Figure 1 Tissue digestion into multiple crypts

Observe the volume of cell pellet collected by centrifugation, add 25 times the volume of Matrigel to resuspend, form a 3D culture space structure, and avoid bubbles during resuspension. The volume of cell pellet is shown in Figure 2. 300 uL, 250 uL, 150 uL, and 100 uL Matrigel are added according to the volume of cell pellet as shown in Figure 2. 
Figure 2 Cell pellet volume

The 24-well cell culture plates were dispensed from 25 uL to 30 uL/well, and the Matrigel was 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 chicken small intestine organoid medium A (preheated in advance) to each well for culture.
3. Organoid subculture (taking 24-well plate as an example)
1. Experimental materials:
Passage buffer G (4 ℃), organoid passage digestion juice D (room temperature or 37 ℃), Matrigel (melted in a 4 ℃ refrigerator 24 h in advance), chicken small intestine organoid medium A (room temperature or 37 ℃), 1.5 mL/15 mL centrifuge tube, 24-well cell culture plate, ice box.
2. Organoid passage:
Select suitable organoids for passage, which usually grows for about one week. More than 20 organoids, or organoids with a size of 100-200 μ, can be seen under a microscope of 10X.
Aspirate the medium, add an equal volume of passage buffer G to each well, gently blow the Matrigel with a pipette gun, collect it in a 15 mL centrifuge tube, transfer it to a centrifuge tube every 6-8 wells, and stand at 4 °C for 10-15 minutes.
2.1 Organoid digestion
According to the growth of organoids, it is decided whether digestive passage is needed. 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.
When the number of organoids is insufficient or the volume is small, centrifuge at 300 g for 5 min and discard the supernatant.
When the number of organoids is large or the volume is large, centrifuge at 300 g for 5 minutes to discard the supernatant, and digestive juice digestion or mechanical digestion can be selected.
Digestive juice digestion: Add 1-2 mL of organoid passage digestive juice D, blow the cell pellet away, digest at room temperature for 1min, pipetting every minute, and observe under a microscope until the digestion reaches the state (Figure A-B). The digestion was terminated by adding 3 times the volume of organoid digestion fluid in passage buffer G, and centrifuged at 300 g for 5 min.

A B
                                                                            

Figure Diagram of passage digestion degree of three types of organs

2.2 Passage organoid culture
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 matrigel volume of organoid precipitate to resuspend organoids. For the volume of matrigel, please refer to "Organoid Primary Culture Operation Figure 2".
The 24-well cell culture plates were dispensed from 25 uL to 30 uL/well, and the Matrigel was 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 chicken small intestine organoid medium A (preheated in advance) to each well for culture.
four, organoid cryopreservation (taking 24-well plate as an example)
1. Experimental materials:
Passage buffer G (4 ℃), organoid cryopreservation solution F (4 ℃), 15 mL centrifuge tube, cell cryopreservation tube, program cooling box, pipette gun.
2. Organoid cryopreservation:
Organoids that are not in use temporarily should be frozen and stored in a low temperature environment.
Aspirate the medium, add an equal volume of passage buffer G to each well, gently blow the Matrigel with a pipette gun, collect it in a 15 mL centrifuge tube, transfer it to a centrifuge tube every 6-8 wells, and stand at 4 °C 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.
Make the mark information, put it in the program cooling box, move it to the-80 ℃ refrigerator, and put liquid nitrogen after 48 hoursPreserved in a jar. 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 store it in a liquid nitrogen tank after 48 hours.
5. Organoid resuscitation culture (taking 24-well plate as an example)
1. Experimental materials:
Passage buffer G, chicken small intestine organoid medium A, Matrigel (melted in a 4 ℃ refrigerator 24 h in advance), 24-well cell culture plate, ice box, 15 mL centrifuge tube, water bath pan, 3 mL pasteurization pipette/pipette gun.
2. Organoid resuscitation culture:
Take out the frozen organoids from the low temperature environment and quickly put them in a 37 ℃ water bath to thaw. During the water bath thawing process, the cryopreservation tube should be gently shaken to ensure that the cryopreservation solution is completely thawed in a short time. The thawed organoids were quickly transferred to a 15 mL centrifuge tube, 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 centrifuge tube and centrifuge 300 g for 5 minutes, and discard the supernatant.
Add 100 uL of Matrigel to resuspend per cryovial, dispense 24-well cell culture plates at 25 uL-30 uL/well, and maintain the Matrigel at 0-4 ℃ throughout the process. Place the cell culture plate in a 39 ℃ incubator for 10-15 minutes. After the Matrigel coagulates, add 500-750 uL of chicken small intestine organoid medium A (preheated in advance) to each well for culture.
6. Use of matrigel
Matrigel was thawed overnight at ambient conditions of 2-8 °C. When using Matrigel, keep it in an ice box to prevent premature setting. The matrigel formed a gel at 37 °C within 20 minutes.
1. Matrigel features:
(1) 4 ℃ can still maintain good fluidity for 14 consecutive days
(2) Put it in a 37 ℃ incubator for 10-15 min to solidify
(3) It is not easy to break during the culture process, and the glue is removed to clean the non-stick culture plate