| Usage | 1. Primary
(1) The normal colon tissue of mice after collection must be placed in a sampling bottle of pre-cooled (2-8 °C) tissue preservation solution E, and quickly transported to a clean laboratory for tissue processing and stem cell separation, photographed and registered information.
(2) Prepare several petri dishes, and add primary culture buffer B pre-cooled at 4 °C for later use.
(3) Disinfect the sampling bottle, put the tissue in a petri dish, wash it three times with primary culture buffer B, remove impurities, and remove the intestinal mucosa of colon tissue with ophthalmic scissors or scalpel, and cut it into a volume of about 1-3mm3The tissue block.
(4) The tissues were digested with digestive juice C of normal colon primary tissue of mice, and digested by shaking at 4 ℃ for 10 minutes (the digestion situation was observed at any time during digestion).
(5) Take a small amount of liquid and observe it under a microscope. After more single cells or cell clusters below 70um are observed under the microscope, add triploid volume primary culture buffer B to terminate digestion.
(6) Filter with a sieve with a pore size of 100um, collect the filtrate, enrich and centrifuge at 300g for 5 minutes, remove the supernatant, add primary culture buffer B and re-suspend and centrifuge.
(7) Matrigel calculation: After step 6, observe the collected tissue volume, add 25 times the tissue volume of Matrigel (abs9495) to resuspend and plate.
(8) Take a 24-well cell culture plate as an example. Each well is dispensed with 25ul tissue matrigel mixture for plating (operation at 4 °C).
(9) Put the laid culture plate into a 37 ℃ incubator for 10-15 minutes to gel, and add mouse normal colon organoid medium A (restored to room temperature) for culture.
2. Organoid subculture
(1) Aspirate the culture medium with a pipette gun, add 1-2ml of 4 ℃ organoid subculture buffer G to each well and leave for 2min.
(2) Gently blow the matrigel with a pipette gun, collect it in a 15ml centrifuge tube, and let it stand at 4 °C for 10 minutes. (groups of 6-8 wells)
(3) a: When the number of organoids is insufficient or the volume is small: centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid subculture buffer G, resuspend it and transfer it into a 1.5 ml centrifuge tube, centrifuge 300g for 5 minutes to discard the liquid for step 4.
b: When the number of organoids is large or the volume is large: centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid subculture juice D for 2-3 minutes, add organoid subculture buffer G to terminate digestion, centrifuge for 5 minutes to discard the mixed solution, add an appropriate amount of organoid subculture buffer G to resuspend and transfer into a 1.5 ml centrifuge tube, centrifuge 300g for 5 minutes to discard the liquid for step 4.
(4) After organ collection, add Matrigel for resuspension, spread 25ul of Matrigel per well in a 24-well cell culture plate, place it in an incubator for 10-15min, and add 500ul of mouse normal colon organoid medium A.
3. Organoid cryopreservation
(1) Aspirate the culture medium with a pipette gun, add 1-2ml of 4 ℃ organoid subculture buffer G to each well and leave for 2min.
(2) Gently blow the matrigel with a pipette gun, collect it in a 15ml centrifuge tube, and let it stand at 4 °C for 10 minutes. (groups of 6-8 wells)
(3) Centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid subculture buffer G to resuspend again, and centrifuge 300g for 5 minutes to discard the liquid.
(4) Add an appropriate amount of organoid cryopreservation solution F, gently blow and resuspend, take a 24-well cell culture plate as an example: the density is 2 wells for cryopreservation in 1 tube, and the volume of each tube is 1.4 ml.
(5) Make good marking information, carry out program cooling, and then move it into liquid nitrogen for long-term storage.
4. Organoid resuscitation
(1) Take 10ml of organoid subculture buffer G in a 15ml centrifuge tube.
(2) Take out the frozen organoid cells from the liquid nitrogen tank and quickly thaw them in a 37 ℃ water bath.
(3) During the water bath melting process, the cryotube needs to be gently shaken to ensure that the cryopreservation solution is completely melted within 1-2 minutes.
(4) Quickly transfer the dissolved organoid cells to a 15ml centrifuge tube, gently pipette 6-8 times with a pipette, centrifuge at 300g for 5min, then remove the supernatant and collect the organoid cell pellet. Add an appropriate amount of organoid subculture buffer G, resuspend it, transfer it into a 1.5 ml centrifuge tube with 300g and centrifuge for 5 minutes.
(5) Resuspend Matrigel, spread 25ul Matrigel per well in a 24-well cell culture plate, place it in an incubator for 10-15min to gel, and add 500ul mouse normal colon organoid medium A. |
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