In vitro Self-organized Mouse Small Intestinal Epithelial Monolayer Protocol

1Biomimetic Systems for Cell Engineering Laboratory, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain; 2Colorectal Cancer Laboratory, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10-12, Barcelona 08028, Spain; 3Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Barcelona, Spain; 4ICREA, Passeig Lluís Companys 23, 08010 Barcelona, Spain; 5Centro de Investigación Biomédica en Red (CIBER), Madrid, Spain; 6Department of Electronics and Biomedical Engineering, University of Barcelona (UB), Barcelona, 08028 Spain #Contributed equally to this work *For correspondence: emartinez@ibecbarcelona.eu; vfernandez@ibecbarcelona.eu

crypt pieces or C. single cells and seeded on Matrigel coated standard tissue culture plates or Transwell inserts to obtain a 2D in vitro intestinal epithelial culture system. 1. Harvest the small intestine from Lgr5-EGFP-IRES-creERT2 mouse.
2. Place the intestine into a Petri dish with PBS and remove the fat and mesentery from the tissue using surgical tweezers and a scalpel or surgical scissors.
Note: Be careful not to rupture the intestinal tissue.
3. Flush the mouse small intestine with PBS using a 10 ml syringe and a 23 G 1" needle. 4. Cut up the tissue longitudinally using surgical scissors.
Note: Make sure that when you cut open the tissue the mucosal side is facing upwards. 5. Remove the villi mechanically by scraping carefully the intestinal mucosa with a glass coverslip.  The Authors; exclusive licensee Bio-protocol LLC. 6 www.bio-protocol.org/e3514 Bio-protocol 10(03): e3514. DOI: 10.21769/BioProtoc.3514 Note: Be careful not to apply too much pressure to preserve the crypts. 6. Rinse 3 times with PBS and place the intestine into a new Petri dish. 7. Cut the tissue into pieces of 1-4 mm thick using surgical scissors (see Figure 2). 8. Transfer the pieces into a sterile 50 ml Falcon tube under the laminar flow hood. 9. Add 10 ml ice-cold PBS and pipette up and down 5-6 times using a 10 ml pipette. Let the pieces settle and discard the supernatant.
Note: It is very important that the PBS is ice cold and that the inner pipettes walls are previously wetted with PBS. If not, the tissue pieces might stick to the pipette. 10. Add fresh 10 ml ice-cold PBS and repeat the Step A9 for 10-20 times until the supernatant becomes almost clear.
Note: The supernatant might still be a bit cloudy after 20 washes, proceed with the next step anyways.
11. Add 25 ml of 2 mM EDTA prepared in cold PBS and incubate it for 30 min at 4 °C. Agitate vigorously the Falcon tube during incubation on a platform shaker.
12. After the incubation, let the tissue pieces settle to the bottom of the Falcon tube and discard the supernatant.
13. Add 10 ml of ice-cold PBS with 10% FBS to the tissue pieces using 10 ml pipette. It is recommended to wet the inner wall of the pipette with ice cold-PBS with 10% FBS to avoid tissue pieces sticking to the pipette.
14. Pipette up and down 3-5 times the tissue pieces using a 10 ml pipette. Let the pieces to settle down and filter the supernatant through a 70 µm pore cell strainer placed on top of a new 50 ml Falcon tube. The eluted solution will correspond to the first crypt elution fraction. 15. Repeat the Steps A13 and A14 for 4-5 times more to obtain the following crypt elution fractions.
Use new 70 µm pore cell strainers and new 50 ml Falcon tubes to collect the subsequent crypt elution fractions. 16. Pipette 5 µl of each of the eluted fractions in a Petri dish and analyze them using a bright field microscope, to determine the purity and the size of the crypts in each fraction. Copyright  Note: Usually, the first fractions contain a lot of debris and some villi pieces. It is advisable to discard these fractions and keep the rest, which mainly contain small crypt pieces (Figure 3). 17. Pool the selected fractions in a 50 ml Falcon tube and centrifuge the suspension at 55 x g for 5 min at 8 °C.
Note: In the case reported in Figure 3, we would discard fraction 1 and pool fractions 2, 3 and

4.
18. Resuspend the pellet containing the crypts in pre-thawed Matrigel by gently pipetting 2-3 times using a P1000 micropipette and cold 1,000 µl pipette tips. The resuspension volume will depend on the number of eluted fractions pooled and therefore the size of the obtained pellet. For example, in the case reported in Figure 3, in which we have polled fractions 2, 3 and 4 ( Figure   3A) and obtained the pellet of cells shown in Figure 3B we would use 500 µl of Matrigel to resuspend the pellet.   Table 1) every other day, in the current culture medium, during the first 4-5 days of the culture and Rho kinase inhibitor (Y-27632) (10 µM) every day for the first 4 days to avoid epithelial cells death by anoikis. Change the whole medium every 2-3 days. Alternatively, add 100 µl fresh medium every other day until passing of the organoids.
Note: Typically, crypts will start budding after 2-3 days in culture (Figure 4). Passage outgrow crypts after one week in culture. In case crypt confluence is very high, an earlier passage of the budding crypts will be required for a proper organoid formation. Typically, in the original drops we obtain around 100-125 organoids per drop. 2. Prepare and pre-warm the organoid growth medium (Table 1) and 24-well plates at 37 °C.
3. Remove the organoid growth media from the wells containing full-grown organoids (see Figure   5, left panels) add 300 µl TrypLE Express 1x to each well and gently detach and disrupt the Matrigel drops by scraping it off using a P1000 micropipette. This step is performed at RT.

Notes:
a. You can use 300 µl TrypLE Express 1x volume to recover organoids from 2 drops. b. TrypLE Express 1x is used in this step to obtain crypts with more homogeneous size and favor Matrigel digestion. 4. Transfer the Matrigel suspension to a 15 ml Falcon tube already placed on ice.
5. Pass the suspension through a 1 ml syringe with a 23 G 1" needle 3-4 times to further mechanically disrupt the organoids. This step is performed on ice.
Note: The number of times you pass the sample through the syringe and the force you apply will depend on the size of the full-grown organoids you intend to break. Be aware that a too gentle procedure would not break the organoids and too harsh will over-digest the crypts. 6. Add 1 ml of 2% FBS in PBS for every 300 µl of suspension and agitate manually the tube to mix well the content.
Note: This step might be repeated in case a further wash is required. 11. Add 400 µl of the intestinal organoid growth medium (Table 1)   b. Mix well the Falcon containing the digested organoid suspension, take 3 times 5 µl samples using a P20 micropipette and place them into a Petri dish.
c. Count and average the number of crypt pieces within the 5 µl volume using a bright field microscope and from that value estimate the total number of crypt pieces in the suspension.
d. Centrifuge to collect the crypt pieces at 72 x g for 3 min at 4 °C.
e. Resuspend the pellet in intestinal organoid growth medium (Recipe 1). Calculate the total medium volume needed for a seeding density of 1,500 crypts/cm 2 . i. Inspect the monolayer formation and growth throughout the culture time using a bright field microscope. After seeding, organoid-derived crypts will rapidly refold on themselves to form crypt-like domains containing Lgr5 + intestinal stem cells (GFP + ). Then, cells spread on the substrate and migrate out of the crypt-like domains, forming epithelial monolayers composed on GFPcells which correspond to the villus-like regions ( Figure 6A).
b. Pass the suspension through 1 ml syringe with a 23 G 1" needle 5-6 times followed by a c. Once single cell suspension is achieved, place the tube on ice and add 1 ml of PBS with 2% FBS for every 300 µl of suspension and agitate manually the tube to mix well the content.

Note: This step might be repeated in case a further wash is required.
d. Count the number of cells in the suspension using a Neubauer counting chamber.
e. Centrifuge at 110 x g for 4 min at 4 °C.
f. Resuspend the pellet in organoid growth medium (Table 1). Calculate the total medium volume needed for a seeding density of 10 5 cells/cm 2 .  Note: This step is important to remove all EDTA. 7. After the incubation, pipette the suspension up and down during 1 min using 10 ml pipette.

Discard the supernatant and add intestinal epithelial myofibroblast digestion medium (
8. Centrifuge at 160 x g for 5 min. 9. Resuspend the pellet with 10 ml of ACK lysing Buffer (pre-warmed at 47 °C).
10. Centrifuge at 160 x g for 5 min.
11. Resuspend the pellet with 10 ml intestinal epithelial myofibroblast culture medium (Table 3)  Note: Tissue pieces will eventually attach to the flask and myofibroblasts will grow out and spread though the flask. Replace the medium every 3-4 days. Usually, 2-3 weeks after isolation flask will be confluent. Figure 7 shows representative bright field microscopy images of this process.
12. Cell are passaged by normal trypsinization for 5 min. Splitting ration 1:3. 2. Harvest the medium and centrifuge it at 160 x g for 5 min to remove debris.
Note: Add fresh intestinal epithelial myofibroblast culture medium (Table 3) to the plates and continue the culture. Passage the cells if required (Step E12).
3. Collect the supernatant and filter it using a 0.22 µm pore size filter and a 10 ml syringe.
6. Pool all ISEMF_CM fraction collected from the same ISEMF isolation and prepare the boosting medium as described in Table 4. 2. Follow the steps described in Step D1a until Step D1d to dissociate the organoids to crypts.
3. Resuspend the pellet in basic-medium (Table 5). Calculate the total medium volume needed for a seeding density of 3,000-3,500 crypts/cm 2 . Notes: a. The surface area of one 6.5 mm diameter Transwell insert is 0.33 cm 2 .
5. Let the crypts attach for 1-2 h and add 200 µl more of basic-medium (Table 5) at the apical (upper) side of the Transwell.
6. Prepare the boosting medium (Table 4) and add 500 µl to the basolateral (bottom) side of the Transwell.
7. Add the corresponding medium to the control (no cells) inserts as well.
8. Place the plate in a 37 °C humidified incubator with 5% CO2. Change the medium of both apical and basolateral sides every other day.
9. After changing medium, let the plate under the hood at RT for 5-10 min so that the medium temperature equilibrates.
10. In the meanwhile, sterilize the EVOM2 epithelial voltohmmeter STX3 electrodes by submerging them into 70% Ethanol (Table 6)  13. Remove the electrodes and reinsert to measure again. Repeat this step one more time to obtain 3 different measurements from the same sample.
14. Wash the electrodes in between measurements by submerging into PBS and then into basic medium.  Add the enzyme just before using the medium. If not prepared fresh, this medium (without adding the ENR_CV and Wnt3a factor) can be stored at 2-8 °C up to 2 weeks. ENR_CV and Wnt3a factor must be added just before used.