Preparation and culture of mouse intestinal organoids

Euthanize mouse in chemical hood with isoflurane followed by cervical dislocation in accordance with IACUC-approved protocol.

Dissect intestine

Collect tissue from desired intestinal regions.

Remove luminal debris

Loosen epithelium

Deplete damaged epithelium

Isolate epithelial units

1.           Use sterile forceps to transfer the intestine from the second tube of Intestinal

Dissociation Solution to the second tube of Crypt Shake Solution

2.           Shake sample at 2.5 cycles per second for 2 minutes

2a.           Can be extended for large samples containing multiple regions or pooled animal samples

2aa.           Tissue fragments float when depleted of epithelium

2aaa.           Adipose attached to the intestine can cause premature floating

3.           Remove a 25 µL aliquot of the Crypt Shake Solution and examine by phase microscopy

3a.           The solution should contain intact villus/crypt units as well as intact villi and

intact crypts

3aa.           The solution can be compared to the contents of the first shake tube

3b.           Use forceps to transfer a piece of digested intestine to the bacterial plate and

examine with a tissue culture microscope. The remaining crypts appear as dense

circles in the epithelium.

3c.           NOTE: If crypt yield is sub-optimal and many crypts are visible in the remaining tissue,

repeat the shaking step using reserved tissue. To avoid damaging crypts

isolated from the first round of shaking, transfer tissue to a new tube with

Epithelium Shake. Pool crypts before proceeding to the next step.

Remove digested tissue using forceps for a sterile cotton swab.

1. Using sterile forceps, transfer digested intestine to a storage tube.

1a. Optional: Reserve on ice in the event of catastrophic failure at downstream steps .

2. Optional: Process intestinal fragment for histology to examine dissociation efficiency.

1.  (Optional) Separate Crypts from isolated whole epithelial units

1. Filter isolated epithelial unit solution produced by SOP: Small Intestine Epithelial

Dissociation through a 100 µm cell strainer

Gently tap the tube to speed up filtration. Two strainers may be necessary if the whole small intestine is used for crypt isolation.

Remove a 25 µL aliquot of the Crypt Shake Solution and examine by phase microscopy

The solution should be enriched for crypts but may contain villi fragments that are similar

to crypt size

2. Filter the flow-through using a 70 µm cell strainer

      Gently tap the tube to speed up filtration. Two strainers may be necessary if the whole small intestine is used for crypt isolation.
   
     Remove a 25 µL aliquot of the Crypt Shake Solution and examine by phase microscopy
   
            The solution should be enriched for crypts but may contain villi fragments that are                   similar to crypt size
   

1. Pellet crypts for plating

1. Remove 3 separate 5 µL aliquots of the filtered crypt solution and examine by phase microscopy
2. Quantify the average crypt contents of the aliquots and extrapolate to determine the total crypt yield
3. Mix the filtered crypt solution by flicking and transfer an adequate volume to a

microcentifuge tube

4. Volume is determined by the amount of Matrigel being utilized

5. Transfer 5 crypts/1 µL Matrigel for plating

6. well then aliquot enough crypts for 5 crypts/1 ul Matrigel to 1.5 mL centrifuge tube or

up to 5000 single cells per well

7. Centrifuge on benchtop centrifuge in a cold room at 2000g for 5 minutes

8. Remove supernatant and examine an aliquot by phase microscopy to ensure crypts are

pelleted efficiently

NOTE: >80% of intact crypts will form organoids. Contaminating single cells that include stem cells can also form organoids.

1. Embed in Matrigel

1. Centrifuge Matrigel at max speed for 15-30 sec in benchtop centrifuge in a cold room.

This will pellet any insoluble collagens that may be in the Matrigel.

2. The amount of insoluble material varies by Matrigel lot.

3. R e-suspend pellet directly into the appropriate volume of Matrigel. Mix very well by pipetting

~25-50 times. *Note: Cells and crypts may be plated in up to a 1:1 dilution in Matrigel. .

Use pre-chilled tubes and tip *Optional: Perform pipetting steps on ice. ice.

4. When pipetting Matrigel from aliquot, pipet up and down one time to coat the inside

of the tip. Or, push the pipette plunger slightly past the stop so that no bubbles will be added to the Matrigel when the depressing plunger

DO NOT introduce bubbles into the Matrigel.

5. Plate by adding Matrigel as a hemispheric ‘dome’ to the center of each well in a

pre-warmed plate

For 48 well plate, use 25-50 uL Matrigel

For 96 well plate, use 8-10 µL Matrige *Note: Place pipette tip on the bottom of the middle of the well, lift slightly, then depress to first stop to plate Matrigel in hemispheric droplet let

6. Carefully transfer the plate to a 37°C incubator and allow the Matrigel to polymerize for

30 minutes

1. Overlay Media

1. Remove the plate from the incubator

2. Add 10-20x Matrigel volume of desired Culture Media

3. Continue to change culture media on an appropriate schedule

Typically, every 2-3 days

4. Transfer into a standard tissue culture incubator

Organoids are ready for passage after 6-12 days or when the lumens begin to burst and expel contents into the Matrigel

1. Digest the matrigel and fragment organoid units

1. Remove culture media from wells being passaged

2. Wash wells in DPBS containing matrigel cultures to remove residual media

3. Add 1.5x culture media volume removed in step 1

4. Remove DPBS and discard

5. Add a small volume of TrypLE +y27 to each well being passaged

6. Add 2x the volume of matrigel in TrypLE +y27

7. Transfer organoids in matrigel for digestion

8. Fragment the matrigel domes in each well using a pipette tip

9. Transfer the fragmented matrigel in the conical tube containing the full volume of TrypLE

+y27 being employed  

10. Digest the matrigel fragments at 37^oC for 5 mins

11. Place the tube upright in a water bath

12. Quench the TrypLE digestion with cold BS

13. Add 10% of the TrypLE solution volume and mix by inverting .

1. Embed organoid fragments in Matrigel

1.Centrifuge matrigel at max speed for 15-30 sec in benchtop centrifuge in a cold room.

2. This will pellet any insoluble collagens that may be in the Matrigel.

3. The amount of insoluble material varies by Matrigel lot.

4. Pellet organoid fragments for plating

5. Centrifuge digestion solution containing organoid fragments at 2000gs for 5 mins

6. Discard supernatant

7. Re-suspend organoid fragments in the appropriate volume of Matrigel.

   Typically 4x the original matrigel volume is used

 Mix very well by pipetting (~40x) without adding bubbles to the system

8. Plate organoid fragments in hemispheric matrigel ‘domes’ in the center of each culture

well

Plate matrigel domes in the center of wells in a new tissue culture plate

For 48 well plate, use 25-50 uL Matrigel

For 96 well plate, use 8-10 µL Matri *Note: Place pipette tip on the bottom of the middle of the well, lift slightly, then depress to first stop to plate Matrigel in hemispheric droplet roplet

9. Transfer plate into tissue culture incubator

10. Incubate at 37^oC for 45 mins to polymerize matrigel

1. Overlay Growth Media on Matrigel Domes

1. Remove the plate from the incubator

2. Add 10-20x Matrigel volume of desired Culture Media

3. Continue to change culture media on an appropriate schedule

Typically, every 2-3 days

4. Transfer into a standard tissue culture incubator