British Journal of Nutrition 108(06):963–73. doi:10.1017/
S0007114511006349.
Kern
eis, S., E. Caliot, H. Stubbe, A. Bogdanova, J.-P. Kraehenbuhl, and
E. Pringault. 2000. Molecular studies of the intestinal mucosal bar-
rier physiopathology using cocultures of epithelial and immune cells:
a technical update. Microbes and Infection 2(9):1119–24.
doi:10.1016/S1286-4579(00)01266-1.
Khor, B., A. Gardet, and R. J. Xavier. 2011. Genetics and pathogenesis
of inflammatory bowel disease. Nature 474(7351):307–17.
doi:10.1038/nature10209.
Khosravi, A., and S. K. Mazmanian. 2013. Disruption of the gut micro-
biome as a risk factor for microbial infections. Current Opinion in
Microbiology 16(2):221–7. doi:10.1016/j.mib.2013.03.009.
Kim, H. J., D. Huh, G. Hamilton, and D. E. Ingber. 2012. Human gut-
on-a-chip inhabited by microbial flora that experiences intestinal
peristalsis-like motions and flow. Lab on a Chip 12(12):2165–74.
doi:10.1039/C2LC40074J.
Kim, H. J., and D. E. Ingber. 2013. Gut-on-a-Chip Microenvironment
induces human intestinal cells to undergo villus differentiation.
Integrative Biology 5(9):1130–40. doi:10.1039/C3IB40126J.
Kim, K.-M., Y.-S. Kim, J. Y. Lim, S. J. Min, H.-C. Ko, S.-J. Kim, and Y.
Kim. 2015. Intestinal anti-inflammatory activity of sasa quelpaerten-
sis leaf extract by suppressing lipopolysaccharide-stimulated inflam-
matory mediators in intestinal epithelial caco-2 cells co-cultured
with RAW 264.7 macrophage cells. Nutrition Research and Practice
9(1):3. doi:10.4162/nrp.2015.9.1.3.
Kim, S. H., M. Chi, B. Yi, S. H. Kim, S. Oh, Y. Kim, S. Park, and J. H.
Sung. 2014. Three-dimensional intestinal villi epithelium enhances
protection of human intestinal cells from bacterial infection by
inducing mucin expression. Integrative Biology 6(12):1122–31.
doi:10.1039/C4IB00157E.
Kimura, H., T. Yamamoto, H. Sakai, Y. Sakai, and T. Fujii. 2008. An
integrated microfluidic system for long-term perfusion culture and
on-line monitoring of intestinal tissue models. Lab on a Chip
8(5):741–6. doi:10.1039/B717091B.
de Kivit, S., E. van Hoffen, N. Korthagen, J. Garssen, and L. E. M.
Willemsen. 2011. Apical TLR ligation of intestinal epithelial cells
drives a Th1-polarized regulatory or inflammatory type effector
response in vitro. Immunobiology 216(4):518–27. doi:10.1016/
j.imbio.2010.08.005.
Kleiveland, C. R. 2015. Peripheral blood mononuclear cells. In The
impact of food bioactives on health. eds. K. Verhoeckx, P. Cotter, I.
L
opez-Exp
osito, C. Kleiveland, T. Lea, A. Mackie, T. Requena, D.
Swiatecka, and H. Wichers, 161–167. New York, NY: Springer
International Publishing.
Kosi
nska, A., and W. Andlauer. 2012. Cocoa polyphenols are absorbed
in caco-2 cell model of intestinal epithelium. Food Chemistry
135(3):999–1005. doi:10.1016/j.foodchem.2012.05.101.
Kosi
nska, A., and W. Andlauer. 2013. Modulation of tight junction
integrity by food components. Food Research International
54(1):951–60. doi:10.1016/j.foodres.2012.12.038.
Kostic, A. D., R. J. Xavier, and D. Gevers. 2014. The microbiome in
inflammatory bowel disease: Current status and the future ahead.
Gastroenterology 146(6):1489–99. doi:10.1053/j.gastro.2014.02.009.
Kuan, S.-F., J. C. Byrd, C. B. Basbaum, and Y. S. Kim. 1987.
Characterization of quantitative mucin variants from a human
Colon cancer cell line. Cancer Research 47(21):5715–24.
Langerholc, T., P. A. Maragkoudakis, J. Wollgast, L. Gradisnik, and A.
Cencic. 2011. Novel and established intestinal cell line models – An
indispensable tool in food science and nutrition. Trends in Food
Science & Technology 22 :S11–S20. doi:10.1016/j.tifs.2011.03.010.
Langhans, S. A. 2018. Three-Dimensional In vitro cell culture models
in drug discovery and drug repositioning. Frontiers in Pharmacology
9:6. doi:10.3389/fphar.2018.00006.
16

M. C. PONCE DE LEON-RODR
IGUEZ ET AL.
Laparra, J. M., R. P. Glahn, and D. D. Miller. 2009. Different responses
of Fe transporters in caco-2/HT29-MTX cocultures than in inde-
pendent caco-2 cell cultures. Cell Biology International 33(9):971–7.
doi:10.1016/j.cellbi.2009.06.001.
Lea, T. 2015. Caco-2 Cell Line. In The impact of food bioactives on
health., ed. K. Verhoeckx, P. Cotter, I. L
opez-Exp

osito, C.
Kleiveland, T. Lea, A. Mackie, T. Requena, D. Swiatecka, and H.
Wichers, 103–111. New York, NY: Springer International
Publishing.
Lefebvre, D. E., K. Venema, L. Gombau, L. G. Valerio, J. Raju, G. S.
Bondy, H. Bouwmeester, R. P. Singh, A. J. Clippinger, E.-M.
Collnot, et al., and V. Stone. 2015. Utility of models of the gastro-
intestinal tract for assessment of the digestion and absorption of
engineered nanomaterials released from food matrices.
Nanotoxicology 9(4):523–42. doi:10.3109/17435390.2014.948091.
Leibovitz, A., J. C. Stinson, W. B. McCombs, C. E. McCoy, K. C.
Mazur, and N. D. Mabry. 1976. Classification of human colorectal
adenocarcinoma cell lines. Cancer Research 36(12):4562–9.
Leonard, F., H. Ali, E.-M. Collnot, B. J. Crielaard, T. Lammers, G.
Storm, and C.-M. Lehr. 2012. Screening of budesonide nanoformula-
tions for treatment of inflammatory bowel disease in an inflamed
3D Cell-Culture Model. ALTEX: Alternatives to Animal Experiments
29(3):275–85. doi:10.14573/altex.2012.3.275.
Leonard, F., E.-M. Collnot, and C.-M. Lehr. 2010. A three-dimensional
coculture of enterocytes, monocytes and dendritic cells to model
inflamed intestinal mucosa in vitro. Molecular Pharmaceutics
7(6):2103–19. doi:10.1021/mp1000795.
Lesuffleur, T., A. Barbat, E. Dussaulx, and A. Zweibaum. 1990. Growth
adaptation to methotrexate of HT-29 human colon carcinoma cells
is associated with their ability to differentiate into columnar absorp-
tive and mucus-secreting cells. Cancer Research 50(19):6334–43.
Lesuffleur, T., N. Porchet, J. P. Aubert, D. Swallow, J. R. Gum, Y. S.
Kim, F. X. Real, and A. Zweibaum. 1993. Differential expression of
the human mucin genes MUC1 to MUC5 in relation to growth and
differentiation of different mucus-secreting HT-29 cell subpopula-
tions. Journal of Cell Science 106(3):771–83.
Li, N., D. Wang, Z. Sui, X. Qi, L. Ji, X. Wang, and L. Yang. 2013.
Development of an improved three-Dimensional In vitro intestinal
mucosa model for drug absorption evaluation. Tissue Eng. Part C
Methods 19(9):708–19. doi:10.1089/ten.tec.2012.0463.
Liang, G. H., and C. R. Weber. 2014. Molecular aspects of tight junc-
tion barrier function. Current Opinion in Pharmacology 19 :84–9.
doi:10.1016/j.coph.2014.07.017.
Liu, T.-C., and T. S. Stappenbeck. 2016. Genetics and pathogenesis of
inflammatory bowel disease. Annual Review of Pathology:
Mechanisms of Disease 11(1):127–48. doi:10.1146/annurev-pathol-
012615-044152.
Loftus, E. V. 2004. Clinical epidemiology of inflammatory bowel dis-
ease: Incidence, prevalence, and environmental influences.
Gastroenterology 126(6):1504–17. doi:10.1053/j.gastro.2004.01.063.
Maccaferri, S., A. Klinder, P. Brigidi, P. Cavina, and A. Costabile. 2012.
Potential probiotic Kluyveromyces marxianus B0399 modulates the
immune response in caco-2 cells and peripheral blood mononuclear
cells and impacts the human gut microbiota in an in vitro colonic
model system. Applied and Environmental Microbiology
78(4):956–64. doi:10.1128/AEM.06385-11.
Madara, J. L., and K. Dharmsathaphorn. 1985. Occluding junction
structure-function relationships in a cultured epithelial monolayer.
Journal of Cell Biology 101(6):2124–33. doi:10.1083/jcb.101.6.2124.
Maher, S., R. J. Mrsny, and D. J. Brayden. 2016. Intestinal permeation
enhancers for oral peptide delivery. Advanced Drug Delivery Reviews
106PartB:277–319. doi:10.1016/j.addr.2016.06.005.
Mahler, G. J., M. B. Esch, R. P. Glahn, and M. L. Shuler. 2009.
Characterization of a gastrointestinal tract microscale cell culture
analog used to predict drug toxicity. Biotechnology and
Bioengineering 104(1):193–205. doi:10.1002/bit.22366.
Mahler, G. J., M. L. Shuler, and R. P. Glahn. 2009. Characterization of
caco-2 and HT29-MTX cocultures in an in vitro digestion/cell cul-
ture model used to predict iron bioavailability. The Journal of
Nutritional Biochemistry 20(7):494–502. doi:10.1016/
j.jnutbio.2008.05.006.
Makon-S
ebastien, N., F. Francis, S. Eric, V. P. Henri, L. J. François, P.
Laurent, B. Yves, and C. Serge. 2014. Lycopene modulates THP1
and Caco2 cells inflammatory state through transcriptional and non-
transcriptional processes. Mediators of Inflammation 2014 :1.
doi:10.1155/2014/507272, 10.1155/2014/507272.
Mallon, D. P., and D. L. Suskind. 2010. Nutrition in pediatric inflam-
matory bowel disease. Nutrition in Clinical Practice 25(4):335–9.
doi:10.1177/0884533610373773.
Manichanh, C., N. Borruel, F. Casellas, and F. Guarner. 2012. The gut
microbiota in IBD. Nature Reviews Gastroenterology & Hepatology
9(10):599–608. doi:10.1038/nrgastro.2012.152.
Martins, I. M., G. A. Macedo, J. A. Macedo, B. S. Roberto, Q. Chen,
J. B. Blumberg, and C.-Y. O. Chen. 2017. Tannase enhances the
anti-inflammatory effect of grape pomace in caco-2 cells treated
with IL-1b. Journal of Functional Foods 29 :69–76. doi:10.1016/
j.jff.2016.12.011.
Maslowski, K. M., Vieira, A. T. Ng, A. Kranich, J. Sierro, F. Yu, D.
Schilter, H. C. Rolph, M. S. Mackay, F. Artis. D., et al. 2009.
Regulation of inflammatory responses by gut microbiota and
chemoattractant receptor GPR43. Nature 461(7268):1282–6.
doi:10.1038/nature08530.
Massironi, S., R. E. Rossi, F. A. Cavalcoli, S. Della Valle, M. Fraquelli,
and D. Conte. 2013. Nutritional deficiencies in inflammatory bowel
disease: Therapeutic approaches. Clinical Nutrition 32(6):904–10.
doi:10.1016/j.clnu.2013.03.020.
Matias, A., S. L. Nunes, J. Poejo, E. Mecha, A. T. Serra, P. J. A.
Madeira, M. R. Bronze, and C. M. M. Duarte. 2014. Antioxidant
and anti-inflammatory activity of a flavonoid-rich concentrate recov-
ered from Opuntia ficus-indica juice. Food & Function
5(12):3269–80. doi:10.1039/C4FO00071D.
Matsusaki, M.,. D. Hikimoto, A. Nishiguchi, K. Kadowaki, K. Ohura,
T. Imai, and M. Akashi. 2015. 3D-fibroblast tissues constructed by a
cell-coat technology enhance tight-junction formation of human
colon epithelial cells. Biochemical and Biophysical Research
Communications 457(3):363–9. doi:10.1016/j.bbrc.2014.12.118.
McCracken, K. W., J. C. Howell, J. M. Wells, and J. R. Spence. 2011.
Generating human intestinal tissue from pluripotent stem cells in
vitro. Nature Protocols 6 (12):1920–8. doi:10.1038/nprot.2011.410.
Middendorp, S., K. Schneeberger, C. L. Wiegerinck, M. Mokry,
R. D. L. Akkerman, S. van Wijngaarden, H. Clevers, and E. E. S.
Nieuwenhuis. 2014. Adult stem cells in the small intestine are
intrinsically programmed with their location-Specific Function. Stem
Cells 32 (5):1083–91. doi:10.1002/stem.1655.
Mijac, D. D., G. L. J. Jankovi
c, J. Jorga, and M. N. Krsti
c. 2010.
Nutritional status in patients with active inflammatory bowel dis-
ease: Prevalence of malnutrition and methods for routine nutritional
assessment. European Journal of Internal Medicine 21(4):315–9.
doi:10.1016/j.ejim.2010.04.012.
Molodecky, N. A., I. S. Soon, D. M. Rabi, W. A. Ghali, M. Ferris, G.
Chernoff, E. I. Benchimol, R. Panaccione, S. Ghosh, H. W. Barkema,
and G. G. Kaplan. 2012. Increasing incidence and prevalence of the
inflammatory bowel diseases with time, based on systematic review.
Gastroenterology 142(1):46–54.e42. doi:10.1053/j.gastro.2011.10.001.
Mori, A., H. Satsu, and M. Shimizu. 2003. New model for studying the
migration of immune cells into intestinal epithelial cell monolayers.
Cytotechnology 43(1-3):57–64. doi:10.1023/B:
CYTO.0000039910.30540.8f.
Moyes, S. M., J. F. Morris, and K. E. Carr. 2010. Macrophages increase
microparticle uptake by enterocyte-like caco-2 cell monolayers.
Journal of Anatomy 217(6):740–54. doi:10.1111/j.1469-
7580.2010.01304.x.
Murakami, H., and H. Masui. 1980. Hormonal control of human colon
carcinoma cell growth in serum-free medium. Proceedings of the
National Academy of Sciences 77(6):3464–8. doi:10.1073/
pnas.77.6.3464.
Nerurkar, M. M., P. S. Burton, and R. T. Borchardt. 1996. The use of
surfactants to enhance the permeability of peptides through caco-2

cells by inhibition of an apically polarized efflux system.
Pharmaceutical Research 13(4):528–34. doi:10.1023/A:1016033702220.
Neurath, M. F. 2014. Cytokines in inflammatory bowel disease. Nature
Reviews Immunology 14(5):329–42. doi:10.1038/nri3661.
Ng, S. C., C. N. Bernstein, M. H. Vatn, P. L. Lakatos, E. V. Loftus, C.
Tysk, C. O’Morain, B. Moum, and J.-F. Colombel. and (ioibd), on
behalf of the E. and N.H.T.F. of the I.O. of I.B.D. 2013. Geographical
variability and environmental risk factors in inflammatory bowel dis-
ease. Gut 62(4):630–49. doi:10.1136/gutjnl-2012-303661.
Nichols, J. M., I. Maiellaro, J. Abi-Jaoude, S. Curci, and A. M. Hofer.
2015. Store-operated” cAMP signaling contributes to Ca2þ-activated
cl  secretion in T84 colonic cells. American Journal of Physiology-
Gastrointestinal and Liver Physiology 309(8):G670–9. doi:10.1152/
ajpgi.00214.2015.
Nollevaux, G., C. Devill
e, B. El Moualij, W. Zorzi, P. Deloyer, Y.-J.
Schneider, O. Peulen, and G. Dandrifosse. 2006. Development of a
serum-free co-culture of human intestinal epithelium cell-lines
(caco-2/HT29-5M21). BMC Cell Biology 7(1):20. doi:10.1186/1471-
2121-7-20.
Nusrat, A., C. V Eichel-Streiber, J. R. Turner, P. Verkade, J. L. Madara,
and C. A. Parkos. 2001. Clostridium difficile toxins disrupt epithelial
barrier function by altering membrane microdomain localization of
tight junction proteins. Infection and Immunity 69(3):1329–36.
doi:10.1128/IAI.69.3.1329-1336.2001.
O’Hara, J. R., and A. G. Buret. 2008. Mechanisms of intestinal tight
junctional disruption during infection. Frontiers in Bioscience 13
:7008–21. doi:10.2741/3206.
Ohnuma, T., H. Arkin, and J. F. Holland. 1986. Effects of cell density
on drug-induced cell kill kinetics in vitro (inoculum effect). British
Journal of Cancer 54(3):415–21. doi:10.1038/bjc.1986.192.
Olejnik, A., K. Kowalska, M. Olkowicz, J. Rychlik, W. Juzwa, K.
Myszka, R. Dembczy
nski, and W. Białas. 2015. Anti-inflammatory
effects of gastrointestinal digested Sambucus nigra L. fruit extract
analysed in co-cultured intestinal epithelial cells and lipopolysac-
charide-stimulated macrophages. Journal of Functional Foods 19
:649–60. Part A: doi:10.1016/j.jff.2015.09.064.
Otte, J.-M., and D. K. Podolsky. 2004. Functional modulation of enter-
ocytes by gram-positive and gram-negative microorganisms.
American Journal of Physiology-Gastrointestinal and Liver Physiology
286(4):G613–26. doi:10.1152/ajpgi.00341.2003.
Ou, G., V. Baranov, E. Lundmark, S. Hammarstr€om, and M.-L.
Hammarstr€ om. 2009. Contribution of intestinal epithelial cells to
innate immunity of the human gut – Studies on polarized mono-
layers of Colon carcinoma cells. Scandinavian Journal of
Immunology 69(2):150–61. doi:10.1111/j.1365-3083.2008.02208.x.
Pampaloni, F., E. G. Reynaud, and E. H. K. Stelzer. 2007. The third
dimension bridges the gap between cell culture and live tissue.
Nature Reviews Molecular Cell Biology 8(10):839–45. doi:10.1038/
nrm2236.
Pandolfi, F., R. Cianci, D. Pagliari, R. Landolfi, and G. Cammarota.
2009. Cellular mediators of inflammation: Tregs and TH cells in
gastrointestinal diseases. Mediators Inflamm 2009 :1. doi:10.1155/
2009/132028.
Parlesak, A., D. Haller, S. Brinz, A. Baeuerlein, and C. Bode. 2004.
Modulation of cytokine release by differentiated CACO-2 cells in a
compartmentalized coculture model with mononuclear leucocytes
and nonpathogenic bacteria. Scandinavian Journal of Immunology
60(5):477–85. doi:10.1111/j.0300-9475.2004.01495.x.
Pinto, M., S. Robineleon, M. Appay, M. Kedinger, N. Triadou, E.
Dussaulx, B. Lacroix, P. Simonassmann, K. Haffen, J. Fogh, and A.
Zweibaum. 1983. Enterocyte-like differentiation and polarization of
the human-Colon carcinoma cell-Line Caco-2 in culture. Biology of
the Cell 47(3):323–30.
Poquet, L., M. N. Clifford, and G. Williamson. 2008. Transport and
metabolism of ferulic acid through the colonic epithelium. Drug
Metabolism and Disposition 36(1):190–7. doi:10.1124/
dmd.107.017558.
Ramadan, Q., H. Jafarpoorchekab, C. Huang, P. Silacci, S. Carrara, G.
Kokl€ u, J. Ghaye, J. Ramsden, C. Ruffert, G. Vergeres, and M. A. M.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 17
Gijs. 2013. NutriChip: Nutrition analysis meets microfluidics. Lab
on a Chip 13(2):196–203. doi:10.1039/C2LC40845G.
Ramadan, Q., and L. Jing. 2016. Characterization of tight junction dis-
ruption and immune response modulation in a miniaturized caco-2/
U937 coculture-based in vitro model of the human intestinal barrier.
Biomedical Microdevices 18(1):11. doi:10.1007/s10544-016-0035-5.
Ranaldi, G., R. Consalvo, Y. Sambuy, and M. L. Scarino. 2003.
Permeability characteristics of parental and clonal human intestinal
caco-2 cell lines differentiated in serum-supplemented and serum-
free media. Toxicology in Vitro 17(5-6):761–7. doi:10.1016/S0887-
2333(03)00095-X.
Raschke, W. C., S. Baird, P. Ralph, and I. Nakoinz. 1978. Functional
macrophage cell lines transformed by abelson leukemia virus. Cell
15(1):261–7. doi:10.1016/0092-8674(78)90101-0.
Ravi, M., V. Paramesh, S. R Kaviya, E. Anuradha, and F. D. P.
Solomon. 2015. 3D cell culture systems: Advantages and applica-
tions. Journal of Cellular Physiology 230(1):16–26. doi:10.1002/
jcp.24683.
Reiff, C., and D. Kelly. 2010. Inflammatory bowel disease, gut bacteria
and probiotic therapy. International Journal of Medical Microbiology
300(1):25–33. doi:10.1016/j.ijmm.2009.08.004.
Des Rieux, A., V. Fievez, I. Th
eate, J. Mast, V. Pr
eat, and Y.-J.
Schneider. 2007. An improved in vitro model of human intestinal
follicle-associated epithelium to study nanoparticle transport by M
cells. European Journal of Pharmaceutical Sciences 30(5):380–91.
doi:10.1016/j.ejps.2006.12.006.
Rodr
ıguez-Ramiro, I., S. Ramos, E. L
opez-Oliva, A. Agis-Torres, L.
Bravo, L. Goya, and M. A. Mart
ın. 2013. Cocoa polyphenols prevent
inflammation in the Colon of azoxymethane-treated rats and in
TNF-a-stimulated caco-2 cells. British Journal of Nutrition
110(02):206–15. doi:10.1017/S0007114512004862.
Ryan, S.-L., A.-M. Baird, G. Vaz, A. J. Urquhart, h Senge, D. J. Richard,
K. J. O’Byrne, and A. M. Davies. 2016. Drug discovery approaches uti-
lizing three-dimensional cell culture. ASSAY and Drug Development
Technologies 14(1):19–28. doi:10.1089/adt.2015.670.
Salerno-Goncalves, R., A. Fasano, and M. B. Sztein. 2011. Engineering of
a multi-cellular organotypic model of the human intestinal mucosa.
Gastroenterology 141(2):e18–20. doi:10.1053/j.gastro.2011.04.062.
Samak, G., S. Aggarwal, and R. K. Rao. 2011. ERK is involved in EGF-
mediated protection of tight junctions, but not adherens junctions,
in acetaldehyde-treated caco-2 cell monolayers. American Journal of
Physiology-Gastrointestinal and Liver Physiology 301(1):G50–9.
doi:10.1152/ajpgi.00494.2010.
Sambuy, Y., I. D. Angelis, G. Ranaldi, M. L. Scarino, A. Stammati, and
F. Zucco. 2005. The caco-2 cell line as a model of the intestinal bar-
rier: influence of cell and culture-related factors on caco-2 cell func-
tional characteristics. Cell Biology and Toxicology 21(1):1–26.
doi:10.1007/s10565-005-0085-6.
Sato, T., D. E. Stange, M. Ferrante, R. G. J. Vries, J. H. van Es, S. van
den Brink, W. J. van Houdt, A. Pronk, J. van Gorp, P. D. Siersema,
and H. Clevers. 2011. Long-Term Expansion of epithelial organoids
from human colon, adenoma, adenocarcinoma, and Barrett’s epithe-
lium. Gastroenterology 141 (5):1762–72. doi:10.1053/
j.gastro.2011.07.050.
Satsu, H., Y. Ishimoto, T. Nakano, T. Mochizuki, T. Iwanaga, and M.
Shimizu. 2006. Induction by activated macrophage-like THP-1 cells
of apoptotic and necrotic cell death in intestinal epithelial caco-2
monolayers via tumor necrosis factor-alpha. Experimental Cell
Research 312(19):3909–19. doi:10.1016/j.yexcr.2006.08.018.
Saxena, K., S. E. Blutt, K. Ettayebi, X.-L. Zeng, R. B. James, E. C. Sue,
and U. Karandikar. 2015. Human intestinal enteroids: A new model
to study human rotavirus infection, host restriction and pathophysi-
ology. Journal of Virology 90(1):43–56. doi:10.1128/JVI.01930-15.
Saxena, K., L. M. Simon, X.-L. Zeng, S. E. Blutt, S. E. Crawford, N. P.
Sastri, and U. C. Karandikar. 2017. A paradox of transcriptional and
functional innate interferon responses of human intestinal enteroids
to enteric virus infection. Proceedings of the National Academy of
Sciences of the United States of America 114(4):E570–79.
doi:10.1073/pnas.1615422114.
18

M. C. PONCE DE LEON-RODR
IGUEZ ET AL.
Schimpel, C., B. Teubl, M. Absenger, C. Meindl, E. Fr€ ohlich, G.
Leitinger, A. Zimmer, and E. Roblegg. 2014. Development of an
advanced intestinal in vitro triple culture permeability model to
study transport of nanoparticles. Molecular Pharmaceutics
11(3):808–18. doi:10.1021/mp400507g.
Selby-Pham, S. N. B., S. A. Osborne, K. S. Howell, F. R. Dunshea, and
L. E. Bennett. 2017. Transport rates of dietary phytochemicals in cell
monolayers is inversely correlated with absorption kinetics in
humans. Journal of Functional Foods 39 :206–14. doi:10.1016/
j.jff.2017.10.016.
Sergent, T., N. Piront, J. Meurice, O. Toussaint, and Y.-J. Schneider.
2010. Anti-inflammatory effects of dietary phenolic compounds in
an in vitro model of inflamed human intestinal epithelium.
Chemico-Biological Interactions 188(3):659–67. doi:10.1016/
j.cbi.2010.08.007.
Shanahan, F., and E. M. M. Quigley. 2014. Manipulation of the micro-
biota for treatment of IBS and IBD—challenges and controversies.
Gastroenterology 146(6):1554–63. doi:10.1053/j.gastro.2014.01.050.
Sheehan, D., C. Moran, and F. Shanahan. 2015. The microbiota in
inflammatory bowel disease. Journal of Gastroenterology
50(5):495–507. doi:10.1007/s00535-015-1064-1.
Shim, K.-Y., D. Lee, J. Han, N.-T. Nguyen, S. Park, and J. H. Sung.
2017. Microfluidic gut-on-a-chip with three-dimensional villi struc-
ture. Biomedical Microdevices 19(2):37. doi:10.1007/s10544-017-0179-
y.
Shimizu, M. 2010. Interaction between food substances and the intes-
tinal epithelium. Bioscience, Biotechnology, and Biochemistry
74(2):232–41. doi:10.1271/bbb.90730.
Shimizu, M. 2017. Multifunctions of dietary polyphenols in the regula-
tion of intestinal inflammation. Journal of Food and Drug Analysis
25(1):93–9. doi:10.1016/j.jfda.2016.12.003.
Simonovic, I., J. Rosenberg, A. Koutsouris, and G. Hecht. 2000.
Enteropathogenic Escherichia coli dephosphorylates and dissociates
occludin from intestinal epithelial tight junctions. Cellular
Microbiology 2(4):305–15. doi:10.1046/j.1462-5822.2000.00055.x.
Smith, P. M., M. R. Howitt, N. Panikov, M. Michaud, C. A. Gallini, M.
Bohlooly-Y, J. N. Glickman, and W. S. Garrett. 2013. The microbial
metabolites, short-Chain Fatty acids, regulate colonic treg cell
homeostasis. Science 341(6145):569–73. doi:10.1126/science.1241165.
Spence, J. R., C. N. Mayhew, S. A. Rankin, M. F. Kuhar, J. E. Vallance,
K. Tolle, E. E. Hoskins, V. V. Kalinichenko, S. I. Wells, A. M. Zorn,
et al. 2011. Directed differentiation of human pluripotent stem cells
into intestinal tissue in vitro. Nature 470 (7332):105–9. doi:10.1038/
nature09691.
Strober, W., I. J. Fuss, and R. S. Blumberg. 2002. The immunology of
mucosal models of inflammation. Annual Review of Immunology
20(1):495–549. doi:10.1146/annurev.immunol.20.100301.064816.
Susewind, J., C. Carvalho-Wodarz, S. de, U. Repnik, E.-M. Collnot, N.
Schneider-Daum, G. W. Griffiths, and C.-M. Lehr. 2016. A 3D co-
culture of three human cell lines to model the inflamed intestinal
mucosa for safety testing of nanomaterials. Nanotoxicology
10(1):1–62. doi:10.3109/17435390.2015.1008065.
Suzuki, M., T. Hisamatsu, and D. K. Podolsky. 2003. Gamma inter-
feron augments the intracellular pathway for lipopolysaccharide
(LPS) recognition in human intestinal epithelial cells through coor-
dinated up-regulation of LPS uptake and expression of the intracel-
lular toll-like receptor 4-MD-2 complex. Infection and Immunity
71(6):3503–11. doi:10.1128/IAI.71.6.3503-3511.2003.
Suzuki, T. 2013. Regulation of intestinal epithelial permeability by tight
junctions. Cellular and Molecular Life Sciences 70(4):631–59.
doi:10.1007/s00018-012-1070-x.
Suzuki, T., S. Tanabe, and H. Hara. 2011. Kaempferol enhances intes-
tinal barrier function through the cytoskeletal association and
expression of tight junction proteins in caco-2 cells. The Journal of
Nutrition 141(1):87–94. doi:10.3945/jn.110.125633.
Tang, J., P. Bouyer, A. Mykoniatis, M. Buschmann, K. S. Matlin, and
J. B. Matthews. 2010. Activated PKCd and PKC inhibit epithelial
chloride secretion response to cAMP via inducing internalization of
the naþ-Kþ-2Cl  cotransporter NKCC1. Journal of Biological
Chemistry 285(44):34072–85. doi:10.1074/jbc.M110.137380.
Tanoue, T.,. Y. Nishitani, K. Kanazawa, T. Hashimoto, and M. Mizuno.
2008. In vitro model to estimate gut inflammation using co-cultured
caco-2 and RAW264.7 cells. Biochemical and Biophysical Research
Communications 374(3):565–9. doi:10.1016/j.bbrc.2008.07.063.
Tom, B. H., L. P. Rutzky, M. M. Jakstys, R. Oyasu, C. I. Kaye, and
B. D. Kahan. 1976. Human colonic adenocarcinoma cells. In Vitro
12(3):180–91. doi:10.1007/BF02796440.
Tsuchiya, S., M. Yamabe, Y. Yamaguchi, Y. Kobayashi, T. Konno, and
K. Tada. 1980. Establishment and characterization of a human acute
monocytic leukemia cell line (THP-1). International Journal of
Cancer 26(2):171–6. doi:10.1002/ijc.2910260208.
Turco, L., T. Catone, F. Caloni, E. D. Consiglio, E. Testai, and A.
Stammati. 2011. Caco-2/TC7 cell line characterization for intestinal
absorption: How reliable is this in vitro model for the prediction of
the oral dose fraction absorbed in human? Toxicology in Vitro
25(1):13–20. doi:10.1016/j.tiv.2010.08.009.
Turner, J. R. 2006. Molecular basis of epithelial barrier regulation:
From basic mechanisms to clinical application. The American
Journal of Pathology 169(6):1901–9. doi:10.2353/ajpath.2006.060681.
Turner, J. R. 2009. Intestinal mucosal barrier function in health and
disease. Nature Reviews Immunology 9(11):799–809. doi:10.1038/
nri2653.
Turpin, W., C. Humblot, M.-L. Noordine, M. Thomas, and J.-P.
Guyot. 2012. Lactobacillaceae and cell adhesion: Genomic and
functional screening. PLoS ONE 7(5):e38034. doi:10.1371/
journal.pone.0038034.
Tyrer, P., A. Ruth Foxwell, J. Kyd, M. Harvey, P. Sizer, and A. Cripps.
2002. Validation and quantitation of an in vitro M-cell model.
Biochemical and Biophysical Research Communications
299(3):377–83. doi:10.1016/S0006-291X(02)02631-1.
Tyrer, P. C., E. G. Bean, A. Ruth Foxwell, and P. Pavli. 2011. Effects of
bacterial products on enterocyte–macrophage interactions in vitro.
Biochemical and Biophysical Research Communications
413(2):336–41. doi:10.1016/j.bbrc.2011.08.100.
Utech, M., A. I. Ivanov, S. N. Samarin, M. Bruewer, J. R. Turner, R. J.
Mrsny, C. A. Parkos, and A. Nusrat. 2005. Mechanism of IFN-
c-induced endocytosis of tight junction proteins: Myosin II-depend-
ent vacuolarization of the apical plasma membrane. Molecular
Biology of the Cell 16(10):5040–52. doi:10.1091/mbc.E05-03-0193.
Vachon, P., and J. Beaulieu. 1992. Transient mosaic patterns of mor-
phological and functional-Differentiation In the caco-2 cell-Line.
Gastroenterology 103(2):414–23. doi:10.1016/0016-5085(92)90829-n.
Vagianos, K., S. Bector, J. McConnell, and C. N. Bernstein. 2007.
Nutrition assessment of patients with inflammatory bowel disease.
Journal of Parenteral and Enteral Nutrition 31(4):311–9. doi:10.1177/
0148607107031004311.
Vamadevan, A. S., M. Fukata, E. T. Arnold, L. S. Thomas, D. Hsu, and
M. T. Abreu. 2010. Regulation of toll-like receptor 4-associated MD-
2 in intestinal epithelial cells: a comprehensive analysis. Innate
Immunity 16(2):93–103. doi:10.1177/1753425909339231.
VanDussen, K. L., J. M. Marinshaw, N. Shaikh, H. Miyoshi, C. Moon,
P. I. Tarr, M. A. Ciorba, and T. S. Stappenbeck. 2014. Development
of an enhanced human gastrointestinal epithelial culture system to
facilitate patient-based assays. Gut 64(6):911–20. doi:10.1136/gutjnl-
2013-306651.
Vergeres, G., Bogicevic, B. Buri, C. Carrara, S. Chollet, M. Corbino-
Giunta, L. Egger, L. Gille, D. Kopf-Bolanz, K. Laederach. K. et al.
2012. The NutriChip project – translating technology into nutri-
tional knowledge. British Journal of Nutrition 108(05):762–8.
doi:10.1017/S0007114512002693.
Viallard, V.,. C. Denis, V. Trocheris, and J. C. Murat. 1986. Effect of
glutamine deprivation and glutamate or ammonium chloride add-
ition on growth rate, metabolism and differentiation of human
Colon cancer cell-line HT29. International Journal of Biochemistry
18(3):263–9. doi:10.1016/0020-711X(86)90116-3.
Vieira, E. L. M., A. J. Leonel, A. P. Sad, N. R. M. Beltr~ao, T. F. Costa,
T. M. R. Ferreira, A. C. Gomes-Santos, A. M. C. Faria, M. C. G.
Peluzio, D. C. Cara, and J. I. Alvarez-Leite. 2012. Oral administra-
tion of sodium butyrate attenuates inflammation and mucosal lesion

in experimental acute ulcerative colitis. International Journal of
Biochemistry 23(5):430–6. doi:10.1016/j.jnutbio.2011.01.007.
Viladomiu, M., R. Hontecillas, L. Yuan, P. Lu, and J. Bassaganya-Riera.
2013. Nutritional protective mechanisms against gut inflammation.
International Journal of Biochemistry 24(6):929–39. doi:10.1016/
j.jnutbio.2013.01.006.
Vindigni, S. M., T. L. Zisman, D. L. Suskind, and C. J. Damman. 2016.
The intestinal microbiome, barrier function, and immune system in
inflammatory bowel disease: a tripartite pathophysiological circuit
with implications for new therapeutic directions. Therapeutic
Advances in Gastroenterology 9(4):606–25. doi:10.1177/
1756283X16644242.
Wallace, D. G., and J. Rosenblatt. 2003. Collagen gel systems for sus-
tained delivery and tissue engineering. Advanced Drug Delivery
Reviews 55(12):1631–49. doi:10.1016/j.addr.2003.08.004.
Walters, W. A., Z. Xu, and R. Knight. 2014. Meta-analyses of human
gut microbes associated with obesity and IBD. FEBS Letters
588(22):4223–33. doi:10.1016/j.febslet.2014.09.039.
Wang, F., W. Vallen Graham, Y. Wang, E. D. Witkowski, B. T.
Schwarz, and J. R. Turner. 2005. Interferon-c and tumor necrosis
factor-a synergize to induce intestinal epithelial barrier dysfunction
by up-regulating myosin light chain kinase expression. The
American Journal of Pathology 166(2):409–19. doi:10.1016/s0002-
9440(10)62264-x.
Wang, F.-C. 2015. Overexpression of HMGB1 A-box reduced lipopoly-
saccharide-induced intestinal inflammation via HMGB1/TLR4 sig-
naling in vitro. World Journal of Gastroenterology 21(25):7764.
doi:10.3748/wjg.v21.i25.7764.
Watson, C. J., C. J. Hoare, D. R. Garrod, G. L. Carlson, and G.
Warhurst. 2005. Interferon-c selectively increases epithelial perme-
ability to large molecules by activating different populations of para-
cellular pores. Journal of Cell Science 118(22):5221–30. doi:10.1242/
jcs.02630.
Watt, F. M. 1988. Effect of seeding density on stability of the differenti-
ated phenotype of pig articular chondrocytes in culture. Journal of
Cell Science 89(3):373–8.
WeR glarz, L., J. Wawszczyk, A. Orchel, M. Jaworska-Kik, and Z.
Dzier_zewicz. 2007. Phytic acid modulates in vitro IL-8 and IL-6
release from colonic epithelial cells stimulated with LPS and IL-1b.
Digestive Diseases and Sciences 52(1):93–102. doi:10.1007/s10620-
006-9320-0.
Wehkamp, J., M. Schmid, K. Fellermann, and E. F. Stange. 2005.
Defensin deficiency, intestinal microbes, and the clinical phenotypes
of Crohn’s disease. Journal of Leukocyte Biology 77(4):460–5.
doi:10.1189/jlb.0904543.
Williams, K. M., K. Gokulan, C. E. Cerniglia, and S. Khare. 2016. Size
and dose dependent effects of silver nanoparticle exposure on intes-
tinal permeability in an in vitro model of the human gut epithelium.
Journal of Nanobiotechnology 14(1):62 doi:10.1186/s12951-016-0214-
9.
Wojtal, K. A., L. Wolfram, I. Frey-Wagner, S. Lang, M. Scharl, S. R.
Vavricka, and G. Rogler. 2013. The effects of vitamin a on cells of
innate immunity in vitro. Toxicology in Vitro 27(5):1525–32.
doi:10.1016/j.tiv.2013.03.013.
Wu, C.-H., H.-W. Huang, J.-A. Lin, S.-M. Huang, and G.-C. Yen.
2011. The proglycation effect of caffeic acid leads to the elevation of
oxidative stress and inflammation in monocytes, macrophages and
vascular endothelial cells. The Journal of Nutritional Biochemistry
22(6):585–94. doi:10.1016/j.jnutbio.2010.05.002.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 19
Wu, G. D., Chen, J. Hoffmann, C. Bittinger, K. Chen, Y.-Y. Keilbaugh,
S. A. Bewtra, M. Knights, D. Walters, W. A. Knight. R. et al. 2011.
Linking long-Term Dietary patterns with gut microbial enterotypes.
Science 334(6052):105–8. doi:10.1126/science.1208344.
Wu, Z., R. Guan, M. Tao, F. Lyu, G. Cao, M. Liu, and J. Gao. 2017.
Assessment of the toxicity and inflammatory effects of different-
sized zinc oxide nanoparticles in 2D and 3D cell cultures. RSC
Advances 7(21):12437–45. doi:10.1039/C6RA27334C.
Xu, C., C. Yang, Y. Yin, J. Liu, and Y. Mine. 2012. Phosphopeptides
(PPPs) from hen egg yolk phosvitin exert anti-inflammatory activity
via modulation of cytokine expression. Journal of Functional Foods
4(4):718–26. doi:10.1016/j.jff.2012.04.011.
Yasumatsu, H., and S. Tanabe. 2010. The casein peptide Asn-Pro-Trp-
Asp-Gln enforces the intestinal tight junction partly by increasing
occludin expression in caco-2 cells. British Journal of Nutrition
104(07):951–6. doi:10.1017/S0007114510001698.
Yi, J., T. A. Knudsen, A.-L. Nielsen, L. Duelund, M. Christensen, P.
Hervella, D. Needham, and O. G. Mouritsen. 2016. Inhibition of
cholesterol transport in an intestine cell model by pine-derived phy-
tosterols. Chemistry and Physics of Lipids 200 :62–73. doi:10.1016/
j.chemphyslip.2016.06.008.
Yu, J., S. Peng, D. Luo, and J. C. March. 2012. In vitro 3D human
small intestinal villous model for drug permeability determination.
Biotechnology and Bioengineering 109(9):2173–8. doi:10.1002/
bit.24518.
Zachos, N. C., O. Kovbasnjuk, J. Foulke-Abel, J. In, S. E. Blutt, H. R.
De Jonge, M. K. Estes, and M. Donowitz. 2015. Human enteroids/
colonoids and intestinal organoids functionally recapitulate normal
intestinal physiology and pathophysiology. The Journal of Biological
Chemistry 291(8):3759–66. doi:10.1074/jbc.R114.635995.
Zhang, H., Y. I. Hassan, J. Renaud, R. Liu, C. Yang, Y. Sun, and R.
Tsao. 2017. Bioaccessibility, bioavailability, and anti-inflammatory
effects of anthocyanins from purple root vegetables using Mono-
and co-culture cell models. Molecular Nutrition & Food Research
61(10):n/a–n/a. doi:10.1002/mnfr.20:1600928.
Zimmer, J., B. Lange, J.-S. Frick, H. Sauer, K. Zimmermann, A.
Schwiertz, K. Rusch, S. Klosterhalfen, and P. Enck. 2012. A vegan or
vegetarian diet substantially alters the human colonic faecal micro-
biota. European Journal of Clinical Nutrition 66(1):53–60.
doi:10.1038/ejcn.2011.141.
Zoumpopoulou, G., E. Tsakalidou, J. Dewulf, B. Pot, and C. Grangette.
2009. Differential crosstalk between epithelial cells, dendritic cells
and bacteria in a co-culture model. International Journal of Food
Microbiology 131(1):40–51. doi:10.1016/j.ijfoodmicro.2008.12.037.
Zucco, F., Batto, A. F. Bises, G. Chambaz, J. Chiusolo, A. Consalvo, R.
Cross, H. Dal Negro, G. de Angelis, I. Fabre. G. et al. 2005. An
inter-laboratory study to evaluate the effects of medium composition
on the differentiation and barrier function of caco-2 cell lines.
ATLA: Alternatives to Laboratory Animals 33(6):603–18.
Zweibaum, A., M. Laburthe, E. Grasset, and D. Louvard. 2011. Use of
cultured cell lines in studies of intestinal cell differentiation and
function. In Comprehensive physiology, 223–255. Hoboken: John
Wiley & Sons, Inc.
Zweibaum, A., M. Pinto, G. Chevalier, E. Dussaulx, N. Triadou, B.
Lacroix, K. Haffen, J.-L. Brun, and M. Rousset. 1985. Enterocytic
differentiation of a subpopulation of the human Colon tumor cell
line HT-29 selected for growth in sugar-free medium and its inhib-
ition by glucose. Journal of Cellular Physiology 122(1):21–9.
doi:10.1002/jcp.1041220105.