This protocol is part of a series of protocols for isolating, culturing, passaging, and cryopreserving mouse small intestinal and colonic crypts using IntestiCult™ Organoid Growth Medium (Mouse). After isolating mouse intestinal crypts, organoid cultures can be established using the procedure below.

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Materials

• IntestiCult™ Organoid Growth Medium (Mouse) (Catalog #06005)
• DMEM/F-12 (e.g. DMEM/F-12 with 15 mM HEPES, Catalog #36254)
• 6-well plate (e.g. Costar® 6-Well Plate, Catalog #38015 or Falcon® 6-Well Plate, Catalog #38016)
• 15 mL conical tubes (e.g. Falcon® Conical Tubes, Catalog # 38009)
• Corning® Matrigel® Matrix, Growth Factor Reduced (GFR), Phenol Red-Free (Corning Catalog #356231)
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• 24-well plate (e.g. Costar® 24-Well Plate, Catalog #38017 or Falcon® 24-Well Plate, Catalog #38021)
• PBS (e.g. D-PBS, Catalog #37350)
• Micropipette (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)
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Protocol

1. Resuspend each isolated mouse intestinal crypt fraction in 10 mL cold (2 - 8°C) DMEM/F-12.
2. Add 1 mL of each fraction to individual wells of a 6-well plate and assess the quality of the fractions using an inverted microscope (these samples can be added back to their respective fractions after evaluation). Select one or two fraction(s) that are enriched for intestinal crypts to carry forward for organoid culture. Crypts desirable for culture can be of various sizes, are typically rectangular or circular in shape with relatively smooth edges, and resemble small, folded sections of an epithelial monolayer (Figure 1A and 1B). Fractions with higher concentrations of villi, single cells, or debris are not suitable for organoid culture. Fractions 3 and 4 often exhibit the greatest enrichment for desirable crypts.
   
   
   

   Figure 1. Assessing Isolated Intestinal Crypt Fractions Prior to Culture

   Light microscope visualizations of intestinal crypt fractions (1 mL in well of 6-well plate) with (A) Fraction 2 at 2X magnification; (B) Fractions 3 and 4 combineed at 10X magnification; (C) Light microscope visualization of intestinal crypt fraction (10 μL on hemocytometer) appropriate for organoid culture. Intestinal crypts circled in green are suitable for organoid culture and should be used to determine crypt concentration in the fraction. Red circles indicate villi, fragments of tissue or debris not suitable for organoid culture.

3. Using a pre-wetted pipette tip, take a 10 μL sample of the selected fraction(s) and deposit on a glass slide or hemocytometer. Using an inverted microscope, count the number of crypts in the aliquot (Figure 1C). Do not count single cells or large, multi-layered tissue fragments. Multiply by 100 to estimate the number of crypts per mL in that fraction.
4. Calculate the volume of the selected fraction(s) containing approximately 500, 1500, and 3000 crypts. Transfer the required volumes to three separate, labeled 15 mL conical tubes and centrifuge at 200 x g and 2 - 8°C for five minutes. Carefully pipette off and discard the supernatant.
   
   

   Example: Crypt Fraction Volume Calculations

   Crypts counted in 10 μL aliquot: 15
   15 x 100 = approximately 1,500 crypts per mL in fraction

   Volumes to centrifuge in step 4:
   0.33 mL for 500 crypts
   1.0 mL for 1500 crypts
   2.0 mL for 3000 crypts

5. Add 150 μL room temperature (15 - 25°C) complete IntestiCult™ Organoid Growth Medium (Mouse) to the pellet in each tube. Do not use cold medium.
   
   

   How to Prepare Complete IntestiCult™ Organoid Growth Medium (Mouse)

   1. On the day of crypt isolation, remove the bottle of IntestiCult™ Basal Medium from the refrigerator and place on the benchtop to warm to room temperature (15 - 25°C). Remove the vials of IntestiCult™ Supplement 1 and Supplement 2 from the freezer and leave them to thaw at room temperature (15 - 25°C). Pipette up and down to mix Supplement 1 and 2 thoroughly.
      
      Note: Once thawed, use immediately.
   2. Make the complete medium by adding 5 mL of Supplement 1 and 5 mL of Supplement 2 to the bottle of Basal Medium. Replace the cap and mix the medium well by inverting the bottle several times.
   3. The medium must equilibrate to room temperature (15 - 25°C) before use. You will require 12 mL of complete medium to initiate organoid cultures in 12 individual culture wells (four wells for each of three plating densities) using crypts isolated from murine small intestine or colon following this protocol.
   4. Immediately before use, add desired antibiotics to complete IntestiCult™ Organoid Growth Medium. We recommend 50 μ/mL gentamicin or 100 units/100 μg per mL penicillin/streptomycin.

   Note: Complete medium can be stored at 2 - 8°C for up to two weeks. To avoid repeated freeze-thaw cycles, aliquot complete medium into appropriate volumes and freeze at -20°C for up to three months. Do not re-freeze aliquots once thawed.

6. Add 150 μL undiluted Matrigel® Matrix to each tube. Using the same pipette tip, carefully pipette up and down ten times to resuspend the pellet. Avoid introducing bubbles.
7. Carefully pipette 50 μL of the 500-crypt suspension into the center of each of four wells of the pre-warmed 24-well plate. To prevent bubbles when plating, dispense to the first stop of the pipette. The samples should form domes in the center of each well (Figure 2). Repeat for the 1500-crypt and 3000-crypt suspensions for a total of 12 wells.
   
   Note: Work quickly as the Matrigel® will begin to solidify
   
   
   

   Figure 2. Matrigel® Domes

   Photos of domes containing crypts suspended in a 1:1 mixture of Matrigel® Matrix and IntestiCult™ Organoid Growth Medium taken from (A) above and (B) the side.

8. Place the plate at 37°C for 10 minutes to set the Matrigel®. Be careful not to disturb the domes when transferring the plate to the incubator.
9. Add 750 μL room temperature (15 - 25°C) complete IntestiCult™ Organoid Growth Medium to each well by pipetting the medium gently down the sidewall of the well. Do not pipette the medium directly onto the domed cultures.
10. Add sterile PBS to any unused wells to ensure proper hydration of the cultures.
11. Place the lid on the culture plate and incubate at 37°C and 5% CO₂.
12. Monitor cultures for organoid growth. Typically, crypts form spherical structures after about three hours of incubation (Figure 3A). After two to four days of incubation, small intestinal organoids typically begin to bud (Figure 3B), and complex, multi-lobed structures form at day five to seven (Figure 3C).
    
    For Colon: Unlike the murine small intestinal system, organoid growth is slower when using colon crypts. By day two small cystic organoids will begin to appear (Figure 3D), and grow in size between days three and seven (Figure 3E). Between days seven and 10 colonic organoids can develop budding structures, though less defined than those of organoids from the small intestine (Figure 3F).
    
    
    

    Figure 3. A Timescale of Organoid Cultures from Small Intestine and Colon After Plating and Incubation

    Light microscope visualizations of intestinal crypts cultured in domes of 1:1 Matrigel® Matrix and IntestiCult™ Organoid Growth Medium are shown following incubation at 37°C and 5% CO₂ for small intestine at (A) Day 1, (B) Day 5, and (C) Day 7, and colon at (D) Day 1, (E) Day 3, and (F) Day 7.

13. Fully exchange the culture medium three times per week by carefully aspirating the existing liquid medium, keeping the pipette tip at the edge of the well bottom. Replace with 750 μL fresh, room temperature (15 - 25°C) complete IntestiCult™ Organoid Growth Medium.
14. Passage organoid cultures using a 1:6 split ratio after seven to 10 days of culture to avoid over-growth and excessive accumulation of debris within the organoid lumen.
    
    For Colon: Passage organoid cultures using a 1:2 split ratio seven to 10 days after plating or when the density reaches 150 organoids per well.

Learn how to passage mouse intestinal organoids >

Frequently Asked Questions

Can the IntestiCult™ Organoid Growth Medium be thawed in a 37°C water bath rather than at room temperature (15 - 25°C)?

Aliquots of IntestiCult™ Organoid Growth Medium can be thawed in a 37°C water bath before use; however, we do not recommend refreezing medium that has been thawed using this method.

Why shouldn’t multi-layered tissue fragments be counted when estimating the number of crypts per ml in the fraction? What are they if not aggregated crypts?

Large, multi-layered fragments are likely debris that were not separated out during the crypt isolation procedure. These pieces likely do not contain stem cells and will not develop into organoids. Such fragments are likely to be enriched in the earlier crypt fractions, and using the later fractions should eliminate most of these multi-layered structures.

Why can’t cold IntestiCult™ medium be used in step 5? What should I do if I accidentally used cold IntestiCult™?

Room temperature medium should be used to mix with the crypts and Matrigel® Matrix as cold medium will dissolve the matrix. If you accidentally use medium that has not warmed to room temperature, allow the mixture to warm to room temperature before plating as domes in the pre-warmed 24-well plate. Note that once the medium and Matrigel® Matrix are combined, you will need to work quickly as it will start to solidify. The four wells should be plated within 30 - 60 seconds. Cooling the mixture briefly on ice will lower the viscosity again if the wells are not plated within this time.