Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

Summary

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

Abstract

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

Introduction

The intestinal epithelium is equipped with various transport proteins (channels, ATPases, co-transporters and exchangers) that perform numerous functions ranging from the absorption of nutrients, electrolytes, and drugs to the secretion of fluid and ions in the lumen. Transport proteins generate electrochemical gradients that permit the movement of ions or molecules in a vectorial manner. This is achieved by the asymmetric distributions of the transport systems in the apical and basolateral membranes of polarized epithelial cells. In addition, tight junctions, which tether adjacent epithelial cells, play an important role in this process by serving as a barrier to the intramembrane diffusion of components between the apical and basolateral membrane domains. Appropriate model systems mimicking these characteristic features of the native intestine (i.e. polarity, differentiation, and tight junction integrity) are critical for the study of the functionality of epithelial transport systems.

With respect to models, the typical cell lines used currently in intestinal epithelial transport research are Caco-2, a model of fully differentiated, absorptive small intestinal epithelial cells; and T84 cells or HT-29 subclones, models of crypt-derived large intestinal epithelial cells¹. Conventionally, these cell lines are grown as monolayers in plastic surfaces or in coated transwell inserts. Trans-well cell culture inserts to some extent resemble the in vivo environment by allowing polarized cells to feed basolaterally. However, a limitation in conventional 2D culture systems is that the cells are forced to adapt to an artificial, flat, and rigid surface. Thus, the physiological complexity of the native epithelia is not accurately reflected in a 2D system. This limitation has been overcome by methods to grow cells in 3D in a specific microenvironment, such as gelatinous protein mixture, containing a variety of extracellular matrix components^2,3. Nevertheless, the in vitro cultures cannot simulate the complexity of the intestinal epithelium, which has multiple cell types and region-specific architecture in the intestine. Thus, the studies using cell culture models require further validation in the native intestine. Using the in vitro 3D cell culture and mouse intestinal mucosa, we describe here simple methods to study the regulation of the intestinal serotonin transporter (SLC6A4, SERT).

The SERT transporter regulates the extracellular availability of an important hormone and neurotransmitter, 5-hydroxytryptamine (5-HT), by rapidly transporting it through a Na⁺/Cl^--dependent process. SERT is a known target of anti-depressants and has recently emerged as a novel therapeutic target of GI disorders, such as diarrhea and intestinal inflammation. Methods to investigate 5-HT uptake in intestinal epithelial cells have been previously described. For example, Caco-2 cells grown on plastic supports or permeable inserts have been shown to exhibit fluoxetine-sensitive ³H-5-HT uptake at both apical and basolateral domains⁴. The measurement of SERT function in isolated Brush Border Membrane Vesicles (BBMVs) prepared from human organ donor small intestines has also been described by us⁵. ³H-5-HT uptake in human BBVMs was shown to be fluoxetine-sensitive and Na⁺/Cl^--dependent, and it exhibited saturation kinetics with a K_m of 300 nM⁵. Utilizing similar methods, we also previously measured SERT function as ³H-5-HT uptake in mouse intestinal BBMVs⁶. However, the preparation of pure plasma membrane vesicles requires a large amount of tissue mucosa. Other methods, such as the radiographic visualization of ³H-5-HT uptake sites, have also been shown previously in guinea pig and rat small intestines⁷.

The Ussing chamber provides a more physiological system to measure the transport of ions, nutrients, and drugs across various epithelial tissues. The main advantage of the Ussing chamber technique is that it enables the precise measurement of the electrical and transport parameters of intact, polarized intestinal epithelium. Further, to minimize the influence of the intrinsic neuromuscular system, seromuscular stripping of intestinal mucosa can be performed to investigate the regulation of transporters in the epithelium⁸.

We demonstrate that Caco-2 cells grown in 3D on gelatinous protein form a hollow lumen expressing distinct apical and basolateral markers. These cells show higher expression of SERT than 2D Caco-2 cells. Methods to grow 3D cells and to perform immunostaining or RNA and protein extraction are described. In addition, we describe methods to study SERT function and regulation by TGF-β1, a pleiotropic cytokine, in small intestinal mucosa by utilizing an Ussing chamber technique.

Protocol

1. Study of Intestinal Transporters in a 3D Culture System of Caco-2: Growing 3D Caco-2 Cell Culture on a Gelatinous Protein Mixture

1. Thaw growth factor reduced gelatinous protein mixture on ice for 8 h (or O/N) at 4 °C. Once thawed, make 1 mL or 500 µL aliquots for use or store at -20 °C for later use.
2. On the day of culture, precool the culture plates (for RNA and protein extraction) or chambered slides (for immunostaining) on ice. Add 30 µL of gelatinous protein mixture to each well of an eight-well chambered-glass slide (or 120 µL per well of a 6-well plate) and spread evenly. Take care not to generate air bubbles during the process.
3. Place the slides/plates inside a cell culture incubator at 37 °C for 15 - 30 min and allow the gelatinous protein mixture to solidify.
4. While the gelatinous protein mixture is solidifying, trypsinize a confluent flask of Caco-2 cells.
5. Count the number of cells and spin them at 500 x g in a tabletop centrifuge for 5 min. Re-suspend the cell pellet in 3D Caco-2 Medium as needed.
6. Seed the glass chamber slides at 4,000 cells per well (for plates, 20,000 per well). Allow the cells to grow in a 5% CO₂ humidified incubator at 37 °C. Change medium every 3 - 4 d.

2. Immunofluorescence Staining for Epithelial Transporters in 3D Caco-2 Cells: Day 1

1. Aspirate the medium and fix the cells with 400 µL of 2% paraformaldehyde (PFA) (in PBS with the pH adjusted to 8.5 with NaOH) for 30 min at RT.
   NOTE: PFA solution should not be stored for more than 1 week at 4 °C, and freshly made solution can be used. CAUTION: PFA is highly toxic and a potential carcinogen. Always handle it with caution in a chemical hood and using personal protective equipment.
2. Rinse the wells twice with 1x PBS and store the slides at 4 °C in PBS (400 µL/well). Once fixed, store them at 4 °C for up to one week.
3. Warm the chamber slides to RT (this will harden the gelatinous protein mixture bed and make it easy to aspirate during the washing steps).
4. Permeabilize the cells with 0.5% Triton in PBS for no longer than 15 min.
5. Prepare PBS-glycine buffer (130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, and 100 mM Glycine). Rinse 3x with 1x PBS-glycine for 10 min each at RT.
6. Prepare immunofluorescence buffer (IF): 130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 100 mM Glycine, 7.7 mM NaN₃, 0.1% BSA, 0.2% TritonX-100, and 0.05% Tween-20.
7. Wash 3x in IF buffer for 10 min each at RT. Block in 5% Normal Goat Serum (NGS) in IF buffer. Incubate with primary antibody diluted in IF + 1% goat serum, 200 µL/well, for 1 - 2 h at RT. Wash with IF buffer 3x for 10 min each.
8. Incubate with secondary antibody (1:200) diluted in IF + 1% NGS, 200 µL/well, for 1 h at RT.
9. Wash with IF buffer three times for 10 min each. Keep the slides in the dark from this step on. Lift the plastic chambers from the glass slides by placing the chamber slide in a black holder and sliding a white lifter through the holder until its edge contacts the edge of the wells. Gently pull up the chambers (the holder and lifter should come with the culture slides).
10. Mount with slow anti-fade medium and allow it to dry for 10 min at RT.
11. Once dried completely, seal the slides with nail polish and image them with confocal microscopy. Store the slides at 4 °C for two weeks or at -20 °C for several months.

3. RNA and Protein Extraction from 3D Caco-2 Cells

1. Treat the cells cultured on gelatinous protein mixture for 12 - 14 d with a test agent, such as TGF- β1 (10 ng/mL, 1 h).
2. After treatment, wash the cells with 1x PBS. Keep the cells on ice for 30 min to liquefy the gelatinous protein mixture.
3. Wash the wells with chilled PBS and collect the cells in individual centrifuge tubes. Collect the cells using low-speed centrifugation at 500 x g and 4 °C for 10 min. Extract the RNA using standard procedures and use real time RT-PCR.
4. For the protein extraction, lyse the cells using 1x cell lysis buffer supplemented with 1x protease cocktail inhibitor. After adding cell lysis buffer to the pellet, lyse the cells by sonication and remove the cell debris by centrifugation at 7,500 x g and 4 °C for 10 min.

4. Measurement of 5-HT Uptake in Mouse Ileum Utilizing an Ussing Chamber: Intestinal Preparation and Seromuscular Stripping

1. Following euthanasia, dissect the mice and remove the small intestines (after the stomach and before the cecum). Divide the small intestine into three parts. Discard the middle third, use the proximal third as jejunum, and use the distal third as ileum. Avoid pulling the intestine from its mesenteric attachment to prevent damage to the epithelium. Keep the tissue section cold by covering it with ice-cold Krebs-Ringer bicarbonate (KBR) buffer.
2. Open the intestine longitudinally using scissors. Incubate freshly excised intestinal sections in ice-cold, gassing KBR containing 1 µM indomethacin for 10 min before the seromuscular stripping.
3. Pin the intestinal section (~1 cm in length) mucosal-side down to a plate containing 7% agarose or cured silicone elastomer (0.5 cm thick).
4. Strip the seromuscular layers under a dissection stereomicroscope with bottom illumination. Cut the seromuscular layer using a feather scalpel blade. Use fine forceps to obtain the edge of the layer along the longitudinal axis of the intestine.
   NOTE: During the stripping procedure, the bottom lighting of the stereomicroscope helps to identify and avoid areas with Peyer's patches.
5. After the completion of the seromuscular stripping, mount the mucosa carefully on the pins of an Ussing half-chamber. During the whole process, take care to hold the stripped mucosal tissue by the edges to avoid tearing it.

5. Equilibration and Tissue Treatment

1. Prepare Kreb's solution: 115 mM NaCl, 25 mM NaHCO₃, 2.4 mM K₂HPO₄, 1.2 mM CaCl₂, 1.2 mM MgCl₂, and 0.4 mM KH₂PO₄ at pH 7.4, gassed with 95% O₂/5% CO₂ at 37 °C. Add 10 mM glucose to the serosal bath to serve as an energy substrate and 10 mM mannitol to the mucosal bath to maintain the osmotic balance.
   NOTE: Kreb's solution is supplemented with L-ascorbic acid (100 µM) and pargyline (100 µM, monoamine oxidase inhibitor) to prevent intracellular 5-HT degradation.
2. Insert the slider into the chamber to expose both the apical (luminal) side and the basolateral (serosal) side of the tissue to Kreb's solution.
3. After an equilibration period of 10 min, pretreat the ileal tissue with fluoxetine (10 µM) for 30 min on the apical side for J_m-s (mucosal-to-serosal flux).
4. Treat the tissue with TGF-β1 (10 ng/mL) for 1 h on basolateral side and then incubate it with ³[H]-5-HT (20 nM) for 30 min on the apical side.

6. Quantification of Mucosal to Serosal Flux (J_m-s) and ³[H]-5-HT Accumulation in the Tissue

1. Collect 0.75 mL aliquots from the serosal reservoir (total of 5 mL) to measure the radioactivity in a liquid scintillation counter and to calculate mucosal-to-serosal (J_m-s) flux rates; fluoxetine-sensitive flux represents SERT-mediated ³[H]-5-HT uptake.
2. To avoid hydrostatic pressure differences across the mucosa during a flux period, take samples from the serosal side and replace them with identical volumes of bath medium.
   NOTE: The flux calculation corrects for the dilution of the end sample (4.25 mL / 5 mL = 0.85).
3. For the quantification of ³[H]-5-HT accumulated in the tissue, dismount the mucosa from the slider, wash it once with ice-cold KRB buffer, and place it in glass culture tubes.
4. Incubate the mucosa in 0.5 mL of 10% KOH overnight at 37 °C. Measure the radioactivity in 150 µL aliquots of the lysates (in triplicates) using a liquid scintillation counter.
5. Use aliquots (3 - 5 µL) of the lysates to measure protein concentration using the Bradford method.
   NOTE: 10 mL of incubation medium containing radioactive serotonin is used as a standard. 5-HT retained in the tissue is expressed as pmol/mg protein/min.

Results

Immunostaining in 3D Caco-2 cysts is shown in Figure 1. A representative image of an XY-plane showing well-demarcated lumen in the 3D cyst stained with phalloidin actin is depicted in Figure 1A. The side facing the lumen denotes the apical side. Epithelial cells cultured in a 3D environment result in a robust epithelial phenotype. Different Caco-2 cysts on day 12, when actin and SERT were co-stained, are shown in Figure 1B. In this repres...

Discussion

Fully differentiated Caco-2 cell monolayers have been used extensively as polarized epithelial monolayers to study intestinal transport^{10,11,12,13,14,15}. However, to mimic the physiological organization of intestinal epithelial cells, there has been considerable interest in the development of a Caco-2 culture in 3D. A number ...

Materials

Name	Company	Catalog Number	Comments
10X PBS	ATCC	ATCC® HTB­37™	Cells
10X PBS	Carl Zeiss		Microsope for confocal Imaging
3[H]-5-HT	LabtekII	152453	Culturing cells for microscopy
5-HT	Gibco	70013-032	Not a hazardous substance
95%O2- 5%Co2 cylinder	KPL	71-00-27	Not a hazardous substance
Agar (for Agar plates)	Fischer	BP151-100	Acute toxicity, Ora
Agar (for electrode)	Sigma	G7126-1KG	Not a hazardous substance
Alexa FluorPhalloidin	Sigma	C3867-1VL	Not a hazardous substance
BSA	Sigma	P6148-500G	Harmful if swallowed or if inhaled
CaCl2	LifeTechnologies	A12380	Not a hazardous substance
CaCo2 Cells	Sigma	A8806-5G	Not a hazardous substance
Cell lysis Buffer	Fischer	BP337-100	Not a hazardous substance
Chabered slides	Sigma	S5886-1KG	Not a hazardous substance
Collagen Type-1Solution	Sigma	S8045-500G
Electrodes	Sigma	S-0876
EMEM	Gibco by Life Technologies	25200-056
Fetal bovine serum	Corning	356234	Used as Extracellular matrix
Fluoxetine	Qiagen	74104
Gentamicin	ATCC	30-2003	Cell culture Media
Glycin	Cell Signaling, Danvers, MA
Hepes	Roche	11836145001
Indomethacin	Gibco by Life Technologies	15070-063
K2HPO4	Gibco by Life Technologies	15710-064
KOH	Gibco by Life Technologies	15630-080
L-ascorbic acid	Gibco by Life Technologies	10082147
Liquid scintillation counter	Gibco by Life Technologies	70013--032
LSM710 Meta	Physiologic Instruments, San Diego, CA	EM-CSYS-8
Mannitol	Physiologic Instruments, San Diego, CA	P2304
Matrigel	Physiologic Instruments, San Diego, CA	VCC MC8
MgCl2	Physiologic Instruments, San Diego, CA	DM MC6
Multichannel Voltage/current Clamp	Physiologic Instruments, San Diego, CA	P2300
NaCl	Physiologic Instruments, San Diego, CA	P2023-100
NaCl	Fisher Scientific, Hampton, NH	BP1423
NaHCO3	Sigma-Aldrich, St Louis, MO	S5886
Normal Goat Serum (10%)	Fisher Scientific, Hampton, NH	S233
Paraformaldehyde	Sigma-Aldrich, St Louis, MO	P288
Pen-Strp	Sigma-Aldrich, St Louis, MO	C3881
Protein assay reagent	Sigma-Aldrich, St Louis, MO	10420
Proteinase Inhibitor Cocktail	MEDOX, Chicago, IL	style G- 12300
RNA easy Mini kit	Sigma-Aldrich, St Louis, MO	M4125
Single Channel Electrode Input Module	Sigma-Aldrich, St Louis, MO	A92902
Sliders for snapwell Chambers	Sigma-Aldrich, St Louis, MO	H9523
Sodium Phosphate (Na2HPO4)	Sigma-Aldrich, St Louis, MO	F132
Sodium-Hydroxide (NaOH)	Sigma-Aldrich, St Louis, MO	T7039
TGF-β1	Perkin-Elmer,Waltham, MA	NFT1167250UC
TritonX-100	Packard Instruments, Downers Grove, IL	B1600
Trypsin EDTA	Sigma-Aldrich, St Louis, MO	221473
Tween-20	Bio Rad Laboratories, Hercules, CA	5000006
Ussing Chamber (chamber only)	Sigma-Aldrich, St Louis, MO	I7378
Ussing Chamber Systems	Sigma-Aldrich, St Louis, MO	A1296