| Instructions | 1, primary
(1) tissue after sampling suggested 2-8° The tissues were stored under C conditions and quickly transferred to a clean laboratory for tissue processing and cell separation. The information was photographed and registered.
(2) Several culture dishes were prepared and primary culture buffer B, precooled at 4 ° C, was added 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 renal cancer primary tissue digestion solution C and shaken at 37℃ for 15-25min (the digestion was observed at any time during the digestion process).
(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. After collecting the filtrate, the supernatant was removed after enrichment and centrifugation at 300g for 5min. The supernatant was resuspended by adding primary culture buffer B, and the supernatant was removed after enrichment and centrifugation at 300g for 5min.
(7) The cell precipitate contained red blood cells, the supernatant was discarded, 1-2 ml of red blood cell lysate was added for 1-2min, diluted to 10mL, and centrifuged at 300g for 5min.
(8) Matrix glue calculation: The collected tissue volume was observed after step 6, and the paving plate was resuspended by adding matrix glue (abs9495) at 25 times the tissue volume.
(9) For the example of 24-well cell culture plate, 25uL-30uL tissue matrix glue mixture was dispensed to each well for paving (Note: matrix glue was maintained at 0-4 ° C for operation).
(10) The paved culture plate was placed in an incubator at 37 ° C for 10-15min. After the matrix gel solidified, 500-750uL of human renal cancer organoid medium A (returned to room temperature) was added for culture.
2, Organoid subculture
(1) The medium was aspirated by pipetting gun, and 1-2mL of 4 ° C 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 10-15min at 4 ° C. (every 6-8 Wells are a group)
(3) a: When the number of organoids is insufficient or the volume is small: Discard the supernatant by centrifugation at 300g for 5min, add 1mL organoid passage culture buffer G, resuspension and transfer into 1.5mL centrifuge tube, and discard the liquid by centrifugation at 300g for 5min for the fourth step.
b: When organoids are large in number or size: Centrifugation at 300g for 5min to discard the supernatant, add 1-2mL organoid passage digestion solution D for 2-4min, add 3 times the volume of organoid passage culture buffer G to terminate digestion, centrifugation at 300g for 5min to discard the supernatant, add an appropriate amount of organoid passage culture buffer G to resuspend and transfer to a 1.5mL centrifuge tube. The supernatant was discarded by centrifugation at 300g for 5min, and the fourth step was carried out.
(4) After organoids were collected, they were resuspended by adding matrigel, and 25uL of Matrigel per well was spread in 24-well cell culture plates and placed in an incubator for 10-15min. After the Matrigel had set, 500 to 750uL of human renal cancer organoid medium A was added to each well.
3, organoids were frozen
(1) The medium was removed by suction with a pipetting gun, and 1-2 ml of 4 ° C 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 2mL organoid cryopreservation solution F to every 3 Wells, gently blow and mix, and transfer to cell cryopreservation tubes, 1mL per tube.
(5) Make the labeling information, after the program cooling, move to -80° C refrigerator, 48h later, transferred to liquid nitrogen for long-term storage. Or put it into the 4℃ refrigerator for 40min, put it into the -20℃ refrigerator for 2h, move it to the -80℃ refrigerator, and put it into the liquid nitrogen tank for storage after 48h.
4, organoid resuscitation
(1) 10mL organoid subculture buffer G was placed in a 15mL 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 in a short time.
(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, then transferred to a 1.5mL centrifuge tube and centrifuged at 300g for 5min, and the supernatant was discarded.
(5) Add 100ul of Matrigel to each tube of cryopreserved tube and resuspend, 25uL of Matrigel in each well was spread in 24-well cell culture plate, placed in the incubator for 10-15min to gel, and 500-750uL of human renal cancer organoid medium A was added. |
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