Carcinoma of the Larynx Organoid Culture

Example: 24 well cell culture plate according to 25 uL-30 uL/ hole Dispensing glue, matrix rubber whole course Maintain 0-4 ℃ operation under conditions 。 cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 uL Human Laryngeal Cancer Organoid Medium (Advance 37 ℃ (Preheating) for cultivation.

III. Organoid subculture
1. Experimental Materials:
（ 1 Materials required: Matrix glue (in advance) 24 h Put in 4 ℃ (Refrigerator melting) 1.5 mL/15 mL EP tube, 3 mL pasteurized straw /1000 uL pipetting gun, 24 Well cell culture plate, metal ice box, organoid subculture buffer ( 4 ℃ ), organoid passage digestive fluid D （ 37 ℃ ）。
（ 2 Reagent: Human laryngeal cancer organoid culture medium (room temperature or 37 ℃ ）。
2. Organoid Passage:
（ 1 Select appropriate organoids for passage, generally after about a week of growth, under a microscope. 10X You can see more below 20 Organoids, or size 100-200 um Organoids. Aspirate off the culture medium, add an equal volume of organoid subculture buffer to each well, gently blow away the matrix gel with a pipette, and collect... 15 mL EP In the tube, each 6-8 The hole is transferred to one EP tube, 4 ℃ standing 10-15 min 。
（ 2 Whether digestion and passage are necessary is determined based on the growth of the organoid. Note: If there is little precipitation at the bottom of the tube after centrifugation, no cells are seen, and the matrix glue is not delaminated, it can be resuspended again to increase the centrifugal force and centrifuge again.
a: When the number of organoids is insufficient or the volume is small, 300 g centrifuge 5 min Abandon the Shangqing Dynasty
b:  When the number of organoids is high or the volume is large, 300 g centrifuge 5 min Discard the supernatant, and you can choose digestive juice digestion or mechanical digestion.
    Digestive juice digestion: Add 1-2 mL organoid passage digestive fluid D After the cell pellet is blown out, it is digested at room temperature. 2-4 min Blow every minute and observe under a microscope until digested (Figure). A-B It can be stopped when in the ) state. Add 3 Organoid subculture buffer with twice the volume of organoid digestive fluid Termination of digestion, 300 g centrifuge 5 min Abandon the Shangqing Dynasty
    Mechanical digestion: Add 1 mL Organoid subculture buffer, 1 mL pipetting gun blowing 20-30 Time, until the blowing and beating reach (picture). C-D It can be stopped and added when in the ) state. 10 mL passage buffer, 300 g centrifuge 5 min Abandon the Shangqing Dynasty

（ 3 Observe the volume of organoid pellets collected by centrifugation. If there is very little pellet, it can be reserved. 1 Dispense the supernatant with twice the precipitation volume; a large amount of precipitated supernatant can be absorbed. Add... 25 The organoids were resuspended in an amount of matrix gel twice the organoid pellet volume. Example: 24 well cell culture plate according to 25 uL-30 uL/ hole Dispensing glue, The matrix adhesive is maintained throughout the 0-4 ℃ operation under conditions 。 cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 uL Human Laryngeal Cancer Organoid Medium (Advance 37 ℃ (Preheating) for cultivation.

IV. Cryopreservation of organoids
1. Experimental Materials:
（ 1 Materials required: 15 mL EP Tubes, cell cryopreservation tubes, programmed cooling cassettes, pipetting guns, organoid subculture buffer ( 4 ℃ ), organoid cryopreservation solution.
2. Cryopreservation of organoids:
Organoids that are temporarily unused should be frozen and stored in a low-temperature environment.
（ 1 Aspirate off the culture medium, add an equal volume of organoid subculture buffer to each well, gently blow the matrix gel with a pipette, and collect... 15 mLEP In the tube, each 6-8 The hole is transferred to one EP tube, 4 ℃ standing 10-15 min 。 300 g centrifuge 5 min Discard the supernatant and add it every three holes. 2 mL Organoid cryopreservation solution, gently blown and mixed, transferred to cell cryopreservation tubes, each tube... 1 mL 。
（ 2 Mark the information, place it in the program cooling box, and move it to... -80 ℃ In the refrigerator, 48 h Afterwards, it was placed in a liquid nitrogen tank for storage. Or put in 4 ℃ refrigerator 40 min Then, put in -20 ℃ in the refrigerator 2 h , move to -80 ℃ In the refrigerator, 48 h Afterwards, it was placed in a liquid nitrogen tank for storage.

V. Organoid resuscitation culture
1. Experimental Materials:
（ 1 Materials required: Matrix glue (in advance) 24 h Put in 4 ℃ (Refrigerator melting) 15 mL EP Pipe, water bath, pipette gun, 24 Well cell culture plate, ice box.
（ 2 Kit reagents: Organoid subculture buffer ( 4 ℃ ), human laryngeal cancer organoid culture medium.
2. Organoid resuscitation culture:
（ 1 The frozen organoids were removed from the cryogenic environment and quickly placed in... 37 ℃ The cryopreservation tube needs to be gently shaken during the water bath thawing process to ensure that the cryopreservation liquid is completely thawed in a short time. Rapid transfer of thawed organoids to 15 mLEP Gently blow on the tube using a pipette. 6-8 next time, 300 g centrifuge 5 min Abandon the Shangqing; Add an appropriate amount of passage buffer G Resuspend, move in 1.5 mL EP tube 300 g centrifuge 5 min They abandoned the Shangqing
（ 2 ) Add per cryopreservation tube 100 uL The matrix glue is resuspended, 24 well cell culture plate according to 25 uL-30 uL/ hole Dispensing adhesive, the matrix adhesive is maintained throughout 0-4 ℃ Operate under these conditions. cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 uL Human Laryngeal Cancer Organoid Medium (Advance 37 ℃ (Preheating) for cultivation.