| Usage | 1. Primary
(1) The extracted tissue must be placed in a pre cooled (2-8° C) tissue preservation solution E sampling bottle, quickly transported to a clean laboratory for tissue treatment and cell separation, photographed, and registered with information
(2) Prepare several culture dishes and add 4 ℃ pre cooled primary culture buffer B for later use
(3) Disinfect the sampling bottle, place the tissue in a culture dish, clean it three times with primary culture buffer B, and then use an ophthalmic scissors or surgical knife to cut the tissue into tissue blocks with a volume of approximately 1-3mm
(4) The tissue was digested with the digestion solution C of the primary tissue of the porcine endometrium, and shaken at 37 ℃ for 10-20 minutes (the digestion process was observed at any time)
(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 pore sieve, collect the filtrate, concentrate and centrifuge at 300g for 5 minutes, remove the supernatant, add primary culture buffer B, and resuspend and centrifuge
(7) Matrix adhesive calculation: After step 6, observe the collected tissue volume and add 25 times the tissue volume of matrix adhesive (ABS9495) to resuspend the board
2. Organ like passage culture
(1) Pipette the culture medium and add 1-2ml of 4 ℃ organ like passage culture buffer G to each well for 2 minutes
(2) Gently blow the matrix adhesive with a pipette, collect it in a 15ml centrifuge tube, and let it stand at 4 ℃ for 10 minutes. (Each 6-8 wells is a group)
(3) a: If the number of organoids is insufficient or the volume is small, centrifuge for 5 minutes to discard the supernatant. Add an appropriate amount of organoid passage culture buffer G and resuspend it into a 1.5ml centrifuge tube. Centrifuge for 5 minutes to discard the liquid and proceed to step 4
b: When the number of organoids is large or the volume is large: centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid passage digestion solution D 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 into a 1.5ml centrifuge tube, centrifuge for 5 minutes to discard the liquid for step 4
3. Organ like cryopreservation
(1) Pipette the culture medium and add 1-2ml of 4 ℃ organ like passage culture buffer G to each well for 2 minutes
(2) Gently blow the matrix adhesive with a pipette, collect it in a 15ml centrifuge tube, and let it stand at 4 ℃ for 10 minutes. (Each 6-8 wells is a group)
(3) Centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid passage culture buffer G, and resuspend again. Centrifuge for 5 minutes at 300g to discard the liquid
(4) Add an appropriate amount of organoid cryopreservation solution F, gently blow and resuspend, taking a 24 well cell culture plate as an example: the density is 2 wells, and one tube is frozen, with a volume of 1.4ml per tube
(5) Mark the information properly, cool down the program, and then transfer it to liquid nitrogen for long-term storage
4. Organ like resuscitation
(1) Take 10ml of organ like passage culture buffer G and place it in a 15ml centrifuge tube<(2) Remove frozen organoid cells from a liquid nitrogen tank and quickly melt them in a 37 ℃ water bath(5) Suspension of matrix adhesive, with 25 ul of matrix adhesive per well laid on a 24 well cell culture plate, placed in a culture chamber for 10-15 minutes to form gel, and 500 ul of porcine endometrial organoid culture medium A. |
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