Generation of intestinal epithelial monolayers from single cells

Background.

Intestinal organoids are generated from intestinal epithelial cells, forming 3D mini-guts characterized by crypts that contain stem cells and transit amplifying cells and inter-crypt regions that contain differentiated cells. While these provide an excellent in vitro model to evaluate and manipulate the regenerative capacities of intestinal epithelial stem cells, their ability to serve as a model for assessing cell-ECM interactions is limited. Plating organoids on different substrates, such as polyacrylamide gels, transforms the 3D organoids into 2D monolayers that self-organize and generate crypt-like and inter-crypt villus-like regions. This provides the opportunity to vary the substrate composition and its physical properties, thus allowing the evaluation of cell-ECM interactions. Here, we describe a protocol to generate such monolayers from a single-cell suspension, resulting in de-novo crypt formation on the desired substrate.

Materials and reagents

• DPBS (Thermo scientific, 14190136)
• Gibco DMEM F12-Glutamax (Thermo-fisher scientific, CAT #11514436)
• Gibco Antibiotic-Antimycotic (Thermo Fisher scientific, CAT #15240096)
• B27 supplement 50X, serum-free (Thermo scientific, CAT #17504001)
• Mouse EGF (peprotech, CAT #17872513)
• Mouse FGF (Peprotech, CAT # 17844573)
• Murine noggin (Peprotech, CAT #250-38)
• R-spondin (R&D systems, 3474-RS-050)
• Nicotinamide (sigma, CAS 98-92-0)
• Chir-99021 (Euromedex, AB-M1692-100MG ; cas 252917-06-9)
• Rock inhibitor Y27632- (ATCC; ATCC-ACS-3030)
• Accumax (sigma-Aldrich, CAT#A7089)
• 20G needle (TERUMO, #AN*2038R1)
• 23G needle (BD microlance, #300800)
• 24G needle (TERUMO, #NN-2425R)
• 10mL syringe (TERUMO, #MDSS10SE)
• 2.5 mL syringe (TERUMO,#SS*02SE1)
• cell strainer 40um (FisherBrand, #22363547)

Previously prepared but not described in this protocol:

1. Intestinal organoids, grown in 50ul Matrigel drops in a 24-well plate. See also note 1
2. 35mm Glass‐bottom dish (WPI FluoroDish FD35‐100) with polyacrylamide gels (PAA), coated with Collagen I and washed several times with PBS. See note 2.

Recipes

Cell dissociation solution

Stop solution

ENR medium 

Plating medium- ENR supplemented with 10uM Y27632; 3uM Chir-99021

ENR-CNY 

Procedure

Media preparation

1. For each gel, you will need 4-6 Matrigel drops containing mature organoids, cultured for 2-3 days. See note 1. 
   For every 12 Matrigel drops, you will need 1ml of dissociation solution. Keep the dissociation solution on ice.
2. You will need 2 volumes of stop solution for every 1 volume of dissociation solution. For example, for 1ml dissociation solution, you will need 2ml of stop solution. Pre-heat the stop solution to 37°C.
3. Prepare ENR media. It can be kept at 4°C for 2-3 weeks. 
4. Prepare ENR-CNY media. Pre-heat to 37°C.

Organoid dissociation

1. Aspirate growth medium from around the Matrigel drop. Add 500µl of PBS on top of each drop.
2. Using a 1ml pipette tip, scrape the Matrigel drop gently to detach the organoids from the Matrigel and the plate. Collect the organoids, broken Matrigel and PBS into a 50mL Falcon.
3. Using a 20G needle and a 10ml syringe, push the solution of organoids up and down 10 times to assist the release of the organoids from the Matrigel and ease their dissociation.
4. Spin for 5 minutes at 300G at 4°C. Discard the supernatant. 
5. Resuspend the pellet with cell dissociation solution. Transfer the cells to a 5mL or 15mL tube. Use 1ml of dissociation solution for every 12 wells.
6. Place the tube containing the resuspended organoids in a dissociation solution at 37°C for 13’. Every 4-5 minutes, flick the tube and pipette the solution up and down to help the dissociation process. 
7. To separate cell clumps into single cells, pass the cell dissociation solution through a 23G needle and a 2.5ml syringe 10 times. Change the needle to a 24G and pass the solution through the syringe an additional 10 times. 
8. Monitor the dissociated cells under the microscope; if cells remain clumped, pass the solution several more times through the 24G needle, pushing the cells against the side of the tube.
9. Once cell dissociation is complete, stop the reaction by adding the stop solution.
10. Filter the cells using a 40um filter to reach a single-cell suspension. 
11. Spin the cells for 5 minutes, 400G, 4°C. 
12. Discard the supernatant; resuspend the pellet in fesh 1 volume stop solution and spin the cells again.

Cell plating

1. Wash the PAA gel with fresh PBS (room temperature); aspirate the PBS carefully so as not to damage the gel. Place the gel to dry lightly in the hood during the last centrifugation of the cells, for approximately 5’. Do not over-dry the gels. 
2. Resuspend the cell pellet from step 13 in the plating medium. For each gel, plate approximately 500,000 cells in 35-45µl of plating medium. See note 3.
3. Place the gels with the drop of cells in a cell-culture incubator, 37°C, 5% CO₂ for 1-2 hours, allowing the cells to attach. 
4. Heat up ENR-CNY medium. Prepare 700µl for each plate.
5. When the initial incubation is over, add the pre-heated ENR-CNY medium to the plate, disturbing the drop of cells as little as possible but ensuring the gel and plate are entirely covered with the medium.
6. 24-48 hours after plating, some cells should have been properly attached to form a monolayer. Dead cells would appear as dots on the gel or will float in the medium. Gently tilt the plate and aspirate the ENR-CNY medium using a pipet. Replace it with a regular, pre-heated ENR medium. Make sure not to disturb the cells.
7. Monitor monolayer growth and differentiation, changing media every 1-2 days. See note 4 and note 5.

Additional notes

1. Organoids can be grown as hyperproliferative cysts, thereby increasing the number of cells with the potential of forming de-novo crypts. To that end, newly split organoids should be grown in ENR containing 10uM Chir990201 and 10mM Nicotinamide. Hyperproliferative crypts have a round, cystic morphology.
2. Generation of PAA gels and protein coating, as well as an alternative method to generate monolayers from single-cell suspension, is described in Pérez-González et al; Nature Cell Biology, 2021. Collagen I coating is necessary for cell attachment and cannot be omitted. Note that gel stiffness, as well as additional protein coats, can effect the efficiency of cell attachment; this protocol has been tried successfully with 2 kPA and 5kPA gels.
3. Cells can be resuspended in higher volumes, though a crowded drop is more efficient for cell attachment. Ensure the gels are not too dry and that the drop does not exceed the size of the gel. If the drop is not sustained on the gel, it means that the gel was not dry enough. Try to aspirate the cells, wash with PBS, dry the gel again and re-apply.
4. Media change should be performed delicately. Tilt the plate and aspirate old media using a pipet; wash gently, releasing the media one drop at a time on top of the monolayer to gently release dead cells. Mature, differentiated monolayers can be kept in culture for over 10 days.
5. Failure of cell attachment is usually caused by a poor collagen coating. However, poor cell attachment can also result from a too high or too low cell density in the plated drop.

Acknowledgments

The work on this protocol was funded by the European Union’s Horizon Europe research and innovation program under the Marie Skłodowska-Curie grant agreement No 101066253. 

The establishment of this protocol was assisted by the work presented in Pérez-González, C. et al, 2021 and Guillaume, J. et al (2022).

References

1. Guillaume, J. et al. Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme. eLife 11:e76541 (2022).
2. Pérez-González, C. et al. Mechanical compartmentalization of the intestinal organoid enables crypt folding and collective cell migration. Nature Cell Biology vol. 23 (2021).