In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

* These authors contributed equally
Biology

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Summary

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

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Li, B. R., Wu, J., Li, H. S., Jiang, Z. H., Zhou, X. M., Xu, C. H., Ding, N., Zha, J. M., He, W. Q. In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability. J. Vis. Exp. (140), e57032, doi:10.3791/57032 (2018).

Abstract

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

Introduction

Intestinal epithelial cells are not only responsible for the absorption of nutrients, but also form an important barrier to defend against pathogenic microorganisms and microbial toxins. This intestinal barrier function is regulated by tight junction permeability and epithelial cell integrity1,2,3, and dysfunction of the epithelial barrier function is associated with inflammatory bowel disease (IBD). The perijunctional actomyosin ring (PAMR) lies within the cell that is closely contiguous to the tight junctions. The contraction of the PAMR, which is regulated by the myosin light chain (MLC), is crucial for the regulation of tight junction permeability4,5,6,7,8,9,10. Tumor necrosis factor (TNF) is central to intestinal barrier loss by upregulating intestinal epithelial MLC kinase (MLCK) expression and inducing occludin internalization11,12,13.

Ions such as Na+ and Cl- can cross the paracellular space by either the pore or leak pathway14. In a "leaky" epithelium, changes in TER primarily reflect altered tight junction permeability. TER measurement is a commonly used electrophysiological approach to quantify tight junction permeability, primarily to Na+ and Cl-, based on the impedance of cell monolayers. Diverse cell types, including intestinal epithelial cells, pulmonary epithelial cells, and vascular endothelial cells, have been reported for TER measurements. Advantages of this method are that TER measurements are non-invasive and can be used to monitor live cells in real-time. In addition, the TER measurement technique is useful for drug toxicity studies15.

Caco-2BBe cells are human epithelial colorectal adenocarcinoma cells with a structure and function similar to the differentiated small intestinal epithelial cells: for example, these cells have microvilli and enzymes associated with small intestinal brush border. Therefore, cultured Caco-2BBe monolayers are utilized as an in vitro model for testing barrier function.

In mice, one way to study intestinal paracellular permeability is by measuring the ability of FITC-dextran to cross from the lumen into the blood. Thus, the intestinal permeability can be assessed by gavaging FITC-dextran directly into mice and measuring the fluorescence within the blood. The following protocol describes two simple methods to assess intestinal epithelium permeability both in vitro and in vivo.

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Protocol

This study was approved by the Animal Care and Use Protocol of Cambridge-Suda Genomic Resource Center (CAM-SU), Soochow University.

1. Plating and Maintenance of Caco-2bbe on Porous Polycarbonate Membranes

  1. Grow cells in a T75 flask with media (DMEM containing 10% FBS). Flasks should be fed regularly, depending on the cell density.
    NOTE: For optimal plating, cells should divide rapidly and have a flat "fried-egg" shape, which indicates that cells are in the growth phase.
  2. Once cells are 80% confluent, take the flask out of the incubator and remove the media. Rinse any residual media with 1-2 mL of sterile PBS (without Ca2+). Pipet 1.5 mL Trypsin-EDTA into the flask and gently rock the flask; then, place the flask in the 37 °C incubator for 20 min without rocking.
  3. While the cells are trypsinizing, place inserts containing porous polycarbonate membranes (pore size, 0.4 µm; surface area, 0.33 cm2; see Table of Materials) into 24-well plates. Add 1.0 mL of culture media in the basal chamber (the lower space of the membrane).
  4. Pipet 5 mL of media into the flask and vigorously pipet the cells against the side of the flask 5-10 times to achieve loose, individual cells or 2-3 cell clumps.
  5. Plate 0.166 mL of cells (achieving a 1:8 dilution ratio) into the apical chamber (the upper space of the membrane). Incubate at 37 °C for up to 3 weeks.
    1. Feed cells three times weekly by carefully aspirating the media from the basal compartment of each well using a pressure pump. Gently drip 1 mL of media into the apical chamber of each insert.

2. Use of Epithelial Meter (Volt/Ohm) for Measuring TER

NOTE: After about 3 weeks of culture on polycarbonate membranes, Caco-2BBe cells are ready for TER measurement.

  1. For cytokine studies, one day before measurement, replace the basal media with media containing 10 ng/mL of IFNγ. On the day of the experiment, replace the media with HBSS containing 2.5 or 7.5 ng/mL of TNF.
    NOTE: IFNγ treatment increases expression of TNF receptor 2 (TNFR2)16.
  2. To correct the meter, insert the correction electrode into the Input port, and choose "Ohm" mode. Adjust the R Adj screw with a screwdriver until the meter displays a reading of 1,000 Ω.
  3. Sterilize the electrodes by placing them in 70% ethanol for 15-30 min, and then allow them to air dry for 15 s. Rinse the electrode in the experimental cell culture media.
  4. Turn on the power, and choose the "Ohm" mode. Carefully place the long ends of the electrode bridges into the basal chamber and the short ends into the apical chamber. Ensure that the longer electrodes touch the bottom of the dish, while keeping the shorter electrodes below the surface of the media but above the tissue culture inserts. Keep the electrodes vertical.
  5. Measure the resistance of the sample inserts and blank inserts (i.e., the culture inserts without cells but with HBSS) at 0, 1, 2, 3, 4 h after cytokine treatment. Record the resistance.
  6. To achieve consistency across different plate formats, calculate the product of the resistance and the effective membrane area:
    Equation
    For 24-well inserts, the effective membrane area is 0.33 cm2.

3. Murine Model of Dextran Sulphate Sodium (DSS)-induced Colitis

  1. Add DSS to autoclaved water to a final concentration of 3.5% (wt/vol).
  2. Administer 3.5% DSS to 8-week-old male C57BL/6 mice for a total of 7 days. Give regular drinking water without DSS to control mice.
    1. Switch the DSS-containing water to regular drinking water after day 7.
  3. Weigh mice and assess clinical scores of each mouse every day. Normalize body weight of each mouse to its initial body weight. Scores are defined according to the disease severity by four parameters: rectal prolapse (0-2), stool consistency (0-2), bleeding (0-2), and activity (0-2)5. Sum the scores from these parameters for a final clinical score.
  4. To analyze the histopathological condition of colon tissue, euthanize mice by intraperitoneal (i.p.) injection with 1.2% (vol/vol) Avertin (0.6 mL/10 g body weight) 7 days post-DSS treatment. To prepare a stock of 100% avertin, mix 10 g of 2,2,2-tribromoethanol with 10 ml of tert-amyl alcohol.  Store in the dark at 4°C. To use, dilute 100% stock to 2% in water.
  5. Isolate the colon and cecum, and measure the length of the colon5.
  6. Cut 0.5 cm segments from the distal colon and fix in a 15 mL falcon tube containing 10 mL of 10% formalin overnight. Wash the fixed tissues with graded ethanol (75, 95, and 100%) and xylene. Embed the tissues in paraffin and cut 6 mm sections for the hematoxylin & eosin staining8.

4. Measuring the Epithelial Barrier Permeability in DSS-induced Colitis Mice

  1. Measure the barrier permeability 7 days after the start of DSS administration.
  2. On the day of the assay, fast mice for 3 h.
  3. Autoclave a gavage needle to ensure sterility, then gavage mice with 150 µL 80 mg/mL 4 kDa FITC-dextran in sterile water, and keep the unused FITC-dextran to measure the standard curve after serum collection. Weigh the mice for the permeability calculation.
    NOTE: The FITC-dextran solution should be made in water.
  4. Using a pair of scissors, clip a 1 cm piece of the tail, and collect 100 µL of blood from the tail into serum collection tubes. Spin the collected blood at 10,000 x g for 10 min at room temperature.
  5. Dilute the serum 1:4 in water. To make a standard curve, dilute unused FITC-dextran with water at 1:300, 1:1,000, 1:3,000, 1:10,000, 1:30,000, 1:100,000, 1:300,000, 1:1,000,000, and 1:3,000,000. Add 100 µL/well of the serum and the standard curve samples into 96-well plates.
  6. Read the fluorescence in a plate reader with 485 excitation/528 emission. Calculate the permeability values based on the standard curve, and multiply by 4 to correct for the dilution.
  7. Divide the concentration of FITC-dextran by the weight to normalize the values (this helps normalize the difference in FITC-dextran delivery if mice are sick and have lost weight).

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Representative Results

In culture, Caco-2BBe cells grow as a monolayer and slowly differentiate into mature absorptive enterocytes that have brush borders. In this protocol, Caco-2BBe cells were plated with a high density on polycarbonate membranes, and cells reached 100% confluency one day after seeding. However, cells are undifferentiated at this stage: To fully differentiate the cells, the media is changed every 2-3 days for 3 weeks. Cells were stained with nuclei and F-actin stains to show the differences between undifferentiated and differentiated cells. Stress fibers are clearly seen in undifferentiated cells. Compared with undifferentiated cells, differentiated cells have a smaller volume, larger nuclei, and fewer stress fibers (Figure 1).

TNF is central to intestinal barrier loss through MLCK-dependent tight junction regulation. TER of cells without or with TNF treatment at different time points was analyzed. As shown in Figure 2, TNF significantly decreases the TER of the Caco-2BBe monolayer in a dose-dependent manner, suggesting that TNF increases epithelial permeability.

Administration of DSS induces acute colitis in mice. Mice treated with DSS lose weight significantly compared to their initial body weight (Figure 3A). The severity of colitis is scored by rectal prolapse, stool consistency, bleeding, and activity (Figure 3B). Cross sections of colonic tissues stained with hematoxylin & eosin show colonic mucosal damage in DSS treated mice (Figure 3C). The length of colon crypt is decreased in DSS treated mice (Figure 3D). The colon length is shortened in DSS treated mice (Figure 3E). As seen in Figure 3F, there is approximately a 2-fold increase in FITC-dextran levels in DSS treated mice compared to control mice.

Figure 1
Figure 1: Caco-2BBe cells cultured on polycarbonate membranes. Cells cultured 1 day (undifferentiated) and 3 weeks (fully-differentiated) after plating are stained with Hoechst 33342 (blue) for the nucleus and Alexa Fluor 594 Phalloidin for F-actin (red). Scale bar = 20 µm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: TNF reduces the TER of the Caco-2BBe monolayer. Cells are primed with 10 ng/mL of IFNγ overnight. On the next day, the culture medium is replaced with HBSS containing 2.5 or 7.5 ng/mL of TNF. TER is measured by an epithelial meter (Volt/Ohm) at the indicated time points. Values are mean ± SEM, n = 4. Significant differences are indicated by * (p <0.05) and ** (p <0.01), as compared to TER values of cells without TNF treatment, by two-tailed t-test. Please click here to view a larger version of this figure.

Figure 3
Figure 3: DSS-induced colitis in mice. C57BL/6 male mice are given 3.5% DSS in drinking water for 7 days. Body weight (A) and clinical scores (B) are assessed daily for each group (mean ± SEM, n = 4 for each group). Control mice received water without DSS. (C) Histological analysis of colonic tissue sections collected on day 7 post–DSS treatment. (D) Colon crypt lengths are measured on day 7 post–DSS treatment. (E) Colon lengths are measured on day 7 post–DSS treatment. (F) Permeability of colon epithelium is measured on day 7 post-DSS treatment by FITC-Dextran gavage. Values are mean ± SEM, n = 4. Significant differences are indicated by * (p <0.05 by two-tailed t-test). Please click here to view a larger version of this figure.

Score Rectal prolapse Stool consistency Bleeding Activity
0 None Normal None Active
1 Sign of prolapse Soft Red Decreased activity
2 Extensive prolapse Diarrhea Gross bleeding Lethargic

Table 1. Disease Clinical Score

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Discussion

There are several critical steps in the protocol. Caco-2BBe (brush border-expressing) cells are always used for TER measurement, selected from the Caco-2 cell line for expression of brush-border proteins. Caco-2BBe cells have a villus absorptive phenotype when fully-differentiated (after about 3 weeks of culture post-confluence)17. It is necessary to avoid contamination during the measurement, and to sterilize the electrode. Because the procedure is non-sterile, measurements can only be performed for up to 8 h in most cases. During the measurement, we determined the resistance value of the two points inside and outside the insert. The resistance measurements at various sites can sometimes greatly differ because of differences in the cell state; This is a major limitation of the method. However, this variation can be significantly reduced by statistical methods and other methods. The TER method can be applied to other cells, such as determining the barrier integrity of pulmonary epithelial cells and vascular endothelial cells. In addition, TER measurements can be used in drug toxicity studies.

Administration of indigestible macromolecules, which enter the intestinal epithelium through diffusion, can be used to evaluate the intestinal permeability. The advantage of this method is that the barrier function of the intestine can be examined in vivo. There are several types of markers that can be used, including radioisotopes, polyethylene glycols, and sugars18. However, several factors should be considered for precise measurement of the permeability: (1) the position of gut barrier abnormality cannot always be precisely determined; and (2) the mucosal blood flow and gastrointestinal motility might affect the absorption and the excretion of the macromolecules.

In this study, 4 kDa FITC-dextran was administered intragastrically in mice and the amount of FITC-dextran in the mouse serum was measured. This method has numerous advantages. Oral gavage reduces possible risks that are associated with using enemas. For instance, unintentional impairment to the colonic epithelium may be caused by rectal instillation, leading to a false-positive result. Additional effects caused by stress should also be eliminated. Permeability is known to increase when the mice are stressed. It is recommended to conduct a preliminary experiment at least one week prior to the actual experiment. Performing a practice experiment will help minimize experimental noise for the real experiment. A wild-type control (no treatment) should always be included.

FITC-dextran travels through the small intestine and begins to enter the colon 3 h after gavaging. Thus, 3 h is an ideal time point for measuring small intestinal permeability. For measurement of colonic permeability, the time can be extended, but this then becomes a balance between the leakage of FITC-dextran into the lumen and clearance of these molecules from the blood stream. 4 h is generally accepted as a good time point for colonic measurements.

In conclusion, two separate methods were described to determine intestinal epithelial cell permeability both in vitro and in vivo. Whereas TER measurement and FITC-dextran gavage methods both measure paracellular permeability, they each provide independent and distinct properties of the paracellular permeability.

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Disclosures

The authors declare no competing financial interests.

Acknowledgements

We thank Dr. Jerrold R. Turner, from Brigham and Women's Hospital, Harvard Medical School, for his generous help in completing this study. This work is supported by the National Natural Science Foundation of China (grant number 81470804, 31401229, and 81200620), the Natural Science Foundation of Jiangsu Province (grant number BK20180838, and BK20140319), The Research Innovation Program for College Graduates of Jiangsu Province (grant number KYLX16-0116), Advanced Research Projects of Soochow University (grant number SDY2015_06), and Crohn's & Colitis Foundation Research Fellowship Award (grant number 310801).

Materials

Name Company Catalog Number Comments
22 G gavage needle VWR 20068-608
4 kDa FITC-dextran Sigma 46944
Avertin Sigma T48402
Black 96-well plates for fluorescence Fisher 14-245-197A
C57/B6 mice Nanjing Biomedical Research Institute of Nanjing University
Caco-2BBe cells ATCC CRL-2102
Dextran sulphate sodium MP Biomedicals 2160110
DMED with high glucose and sodium pyruvate Hyclone SH30243.01B
Epithelial (Volt/Ohm) Meter Millicell-ERS MERS00002
Ethanol Sinopharm ChemicalReagent 10009218
Falcon tube (15 mL) Corning 430791
FBS Gibco 10437-028
Fluorescence microscope Olympus FV1000
Fluorometer Biotek Synergy 2
HBSS 138 mM NaCl, 0.3 mM Na2HPO4, 0.4 mM MgSO4, 0.5 mM MgCl2, 5.0 mM KCl, 0.3 mM KH2PO4, 15.0 mM HEPES, 1.3 mM CaCl2, 25 mM glucose 
IFNg PeproTech 315-05-20
Modular Tissue Embedding Center Leica EG1150H
Serum collection tubes Sarstedt 41.1378.005
T75 flask corning 430641
TNF PeproTech 315-01A
Parraffin Sigma A6330-1CS
Polycarbonate membranes (Transwell) Costar 3413
Pressure pump AUTOSCIENCE AP-9925
Rotary Microtomy Leica RM2235
Trypsin-EDTA Gibco 25200-056
Xylene Sinopharm ChemicalReagent 10023418

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References

  1. Turner, J. R. Intestinal mucosal barrier function in health and disease. Nature reviews. Immunology. 9, 799-809 (2009).
  2. Odenwald, M. A., Turner, J. R. The intestinal epithelial barrier: a therapeutic target? Nature reviews. Gastroenterology & hepatology. 14, 9-21 (2017).
  3. Clarke, H., Soler, A. P., Mullin, J. M. Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets. J. Cell Sci. 113, (Pt 18), 3187-3196 (2000).
  4. Clayburgh, D. R., et al. A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability. J.Biol. Chem. 279, 55506-55513 (2004).
  5. Su, L., et al. TNFR2 activates MLCK-dependent tight junction dysregulation to cause apoptosis-mediated barrier loss and experimental colitis. Gastroenterology. 145, 407-415 (2013).
  6. Su, L., et al. Targeted epithelial tight junction dysfunction causes immune activation and contributes to development of experimental colitis. Gastroenterology. 136, 551-563 (2009).
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  9. He, W. Q., et al. Myosin light chain kinase is central to smooth muscle contraction and required for gastrointestinal motility in mice. Gastroenterology. 135, 610-620 (2008).
  10. Li, H. S., et al. Myosin regulatory light chain phosphorylation is associated with leiomyosarcoma development. Biomed. Pharmacother. 92, 810-818 (2017).
  11. Clayburgh, D. R., et al. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115, 2702-2715 (2005).
  12. Wang, F., et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am. J. Pathol. 166, 409-419 (2005).
  13. Ye, D., Ma, T. Y. Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity. J. Cell Mol. Med. 12, 1331-1346 (2008).
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  17. Peterson, M. D., Mooseker, M. S. Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line Caco-2. J. Cell Sci. 102, (Pt 3), 581-600 (1992).
  18. Wang, L., et al. Methods to determine intestinal permeability and bacterial translocation during liver disease. J. Immunol. Methods. 421, 44-53 (2015).

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