Small Intestinal Organoid Cytokine Set, Mouse

Preparation of Complete Culture Medium for Mouse Small Intestinal Organoids: Mix the basal culture medium, cytokines (1-4), and various additives in the predetermined proportions to prepare the complete culture medium.

1. Primary Culture
- (1) In the laminar flow cabinet, completely remove the mouse small intestine tissue and place it in pre - cooled PBS (with penicillin, streptomycin, and primary antibiotics). Using sterilized scissors, longitudinally cut open the intestinal segments, then unfold them with the intestinal cavity facing up. Scrape the intestinal impurities and residues with a cover slip, back and forth two or three times, and rinse the small intestine with pre - cooled PBS.
- (2) Hold one end of the small intestine with tweezers, cut it into small segments of about 3 - 5mm with scissors, and collect and transfer them to a 50ml sterile centrifuge tube. Then add pre - cooled PBS to wash two or three times.
- (3) Add 30ml of 5mM EDTA/PBS solution (30ml PBS + 300ul 0.5M EDTA) to the centrifuge tube. Place the small intestine tissue into the centrifuge tube for digestion. Digest on a shaking bed at 4℃ for 20 - 30min. Frequently observe the situation under a microscope during this period. The detachment of crypts is the signal to terminate the digestion. The total digestion time should not exceed 40min.
- (4) Discard the digestion solution, add pre - cooled PBS, and gently shake to remove EDTA.
- (5) Add 30ml of pre - cooled PBS containing 0.1% BSA, and vortex to shake off the crypts from the small intestine.
- (6) Retain the supernatant, pass it through a 70µm cell strainer, and evenly distribute the filtered cell suspension into two 15ml centrifuge tubes. Centrifuge at 1000rpm for 5min, and then discard the supernatant after centrifugation.
- (7) Repeat steps 5 - 6 twice to increase the number of crypts obtained.
- (8) Resuspend the precipitate with an appropriate amount of basic culture medium or PBS.
- (9) Mix the matrix gel and crypts in a suitable proportion. For example, for a 24 - well cell culture plate, spot 25 - 30ul of the matrix gel mixture into each well for coating (operate at 4℃).
- (10) Place the coated culture plate in a 37℃ incubator for 20 - 30min to gel. Add an appropriate amount of mouse small intestine organoid complete culture medium (at room temperature) for culture.

2. Organoid Passaging Culture
- (1) Aspirate the culture medium with a pipette, add 1 - 2ml of 4℃ PBS to each well, and leave it for 2min.
- (2) Gently pipette the matrix gel to collect it in a 15ml centrifuge tube. Adjust the volume with PBS to 10 - 14ml, and leave it at 4℃ for 20 - 30min to dissolve the gel (group every 3 - 5 wells). Centrifuge at 1000rpm for 5min to discard the liquid and retain the precipitate.
- (3) Resuspend the organoids with an appropriate amount of matrix gel, and spot 25 - 30ul of matrix gel into each well of a 24 - well cell culture plate. Place it in the incubator for 20 - 30min to gel, and then add an appropriate amount of mouse small intestine organoid complete culture medium.

3. Organoid Cryopreservation
- (1) Aspirate the culture medium with a pipette, add 1 - 2ml of 4℃ PBS to each well, and leave it for 2min.
- (2) Gently pipette the matrix gel to collect it in a 15ml centrifuge tube. Adjust the volume with PBS to 10 - 14ml, and leave it at 4℃ for 20 - 30min to dissolve the gel (group every 3 - 5 wells). Centrifuge at 1000rpm for 5min to discard the liquid and retain the precipitate.
- (3) Add an appropriate amount of organoid cryopreservation solution, and gently pipette to resuspend. For example, for a 24 - well cell culture plate, cryopreserve 2 - 3 wells in one tube, with a volume of 1ml per tube.
- (4) Label the information properly, and then transfer it to liquid nitrogen for long - term storage after programmed cooling.

4. Organoid Thawing
- (1) Take 10ml of DMEM/F12 basic culture medium in a 15ml centrifuge tube.
- (2) Remove the cryopreserved organoids from the liquid nitrogen tank, and quickly place them in a 37℃ water bath to thaw.
- (3) During the water bath thawing process, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1 - 2min.
- (4) Quickly transfer the thawed organoids to a 15ml centrifuge tube, gently pipette 6 - 8 times with a pipette, centrifuge at 1000rpm for 5min, and then remove the supernatant to collect the organoid precipitate.
- (5) Resuspend with matrix gel, spot 25 - 30ul of matrix gel into each well of a 24 - well cell culture plate, place it in the incubator for 20 - 30min to gel, and then add an appropriate amount of mouse small intestine organoid complete culture medium.

Avoid vortex oscillation; Store in aliquots to reduce the number of freeze-thaw cycles