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Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

Published: July 29, 2021 doi: 10.3791/62074
Wies T. M. van Dooremalen1, Merel Derksen1, Jamie Lee Roos1, Celia Higuera Barón1, Carla S. Verissimo1, Robert G. J. Vries1, Sylvia F. Boj1, Farzin Pourfarzad1


Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.


In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport, antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.


The intestinal epithelium acts as a physical barrier between the luminal content of the intestines and the underlying tissue. This barrier comprises a single epithelial layer of mainly absorptive enterocytes that are connected by tight junctions, which establish strong intercellular connections between adjacent cells. These cells form a polarized epithelial lining that separates the apical (lumen) and basolateral sides of the intestine, while simultaneously regulating paracellular transport of digested nutrients and metabolites. In addition to enterocytes, other important epithelial cells such as goblet, Paneth, and enteroendocrine cells also contribute to intestinal homeostasis by producing mucus, antimicrobial peptides, and hormones, respectively. The intestinal epithelium is constantly replenished by dividing leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) stem cells in the bottom of intestinal crypts producing transit-amplifying (TA) cells that migrate upwards and differentiate into other cell types1. Disruption of intestinal epithelial homeostasis by genetic and environmental factors, such as exposure to food allergens, medicinal compounds, and microbial pathogens, leads to disruption of intestinal barrier function. These conditions cause several intestinal diseases including inflammatory bowel disease (IBD), celiac disease, and drug-induced GI toxicity2.

Studies on the intestinal epithelium are performed using several in vitro platform systems such as membrane inserts, organs-on-a-chip systems, Ussing chambers, and intestinal rings.These platforms are suitable for establishing polarized epithelial monolayers with access to both apical and basolateral sides of the membrane, using transformed cell lines or primary tissue as models. Although transformed cell lines, such as the colorectal (adeno)carcinoma cell lines Caco-2, T84, and HT-29, are able to differentiate into polarized intestinal enterocytes or mucus-producing cells to some extent, they are not representative of the in vivo epithelium as several cell types are missing, and various receptors and transporters are aberrantly expressed3. In addition, as cell lines are derived from a single donor, they do not represent interpatient heterogeneity and suffer from reduced complexity and physiological relevance. Although primary tissues used in Ussing chambers and as intestinal rings are more representative of the in vivo situation, their limited availability, short-term viability, and lack of expandability make them unsuitable as a medium for high-throughput (HT) studies.

Organoids are in vitro epithelial cultures established from different organs such as the intestine, kidney, liver, pancreas, and lung. They are proven to have long-term, stable expandability as well as genetic and phenotypic stability and therefore are representative biological miniatures of the epithelium of the original organ with faithful responses to external stimuli4,5,6,7,8,9. Organoids are efficiently established from either resected or biopsied normal, diseased, inflamed, or cancerous tissue, representing heterogeneous patient-specific responses10,11,12,13,14,15,16. This paper demonstrates how to establish intestinal epithelial monolayers derived from organoid cultures. Monolayers have been successfully established from small intestinal as well as colonic and rectal organoid cultures. This model creates an opportunity to study the transport and permeability of the epithelial cells to drugs as well as their toxicological effects on the epithelium. Moreover, the model allows co-culture with immune cells and bacteria to study their interactions with the intestinal epithelium17,18,19. Furthermore, this model can be used to study responses to therapies in a patient-specific manner and initiate screening efforts to look for the next wave of epithelial barrier-focused therapeutics. Such an approach could be extended to the clinic and pave the way toward personalized treatments.

Although the epithelial monolayers in this protocol are prepared from human normal intestinal organoids, the protocol can be applied and optimized for other organoid models. Epithelial organoid monolayers are cultured in intestinal organoid expansion medium containing Wnt to support stem cell proliferation and represent intestinal crypt cellular composition. Intestinal organoids can be enriched to have different intestinal epithelial fates, such as enterocytes, Paneth, goblet, and enteroendocrine cells, by modulating Wnt, Notch, and epidermal growth factor (EGF) pathways. Here, after the establishment of monolayers in expansion medium, they are driven toward more differentiated intestinal epithelial cells, as described previously20,21,22,23,24,25. For screening purposes, depending on the mode of action of the compound of interest, its target cells, and the experimental conditions, the monolayers can be driven toward the cellular composition of choice to measure the effects of the compound with relevant functional readouts.

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1. Preparing reagents for culture

NOTE: Perform all steps inside a biosafety cabinet and follow standard guidelines for working with cell cultures. Ultraviolet light is used for 10 min before starting up the biosafety cabinet. Before and after use, the surface of the biosafety cabinet is cleaned with a tissue paper drenched in 70% ethanol. To facilitate the formation of three-dimensional drops of extracellular matrix (ECM), keep a prewarmed stock of 96-, 24-, and 6-well plates ready in the incubator at 37 °C.

  1. Basal medium preparation
    1. Prepare basal medium (BM) in a 500 mL Advanced Dulbecco's Modified Eagle Medium with Ham's Nutrient Mixture F-12 (Ad-DF) medium bottle by adding 5 mL of 200 mM glutamine, 5 mL of 1 M 4-(2-hydroxyethil)-1piperazineethanesulfonic acid (HEPES), and 5 mL of penicillin/streptomycin (pen/strep) solutions (10,000 U/mL or 10,000 µg/mL). This can be stored in the refrigerator at 4 °C for at least 4 weeks.
  2. Wnt sources
    1. Prepare Wnt3a-conditioned medium (Wnt3aCM) according to the previously described method26.
      NOTE: Recently, a next-generation surrogate Wnt (NGS-Wnt), which also supports expansion of human intestinal organoids, has been generated27.
  3. Intestinal organoid base medium preparation
    NOTE: Use all growth factors and reagents according to the manufacturer's recommendations. Use small aliquots and avoid freeze-thaw cycles; functional growth factors are essential for successful organoid culture.
    1. Prepare concentrated 2x intestinal organoid base medium (2x IBM) by supplementing BM with 1 µM A83-01, 2.5 mM N-acetylcysteine, 2x B27 supplement, 100 ng/mL human epidermal growth factor (hEGF), 10 nM gastrin, 200 ng/mL hNoggin, and 100 µg/mL of an antimicrobial formulation for primary cells (see the Table of Materials).
    2. Aliquot the 2x IBM and freeze at -20 °C for up to 4 months. When needed, thaw an aliquot overnight at 4 °C or for several hours at room temperature (RT).
    3. To prepare intestinal organoid expansion medium (IEM), supplement 2x IBM with either 50% Wnt3aCM or 50% BM and 0.5 nM NGS-Wnt, 250 ng/mL human Rspondin-3 (hRspo3), 10 mM nicotinamide, and 10 µM SB202190.
  4. Intestinal organoid differentiation medium preparation
    1. Prepare enterocyte differentiation medium (eDM) by supplementing 2x IBM with 50% BM, 250 ng/mL hRspo3, and 1.5 µM Wnt pathway inhibitor (IWP-2). Store eDM at 4 °C for up to 10 days.
    2. Prepare combination differentiation medium (cDM) by supplementing 2x IBM with either 40% BM and 10% Wnt3aCM or 50% BM and 0.1 nM NGS-Wnt, 250 ng/mL hRspo3, 10 µM DAPT and 100 nM PD0325901. Store cDM at 4 °C for up to 10 days.
  5. Manipulation of extracellular matrix (ECM)
    NOTE: Prepare the extracellular matrix (ECM) (see the Table of Materials) according to the manufacturer's recommendation.
    1. Thaw ECM overnight on ice; transfer the ECM from the bottle to a 15 mL conical tube using a 5 mL pipette, both pre-cooled at -20 °C. Refreeze aliquots only once at -20 °C. Once thawed, store the ECM in a refrigerator at 4 °C for up to 7 days. Incubate for at least 30 min on ice before use.
      ​NOTE: Mix ECM properly and ensure that it is cold before embedding crypts or organoids.

2. Organoid cultures

  1. Establishing cultures from frozen organoids
    NOTE: Let BM reach RT, and keep a 12 mL aliquot, warmed to 37 °C, ready before starting the procedure of thawing one cryovial containing frozen organoids.
    1. Thaw the organoid cryovial rapidly by agitating in a 37 °C water bath until only a sliver of ice remains. Immediately add 500 µL of warm BM dropwise to the cryovial, and pipet up and down a few times to dilute the freezing medium and mix the contents carefully.
    2. Using a P1000 pipette, transfer the organoids to a 15 mL conical tube, and add another 1 mL of warm BM dropwise while gently mixing the bottom of the tube. Pipet up and down a few times to dilute the freezing medium and mix the contents carefully.
    3. Add up to 12 mL of warm BM dropwise to the 15 mL conical tube containing the organoids, and pipet up and down with a 10 mL sterile pipette to gently resuspend the organoids.
    4. Centrifuge the organoid suspension for 5 min at 85 × g and 8 °C. Discard the supernatant carefully without disturbing the pellet, and resuspend the organoids in 30% v/v of IEM supplemented with 10 µM Y27632 or other rho-associated coiled-coil-forming protein serine/threonine kinase inhibitor (ROCK inhibitor). Place the tube on ice.
    5. Add 70% v/v of ECM in the 15 mL conical tube containing the organoids. Mix the organoid suspension keeping the 15 mL conical tube on ice, and seed 5 µL of the suspension to check the density (Figure 1A). Continue plating if the density is appropriate; if the density is too high, add more IEM/ECM solution in the same ratio of 30-70% v/v, respectively.
    6. In each well of a prewarmed 24-well plate, seed 50 µL of the organoid suspension by pipetting 5 separate drops of 10 µL (Figure 1B). Turn the plate upside down, and leave it in the biosafety cabinet for 5 min. Transfer the plate still upside down to the 37 °C incubator, and leave it for another 30 min.
    7. Add 500 µL of IEM with 10 µM ROCK inhibitor to each well, and transfer the plate to the incubator. Image one drop regularly to monitor the growth, and refresh IEM every 2-3 days by aspirating the old medium and adding 500 µL of fresh IEM.
    8. Passage the organoids once they have recovered properly from thawing and have reached the right size to be processed (Figure 1C), as described in section 2.2.
  2. Passaging of intestinal organoids
    ​NOTE: Chill the ECM on ice for at least 30 min, and keep the IEM at RT for at least 1 h before use.
    1. Use the medium from one culture well to break up the organoid domes using a 1250 µL low-retention filter-tip, and transfer the well contents to a labeled 15 mL conical tube. Wash the well with 1 mL of BM, and transfer it to the same 15 mL conical tube.
    2. Repeat steps 2.2.1 and 2.2.2 with all other wells (a maximum of half a plate or 600 µL of ECM drops can be washed and added to one 15 mL conical tube).
    3. Add BM to fill the tube up to 12 mL, and pipet up and down 10x using a 10 mL pipette. Centrifuge at 85 × g for 5 min at 8 °C.
    4. Before removing the BM, check under the microscope to see whether all organoids are pelleted at the bottom of the 15 mL conical tube (Figure 1D). If there is no ECM overlaying the organoid pellet or the ECM layer is either clean or contains just debris, single cells, or very few organoids compared with the pelleted organoids, aspirate the supernatant and pipet out the ECM overlaying the organoid pellet very carefully using a P200 pipette.
      NOTE: Organoids can get trapped in the ECM and do not sediment as a compact pellet due to low centrifugation force. If the ECM contains organoids, centrifuge the tube again at 450 × g for 5 min at 8 °C, and carefully remove the supernatant as described in 2.2.4. If there are multiple 15 mL conical tubes, they can be pooled after step 2.2.4.
    5. Add 1 mL of BM to each pellet (of volume of 50-200 µL, depending on the organoid culture and density), and resuspend carefully. Pipet the organoids up and down at least 5x to shear them, avoiding foam formation. Check under the microscope to see whether the organoids are disrupted (Figure 2A). If the organoids are disrupted, proceed to step 2.2.7; if the organoids are not disrupted, pipet them another 5x. This time, touch the wall of the plastic tube with the pipette tip to exert more mechanical force to disrupt the organoids.
      ​NOTE: Mechanical shearing of cystic (Figure 1C) and budding (Figure 1E) organoids is possible with either a 200 µL or 10 µL plastic pipette tip fitted on a low-retention 1250 µL filter-tip (Figure 1F), depending on the volume required for disrupting the organoids. The use of a narrowed glass pipette (Figure 1F) is recommended when more than 200 µL of ECM containing the organoids are processed (one well of a 6-well plate or 4 wells of a 24-well plate).
    6. Check under the microscope again to see whether the organoids are disrupted. If disrupted, proceed with the next step; if not, pipet the organoids up to 20x, checking the organoids under the microscope regularly. If the organoids are still not disrupted, add 25% v/v cell dissociation reagent 1 (see the Table of Materials) to the suspension, incubate in the water bath at 37 °C for 2 min, and pipet the organoids up to 20x, checking the organoids under the microscope regularly to make sure they are not digested to single cells.
    7. Add up to 12 mL of BM to the 15 mL conical tube, and wash the organoid pellet by pipetting up and down. Centrifuge at 85 × g for 5 min at 8 °C. Discard the supernatant, and adjust the final concentration to 70% v/v ECM by adding IEM and ECM to the organoid pellet.
    8. Start resuspending the organoid pellet with double the volume of IEM/ECM collected for passaging, and seed 5 µL of the suspension to check the density. Continue plating if the density is appropriate (Figure 2B); add more IEM/ECM solution if the density is too high. Add 200 µL of the suspension to each well of a prewarmed 6-well plate, making separate drops of 10 µL volume.
    9. Turn the plate upside down, and leave it in the biosafety cabinet for 5 min. Transfer the plate still upside down to the 37 °C incubator, leaving it for another 30 min. Add 2 mL of IEM with 10 µM ROCK inhibitor to each well, and transfer the plate to the incubator.
    10. Image one drop regularly to monitor the growth, and refresh IEM every 2-3 days by aspirating the old medium and adding 2 mL of fresh IEM.
  3. Passaging of intestinal organoids for epithelial monolayer preparation
    1. Passage organoids 3 days prior to harvesting for monolayer preparation by following the same passaging protocol described in section 2.2 with one exception. In step 2.2.7, resuspend the organoids in 1-1.5x the starting volume of IEM/ECM to have a higher density and expansion potential when they are harvested for monolayer preparation (Figure 3A).

3. Epithelial monolayer preparation

  1. Culture epithelial monolayers on both 24-well and 96-well membrane inserts with a variety of available plate types (Table 1). Use high-throughput system (HTS) membrane inserts for both sizes as these contain an integral tray with the membrane inserts and a receiver plate. For the 24-well format, the use of plates with separate removable membrane inserts is also possible.
    NOTE: Different membrane types (polyethylene terephthalate (PET) or polycarbonate) and pore sizes (0.4-8.0 µm) are available and can be used depending on experimental needs. Monolayers can only be imaged by brightfield when inserts with PET membranes are used. Light-tight membranes block fluorescent light leakage from the apical to the basolateral compartment and can be considered when dynamic transport or permeability of fluorescently labeled substrates is studied. The current protocol uses 24-well membrane inserts; adaptations for 96-well membrane inserts are described in section 5. Depending on the density, morphology, and size of the organoids (Figure 3A) , 6 wells of a 6-well plate (as seeded in section 2.3) are enough for seeding a full 24-well plate of membrane inserts.
  2. Coating membrane inserts with ECM
    NOTE: If there are doubts about having enough cells, coat the inserts after counting the cells. This is to prevent unnecessary coating and loss of the expensive membrane inserts.
    1. Place the membrane inserts into the support plate in the biosafety cabinet. Dilute the ECM 40x in ice-cold Dulbecco's phosphate-buffered saline (DPBS) with Ca2+ and Mg2+, and pipet 150 µL of the diluted ECM into the apical compartment of each insert. Incubate the plate at 37 °C for at least 1 h.
  3. Preparation of cells for seeding
    1. Prewarm aliquots of the cell dissociation reagent 2 in the water bath (37 °C). Prepare 2 mL of the reagent for each well of a 6-well plate.
    2. Transfer the culture plate containing the organoids (prepared in section 2.3) from the incubator to the biosafety cabinet. Process the organoids, as described in steps 2.2.1.-2.2.4. Do not pool multiple tubes into one tube.
    3. Fill the tube, containing organoids from a maximum of 3 wells of a 6-well plate, up to 12 mL with DPBS (without Ca2+ and Mg2+), and pipet up and down 10x using a 10 mL pipette. Centrifuge at 85 × g for 5 min at 8 °C, and aspirate the supernatant without disturbing the organoid pellet.
    4. Add 2 mL of the prewarmed cell dissociation reagent 2 per well of a 6-well plate used as the starting material and resuspend. Incubate the tubes diagonally or horizontally for 5 min in the water bath at 37 °C, to prevent the sinking of the organoids to the bottom of the tube.
    5. Pipet up and down 10x using a 5 mL sterile plastic pipette or a P1000 pipette, depending on the total volume of the cell dissociation reagent. Check the organoid suspension under the microscope to see if a mixture of single cells and some cell clumps consisting of 2-4 cells has formed (Figure 3B). If needed, continue the digestion by repeating steps 3.3.4-3.3.5 (do not increase volume of cell dissociation reagent) until the mixture looks similar to Figure 3B.
      ​NOTE: Avoid digesting the organoids fully to single cells. It is necessary to have some small groups of cells (i.e., groups of 2-4 cells).
    6. Stop cell dissociation by adding up to 12 mL of BM including 10 µM ROCK inhibitor to the cell suspension. Centrifuge at 450 × g for 5 min at 8 °C, and aspirate the supernatant without disturbing the cell pellet. When handling the same organoid culture in several 15 mL conical tubes, pool the cell pellets and resuspend them in 12 mL of BM.
    7. Filter the cell suspension through a 40 µm strainer prewetted with BM, and harvest the flow-through into a 50 mL conical tube. Wash the strainer with 10 mL of BM, and harvest the flow-through into the same 50 mL conical tube.
    8. Transfer the strained cell suspension into two new 15 mL conical tubes. Centrifuge at 450 × g for 5 min at 8 °C, and aspirate the supernatant without disturbing the cell pellet. Resuspend the cells in 4 mL of IEM supplemented with 10 µM ROCK inhibitor per full culture plate used as starting material.
    9. Mix a small amount of cell suspension in a 1:1 ratio with trypan blue for counting. Count the live, not blue, cells (Figure 3C), and calculate the total number of live cells. In small clumps, count each individual cell.
    10. Prepare a cell suspension containing 3 × 106 live cells per mL of IEM supplemented with 10 µM ROCK inhibitor.
  4. Seeding cells on polyester membrane inserts
    1. Carefully aspirate DPBS from the ECM-coated inserts (step 3.2.1), whilst keeping the plate horizontally. Pipet 800 µL of IEM supplemented with ROCK inhibitor into each basolateral compartment. Pipet 150 µL of the cell suspension prepared in step 3.3.10 onto the ECM-coated membrane in the apical compartment dropwise. Per plate, be sure to have at least one "blank" well with BM only.
    2. Once the cells have sedimented onto the membrane, measure transepithelial electrical resistance (TEER), as described in section 4.1, and image the membrane inserts using a microscope. Place the plate in the incubator at 37 °C and 5% CO2. Measure TEER every day, and acquire images regularly to monitor monolayer formation (Figure 4A-D).
  5. Refreshing monolayers
    ​NOTE: Refresh the medium every 2-3 days, adhering to the following order to maintain a positive hydrostatic pressure above the cells and prevent cells from being pushed off the membrane. While refreshing the medium, make sure the monolayer, which is visible upon aspiration of the medium, is not damaged by the pipette tip.
    1. Remove the medium from the basolateral compartments of the plate containing the membrane inserts. Then, carefully aspirate the medium from the apical compartments of the membrane inserts.
    2. Add 150 µL of fresh IEM dropwise to each apical compartment, and then add 800 µL of fresh IEM to each basolateral compartment.
  6. Enrichment of the monolayer for desired intestinal epithelial cell types
    1. Allow the monolayer to become confluent in IEM, corresponding to a TEER value of around 100 Ω·cm² (as calculated in step Check under the microscope to determine whether the monolayers have completely formed (Figure 4D) and for the absence of holes (as seen in Figure 4B,C).
    2. Carefully remove IEM from the basolateral and apical compartments of the membrane inserts, and replace with either eDM or cDM as prepared in section 1.4. Culture the monolayer for another 3-4 days in the specific differentiation medium to get the organoid cells enriched with the desired specific cell type. Refresh the medium every 2-3 days, as described in section 3.4.
    3. Measure TEER daily, and acquire images regularly if desired (Figure 5A-C).
      NOTE: The TEER value that indicates a fully organized enriched monolayer varies per organoid culture; typically TEER values increase to 600 and can increase up to 1000 Ω·cm2 (as calculated in step after 3 days in differentiation media and are stable for 3-5 days.

4. Epithelial monolayer assay readouts

  1. Measurement of transepithelial electrical resistance (TEER)
    ​NOTE: TEER measurements are widely accepted as a method to analyze tight junction dynamics and barrier function integrity in biological models of physiological barriers, such as epithelial monolayers28, 29. Increase in TEER after differentiation because of increased cellular interaction at tight junctions can be measured using a manual TEER meter or an automated TEER measurement robot.
    1. Measurement of TEER using a manual TEER meter
      1. Clean the electrode with 70% ethanol, and let it air-dry inside the biosafety cabinet. Place the electrode in a tube containing BM. Connect the electrode to the manual TEER meter. Turn the Function switch to measure in Ohms (Ω). Turn the Power switch on.
      2. Place the short electrode in the apical compartment of the insert, while the long electrode is positioned in the basolateral compartment (Figure 6A). Avoid touching the monolayer.
      3. Measure resistance in the blank well (Rblank), and then measure the remaining samples (Rsample) in the same way. Wash the electrode with BM between samples with different conditions. Clean the electrode first with demi water and then with 70% ethanol and let it air-dry.
      4. Calculate TEER (Ω·cm2): [Rsample (Ω) - Rblank (Ω)] × membrane area (cm2) (Table 1 and Figure 6B).
    2. Measurement of TEER using an automated TEER measurement robot (Table of Materials)
      1. Perform automated TEER measurements when using HTS systems for 96-well and 24-well HTS plates containing membrane inserts. Use different electrodes for TEER measurement for both types (24- and 96- HTS membrane inserts). To measure TEER using an automated TEER measurement robot, follow the manufacturer's instructions.
  2. Measurement of epithelial barrier integrity and permeability
    NOTE: This protocol introduces Lucifer Yellow permeability from the apical to basolateral compartment as an indication of monolayer integrity. This section describes fluorescence measurement in the basolateral compartment after a 1 h incubation step to evaluate monolayer permeability and thus, barrier integrity. This measurement is an end-point assay and is especially useful when testing compounds for their effect on barrier integrity.
    1. Thaw Lucifer Yellow on ice, and let BM equilibrate to RT. For one 24-well plate of membrane inserts, prepare 5 mL of working solution of 60 µM Lucifer Yellow in BM.
      ​NOTE: Lucifer Yellow is light-sensitive. Prepare dilutions in dark 1.5 mL sterile tubes and perform all steps with the biosafety cabinet light switched off.
    2. Carefully remove the medium from the basolateral and apical compartments of the membrane inserts, as described in step 3.5.1. If desired, scratch one untreated monolayer using a pipette tip as a positive control for Lucifer Yellow leakage through a damaged barrier.
    3. Add 150 µL of BM with 60 µM Lucifer Yellow to each apical compartment, and add 800 µL of BM without Lucifer Yellow to each basolateral compartment. Place the plate on a shaker at 37 °C, 50 rpm for 60 min.
    4. In the meantime, prepare a standard curve of Lucifer Yellow in BM starting with the working solution prepared in step 4.2.1. Dilute 1:3 in each step until a concentration of 3 nM is reached. Include a negative control (BM only).
    5. Transfer 100 µL of each standard in triplicate to a 96-well transparent plate. After 60 min incubation, remove the membrane inserts and transfer 100 µL from each basolateral well (step 4.2.3) in triplicate to the 96-well transparent plate. Measure fluorescence of the plate using a plate reader at an excitation wavelength of 430 nm and an emission wavelength of 530 nm.
    6. After correcting for the negative control value (BM only), use the standard curve values to calculate the Lucifer Yellow concentration in the basolateral compartment (final receiver concentration (µM)).
    7. Calculate the apparent permeability coefficient (Papp) according to the following formula (Figure 6C):
      Equation 1
    8. For a 24-well plate containing membrane inserts, use the following formula:
      Equation 2
  3. Fixing monolayers and preparing paraffin blocks for histology
    NOTE: Epithelial monolayers can be used for histologic staining for evaluation of their cellular composition, polarity, and expression of different proteins of interest such as junctional proteins, proliferation, or differentiation markers. This section describes paraffin block preparation for histologic staining.
    1. Carefully remove the medium from the basolateral and apical compartments of the membrane inserts, as described in steps 3.5.1.
    2. Wash the monolayers by adding 150 µL of DPBS (without Ca2+ and Mg2+) to each apical compartment and 800 µL to each basolateral compartment. Carefully aspirate the DPBS again, first from the basolateral compartment and then from the apical compartment.
      ​NOTE: The basolateral compartment will stay empty from this step on.
    3. In a fume hood, add 150 µL of 4% paraformaldehyde to each apical compartment, and incubate for 30 min at RT.
      ​NOTE: From this step onwards, perform all actions in this section inside a fume hood, as paraformaldehyde is toxic.
    4. Carefully aspirate the fixative from the apical compartments of the membrane inserts, and dispose of it as liquid halogen waste.
      NOTE: From this step onwards, dispose of all liquid waste as liquid halogen waste.
    5. Wash the monolayers by adding 200 µL of DPBS (without Ca2+ and Mg2+) to each apical compartment, and carefully aspirate the DPBS again. Repeat this step one more time.
    6. Add 200 µL of 25% ethyl alcohol (EtOH) to each apical compartment, and incubate for 15 min at RT. After 15 min, carefully aspirate the 25% EtOH from the apical compartments of the membrane inserts. Repeat with 50% EtOH solution and subsequently with 70% EtOH solution.
    7. Add 200 µL of 70% EtOH to each apical compartment, and wrap the plate with parafilm. Store it at 4 °C until further use.
    8. Carefully aspirate the 70% EtOH, and use a scalpel to carefully cut the monolayer membranes from the inserts. Cut from the basolateral side, around the edge of the insert.
    9. Prepare paraffin blocks following the standard procedure.
      1. When the paraffin is still warm, take the monolayer from the paraffin with tweezers, and place it on a precooled surface.
      2. Be careful not to damage the monolayer. Cut the monolayer in half using a single edge blade.
      3. When the paraffin in the bottom of the cassette starts to solidify, use heated tweezers to place the two monolayer parts in the paraffin, next to each other with the straight side down and in a vertical direction to ensure that the monolayer will be vertical in the coupe.
    10. When paraffin blocks are ready, cut the blocks using a microtome, and make slides of 4 µm thick sections following standard procedure. Make sure that the monolayers end up vertical in the coupe.
    11. Perform histologic stains as described previously7,9. Use hematoxylin and eosin (H&E), Ki67, mucin-2 (MUC2), and Alcian Blue to show general morphology, proliferative cells, mucus production and goblet cells, respectively (Figure 6E).
      NOTE: Additional differentiation markers, such as lysozyme for Paneth cells, can be used as well. This marker is not presented in Figure 6E as Paneth cells are present in small intestinal epithelium rather than colon epithelium.
  4. Secreted protein measurement in medium supernatant
    1. Measure lysozyme levels in the apical supernatant of ileal monolayers (see Figure 6D) using the kit listed in the Table of Materials. If desired, measure levels of different cytokines and other proteins of interest.
  5. Gene expression analysis
    1. Quantify the effects of the differentiation media on the expression of epithelial cell marker genes using quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
      1. Lyse the monolayers in 350 µL of RNA lysis buffer followed by RNA isolation according to the manufacturer's instructions. Perform cDNA synthesis and qPCRs, as described earlier7,9, using the cDNA synthesis kits, master mix, and oligonucleotides listed in the Table of Materials.

5. Upscaling to 96-well plates containing membrane inserts

NOTE: Prepare epithelial monolayers for higher throughput drug screenings or multiple medium conditions using HTS 96-well plates containing membrane inserts.

  1. Adaptations when preparing monolayers in 96-well format
    1. Follow all steps described in this protocol for 24-well plates containing membrane inserts, changing volumes and cell numbers to those described in Table 1. For preparing monolayers on 96-well plates with membrane inserts, proceed as described in section 3 with the following differences.
      1. Approximately 9 wells of a 6-well culture plate with organoid density represented in Figure 3A are needed to seed a full 96-well plate with membrane inserts. In step 3.2.1, precoat the membranes with 67 µL of 40x diluted ECM in DPBS (with Ca2+ and Mg2+).
      2. In section 3.5, first transfer the integral plate of membrane inserts to another 96-well plate to allow medium refreshment of both apical and basolateral compartments.

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Representative Results

Figure 1A shows a representative brightfield image of intestinal organoids after thawing them from a cryovial. It is important to thaw organoids at a high density to ensure optimal recovery. Organoids are plated in 24- or 6-well plates in ECM domes of approximately 10 µL (Figure 1B). Most normal intestinal organoids have a cystic morphology. After recovering from the thawing process, the organoids grow to a bigger size and are ready to be passaged after 3-7 days depending on the organoid culture (Figure 1C). After harvesting the organoids and washing the ECM away (Figure 1D), organoids can be disrupted to a small size by mechanical shearing. Depending on the morphology of the organoids (cystic Figure 1C, budding Figure 1E), organoids can be disrupted using plastic or glass pipettes (Figure 1F). Organoids are disrupted in a suspension (Figure 2A), which should be regularly monitored under the microscope. It is important to avoid making them too small, as groups of cells need to stay together to ensure organoid growth. Figure 2B shows the organoids in ECM drops just after passaging. In general, cystic organoids are plated at a relatively high density while budding organoids are plated in a low density; however, this can differ between different organoid cultures.

When passaging organoids for the preparation of monolayers, be sure to plate them at a high density, and let them grow for three days so they are in optimal expansion conditions. Organoids can be harvested for monolayer preparation when they are comparable with Figure 3A in size and density, where 6 wells of a 6-well plate, each containing 200 µL of organoid domes, are typically enough for seeding a full 24-well plate of membrane inserts. After the preparation of a single-cell suspension with the cell dissociation reagent, single cells and small clumps of cells should be visible (Figure 3B), and live cells can be counted (Figure 3C). The arrows indicate dead cells stained with trypan blue, which should be excluded from counting. The single cells and small clumps are then seeded in the membrane inserts as seen in Figure 4A. Monolayer formation is visible after 1-3 days (Figure 4B,C), and the monolayers will be generally be confluent after 3-6 days depending on the organoid culture (Figure 4D). Monolayers stay in expansion medium until they are confluent, after which they can be enriched with, amongst others, enterocytes or goblet cells using different enrichment media. Figure 5A shows a monolayer that was cultured for 8 days in expansion medium (IEM). When enriched with enterocytes (eDM), a structure is seen, as in Figure 5B, while monolayers exposed to combination medium (cDM) show a smoother structure (Figure 5C).

Monolayer formation can be quantitatively followed by measuring TEER (Figure 6A). A completely confluent monolayer has a TEER value of ~100 Ω·cm2, which increases to ~1000 Ω·cm2 when exposed to either differentiation medium (Figure 6B). Monolayers in all medium conditions show a low apparent permeability (Papp) to Lucifer Yellow (0.45 kDa). Lysozyme secretion by ileal monolayers cultured in IEM was higher than that of monolayers cultured in IEM until confluent and for another 4 days in eDM or cDM (denoted as + subsequent eDM or cDM) (Figure 6D). Monolayers cultured in IEM, IEM + subsequent eDM or IEM + subsequent cDM show different morphology, as can be observed with H&E staining (Figure 6E). While colon organoid-derived epithelial monolayers in IEM and cDM media have a smooth apical surface, enterocyte-differentiated monolayers present an invaginated apical morphology in the absence of Wnt. Ki67-positive proliferative cells can be detected in expansion conditions only. Alcian Blue and MUC2 stain mucus produced by goblet cells, which is visualized in the monolayers differentiated in eDM and more prominently in cDM when Wnt, Notch, and EGF signaling are inhibited, respectively (Figure 6E). Upon differentiation, proliferative cells decrease while goblet cell and enterocyte marker gene expression increases in comparison to that observed under IEM conditions, as shown by LGR5, MUC2, and ALPI gene expression quantification by qRT-PCR, respectively (Figure 6F).

Figure 1
Figure 1: Establishing an intestinal organoid culture from frozen organoids. (A) Representative brightfield image of an intestinal organoid culture after thawing. (B) ECM domes (50 µL) seeded in each well of a 24-well culture plate. (C) Representative image of a normal intestinal organoid culture ready for passaging. (D) Representative image on how to check the presence of ECM in a 15-mL tube containing organoids under a light microscope. (E) Representative image of a budding intestinal organoid culture ready for passaging. (F) A 10 µL plastic pipette tip fitted on a low-retention 1250 µL filter tip (left) and a narrowed glass pipette (right) for mechanical shearing of organoids. Scale bars = 100 µm. Abbreviation: ECM = extracellular matrix. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Processing intestinal organoids for maintenance or preparation of monolayers. (A) Representative image of intestinal organoids after mechanical disruption. (B) Representative image of an intestinal organoid culture seeded after passaging. Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Preparing single cells from intestinal organoids for monolayer preparation. (A) Intestinal organoids ready to harvest for monolayer preparation. (B) Single cells and small clumps of cells after single cell preparation. (C) Visible single cells and small clumps during counting in a grid chamber. Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Monolayer formation after seeding single cells on membranes. (A) Single cells just after seeding on membranes. On average, (B) the monolayer is around 50% confluent 1-3 days after seeding, (C) ~90% confluent at day 3-5, and (D) the complete monolayer has formed around day 4-7. Scale bars = 100 µm. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Enrichment of specific cell types in the monolayer. (A) Monolayer after 8 days in IEM. (B) Monolayer enriched with enterocytes after 4 days in IEM and another 4 days in eDM. (C) Monolayer enriched with goblet cells and other cell types after 4 days in IEM and another 4 days in cDM. Scale bars = 100 µm. Abbreviations: IEM = intestinal organoid expansion medium; eDM = enterocyte differentiation medium; cDM = combination differentiation medium. Please click here to view a larger version of this figure.

Figure 6
Figure 6: A variety of possible readouts using epithelial organoid monolayers. (A) Electrode in the membrane insert to measure TEER. (B) TEER values increase in time with a value of ~100 Ω·cm2 when the monolayer reaches confluence. After enriching monolayers with enterocytes or a combination of different epithelial cells, TEER increases to 1000 Ω·cm2 or higher. (C) Monolayers in all medium conditions (IEM + 4 days IEM/eDM/cDM) are impermeable to Lucifer Yellow. (D) Expression of lysozyme is higher in ileum monolayers when grown in expansion medium than in either type of differentiation medium (IEM + 4 days IEM/eDM/cDM). (E) Colon monolayers show different morphologies when exposed to different medium conditions (IEM + 4 days IEM/eDM/cDM) as visualized by H&E, Ki67, Alcian Blue, and MUC2 stains. As expected, monolayers cultured in expansion medium are very proliferative, as shown by Ki67 staining. Monolayers differentiated with eDM show a columnar epithelium without proliferative cells. Monolayers exposed to cDM are also not proliferative and develop more goblet cells. Scale bar = 100 µm. (F) Stem cell (LGR5), goblet cell (MUC2), and enterocyte (ALPI) marker gene expression in colon monolayers by qRT-PCR. Abbreviations: TEER = transepithelial electrical resistance; IEM = intestinal organoid expansion medium; eDM = enterocyte differentiation medium; cDM = combination differentiation medium; Papp = apparent permeability coefficient; LGR5 = leucine-rich repeat-containing G-protein-coupled receptor 5; H&E = hematoxylin and eosin; AB = Alcian Blue; MUC2 = mucin-2; ALPI = intestinal alkaline phosphatase; qRT-PCR = quantitative reverse-transcription polymerase chain reaction. Please click here to view a larger version of this figure.

Membrane inserts
24-well plates 96-well plates
Membrane surface (cm2) 0.33 0.143
Apical volume (µL) 150 100
Basolateral volume (µL) 800 300
# Cells to seed per well 450,000 200,000
HTS plates with membrane inserts PET PET
Plates with separate inserts PET NA
Light-tight plates with membrane inserts
Electrodes are different for the two formats Refer to the Table of Materials

Table 1

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This protocol describes the general manipulation and maintenance of intestinal organoids as well as the preparation and possible applications of epithelial monolayers derived from these organoids. To date, monolayers have been successfully prepared from the duodenum, ileum, and different regions of colon organoids derived from normal as well as previously and actively inflamed intestinal tissue (unpublished data). The application of patient-derived organoid monolayers facilitates the study of barrier function in a disease- and patient-specific manner as well as the study of patient-specific responses to a variety of drug treatments. Although cell lines can form differentiated and polarized monolayers containing intestinal enterocytes and goblet-like cells, many different enzymes and transporters are aberrantly expressed in these cell lines, resulting in reduced complexity and physiological relevance3,30. Organoid culture is technically more demanding and laborious than cell culture; however, organoids are more representative of the in vivo situation, whereas cell lines have repeatedly failed to represent tissue responses in complex setups.

Although primary tissue-based models can be representative of the in vivo situation, they require the use of test animals or access to human material, which is associated with limited availability and ethical constraints. Additionally, primary tissue has no or limited expandability and is not stable in extended experimental timeframes. Organoid technology requires limited amount of primary tissue to establish genetically and physiologically stable long-term expanding cultures while still representing epithelial tissue complexity and patient heterogeneity. Different cell lines, such as Caco-2, are often used to prepare epithelial monolayers. Caco-2 cells take 21 days to establish a polarized epithelial monolayer that can be used for any experiment30. Organoid-derived epithelial monolayers are prepared from organoid single cells as described in the current protocol, and after 3-6 days, form a polarized epithelium that can be further differentiated to enrich them with enterocytes or goblet cells.

Monolayers are a static model lacking a microfluidic flow or mechanic stretch as is seen in organs-on-a-chip. They do, however, offer the opportunity for co-culturing with (autologous) immune cells, as well as bacteria or parasites17,18,19, 31. Organoids are suitable for monolayer preparation when they are in their expansion phase; for intestinal organoids, this is usually 3 days after passaging. Dissociation of organoids to single cells should be performed quickly to avoid long-term exposure to digestive enzymes. To increase the survival of stem cells after the preparation of a single-cell suspension, the Rho-associated protein kinase inhibitor (ROCK inhibitor), Y27632, is added to the cells to prevent anoikis-induced cell death32. Furthermore, it is critical to culture the monolayers on a membrane precoated with ECM to ensure that they maintain their organoid characteristics and polarization. For functional screening assays that quantify GI tract epithelial cell responses to external stimuli, it is important that organoid cultures represent the required cellular complexity depending on the type of assay to be developed and eventual readouts.

Intestinal organoids represent different epithelial cell types present in vivo, such as stem, TA, enterocyte, Paneth, goblet, and enteroendocrine cells4,5, and are prepared and maintained using a defined organoid medium prepared as described in this protocol. Enterocyte differentiation can be promoted by culturing organoids for an additional four days using culture conditions that dually inhibit the Wnt pathway, by removal of Wnt molecules/activators, and the addition of the Porcupine inhibitor, IWP-2 (enterocyte colon differentiation medium, eDM). As mucus-producing goblet cells are essential for barrier function homeostasis as well, a second culture condition aims to produce a more heterogeneous cell population containing stem, enterocyte, and goblet cells, in which Notch and EGF pathways are inhibited while Wnt signaling is kept partially active by reducing Wnt3aCM to 10% (or 0.1 nM for NGS-Wnt) instead of 50% (0.5 nM NGS-Wnt) used in IEM. In contrast to eDM, which enriches for enterocyte differentiation, this second condition supports the presence of several cell types and is therefore called combination differentiation medium (cDM).

Applications of organoid-derived monolayers include the monitoring of epithelial barrier integrity as well as examining tight junction and transporter expression. Barrier integrity, permeability, and transport can be analyzed using the readouts introduced in this protocol. While TEER measures ionic conductance through tight junctions, the permeability assay measures water flow and thus paracellular permeability through the tight junctions28,29. Epithelial barrier integrity, permeability, and transport functionality of the monolayers can be evaluated by measuring the traffic of different fluorescent or radioactive substrates across apical and basolateral compartments of the membrane inserts. TEER measurements allow for the quantification of barrier integrity, showing an increase in values when differentiating towards polarized enterocytes and goblet cells, and a decrease after inducing injury to the monolayers.

The Lucifer Yellow permeability assay can be used for initial barrier integrity assessment as well as the confirmation of reduced integrity after inducing injury to the monolayers. This protocol introduces Lucifer Yellow permeability from the apical to basolateral compartment as an indication of monolayer integrity. Similarly, other fluorescently labeled reagents, such as 4 or 40 kDa dextran, can be employed to evaluate increased paracellular permeability as the result of barrier damage-inducing agents. Fluorescently labeled substrates, such as Rhodamine 123, can be used for measuring the activity of different transporters, such as P-glycoprotein-1. The fluorescence assay described in this protocol allows the measurement of the levels of proteins, such as lysozyme, that are secreted into the apical compartment. Responses to injury induction by pro-inflammatory cytokines can be measured with these readouts, as well as the potential effects of barrier-restoring compounds.

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The authors declare no conflict of interest.


This work is supported by the Topsector Life Sciences & Health - Topconsortium voor Kennis en Innovatie Health~Holland (LSH-TKI) public-private partnerships (PPP) allowance of the Dutch LSH sector with Project number LSHM16021 Organoids as novel tool for toxicology modelling to Hubrecht Organoid Technology (HUB) and HUB internal funding to Disease Modeling and Toxicology department. We thank the laboratories of Sabine Middendorp (Division of Pediatric Gastroenterology, Wilhelmina Children's Hospital, UMC, Utrecht) and Hugo R. de Jonge and Marcel J.C. Bijvelds (Department of Gastroenterology and Hepatology, Erasmus MC, Rotterdam) for providing initial technical support to set up monolayers on membrane inserts.


Name Company Catalog Number Comments
100% ethanol Fisher Emergo 10644795
1250, 300, and 20 µL low-retention filter-tips Greiner bio-one 732-1432 / 732-1434 / 732-2383
15 mL conical tubes Greiner bio-one 188271
24-well cell culture plates Greiner bio-one 662160
24-well HTS Fluoroblok Transwell plate (light-tight) Corning 351156
24-well HTS Transwell plates (Table 1) Corning 3378
24-well plate with Transwell inserts Corning 3470
40 µm cell strainer PluriSelect 43-50040-01
50 mL conical tubes Greiner bio-one 227261
6-well cell culture plates Greiner bio-one 657160
96-well black plate transparent bottom Greiner bio-one 655090
96-well fast thermal cycling plates Life Technologies Europe BV 4346907
96-well HTS Fluoroblok Transwell plate Corning 351162
96-well HTS Transwell plates (Table 1) Corning 7369
96-well transparent culture plate Greiner bio-one 655180
A83-01 Bio-Techne Ltd 2939
Accutase Cell Dissociation Reagent Life Technologies Europe BV A11105-01 Cell dissociation reagent 2
Advanced DMEM/F-12 Life Technologies Europe BV 12634028
B27 supplement Life Technologies Europe BV 17504001
Cell culture microscope (light / optical microscope) Leica
CellTiter-Glo Promega G9683
Centrifuge Eppendorf
CO2 incubator PHCBI
DAPT Sigma-Aldrich D5942
DEPC treated H2O Life Technologies Europe BV 750024
Dulbecco's phosphate-buffered saline (DPBS) with Ca2+ and Mg2+ Life Technologies Europe BV 14040091
DPBS, powder, no calcium, no magnesium Life Technologies Europe BV 21600069
EnzChek Lysozyme Assay Kit Life Technologies Europe BV E22013
EVOM2 meter with STX electrode WTI
Gastrin Bio-Techne Ltd 3006
Glass pipettes Volac
GlutaMAX Life Technologies Europe BV 35050038
hEGF Peprotech AF-100-15
HEPES Life Technologies Europe BV 15630056
Human Noggin Peprotech 120-10C
Human Rspo3 Bio-Techne Ltd 3500-RS/CF
IWP-2 Miltenyi Biotec 130-105-335
Ki67 primary antibody Sanbio BSH-7302-100
Ki67 secondary antibody Agilent K400111-2
Kova International Glasstic Slide with Counting grids Fisher Emergo 10298483
Laminar flow hood Thermo scientific
Lucifer Yellow CH dilithium salt Sigma-Aldrich L0259
Matrigel, Growth Factor Reduced (GFR) Corning 356231 extracellular matrix (ECM)
MicroAmp Fast 8-Tube Strip, 0.1 mL Life Technologies Europe BV 4358293
MicroAmp Optical 8-Cap Strips Life Technologies Europe BV 4323032
Microcentrifuge tubes Eppendorf 0030 120 086
Micropipettes (1000, 200, and 20 µL) Gilson
Microtome Leica
MUC2 primary antibody Santa Cruz Biotechnology sc-15334
MUC2 secondary antibody VWR VWRKS/DPVR-HRP
Multichannel pipette (200 µL) Gilson
N-acetylcysteine Sigma-Aldrich A9165
NGS Wnt U-Protein Express N001-0.5mg
Nicotinamide Sigma-Aldrich N0636
Oligonucleotide ALPI1/Forward Custom-made GGAGTTATCCTGCTCCCCAC
Oligonucleotide ALPI1/Reverse Custom-made CTAGGAGGTGAAGGTCCAACG
Oligonucleotide LGR5/Forward Custom-made ACACGTACCCACAGAAGCTC
Oligonucleotide LGR5/Reverse Custom-made GGAATGCAGGCCACTGAAAC
Oligonucleotide MUC2/Forward Custom-made AGGATCTGAAGAAGTGTGTCACTG
Oligonucleotide MUC2/Reverse Custom-made TAATGGAACAGATGTTGAAGTGCT
Oligonucleotide TBP/Forward Custom-made ACGCCGAATATAATCCCAAGCG
Oligonucleotide TBP/Reverse Custom-made AAATCAGTGCCGTGGTTCGTG
Optical adhesive covers Life Technologies Europe BV 4311971
PD0325901 Stemcell Technologies 72184
Penicillin/streptomycin Life Technologies Europe BV 15140122
Plate shaker Panasonic
PowerUp SYBR Green Master Mix Fisher Emergo A25776
Primocin InvivoGen ANT-PM-2 antimicrobial formulation for primary cells
Qubit RNA HS Assay Kit Life Technologies Europe BV Q32852
Reagent reservoir for multichannel pipet Sigma-Aldrich CLS4870
REMS AutoSampler with 24-probe or 96C-probe WTI
Richard-Allan Scientific Alcian Blue/PAS Special Stain Kit Thermo scientific 87023
RNase-Free DNase Set Qiagen 79254
RNeasy Mini Kit Qiagen 74106
SB202190 Sigma-Aldrich S7076
Serological pipettes Greiner bio-one 606180 / 607180 / 760180
Serological pipettor (Pipet-Aid) Drummond
Single edge razor blade GEM Scientific
Superscript 1st strand system for RT-PCR Life Technologies Europe BV 11904018
Tecan Spark 10M plate reader Tecan
Trypan Blue Solution, 0.4% Life Technologies Europe BV 15250-061
TrypLE Express Enzyme (1x) Life Technologies Europe BV 12605-010 Cell dissociation reagent 1
Water bath Grant
Y27632 (ROCK inhibitor) AbMole M1817



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Cite this Article

van Dooremalen, W. T. M., Derksen, M., Roos, J. L., Higuera Barón, C., Verissimo, C. S., Vries, R. G. J., Boj, S. F., Pourfarzad, F. Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function. J. Vis. Exp. (173), e62074, doi:10.3791/62074 (2021).More

van Dooremalen, W. T. M., Derksen, M., Roos, J. L., Higuera Barón, C., Verissimo, C. S., Vries, R. G. J., Boj, S. F., Pourfarzad, F. Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function. J. Vis. Exp. (173), e62074, doi:10.3791/62074 (2021).

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