1. Reagent arrival processing:
1. Cervical organoid culture medium A can be stored at 4 ℃ for 3 months. It is stored at 4 ℃ after receiving the goods. It is recommended to use it within 1 month. It is recommended to store it at-20 ℃ for a long time to avoid repeated freezing and thawing more than 2 times.
2. Tissue preservation solution E and primary tissue digestion solution C contain nutrients to maintain cell activity. In order to maintain the activity of reagent nutrients, it is not recommended to store them at-20 °C for a long time to avoid repeated freezing and thawing more than 2 times.
2. Operation steps and methods:
1. Tissue pretreatment
(1) Experimental materials
Primary buffer B (pre-cooled at 4 ℃), tissue preservation solution E, sampling tube, tissue transport box and ice pack should be prepared in advance.
(2) Acquisition and transportation of organizations
Tissue sampling and transportation is the first and most easily neglected link in the successful construction of organoids. If the tissue is not preserved properly in the early stage, it will lead to problems such as poor cell activity, pollution, and few normal cells, which will reduce the success rate of organoid construction.
The tissue should be put into tissue preservation solution E within 30 minutes of isolation, and the primary buffer solution B should be washed for 3-5 times. The blood on the tissue surface should be washed clean, and put into tissue preservation solution E. The sampling tube should be stored and transported at 4 ℃ at low temperature (within 72 hours).
2. Primary culture of organoids (taking 24-well plates as an example)
(1) Experimental materials
Primary buffer B (4 ℃), primary tissue digestion juice C (37 ℃), Matrigel (melted in 4 ℃ refrigerator 24 h in advance), cervical organoid culture medium A (room temperature or 37 ℃), tweezers (10 ㎝), pointed ophthalmic surgical scissors/blade, disposable 60 mm culture dish, 1.5 mL/15 mL/50 mL centrifuge tube, 100 μ cell strainer, 3 mL pasteurization pipette/1000 ul pipette gun, 24-well cell culture plate, metal ice box, water bath.
(2) Cervical organoid construction
The handling of the organization
It is recommended that the cervical tissue after sampling be stored and transported at 2-8 ℃, and quickly transported to a clean laboratory for the experimental process of cervical organoid construction, organize photos and register detailed information.
   ① Cleaning of tissues
After the sampling tube is disinfected, the tissue is removed from the ultra-clean table, placed into a culture dish, added primary buffer B, blown and cleaned with a 3 mL pasteurization pipette or a 1000 uL pipette gun, and the cleaning operation is repeated three times or more.
   ② Dissociation and digestion of tissues                                                                         
Remove tissue impurities with ophthalmic scissors or surgical blade, transfer forceps to 1.5 mL EP tube, and further mechanically dissociate tissue with ophthalmic scissors to a volume of about 1 ~ 3 mm³Transfer the tissue block into a 15 mL EP tube, add 5 mL of primary tissue digestion juice and shake it at 37 ℃ for 15-25 min. During the digestion process, the tissue digestion was observed under a microscope every 10 minutes, and a small amount of digestive juice was taken for observation under a microscope. After more cell clusters or single cells below 70μm were observed, the next operation was carried out. The degree of tissue digestion is shown in Figure 1.
If the amount of tissue is too small or the biopsy tissue is used1 mLPrimary tissue digestive juiceCIn1.5 mL EPTube digestion.

Figure 1 Tissue digestion into more cell clusters or more single cells

     ③ Filtering of tissues
After the digestion is completed, the tissue vortexer is vortexed for 10 seconds, 3 times the volume of primary buffer B is added to terminate the digestion, the digested tissue mixture is filtered through a cell screen with a pore size of 100 μm, and the tissue is continuously rinsed with 10-20 ml of primary buffer B to collect more tissue mass cells, and the supernatant is discarded after enriching and centrifuging at 300 g for 5 minutes; Resuspend the centrifugation pellet with 8-10 ml of primary buffer B (remove more impurities), enrich 300 g centrifuge for 5 min, and then discard the supernatant.
If the cell pellet contains red blood cells, add 1-2 mL of red blood cell lysate for 1-2 minutes, dilute to 10 mL, enrich 300 g and centrifuge for 5 minutes, and then discard the supernatant.Organoid culture was carried out directly when there were too little or no red blood cells precipitated.
④ Organoid culture
Observe the volume of cell pellets collected by centrifugation, add 25 times the volume of Matrigel to resuspend, form a 3D culture space structure, and avoid bubbles during resuspension. The volume of cell pellet is shown in Figure 2. 300 ul, 250ul, 150 ul, and 100 ul Matrigel are added according to the volume of cell pellet as shown in the figure.
Figure 2 Cell pellet volume
The 24-well cell culture plate is dispensed according to 25ul-30ul/well,Matrigel is maintained throughout0-4 ℃Operation under conditions。 Place the cell culture plate in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulates, add 750μl of human cervical organoid medium A (room temperature) to each well of the 24-well cell culture plate and place it in a 37 ℃ incubator for culture.
3. Organoid subculture (taking 24-well plate as an example)
(1) Experimental materials
Passage buffer G (4 ℃), organoid digestive juice D (room temperature or 37 ℃), Matrigel (melted in a 4 ℃ refrigerator 24 h in advance), cervical organoid medium A (room temperature or 37 ℃), 1.5 mL/15 mL centrifuge tube, 24-well cell culture plate, ice box.
(2) Organoid passage
Select suitable organoids for passage, which usually grows for about one week. More than 20 organoids, or organoids with a size of 100-200 μ, can be seen under a microscope of 10X.
Aspirate the medium, add an equal volume of passage buffer G to each well, gently blow the Matrigel with a pipette gun, collect it in a 15 mL centrifuge tube, transfer it to a centrifuge tube every 6-8 wells, and stand at 4 °C for 10-15 minutes.
① Organoid digestion
According to the growth of organoids, it is decided whether digestive passage is needed.After centrifugation, if there is little sediment at the bottom of the tube, no cells are seen, and the matrigel is not stratified, it can be resuspended again, the centrifugal force is increased, and centrifuged again。
When the number of organoids is insufficient or the volume is small, centrifuge at 300 g for 5 min and discard the supernatant.
When the number of organoids is large or the volume is large, centrifuge at 300 g for 5 minutes to discard the supernatant, and digestive juice digestion or mechanical digestion can be selected.
Digestive juice digestion: Add 1-2 mL of organoid digestive juice D, blow away the cell pellet, digest at room temperature for 2-4 min, pipetting every minute, 20 times each time, and observe under a microscope until the digestion reaches the state (Figure A-B). Digestion was terminated by adding 3 times the volume of organoid digest fluid in passage buffer G, and centrifuged at 300 g for 5 min.

Figure Diagram of passage digestion degree of three types of organs

② Passage organoid culture
Observe the volume of organoid precipitate collected by centrifugation. If there is little precipitate, reserve 1 time the precipitate volume of supernatant for dispensing. If a lot of precipitate supernatant can be aspirated. Add 25 times the matrigel volume of organoid precipitate to resuspend organoids. For the volume of matrigel, please refer to "Organoid Primary Culture Operation Figure 2".
The 24-well cell culture plate is dispensed according to 25ul-30ul/well,Matrigel is maintained throughout0-4 ℃Operation under conditions。 Place the cell culture plate in a 37 ℃ incubator for 10-15 minutes. After the Matrigel coagulates, add 750μl of human cervical organoid medium A (room temperature) to each well of the 24-well cell culture plate and place it in a 37 ℃ incubatorRaise.
4. Cryopreservation of organoids (taking 24-well plate as an example)
(1) Experimental materials
Passage buffer G (4 ℃), organoid cryopreservation solution F (4 ℃), 15 mL centrifuge tube, cell cryopreservation tube, program cooling box, pipette gun.
(2) Organoid cryopreservation
Organoids that are not in use temporarily should be frozen and stored in a low temperature environment.
Aspirate the medium, add an equal volume of passage buffer G to each well, gently blow the Matrigel with a pipette gun, collect it in a 15 mL centrifuge tube, transfer it to a centrifuge tube every 6-8 wells, and stand at 4 °C for 10-15 minutes. Centrifuge at 300 g for 5 minutes to discard the supernatant, add 2 mL of organoid cryopreservation solution F every three wells, mix well by gently pipetting, and transfer to cell cryopreservation tubes, each tube containing 1 mL.
Make the mark information, put it in the program cooling box, move it to the-80 ℃ refrigerator, and store it in the liquid nitrogen tank after 48 hours. Or put it in a 4 ℃ refrigerator for 40 minutes, put it in a-20 ℃ refrigerator for 2 hours, move it to a-80 ℃ refrigerator, and after 48 hours, put it in a liquid nitrogen tank for storage.
5. Organoid resuscitation culture (taking 24-well plate as an example)
(1) Experimental materials
Passage buffer G, cervical organoid medium A, Matrigel (melted in a 4 ℃ refrigerator 24 h in advance), 24-well cell culture plate, ice box, 15 mL centrifuge tube, water bath pan, 3 mL pasteurization pipette/pipette gun.
(2) Organoid resuscitation culture
Take out the frozen organoids from the low temperature environment and quickly put them in a 37 ℃ water bath to thaw. During the water bath thawing process, the cryopreservation tube should be gently shaken to ensure that the cryopreservation solution is completely thawed in a short time. The thawed organoids were quickly transferred to a 15 mL centrifuge tube, gently pipetted 6-8 times with a pipette gun, and centrifuged at 300 g for 5 minutes to discard the supernatant; Add an appropriate amount of passage buffer G to resuspend, transfer it into a 1.5 mL centrifuge tube and centrifuge 300 g for 5 minutes, and discard the supernatant.
Add 120ul Matrigel to each cryopreservation tube for resuspension, dispense 24-well cell culture plates according to 25ul-30ul/well, and maintain the Matrigel at 0-4 ℃ throughout the process. Place the cell culture plate in a 37 ℃ incubator for 10-15 minutes. After the matrigel coagulates, add 750μl of human cervical organoid medium A (room temperature) to each well of the 24-well cell culture plate and place it in a 37 ℃ incubator for culture.
6. Use of matrigel
Matrigel was thawed overnight at ambient conditions of 2-8 °C. When using Matrigel, keep it in an ice box to prevent premature setting. The matrigel formed a gel at 37 °C within 20 minutes.
Matrigel features:
(1) 4 ℃ can still maintain good fluidity for 14 consecutive days
(2) Put it in a 37 ℃ incubator for 10-15 min to solidify
(3) It is not easy to break during the culture process, and the glue is removed to clean the non-stick culture plate