BACKGROUND
intestinal epithelium is organized as a single layer which covers the luminal side of this part of the digestive tract.
cells that form this inner cover present specialisations according to their role in the digestive function but they have also common functions and are continuously renewed by the stem cell proliferation.
in vivo, these stem cells, known as progenitors of all cell types, are mostly located in the lower third of the epithelium crypts, which are epithelial invaginations into the lamina propria.
cells differentiation is associated with their migration from the depth of the crypts to the top of the villi and is followed by the cells death and desquamation into the intestinal lumen .
enterocyte cultures represent valuable tools to assess the passage and/or toxicity of drugs, as well as the molecular mechanisms operating in pathologies caused by infectious agents known to affect the intestinal epithelium integrity .
however, the successful establishment of an intestinal cell culture is hampered by the high rate of cell death occurring when isolating them from the epithelium  and the difficulty to select proliferating cells to ensure several cell generations in vitro.
although several investigators have already developed culture methods of intestinal cells for animal species such as the mouse  <cit> , the rabbit  <cit>  or the pig  <cit> , until now only two studies reported the production of primary cultures from intestinal bovine cells.
more precisely, dibb-fuller and coworkers  <cit>  established a procedure for obtaining such cultures from ileum and colon.
föllmann and collaborators  <cit>  also used the bovine colon as a source of intestinal cell cultures.
various strategies were reported in order to isolate enterocytes from the colon or the small bowel.
among the oldest methods, the mechanical dissociation  <cit>  provided rapidly a viable cell preparation but was frequently associated with a fibroblast contamination.
the chelating methods also generated isolated epithelial cells retaining their morphologic characteristics.
however, while first attempts seemed to affect cell surface receptors  <cit> , further applications of chelating agents for a short time were suitable for the production of uncontaminated cultures of human colonocytes  <cit> .
matrisperse, a non enzymatic solution initially designed to isolate epithelial cells grown on engelbreth-holm-swarm  biomatrix, also allowed the dissociation of the integral villus epithelial lining from human intestinal biopsies that produced a confluent monolayer in vitro  <cit> .
the enzymatic digestion provided a cell preparation made up mainly of organoids  that were shown to successfully reconstitute either a monolayer of epithelial cells in vitro or crypt-villus structures in vivo following grafting in various tissues  <cit> .
in spite of these numerous efforts, little is known about the cell differentiation evolution over culture generations.
the present study describes several complementary approaches to characterize bovine primary cultures of intestinal epithelial cells called jejunocytes and colonocytes as they were respectively isolated from adult jejunum and spiralled colon.
cultures were initiated using an organoid-enriched suspension obtained by a multi-step method.
results of vimentin expression in both types of enterocyte primary cultures are discussed in terms of differentiation status of cells.
RESULTS
morphological features of jejunocyte and colonocyte cultures
the method developed to isolate cell material from the bovine jejunum and spiraled colon led to the production of suspension enriched in undissociated cell aggregates  adhering to collagen coated culture flasks.
they give rise to circular proliferating foci .
within  <dig> days the initial foci enlargement had led to the fusion of cell plaques in one confluent layer .
confluent monolayers arising from the initial seeding of cultures presented a heterogeneous aspect  due to the presence of residual multicellular organoids dispersed in the newly formed monolayer.
with the first passage jejunocyte and colonocyte cultures acquired a homogeneous pavement-like aspect typical of epithelial sheets.
we generally carried out  <dig> to  <dig> passages  over  <dig> to  <dig> weeks in both cell series  without major changes of cell morphology.
during early passages , the confluence recovery was achieved within  <dig> to  <dig> days and then a progressive slowdown in the cell proliferation rate was observed until cessation.
confluent monolayers of both cell types exhibited domes, appearing as round blurred arrays corresponding to fluid entrapment between the flask wall and the basolateral side of the monolayer, resulting from ionic regulations by functional epithelial cells .
this observation is in agreement with dome formation in cultures of the human intestinal cell lineage caco- <dig>  <cit> .
ultrastructural analyses of cultured cells revealed apical tight junctions  between cells forming the monolayer of both cell types.
cultured jejunocytes and colonocytes also exhibited a few apical microvilli .
to ascertain the identity and functionality of cultured monolayers, cells components from various culture passages were submitted to immunolabelling .
these data were compared to results from gene expression studies  and measurements of specific activities of brush border associated enzymes.
cell characterization using immunodetection
antibodies directed against cytokeratins  and vimentin  were used to distinguish cells of epithelial origin from contaminating fibroblasts.
double immunolabeling of cryosections of the bowel wall showed the presence of the cytokeratin positive cells all over the lining epithelium and vimentin positive cells in the submucosa .
cytokeratins showing the characteristic disposition of intermediate filaments were also recognized in obtained monolayers.
surprisingly, double staining vimentin-cytokeratin  clearly showed that cultured cells expressed both epithelial and mesenchymal markers.
however, the double staining cytokeratin-α-actin  distinguished separate cells populations.
the few α-actin positive cells, probably contaminant myofibroblasts  were released from the gut wall during the dissociation procedure and not completely eliminated by the sorbitol centrifugation or the selective attachment.
indirect immunofluorescence revealed also the membrane distribution of the epithelial specific antigen  in cultured enterocytes  and intracellular distribution of the cytokeratin  <dig> .
e-cadherin representing one of the proteins located at the adherence junctions was also detected, but a mis-localization was observed.
the cells did not present the characteristic staining pattern to the cell periphery, but a granular distribution into the cytoplasm .
western blot analyses also confirmed the presence of epithelial characters in cultured cells.
pan-anti-cytokeratin and anti-cytokeratin  <dig> antibodies recognised the same major  <dig> kda protein  in homogenates from:  freshly scrapped intestinal epithelium,  organoid suspensions used to seed cultures,  intestinal cell cultures of both types, and in the  positive control caco- <dig> cells.
no cytokeratin staining was noticeable in 3t <dig> fibroblasts.
the size uniformity of stained products in all tested epithelial cells was also confirmed using the pan-cytokeratin antibody .
in addition, blots were also positive for another epithelial cell marker, the e-cadherin .
compared to the expected size , the anti-e cadherin target likely presented the size of a protein domain .
the antibody used was directed against the intracellular domain of this protein.
in agreement with immunocytochemistry results, vimentin and α-actin presented distinct distribution in sample homogenates.
indeed, the anti-vimentin targeted the same proteins in all bovine intestinal cell cultures and in the control 3t <dig> fibroblasts , while α-actin was scarcely detected in more advanced passages .
taken together, western blot and immunocytochemistry results strongly suggested that vimentin is expressed in primary cultures of epithelial intestinal cells from bovine jejunum and colon.
however, vimentin was not detected by western-blot in caco- <dig> cell proteins .
although immunocytochemistry results for α-actin indicated a very low contamination of bovine epithelial cell cultures by mesenchymal cells, western blot results indicated that this phenomenon is to low to be detected in most culture stages.
gene expression analyses of enterocyte markers
rt-pcr analyses allowed us to investigate the expression of bovine enterocyte markers namely: villin , zonula occludens , fatty acid binding protein , small intestine peptidase  and e-cadherin.
expressions studies were also used to confirm the presence of the vimentin transcript in bovine cells.
similarly to western blot analyses, gene expressions studies were carried out on total rna extracted from bovine samples of:  freshly scrapped epithelia,  organoid suspensions used to seed cultures and  intestinal cell cultures of both types.
as can be seen in figure  <dig> , cdna amplification products presented the expected size  using specific primers for coding regions of the epithelial markers, such as the villin gene , the zo <dig> gene  and the e-cadherin , that were uniformly expressed in cells from primary cultures of bovine enterocytes.
besides these structural components expected in any epithelium, we also investigated the expression of functional markers of digestive epithelia.
among them, the gene coding for the fatty acid binding protein , presented a constant expression in both jejunocytes and colonocytes in vitro.
the second tissue specific marker studied was the gene coding for the intestinal peptidase .
in this case, the expression was restricted to jejunocyte samples, as logically expected on the basis of the functional specialization of the jejunum .
the ip expression could then be considered as suitable jejunocyte marker.
in accordance with immunostaining results, samples from jejunocyte and colonocyte cultures expressed the vimentin gene .
interestingly, while western blots analyses failed to reveal vimentin either in freshly removed epithelia or in organoids suspensions used to seed cultures, the transcript of vimentin gene was clearly detected in those samples, indicating that the vimentin is constitutively expressed in jejunum, as well as in colon from the bovine intestine.
however, western blot results suggested that vimentin synthesis seemed to be restricted in cultured cells with a progressive increase over the culture passage number .
specific activities of two brush border-associated enzymes
the chosen enzymes were a disaccharidase  and the alkaline phosphatase.
as for gene expression studies, enzyme activities were investigated in bovine samples of:  freshly scrapped epithelia,  organoid suspensions used to seed cultures and  intestinal cell cultures of both types.
assays performed on fresh epithelial tissue confirmed that jejunum and colon differed in respect to enzyme specific activities , the jejunum presenting the highest level.
maltase sa  clearly decreased in primary cultures of both cells types .
furthermore, the cell isolation procedure selected disaccharidase depleted material, since organoid suspensions presented a 50% reduction in regard to the fresh epithelium preparation.
it is interesting to note that, compared to the organoids suspension, jejunocytes from the first culture passage did not present a drastic reduction of the maltase sa.
however, with subsequent culture passages the decreasing of disaccharidase sa was progressing toward a stable low level.
similar results were obtained from measurements of the intestinal alkaline phosphatase sa , a ubiquitous enzyme that is a marker of brush border in intestinal epithelial cultures.
indeed, as expected from functional differences between jejunum and colon in vivo, epithelia homogenates, organoids suspensions, and cultures at first passages from bovine jejunum presented higher sa for this enzyme.
moreover, similarly to what was noted for maltase sa, iap presented a strong decrease from fresh tissue to subcultured cells, so that jejunocyte and colonocyte sa values joined together to the same low level as the culture passage number increased.
regarding a given passage number, the maltase sa decreased over the culture duration .
these results reflected a loss of cell differentiation in vitro compared to the in vivo level.
adjustments of cell culture medium were made to improve the differentiation status of jejunocytes and colonocytes in vitro.
to this end the glucose was substituted by inosin in the culture medium; this condition was described to stimulate the acquisition of the enterocyte differentiated phenotype in vitro  <cit> .
as seen in figure 8b, the use of a inosin-containing glucose-free culture medium during  <dig> days, led to an increase of maltase activity.
however this effect diminished as the passage number increased, suggesting that glucose substitution by inosin should be complemented by other culture medium modifications.
addition of sodium butyrate , a substance thought to promote enterocyte differentiation through a stimulation of the cdx <dig> homeobox gene expression  <cit> , proved to be efficient to stimulate maltase sa .
discussion
morphological data presented in the present study indicated that primary cultures of bovine enterocytes isolated from colon and jejunum presented characteristics of epithelial cells, such as a typical pavement-like aspect, the formation of domes and apical tight junctions and microvilli in confluent cultures.
these bovine intestinal cells were shown to express in vitro epithelial cell markers such as brush border enzymes  and the epithelium typical cytoskeleton proteins, the cytokeratins.
the enterocyte isolation procedure developed in the present study proved efficient to avoid a noticeable contamination from α-actin-positive cells  in the two first steps of each type of culture .
the first passage also corresponded to cells presenting still a substantial differentiation level .
to this respect, the first passage of each type of cultures appeared to be suitable to establish immortalized cell lines, a task which is now underway in our laboratory.
the functional differentiation state of the colon cells in culture could be estimated by the activities of drug metabolizing enzymes  <cit> .
indeed, bovine colon epithelial cell culture were characterized, as freshly isolated cells, by cytochrome p <dig> 1a1-associated 7-ethoxyresorufin o-deethylase activity as well as by prostaglandin h-synthase-mediated production of prostaglandin e <dig>  activities of phase ii enzymes  were also observed in colon epithelial cell cultures.
by contrast to most reports about mammalian enterocyte primocultures, data accumulated in the present study using  cell immunocytochemistry,  western blot and  gene expression analyses, showed that intestinal cell cultures from bovine jejunum and colon co-expressed epithelial markers and vimentin, an embryonic cytoskeleton filament that is expressed only in mesenchymal cells after birth.
a second fibroblast marker, the α-actin was scarcely detected, indicating that the vimentin strong expression of culture samples did not reflect a culture contamination by mesenchymal cells.
in addition, the vimentin distribution pattern did not exactly fit with the gene expression of this protein.
indeed, the immunodetection was negative in homogenates of fresh epithelia from jejunum and colon, as well as in organoid suspension used to seed each culture type, while the protein was essentially detected in samples of culture after the second passage.
by contrast, the vimentin gene transcript appeared in all bovine samples analysed, including the undissociated epithelium.
it seemed then that a post-transcriptional basal inhibition of vimentin synthesis has been suppressed in vitro.
in agreement with this hypothesis, a previous research dedicated to the development of an intestinal lineage from the porcine intestine  <cit>  led to the production of vimentin-positive cultures.
in view of this result, authors postulated that cultured cells had undergone a "mesenchymal transformation" in vitro.
similarly, a re-expression of vimentin has been reported in epithelial cells under pathological conditions in vivo, as well as in primary culture  <cit> .
vimentin re-expression in vitro could be instrumental in the maintenance of cell structure and/or functions of specific proteins such as the ones associated to membrane lipid rafts  <cit> .
besides the possible mesenchymal transformation of epithelial cells in vitro, accumulating data are now in favour of a natural incidence of vimentin in the undissociated intestine epithelium.
for instance, specialized m-cells found in the epithelium covering the intestine peyer's patches were identified, among other criteria, by vimentin immunostaining  <cit> .
these "microfold-cells" are almost devoid of microvilli at their apical side as they are specialized in the transport of particulate antigens from the gut lumen to the underlying lymphoid tissue, where specific immune responses could take place.
although the structure and functions of these cells seem to be broadly admitted it is still unclear whether epithelial cells leaving the epithelium crypts are predetermined as m-cells or whether their particular phenotype developed from differentiated enterocytes at the lymphoid tissue vicinity.
in favour of a possible local induction of m-cells differentiation, in vitro experiments using caco- <dig> cells showed that the enterocyte typical phenotype could be converted in a m-like phenotype adding b lymphocytes to the basolateral side of the epithelium  <cit> .
regardless the aforementioned debate concerning the m-cells differentiation, enterocyte-lymphocyte co-culture experiments clearly demonstrated that a non-bacterial environmental factor could cause the brush border effacement of a vimentin-negative enterocyte  leading to the phenotype of an intestinal cell type with a vimentin synthesis potential .
supporting the role of lymphocytes as phenotype conversion inducers, vimentin-positive cells were observed scattered throughout the villus epithelium of the rabbit small intestine, with the protein location extending from the perinuclear region to the cell membrane touching intraepithelial lymphocytes  <cit> .
additional indications that vimentin could be a marker of "differentiation variants" of enterocytes, came from a recent study that pointed out, in the ordinary epithelium villi of the rabbit ileum, a vimentin-positive enterocyte type sharing m-cell morphological features , but most probably representing a distinct cell type.
indeed, these so-called "cup-cells", differed from m-cells at two levels: they bound distinctive lectins and they did not take up microbeads instilled in the ileal lumen  <cit> .
CONCLUSIONS
altogether, m-cells and cup-cells features in vivo, as well as the experimental effacement of the enterocyte brush-border in vitro suggest that a vimentin re-expression could be a marker of cellular dedifferentiation.
in accordance with this hypothesis, primary cultures of bovine enterocytes obtained in the present study exhibited a low differentiation level.
a poor differentiated level of cultured intestinal cells has already been described by several authors using intestinal biopsies from the human  <cit> , the mouse  <cit>  or the rat  <cit> .
this differentiation deficiency could stem from the preferential selection of less differentiated cells  by the isolation procedure.
a low differentiation level in vitro may also be due to the suboptimal conditions of culture.
accordingly, obtaining a primary culture of fully differentiated intestinal cells from human foetal gut, perreault and beaulieu  <cit>  failed to detect vimentin neither by immunocytochemistry, nor by western-blot.
further studies would then be designed to improve the differentiation level of bovine enterocytes in vitro and then allow us to verify if this is associated with an inhibition of vimentin synthesis.
results presented in the present study, suggest that the combination of at least two culture medium modifications would be the first step of this study: the glucose substitution by inosin and the addition of butyrate in the culture medium.

 intestinal epithelium is organized as a single layer which covers the luminal side of this part of the digestive tract. cells that form this inner cover present specialisations according to their role in the digestive function but they have also common functions and are continuously renewed by the stem cell proliferation. in vivo, these stem cells, known as progenitors of all cell types, are mostly located in the lower third of the epithelium crypts, which are epithelial invaginations into the lamina propria. cells differentiation is associated with their migration from the depth of the crypts to the top of the villi and is followed by the cells death and desquamation into the intestinal lumen. enterocyte cultures represent valuable tools to assess the passage and/or toxicity of drugs, as well as the molecular mechanisms operating in pathologies caused by infectious agents known to affect the intestinal epithelium integrity. however, the successful establishment of an intestinal cell culture is hampered by the high rate of cell death occurring when isolating them from the epithelium and the difficulty to select proliferating cells to ensure several cell generations in vitro. although several investigators have already developed culture methods of intestinal cells for animal species such as the mouse, the rabbit or the pig, until now only two studies reported the production of primary cultures from intestinal bovine cells. more precisely, dibb-fuller and coworkers established a procedure for obtaining such cultures from ileum and colon. föllmann and collaborators also used the bovine colon as a source of intestinal cell cultures. various strategies were reported in order to isolate enterocytes from the colon or the small bowel. among the oldest methods, the mechanical dissociation provided rapidly a viable cell preparation but was frequently associated with a fibroblast contamination. the chelating methods also generated isolated epithelial cells retaining their morphologic characteristics. however, while first attempts seemed to affect cell surface receptors, further applications of chelating agents for a short time were suitable for the production of uncontaminated cultures of human colonocytes. matrisperse, a non enzymatic solution initially designed to isolate epithelial cells grown on engelbreth-holm-swarm biomatrix, also allowed the dissociation of the integral villus epithelial lining from human intestinal biopsies that produced a confluent monolayer in vitro. the enzymatic digestion provided a cell preparation made up mainly of organoids that were shown to successfully reconstitute either a monolayer of epithelial cells in vitro or crypt-villus structures in vivo following grafting in various tissues. in spite of these numerous efforts, little is known about the cell differentiation evolution over culture generations. the present study describes several complementary approaches to characterize bovine primary cultures of intestinal epithelial cells called jejunocytes and colonocytes as they were respectively isolated from adult jejunum and spiralled colon. cultures were initiated using an organoid-enriched suspension obtained by a multi-step method. results of vimentin expression in both types of enterocyte primary cultures are discussed in terms of differentiation status of cells.  morphological features of jejunocyte and colonocyte cultures the method developed to isolate cell material from the bovine jejunum and spiraled colon led to the production of suspension enriched in undissociated cell aggregates adhering to collagen coated culture flasks. they give rise to circular proliferating foci. within days the initial foci enlargement had led to the fusion of cell plaques in one confluent layer. confluent monolayers arising from the initial seeding of cultures presented a heterogeneous aspect due to the presence of residual multicellular organoids dispersed in the newly formed monolayer. with the first passage jejunocyte and colonocyte cultures acquired a homogeneous pavement-like aspect typical of epithelial sheets. we generally carried out to passages over to weeks in both cell series without major changes of cell morphology. during early passages, the confluence recovery was achieved within to days and then a progressive slowdown in the cell proliferation rate was observed until cessation. confluent monolayers of both cell types exhibited domes, appearing as round blurred arrays corresponding to fluid entrapment between the flask wall and the basolateral side of the monolayer, resulting from ionic regulations by functional epithelial cells. this observation is in agreement with dome formation in cultures of the human intestinal cell lineage caco-. ultrastructural analyses of cultured cells revealed apical tight junctions between cells forming the monolayer of both cell types. cultured jejunocytes and colonocytes also exhibited a few apical microvilli. to ascertain the identity and functionality of cultured monolayers, cells components from various culture passages were submitted to immunolabelling. these data were compared to results from gene expression studies and measurements of specific activities of brush border associated enzymes. cell characterization using immunodetection antibodies directed against cytokeratins and vimentin were used to distinguish cells of epithelial origin from contaminating fibroblasts. double immunolabeling of cryosections of the bowel wall showed the presence of the cytokeratin positive cells all over the lining epithelium and vimentin positive cells in the submucosa. cytokeratins showing the characteristic disposition of intermediate filaments were also recognized in obtained monolayers. surprisingly, double staining vimentin-cytokeratin clearly showed that cultured cells expressed both epithelial and mesenchymal markers. however, the double staining cytokeratin-α-actin distinguished separate cells populations. the few α-actin positive cells, probably contaminant myofibroblasts were released from the gut wall during the dissociation procedure and not completely eliminated by the sorbitol centrifugation or the selective attachment. indirect immunofluorescence revealed also the membrane distribution of the epithelial specific antigen in cultured enterocytes and intracellular distribution of the cytokeratin. e-cadherin representing one of the proteins located at the adherence junctions was also detected, but a mis-localization was observed. the cells did not present the characteristic staining pattern to the cell periphery, but a granular distribution into the cytoplasm. western blot analyses also confirmed the presence of epithelial characters in cultured cells. pan-anti-cytokeratin and anti-cytokeratin antibodies recognised the same major kda protein in homogenates from: freshly scrapped intestinal epithelium, organoid suspensions used to seed cultures, intestinal cell cultures of both types, and in the positive control caco- cells. no cytokeratin staining was noticeable in 3t fibroblasts. the size uniformity of stained products in all tested epithelial cells was also confirmed using the pan-cytokeratin antibody. in addition, blots were also positive for another epithelial cell marker, the e-cadherin. compared to the expected size, the anti-e cadherin target likely presented the size of a protein domain. the antibody used was directed against the intracellular domain of this protein. in agreement with immunocytochemistry results, vimentin and α-actin presented distinct distribution in sample homogenates. indeed, the anti-vimentin targeted the same proteins in all bovine intestinal cell cultures and in the control 3t fibroblasts, while α-actin was scarcely detected in more advanced passages. taken together, western blot and immunocytochemistry results strongly suggested that vimentin is expressed in primary cultures of epithelial intestinal cells from bovine jejunum and colon. however, vimentin was not detected by western-blot in caco- cell proteins. although immunocytochemistry results for α-actin indicated a very low contamination of bovine epithelial cell cultures by mesenchymal cells, western blot results indicated that this phenomenon is to low to be detected in most culture stages. gene expression analyses of enterocyte markers rt-pcr analyses allowed us to investigate the expression of bovine enterocyte markers namely: villin, zonula occludens, fatty acid binding protein, small intestine peptidase and e-cadherin. expressions studies were also used to confirm the presence of the vimentin transcript in bovine cells. similarly to western blot analyses, gene expressions studies were carried out on total rna extracted from bovine samples of: freshly scrapped epithelia, organoid suspensions used to seed cultures and intestinal cell cultures of both types. as can be seen in figure, cdna amplification products presented the expected size using specific primers for coding regions of the epithelial markers, such as the villin gene, the zo gene and the e-cadherin, that were uniformly expressed in cells from primary cultures of bovine enterocytes. besides these structural components expected in any epithelium, we also investigated the expression of functional markers of digestive epithelia. among them, the gene coding for the fatty acid binding protein, presented a constant expression in both jejunocytes and colonocytes in vitro. the second tissue specific marker studied was the gene coding for the intestinal peptidase. in this case, the expression was restricted to jejunocyte samples, as logically expected on the basis of the functional specialization of the jejunum. the ip expression could then be considered as suitable jejunocyte marker. in accordance with immunostaining results, samples from jejunocyte and colonocyte cultures expressed the vimentin gene. interestingly, while western blots analyses failed to reveal vimentin either in freshly removed epithelia or in organoids suspensions used to seed cultures, the transcript of vimentin gene was clearly detected in those samples, indicating that the vimentin is constitutively expressed in jejunum, as well as in colon from the bovine intestine. however, western blot results suggested that vimentin synthesis seemed to be restricted in cultured cells with a progressive increase over the culture passage number. specific activities of two brush border-associated enzymes the chosen enzymes were a disaccharidase and the alkaline phosphatase. as for gene expression studies, enzyme activities were investigated in bovine samples of: freshly scrapped epithelia, organoid suspensions used to seed cultures and intestinal cell cultures of both types. assays performed on fresh epithelial tissue confirmed that jejunum and colon differed in respect to enzyme specific activities, the jejunum presenting the highest level. maltase sa clearly decreased in primary cultures of both cells types. furthermore, the cell isolation procedure selected disaccharidase depleted material, since organoid suspensions presented a 50% reduction in regard to the fresh epithelium preparation. it is interesting to note that, compared to the organoids suspension, jejunocytes from the first culture passage did not present a drastic reduction of the maltase sa. however, with subsequent culture passages the decreasing of disaccharidase sa was progressing toward a stable low level. similar results were obtained from measurements of the intestinal alkaline phosphatase sa, a ubiquitous enzyme that is a marker of brush border in intestinal epithelial cultures. indeed, as expected from functional differences between jejunum and colon in vivo, epithelia homogenates, organoids suspensions, and cultures at first passages from bovine jejunum presented higher sa for this enzyme. moreover, similarly to what was noted for maltase sa, iap presented a strong decrease from fresh tissue to subcultured cells, so that jejunocyte and colonocyte sa values joined together to the same low level as the culture passage number increased. regarding a given passage number, the maltase sa decreased over the culture duration. these results reflected a loss of cell differentiation in vitro compared to the in vivo level. adjustments of cell culture medium were made to improve the differentiation status of jejunocytes and colonocytes in vitro. to this end the glucose was substituted by inosin in the culture medium; this condition was described to stimulate the acquisition of the enterocyte differentiated phenotype in vitro. as seen in figure 8b, the use of a inosin-containing glucose-free culture medium during days, led to an increase of maltase activity. however this effect diminished as the passage number increased, suggesting that glucose substitution by inosin should be complemented by other culture medium modifications. addition of sodium butyrate, a substance thought to promote enterocyte differentiation through a stimulation of the cdx homeobox gene expression, proved to be efficient to stimulate maltase sa. discussion morphological data presented in the present study indicated that primary cultures of bovine enterocytes isolated from colon and jejunum presented characteristics of epithelial cells, such as a typical pavement-like aspect, the formation of domes and apical tight junctions and microvilli in confluent cultures. these bovine intestinal cells were shown to express in vitro epithelial cell markers such as brush border enzymes and the epithelium typical cytoskeleton proteins, the cytokeratins. the enterocyte isolation procedure developed in the present study proved efficient to avoid a noticeable contamination from α-actin-positive cells in the two first steps of each type of culture. the first passage also corresponded to cells presenting still a substantial differentiation level. to this respect, the first passage of each type of cultures appeared to be suitable to establish immortalized cell lines, a task which is now underway in our laboratory. the functional differentiation state of the colon cells in culture could be estimated by the activities of drug metabolizing enzymes. indeed, bovine colon epithelial cell culture were characterized, as freshly isolated cells, by cytochrome p 1a1-associated 7-ethoxyresorufin o-deethylase activity as well as by prostaglandin h-synthase-mediated production of prostaglandin e activities of phase ii enzymes were also observed in colon epithelial cell cultures. by contrast to most reports about mammalian enterocyte primocultures, data accumulated in the present study using cell immunocytochemistry, western blot and gene expression analyses, showed that intestinal cell cultures from bovine jejunum and colon co-expressed epithelial markers and vimentin, an embryonic cytoskeleton filament that is expressed only in mesenchymal cells after birth. a second fibroblast marker, the α-actin was scarcely detected, indicating that the vimentin strong expression of culture samples did not reflect a culture contamination by mesenchymal cells. in addition, the vimentin distribution pattern did not exactly fit with the gene expression of this protein. indeed, the immunodetection was negative in homogenates of fresh epithelia from jejunum and colon, as well as in organoid suspension used to seed each culture type, while the protein was essentially detected in samples of culture after the second passage. by contrast, the vimentin gene transcript appeared in all bovine samples analysed, including the undissociated epithelium. it seemed then that a post-transcriptional basal inhibition of vimentin synthesis has been suppressed in vitro. in agreement with this hypothesis, a previous research dedicated to the development of an intestinal lineage from the porcine intestine led to the production of vimentin-positive cultures. in view of this result, authors postulated that cultured cells had undergone a "mesenchymal transformation" in vitro. similarly, a re-expression of vimentin has been reported in epithelial cells under pathological conditions in vivo, as well as in primary culture. vimentin re-expression in vitro could be instrumental in the maintenance of cell structure and/or functions of specific proteins such as the ones associated to membrane lipid rafts. besides the possible mesenchymal transformation of epithelial cells in vitro, accumulating data are now in favour of a natural incidence of vimentin in the undissociated intestine epithelium. for instance, specialized m-cells found in the epithelium covering the intestine peyer's patches were identified, among other criteria, by vimentin immunostaining. these "microfold-cells" are almost devoid of microvilli at their apical side as they are specialized in the transport of particulate antigens from the gut lumen to the underlying lymphoid tissue, where specific immune responses could take place. although the structure and functions of these cells seem to be broadly admitted it is still unclear whether epithelial cells leaving the epithelium crypts are predetermined as m-cells or whether their particular phenotype developed from differentiated enterocytes at the lymphoid tissue vicinity. in favour of a possible local induction of m-cells differentiation, in vitro experiments using caco- cells showed that the enterocyte typical phenotype could be converted in a m-like phenotype adding b lymphocytes to the basolateral side of the epithelium. regardless the aforementioned debate concerning the m-cells differentiation, enterocyte-lymphocyte co-culture experiments clearly demonstrated that a non-bacterial environmental factor could cause the brush border effacement of a vimentin-negative enterocyte leading to the phenotype of an intestinal cell type with a vimentin synthesis potential. supporting the role of lymphocytes as phenotype conversion inducers, vimentin-positive cells were observed scattered throughout the villus epithelium of the rabbit small intestine, with the protein location extending from the perinuclear region to the cell membrane touching intraepithelial lymphocytes. additional indications that vimentin could be a marker of "differentiation variants" of enterocytes, came from a recent study that pointed out, in the ordinary epithelium villi of the rabbit ileum, a vimentin-positive enterocyte type sharing m-cell morphological features, but most probably representing a distinct cell type. indeed, these so-called "cup-cells", differed from m-cells at two levels: they bound distinctive lectins and they did not take up microbeads instilled in the ileal lumen.  altogether, m-cells and cup-cells features in vivo, as well as the experimental effacement of the enterocyte brush-border in vitro suggest that a vimentin re-expression could be a marker of cellular dedifferentiation. in accordance with this hypothesis, primary cultures of bovine enterocytes obtained in the present study exhibited a low differentiation level. a poor differentiated level of cultured intestinal cells has already been described by several authors using intestinal biopsies from the human, the mouse or the rat. this differentiation deficiency could stem from the preferential selection of less differentiated cells by the isolation procedure. a low differentiation level in vitro may also be due to the suboptimal conditions of culture. accordingly, obtaining a primary culture of fully differentiated intestinal cells from human foetal gut, perreault and beaulieu failed to detect vimentin neither by immunocytochemistry, nor by western-blot. further studies would then be designed to improve the differentiation level of bovine enterocytes in vitro and then allow us to verify if this is associated with an inhibition of vimentin synthesis. results presented in the present study, suggest that the combination of at least two culture medium modifications would be the first step of this study: the glucose substitution by inosin and the addition of butyrate in the culture medium. 
the present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. the low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate or using a glucose-deprived culture medium. in vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes.
 cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. in vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract.  cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. accordingly, cells expressed several markers of enterocyte brush border as well as an epithelial cytoskeleton component. however, enterocyte primocultures were also positive for the vimentin immunostaining. vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. the low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate or using a glucose-deprived culture medium.  the present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. cultured cells kept their morphologic and functional characteristics during several generations. 