HI All,

Could some provide some suggestions on improving the mouse intestinal organoid culture? here are the steps we did: 1. flush out the feces and wash intestinal pieces in cold PBS 5 times. 2. incubate in 2.5mM EDTA/PBS for 30 minutes. 3. Use a transfer pipette to pipette up and down 10 times, and filter fraction 3 and 4 through a 70 μm filter. 4. Centrifuge filtered fractions at 200g, 4 degree for 5 min to separate crypts from single cells. 5. wash and then Resuspend crypt fractions in 5 mL cold (2 - 8°C) DMEM/F-12. 6. count crypts and transfer needed crypt numbers and recentrifuge then resuspend in matrigel. 7. Finally culture them in IntestiCult™ Organoid Growth Medium. The problems we have are: 1) too many debris, such as those small pieces that can filter through 70 μm stainer. 2) the crypts do not bud very well. The growth of organoids is not consistent between each culture. I greatly appreciate if you could provide suggestions to resolve these problems.