TITLE: Preparation and culture of mouse intestinal organoids
AUTHORS: arpine.sokratian, Ian Williamson, Ramos Chavez Katherine, Liddle Roger

[DESCRIPTION]
This protocol details an approach for the dissociation of colon epithelial cells from the murine samples. The key goal is to yield intact crypt samples suitable for culture and further dissociation into organoids. The protocols outline specific tools and reagents required for each step ensuring the isolation of crypt units with minimal contamination from other cell types. 

[STEPS]
SECTION: Dissect Mouse Intestine
1. 
 

SECTION: Dissect Mouse Intestine
2. Euthanize mouse in chemical hood with isoflurane followed by cervical dislocation in accordance with IACUC-approved protocol.
SECTION: Murine Small Intestine Epithelial Dissociation
6. 
 

SECTION: Dissect Mouse Intestine
3. Dissect intestine
 

SECTION: Dissect Mouse Intestine
4. Collect tissue from desired intestinal regions.
 

SECTION: Dissect Mouse Intestine
5. Remove luminal debris
 

SECTION: Murine Small Intestine Epithelial Dissociation
10. 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.
SECTION: Murine Small Intestine Epithelial Dissociation
7. Loosen epithelium
 

SECTION: Murine Small Intestine Epithelial Dissociation
8. Deplete damaged epithelium
 

SECTION: Murine Small Intestine Epithelial Dissociation
9. 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.
SECTION: Embedding Cypts in Matrigel for Organoid Culture
11. 
 

SECTION: Embedding Cypts in Matrigel for Organoid Culture
12. 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
SECTION: Embedding Cypts in Matrigel for Organoid Culture
13. 1.   Pellet crypts for plating
  Remove 3 separate 5 µL aliquots of the filtered crypt solution and examine by phase
microscopy
   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. 
SECTION: Embedding Cypts in Matrigel for Organoid Culture
14. 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. Re-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 tips.
*Optional: Perform pipetting steps on 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 Matrigel
*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
6. Carefully transfer the plate to a 37°C incubator and allow the Matrigel to polymerize for
30 minutes
SECTION: Embedding Cypts in Matrigel for Organoid Culture
15. 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

 
SECTION: Passage Organoids Grown in Matrigel
16. 
 

SECTION: Passage Organoids Grown in Matrigel
17. 1.   Digest the matrigel and fragment organoid units 
  Remove culture media from wells being passaged
   Wash wells in DPBS containing matrigel cultures to remove residual media
   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 37oC 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.

SECTION: Passage Organoids Grown in Matrigel
18. 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 Matrigel
*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
9. Transfer plate into tissue culture incubator
10. Incubate at 37oC for 45 mins to polymerize matrigel
SECTION: Passage Organoids Grown in Matrigel
19. 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