Organotial Human laryngeal carcinoma organoid Culture Kit

example: 24 hole cell culture plate according to the uL 25-30 uL/hole point glue, matrix strong >all strong >at 0 to 4 strong >& have spent operation under the condition of ℃. The cell culture plate was placed in A 37 ° C incubator for 10-15 min, and after the matrix gel had solidified, 500-750 uL of human laryngeal cancer organoid medium A (preheated at 37 ° C) was added to each well for culture.

III. Organoid subculture
1. Experimental materials:
(1) Materials to be prepared: matrix glue (melt in 4 ℃ refrigerator 24 hours in advance), 1.5 mL/15 mL EP tube, 3 mL Papanicolaek pipette /1000 uL pipette gun, 24-well cell culture plate, metal ice box.
(2) Kit reagent: organoid passage culture buffer G (4 ℃), organoid passage digestion solution D (37 ℃), human laryngeal cancer organoid culture medium A (room temperature or 37 ℃).
2. Organoid passage:
(1) Select appropriate organoids for passage, generally grow for about a week, more than 20 organoids can be seen under a microscope at 10X, or organoids with a size of 100-200 um. The same volume of organoid passage culture buffer G was added to each well. The matrix glue was gently blown off by a pipetting gun, collected in 15 mL EP tubes, transferred to an EP tube every 6-8 Wells, and left for 10-15 min at 4 ° C.
(2) Determine whether digestion and passage are necessary according to the growth of the organoids. Note: After centrifugation, if there is less precipitation at the bottom of the tube, no cells are seen, and the matrix glue is not stratified, it can be resuspended again, the centrifugal force can be increased, and then centrifuged again.
a: When the number of organoids was insufficient or the volume was small, the supernatant was discarded by centrifugation at 300 g for 5 min.
b:  When the number of organoids is large or large, 300  g centrifuge 5  The supernatant was discarded in min and either digested with digester or mechanically.
    Digestion with digestive fluid: Add 1-2 mL organoid passage digestion solution D, blow off the cell precipitate, digest at room temperature for 2-4 min, blow every minute, observe under the microscope, until the digestion to the state (FIG. A-B) can be stopped. The digestion was terminated by adding 3 times the volume of organoid digestion liquid in organoid passage culture buffer G, and the supernatant was discarded by centrifugation at 300 g for 5 min.
    Mechanical digestion: Add 1 mL organoid passage culture buffer G, blow with 1 mL pipettes for 20-30 times until blowing to (FIG. C-D) state, add 10 mL passage buffer, centrifugation at 300 g for 5 min and discard the supernatant.

(3) Observe the volume of organoid precipitation collected by centrifugation. If the precipitation is very small, 1 times the volume of precipitation supernatant can be reserved for gelatin injection, a lot of precipitation supernatant can be absorbed, add 25 times the volume of organoid precipitation matrix glue to resuspend the organoid. Example: The 24-well cell culture plate was dispensed at 25 ul-30 uL/ well, and the matrix glue was maintained at 0-4 ℃ throughout the operation . The cell culture plate was placed in A 37 ℃ incubator for 10-15 min, and after the matrix gel solidified, 500-750 uL of human laryngeal cancer organoid medium A (preheated at 37 ℃) was added to each well for culture.

IV. Organoid Cryopreservation
1. Experimental materials:
(1) Self-supplied materials: 15 mL EP tube, cell cryopreservation tube, program cooling box, pipeting gun.
(2) Kit reagent: organoid passage culture buffer G (4 ℃), organoid freezing solution F.
Organoid cryopreservation:
organoids not in use should be frozen and stored in a low temperature environment.
(1) The medium was sucked out, and the equal volume of organoid passage culture buffer G was added to each well. The matrix glue was gently blown off by a pipetting gun, collected in 15 mLEP tubes, transferred to an EP tube every 6-8 Wells, and left for 10-15 min at 4 ℃. The supernatant was discarded by centrifugation at 300 g for 5 min. Then 2 mL organoid cryopreservation solution F was added to every three Wells, gently blown and mixed, and transferred to cell cryopreservation tubes (1 mL per tube).
(2) The labeled information was put into the program cooling box, transferred to the refrigerator at -80 ℃, and stored in a liquid nitrogen tank after 48 hours. Or put it in the 4 ℃ refrigerator for 40 min, put it in the -20 ℃ refrigerator for 2 h, move it to the -80 ℃ refrigerator, and put it in the liquid nitrogen tank after 48 h.

V. Organoid resuscitation and culture
1. Experimental materials:
(1) Materials to be prepared: matrix glue (melt in 4 ℃ refrigerator 24 hours in advance), 15 mL EP tube, water bath, pipettes gun, 24-well cell culture plate, ice box.
(2) Kit reagent: organoid passage culture buffer G (4 ℃), human laryngeal cancer organoid culture medium A.
2. Resuscitation culture of organoids:
(1) The frozen organoids were removed from the low temperature environment and quickly thawed in a 37 ℃ water bath. During the process of water bath melting, it was necessary to gently shake the frozen tube to ensure that the frozen solution was completely thawed in a short time. The thawed organoids were quickly transferred to a 15 mLEP tube, gently blown by a pipettes gun for 6-8 times, and centrifuged at 300 g for 5 min to discard the supernatant. An appropriate amount of passage buffer G was added and resuspended, transferred to a 1.5 mL EP tube and centrifuged at 300 g for 5 min, and the supernatant was discarded.
(2) 100 uL of Matrigel was added to each cryopreservation tube and resuspended. The 24-well cell culture plate was dispensed at 25 ul-30 uL/ well, and the matrigel was maintained at 0-4 ℃ throughout the operation. The cell culture plate was placed in A 37 ℃ incubator for 10-15 min, and after the matrix gel solidified, 500-750 uL of human laryngeal cancer organoid medium A (preheated at 37 ℃) was added to each well for culture.