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Culturing Colon Organoid Monolayers using Millicell® Hanging Inserts

Section Overview

Overview and Benefits of Organoid-Derived Monolayers

Organoid-derived monolayers cultured on permeable inserts, also known as 2.5D cell culture, bridge the gap between 3D organoid models and more traditional two-dimensional in vitro methods. Derived from patient tissues (PDOs) or induced pluripotent stem cells (iPSCs), organoids provide a physiologically relevant model that closely mimics native epithelial tissue. By culturing organoids as monolayers on permeable inserts, researchers can establish a uniform and well-defined epithelial layer that is conducive to transepithelial electrical resistance (TEER) measurement.

Compared to traditional monolayers, organoid-derived monolayers offer several advantages. They exhibit enhanced barrier properties, including tighter junctions and heightened TEER, resulting in more accurate assessments of barrier integrity. Additionally, organoids retain cellular diversity, better representing the complexity of native epithelial tissue. Organoid-derived monolayers cultured on permeable membrane inserts enable precise experimental manipulation and measurement, facilitating studies of drug absorption mechanisms, disease pathologies, and interactions with the gut microbiota. Here we present comprehensive guidelines for generating, optimizing, and assessing the generation of organoid-derived monolayers cultured on Millicell® 24-well hanging cell culture inserts.

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Generating Organoid-derived Monolayers

Single cell dissociation of iPSC-derived Colon Organoids (SCC300)

For this single cell dissociation experiment Millicell® 24-well hanging inserts (PTRP24H48) were used with a cell density of 2x105 cells per insert. The inserts were coated with 2% GFR Matrigel® in 1X PBS for at least 1 h at 37°C or overnight at 4°C.

Note: Coat inserts before starting the dissociation protocol. This protocol is based on hiPSC-derived colon organoids (SCC300) P21 Day 6, 120 x 25 µL domes from 2 x 10 cm dishes. Add 1X ROCKi (SCM075) at 10 µM to all reagents such as Accumax (SCR006) and 1X PBS.

  1. Detach and break up the 120 x 25 µL domes with a 10 mL pipet in 10 mL cold 1X PBS+1X ROCKi.
  2. Transfer 2.5 mL of organoid suspension into each of 4 x 50 mL conical tubes (30-40 x 25 µL domes per 50 mL conical tube) and discharge suspension against the side 5-10 times to further releasing organoids from Matrigel® domes.
  3. Fill each tube to 30 mL with ice-cold 1X PBS+1X ROCKi and spin down at 2000 rpm (872 x g) for 10 minutes.
  4. Carefully aspirate off supernatant and resuspend each pellet in the 4 x 50 mL conical tubes in 5 mL ice-cold 1X PBS+1X ROCKi.
  5. Pool suspensions from 2 x 50 mL conical tubes into a 15 mL conical tube. Repeat for the suspensions in the other 2 x 50 mL tubes. NOTE: Transferring the suspension at this step to 15 mL conical tubes will help to remove residual Matrigel® without losing some of the pellet.
  6. Spin down at 2000 rpm (872 x g) for 10 minutes and carefully aspirate off supernatant from each of the 2 x 15 mL conical tubes. NOTE: Try to remove as much Matrigel® as possible from the pellets by attaching a P-2OO tip to the aspirating pipet.
  7. Resuspend each pellet with 4 mL Accumax+1X ROCKi by pipetting up and down four times and then pool two pellets into one tube (8 mL Accumax+1X ROCKi total).
  8. Resuspend the suspension further by discharging against the side of the tube 4 to 6 times.
  9. Place tubes in 37°C w/ 5% CO2 incubator for 10 minutes. Remove tubes at 5-minute intervals and transfer to the hood to resuspend by pipetting 8-10 times against the wall of each tube.
  10. Place tubes back in the incubator for another 5 minutes.
  11. Remove tubes from the incubator and pipet for another 8-10 times against the side of the tube.
  12. Observe 10 µL of cell suspension under the microscope to check for single cell dissociation. NOTE: 10 minutes incubation should be sufficient to get single cells with some small clumps.
  13. Transfer and pool suspensions into a 50 mL conical tube.
  14. Add 25 mL of ice-cold 1X PBS + 1X ROCKi to the 50 mL conical tube containing cell suspension and spin down at 2000 rpm or 872 x g for 10 minutes.
  15. Aspirate off supernatant and resuspend cell pellet in 3 mL 3dGRO® Human Colon Organoid Expansion Medium (SCM304) + Penicillin/Streptomycin (P/S) + 1X ROCKi with a 5 mL pipette by pipetting up and down 6 to 8 times.
  16. Take 50 µL of cells out to count on hemocytometer or cell counter, such as the Scepter 3.0.
  17. If necessary, spin down cells. Resuspend cells to 1x106 cells/mL with SCM304 + P/S + 1X ROCKi.
  18. Prepare two additional samples of cells at 1x106 cells/mL and 0.5x106 cells/mL and seed cells.
    a. Seed 200 µL of cells at 2x106 cells/mL into each of the pre-coated 2% GFR Matrigel® inserts for 400k/insert.
    b. Seed 200 µL of cells at 1x106 cells/mL into each of the pre-coated 2% GFR Matrigel® inserts for 200k/insert.
    c. Seed 200 µL of cells at 0.5x106 cells/mL into each of the pre-coated 2% GFR Matrigel® inserts for 100k/insert.
  19. Add 1000 µL of growth medium + 1X ROCK inhibitor in each well on the basal side of the insert.
  20. Place plate in a 37°C incubator with 5% CO2 to culture.
  21. Monitor cells seeded on inserts daily under a microscope for confluency of monolayers.
  22. Perform Live/Dead Viability staining 1 day after seeding using Live Dead Viability Assay Kit for 3D and 2D cell culture (CBA415).
  23. Change media every 2 days with SCM304+P/S+1X ROCKi until monolayers reach 100% confluency.
  24. Obtain TEER measurements in a tissue culture hood a day before monolayers become confluent and every day thereafter until the desired timepoint has been reached. Refer to the manufacturing instructions to setup the TEER instrument before use.

Single cell dissociation of Colon PDO (SCC321)

For this single cell dissociation experiment Millicell® 24-well hanging inserts (PTRP24H48) were used with a cell density of 2x105 cells per insert. The inserts were coated with 2% GFR Matrigel® in 1X PBS for at least 1 h at 37°C or overnight at 4°C. This protocol is based on 50-60 x 10 µL domes of colon patient-derived organoids (PDOs) (SCC321) at day 5 in culture. Add 1X ROCKi (SCM075) to all reagents such as TrypLE Express and 1X PBS.

  1. Detach and break up the Matrigel® domes in cold 1X PBS+1X ROCKi several times with a P-1000 pipette.
  2. Transfer organoid suspension into 3 x 15 mL conical tubes (20 x 10 µL domes max per 15 mL conical tube) and discharge suspension against the side several times to further releasing organoids from Matrigel® domes. NOTE: For greater than 60 x 10 µL domes, use additional 15 mL tubes.
  3. Centrifuge at 2000 rpm (872 x g) for 10 minutes at 4°C.
  4. Carefully aspirate off supernatant and resuspend each pellet in the 3 x 15 mL conical tubes in 5 mL ice-cold 1X PBS+1X ROCKi.
  5. Centrifuge at 2000 rpm (872 x g) for 10 minutes at 4°C.
  6. Carefully aspirate off supernatant and resuspend each pellet in the 3 x 15 mL conical tubes in 2 mL TrypLE Express+1X ROCKi. Add more TrypLE Express if suspension is very vicious.
  7. Incubate for 10 minutes at 37°C incubator and resuspend by discharging against the side of each tube several times with a pipette every 10 minutes.
  8. Check under a microscope for single cells in suspension. Only few groups of 2-3 cells should be present. If organoids are still not dissociated, continue incubation at 37°C and check every 5 minutes for dissociation.
  9. Once organoids are dissociated, add 5 mL chilled DMEM/F-12 PLUS media (SCM162) + 1X ROCK inhibitor or 1X PBS + 1X ROCK inhibitor to cell suspension.
  10. Centrifuge cell suspension for 5 minutes at 500 x g.
  11. Remove supernatant and carefully remove any residual Matrigel® layer above the cell pellet.
  12. Resuspend cell pellet in growth medium (SCM105) + 1X ROCK inhibitor at a density of 1x105 cells/mL and pipet up and down several times to break up any clumps of cells.
  13. Seed 2x105 cells/insert in 300 µL of growth medium.
  14. Add 1000 µL of growth medium + 1X ROCK inhibitor in each well on the basal side of the insert.
  15. Place plate in a 37°C incubator with 5% CO2 to culture.
  16. Monitor cells seeded on inserts daily under a microscope for confluency of monolayers.
  17. Perform Live/Dead Viability staining 1 day after seeding using Live Dead Viability Assay Kit for 3D and 2D cell culture (CBA415).
  18. Change media every 2 days with SCM304+P/S+1X ROCKi until monolayers reach 100% confluency.
  19. Obtain TEER measurements in a tissue culture hood a day before monolayers become confluent and every day thereafter until the desired timepoint has been reached. Refer to the manufacturing instructions to setup the TEER instrument before use.

Determine the Optimal Percentage of GFR Matrigel® for Coating Millicell® Inserts

For this single cell dissociation experiment the Millicell® 24-well hanging inserts (PTRP24H48) with 1 µm pore size for Greiner CELLSTAR® 24-well plate (M8812) was used with a cell density of 200k cells per insert (24-well). The inserts were coated with 1, 2, 4, 6, 8 or 10% GFR Matrigel® in 1X PBS.

  1. Prepare 1%, 2%, 4%, 6%, 8% and 10% of GFR Matrigel® in ice-cold 1X PBS or DMEM/F-12 Plus (SCM162) for coating Millicell® inserts. NOTE: Store dilutions on ice until ready to use.
  2. Using sterile technique, remove Millicell® hanging inserts from their blister packaging and place into 24-well cell culture plates.
  3. Remove diluted GFR Matrigel® from ice and transfer to the tissue culture hood.
  4. Add between 200-400 µL of diluted GFR Matrigel® into the appropriate inserts and place at 37°C incubator for at a minimum of 1 hour before seeding cells or overnight in the fridge.
    a. Refer to the above protocol on how to single cell dissociate iPSC-derived Organoids (SCC300) with Accumax for seeding cells onto Millicell® inserts.
    b. Refer to the above protocol on how to single cell dissociate patient-derived organoids (SCC321) with TrypLE Express for seeding cells onto Millicell® inserts.

Live-Dead Cell Viability Staining of Organoid-derived Monolayers on Millicell® Inserts

  1. Follow staining protocol for using Live Dead Cell Viability Assay Kit for 3D and 2D Cell Culture (CBA415) under the section titled “Protocol for Spheroid and Organoid Culture”.
  2. Remove 24-well insert plate from a humidified 37°C incubator.
  3. Carefully aspirate the media from the apical side with an aspirating pipette with its tip attached to a P-200 tip without filter. NOTE: Be careful not to scratch the surface of the insert (monolayer).
  4. Add 5 µL Calcein AM, 20 µL Propidium Iodine, and 8 µL of Hoechst 33342 per 12 mL solution (1:1 culture medium to 1X PBS).
  5. Vortex the staining solution to thoroughly mix the components.
  6. Wash monolayers on inserts twice with 1X PBS.
  7. Add 200-400 µL of staining solution to each insert containing cell monolayer and incubate for 60 minutes at 37°C.
  8. Replace staining solution with 1X PBS and image on a fluorescence microscope with the appropriate excitation and emission filters.
  9. For cells used in continued culture, wash twice with media and replace with fresh medium.
  10. Put plate back in a humidified 37°C incubator to continue culture.

Monitoring the Formation of Monolayers on Millicell® Inserts

Monitor cells seeded on inserts daily under a microscope to determine the confluency of monolayers and start TEER measurement in the tissue culture hood a day before monolayers become confluent and every day thereafter. Refer to the manufacturing instructions to setup the TEER instrument before use.

Differentiating Monolayers on Millicell® Inserts

  • Start differentiation only when monolayers reach 100% confluency.
  • Differentiate the monolayers with ENR differentiation media formulation by adding 300 µL to the insert (apical side) and 1000 µL to the well (basal side).

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  • Change media every other day.
  • Continue to take TEER measurement daily or every other day until the desired endpoint of the experiment is reached.

ICC Staining and Imaging of Monolayers on Millicell® Inserts

  1. Remove monolayers from 37°C with 5% CO2 incubator at the desired time-point and place in a tissue culture hood.
  2. Aspirate off media from the apical side and the basolateral side (the well) of each insert with an aspirating pipette. TIP: attach a P-200 tip without the filter to the tip of an aspirating pipette or use a micropipette and slowly aspirate off media from the apical and basolateral side. Be careful not to scratch the membrane surface of the inserts during this and following steps.
  3. Wash the apical side with 0.4 mL and the basal lateral side with 1 mL 1X PBS once.
  4. Slowly aspirate off 1X PBS from the apical and basal lateral sides with an aspirating pipette with its tip attached to a P-200 tip without filter.
  5. Fix each 24-well insert with 0.4 mL of 4% Paraformaldehyde in 1X PBS for 30 minutes at room temperature.
  6. Remove the 4% Paraformaldehyde with a P-1000 pipette and discard of it appropriately.
  7. Wash the apical side with 0.4 mL and the basal lateral sides with 1 mL 1X PBS twice.
  8. Slowly aspirate off 1X PBS from the apical and basal lateral sides with an aspirating pipette with its tip attached to a P-200 tip without filter.
  9. Permeabilize each monolayer with 200-400 µL of 0.3% Triton-X-100 + 1% BSA in 1X PBS for 15 minutes.
  10. Slowly aspirate off permeabilization buffer from the apical side with an aspirating pipette with its tip attached to a P-200 tip without filter.
  11. Add 0.4 mL of blocking buffer (1X PBS + 5% normal donkey serum + 1% BSA) for 1 hour at RT or overnight at 4°C.
  12. Slowly aspirate off blocking buffer from the apical side with an aspirating pipette with its tip attached to a P-200 tip without filter. Be careful not to scratch the surface of the inserts during the process.
  13. Stain inserts with 0.2-0.4 mL of primary unconjugated or conjugated antibodies in diluent buffer (1% BSA in 1X PBS) for 2-4 hours at RT or overnight at 4°C while shaking.
  14. Remove staining solution from insert with a P-1000 pipette and change tip with each insert.
  15. Wash each insert 3X with 1X PBS for 5 minutes each.
  16. Slowly aspirate off 1X PBS from the apical side with an aspirating pipette with its tip attached to a P-200 tip without filter. Be careful not to scratch the surface of the inserts during the process.
  17. Stain inserts with the appropriate secondary antibodies if necessary in diluent buffer (1% BSA in 1X PBS) for 2-3 hours at RT or overnight at 4°C while shaking.
  18. Wash each insert 3X with 1X PBS for 5 minutes each.
  19. Slowly aspirate off 1X PBS from the apical side with an aspirating pipette with its tip attached to a P-200 tip without filter. Be careful not to scratch the surface of the inserts during the process.
  20. Counterstain with 0.2-0.4 mL of DAPI at 2-10 µg/mL in 1X PBS for 15 minutes at RT while shaking
  21. Slowly aspirate off DAPI solution from the apical side with an aspirating pipette with its tip attached to a P-200 tip without filter. Be careful not to scratch the surface of the inserts during the process.
  22. Wash once with 1X PBS.
  23. Slowly aspirate off 1X PBS from the apical side with an aspirating pipette with its tip attached to a P-200 tip without filter. Be careful not to scratch the surface of the inserts during the process.
  24. Add 0.4 mL of 1X PBS to the apical side and 1 mL of 1X PBS to the basal lateral side. Store at 4°C.
  25. Image the insert directly in the plate or carefully cut out insert and mount on a glass-bottom plate.

Optimizing PDO and hi-PSC Organoid-derived Monolayer Workflow

PDO colon organoids (SCC321) require 5-7 days and hiPSC-derived colon organoids (SCC300) require 10 days in culture before passaging. The optimal time to harvest for single cell dissociation and seeding on Millicell® inserts is during log phase growth: Day 5 for SCC321 and Day 7 for SCC300. Usually, log phase growth is around 2-3 days before the culture is ready to be passaged, but this may be different for individual organoid systems.

Next, determine the approximate yield of single cells from a certain number of organoid domes - it’s recommended to keep the occupancy of organoids in each dome consistent during every passage. Finally, determine how many single cells are required to seed a Millicell® 24-well hanging cell culture insert (PTRP24H48) in order to reach 100% confluency by day 3 or 4. The total number of organoid domes required for Millicell® 24-well hanging cell culture inserts can be then be calculated (Table 1).

 

 Optimal Time to Harvest Approximate Yield Number of Organoid Domes Required per Insert Number of Organoid Domes Required for 24 Inserts

PDO Colon

Day 5

~10 x 10µL domes yield 434k cells

200k/insert (~5 x 10µL domes)

 

Need ~120 x 10µL domes for 24 inserts

hiPSC-Colon

Day 7

~25 x 10µL domes yield 525k cells

200k/insert (~10 x 10µL domes)

~240 x 10µL domes for 24 inserts

Table 1: Guidelines for harvesting and seeding PDO colon organoids (SCC321) and hiPSC-derived colon organoids (SCC300)

The optimal time for dissociation is the shortest incubation that yields a single cell suspension. We found that 20 to 30 minutes incubation with TrypLE works best for SCC321 and about 10 minutes incubation with Accumax (SCR006) works best for SCC300 (Figure 1). Incubating SCC321 with TrypLE Express for 20-30 minutes is sufficient to perform single cell dissociation and seeding at 200 k cells/insert will generate a confluent monolayer by Day 4.

The optimized seeding density to reach confluency 200k cells/insert yielding confluent monolayers for both SCC321 and SCC300 between Days 3-5 (Figure 1). 

Figure 1. Comparison of dissociation reagents and incubation periods. A. SCC300 was incubated with TrypLE Express for 15 minutes at 200k cells/insert and did not result in a confluent monolayer by Day 5. B. SCC300 was incubated with TrypLE Express for 45 minutes at 200k cells/insert and did not result in a confluent monolayer by Day 5. C. SCC300 incubated with Accumax for 10 minutes at 37°C is sufficient to perform single cell dissociation. Seeding at 200 k cells/insert generated a confluent monolayer by Day 5. 

Determining the optimal percentage of Matrigel® to coat inserts is crucial for the successful generation of organoid-derived monolayers. We tested inserts that were coated with 2% and 4% GFR Matrigel® and found that too high of a percentage of GFR Matrigel® can lead to longer times for the monolayers to reach confluency, uneven monolayer thickness, and the production of 3D structures (Figure 2B).

Figure 2: Determining the optimal percentage of GFR Matrigel® for coating inserts cultured with hiPSC-colon organoids seeded at 100k. A. 2% GFR Matrigel® worked best in forming a confluent monolayer by Day 8. B. 4% GFR Matrigel® led to the formation of 3D structures on the membrane insert by Day 8.

After 24 hours of seeding single cell suspension onto Millicell® inserts, cell viability can be determined by using the Live-Dead Cell Viability Assay Kit (CBA415). Around 85-90% of cells on insert are stained green with Calcein-AM (green), indicating viable cells, while a very small percentage is stained with propidium iodide (PI) (dead cells, red) (Figure 3).

Figure 3: Post-seeding cell viability on insert. A. hiPSC colon cells (SCC300) on membrane insert stained green with Calcein-AM (green), indicating viable cells. B. Cells on membrane insert stained with PI (dead cells, red). Monolayers were washed twice with 1X PBS before imaging.

Organoids grown in expansion media are enriched with proliferating cells and have fewer differentiated cell types such as goblet cells, enterocytes, and enteroendocrine cells. When grown in differentiation media containing EGF, Noggin, and R-Spondin (ENR), organoids are less enriched with proliferative cells and have a higher proportion of mature cell types. These mature cell types are important for the formation of tight junctions that act as a barrier in the intestinal linings by regulating what goes in and out (Figure 4).

Figure 4. Representative images of confluent organoid-derived monolayers on Millicell® inserts. A. Bright field images at Day 4 after plating for SCC321 at 200k cells/insert. B. Bright field images at Day 3 after plating for SCC300 at 200k cells/insert. C. Hematoxylin eosin staining of organoids monolayer at Day 12.

SCC321 required 9+ days of differentiation in ENR media to reach peak TEER measurement, while SCC300 required 8-9 days in ENR media and 11-12 days in 3dGRO® Human Colon Organoid Expansion Medium (SCM304) to reach peak TEER measurement.

TEER measurements evaluate the integrity and tightness of epithelial cell layers, like those in the intestinal lining. This method typically employs epithelial voltohmmeters like the Millicell® ERS 3.0 Voltohmmeter (MERS03000), which applies a low-level electrical current across the cultured epithelial cell layer on a permeable support membrane, such as Millicell® hanging inserts (PTRP24H48).

TEER measurements conducted daily on PDO colon organoids (SCC321) and hiPSC-derived colon organoids (SCC300) achieved confluency by Day 3 and Day 4 respectively. The cultures were maintained either in expansion media (3dGRO® L-WRN Conditioned Media Supplement (SCM105) for SCC321 or 3dGRO® human colon organoid expansion media (SCM304) for SCC300) or in differentiation media (ENR). Colon PDO (SCC321) monolayers cultured in ENR displayed a consistent increase in TEER values, while those cultured in L-WRN failed to maintain this trend (Figure 5A). In contrast, iPSC-derived colon organoid (SCC300) monolayers showed a steady increase in TEER values regardless of whether they were cultured in SCM304 or ENR media (Figure 5B).

Figure 5. TEER measurements of organoid-derived monolayers grown in Millicell® inserts (PTRP24H48) over 12 days. A. Patient-derived colon organoids (SCC321) were seeded and cultured in expansion media for 3 days. On Day 4 Millicell® inserts containing monolayers were either switched to differentiation media (ENR) or maintained in expansion media (L-WRN). B. Human iPSC-derived colon organoids (SCC300) were seeded and cultured in 3dGRO® human colon organoid expansion media (SCM304) or differentiation media (ENR) starting at Day 3 after seeding.

This discrepancy may be attributed to the more mature phenotype observed in iPSC-derived colon organoids. Relative gene expression analysis revealed higher levels of chromogranin A (CHGA) and MUC2 mucin in iPSC-derived colon organoids compared to colon PDO (Figure 6). These findings suggest that iPSC-derived colon organoids harbor specialized cell lineages, while further differentiation with ENR media is necessary for colon PDOs.

Figure 6. RNAseq relative gene expression for chromogranin A (CHGA) and MUC2 mucin in iPSC-derived colon organoids compared with colon PDO that were maintained in expansion media (L-WRN).

Elevated TEER values signify a strong epithelial barrier characterized by intact tight junctions and minimal paracellular permeability. This correlates with the presence of the tight junction protein ZO-1 localized apically. Immunofluorescence staining of ZO-1 in monolayers of colon PDO (SCC321) and iPSC-derived colon organoids (SCC300) demonstrated proper apical localization of the protein (Figure 7).

Figure 7. Confocal immunofluorescence images of organoid derived monolayers grown on Millicell® inserts stained with anti ZO-1 in green (MABT339) and DAPI (D9542). A. Patient-derived colon organoids (SCC321) were used to generate monolayers and switched to differentiation media (ENR) on Day 4. Staining was performed at Day 12. B. iPSC-derived colon organoids (SCC300) were used to generate monolayers and switched to differentiation media (ENR) on Day 3. Staining was performed at Day 5.

Tips and Tricks for Coating and Seeding Millicell® Inserts

  • Attach a P-200 tip without the filter to the tip of an aspirating pipet or use a P-200 pipette and slowly aspirate off PBS from the inserts. Be careful not to scratch the surface of the inserts.
  • GFR Matrigel® concentration may need to be optimized for different model systems. If 3D structures form on membrane surface, consider reducing Matrigel® percentage.
  • Add 10 µM ROCK inhibitor to dissociation reagent to enhance viability of cells during incubation (see dissociation protocol above).
  • Seed single cell suspensions at 200k cells/24-well insert to achieve confluent monolayers in 3-4 days.
  • Use 300 µL for the apical side and 1000 µL for the basolateral side of a Millicell® 24-well insert in a Greiner 24-well culture plates (M8812). The optimal basolateral volume varies by brand of plates. More information on recommended media volumes can be found in the Millicell® insert user guide.
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