This protocol describes how to freeze mouse or human intestinal organoids at 200 organoids per cryovial using CryoStor® CS10. For optimal results, cryopreservation is best performed when organoids are mature.
Intestinal organoids are suitable for cryopreservation after two passages from primary culture or from organoids that have been cryopreserved, thawed, and cultured. Larger cystic or budded organoids result in a higher yield of viable fragments than smaller, dark, or collapsed organoids (Figure 1). Organoids from small intestine reach maturity between five to seven days of culture (Figure 2A). Colonic organoids mature more slowly, with less defined budding, reaching maturity between seven to 10 days of culture (Figure 2B).
Figure 1. Human Intestinal Organoids for Cryopreservation
Human intestinal organoids should be cryopreserved when they are mature, approximately 7 - 10 days post-passage. For best results, larger cystic or budded organoids should be used (red circles). Smaller, dark, or collapsed organoids (blue circles) may not provide acceptable viability post-thaw.
Figure 2. Mouse Intestinal Organoids for Cryopreservation
Light microscope visualization (10X) of mature intestinal organoids cultured in domes of 1:1 Matrigel® Matrix and IntestiCult™ Organoid Growth Medium and incubated at 37°C and 5% CO2. (A) Small intestinal organoid after five days of culture and (B) colon organoids after 10 days of culture.
Prepare all media and reagents required for this protocol. Place PBS without magnesium or calcium, DMEM/F-12 with 15 mM HEPES, and CryoStor® CS10 to cool on ice. Retrieve the plate containing the organoids to freeze.
Using an inverted microscope, count the number of mature organoids found in each well. Combine the contents of multiple wells as needed to achieve 200 organoids in each cryovial.
Remove the IntestiCult™ Organoid Growth Medium from each well containing organoids. Replace it with 1 mL of cold (2 - 8°C) PBS.
Break up the Matrigel® Matrix by pipetting up and down ten to twenty times with a PBS-wetted 1000 μL pipette tip. Transfer suspensions containing 200 organoids, combining wells if necessary, to a single 15 mL conical tube.
Wash each well with 1 mL of cold (2 - 8°C) PBS by pipetting up and down five times with a pre-wetted 1000 μL pipette tip and transfer to the 15 mL conical tube.
Pellet the organoids by centrifuging at 290 x g for five minutes at 2 - 8°C. Remove and discard the supernatant, being careful not to disturb the organoid pellet.
Wash the organoid pellet by resuspending in 7 to 10 mL of cold DMEM/F-12 with 15 mM HEPES. Gently flick the tube, or gently pipette the contents, to help break down the pellet if needed. Centrifuge the suspension at 200 x g for five minutes at 2 - 8°C then carefully remove and discard the supernatant.
Resuspend the organoid pellet in cold (2 - 8°C) CryoStor® CS10 freezing medium using 1 mL of freezing medium per cryovial of 200 organoids.
Using the same pipette tip, move the organoids suspended in CryoStor® CS10 to a labeled cryovial. Place the cryovial in a freezing container with 500 mL of isopropyl alcohol, or in an IPA-free freezing container.
Transfer the freezing container to a -80°C freezer for 24 hours then transfer the cryovial to liquid nitrogen (-135°C) for long-term storage. Intestinal organoids can be stored at -135°C for six months. Long-term storage at -80°C is not recommended.
Note: Work quickly to avoid prolonged exposure of non-frozen organoids to CryoStor® CS10.
Will organoids recover if they have been degraded before or after cryopreservation?
We have been able to show that as long as there are healthy, single, intestinal stem cells in the culture, previously degraded organoids recover after one passage (Figure 3). These organoids generally take an additional three to five days to establish and will likely require multiple passages to generate an adequate population of organoids.
Figure 3. Light Microscope Visualizations of Intestinal Organoid Cultures from Frozen Organoids That Had Been Degraded Either Before or After Cryopreservation
Organoids cultured in domes of 1:1 Matrigel® Matrix and IntestiCult™ Organoid Growth Medium are shown following incubation at 37°C and 5% CO2 at (A) Passage 0, Day 5 and (B) Passage 1, Day 6.
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