BACKGROUND
among human intestinal diseases, intestinal inflammatory disorders, such as food allergies and crohn's disease, are characterized by an increase in intestinal permeability  <cit> .
the in vitro study of these pathologies as well as of permeability modulators requires the use of a cell model representing as closely as possible the physiological conditions.
the small intestinal epithelium is the main barrier preventing the molecules from the lumen  to reach the blood compartment  <cit> .
this epithelium is composed of several cell types: enterocyte, goblet cell, paneth cell, endocrine cell and stem cell  <cit> .
the absorptive and goblet cells are the two main cellular types in the intestinal epithelium.
the apical side of the enterocytes is characterized by a brush border which contains several enzymes and which increases the surface for nutrient absorption.
the goblet cells secrete a mucus  <cit> , which covers the apical membrane of intestinal cells and limits molecule absorption  <cit> .
the cellular phenotypes in the epithelium are influenced by cell-cell and cell-matrix interactions and are defined by the expression of specific genes  <cit> .
in vitro models are increasingly developed to study drug and nutrient transport across the intestinal epithelium  <cit> .
the intestinal cell lineage caco- <dig> is the most commonly used cell model  <cit> .
however, some authors report in caco- <dig> a low paracellular permeability  <cit>  and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus  <cit> .
caco- <dig> cells were obtained from human colon adenocarcinoma  <cit> .
it differentiates spontaneously when it grows to confluence  <cit> .
the alkaline phosphatase , coded by the iap gene, is an enzyme widely used as a marker of differentiation in the caco- <dig> cell type  <cit> .
the parental ht <dig> cell line was obtained from human colorectal cancer  <cit> .
the ht29-5m <dig> cell line results from the isolation of ht <dig> cells adapted to methotrexate   <cit> .
the differentiation of goblet cells is characterized by the secretion of several mucins  <cit> .
the muc5ac mucin gene, usually expressed in the stomach, accounts for the major expressed mucin gene in ht29-mtx  <cit> .
the aim of our study is the development of a serum-free co-culture of human intestinal epithelium cell lines.
this reproducible co-culture will form an epithelial monolayer exhibiting the two main cellular types encountered in the human intestinal epithelium.
the systematic use of co-culture systems requires the characterization of the monolayer under a given experimental procedure.
in this study, we investigated the activity and localization of alkaline phosphatase and the expression of iap and muc5ac genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of caco- <dig> and ht29-5m <dig> intestinal cells.
RESULTS
co-culture characterization
microscopy analysis of the caco-2/ht29-5m <dig>  co-cultures showed a monolayer of differentiated cells with microvilli .
monolayer formed domes which have been shown to exhibit signs of enterocytic differentiation and transport properties.
viability of the caco-2/ht29-5m <dig>  co-cultures was assayed by fluorescent dye exclusion .
living cells were observed with a green fluorescent nuclei.
at the end of the culture  few cells were observed with an orange fluorescent nuclei/cytoplasm.
permeability properties
transepithelial electrical resistance  measurement was assayed  <dig> days after confluence on caco-2/ht29-5m <dig> co-cultures at different seeding ratio .
results showed a decrease of the teer with the decrease of the caco- <dig> proportion.
monolayer permeability to lucifer yellow and to  <dig> kda fluorescent-dextran was assayed  <dig> days after confluence on caco-2/ht29-5m <dig> co-cultures at different seeding ratio .
permeability of caco-2/ht29-5m <dig> co-cultures was 10-fold higher to lucifer yellow than to  <dig> kda-dextran.
results showed a decrease of the permeability to these molecules with the increase of the caco- <dig> proportion in the co-culture.
enzyme activity and localization
in order to study the relationship between enzyme activity and the cellular composition of the monolayer, experiments concerning the specific activity of alkaline phosphatase and enzyme localization were conducted in cell cultures.
we used caco- <dig> cells, ht29-5m <dig> cells and co-cultures of these two cell types .
the alkaline phosphatase specific activity  was detected in the caco- <dig> cultures, whereas no activity was detected in the ht29-5m <dig> cultures.
in the co-cultures and in the cell homogenate mixtures, the sa of alkaline phosphatase increased with the proportion of caco- <dig> .
the alkaline phosphatase sa was significantly  greater in the co-cultures than in the mixtures of caco- <dig> and ht29-5m <dig> homogenates .
comparisons of the pairs showed that this general effect was the result of the 2: <dig> and 3: <dig> ratios, where the alkaline phosphatase sa was significantly  greater in the co-cultures than in the mixtures of caco- <dig> and ht29-5m <dig> homogenates.
the specific detection of alkaline phosphatase activity was further investigated by cytochemistry.
results showed a significant linear correlation  between alkaline phosphatase positive-cells and caco- <dig> proportion in the culture.
however, caco- <dig> culture did not show an alkaline phosphatase localization on the whole culture surface .
a progressive bias towards the caco- <dig> proportion and the alkaline phosphatase-positive area resulted from a 2: <dig> caco-2/ht29-5m <dig> seeding ratio.
specificity of the primers
the specificity of the primer pairs  was tested on caco- <dig> and ht29-5m <dig> cdna.
caco- <dig> cdna gave rise to a  <dig> bp amplicon with iap hu fo/ba primers and was not amplified with muc5ac fo/ba primers .
ht29-5m <dig> cdna gave rise to a  <dig> bp amplicon with muc5ac fo/ba primers and was not amplified with iap hu fo/ba primers .
in the caco-2/ht29-5m <dig> co-culture a  <dig> or a  <dig> bp amplicon was detected with, respectively, iap hu fo/ba and muc5ac fo/ba primer pairs .
the bact hu fo/ba primer pairs amplified a  <dig> bp fragment in caco- <dig> and ht29-5m <dig> cdna .
expression of iap and muc5ac genes
the expression level of iap and muc5ac genes was determined by real-time pcr in the caco- <dig> and ht29-5m <dig> co-cultures.
after  <dig> pcr cycles, the fluorescence, corresponding to syber green® binding, was recorded and plotted against the cycle number .
our results showed that the threshold cycle number  value, corresponding to the amount of iap cdna, is the same in the caco- <dig> culture and in co-cultures at different seeding caco-2/ht29-5m <dig> proportions .
with regard to the ht29-5m <dig> cdna, no amplification product was detected until a ct value corresponding to the value of the negative control was obtained without template.
the ct value did not differ significantly with the proportion of caco- <dig> cells in the culture.
the same results  were obtained for muc5ac .
discussion
to understand the cellular events leading to a leaky gut in pathologies such as inflammatory bowel diseases, an in vitro approach using cell models is required.
the intestinal cell lineage caco- <dig> is the cell model most commonly used to reproduce the features of the human small intestine epithelium  <cit> .
nevertheless, the caco- <dig> cell model is different in some respects to the human epithelium.
for example, it has a low paracellular permeability and allows access of highly diffusible small molecules to the microvilli too easily  <cit> , owing to the lack of an adequate mucus layer.
as an alternative, therefore, we analyzed the properties of a co-culture of caco- <dig> cells and ht29-5m <dig> cells differentiating spontaneously into mucus-secreting cells.
this co-culture system exhibits the two main important cellular types encountered in the human intestinal epithelium.
in order to make this procedure reliable and reproducible, we replaced the usual serum-containing culture medium with a serum-free medium supplemented with hormones and amino acids.
although the proportions of both cellular types are determined at culture seeding, they can rapidly modify during culture owing to the ability of each cell line to adhere to and to proliferate on the culture substrate.
more, the heterogeneity of caco- <dig> cells between  <dig> and  <dig> days of confluence  <cit>  can deeply influence the co-culture composition.
a brief characterization of the caco-2/ht29-5m <dig> co-culture, initially seeded with a 3: <dig> ratio, was performed.
results showed a monolayer containing few cells with condensed dna .
the ethidium bromide accumulation may reflect an early proliferative/apoptotic activity and modified nucleic acid expression.
indeed, an increase in dna content is a feature of actively-proliferating cells that would therefore exhibit higher fluorescence peaks when labeled by ethidium bromide  <cit> .
moreover, previous studies reported the appearance of an increase in dna staining by ethidium bromide during the early phases of apoptosis, known as the hyperdiploid peak,  <cit> .
on the other hand, intracellular ethidium bromide fluorescence could also be enhanced by an increase in dna base pair accessibility associated with the differentiation state  <cit> .
then, a recent study indeed suggests that cell proliferation and apoptosis can co-exist  <cit> .
in our case we can not speculate between these possibilities.
whatever the solution may be, our results show that our co-culture has a high proportion of living cells.
for this study, we used alkaline phosphatase as an indicator of the cell proportion in co-culture models.
the alkaline phosphatase is a membrane-bound enzyme localized in the brush border of enterocytes in the human intestinal epithelium and was used previously by matsumoto et al.
<cit>  to monitor the differentiation of enterocyte-like cells.
in order to analyze the correlation between the proportion of the enterocyte-like cells and the alkaline phosphatase in the caco-2/ht29-5m <dig> co-cultures, we estimated the enzyme sa.
this enzyme activity is increased with the caco- <dig> proportion.
however, the alkaline phosphatase sa in co-cultures seems to be higher than in cell mixtures with the same proportions .
this result suggests a positive interaction between caco- <dig> and ht29-5m <dig> cells.
this interaction could lead to an increase of the gene expression, an increase of the translation rate or a decrease of enzyme degradation.
this last possibility was already proposed by matsumoto et al.
<cit>  during cell differentiation.
the alkaline phosphatase activity was detected in co-cultures by the cytochemistry method.
our results show that the alkaline phosphatase was restricted to the enterocyte-like cell and could then be used as a potential marker for the monitoring of these cells under several co-culture conditions.
the discrepancy between the alkaline phosphatase-positive area and the caco- <dig> seeding proportion in culture could be explained by the heterogeneity of the cell lineage  <cit> .
indeed, this cell line differentiates spontaneously in enterocyte-like cells, but also in other cell types which do not express alkaline phosphatase.
our results underline this heterogeneity, as we observed, by cytochemistry at cellular level, several red-scales  in the same culture.
this differentiation seems to be influenced by the caco-2/ht29-5m <dig> co-culture and thus by a cell-cell interaction.
in order to collect more data on the proportionate composition of the monolayer, we investigated the expression level of the alkaline phosphatase coding gene and of the muc5ac gene.
though the two cell lineages were both of an adenocarcinoma origin  <cit> , we showed that the iap and muc5ac gene expressions are restricted, respectively, to caco- <dig> cells and ht29-5m <dig> cells.
we showed by real-time pcr that the expression level of the iap and muc5ac genes in the monolayer is independent of the cell proportion and thus of the caco- <dig> or ht29-5m <dig> abundancy.
however, the specific activity of the alkaline phosphatase was dependant on the caco- <dig> proportion.
consequently, there is a complex control of the gene expression, enzyme translation and degradation regarding the cell-cell interaction.
monolayer permeability is influenced by the presence of ht29-5m <dig> cells in the system.
this influence is in accordance with an increase of the permeability.
indeed, we showed a decrease of the teer and an increase of the lucifer yellow and  <dig> kda dextran permeability with the increase of ht29-5m <dig> proportion in the monolayer at seeding.
the cell ratio is established at seeding and is probably not indicative of the situation when permeability is measured.
however, ht29-5m <dig> cells seem to remain in sufficient proportion in the culture to increase cell permeability.
the range of molecular masses  used in this study to explore monolayer permeability allow to characterize para-cellular permeability  <cit> .
lucifer yellow is not absorbed by epithelial cell but is able to pass through cell junctions as mannitol does.
dextrans are largely used as macromolecule permeability tracer.
CONCLUSIONS
the phosphatase alkaline, a well-established marker for enterocyte-like cells, could be proposed, in combination with muc5ac, as a new marker of great interest for monitoring the cellular composition of the co-culture.
we are currently developing an immuno-labelling system to quantify the level of this protein.
on the basis of the enterocyte/goblet cell ratio observed in vivo  <cit> , the cell proportion 3: <dig> allows us to decrease the permeability to macromolecules who becomes closer to the situation observed in a human intestinal epithelium.
permeability studies, using this co-culture, indicate that caco-2/ht29-5m <dig> co-culture is more permissive than caco- <dig> cell culture regarding to para-cellular permeability.
this characteristic is probably in accordance with the in vivo permeability.
we can expect to use the model to study some human diseases involving permeability alterations as coeliac disease.

 among human intestinal diseases, intestinal inflammatory disorders, such as food allergies and crohn's disease, are characterized by an increase in intestinal permeability. the in vitro study of these pathologies as well as of permeability modulators requires the use of a cell model representing as closely as possible the physiological conditions. the small intestinal epithelium is the main barrier preventing the molecules from the lumen to reach the blood compartment. this epithelium is composed of several cell types: enterocyte, goblet cell, paneth cell, endocrine cell and stem cell. the absorptive and goblet cells are the two main cellular types in the intestinal epithelium. the apical side of the enterocytes is characterized by a brush border which contains several enzymes and which increases the surface for nutrient absorption. the goblet cells secrete a mucus, which covers the apical membrane of intestinal cells and limits molecule absorption. the cellular phenotypes in the epithelium are influenced by cell-cell and cell-matrix interactions and are defined by the expression of specific genes. in vitro models are increasingly developed to study drug and nutrient transport across the intestinal epithelium. the intestinal cell lineage caco- is the most commonly used cell model. however, some authors report in caco- a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. caco- cells were obtained from human colon adenocarcinoma. it differentiates spontaneously when it grows to confluence. the alkaline phosphatase, coded by the iap gene, is an enzyme widely used as a marker of differentiation in the caco- cell type. the parental ht cell line was obtained from human colorectal cancer. the ht29-5m cell line results from the isolation of ht cells adapted to methotrexate. the differentiation of goblet cells is characterized by the secretion of several mucins. the muc5ac mucin gene, usually expressed in the stomach, accounts for the major expressed mucin gene in ht29-mtx. the aim of our study is the development of a serum-free co-culture of human intestinal epithelium cell lines. this reproducible co-culture will form an epithelial monolayer exhibiting the two main cellular types encountered in the human intestinal epithelium. the systematic use of co-culture systems requires the characterization of the monolayer under a given experimental procedure. in this study, we investigated the activity and localization of alkaline phosphatase and the expression of iap and muc5ac genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of caco- and ht29-5m intestinal cells.  co-culture characterization microscopy analysis of the caco-2/ht29-5m co-cultures showed a monolayer of differentiated cells with microvilli. monolayer formed domes which have been shown to exhibit signs of enterocytic differentiation and transport properties. viability of the caco-2/ht29-5m co-cultures was assayed by fluorescent dye exclusion. living cells were observed with a green fluorescent nuclei. at the end of the culture few cells were observed with an orange fluorescent nuclei/cytoplasm. permeability properties transepithelial electrical resistance measurement was assayed days after confluence on caco-2/ht29-5m co-cultures at different seeding ratio. results showed a decrease of the teer with the decrease of the caco- proportion. monolayer permeability to lucifer yellow and to kda fluorescent-dextran was assayed days after confluence on caco-2/ht29-5m co-cultures at different seeding ratio. permeability of caco-2/ht29-5m co-cultures was 10-fold higher to lucifer yellow than to kda-dextran. results showed a decrease of the permeability to these molecules with the increase of the caco- proportion in the co-culture. enzyme activity and localization in order to study the relationship between enzyme activity and the cellular composition of the monolayer, experiments concerning the specific activity of alkaline phosphatase and enzyme localization were conducted in cell cultures. we used caco- cells, ht29-5m cells and co-cultures of these two cell types. the alkaline phosphatase specific activity was detected in the caco- cultures, whereas no activity was detected in the ht29-5m cultures. in the co-cultures and in the cell homogenate mixtures, the sa of alkaline phosphatase increased with the proportion of caco-. the alkaline phosphatase sa was significantly greater in the co-cultures than in the mixtures of caco- and ht29-5m homogenates. comparisons of the pairs showed that this general effect was the result of the 2: and 3: ratios, where the alkaline phosphatase sa was significantly greater in the co-cultures than in the mixtures of caco- and ht29-5m homogenates. the specific detection of alkaline phosphatase activity was further investigated by cytochemistry. results showed a significant linear correlation between alkaline phosphatase positive-cells and caco- proportion in the culture. however, caco- culture did not show an alkaline phosphatase localization on the whole culture surface. a progressive bias towards the caco- proportion and the alkaline phosphatase-positive area resulted from a 2: caco-2/ht29-5m seeding ratio. specificity of the primers the specificity of the primer pairs was tested on caco- and ht29-5m cdna. caco- cdna gave rise to a bp amplicon with iap hu fo/ba primers and was not amplified with muc5ac fo/ba primers. ht29-5m cdna gave rise to a bp amplicon with muc5ac fo/ba primers and was not amplified with iap hu fo/ba primers. in the caco-2/ht29-5m co-culture a or a bp amplicon was detected with, respectively, iap hu fo/ba and muc5ac fo/ba primer pairs. the bact hu fo/ba primer pairs amplified a bp fragment in caco- and ht29-5m cdna. expression of iap and muc5ac genes the expression level of iap and muc5ac genes was determined by real-time pcr in the caco- and ht29-5m co-cultures. after pcr cycles, the fluorescence, corresponding to syber green® binding, was recorded and plotted against the cycle number. our results showed that the threshold cycle number value, corresponding to the amount of iap cdna, is the same in the caco- culture and in co-cultures at different seeding caco-2/ht29-5m proportions. with regard to the ht29-5m cdna, no amplification product was detected until a ct value corresponding to the value of the negative control was obtained without template. the ct value did not differ significantly with the proportion of caco- cells in the culture. the same results were obtained for muc5ac. discussion to understand the cellular events leading to a leaky gut in pathologies such as inflammatory bowel diseases, an in vitro approach using cell models is required. the intestinal cell lineage caco- is the cell model most commonly used to reproduce the features of the human small intestine epithelium. nevertheless, the caco- cell model is different in some respects to the human epithelium. for example, it has a low paracellular permeability and allows access of highly diffusible small molecules to the microvilli too easily, owing to the lack of an adequate mucus layer. as an alternative, therefore, we analyzed the properties of a co-culture of caco- cells and ht29-5m cells differentiating spontaneously into mucus-secreting cells. this co-culture system exhibits the two main important cellular types encountered in the human intestinal epithelium. in order to make this procedure reliable and reproducible, we replaced the usual serum-containing culture medium with a serum-free medium supplemented with hormones and amino acids. although the proportions of both cellular types are determined at culture seeding, they can rapidly modify during culture owing to the ability of each cell line to adhere to and to proliferate on the culture substrate. more, the heterogeneity of caco- cells between and days of confluence can deeply influence the co-culture composition. a brief characterization of the caco-2/ht29-5m co-culture, initially seeded with a 3: ratio, was performed. results showed a monolayer containing few cells with condensed dna. the ethidium bromide accumulation may reflect an early proliferative/apoptotic activity and modified nucleic acid expression. indeed, an increase in dna content is a feature of actively-proliferating cells that would therefore exhibit higher fluorescence peaks when labeled by ethidium bromide. moreover, previous studies reported the appearance of an increase in dna staining by ethidium bromide during the early phases of apoptosis, known as the hyperdiploid peak,. on the other hand, intracellular ethidium bromide fluorescence could also be enhanced by an increase in dna base pair accessibility associated with the differentiation state. then, a recent study indeed suggests that cell proliferation and apoptosis can co-exist. in our case we can not speculate between these possibilities. whatever the solution may be, our results show that our co-culture has a high proportion of living cells. for this study, we used alkaline phosphatase as an indicator of the cell proportion in co-culture models. the alkaline phosphatase is a membrane-bound enzyme localized in the brush border of enterocytes in the human intestinal epithelium and was used previously by matsumoto et al. to monitor the differentiation of enterocyte-like cells. in order to analyze the correlation between the proportion of the enterocyte-like cells and the alkaline phosphatase in the caco-2/ht29-5m co-cultures, we estimated the enzyme sa. this enzyme activity is increased with the caco- proportion. however, the alkaline phosphatase sa in co-cultures seems to be higher than in cell mixtures with the same proportions. this result suggests a positive interaction between caco- and ht29-5m cells. this interaction could lead to an increase of the gene expression, an increase of the translation rate or a decrease of enzyme degradation. this last possibility was already proposed by matsumoto et al. during cell differentiation. the alkaline phosphatase activity was detected in co-cultures by the cytochemistry method. our results show that the alkaline phosphatase was restricted to the enterocyte-like cell and could then be used as a potential marker for the monitoring of these cells under several co-culture conditions. the discrepancy between the alkaline phosphatase-positive area and the caco- seeding proportion in culture could be explained by the heterogeneity of the cell lineage. indeed, this cell line differentiates spontaneously in enterocyte-like cells, but also in other cell types which do not express alkaline phosphatase. our results underline this heterogeneity, as we observed, by cytochemistry at cellular level, several red-scales in the same culture. this differentiation seems to be influenced by the caco-2/ht29-5m co-culture and thus by a cell-cell interaction. in order to collect more data on the proportionate composition of the monolayer, we investigated the expression level of the alkaline phosphatase coding gene and of the muc5ac gene. though the two cell lineages were both of an adenocarcinoma origin, we showed that the iap and muc5ac gene expressions are restricted, respectively, to caco- cells and ht29-5m cells. we showed by real-time pcr that the expression level of the iap and muc5ac genes in the monolayer is independent of the cell proportion and thus of the caco- or ht29-5m abundancy. however, the specific activity of the alkaline phosphatase was dependant on the caco- proportion. consequently, there is a complex control of the gene expression, enzyme translation and degradation regarding the cell-cell interaction. monolayer permeability is influenced by the presence of ht29-5m cells in the system. this influence is in accordance with an increase of the permeability. indeed, we showed a decrease of the teer and an increase of the lucifer yellow and kda dextran permeability with the increase of ht29-5m proportion in the monolayer at seeding. the cell ratio is established at seeding and is probably not indicative of the situation when permeability is measured. however, ht29-5m cells seem to remain in sufficient proportion in the culture to increase cell permeability. the range of molecular masses used in this study to explore monolayer permeability allow to characterize para-cellular permeability. lucifer yellow is not absorbed by epithelial cell but is able to pass through cell junctions as mannitol does. dextrans are largely used as macromolecule permeability tracer.  the phosphatase alkaline, a well-established marker for enterocyte-like cells, could be proposed, in combination with muc5ac, as a new marker of great interest for monitoring the cellular composition of the co-culture. we are currently developing an immuno-labelling system to quantify the level of this protein. on the basis of the enterocyte/goblet cell ratio observed in vivo, the cell proportion 3: allows us to decrease the permeability to macromolecules who becomes closer to the situation observed in a human intestinal epithelium. permeability studies, using this co-culture, indicate that caco-2/ht29-5m co-culture is more permissive than caco- cell culture regarding to para-cellular permeability. this characteristic is probably in accordance with the in vivo permeability. we can expect to use the model to study some human diseases involving permeability alterations as coeliac disease. 
the absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. indeed, some authors report in caco- a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. the systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. in this study, we investigated the activity and localization of the alkaline phosphatase and the expression of iap and muc5ac genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of caco- and ht29-5m cells. the culture formed by 75% caco- and 25% ht29-5m produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. the alkaline phosphatase expression characterizing caco- cells was influenced by the presence of ht29-5m cells. a co-culture system carried out in a serum-free medium and comprising both caco- and ht29-5m cells was developed.
 the absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. the cell lineage caco- is a model commonly used to reproduce the features of the bowel epithelium. however, there is a strong debate regarding the value of caco- cell culture to mimick in vivo situation. indeed, some authors report in caco- a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. the ht29-5m intestinal cell lineage is a mucin-secreting cellular population. a co-culture system carried out in a serum-free medium and comprising both caco- and ht29-5m cells was developed. the systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure.  in this study, we investigated the activity and localization of the alkaline phosphatase and the expression of iap and muc5ac genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of caco- and ht29-5m cells. we observed that the culture conditions used did not change the phenotype of each cell type, and produced a reproducible model. the alkaline phosphatase expression characterizing caco- cells was influenced by the presence of ht29-5m cells.  the culture formed by 75% caco- and 25% ht29-5m produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. 