Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

All procedures described below have been approved and conducted in accordance with the guidelines set by University of Michigan Institutional Animal Care and Use Committee.

1. Preparation of reagents for crypt isolation and culture (prepare in tissue culture hood)

1. 50 mM ethylenediamine tetraacetic acid (EDTA): Add 50 mL 0.5 M stock to 450 mL of phosphate buffered saline, without calcium (Ca²⁺) and magnesium (Mg²⁺) to prepare 500 mL. In this protocol PBS will refer to PBS without calcium and magnesium unless otherwise stated.
2. Shake buffer: Dissolve 7.4 g sucrose (43.3 mM) and 5 g sorbitol (54.9 mM) in PBS to prepare 500 mL.
3. L-WRN (L-Wnt-3A, R-spondin and Noggin) media: Supplement Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 (DMEM/F12) (780 mL) with 20% Fetal Bovine Serum (FBS) (200 mL), 1x commercially available glutamine supplement (10 mL), 100 U/mL penicillin and, 100 g/mL streptomycin (10 mL), and filter sterilize with 0.22 µm filter.
   1. Get L-WRN cells through ATCC, grow in T175 flasks, and select using Geneticin and Hygromycin. Media is changed and collected for 12 days.
      NOTE: Every batch of media is tested for Wnt activity using a TOPflash Wnt Reporter assay. In this case the Michigan Medicine Translational Tissue Modeling Laboratory protocols (https://www.umichttml.org/protocols) were followed. The TOPflash HEK 293 cell line is grown to confluency in a T75 flask, trypsinized and plated onto a 96-well plate. The following day different dilutions of the collected media is added to cells and incubated in a 5% CO₂ incubator at 37 °C overnight. Next day, the cells are lysed, and Firefly Luciferase assay is performed according to manufacturer's instructions. The assay is normalized using recombinant Wnt-3A. The media is divided into 25 mL aliquots in 50 mL conical tubes and store at -80 °C.
4. Base media: For 500 mL, supplement Advanced DMEM/F12 (448 mL) with 2x commercially available glutamine supplement (10 mL), 20 mM HEPES (10 mL), 100 U/mL penicillin and, 100 g/mL streptomycin (10 mL), 2 mM N-acetyl-L-cysteine (2 mL), N2 supplement (10 mL) and B27 supplement (20 mL), filter sterilize with 0.22 µm filter. Divide the media into 25 mL aliquots in 50 mL conical tube and store at -80 °C.
5. LWRN complete media: Combine 25 mL LWRN media with 25 mL of base media and supplement with 200 ng/mL Epidermal growth factor (EGF) (20µL) and 2x antibiotic-antimycotic solution (1 mL). Store the complete media at 4 °C.
6. Collagen and Laminin: Dissolve 5 mg powder in 5 mL of filter sterilized 100 mM acetic acid (add 60 µL of acetic acid stock to 9.94 mL of molecular grade water) to produce a stock concentration of 1 mg/mL. Rotate at 4 °C for 4 h and make 100 µL aliquots in 0.2 mL tubes. Freeze at -20 °C. Laminin is purchased at a stock concentration of 100 µg/mL.
7. Complete media without growth factors (CMGF-): Supplement Advanced DMEM/F12 (500 mL) with 1x commercially available glutamine supplement (5 mL), 10 mM HEPES (5 mL), 100 U/mL penicillin and, 100 g/mL streptomycin (5 mL).
8. Differentiation media: To 9.2 mL of CMGF- media, add 200 µL of B27 supplement, 100 µL of N2 supplement, 20 µL of N-acetyl-L-cysteine, 500 µL of Noggin media¹² (made from Noggin-producing cells) and 2 µL of EGF to make 10 mL of differentiation media.

2. Preparation of plates, chamber slides and cell culture membrane inserts

1. Coating 48-well plate and chamber slides for plating 2D monolayer: Use the coating solution which constitutes laminin (1:40 dilution, see Table of Materials) and collagen (1:30 dilution) in cold Dulbecco's Phosphate buffered saline, with Ca²⁺ and Mg²⁺ (DPBS). Add 200 µL of coating solution to each well and pre-incubate the plate/chamber slide in a 5% CO₂ incubator at 37 °C for at least 2 h.
2. Coating 0.4 µm cell culture membrane inserts: Make 1:30 dilution of collagen in molecular grade water and add 200 µL to each insert. Keep the plate containing the membrane insert on ice at 4 °C for 30 min. Following 30 min incubation, keep the plate in a 5% CO₂ incubator at 37 °C for 1.5-2 h. Polyester and polycarbonate membrane inserts yield comparable results.
   NOTE: Any tissue culture plate can be seeded (up and downscaling can be performed) using this protocol by adjusting the collagen/laminin volume to obtain full coverage of the plating surface.

3. Crypt isolation

NOTE: Before starting the dissection, prepare collagen and/or laminin coated plate/membrane inserts/chamber slide and leave them in a 5% CO₂ incubator at 37 °C. Prepare a clean work bench and sterile surgical instruments appropriate for surgery, and a biological safety cabinet for culturing 2D monolayers. Confirm all other standard equipment for 2D monolayer culturing such as humidified CO₂ incubator, tabletop centrifuges (maintained at 4 °C), microscope and pipettes (including serological pipettes) are ready.

1. Use C57Bl/6 mice, 8-12 weeks old. Euthanize mice using an approved method of euthanasia.
2. Spray the mice carcasses with 70% ethanol (EtOH) solution to clean the dissection area and remove excess EtOH using paper tissue.
   NOTE: Ensure that the crypt isolation reagents (such as PBS, 50 mM EDTA, shake buffer) crypts are kept cold. These reagents can be prepared one-day prior and are good to use for at least 3 months when stored at 4 °C. Also, make sure that the LWRN complete media is kept in a bead/water bath maintained at 37 °C until use.
3. Using clean dissection scissors and forceps, dissect out the colon from the rectum to the cecum. Hold the end of the colon using forceps and very gently flush off the feces using ice cold PBS in a 10 mL syringe, fitted with a 20 G feeding tube (Figure 1A). Make sure not to rupture the colon.
   1. Remove the proximal colon. This will be the portion of the colon closest to the ileocecal junction.
4. With forceps, slide the distal colon gently onto the 20 G feeding tube, tying off the colon at the end of the tube by the tip with 4-0 silk suture thread (Figure 1B).
5. Invert the colon inside out using one's fingers over the end tied off and tie the other end with 4-0 silk suture thread. Using surgical scissors, cut colon below the tip of the feeding tube (Figure 1C,D,E).
6. Using the plunger of a 1.25 mL repeat syringe, gently open the untied end of the inverted colon onto the tip of a 1.25 mL repeat syringe. Slide the inverted colon onto the end of the syringe and tie tightly with 4-0 silk suture thread (Figure 1F,G).
7. Insert the plunger into the syringe and inflate the colon to form a sausage. Inflate until the colon sausage looks turgent with no visible wrinkles (Figure 1H).
8. Place syringe/colon in 15 mL tube with 5 mL of Cell Recovery Solution on ice for 20 min, inflate and deflate the colon once every 5 min (Figure 1I). The sausage must remain inflated during the incubation.
9. Tie off using 4-0 silk suture thread below the tip of the repeat syringe with the colon inflated. Cut the sausage off the repeat syringe and place in 15 mL tube containing 10 mL of 50 mM (2mM for small intestine) EDTA for 40 min and rotate at 4 °C (Figure 1J,K).
10. Decant off the EDTA solution and replace with 5 mL of shake buffer. Shake the sausage manually in vertical position (vigorously) for 2 min.
11. Decant the shaking solution into a new 15 mL tube and repeat shaking step for a total of 10 mL of crypts in shaking buffer.
12. Take 20 µL of the crypt suspension in a Petri dish and count the number of crypts under a microscope. Calculate the crypt concentration in crypts/µL. Depending on the concentration, dilute the samples to obtain 5 crypts/ µL at the moment of plating (1000 crypts/cm²).
13. Spin the tube with isolated crypts using tabletop centrifuge at 400 x g for 10 min at 4 °C.
14. In the meantime, remove 48-well plate/insert/chamber slide from the incubator and place it in biosafety cabinet. Aspirate the coating solution using P200 and leave the plate with the lid slightly offset until cells are ready to be plated.

4. Culturing 2D monolayer

NOTE: For a detailed protocol on how to generate intestinal epithelial monolayers from 3D colonoids check protocols by Estes and Kovbasnjuk labs (^7,11).

1. Remove the shaking buffer using 10 mL serological pipette. Make sure that the pellet is intact and may using P1000 to remove any liquid left. Re-suspend the pellet in 3 mL of LWRN complete media and pipette up and down with P1000. Add 200 µL of crypts to each well of a pre-coated 48-well plate/chamber slide and incubate in a 5% CO₂ incubator at 37 °C.
2. The following day aspirate the media using P200 and add fresh media. The cells become confluent in 24-48 h.
3. For cell culture membrane inserts, add 200 µL crypts (5 crypts/µL) to top of the inserts and 600 µL of complete L-WRN media to the bottom part. The following day aspirate the media using P200 and add fresh media only to the top chamber. Incubate the plate in a 5% CO₂ incubator at 37 °C. Transepithelial Electrical Resistance (TEER) is measured every day using Epithelial Volt/Ohm meter (EVOM).
   NOTE: If the culture has a TEER reading greater than 300 Ω.cm², they are meant to be confluent. Confluency is achieved in 3-4 days. Change media every 2 days.