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Optimization of Caco-2 and HT29 co-culture in vitro

cell models for permeability studies
a a a a a
Fengguang Pan , Lu Han , Yan Zhang , Yiding Yu & Jingbo Liu
a
Laboratory of Nutrition and Functional Food, Jilin University, Changchun, PR China
Published online: 21 Aug 2015.

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To cite this article: Fengguang Pan, Lu Han, Yan Zhang, Yiding Yu & Jingbo Liu (2015): Optimization of Caco-2 and HT29 co-
culture in vitro cell models for permeability studies, International Journal of Food Sciences and Nutrition

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ISSN: 0963-7486 (print), 1465-3478 (electronic)

Int J Food Sci Nutr, Early Online: 1–6

! 2015 Taylor & Francis. DOI: 10.3109/09637486.2015.1077792

RESEARCH ARTICLE

Optimization of Caco-2 and HT29 co-culture in vitro cell models for

permeability studies
Fengguang Pan, Lu Han, Yan Zhang, Yiding Yu, and Jingbo Liu

Laboratory of Nutrition and Functional Food, Jilin University, Changchun, PR China

Downloaded by [University of Nebraska, Lincoln] at 06:50 07 September 2015

Abstract Keywords
The purpose of this study was to investigate the appropriate proportion of Caco-2 and HT29 Cell seeding ratio, co-culture, gene
co-culture in vitro cell models for permeability studies. The results showed that the expression, monolayer, optimization
transepithelial electrical resistance values of 9:1 and 1:0 groups (263 ± 3.61 and 300 ± 7.55)
after 21-day culture were 4250 V cm2, which were suitable for further experiments. The History
confocal laser microscopy showed that the group of 9:1 (Caco-2:HT29) had the highest
integrity, whereas the group of 0:1 (Caco-2:HT29) exhibited the lowest. The staining study Received 12 April 2015
confirmed that mucus was successfully produced by HT29 cells, and it was also produced in co- Revised 16 June 2015
cultures with Caco-2 cells model, but the Caco-2 monocultures did not have any blue staining, Accepted 12 July 2015
which made us affirm that mucus is only produced in the presence of HT29 cells. The real-time Published online 21 August 2015
PCR results showed that the total highest expression level of ALPi and MUC5AC was the ratio of
9:1 (Caco-2:HT29) and lowest is 1:1 (Caco-2:HT29). So we concluded that 9:1 (Caco-2:HT29) is
the optimal Caco-2 to HT29 ratio in the in vitro model co-culture for permeability studies.

Introduction in human intestinal epithelium (Behrens et al., 2001; Leonard

et al., 2010).
The intestine, particularly the small intestinal epithelium, is the
To modify and improve the Caco-2 cell model, co-cultures of
primary area for absorption and metabolism in the human body.
Caco-2 cells with other cell types, such as the mucus-secreting
This epithelium is the main barrier preventing molecules from
HT29 cells, have been established to simulate human intestines
intestinal lumen to reach the blood compartment (Baumgart &
(Antunes et al., 2013; Chen et al., 2010; Mahler et al., 2009;
Dignass, 2002). Therefore, in vitro models, particularly cell
Wood et al., 2010). HT29 are also human colorectal carcinoma
models, are increasingly developed to study the transport across
cells. As a goblet cell, HT29 could secrete several mucins mainly
the intestinal epithelium (Agarwal et al., 2007; Hilgendorf et al.,
expressed by the MUC5AC mucin gene (Gouyer et al., 2001).
2000; Walter et al., 1996).
HT29 cell line grows with an apical brush border in a monolayer,
Undoubtedly, the human colon carcinoma cell lines (so-called
and mucus production is similar to that in the human intestinal
Caco-2) have been the most widely used in vitro cell model in the
mucosa (Behrens et al., 2001; Mahler et al., 2009). Compared
study of intestinal peptide permeability over the last 20 years
with the Caco-2 or HT29 single cell culture model, the
(Artursson et al., 2012; Ding et al., 2014; Sambuy et al., 2005).
characteristics of co-culture model, such as the generation of
Caco-2 secretes alkaline phosphatase, an enzyme coded by the
mucus and the TEER value, are more similar to that of human
ALPi gene, which could be marked during growth (Matsumoto
small intestine. Moreover, the Caco-2:HT29 co-culture model
et al., 1990). Although these cells are similar to human intestinal
could accurately imitate the small intestine because the tight
epithelial cells in morphology and function (Smetanova et al.,
junctions of HT29 cells are not as tight as the Caco-2 cells (Li
2011), a number of authors have reported that Caco-2 lack mucus
et al., 2013; Pontier et al., 2001).
production, which is an important property of intestinal mucosa,
To accurately simulate the human intestinal barrier, the
thereby resulting in low paracellular permeability (Artursson
proportions of the two types of cells in co-culture are significant
et al., 2012; Menon & Barr, 2003). Furthermore, several
factors that should be determined (Hilgendorf et al., 2000;
transporters are differently expressed in Caco-2 and human
Nollevaux et al., 2006).
small intestines (Lin et al., 2011). Moreover, Caco-2 cells
On the basis of previous studies (Beduneau et al., 2014; Carr &
form tight junctions of the monolayer that resemble the colon
Peppas, 2010; Martinez-Augustin et al., 2014), we established a
in contrast to the small intestine; thus, the transepithelial
cellular model of intestinal epithelium with Caco-2 and HT29
electrical resistance (TEER) detected is significantly higher in
cells and investigated the optimal cell ratio.
Caco-2 models than in the human intestines (Le Ferrec et al.,
2001). Caco-2 cells are simply formed by enterocytes; a
considerable number of complex interactions occur among cells Materials and methods
Materials
Correspondence: Jingbo Liu, Laboratory of Nutrition and Functional
Food, Jilin University, Changchun, Jilin 130062, China. Tel: +86 431 Caco-2 cell lines were obtained from the American Type Culture
87836351. E-mail: ljb168@sohu.com Collection (Rockefeller, MD). HT29 cell lines were purchased
 2 F. Pan et al. Int J Food Sci Nutr, Early Online: 1–6

from the Chinese Academy of Science Type Research Products Alcian blue staining
(Shanghai, China) Culture Collection. Dulbeco’s Modified
This experiment was conducted to evaluate if there was mucus
Eagle’s Medium (DMEM), fetal bovine serum, non-essential
secretion by the HT29 cells and the co-culture model.
amino acid, penicillin–streptomycin and phosphate-buffered
To demonstrate mucus production, co-culture Caco-2 and HT29
saline (PBS) were purchased from Gibco BRL Life Technology
cells were seeded on Transwell inserts at an initial cell density of
(Gaithersburg, MD). Trypsin, dimethyl sulfoxide, paraformalde-
6  104 cells/cm2 and cultured for 21 days. Alcian blue was used
hyde and ethylene diamine tetraacetic acid (EDTA) were
to stain mucus that produced by HT29 cells. Briefly, culture
purchased from Sigma–Aldrich (Milan, Italy). FITC-Phalloidin
media were discarded and the cells were washed twice with PBS
was purchased from MultiSciences Biotech Co., Ltd (Hangzhou,
(Chen et al., 2010). And then, fixed the cells with paraformalde-
China). Alcian blue 8GX were obtained from Amresco
hyde (4%) for 30 min at room temperature. After washed twice
Biochemicals and Life Science Research Products (Shanghai,
with PBS, 0.1% HCl was added to reduce the pH of the cells.
China). TriPure Isolation Reagent and FastStart Universal SYBR
Further, added Alcian blue, which was prepared with glacial
Green Master (ROX) were purchased from Roche Diagnostics
acetic acid, to stain the mucus for 30 min at room temperature.
GmbH (Mannheim, Germany). PrimeScriptTM Reverse
Cell monolayers were washed twice with PBS.
Transcription System reagent kit with gDNA Eraser was
purchased from TaKaRa (Dalian, China). GoTaqÕ Green Master
Mix was obtained from Promega Biotechnology Co., Ltd. DNA RNA extraction
maker was purchased from TIANGEN Biotech Co., Ltd (Beijing, This study used the Trizol method for RNA extraction. Cells were
China). BiowestÕ Regular Agarose G-10 was purchased from
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split with trypsin-EDTA solution and centrifuged to obtain the cell

Gene Company Ltd (Hong Kong, China). PCR primers were precipitates. The 2  106 cells were dissolved in 1 ml Trizol.
obtained from Shanghai Generay Biotech Co., Ltd (Shanghai, Chloroform (0.2 ml/1 ml) was added to each sample. The samples
China). All other reagents were of analytical grade. were vigorously shaken for 15 s. After 5 min of incubation, the
tubes were centrifuged at 12 000  g for 15 min at 4  C. The
Cell culture colorless upper aqueous phase was transferred to a new polypro-
Caco-2 (passage 30–40) and HT29 (passage 30–40) cells were pylene centrifuge tube. Isopropanol (0.5 ml/1 ml Trizol) was added
separately grown in tissue culture flasks in DMEM supplemented to the aqueous phase and centrifuged at 12 000  g for 10 min at
with 10% fetal bovine serum, 1% non-essential amino acids, 1% L- 4  C after incubation for 5 min at 20  C to precipitate the RNA from
glutamine and 1% penicillin and streptomycin, at 37  C under an the colorless aqueous phase. Then, 1 ml 75% ethanol/1 ml Trizol
atmosphere of 5% CO2 with 90% relative humidity. Caco-2 and was added to each centrifuge tube. After centrifugation at 7500  g
HT29 cells were grown in 25 cm2 tissue culture flasks and split at for 5 min at 4  C, excess ethanol from the RNA pellet was removed.
80–90% confluence (4 days) using 0.25% trypsin and 0.02% Finally, the RNA pellet was resuspended in 20 ml diethylpyrocar-
EDTA solution. The cells were seeded at a density of 6  104 cells bonate (DEPC)-treated RNase-free water. RNA concentration was
per 25-cm2 flask. The culture medium was replaced every estimated by a microplate reader at 260 nm.
other day.
Reverse transcription
Cell co-cultures cDNA was synthesized using PrimeScriptÔ Reverse Transcription
For the following experiments, monocultures of Caco-2 and System reagent kit according to the reverse transcription experi-
HT29 cells were seeded on the apical chamber of Transwell ment kit.
inserts with different proportions (1:0, 1:1, 1:3, 3:1, 9:1 and 0:1),
and grown in 12-well TranswellÔ plates with a final density of PCR primers
1  105 cells/cm2 in each insert. Cells were maintained in the ALPi was the specific gene primer for Caco-2 cell; MUC5AC was
same conditions and allowed to grow for 21 days with medium the specific gene primer for HT29 cell and GAPDH was a
(0.5 ml in the apical side and 1.5 ml in the basolateral side) housekeeping gene (Gouyer et al., 2001). In this study, the primer
changes every other day. sequences used are described in Table 1.
Cell monolayer integrity
PCR and agarose gel electrophoresis
The cell monolayer integrity during the experiments was
In this research, PCR was performed on the cDNA using GoTaqÕ
determined by TEER measurement using an EVOM epithelial
Green Master Mix kit. Each primer was assigned a temperature
volt-ohm meter equipped with chopstick electrodes to monitor the
gradient to determine the optimal PCR temperature.
evolution of confluence and integrity (Yamashita et al., 2000). We
Agarose gel (1%) electrophoresis was used to detect the
chose three different areas to detect the TEER values in each well
expression of cDNA (Stellwagen, 2009). The samples included
and the averages were the final results. TEER values 4250 V cm2
the DNA maker and the six groups of different cell percentages of
were used for the permeability studies.
cDNA obtained above. The voltage was adjusted to 90 V for
35 min.
Cell monolayer stain
FITC-Phalloidin was used to stain the cells that adhered to the
Real-time PCR
Transwell membranes (Kim et al., 2001). The cells were fixed for
10 min at room temperature after washing twice with PBS (37  C), Real-time PCR was carried out using the real-time PCR system
and then washed again twice. Further, 200 ml of diluted solution of with a final volume of 20 ml containing 10 ml of SYBR Green
FITC-Phalloidin (5 mg/ml) was added to the cell monolayers to Master (Huber et al., 2003; Komatsu et al., 2013), 1 ml of cDNA
label cell nuclei and cell actin. The cells were maintained in the and 1 ml of each primer. DEPC-treated RNase-free water accounted
stain for 10 min. Samples were rinsed twice with PBS and for the remaining volume. After complete blending, the samples
observed using FV1000 Laser Scanning Confocal Microscope were added to the PCR plate. Then, the whole plate was centrifuged
(Olympus, Tokyo, Japan) and images were taken. at 1000  g for 1 min, and the sample was subjected to the real-time
 DOI: 10.3109/09637486.2015.1077792 Caco-2 and HT29 co-culture in vitro cell models 3
Table 1. List of primers used in the study.

Predicted PCR
product Temperature
Primer Sequences 50 !30 size (bp) ( C)

ALPi FP CCTGGAGACATGAAATACG 461 56

ALPi RP GCTGACTGCTGCCGATA 59
MUC5AC FP GGCAACAACGTCATCTCC 239 59
MUC5AC RP CCATACACGGGCACAAT 57
GAPDH FP ATCCCATCACCATCTTCC 382 56
GAPDH RP CCATCACGCCACAGTTT 58

The sequences, predicted PCR product size and temperature of the

primers.

PCR system. For specific steps, refer to the instruction of FastStart

Universal SYBR Green Master (ROX).

Statistics Figure 1. TEER values of cell monolayers. TEER values of cell

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monolayers monitored as a function of time during the 21 days of cell

The data of real-time PCR was analyzed by 7500 software v2.0.6 cultures in TranswellÔ membranes. All values were the means ± SD
(Beijing, China). All data were reported as the mean ± SD. (n ¼ 3).
ANOVA was used to evaluate differences between groups. All the
statistical analyses were conducted with SPSS 19.0 (Beijing,
Specificity of the primers
China).
The specificity of the primer pairs (ALPi, MUC5AC, GAPDH)
Results was tested on Caco-2 and HT29 cDNA. ALPi primers amplified
Caco-2 cDNA to 461 bp, but HT29 cDNA was not amplified.
Cell monolayer integrity
MUC5AC primers amplified HT29 cDNA to 239 bp, but Caco-2
Transepithelial electrical resistance values indicate cell monolayer cDNA was not amplified. The GAPDH primers amplified HT29
tightness, which can resemble the human intestinal epithelium and Caco-2 cDNA to a 382-bp fragment. Results are presented in
better in the established model. From Figure 1, all the TEER Figure 4, the expression level of ALPi increased with the raising
values increase with the culture time, being highest in Caco- of the proportion of Caco-2, the expression level of MUC5AC
2:HT29 of 1:0 and lowest in 0:1. Meanwhile, the TEER values of increased with the raising of the proportion of HT29, and the
1:1, 1:3, 3:1 and 0:1 groups after 21-day culture were 210 ± 6.24, expression level of the housekeeping gene GAPDH is stable. At
150 ± 2.00, 240 ± 4.28 and 150 ± 3.61, which were 5250 V cm2. the same time, the primers have a good specificity.
The TEER values of 9:1 and 1:0 groups after 21-day culture were
263 ± 3.61 and 300 ± 7.55, which were 4250 V cm2 that suitable Expression of ALPi and MUC5AC genes
for further experiments. The continuous proliferation of cells can
The expression of ALPi and MUC5AC genes in the Caco-2 and
enhance the integrity of monolayer. In Figure 1, the TEER values
HT29 co-cultures model was determined by real-time PCR. 7500
increased with the culture time. As the ratio of Caco-2 cells
software v2.0.6 was used to analyze the data. After 40 PCR
decreased, the TEER values also decreased. However, as the ratio
cycles, the 7500 software v2.0.6 was applied to analyze the data of
of HT29 cells decreased, the TEER values increased. These
the fluorescence and the cycle number of different groups, and
results could be due to the presence of HT29 cells.
then the expression level of corresponding genes were calculated.
The expression level of genes is shown in Figure 5. From
Cell monolayer stain
Figure 5, we can see that the expression of ALPi in 1:0, 1:1, 3:1,
Cell monolayer morphology of co-cultures was observed by 1:3, 9:1 and 0:1 groups were 1 ± 0.11, 0.36 ± 0.13, 0.61 ± 0.15,
confocal laser microscopy (Figure 2). The proportion of Caco- 0.28 ± 0.06, 0.83 ± 0.12 and 0.001 ± 0.02, respectively. The
2:HT29 of 1:0 showed the highest integrity, whereas the expression of MUC5AC in 1:0, 1:1, 3:1, 1:3, 9:1 and 0:1
proportion of Caco-2:HT29 of 0:1 exhibited the lowest. On the groups were 0.0002 ± 0.13, 0.36 ± 0.13, 0.30 ± 0.18, 0.62 ± 0.12,
basis of the dates, Figure 2(d–f) shows good integrity. But, 0.30 ± 0.13 and 1 ± 0.67, respectively. The highest total expres-
the Caco-2 single model was not conform to in vivo situations. So, sion of ALPi and MUC5AC was the ratio of Caco-2:HT29 is 9:1
the proportions 9:1 and 3:1 are suitable for further experiments. and lowest is 1:1.

Alcian blue staining Discussion

From Figure 3(a), it is possible to confirm that mucus was In this research, we have established a co-culture model composed
successfully produced by HT29 cells. It was also produced in co- of Caco-2 cells and mucus-secreting HT29 cells. On the basis of
cultures with Caco-2 cells model (Figure 3b–e). At the same time, previous studies (Calatayud et al., 2012; Carr & Peppas, 2010;
the ratio of Caco-2:HT29 of 1:3 (Figure 3b) staining area was Chen et al., 2010), we have studied the seeding proportion of two
larger than the ratio of 1:1 (Figure 3c), and the staining area of 1:1 types of cells.
group was larger than the 3:1 group (Figure 3d), and the staining To accurately imitate the features of the human intestinal
area of 3:1 group was larger than the 9:1 group (Figure 3e). At the epithelium tightness in a monolayer, TEER values that represent
same time, as the increasing of the ratio of HT29 cells, the amount the epithelial integrity of the monolayers of six different ratios of
of mucus produced was also increased. The Caco-2 monocultures Caco-2 and HT29 cells in co-culture were measured. In the
(Figure 3f) did not have any blue staining, which affirm that permeability experiment, the Caco-2 single cell model had an
mucus is only produced in the presence of HT29 cells. increased permeability as the TEER values are decreased by the
 4 F. Pan et al. Int J Food Sci Nutr, Early Online: 1–6
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Figure 2. Confocal laser scanning microscopy revealed growth of co-culture cells. The proportion of Caco-2:HT29 were 0:1 (a), 1:3 (b), 1:1 (c), 3:1
(d), 9:1 (e) and 1:0 (f). Bar ¼ 100 mm.

Figure 3. Alcian Blue mucus stain. Cells stained with Alcian blue highlight the mucin produced by HT29 cells not only in monocultures (a) but also
when they grow together with Caco-2 cells (b–e). The lack of staining in Caco-2 (f) confirms that mucus is only produce by HT29 cells. Bar ¼ 50 mm.

tight junctions (Silva et al., 2006). However, this result does not the TEER values, the proportion of 9:1 is suitable for further
conform to in vivo situations. Therefore, it is very necessary to experiments.
determine the most similar range of TEER values between 150 FITC-Phalloidin is a poisonous alkaloid isolated from toxic
and 1600 V cm2 (Pontier et al., 2001; Silva et al., 2006; Walter mushrooms. FITC-Phalloidin is a fluorescent dye that combines
et al., 1996) to accurately predict in vivo nutrition absorption with cell actin; thus, shows the distribution of the microfilament
(Balimane & Chong, 2005). The average pore radius of the tight skeleton in cells. It was an important and intuitive result to verify
junction of the human small intestine is 6–13 Å, whereas Caco-2 the integrity of cell monolayer. To determine morphological
cells are only 5 Å (Calatayud et al., 2012; Han et al., 2012; differences among various ratios of Caco-2 and HT29, fluores-
Tavelin et al., 2003). As expected, the TEER values of Caco-2 and cence microscopy was used for observation after 21-day co-
HT29 cells co-cultures are lower than that of Caco-2 monolayer culture. Previous studies have demonstrated that when comparing
culture because of the presence of mucus-secreting HT29 cells. the Caco-2 cell model to the Caco-2/HT29 co-culture (Caco-
Therefore, modulation of tight junctions HT29 cells leads to 2:HT29 is 9:1), there seems to be similarity on the cell membrane
reduced TEER values and thus more inter-cellular space exist morphology and monolayer integrity, but slight enlargement of
(Calatayud et al., 2012; Hilgendorf et al., 2000; Rocha et al., the cell membrane staining intensity in the co-culture model,
2012). Some results have shown that the pore radius of the Caco-2 probably due to the specificities of goblet cells presented in the
and HT29 co-cultures in various proportions is 6–8 Å; these co-culture model (Antunes et al., 2013).
data are more similar to human intestinal epithelium than to Caco- In order to confirm the presence of mucus, we stained the
2 cells (Calatayud et al., 2012). From an experimental point of monolayers surface by Alcian blue. Firstly, it was confirmed that
view, the TEER value should be 250 V cm2. Thus, according to the mucus secretion by the HT29 cells, were existence either in
 DOI: 10.3109/09637486.2015.1077792 Caco-2 and HT29 co-culture in vitro cell models 5

Figure 5. The expression level of genes. The expression level of six

groups genes of different proportion. The data of reference genes are not
Downloaded by [University of Nebraska, Lincoln] at 06:50 07 September 2015

shown. All values were the means ± SD (n ¼ 3).

cells have different expression of genes after 21-day cell culture. In

order to optimize the Caco-2 and HT29 co-culture in vitro cell
models for permeability studies, the final cell co-culture model
must have a good cell viability than single cell culture or any other
ratios. The high expression level of genes had good cell viability.
The expression level of ALPi and MUC5AC was highest in Caco-
2:HT29 of 9:1 and lowest in 1:1. So in view of gene expression, the
ratio of 9:1 is the optimum proportion.
The final proportion of co-culture cell model has been reported
recently that the co-culture ratios between cells slightly changed
after 21 days of cell differentiation because Caco-2 cells have a
higher doubling time than HT29-MTX, and that the ratio of 90/10
was found to be the most physiologically relevant by flow
cytometry. But it is not known about the TEER value, microscopy,
staining and any molecular biology study of co-culture model
(Araujo & Sarmento, 2013). Moreover, it has also demonstrated the
importance of selecting an appropriate cell model for in vitro
investigation of Fusarium mycotoxin effects either alone or in
combinations, and it has showed that in co-cultures, lower
Figure 4. Electrophoresis results of different genes and groups. Different
MUC5AC and MUC5B mRNA, protein and total mucin production
proportions of cells were screened by PCR with ALPi, MUC5AC and occurred following exposure, which might suggest higher intestinal
GAPDH gene. Products represented by agarose gel electrophoresis. M permeability and susceptibility to toxin exposure (Wan et al.,
represented the DNA maker. Analysis was repeated 3 times. 2014). These conclusions are consistent with our conclusion. In
addition, the co-culture in vitro cell models (Caco-2:HT29 is 9:1)
were also used by Filipa Antunes to study intestinal absorption of
HT29 cells or in the co-cultures with Caco-2 cells. Alcian blue is a
peptide drugs, and the membranes tested were differentiated and
cationic dye, mostly used in histology, with specific for acidic
presented good integrity and confluence also confirmed by the
mucin glycoproteins; it can form salt bond when comes into contact
TEER measurements (Antunes et al., 2013).
with mucin glycoproteins, showing a deep blue color. From the
Through our comprehensive research, we conclude that the
results of Figure 3, we obtained that HT29 cells producing mucus
optimal co-culture model of Caco-2 and HT29 should have a ratio
not only when cultured alone but also when grown together with
of 9:1 based on the method used in this study. The results have
Caco-2 cells. The findings suggesting that the primary function of
important significance for further research on permeation and
HT29 is not influenced by the presence of the other cells. The areas
metabolism. But the study also has a limitation. Although the cells
of mucus that stained blue probably close to the areas of HT29
model is similar to human intestinal, it is not completely imitate
placement. Similar to the conclusion of our experiment results,
the human intestinal. More details will be studied for future
Francisca Araujo was observed that HT29-MTX cells are able to
research.
maintain their intrinsic properties, producing mucus not only when
in monocultures but also when co-culture. Their growth in presence
Conclusion
of other cells is possible, thus suggesting that the primary function
of HT29-MTX is not influenced by the presence of the other cells In this study, we established a cellular model of intestinal
(Araujo & Sarmento, 2013). epithelium with Caco-2 cells that resemble enterocytes and the
On the basis of the results below, the six cell model groups were mucus producer HT29 cell that look alike goblet cells, and
studied by real-time PCR. The real-time PCR uses the variation of optimized the cells ratio of Caco-2 and HT29. The TEER values,
fluorescent signals and monitors changes of each PCR amplifica- cell monolayer stain, Alcian blue staining and the expression of
tion reaction in real time. From Figure 5, different seeding ratios of ALPi and MUC5AC genes were performed to determine the
 6 F. Pan et al. Int J Food Sci Nutr, Early Online: 1–6

cellular composition of co-culture monolayer. As can be seen Komatsu H, Tsunoda T, Inui A, Sogo T, Fujisawa T, Imura M, Tateno A.
from the above research, the proportions of 9:1 between the 2013. Successful use of saliva without DNA extraction for detection of
different cell types were showed the best result in many respects. macrolide-resistant Mycoplasma pneumoniae DNA in children using
LNA probe-based real-time PCR. J Infect Chemother 19:1087–1092.
It is expected that the Caco-2 and HT29 cells co-culture model Le Ferrec E, Chesne C, Artusson P, Brayden D, Fabre G, Gires P, Guillou
could serve as a good alternative to the in vitro methods already F, et al. 2001. In vitro models of the intestinal barrier – the report and
existent and has progressive significance for further metabolism recommendations of ECVAM Workshop 46. ATLA-Altern Lab Anim
and permeability test. 29:649–668.
Leonard F, Collnot EM, Lehr CM. 2010. A three-dimensional coculture
Declaration of interest of enterocytes, monocytes and dendritic cells to model inflamed
intestinal mucosa in vitro. Mol Pharm 7:2103–2119.
This work was supported by Project of National Key Technology Li N, Wang DD, Sui ZG, Qi XY, Ji LY, Wang XL, Yang L. 2013.
Research and Development Program for The 12th Five-year Plan (No. Development of an improved three-dimensional in vitro intestinal
2012BAD33B03). At the same time, this work was also supported by mucosa model for drug absorption evaluation. Tissue Eng C Methods
Graduate Innovation Fund of Jilin University (No. 2015052). 19:708–719.
Lin XN, Skolnik S, Chen XH, Wang JL. 2011. Attenuation of intestinal
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