Mouse Intestinal Organoid Kit Plus

Product specifications：500mL;100mL
Product Information

Preparation of Complete Culture Medium for Mouse Small Intestinal Organs:
Prepare complete culture medium for mouse small intestine organs using aseptic manipulation techniques. The following is an example of preparing 10mL of complete culture medium. If the required amount is different, the amount can be adjusted accordingly.
1. Thaw on ice with Mouse Intrinsic Organoid Supplement B (50x) and Mouse Intrinsic Organoid Supplement C (250x).
Note: After thawing, it is recommended to separate and store the Mouse Intrinsic Organoid Supplement B (50x) and Mouse Intrinsic Organoid Supplement C (250x) separately to avoid repeated freeze-thaw cycles.
2. Add 200uL Mouse Intrinsic Organoid Supplement B (50x) and 40uL Mouse Intrinsic Organoid Supplement C (250x) to 9.76mL Mouse Intrinsic Organoid Basal Medium, mix thoroughly, and prepare 10mL mouse small intestine organoid complete culture medium.
Note: The prepared mouse small intestine organ complete culture medium can be stored at 2-8 ° C and is recommended to be used within two weeks. Mouse Intellectual Organoid Supplement B (50x) contains bacterial and fungal antibiotics (50x).

Primary Culture of Small Intestinal Organs in Mice
1. Sacrifice mice in accordance with the ethical and operational guidelines for experimental animals approved by the unit.
2. Prepare several culture dishes and add DPBS pre cooled at 4 ℃ for later use.
3. The standard surgical procedure involves taking small intestine segments from mice, with a total length of approximately 3 to 20 centimeters, according to experimental requirements, and placing them in a culture dish containing DPBS.
4. Use a pipette or syringe to inject DPBS into one end of the small intestine to rinse the intestinal contents. After rinsing, place the contents in a new culture dish containing DPBS and repeat rinsing several times until the contents are completely rinsed. Place the contents in a new culture dish containing DPBS.
5. Use surgical scissors to cut open the intestinal canal, with the intestinal cavity facing upwards. With one hand, use surgical forceps to clamp one end of the intestinal tissue, and the other hand, use a surgical blade to gently scrape off the intestinal villi on the surface of the intestinal cavity. After the intestinal villi have been scraped clean, place the intestinal tissue in a new culture dish containing DPBS and wash it again.
6. Cut the cleaned small intestine tissue into 2mm wide pieces and transfer it to pre cooled DPBS containing 5mmol/L EDTA for digestion. Incubate at 4 ℃ for 30 minutes.
After digestion is completed, transfer the tissue fragments to a new culture dish containing DPBS for cleaning and repeat once to remove EDTA.
8. Use a 5mL pipette to blow and resuspend tissue fragments in a culture dish containing cold DPBS or a 50mL centrifuge tube, allowing the tissue to repeatedly pass through the pipette tip to generate mechanical shear force and separate the crypt from the basal layer. Take a portion of the suspension for microscopic examination, and when a large number of crypt like structures can be seen, stop blowing and perform 70% of the blown tissue suspension μ Filter with m filter.
9. Collect the tissue suspension passing through the filter screen and centrifuge at 4 ℃ for 3 minutes with a centrifugal force of 150g.
10. Discard the supernatant, use 1mLDPBS to resuspend tissue precipitation, take 20uL of suspension for microscopic examination and crypt counting. After counting, extract the suspension containing the required amount of crypts, centrifuge at 4 ℃ for 3 minutes at 150g, and discard the supernatant before placing it on ice.
11. Use an appropriate amount of Matrigel to resuspend tissue precipitation. The recommended resuspension density is 200 to 600 pits per 10uL of Matrigel suspension. After resuspension, place it on ice for no more than 30 seconds to avoid premature solidification of Matrigel.
Note: The dilution ratio of Matrigel should be above 70% to ensure the structural stability of Matrigel during the cultivation process.
12. Pour the mixed suspension of Matrigel and tissue cells into the center of the bottom of the 24 well plate, with a volume of about 30uL per well, to avoid contact with the sidewall of the plate.
Note: To prevent Matrigel from solidifying at room temperature, this step should be completed as soon as possible.
13. Place the inoculated culture plate in a 37 ℃ carbon dioxide constant temperature incubator and incubate for about 15 minutes until the matrix solidifies.
14. Prepare complete culture medium for mouse small intestine organs.
15. After the Matrigel has completely solidified, add the prepared mouse small intestine organ complete culture medium, with a 24 well plate of 500uL per well.
Attention: Please slowly add along the wall to avoid damaging the solidified structure.
16. Place the 24 well plate in a 37 ℃ carbon dioxide incubator for cultivation.
17. Change the culture medium every 3 days, and avoid damaging the matrix when changing the culture medium.
18. Close monitoring of organ growth status. Ideally, mouse small intestine organs should be established within 5 to 7 days.
Passage Culture of Small Intestinal Organs in Mice
19. Blow and scrape the organoids using a gun head that has been moistened with the Anti Adhesion Rinsing Kit (E238002), and transfer the suspension of the organoids and culture medium to a 1.5mL EP tube that has been moistened with the Anti Adhesion Rinsing Kit.
20. Use a gun head that has been moistened by the Anti Adhesion Rinsing Kit to resuspend the organoid suspension, separating the organoid from the matrix.
21. Centrifuge at 4 ℃ for 3 minutes with 150g centrifugal force. Discard the supernatant and resuspend the bottom organs with DPBS for sedimentation. Centrifuge again at 4 ℃ for 3 minutes with 150g centrifugal force. Discard the supernatant and place it on ice.
22. Resuspend an appropriate amount of Matrigel to precipitate the organs, and then place it on ice for no more than 30 seconds to avoid premature solidification of Matrigel.
Note: The dilution ratio of Matrigel should be above 70% to ensure the structural stability of Matrigel during the cultivation process.
23. Pour the mixed suspension of Matrigel and organoids into the center of the bottom of the 24 well plate to avoid contact with the sidewall of the plate, with a volume of about 30uL per well.
Note: To prevent Matrigel from solidifying at room temperature, this step should be completed as soon as possible.
24. Place the inoculated culture plate in a 37 ℃ carbon dioxide constant temperature incubator and incubate for about 15 minutes until the matrix solidifies.
25. Prepare complete culture medium for mouse small intestine organs.
26. After the Matrigel has completely solidified, add the prepared mouse small intestine organ complete culture medium, with a 24 well plate of 500uL per well.
27. Place the 24 well plate in a 37 ℃ carbon dioxide incubator for cultivation.