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Use of Cultured Cell Lines in Studies of Intestinal Cell Differentiation and Function

Alain Zweibaum, Marc Laburthe, Etienne Grasset, Daniel Louvard

10.1002/cphy.cp060407

Source: Supplement 19: Handbook of Physiology, The Gastrointestinal System, Intestinal Absorption and Secretion

Originally published: 1991

Published online: January 2011

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Cellular Models
1.1 Cell Lines From Normal Tissues
1.2 Cell Lines From Chemically Induced Tumors
1.3 Human Colon Carcinoma Cell Lines
2 Use of Cultured Cells for Study of Enterocytic Differentiation and Polarization
2.1 Polarized Organization of Enterocytes: Current Knowledge and Limitations
2.2 HT‐29 and Caco‐2 Cells as in Vitro Models: Advantages and Limitations
2.3 What Can We Learn From HT‐29 and Caco‐2 Cells?
3 Use of Cultured Cells for Study of Neurohormonal Receptors
3.1 Expression of Neurohormonal Receptors: Normal Intestine Versus Cell Line
3.2 Receptor Studies
4 Use of Cultured Cells for Study of Intestinal Transport
4.1 Transport Properties of Cultured Intestinal Cells
4.2 Regulation of Intestinal Transport: Effect of Secretagogues
4.3 Comparison With Other Existing Models: Possible Future Developments
5 Use of Cultured Cells for Study of Metabolic Functions
5.1 Colonic Malignant Cells as Models for Study of Glucose Metabolism
5.2 HT‐29 Glucose‐Negative Cells: Model for Study of Gluconeogenesis
6 Potential Use of Cultured Cells for Other Studies
7 Conclusion
Figure 1. Figure 1.

Transmission electron micrograph of Caco‐2 cells (passage 70) in relation to cell growth. Sections of monolayer are perpendicular to bottom of flask. A: exponentially growing cells (day 5). × 3,400. B: postconfluent stationary cells (day 15). × 3,400. Note presence in A of only sparse irregular microvilli on apical side of cells and presence in B of well‐developed apical brush border. However, note that cells are polarized (A and B) with presence of tight junctions. Also note absence in A of distinguishable intercellular spaces that are considerably enlarged in B; these changes in appearance of intercellular junctions most likely depend on growth‐related variations of intracellular accumulation of cAMP, which is higher in exponentially growing than in stationary cells 204.

(Courtesy of G. Chevalier.)
Figure 2. Figure 2.

Higher magnification of brush‐border microvilli of postconfluent Caco‐2 cells (day 15) showing core of microfilaments that extends into cytoplasm and presence of poorly developed glycocalyx and of tight junctions, × 5,500.

(Courtesy of G. Chevalier.)
Figure 3. Figure 3.

Immunofluorescent staining of monolayer of postconfluent Caco‐2 cells (day 10) obtained with rabbit antiserum specific for sucrase‐isomaltase from adult human jejunum. Positive staining corresponds to binding of antibodies to apical brush‐border microvilli. × 430.

(Courtesy of N. Triadou). (Courtesy of E. Dussaulx.)
Figure 4. Figure 4.

Variations of sucrase activity during growth in culture of Caco‐2 cells. Sucrase activity (filled triangles) is measured in brush‐border–enriched fraction at indicated days and is expressed as milliunits per milligrams of cell protein 181, 204. Caco‐2 cells (passage 70) are plated in 25 cm2 plastic flasks (3 × 105 cells per flask) 204; growth curve is expressed as milligrams of cell protein per flask (open triangles). Note that net increase in sucrase activity is concomitant with entry of cells in stationary phase of growth. Similar pattern of growth‐related variations of activity is observed for other hydrolases associated with brush‐border membrane of Caco‐2 cells 181, 204.

(Courtesy of M. Rousset.)
Figure 5. Figure 5.

Transmission electron micrograph of HT‐29 cells (day 30 in culture) in relation to culture conditions. A: cells grown with 25 mM glucose. Section is perpendicular to bottom of flask, and cells are organized into multilayer of unpolarized cells with presence of only sparse irregular microvilli on apical surface of cell layer. × 2,300. B: detail of apical surface of enterocyte‐like differentiated HT‐29 cells grown for 5 passages in glucose‐free medium supplemented with 2.5 mM inosine 248. Note presence of organized brush border and tight junctions. × 13,500.

(Courtesy of G. Chevalier.)
Figure 6. Figure 6.

Isolation of subclones of HT29–18 cells. Starting from culture of undifferentiated cells, differentiated culture can be obtained in media containing galactose; cells must be progressively adapted to decreasing concentrations of glucose and increasing concentrations of galactose to avoid cell death. Cultures were then maintained in 5 mM galactose medium for more than 20 generations and subsequently replated in glucose‐containing media. Cultures then displayed obvious heterogeneity when observed with an inverted microscope. Cells were replated and grown at low density; individual colonies were separated. Each subclone displayed a homogeneous differentiated phenotype (polarized monolayer) in glucose‐containing media. Enhanced differentiation characteristics (brush border or mucous granules) can be seen when cultures are grown in galactose. When transferred to galactose‐containing medium, subclones do not require progressive adaptation to new medium.
Figure 7. Figure 7.

Mucous secretion in HT29–18‐N2 cells. Specific monoclonal antibodies against fetal mucins were used to detect mucous content of this subclone (using immunofluorescence techniques). A: culture was fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X‐100. Cells loaded with immunoreactive material can be seen. No significant staining is obtained when cells are not permeabilized, thus demonstrating intracellular localization of mucins. B: culture was incubated at 37°C for 30 min with 20 μM carbachol. Long wisps of mucosal secretions are seen without permeabilization of formaldehyde‐fixed cells monolayers, thus demonstrating the exocytosis of granular contents induced by carbachol (bar = 13 μm).

(courtesy of J. Bara) (Courtesy of C. Huet).
Figure 8. Figure 8.

Structure of the mucus‐secreting clonal cell line HT29–16E. A: low‐magnification electron micrograph of vertical section of postconfluent HT29–16E cells showing polarized mucous secretion. B: ultrastructure of mucus‐secreting cell exhibiting features of a typical goblet cell; supranuclear cytoplasm is filled with many vacuoles of variable electron density. C: electron micrograph of section cut parallel to superficial layer of cultured cells. All superficial cells exhibit abundant mucous secretion.

[From Augeron and Laboisse 5].
Figure 9. Figure 9.

Immunological detection of sucrase‐isomaltase in brush‐border–enriched fractions analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis transferred onto nitrocellulose sheet (for details of methods see ref. 256). A: control sucrase‐isomaltase complex from adult jejunum showing high‐molecular‐weight proform of enzyme (top band) and 2 subunits of sucrase and isomaltase (bottom bands). B: Caco‐2 cells (passage 70, day 16). C: colon from a 16‐wk‐old fetus. Note that in B and C, only high‐molecular‐weight band is present and migrates faster than corresponding adult intestinal proform.

(Courtesy of I. Chantret.)
Figure 10. Figure 10.

Immunological detection of villin and actin in HT‐29 cell extracts. Immunoblotting procedures were applied to total cell extracts derived from undifferentiated and differentiated HT29–18 cells. Monoclonal or polyclonal antibodies against villin give identical results. Specific band at 90 kDa (villin) is visualized and strongly enhanced (at least 10 times) in differentiated HT29–18 cells. Polyclonal antibody against actin was applied and detected simultaneously on these immunoblots. No significant increase in amount of actin during HT29–18 cell differentiation can be observed, thus providing an internal control. A: proteins staining. B: immunoreplica. Glu, cells grown in glucose; gal, cells grown in galactose. Molecular‐size markers: myosin heavy chain, 180 kDa; β‐galactosidase, 116 kDa; phosphorylase b, 92 kDa; bovine serum albumin, 62 kDa.

(Courtesy of E. Coudrier and S. Robine.)
Figure 11. Figure 11.

Comparison of development of VIP receptor concentration and sucrase activity during the growth in culture of Caco‐2 cells (passage 70). Assays were performed for both sucrase and VIP receptors with crude plasma membranes prepared as described in reference 45.

(M. Laburthe and M. Rousset, personal communication.)
Figure 12. Figure 12.

Bioassay of VIP, which is highly sensitive 127 and based on VIP‐stimulated accumulation of cAMP in HT‐29 cells 132. Standard VIP (left panel) and plasma from two patients with watery diarrhea syndrome (middle and right panels) are tested either alone (filled symbols) or after previous exposure to a specific anti‐VIP antibody (open symbols).

[From Laburthe et al. 127.]
Figure 13. Figure 13.

Phase‐contrast micrograph of dome formed by monolayer of confluent Caco‐2 cells grown on plastic (day 14) focused on monolayer (A) and focused on top of dome (B).

[From Pinto et al. 181.]
Figure 14. Figure 14.

Measurement of electrical parameters in dome cells. Current‐conveying microelectrode is inserted between monolayer and plastic dish. Second microelectrode is advanced from culture medium (position 1) toward cell, first crossing mucosal membrane (position 2) and then serosal membrane (position 3). Upper right, schematic representation of potential difference recorded in 3 positions. This difference allowed calculation of intracellular potential and mucosal membrane fractional resistance. Mucosal solution is taken as electrical reference

[From Grasset et al. 89.]
Figure 15. Figure 15.

Effect of mucosal addition of 5 μg/ml amphotericin B on short‐circuit current (Isc) in filter‐grown Caco‐2 cells. At initial peak, scale had to be divided by 2 during first few minutes. Application of sodium‐free solution (Na 0) on both sides of tissue abolished Isc. Serosal ouabain (0.1 mM) also abolished Isc within a maximum of 30 min.

[From Grasset et al. 89.]
Figure 16. Figure 16.

Filter‐grown HT29–19A cells: stimulation of short‐circuit current (Isc) by serosal dibutyryl cAMP and inhibition by serosal bumetanide (bipolar current pulses were used to monitor transepithelial electrical resistance).

[From Augeron et al. 6.]
Figure 17. Figure 17.

Time course of effect of VIP (A), Ca2+ ionophore A23187 (B), and VIP plus A23187 (C) on net ion transport and short‐circuit current and Isc across T84 cell monolayers (grown on collagen‐coated monolayers). Open bars, net Cl− transport; solid bars, net Na+ transport over 10‐min flux period. Circles and lines, Isc. Hatched bars, open circles, and dashed line (C), predicted additive response from results shown in A and B. Results are means + SE. Values above dashed line represent net secretion and those below the line net absorption. [(From Cartwright et al. 30.]


Figure 1.

Transmission electron micrograph of Caco‐2 cells (passage 70) in relation to cell growth. Sections of monolayer are perpendicular to bottom of flask. A: exponentially growing cells (day 5). × 3,400. B: postconfluent stationary cells (day 15). × 3,400. Note presence in A of only sparse irregular microvilli on apical side of cells and presence in B of well‐developed apical brush border. However, note that cells are polarized (A and B) with presence of tight junctions. Also note absence in A of distinguishable intercellular spaces that are considerably enlarged in B; these changes in appearance of intercellular junctions most likely depend on growth‐related variations of intracellular accumulation of cAMP, which is higher in exponentially growing than in stationary cells 204.

(Courtesy of G. Chevalier.)


Figure 2.

Higher magnification of brush‐border microvilli of postconfluent Caco‐2 cells (day 15) showing core of microfilaments that extends into cytoplasm and presence of poorly developed glycocalyx and of tight junctions, × 5,500.

(Courtesy of G. Chevalier.)


Figure 3.

Immunofluorescent staining of monolayer of postconfluent Caco‐2 cells (day 10) obtained with rabbit antiserum specific for sucrase‐isomaltase from adult human jejunum. Positive staining corresponds to binding of antibodies to apical brush‐border microvilli. × 430.

(Courtesy of N. Triadou). (Courtesy of E. Dussaulx.)


Figure 4.

Variations of sucrase activity during growth in culture of Caco‐2 cells. Sucrase activity (filled triangles) is measured in brush‐border–enriched fraction at indicated days and is expressed as milliunits per milligrams of cell protein 181, 204. Caco‐2 cells (passage 70) are plated in 25 cm2 plastic flasks (3 × 105 cells per flask) 204; growth curve is expressed as milligrams of cell protein per flask (open triangles). Note that net increase in sucrase activity is concomitant with entry of cells in stationary phase of growth. Similar pattern of growth‐related variations of activity is observed for other hydrolases associated with brush‐border membrane of Caco‐2 cells 181, 204.

(Courtesy of M. Rousset.)


Figure 5.

Transmission electron micrograph of HT‐29 cells (day 30 in culture) in relation to culture conditions. A: cells grown with 25 mM glucose. Section is perpendicular to bottom of flask, and cells are organized into multilayer of unpolarized cells with presence of only sparse irregular microvilli on apical surface of cell layer. × 2,300. B: detail of apical surface of enterocyte‐like differentiated HT‐29 cells grown for 5 passages in glucose‐free medium supplemented with 2.5 mM inosine 248. Note presence of organized brush border and tight junctions. × 13,500.

(Courtesy of G. Chevalier.)


Figure 6.

Isolation of subclones of HT29–18 cells. Starting from culture of undifferentiated cells, differentiated culture can be obtained in media containing galactose; cells must be progressively adapted to decreasing concentrations of glucose and increasing concentrations of galactose to avoid cell death. Cultures were then maintained in 5 mM galactose medium for more than 20 generations and subsequently replated in glucose‐containing media. Cultures then displayed obvious heterogeneity when observed with an inverted microscope. Cells were replated and grown at low density; individual colonies were separated. Each subclone displayed a homogeneous differentiated phenotype (polarized monolayer) in glucose‐containing media. Enhanced differentiation characteristics (brush border or mucous granules) can be seen when cultures are grown in galactose. When transferred to galactose‐containing medium, subclones do not require progressive adaptation to new medium.



Figure 7.

Mucous secretion in HT29–18‐N2 cells. Specific monoclonal antibodies against fetal mucins were used to detect mucous content of this subclone (using immunofluorescence techniques). A: culture was fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X‐100. Cells loaded with immunoreactive material can be seen. No significant staining is obtained when cells are not permeabilized, thus demonstrating intracellular localization of mucins. B: culture was incubated at 37°C for 30 min with 20 μM carbachol. Long wisps of mucosal secretions are seen without permeabilization of formaldehyde‐fixed cells monolayers, thus demonstrating the exocytosis of granular contents induced by carbachol (bar = 13 μm).

(courtesy of J. Bara) (Courtesy of C. Huet).


Figure 8.

Structure of the mucus‐secreting clonal cell line HT29–16E. A: low‐magnification electron micrograph of vertical section of postconfluent HT29–16E cells showing polarized mucous secretion. B: ultrastructure of mucus‐secreting cell exhibiting features of a typical goblet cell; supranuclear cytoplasm is filled with many vacuoles of variable electron density. C: electron micrograph of section cut parallel to superficial layer of cultured cells. All superficial cells exhibit abundant mucous secretion.

[From Augeron and Laboisse 5].


Figure 9.

Immunological detection of sucrase‐isomaltase in brush‐border–enriched fractions analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis transferred onto nitrocellulose sheet (for details of methods see ref. 256). A: control sucrase‐isomaltase complex from adult jejunum showing high‐molecular‐weight proform of enzyme (top band) and 2 subunits of sucrase and isomaltase (bottom bands). B: Caco‐2 cells (passage 70, day 16). C: colon from a 16‐wk‐old fetus. Note that in B and C, only high‐molecular‐weight band is present and migrates faster than corresponding adult intestinal proform.

(Courtesy of I. Chantret.)


Figure 10.

Immunological detection of villin and actin in HT‐29 cell extracts. Immunoblotting procedures were applied to total cell extracts derived from undifferentiated and differentiated HT29–18 cells. Monoclonal or polyclonal antibodies against villin give identical results. Specific band at 90 kDa (villin) is visualized and strongly enhanced (at least 10 times) in differentiated HT29–18 cells. Polyclonal antibody against actin was applied and detected simultaneously on these immunoblots. No significant increase in amount of actin during HT29–18 cell differentiation can be observed, thus providing an internal control. A: proteins staining. B: immunoreplica. Glu, cells grown in glucose; gal, cells grown in galactose. Molecular‐size markers: myosin heavy chain, 180 kDa; β‐galactosidase, 116 kDa; phosphorylase b, 92 kDa; bovine serum albumin, 62 kDa.

(Courtesy of E. Coudrier and S. Robine.)


Figure 11.

Comparison of development of VIP receptor concentration and sucrase activity during the growth in culture of Caco‐2 cells (passage 70). Assays were performed for both sucrase and VIP receptors with crude plasma membranes prepared as described in reference 45.

(M. Laburthe and M. Rousset, personal communication.)


Figure 12.

Bioassay of VIP, which is highly sensitive 127 and based on VIP‐stimulated accumulation of cAMP in HT‐29 cells 132. Standard VIP (left panel) and plasma from two patients with watery diarrhea syndrome (middle and right panels) are tested either alone (filled symbols) or after previous exposure to a specific anti‐VIP antibody (open symbols).

[From Laburthe et al. 127.]


Figure 13.

Phase‐contrast micrograph of dome formed by monolayer of confluent Caco‐2 cells grown on plastic (day 14) focused on monolayer (A) and focused on top of dome (B).

[From Pinto et al. 181.]


Figure 14.

Measurement of electrical parameters in dome cells. Current‐conveying microelectrode is inserted between monolayer and plastic dish. Second microelectrode is advanced from culture medium (position 1) toward cell, first crossing mucosal membrane (position 2) and then serosal membrane (position 3). Upper right, schematic representation of potential difference recorded in 3 positions. This difference allowed calculation of intracellular potential and mucosal membrane fractional resistance. Mucosal solution is taken as electrical reference

[From Grasset et al. 89.]


Figure 15.

Effect of mucosal addition of 5 μg/ml amphotericin B on short‐circuit current (Isc) in filter‐grown Caco‐2 cells. At initial peak, scale had to be divided by 2 during first few minutes. Application of sodium‐free solution (Na 0) on both sides of tissue abolished Isc. Serosal ouabain (0.1 mM) also abolished Isc within a maximum of 30 min.

[From Grasset et al. 89.]


Figure 16.

Filter‐grown HT29–19A cells: stimulation of short‐circuit current (Isc) by serosal dibutyryl cAMP and inhibition by serosal bumetanide (bipolar current pulses were used to monitor transepithelial electrical resistance).

[From Augeron et al. 6.]


Figure 17.

Time course of effect of VIP (A), Ca2+ ionophore A23187 (B), and VIP plus A23187 (C) on net ion transport and short‐circuit current and Isc across T84 cell monolayers (grown on collagen‐coated monolayers). Open bars, net Cl− transport; solid bars, net Na+ transport over 10‐min flux period. Circles and lines, Isc. Hatched bars, open circles, and dashed line (C), predicted additive response from results shown in A and B. Results are means + SE. Values above dashed line represent net secretion and those below the line net absorption. [(From Cartwright et al. 30.]

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Article Title: Use of Cultured Cell Lines in Studies of Intestinal Cell Differentiation and Function
 

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Alain Zweibaum, Marc Laburthe, Etienne Grasset, Daniel Louvard. Use of Cultured Cell Lines in Studies of Intestinal Cell Differentiation and Function. Compr Physiol 2011, Supplement 19: Handbook of Physiology, The Gastrointestinal System, Intestinal Absorption and Secretion: 223-255. First published in print 1991. doi: 10.1002/cphy.cp060407