| Usage | 1. Primary
(1) After sampling, the tissue must be placed in a sampling bottle containing pre-cooled (2-8°C) tissue preservation solution E and quickly transported to a clean laboratory for tissue processing and cell separation, and photographed and registered.
(2) Prepare several culture dishes and add 4°C pre-cooled primary culture buffer B for use.
(3) Disinfect the sampling bottle, place the tissue in the culture dish, wash it three times with primary culture buffer B, and use ophthalmic scissors or a scalpel to cut the tissue into tissue blocks of approximately 1-3 mm3 in volume.
(4) Digest the tissue with human liver cancer primary tissue digestion solution C at 37°C for 10-20 minutes (observe the digestion progress at any time during the digestion process).
(5) Take a small amount of liquid and observe it under a microscope. When a large number of single cells or cell clusters less than 70 μm are observed under the microscope, add three times the volume of primary culture buffer B to stop the digestion.
(6) Filter using a 100 μm pore size mesh, collect the filtrate, concentrate and centrifuge at 300 g for 5 minutes, remove the supernatant, add primary culture buffer B and resuspend and centrifuge.
(7) Matrigel calculation: After step 6, observe the volume of tissue collected, add 25 times the volume of tissue Matrigel (ABS9495) to resuspend and plate.
(8) For a 24-well cell culture plate, dispense 25 μl of tissue-matrix gel mixture into each well for plating (operate at 4°C).
(9) Place the plated culture plate in a 37°C incubator for 10-15 minutes to gel, add human liver cancer organoid culture medium A (return to room temperature) and culture.
2. Organoid subculture
(1) Remove the culture medium with a pipette, add 1-2 ml of 4°C organoid subculture buffer G to each well and let it sit for 2 minutes.
(2) Gently blow the matrix gel with a pipette, collect it in a 15ml centrifuge tube, and let it stand at 4℃ for 10 minutes. (Each 6-8 wells are a group)
(3)a: When the number of organoids is insufficient or the volume is small: Centrifuge for 5 minutes and discard the supernatant, add an appropriate amount of organoid subculture buffer G, resuspend and transfer to a 1.5ml centrifuge tube, centrifuge at 300g for 5 minutes, discard the liquid and proceed to step 4.
b: When the number of organoids is large or the volume is large: Centrifuge for 5 minutes and discard the supernatant, add an appropriate amount of organoid subculture buffer D and digest for 2-3 minutes, add organoid subculture buffer G to stop digestion, centrifuge for 5 minutes and discard the mixed solution, add an appropriate amount of organoid subculture buffer G and resuspend and transfer to a 1.5ml centrifuge tube, centrifuge at 300g for 5 minutes, discard the liquid and proceed to step 4.
(4) After the organoids are collected, add Matrigel to resuspend them. 25ul of Matrigel is spread on each well of a 24-well cell culture plate. Place it in the incubator for 10-15 minutes and then add 500ul of human liver cancer organoid culture medium A.
3. Organoid freezing
(1) Aspirate the culture medium with a pipette and add 1-2ml of 4℃ organoid subculture buffer G to each well and let it stand for 2 minutes.
(2) Gently pipette the Matrigel and collect it in a 15ml centrifuge tube and let it stand at 4℃ for 10 minutes. (Each 6-8 wells are a group)
(3) Centrifuge for 5 minutes and discard the supernatant. Add an appropriate amount of organoid subculture buffer G and resuspend again. Centrifuge at 300g for 5 minutes and discard the liquid.
(4) Add an appropriate amount of organoid freezing medium F and gently pipette to resuspend. Taking a 24-well cell culture plate as an example: the density is 2 wells and freeze 1 tube. The volume of each tube is 1.4ml.
(5) Label the cells, cool them down, and transfer them to liquid nitrogen for long-term storage.
4. Organoid Recovery
(1) Place 10 ml of Organoid Subculture Buffer G in a 15 ml centrifuge tube.
(2) Remove the frozen organoid cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath.
(3) During the thawing process in the water bath, gently shake the cryovial to ensure that the cryopreservative solution is completely thawed within 1-2 minutes.
(4) Quickly transfer the dissolved organoid cells to a 15 ml centrifuge tube, gently pipette 6-8 times, centrifuge at 300 g for 5 minutes, then remove the supernatant and collect the organoid cell pellet. Add an appropriate amount of Organoid Subculture Buffer G, resuspend, transfer to a 1.5 ml centrifuge tube, and centrifuge at 300 g for 5 minutes. (5) Resuspend the matrix gel and spread 25ul matrix gel per well in a 24-well cell culture plate. Place it in the incubator for 10-15 minutes to gel, and add 500ul human liver cancer organoid culture medium A. |
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