TITLE: Generation of human colon organoids from healthy and inflammatory bowel disease  mucosa 
AUTHORS: Isabella Dotti, Aida Mayorgas, Azucena  Salas

[DESCRIPTION]
Ulcerative colitis and Crohn’s Disease are chronic inflammatory bowel diseases (IBD) of unknown cause characterized by a relapsing-remitting behavior. Growing evidence supports the idea that the epithelial barrier plays a central role in the pathogenesis of IBD as well as in its evolution over time, thus representing a potential target for novel therapeutic options. 
In the last decade, the introduction of 3D epithelial cultures from ex vivo-expanded intestinal adult stem cells (ASCs) has impacted our ability to study the function of the epithelium in several gastrointestinal disorders, including IBD.
Here, we describe in detail a reproducible protocol to generate Matrigel-embedded epithelial organoids from ASCs of non-IBD and IBD donors using small colonic biopsies, including steps for its optimization. A slightly modified version of this protocol is also provided in case surgical samples are used.
With this method, epithelial organoids can be expanded over several passages, thereby generating a large quantity of viable cells that can be used in multiple downstream analyses including genetic, transcriptional, proteomic and/or functional studies. In addition, 3D cultures generated using our protocol are suitable for the establishment of 2D cultures, which can model relevant cell-to-cell interactions that occur in IBD mucosa.

[GUIDELINES]
Approval from the ethics committee and signed consent by the donor must be obtained prior to human sample processing. In case of underage donors, the consent must be signed by the legal representatives.

[STEPS]
SECTION: 1. Intestinal crypt isolation (day 1)
1. Collect 4-8 colonic biopsies (from non-IBD or IBD subject) obtained during the colonoscopy procedure, in a 15 mL Falcon tube containing 10 mL HBSS, preferably cold. 

 

 

ALTERNATIVE PROTOCOL: For isolation of intestinal crypts from a surgical sample, follow Steps 14-31
SECTION: 1. Intestinal crypt isolation (day 1)
2. In this protocol, crypts are embedded in 25 µL-Matrigel drops seeded in a 48-well plate, one drop per well. 

Based on the estimated number of crypts of the experiment (as determined in Steps 32-33), place the required number of Matrigel aliquots on ice for 1-2 hours before use, pre-warm the required number of 48-well plates in the cell incubator (5% CO2, 37°C) about 1-2 hours before use, and prepare the required volume of organoid growth medium.

 


SECTION: 1. Intestinal crypt isolation (day 1)
3. Wash the biopsies 1-2 times with ≈ 10 mL cold DPBS by gently flipping the tube for a few seconds.
SECTION: 1. Intestinal crypt isolation (day 1)
4. Incubate the sample in 10 mL of the antimicrobial cocktail for 15 min at room temperature under gentle agitation on a platform rocker.


SECTION: 1. Intestinal crypt isolation (day 1)
5. Wash 1-2 times with ≈ 10 mL DPBS at room temperature by gentle manual flipping for a few seconds to remove the antimicrobial cocktail.
SECTION: 1. Intestinal crypt isolation (day 1)
6. Incubate the biopsies with 10 mL of the crypt isolation buffer for 45 min at 4°C under gentle agitation on a platform rocker.
SECTION: 1. Intestinal crypt isolation (day 1)
7.  Wash once with ≈ 10 mL cold DPBS by gentle manual flipping for a few seconds to remove the crypt isolation buffer.
SECTION: 1. Intestinal crypt isolation (day 1)
8. To start collecting the fractions enriched with viable crypts, add 10 mL cold DPBS and shake vigorously by hand for 15-20 seconds. Depending on the sample, isolated crypts released from the tissue can be visible in the suspension in this first wash. Regardless, proceed to the next step.
SECTION: 1. Intestinal crypt isolation (day 1)
9. Leave the biopsies to settle at the bottom of the tube and collect about 10 mL of the suspension in a new cold 15 mL Falcon tube using a 5 mL pipette. Add FBS to the suspension to a 5% final concentration to maintain cell viability.

 

SECTION: 1. Intestinal crypt isolation (day 1)
10. Repeat Steps 8-9 until no additional crypts are released. Usually, no crypts are collected after 5-6 shaking cycles.
SECTION: 1. Intestinal crypt isolation (day 1)
11. Spin the crypt fractions at 200 x g for 3 min at 4°C.
SECTION: 1. Intestinal crypt isolation (day 1)
12. Carefully remove the supernatant from each fraction using a 5 mL pipette and gently resuspend each crypt pellet in 1 mL cold washing medium. Merge the fractions in one 15 mL Falcon tube and spin again at 200 x g for 3 min at 4°C.
SECTION: 1. Intestinal crypt isolation (day 1)
13. Resuspend the crypt pellet in cold washing medium. 1-2 mL washing medium are usually enough for an optimal crypt count. At this point, jump to Step 32 or Step 33 depending if crypt count is desired or not.

   

SECTION: 1. Intestinal crypt isolation (day 1)
14. Collect the fresh colonic surgical sample ( 1-2 cm2-size) in a 50 mL Falcon tube containing  20 mL HBSS, preferably cold.
 

 

SECTION: 1. Intestinal crypt isolation (day 1)
15. In this protocol, crypts are embedded in 25 µL-Matrigel drops seeded in a 48-well plate, one drop per well. 

Based on the estimated number of crypts of the experiment (as determined in Steps 32-33), place the required number of Matrigel aliquots on ice for 1-2 hours before use, pre-warm the required number of 48-well plates in the cell incubator (5% CO2, 37°C) about 1-2 hours before use, and prepare the required volume of organoid growth medium.

 

SECTION: 1. Intestinal crypt isolation (day 1)
16. Wash the surgical sample with ≈ 20 mL cold DPBS by gentle manual flipping for a few seconds. Discard the DPBS and repeat the washing until the DPBS becomes clear.
SECTION: 1. Intestinal crypt isolation (day 1)
17. Transfer the sample to a 10 cm Petri dish and remove the submucosa and larger contaminating blood vessels with scissors and scalpel.
SECTION: 1. Intestinal crypt isolation (day 1)
18. Cut the sample into pieces of 0.2-0.3 cm2, transfer them to a new 50 mL Falcon tube and perform a couple of additional washes with ≈ 20 mL cold DPBS by gentle manual flipping for a few seconds.

 

SECTION: 1. Intestinal crypt isolation (day 1)
19. Incubate the sample in 20 mL of the antimicrobial cocktail for 20 min at room temperature under gentle agitation on a platform rocker.


SECTION: 1. Intestinal crypt isolation (day 1)
20. Wash 1-2 times with ≈ 20 mL DPBS at room temperature to remove the antimicrobial cocktail.
SECTION: 1. Intestinal crypt isolation (day 1)
21. Incubate with 10 mM DTT for 8 min at room temperature under gentle agitation on a platform rocker to remove the mucus.
SECTION: 1. Intestinal crypt isolation (day 1)
22. Wash 1-2 times with ≈ 20 mL cold DPBS to remove DTT.
SECTION: 1. Intestinal crypt isolation (day 1)
23. Incubate the sample pieces with 20 mL of crypt isolation buffer for 45 min at 4°C under gentle agitation on a platform rocker.
SECTION: 1. Intestinal crypt isolation (day 1)
24. Remove the EDTA buffer and wash once with 20 mL cold DPBS by gentle flipping.
SECTION: 1. Intestinal crypt isolation (day 1)
25. Resuspend the sample pieces in 20 mL cold DPBS and perform 1-2 manual vigorous shakes for 10-15 seconds each to remove the mucus, debris and dead cells. Usually, these supernatants are discarded since no viable crypts are detectable at this point.
SECTION: 1. Intestinal crypt isolation (day 1)
26. To start collecting the fractions enriched with viable crypts, add 20 mL cold DPBS and shake vigorously by hand for 15-20 seconds. Crypts released from the tissue will be detectable in the suspension to the naked eye.


SECTION: 1. Intestinal crypt isolation (day 1)
27. After each shake cycle, leave the sample pieces to settle at the bottom of the tube and collect about 20 mL of the suspension enriched in crypts in a new cold 50 mL Falcon tube using a 10 mL pipette. Add FBS to a 5% final concentration to maintain cell viability.
 

SECTION: 1. Intestinal crypt isolation (day 1)
28. Repeat Steps 26-27 until the desired number of crypts is obtained.

 

SECTION: 1. Intestinal crypt isolation (day 1)
29. Spin the crypt fractions at 150 x g for 3 min at 4°C.
SECTION: 1. Intestinal crypt isolation (day 1)
30. Carefully remove the supernatant from each fraction using a 10 mL pipette and gently resuspend each crypt pellet in 1 mL cold washing medium. Merge the fractions in one 50 mL Falcon tube and spin again at 150 x g for 3 min at 4°C.
SECTION: 1. Intestinal crypt isolation (day 1)
31. Resuspend the crypt pellet in cold washing medium. Depending on the size of the obtained crypt pellet, between 5- and 10-mL washing medium are usually necessary for an optimal crypt count. 
SECTION: 2. Intestinal crypt culture (day 1 to day 3)
32. Place two or three 50 µL-drops of the crypt suspension on a glass slide to check crypt integrity and estimate the average number of crypts in the sample by bright-field microscopy examination.
 

  

SECTION: 2. Intestinal crypt culture (day 1 to day 3)
33. Take out the volume of crypts required for the planned experiment and transfer it to a new tube. We suggest embedding 80-100 crypts/25 µL-Matrigel drop seeded in a 48-well plate. 

Since the amount of crypts isolated from biopsy samples is limited, Step 32 may be skipped when using this type of specimen. Based on our experience, indeed, we calculate that on average 12-16 25µL-Matrigel drops are seeded in a 48-well plate when starting with 6-8 biopsies.

SECTION: 2. Intestinal crypt culture (day 1 to day 3)
33.1. At this point the isolated crypts can be also used to prepare a short-term crypt culture. In this case we recommend using a lower number of crypts per Matrigel drop (about 50 crypts/25µl-drop). Refer to the following publication as an example of overnight crypt culture:

Calderón-Gómez E, et al. Commensal-Specific CD4(+) Cells From Patients With Crohn's Disease Have a T-Helper 17 Inflammatory Profile. Gastroenterology. 2016 Sep;151(3):489-500.e3.
SECTION: 2. Intestinal crypt culture (day 1 to day 3)
34. Wash the tube containing the crypts with washing medium 2 times at 150-200 x g for 3 min at 4°C. Perform the second and last wash in a 1.5 mL Eppendorf tube if the required final volume of Matrigel fits in. Carefully aspirate the supernatant from the crypt pellet using a p1000 pipette.


SECTION: 2. Intestinal crypt culture (day 1 to day 3)
35. Carefully resuspend the crypt pellet in the proper volume of ice-cold Matrigel in accordance with the number of crypt-Matrigel drops defined in the experimental plan. Avoid bubble formation.

 

SECTION: 2. Intestinal crypt culture (day 1 to day 3)
36. Plate a 25 µL crypt-Matrigel drop per well in a 48-well plate (or adjust depending on the well size) and return the plate to the incubator for 15-20 min to allow Matrigel solidification.
SECTION: 2. Intestinal crypt culture (day 1 to day 3)
37. Overlay each Matrigel dome with 250 µL (or an appropriate volume depending on the well size) organoid growth medium. The crypt culture is usually ready to be expanded after 2-3 days, thus no medium change is usually required.

   

SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
38. 
This protocol is suitable for expanding crypt cultures as well as previously passaged organoid cultures. 

Observe the crypt/organoid culture under the microscope to define the optimal dilution factor for expansion. Volumes indicated here are adjusted to 48 well-plates.

 

SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
39. Thaw the required number of Matrigel aliquots by placing them on ice 1-2 hours before use. Pre-warm the required number of 48-well plates in a cell incubator (5% CO2, 37°C) for about 2-3 hours before use. Prepare organoid growth medium and warm it at room temperature before use. Work with ice-cold tubes and refrigerated centrifuges. Perform all the steps in a sterile biosafety cabinet.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
40. Remove the organoid growth medium and wash the wells once with ≈ 250 µL cold DPBS per well.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
41. Add 1 mL cold Recovery solution for every ≈ 3 drops of 25 µL Matrigel. Scrape the drops from the well surface with the P1000 micropipette tip and collect them in ice-cold 15 mL Falcon tubes. Avoid excessive pipetting since the organoids can stick to the tip and tube walls. Fill one Falcon tube with approximately 20 Matrigel drops maximum.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
42. Incubate for 30-40 min on ice, gently inverting the tubes every 5-10 min to facilitate Matrigel depolymerization.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
43. During Step 42, prepare the organoid dissociation solution (without yet adding the dispase to prevent loss of enzymatic activity) and pre-warm it in a water bath at 37°C. Consider that 5 mL solution will be prepared per Falcon tube containing the organoids/Recovery solution mix.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
44. Fill each tube containing the Recovery solution and organoids with additional 5-7 mL cold WM and spin at 400 x g for 3 min at 4°C.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
45. During the spin, add dispase to the organoid dissociation solution.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
46. Discard the supernatant with a 5 mL pipette first, and then with a P1000 micropipette to completely remove the solution. To resuspend the organoid pellet, first gently add 1 mL of the pre-warmed organoid dissociation solution, pipet gently 2-3 times, and then add the remaining 4 mL.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
47. Leave the Falcon tubes in the cell incubator or in a water bath at 37°C for 15-20 min gently inverting the tubes every 5-10 min.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
48. Mechanically dissociate the organoids with a G20-G21 needle mounted on a 5 mL syringe. It will take 2-5 min per tube, depending on weather the culture is derived from crypts or from a previously expanded organoid culture. Avoid generating too many bubbles in the solution while syringing.
 

SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
49. Spin dissociated organoids at 600-800 x g for 4 min at 4°C.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
50. Discard the supernatant with a 5 mL pipette first, and then with a P1000 micropipette to completely remove the solution.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
51. Perform 3 additional washes with ≈ 5 mL cold washing medium at 600-800 x g for 4 min at 4°C to completely remove the dispase. Perform the first two washes in the 15 mL Falcon tube and the last wash in a 1.5 mL Eppendorf tube if the required final volume of Matrigel fits in. At this point single cells from dissociated organoids can be cryopreserved (Step 51.1) or used for the generation of 2D cultures (Step 51.2).


SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
51.1. For cryopreservation, perform the following:
- Transfer a volume of single cells corresponding to 4-6 original Matrigel drops to a new cold cryopreservation tube.
- Spin at 600-800 x g for 4 min at 4°C.
- Resuspend the cell pellet in 1 mL ice-cold Freezing Medium and transfer the sample to a freezing container.
- Store the sample at -80°C for 24h and transfer to a liquid nitrogen container for long-term storage.
- To thaw, resuspend the cell-containing cryopreservation tube in 5-10 mL warm washing medium.
- Spin at 700 x g for 4 min at 4°C, resuspend in 1 mL cold washing medium and transfer to a new cold Eppendorf tube.
- Spin again at 700 x g for 4 min at 4°C.
- At this point, go to  Step 52. 
 

SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
52. Resuspend the cell pellet in Matrigel supplemented with 10 µM Y-27632. The volume of Matrigel must be adjusted to the total number of drops (22-25µL Matrigel/drop) that will be seeded. Avoid bubbles while resuspending.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
53. Seed the required Matrigel drops in the pre-warmed 48-well plate and put the plate back in the incubator for 15-20 min to allow the Matrigel to solidify.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
54. Overlay each Matrigel-embedded dome with 250 µL of organoid growth medium supplemented with 10 µM Y-27632.
SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
55. Change the medium with fresh organoid growth medium (without Y-27632) every 48-72h hours. Organoid cultures grown for 5-6 days are typically ready to be used for differentiation (Steps 56-59) or for further expansion by repeating the procedure described in this section (Steps 38-55).

  


SECTION: 4. Organoid differentiation (day 8 to day 13)
56. Carefully aspirate the organoid growth medium from the culture generated in the previous section. Avoid detaching the Matrigel drops from the bottom of the wells.
SECTION: 4. Organoid differentiation (day 8 to day 13)
57. Perform two washes of ≈ 250 µL/well for 10 min each with warm basic medium to remove the culture components by diffusion through the Matrigel dome. Meantime, prepare the organoid differentiation medium.
SECTION: 4. Organoid differentiation (day 8 to day 13)
58. Add 250 µL of the organoid differentiation medium to each well. 


SECTION: 4. Organoid differentiation (day 8 to day 13)
59. After 2 days, add fresh organoid differentiation medium deprived of RSPO1. Terminal differentiation is usually achieved on day 5.

  

SECTION: 3. Crypt/organoid expansion (day 3 to day 8)
51.2. For the establishment of a 2D culture, refer for example to the following publication describing in detail how to set a monolayer on conventional culture plates using organoid cultures generated by the proposed protocol:

Mayorgas A, et al. A Novel Strategy to Study the Invasive Capability of Adherent-Invasive Escherichia coli by Using Human Primary Organoid-Derived Epithelial Monolayers. Front Immunol. 2021 Mar 29;12:646906.