TITLE: Overcoming problematic growth phenotypes in organoids from patients with monogenic GI disease
AUTHORS: Katlynn Bugda Gwilt, Jay Thiagarajah

[DESCRIPTION]
Patient-derived organoids provide a unique model system to explore disease causing mutations ex vivo. By use of organoids from duodenal or colonic biopsies of pediatric patients with intestinal epithelial disorders, we can directly assay the patient cells to tailor treatment to their unique disease state. The advent of organoid technology from patients with severe intestinal disorders such as either Congenital Diarrhea Enteropathies (CoDE) and Very-Early-Onset Inflammatory Bowel Disease (VEO-IBD) has allowed for rapid advances in the understanding of and the treatment of these monogenic disorders. Still, the expansion of these lines for scalable studies is not trivial, and success rates of expansion are variable between groups, and even lab members within the same group. These protocols have been validated on patients with CoDE or VEO-IBD and age matched control patients. Here, we present our recommended protocols for the cultivation of organoids from pediatric patients with CoDE and VEO-IBD.  These protocols have been validated on organoids generated from the duodenum (duodenoids), ileum (ileoids), colon (colonoids) and iPSC derived intestinal colonoids from pediatric healthy donors or donors with CoDE or VEO-IBD1. Using our modified culture media, extended culture times from biopsy preparation and thawing frozen lines, gentle passaging techniques with the incomplete removal of the organoids from the matrigel, and modified monolayer protocols2,3, we have been able to successfully culture and expand several lines for >5 years. The conditions and protocols used here provide a basis for reproducible phenotypes, scaling for larger functional studies on patient lines, and for reproducibility of results between several investigators. We provide a useful starting point and troubleshooting guidelines for optimization of culturing organoids from any patient with novel disease pathology

[BEFORE_START]
Ensure that your lab has an up-to-date IRB allowing for human subject research.

[STEPS]
SECTION: Thawing Frozen Lines
8. 300 x g Centrifuge at 300 x g for 5 minutes

SECTION: Thawing Frozen Lines
9. Aspirate media leaving all of the residual matrigel from freezing behind
SECTION: Thawing Frozen Lines
10. Add 200ul of fresh matrigel to the organoid/matrigel pellet
SECTION: Thawing Frozen Lines
11. Gently resuspend using P1000 and 1000ul tip
SECTION: Thawing Frozen Lines
12. Plate 40ul/well of a 24-well plate and allow to polymerize for 20 minutes at 37°C
SECTION: Thawing Frozen Lines
13. Add 1ml of Plating Media per well
SECTION: Thawing Frozen Lines
14. Keep in the 1ml Plating Media for ~72 hrs (i.e. Fri-Mon)
SECTION: Thawing Frozen Lines
15. Resume every other day media changes after the initial 72 hour period
SECTION: Organoid Expansion
16. Room temperature When organoids are highly stem like, add 1.0 mL of RT PBS to each well containing matrigel and organoids.

SECTION: Organoid Expansion
18. 300 x g Centrifuge the organoids at 300 x g for 5 minutes.

SECTION: Organoid Expansion
19. Aspirate the PBS and the upper most layer of matrigel.
 

SECTION: Organoid Expansion
20. Room temperature Wash the remaining pellet and matrigel once more with RT PBS and pellet organoids.

SECTION: Organoid Expansion
21. 300 x g Centrifuge the organoids at 300 x g for 5 minutes.

SECTION: Organoid Expansion
22. Aspirate the PBS and remove as much of the matrigel as possible leaving only the lower-most layer of matrigel that has organoids suspended.
SECTION: Organoid Expansion
23. Room temperature Add 1ml of complete base medium.

SECTION: Organoid Expansion
24. Using P200 pipet and pipette tip, triturate at medium intensity.  Triturate approximately 30 times.  Organoids should be fluffy floating in the media along the edges. For diseased lines, pipette with less force to retain viability.
SECTION: Organoid Expansion
25. on ice Add 4 ml of ice-cold PBS to the fragmented organoids to facilitate the removal of debris.

SECTION: Organoid Expansion
26. 300 x g Centrifuge the organoids at 300 x g for 5 minutes.

SECTION: Organoid Expansion
27. Aspirate JUST the PBS/base media mixture leaving almost no media on top of the matrigel pellet
SECTION: Organoid Expansion
28. Add 50ul of full growth media to the pellet and resuspend
SECTION: Organoid Expansion
29. Add 35ul of fresh matrigel per expected wells to the pellet and resuspend slowlyto avoid bubbles.
 
   

SECTION: Organoid Expansion
17. Gently scrape the matrigel from the bottom of the well using a P1000 tip and transfer the entire contents of the well to to a 15ml conical tube. 

SECTION: Plating Patient-derived organoids as monolayers
35. To coat each transwell well with collagen, add 100ul of working concentration (33ug/ml) of collagen to each transwell. Be sure the entire area of the transwell is coated with collagen. 
SECTION: Plating Patient-derived organoids as monolayers
36.   Be sure the plate is covered and incubate at 4°C overnight. 
SECTION: Plating Patient-derived organoids as monolayers
37. On the day of plating wash both the apical and basolateral chamber with base media prior to plating organoids. 

SECTION: Plating Patient-derived organoids as monolayers
38. Aspirate medium from each well of the 24 plate and add 1.0 mL of cell recovery/dissociation solution directly to the matrigel. 
SECTION: Plating Patient-derived organoids as monolayers
40. 300 x g Centrifuge the organoids at 300 x g for 5 minutes.

SECTION: Plating Patient-derived organoids as monolayers
41. Aspirate the cell recovery solution and wash the remaining pellet once more with ice cold cell recovery media.
SECTION: Plating Patient-derived organoids as monolayers
43. Aspirate the cell recovery solution and remove as much of the supernatant as possible
SECTION: Plating Patient-derived organoids as monolayers
44. Add undiluted Trypsin+EDTA to enteroids; mix gently 5X with P200.
SECTION: Plating Patient-derived organoids as monolayers
45. 37 °C Place in a 37C water bath for absolutely no greater than 90seconds.

SECTION: Plating Patient-derived organoids as monolayers
46. Spray with ethanol prior to bringing back into the hood.
SECTION: Plating Patient-derived organoids as monolayers
47. Immediately add 5ml of chilled complete base media.
SECTION: Plating Patient-derived organoids as monolayers
48. Triturate with P200 to finish the fragmenting of the organoids.
SECTION: Plating Patient-derived organoids as monolayers
50. Aspirate the supernatant and gently suspend the organoid fragments in the plating media using a P200. Add 160ul for every transwell to be plated.

SECTION: Plating Patient-derived organoids as monolayers
51. After the transwells are washed, add 1000μl of plating media to the basolateral chamber. 
SECTION: Plating Patient-derived organoids as monolayers
52. Make sure the organoid pellet is evenly dispersed when resuspended. Pipet 155μl of organoids to the center of each transwell SLOWLY.
SECTION: Plating Patient-derived organoids as monolayers
53. GENTLY place the plate in the incubator. We recommend placing at the back of your incubator shelf at 37°C. 

 

SECTION: Plating Patient-derived organoids as monolayers
54. On day 4 post plating, gently bring the plate out of the incubator and into the TC hood, and aspirate the basolateral media without disturbing the apical media. Add 1000μl of fresh media to each transwell.
55. Do not change the apical media until day 6 post plating.
56. Proceed with every other day media changes as your monolayer becomes stable. The monolayer will take 7-14 days to become confluent. 
SECTION: Plating Patient-derived organoids as monolayers
49. 300 x g Centrifuge the organoids at 300 x g for 5 minutes.

57. Once the monolayer is fully confluent, switch to differentiation media for 5 days. 
58. On day 5, post-differentiation media, take the transepithelial electrical resistance (TEER) of the monolayer. 

 
  

SECTION: Preparation of Media
1. Prepare conditioned media (CM) from L-WRN cells, L-Wnt3a cells, 293T-R-Spondin-1 Cells, and 293T-Noggin Cells as described previously. 
 

SECTION: Preparation of Media
1.1. Aliquot L-WRN media into single use aliquots - recommended at 25ml each for duodenum expansion media or 37.5ml aliquots for colonoid expansion media.
 

SECTION: Preparation of Media
1.2. Create a Wnt3A, R-Spondin-1 and Noggin single use aliquot by combining each component into one tube as follows:
10 ml Noggin CM
5 ml Wnt3A CM
10 ml R-Spondin 1 
SECTION: Preparation of Media
2. Prepare base media as follows:
Base Media
Advanced DMEM/F12 	~80% (v/v)
FBS (as supplied)	~20% (v/v)
Pen/Strep (100x)*	~1% (v/v)

SECTION: Preparation of Media
3. Prepare Colonoid or Duodenoid Expansion Media as follows:
Colonoid Expansion Media
L-WRN Conditioned Media 	50% (v/v)
Wnt/Rspondin-1/Noggin CM Mix	25% (v/v)
Base Media	25% (v/v)
GlutaMax 	1x
HEPES 	(10mM)
Primocin	1x
Normocin 1x
B27 	1X
N2 	1X
Nicotinamide 	10mM
N-Acetyl-Cysteine 	500μM
A8301	500nM
SB202190	10μM
EGF	100ng/mL
Gastrin	10nM
Prostaglandin E2	100nM



Duodenoid Expansion Media
L-WRN Conditioned Media 	40% (v/v)
Wnt/Rspondin-1/Noggin CM Mix	20% (v/v)
Base Media	32% (v/v)
GlutaMax 	1x
HEPES 	(10mM)
Primocin	1x
Normocin 1x
B27 	1X
N2 	1X
Nicotinamide 	10mM
N-Acetyl-Cysteine 	500μM
A8301	500nM
SB202190	10μM
EGF	100ng/mL
Gastrin	10nM
 
    

SECTION: Preparation of Media
4. Prepare Plating Media as follows:
Plating Media:
Add:
Y-27632	10μM & CHIR 99021 250nM
to Duodenoid or Colonoid Expansion media as prepared above
 

SECTION: Thawing Frozen Lines
7. Using P1000 move frozen cells from vial to conical tube of chilled complete base media
SECTION: Preparation of Media
5. Prepare Differentiation Media as follows:

Differentiation Media:
Base Media 90% (v/v)
Noggin Conditioned Media 10% (v/v)
Glutamax 1x
HEPES 10mM
Primocin 1x
Normocin 1x
B27 1X
A8301 500nM
EGF 50ng/ml
Gastrin 10nM
SECTION: Thawing Frozen Lines
6. Thaw organoid vial in a 37C water bath until a small ice chunk remains
59. Proceed with functional experiment. 
SECTION: Organoid Expansion
30. Plate matrigel and organoids in 35ul droplets in the center of the well of a 24-well plate. 
SECTION: Organoid Expansion
31. Add 500 ul of media. Continue with media changes every other day until experiment.
SECTION: Plating Patient-derived organoids as monolayers
33. Prepare stock collagen by dissolving to a final concentration of 1mg/ml in 100 mM acetic acid. Aliquot and store at -20C until use.
SECTION: Plating Patient-derived organoids as monolayers
34. Prepare working concentration of collagen by dissolving the stock collagen to a working concentration of 33ug/ml collagen in cell culture water
SECTION: Plating Patient-derived organoids as monolayers
32. Expand organoids to plate in a ratio of 1.5 wells/2 transwells (Figure 2B) or 1.5 well/2 transwells (Figure 2C/D). Ensure that the organoids are still cyst like with little differentiation.  
SECTION: Plating Patient-derived organoids as monolayers
39. Scrape the matrigel from the bottom of the plate and transfer the entire contents of the well to a 15ml conical tube.
SECTION: Plating Patient-derived organoids as monolayers
42. 300 x g  Centrifuge the organoids at 300 x g for 5 minutes.