Example: 24-well cell culture plates were dispensed according to 25 uL-30 uL/well,Matrigelwhole courseMaintain at 0-4 Operation at ℃。 Place the cell culture plate in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulates, add 500-750 uL of bovine normal intestinal organoid medium A (preheated at 37 ℃ in advance) to each well for culture.

3. Organoid subculture
1. Experimental materials:
(1) Materials to be brought by yourself: Matrigel (melted in a 4 ℃ refrigerator 24 hours in advance), 1.5 mL/15 mL EP tube, 3 mL pasteurization pipette/1000 uL pipette gun, 24-well cell culture plate, metal ice box.
(2) Kit reagents: bovine normal intestinal organoid culture medium A (room temperature or 37 ℃), organoid subculture digestion juice D (37 ℃), and organoid subculture buffer G (4 ℃).
2. Organoid passage:
(1) Select suitable organoids for passage, generally for about one week of growth. More than 20 organoids, or organoids with a size of 100-200 um, can be seen under a microscope of 10X. Aspirate the medium, add an equal volume of organoid subculture buffer G to each well, gently blow the Matrigel with a pipette gun, collect it in a 15 mL EP tube, transfer it to an EP tube every 6-8 wells, and stand at 4 ℃ for 10-15 minutes.
(2) Decide whether digestion and passage are needed according to the growth of organoids.Note:After centrifugation, if there is little sediment at the bottom of the tube, no cells are seen, and the matrigel is not stratified, it can be resuspended again, the centrifugal force can be increased, and centrifuged again.
A: When the number of organoids is insufficient or the volume is small, centrifuge at 300 g for 5 min and discard the supernatant.
b: When the number of organoids is large or the volume is large, centrifuge at 300 g for 5 minutes to discard the supernatant, and select digestive juice for digestion.
Digestive juice digestion: Add 1-2 mL of organoid passage digestive juice D, blow the cell pellet away, digest at room temperature for 2-4 min, pipetting every minute, and observe under a microscope until the digestion reaches the state (Figure A-B). Organoid subculture buffer G was added 3 times the volume of organoid digestion fluid to terminate digestion, and the supernatant was discarded by centrifugation at 300 g for 5 min.
Mechanical digestion: Add 1 mL of organoid subculture buffer G, pipetting 1 mL of gun tip for 20-30 times until pipetting reaches the state (Figure C-D), and then stop. Add 10 mL of organoid subculture buffer G, centrifuge at 300 g for 5 minutes, and discard the supernatant.

(3) Observe the volume of organoid precipitate collected by centrifugation. If there is little precipitate, reserve 1 time the precipitate volume of supernatant for dispensing. If a lot of precipitate supernatant can be aspirated. Add 25 times the volume of matrigel of organoid precipitate to resuspend organoids. Example: 24-well cell culture plates were dispensed according to 25 uL-30 uL/well,The Matrigel is maintained at 0-4 ℃ throughout the process。 Place the cell culture plate in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulates, add 500-750 uL of bovine normal intestinal organoid medium A (preheated at 37 ℃ in advance) to each well for culture.

4. Organoid cryopreservation
1. Experimental materials:
(1) Materials to be prepared by yourself: 15 mL EP tube, cell cryopreservation tube, program cooling box, pipette gun.
(2) Kit reagents: organoid subculture buffer G (4 ℃), organoid cryopreservation solution F.
2. Organoid cryopreservation:
Organoids that are not in use temporarily should be frozen and stored in a low temperature environment.
(1) Aspirate the medium, add an equal volume of organoid subculture buffer G to each well, gently blow the matrigel with a pipette gun, collect it in a 15 mLEP tube, transfer it to an EP tube every 6-8 wells, and stand at 4 ℃ for 10-15 minutes. Centrifuge at 300 g for 5 minutes to discard the supernatant, add 2 mL of organoid cryopreservation solution F every three wells, mix well by gently pipetting, and transfer to cell cryopreservation tubes, each tube containing 1 mL.
(2) Make the mark information, put it in the program cooling box, move it to the-80 ℃ refrigerator, and put it in the liquid nitrogen tank for storage after 48 hours. Or put it in a 4 ℃ refrigerator for 40 minutes, put it in a-20 ℃ refrigerator for 2 hours, move it to a-80 ℃ refrigerator, and after 48 hours, put it in a liquid nitrogen tank for storage.

5. Organoid resuscitation culture
1. Experimental materials:
(1) Materials to be brought by yourself: Matrigel (melted in a 4 ℃ refrigerator 24 hours in advance), 15 mL EP tube, water bath, pipette gun, 24-well cell culture plate, ice box.
(2) Kit reagents: organoid subculture buffer G (4 ℃), bovine normal intestinal organoid culture medium A.
2. Organoid resuscitation culture:
(1) Take out the frozen organoids from the low temperature environment and quickly put them in a 37 ℃ water bath to thaw. During the thawing process of the water bath, the cryopreservation tube should be gently shaken to ensure that the cryopreservation solution is completely melted in a short time. The thawed organoids were quickly transferred to 15 mLEP tubes, gently pipetted 6-8 times with a pipette gun, and centrifuged at 300 g for 5 minutes to discard the supernatant; Add an appropriate amount of passage buffer G to resuspend, transfer it into a 1.5 mL EP tube at 300 g and centrifuge for 5 minutes, and discard the supernatant.
(2) Add 100 uL of Matrigel to each cryopreservation tube for resuspension, dispense 24-well cell culture plates according to 25 uL-30 uL/well, and maintain the Matrigel at 0-4 ℃ throughout the process. Place the cell culture plate in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulates, add 500-750 uL of bovine normal intestinal organoid medium A (preheated at 37 ℃ in advance) to each well for culture.