| Instructions | 1, primary
(1) tissue after harvesting should be stored in pre-cooled (2-8° C) In the sampling bottle of tissue preservation solution E, the tissue was quickly transferred to a clean laboratory for tissue processing and cell separation, photographed and recorded.
(2) Prepare several culture dishes and add primary culture buffer B precooled at 4 ° C for later use.
(3) The sampling bottle was disinfected, and the tissue was placed in the Petri dish. After washing three times with primary culture buffer B, the tissue was cut into tissue blocks of about 1-3mm3in volume using ophthalmic scissors or scalpel.
(4) The tissues were digested with human esophageal cancer primary tissue digestion solution C and shaken at 37℃ for 10-20min (the digestion was observed at any time during the digestion).
(5) A small amount of liquid was observed under the microscope. When more single cells or cell clusters below 70um were observed under the microscope, triploid volume primary culture buffer B was added to terminate the digestion.
(6) The filtrate was filtered by a sieve with a pore size of 100um, and the filtrate was collected. After enrichment and centrifugation at 300g for 5min, the supernatant was removed, and the primary culture buffer B was added and resuspended for centrifugation.
(7) Matrix glue calculation: The volume of collected tissue was observed after step 6, and matrix glue (abs9495) was added 25 times the tissue volume to resuspend the paving plate.
(8) For the example of 24-well cell culture plates, 25ul of tissue matrix glue mixture was dispensed to each well for paving (operation at 4 ° C).
(9) The paved culture plate was placed in an incubator at 37 ° C for 10-15min to form gelatin, and human esophageal cancer organoid medium A was added for culture (return to room temperature).
2, Organoid subculture
(1) The medium was sucked out by pipetting gun, and 1-2 mL of 4℃ organoid subculture buffer G was added to each well for 2min.
(2) The matrix glue was gently blown by a pipetting gun, collected in a 15ml centrifuge tube and allowed to rest for 10min at 4 ° C. (every 6-8 Wells were a group)
(3) a: When the number of organoids was insufficient or the volume was small: the supernatant was discarded by centrifugation for 5min, then an appropriate amount of organoid passage culture buffer G was added and resuspended into a 1.5ml centrifuge tube, and the liquid was discarded by centrifugation at 300g for 5min for the fourth step.
b: When organoids are large in number or size: The supernatant was discarded by centrifugation for 5min, then the organoid passage digestion solution D was added for 2-3min, then the organoid passage culture buffer G was added to terminate the digestion, the mixture was discarded by centrifugation for 5min, then the organoid passage culture buffer G was added and the mixture was resuspended and transferred to a 1.5ml centrifuge tube, and the liquid was discarded by centrifugation at 300g for 5min for step 4.
(4) After the organoid was collected, 25ul of Matrigel was added to the 24-well cell culture plate, and 500ul of human esophageal cancer organoid medium A was added to the incubator for 10-15min.
3. Organoids were frozen
(1) The culture medium was removed by pipetting gun, and 1-2ml of 4℃ organoid passage culture buffer G was added to each well and placed for 2min.
(2) The matrix glue was gently blown by a pipetting gun, collected in a 15ml centrifuge tube and allowed to rest for 10min at 4 ° C. (every 6-8 Wells were a group)
(3) The supernatant was discarded by centrifugation for 5min, then an appropriate amount of organoid passage culture buffer G was added and resuspended again, and the liquid was discarded by centrifugation at 300g for 5min.
(4) Add appropriate amount of organoid cryopreservation solution F, gently blow and resuspend, take 24-well cell culture plate as an example: the density is 2 Wells and freeze 1 tube, each tube volume is 1.4ml.
(5) After making the labeling information and cooling, the samples were transferred to liquid nitrogen for long-term storage.
4, organoid resuscitation
(1) 10 mL organoid subculture buffer G was placed in a 15 mL centrifuge tube.
(2) The frozen organoid cells were removed from the liquid nitrogen tank and quickly thawed in a 37℃ water bath.
(3) During the water bath melting process, it is necessary to gently shake the freezing tube to ensure that the frozen solution is completely melted within 1-2min.
(4) The dissolved organoid cells were quickly transferred to a 15ml centrifuge tube, gently blown with a pipette for 6-8 times, centrifuged at 300g for 5min, and then the supernatant was removed and the organoid cell precipitate was collected. An appropriate amount of organoid passage culture buffer G was added and resuspended into a 1.5ml centrifuge tube and centrifuged at 300g for 5min.
(5) Matrigel was resuspended, 25ul of Matrigel per well was spread in A 24-well cell culture plate, placed in an incubator for 10-15min to gel, and 500ul of human esophageal cancer organoid medium A was added. |
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