Yes, antibiotics should be added because the organoids are generated from the crypts isolated from the intestine, which are lined with microbiota. The addition of antibiotics can minimize the chances of bacterial contamination. We advise adding antibiotics immediately before using the medium.

We recommend TC-treated plates. With TC-treated plates, the domes are more spread out; with non-TC-treated plates, the domes are more compact and stand higher, which can increase the risk of shearing at the top of the dome when performing a medium change.

We used C57BL6, 8-week old males for standardization; however, females can be used if desired.

No, all BSA sources should suffice.

Aliquots of IntestiCult™ OGM (Mouse) 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.

Morphology is the key determinant; you want a dark lumen with lots of buds. However, a darker lumen also means that more cells are dying and creating a toxic environment, which can cause deterioration of the buds. Therefore, passage the organoids before the lumen gets too dark. Note that the morphology of mouse intestinal organoids from colonic samples may look different and have fewer budding structures.

We recommend using the same amount of IntestiCult™ OGM (Mouse) for all densities. High-density domes of 150 organoids are optimal. With more organoids per dome, the rate of nutrient consumption is faster and the media turns yellow due to increased metabolism and will therefore need to be changed more frequently. If organoids are sparse (20 per well), less nutrients are consumed and you can increase the time between media changes.

Bigger fragments grow better, whereas single cells or small fragments have much lower growth efficiency (approximately 10 - 15%) but will still grow.

Growth % = (Day 5 mature organoids counts)/ (Day 1 total counts)*100
Example: If you counted 20 fragments at Day 1, and 12 of these grew into mature* organoids by Day 5 then growth % = 12/20 = 60%

Yes, and room temperature media is also okay.

Organoids need to be seeded in a dome to prevent them from sinking to the bottom of the plate. If organoids make contact with the plastic they tend to differentiate and die. In addition, nutrients from the medium will find the center of the dome more readily versus a flat matrix.

First 10 cm, but the whole small intestine should work well.

The optimal density is 150 organoids per 50 μl Matrigel® dome. If the dome appears too dense, you can passage the organoids earlier as subsequent passages will rescue the organoids.

Cold GCDR will help further dissolve the Matrigel®; however, it can also be used at room temp for our protocol because the mechanical pipetting up and down also dissolves the Matrigel®.

Passage the organoids when they look mature, not based on the level of debris. Mature = dark lumen and multiple buds. If you have lots of debris, consider doing an extra wash step when passaging.

Yes, you can scale up and make a relative calculation. There is no limit on how many domes to combine. Generally, we combine a maximum of eight domes into one 15 mL tube.

Try to create a flow of liquid; you can use an orbital shaker or use your hand to rock. You want to avoid having the organoids settle to the bottom of the tube during this step.

Yes, high-throughput robotic systems work. Mechanical disruption without GCDR works but not as well because GCDR helps keep the fragments more consistent in size.

One vial of organoids post-thaw (P0) can be split into four domes. This can be considered as the “recovery” phase and is not for expansion of the organoids. You would then passage these wells at a 1:1 split ratio; this is the growth phase (P1). The next passage is for expansion (P2) using a 1:4 or 1:6 split ratio. We suggest adding enough media/Matrigel® to prepare 6 domes to account for pipetting.

Yes, there is a time range of being able to cryopreserve the organoids. Day 6 has maximum budding within each organoid (LGR5+ stem cells and Paneth cells are contained in the buds, which will develop into new organoids). You can freeze down organoids at days 2 - 3 if there is a clear LGR5+ stem cell component, however, they may regrow at a lower efficiency.

This is not recommended, entire organoid structures will break down and fragments will die off during the thawing process and you will lose the LGR5+ stem cell components.