Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

Summary

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

Abstract

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

Introduction

The GIT plays a central role in digestion, nutrient absorption, and waste excretion through feces¹. Additionally, it functions as a barrier against pathogens, a role supported by a diverse cellular composition, including stem cells, mucus-producing goblet cells, enteroendocrine cells, and absorptive enterocytes². Intestinal homeostasis can be disrupted by various factors, such as bacterial infections³ or inflammatory processes⁴, leading to severe consequences for the organism, such as malabsorption, diarrhea, or even death⁵. Investigating such pathophysiological scenarios is commonly done using laboratory animals or, in accordance with the 3R principle⁶, cell cultures derived from various species. Accurate prediction and transferability of results are crucial when employing species-specific models⁷. Despite this need, there is a notable lack of pig-derived cell cultures that adequately replicate the complexity and functionality of the intestinal tract.

To address this challenge, also relevant to other species, three-dimensional (3D) organoids have been developed in an attempt to replicate the physiological complexity of the GIT⁸. Initially, organoids were created from human and mice intestines; to date, porcine organoids from juvenile and adult pigs have also been successfully developed and cultivated^9,10. Since their inception, these porcine organoids have been utilized in several studies, primarily focusing on intestinal infections^11,12,13,14. Research aimed at characterizing physiological properties, such as nutrient transport or secretory processes, remains limited¹⁵. This may be due to the orientation of intestinal organoids, with the apical surface facing inward and the basolateral side outward, limiting accessibility to the apical surface. This limitation was addressed by successfully cultivating porcine organoids in a two-dimensional format¹⁶, a method that has been further advanced through the use of frozen tissue to generate them¹⁷.

The 2D cultivation of porcine organoids provides access to both sides of the epithelium, enabling the application of well-established methods to study transport processes across the epithelial layer. One such method is the Ussing chamber¹⁸, which allows real-time observation of electrogenic absorptive and secretory processes across the epithelium. Extensive use of this system has provided a comprehensive understanding of the porcine intestinal function in vivo, covering the entire intestinal axis. This includes studies on monosaccharide transport or transport of short-chain fatty acids or responses to secondary plant metabolites such as resveratrol that influence intestinal transport characteristics^{19,20,21,22,23,24}. The substantial body of data from these studies facilitates direct comparisons between the well-characterized in vivo conditions and the in vitro environment of porcine organoids, enhancing our understanding of their physiological relevance.

In this study, we present a protocol for generating and cultivating 2D monolayers from 3D porcine organoids. Additionally, we detail the methodological approach for quantifying intestinal transport processes using the Ussing chamber technique. The protocol offers tools to study absorptive and secretory characteristics in vitro in jejunum and colon organoids, allowing for direct comparison with well-characterized in vivo conditions. Future applications of this protocol may include investigating the effects of pharmacological or toxicological substances, as well as exploring interactions between the epithelium and pathogens.

Protocol

For this protocol, two healthy pigs (Bentheim Blacked Pied pig; 1 male, 1 female; 4.5 months old; approximately 65 kg) were sacrificed by captive bolt shoot and bleeding. According to the Animal Protection Law, this (slaughter and removal of tissues) is not classified as an animal experiment but has to be announced to the animal welfare officer (registration no. TiHo-T-2023-15) of the University of Veterinary Medicine Hannover Foundation.

1. Coating of inserts

NOTE: All steps are carried out with sterile materials under a safety cabinet. All steps of the protocol are performed on ice if not stated otherwise.

1. Thaw the frozen basement membrane on ice for at least 1 h at room temperature or overnight on the ice at 4 °C. Mix the basement membrane 1:40 (v/v) with ice-cold sterile phosphate-buffered saline (PBS) in a conical tube.
2. Remove the sterile plates with inserts from the packaging. Add 200 µL of basement membrane mix to the apical compartment of each insert.
3. Replace the lid of the well plate and incubate for at least 1.5 h at 37 °C, 5% CO₂ in an incubator.
4. Aspirate the solution carefully before seeding the cells. Ensure that the tip does not touch the membrane during aspiration.

2. Generation of 2D organoid monolayers

NOTE: Porcine colon organoids are generated and cultivated as described for porcine jejunal organoids²⁵. After generation of 3D organoids these should be cultivated for at least 3-4 weeks while weekly passaging to ensure consistent growth. The number of cells within each dome containing 3D organoids, which are dissolved in the subsequent steps, is sufficient to cover a single transmembrane filter. Before monolayer generation, the 3D organoids were subjected to optical quality control to check the previous growth and possible contamination (Figure 1).

Figure 1: Representative 3D organoids. Three-dimensional (A) jejunum and (B) colon organoids are carefully examined under the microscope prior to generating monolayers. Special attention is given to assessing previous growth patterns, structural integrity, and the presence of any impurities or contamination. Please click here to view a larger version of this figure.

1. Remove the organoid medium (Table 1) from the wells with 3D crypt organoids. Add 1 mL of ice-cold PBS per well and gently dissolve the basement membrane by pipetting up and down with a p1000 tip.
2. Collect all dissolved organoids in a 15 mL tube pre-filled with 10 mL of ice-cold PBS. Centrifuge the tube at 250 x g, 10 min at 4 °C. Aspirate the supernatant.
3. Resuspend pellet in 1 mL of warm (37 °C) 0.05 % (v/v) trypsin/EDTA (2 tubes can be pooled at this stage). Incubate for 5 min at 37 °C in a water bath and put directly on ice afterward to stop the reaction.
4. Resuspend on ice 20x with p1000 tip, and another 15x with p1000 tip + p200 tip on top. Add 10 mL of ice-cold DMEM supplemented with 10% (v/v) fetal calf serum (FCS).
5. Centrifuge tube at 1,000 x g, 10 min at 4 °C. Discard supernatant and resuspend pellet in 1 mL of monolayer medium (Table 1).
6. Determine the number of living cells per mL using a Neubauer chamber according to the manufacturer's instructions.
7. Remove the coating solution at this point from the apical compartment and replace it with 500 µL of warm (37 °C) monolayer medium. Add 3 mL of monolayer medium to the basolateral compartment.
8. Determine the blank resistance (TEER) of each empty transwell insert, as described in detail in step 3. The time in which no medium is present in the apical chamber should be as short as possible to prevent the cells from drying out.
9. Remove the apical monolayer medium and add for the jejunal 2D culture 2 x 10⁵ epithelial cells and for the colonic 2D culture 1.5 x 10⁵ epithelial cells in 500 µL of monolayer medium in the apical chamber of each transwell.
10. Change medium and measure TEER every 2-3 days. Measure TEER before the medium is changed. Carefully aspirate the medium of the apical and basal compartment and replace it with 500 µL or 3 mL of fresh, warm (37 °C) medium.
    1. For jejunum organoids: on day 16, after seeding, change from monolayer medium to differentiation medium (Table 1). For colon organoids: change medium on day 7 to differentiation medium.
11. Change the differentiation medium every day and perform TEER measurement during each medium change. On day 18 (for jejunal organoids) or day 9 (for colon organoids), after seeding, proceed with functional experiments.

3. Measurement of transepithelial electrical resistance (TEER)

NOTE: All measurements are performed under a safety cabinet to avoid contamination. Before seeding the cells, every empty coated transwell is measured to get individual blank values. The volt-ohm meter stores values on an introduced USB device.

1. Clean the chopstick electrode with 70% (v/v) ethanol and allow the electrode to dry completely. Meanwhile, remove the plate from the incubator and place it under the cabinet.
2. Introduce the short arm of the chopstick electrode to the apical compartment of the inserts and the long arm to the basolateral compartment of the insert. Avoid the contact of the short arm with the cell layer.
3. Measure the transepithelial electrical resistance of each transwell. Allow the volt-ohm meter to equilibrate to ensure a stable measurement. Measure TEER values by clicking the Store button.
4. Continue steps 3.2 and 3.3 with the remaining wells. After reaching the last well, the volt-ohm meter opens a request to store the data on a USB device. Click Save.
5. Clean the electrode after each plate and at the end of the measurement and allow it to dry completely before storage.
6. Subtract blank values determined before seeding the cells from the measured cellular values.

4. Electrophysiological transport studies using the Ussing chamber technique

NOTE: Determination of electrophysiological transport studies is performed by using a Ussing chamber consisting of two chamber compartments, which are divided by the epithelium. This chamber is connected to a voltage clamp by Ag/AgCl Electrodes. This technique allows the tracking of active electrogenic transport processes of the epithelium through the changes in the short-circuit current (I_sc) induced by the voltage clamp as well as the tissue resistance (R_t) calculated by Ohm´s law. I_sc and R_t are recorded every 6 s during the whole experiment. During the experiment the investigated tissue is aerated with carbogen and incubated with modified Krebs-Henseleit solutions to ensure viable conditions. Indomethacin (10 µM) is added to buffer solutions to inhibit prostaglandin synthesis²⁶.

1. Warm-up mucosal and serosal buffer solutions to 37 °C and aerate with carbogen. Assemble the single chambers using an empty insert for each individual chamber. Make sure the apical sides of the transwells are all facing in the same direction.
2. Fill all chambers with 5 mL of pre-warmed mucosal buffer solution (Table 2). Connect all electrodes from the voltage clamp to the individual chambers according to the manufacturer's instructions. Make sure no gas bubbles are interfering with the electrodes to ensure correct measurement.
3. Calibrate the Ussing chamber software with these conditions by clicking the Rf/dpI button in the voltage-clamp software. Resistance of all used empty inserts should be equal (~ 70 Ohm), and the current should be around 0 mV (± 5 mV). If this is not the case for some chambers, check for the proper placement of electrodes or bubbles in the system.
4. Remove all electrodes and discard the used buffer solution. Open all individual chambers and remove the empty inserts. Make sure that the order of the individual chambers is maintained.
5. Move the well plate with inserts from the incubator to the safety cabinet. Carefully aspirate the basolateral and apical medium.
6. Wash the cells by adding 500 µL of warm (37 °C) mucosal buffer to the apical chamber and 3 mL of serosal buffer to the basolateral chamber. Aspirate the buffers and repeat 2x.
7. Remove the inserts from the plate. Gently remove the supports of the inserts. Place the inserts into the Ussing chambers, and make sure the orientation of the inserts is the same as during the calibration phase. Assemble the individual chambers as displayed in Figure 2.
8. Fill the chambers facing the basolateral side of the cells with 5 mL of serosal buffer (Table 2) and the chamber facing the apical chamber with 5 mL of mucosal buffer.
9. Connect the electrodes and the aeration tubes to each individual chamber. Start measurement in the Ussing software. Allow equilibration for 15 min.
10. Change the conditions from open circuit to short-circuit conditions. Equilibrate for 5 min to 10 min. Add 10 µM of forskolin to the serosal chamber.
    NOTE: The addition of forskolin may be replaced by other agents/substances depending on individual intestinal segments and research questions.
11. After 15 min, either add more agents in the same manner as forskolin or stop the measurement. Remove aeration tubes and electrodes from individual chambers. Pour the buffer solutions of both chambers into a bowl.
12. Disassemble the chambers and either discard the inserts or use them for follow-up analysis (e.g., immunohistochemistry or expression analysis)

Figure 2: Schematic structure of the Ussing chamber. Displayed are both chambers divided by the membrane with the grown 2D monolayer. Both chambers are aerated with carbogen; voltage and current are monitored by two electrodes per chamber. Please click here to view a larger version of this figure.

5. Analysis of data generated by the Ussing chamber setup

1. Import the data generated by the voltage clamp software into a suitable spreadsheet editor.
2. To determine basal I_sc and R_t values, calculate the mean of 10 consecutive data points taken 10 min after initializing the short-circuit conditions. Each individual chamber serves as a technical replicate, while every passage of organoids serves as a biological replicate.
3. To assess the effects of applied agents, calculate the mean of 10 consecutive data points immediately before the agent's addition. Compare this mean to the maximum (or minimum) value observed after the agent is applied. Data obtained is presented as mean ± standard deviation (SD).

Results

This protocol facilitates the reliable generation of porcine 2D monolayers by disaggregating 3D organoids derived from the jejunum and colon of pigs. Over a cultivation period of 16 days for jejunum organoids and 9 days for colon organoids, intact monolayers are formed. These monolayers can subsequently be used to assess electrogenic and physiological transport properties using the Ussing chamber technique.

After disintegrating the 3D organoids, single cells are seeded onto coated inserts. Cel...

Discussion

This protocol describes a method for converting established porcine 3D organoids into single cells, which are then seeded onto transwell membranes to form an intact monolayer. This configuration grants access to the apical side of the cells, facilitating the use of Ussing chambers to monitor absorptive and secretory processes.

The initial and crucial step in this multi-step process is the precise disintegration of the 3D organoids. Achieving uniform seeding of the single cells is essential for...

Materials

Name	Company	Catalog Number	Comments
24 well plate	SARSTEDT AG & Co. KG	8,33,922
A83-01	MedChemExpress, New Jersey, USA	HY-10432	Store at -20 °C. Thaw when needed
accujet S	Brand GmbH + Co KG, Wertheim, Germany	26351
Advanced DMEM/F12 Medium	Thermo Fisher Scientific, Waltham, USA	12634010	Store at 4 °C
B27 supplement	Thermo Fisher Scientific, Waltham, USA	17504044	Store at -20 °C. Thaw when needed
CaCl2.2 H2O	Merck KGaA, Darmstadt, Germany	C3306	Store at room temperature
D(+)-Glucose (wasserfrei)	Merck KGaA, Darmstadt, Germany	1.08337	Store at room temperature
DAPT	MedChemExpress, New Jersey, USA	HY-13027	Store at -20 °C. Thaw when needed
D-Mannitol	Merck KGaA, Darmstadt, Germany	M4125	Store at room temperature
DMSO	Sigma-Aldrich, Schnelldorf, Germany	154938	Store at room temperature
Electrode-Set (AgCl/PtIr/Std.)	Scientific Instruments, Simmerath, Germany	#1316
Eppendorf Research plus	Eppendorf SE, Hamburg, Gemany	3123000063
Eppendorf Research plus	Eppendorf SE, Hamburg, Gemany	3123000047
EVOM3 Manual Epithelial Volt Ohm Meter	World precision instruments, Sarasota, USA	EVM-MT-03-01
FBS	Sigma-Aldrich, Schnelldorf, Germany	F7524	Store at -20 °C. Thaw when needed
Forskolin	Sigma-Aldrich, Schnelldorf, Germany	F6886	Store at -20 °C. Thaw when needed
gasprofi 1 SCS micro	WLD-TEC GmbH, Arsenhausen, Germany	60,04,000
Gastrin 1	MedChemExpress, New Jersey, USA	HY-P1097	Store at -20 °C. Thaw when needed
Glutamax	Thermo Fisher Scientific, Waltham, USA	35050061	Store at 4 °C.
HCl	Sigma-Aldrich, Schnelldorf, Germany	1090571000	Store at room temperature
HEPES	Sigma-Aldrich, Schnelldorf, Germany	H0887	Store at 4 °C
Herasafe 2025 Class II Biological Safety Cabinet	Thermo Fisher Scientific, Waltham, USA	51033316
Incubator ICO105	Memmert GmbH + Co.KG, Schwabach, Germany	62,20,143
Indomethacin	Merck KGaA, Darmstadt, Germany	I7378	Store at room temperature
KCl	Merck KGaA, Darmstadt, Germany	1.04936	Store at room temperature
L-Glutamin	Sigma-Aldrich, Schnelldorf, Germany	G7513	Store at -20 °C. Thaw when needed
LWRN Supernatant	selfmade		Store at -20 °C. Thaw when needed. LWRN supplement is produced according to Miyoshi et al. (2012)
Matrigel Basement Membrane Matrix, LDEV-free, 10 mL	Corning Incorporated - Life Sciences	354234	Store at -20 °C. Thaw carefully on ice when needed
Megafuge 1.OR	Heraeus Instruments, Osterode, Germany	75003060
MgCl2.6 H2O	Merck KGaA, Darmstadt, Germany	1.05833	Store at room temperature
Na2HPO4.2H2O	Merck KGaA, Darmstadt, Germany	1.06580	Store at room temperature
N-Acetyl-L-cysteine	Sigma-Aldrich, Schnelldorf, Germany	A7250	Store at -20 °C. Thaw when needed
NaCl	Merck KGaA, Darmstadt, Germany	1.06404	Store at room temperature
NaH2PO4.H2O	Merck KGaA, Darmstadt, Germany	1.06346	Store at room temperature
NaHCO3	Merck KGaA, Darmstadt, Germany	1.06329	Store at room temperature
Neubauer improved chamber	Glaswarenfabrik Karl Hecht, Sondheim vor der Rhön, Germany	40442712
Olympus IX70 iverted Microscope	Olympus Corporation, Hamburg, Germany
Pen/Strep	Thermo Fisher Scientific, Waltham, USA	15140122	Store at -20 °C. Thaw when needed
PolymyxinB	Sigma-Aldrich, Schnelldorf, Germany	P4932-1MU	Store at -20 °C. Thaw when needed
Primovert microscope stand with binocular phototube	Zeiss	415510-1101-000
rm EGF	Prepotech, New Jersey, USA	315-09	Store at -20 °C. Thaw when needed
SB202190	MedChemExpress, New Jersey, USA	HY-10295	Store at -20 °C. Thaw when needed
Snapwell 3801	Corning Incorporated - Life Sciences	3801
Trypsin/EDTA	Thermo Fisher Scientific, Waltham, USA	25300054
Ussing Base System	Scientific Instruments, Simmerath, Germany	#1317
Ussing Diffusion Chamber	Scientific Instruments, Simmerath, Germany	SKU 1307
Voltage/Current Clamp VCC6	Scientific Instruments, Simmerath, Germany	SKU 1310
Y27632	MedChemExpress, New Jersey, USA	HY-10583	Store at -20 °C. Thaw when needed