Organoid culture kit (liver cancer, colon cancer, kidney cancer)

Instructions for use

1. Original generation
(1) The collected tissue must be placed in a pre cooled (2-8 ° C) tissue preservation solution E sampling bottle, quickly transferred to a clean laboratory for tissue processing and cell separation, photographed, and recorded for information.
(2) Prepare several culture dishes and add primary culture buffer B pre cooled at 4 ℃ for later use.
(3) Disinfect the sampling bottle, place the tissue in a culture dish, wash it three times with primary culture buffer B, and then use ophthalmic or surgical methods to cut the tissue into volumes of approximately 1-3mm3The organizational block.
(4) The tissue was digested with the primary human breast cancer tissue, Ye C, and digested with shock at 37 ℃ for 10-20min (observe the digestion at any time during digestion).
(5) Take a small amount of liquid and observe under a microscope. After observing a large number of individual cells or cell clusters below 70um, add three times the volume of primary culture buffer B to terminate digestion.
(6) Filter using a 100um sieve, collect the filtrate, centrifuge at 300g for 5 minutes, remove the supernatant, add primary culture buffer B and resuspend for centrifugation.
(7) Matrix gel calculation: After step 6, observe the collected tissue volume and add 25 times the tissue volume of matrix gel（abs9495）Suspend the flooring.
(8) Taking a 24 well cell culture plate as an example, apply 25ul of tissue matrix adhesive mixture to each well for plate laying (operated at 4 ℃).
(9) Put the paved culture plate into the 37 ℃ incubator for 10-15min to form glue, and add human breast cancer like organ culture medium A (to restore the room temperature) for culture.
2. Subculture of organoids
(1) Remove the culture medium with a pipette, add 1-2ml of 4 ℃ organoid passage culture 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. (Set every 6-8 holes)
(3) 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 passage culture buffer G and resuspend in a 1.5ml centrifuge tube. Centrifuge 300g for 5 minutes and discard the liquid for step 4.
b: When there are a large number or volume of organoids: centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid passage digestion buffer D for digestion for 2-3 minutes, add organoid passage culture buffer G to terminate digestion, centrifuge for 5 minutes to discard the mixture, add an appropriate amount of organoid passage culture buffer G and resuspend in a 1.5ml centrifuge tube. Centrifuge 300g for 5 minutes and discard the liquid for step 4.
(4) After the collection of similar organs, add matrix glue for resuspension. Each hole of 25ul matrix glue is laid on a 24 hole cell culture plate. Place it in the incubator for 10-15min and add 500ul human breast cancer like organ culture medium A.
3. Cryopreservation of organoids
(1) Remove the culture medium with a pipette, add 1-2ml of 4 ℃ organoid passage culture 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. (Set every 6-8 holes)
(3) Centrifuge for 5 minutes and discard the supernatant. Add an appropriate amount of organoid passage culture buffer G and resuspend. Centrifuge 300g for 5 minutes and discard the liquid.
(4) Add an appropriate amount of organoid cryopreservation solution F, gently blow and resuspend, using a 24 well cell culture plate as an example: freeze 1 tube with a density of 2 wells and a volume of 1.4ml per tube.
(5) Mark the information properly, cool down the program, and store it in liquid nitrogen for a long time.
4. Organoid resuscitation
(1) Take 10ml of organoid passage culture buffer G and transfer it to a 15ml centrifuge tube.
(2) Take out the frozen organoid cells from the liquid nitrogen tank and quickly melt them in a 37 ℃ water bath.
(3) During the water bath melting process, gently shake the freezing tube to ensure that the frozen solution melts within 1-2 minutes.
(4) Quickly transfer the dissolved organoid cells to a 15ml centrifuge tube, gently blow 6-8 times with a pipette, centrifuge at 300g for 5 minutes, then remove the supernatant and collect the organoid cell precipitate. Add an appropriate amount of organoid passage culture buffer G and resuspend in a 1.5ml centrifuge tube at 300g for 5 minutes.
(5) The matrix glue was suspended again. Each hole of 25ul matrix glue was placed in a 24 hole cell culture plate, placed in the incubator for 10-15min to form glue, and 500ul human breast cancer like organ culture medium A was added.