Human Gastric Organoid Culture Medium

1. Primary culture
(1) The collected tissue is placed in a sampling bottle of pre-cooled (2-8 °C) tissue preservation solution, and quickly transferred to a clean laboratory for tissue processing and cell separation, taking photos and registering information.
(2) Prepare several petri dishes, and add 4 ℃ pre-cooled human normal gastric organoid tissue primary culture buffer # abs9731 for later use.
(3) Sterilize the sampling bottle, put the tissue into a petri dish, wash it three times with the primary culture buffer of human normal gastric organoid tissue, and cut the tissue into a volume of about 1-3mm with ophthalmic scissors or scalpel³The tissue block.
(4) The tissues were digested with human normal stomach primary tissue digestive juice # abs9783, and digested by shaking at 4 ℃ for 15-20min (observe the digestion at any time during the digestion process).
(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 human normal gastric organoid tissue primary culture buffer to stop digestion.
(6) Use a sieve with a pore size of 100um for filtration, collect the filtrate, enrich and centrifuge at 300g for 5 minutes, remove the supernatant, add human normal gastric organoid tissue primary culture buffer 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 form a gel, and add human normal gastric organoid culture medium (restored to room temperature) for culture.
2. Organoid subculture
(1) Aspirate the medium with a pipette gun, add 1-2ml of 4 ℃ organoid subculture buffer # abs9730 to each well 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 human normal gastric organoid subculture buffer to resuspend 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 # abs9520 for 2-3 minutes, add human normal gastric organoid subculture buffer to terminate digestion, centrifuge for 5 minutes to discard the mixed solution, add an appropriate amount of human normal gastric organoid subculture buffer to resuspend and transfer into a 1.5 ml centrifuge tube, and centrifuge 300g for 5 minutes to discard the liquid for step 4.
(4) After organoid 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 human normal gastric organoid culture medium.
3. Organoid cryopreservation
(1) Aspirate the culture medium with a pipette gun, add 1-2ml of 4 ℃ organoid subculture buffer to each well and place it for 2 minutes.
(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 human normal gastric organoid subculture buffer to resuspend again, and centrifuge 300g for 5 minutes to discard the liquid.
(4) Add an appropriate amount of organoid cryopreservation solution # abs9519, gently blow and resuspend, take a 24-well cell culture plate as an example: the density is 2 wells and cryopreserve 1 tube, each tube has a volume of 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 human normal gastric organoid subculture buffer 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 appropriate amount of human normal gastric organoid subculture buffer, resuspend, transfer into 1.5 ml centrifuge tube 300g and centrifuge for 5min.
(5) Resuspend Matrigel, spread 25ul Matrigel per well in a 24-well cell culture plate, place it in an incubator for 10-15min to form a gel, and add 500ul human normal gastric organoid culture medium.