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1. Reagent treatment:
1 Cervical organoid culture medium A In 4 ℃ Can be saved 3 Months after receiving the goods 4 ℃ Save, suggest 1 It should be used within a month; it is not recommended to store it for a long time without use. -20 ℃ Store to avoid repeated freezing and thawing exceeding 2 Next 。
2 、 tissue preservation solution E Primary tissue digestive juice C It contains nutrients that maintain cell activity. To maintain the activity of the reagent nutrients, it is recommended not to store them for a long time. -20 ℃ Store to avoid repeated freezing and thawing exceeding 2 Next 。
II. Operating Steps and Methods:
1 Pre-organization treatment
（ 1 Experimental materials:
Primary buffer needs to be prepared in advance B （ 4 ℃ Pre-cooling), tissue preservation solution E Sampling tube, tissue transport box, ice pack.
（ 2 Organizational acquisition and transportation:
Tissue sampling and transportation are the first and most easily overlooked steps in the successful construction of organoids. Improper preservation of tissues in the early stages can lead to problems such as poor cell activity, contamination, and few normal cells, reducing the success rate of organoid construction.
The tissue should be in vitro 30 min Place tissue preservation solution inside E , primary buffer B cleaning 3-5 Next, the blood on the tissue surface is washed clean and placed in tissue preservation solution. E In the sampling tube 4 ℃ Cryogenic storage and transportation ( 72 h inside).
2 Organoid primary culture (with 24 (Taking orifice plates as an example)
（ 1 Experimental materials:
Primary buffer needs to be prepared in advance B （ 4 ℃ ), primary tissue digestive juice C （ 37 ℃ ), matrix glue (in advance 24 h Put in 4 ℃ Refrigerator thawing), cervical organoid culture medium A (Room temperature or 37 ℃ ), tweezers ( 10cm ), pointed ophthalmic surgical scissors / Surgical blade, disposable 60 mm Petri dishes, 1.5 mL/15 mL/50 mL centrifuge tube, 100μm a cell screen, 3 mL pasteurized straw /1000μL pipetting gun, 24 Well cell culture plate, metal ice box, water bath.
（ 2 Cervical organoid construction
① Treatment of tissues
It is recommended that the cervical tissue after sampling be... 2 – 8 ℃ Store and transport under these conditions, and quickly transfer to a clean laboratory for further processing. Cervical organoid construction experimental procedure, organizing photography and registering detailed information.
② Tissue cleansing
After the sampling tube is sterilized, the tissue is removed from the ultra-clean table, placed in a petri dish, and primary buffer is added. B , use 3 mL pasteurized straw or 1000μL The pipette gun is blown and cleaned, and the cleaning operation is repeated three or more times.
③ Tissue dissociation and digestion
Tissue impurities are removed with ophthalmic scissors or a surgical blade, and the forceps are transferred to 1.5 mL EP In the tube, ophthalmic scissors are used to further mechanically dissociate the tissue into a volume of approximately 1~3 mm3 The tissue block was transferred to 15 mL EP In the tube, add 5 mL primary tissue digestive fluid 37 ℃ oscillation digestion 15-25 min 。 Each digestive process 10 min The tissue digestion was observed under a microscope. A small amount of digestive juice was taken and observed under the microscope, and a larger amount was observed. 70 μm The following cell clusters or individual cells are followed by the next step. The degree of tissue digestion is shown in the figure 1 。
If the amount of tissue is too small or the biopsy tissue is used 1 mL primary tissue digestive fluid C In 1.5 mL EP Tubular digestion.
Figure 1 Tissue digestion into more cell clusters or more single cells
（ 3 ) Tissue filtration
After digestion is complete, the tissue vortex is scrolled. 10s , join 3 Double the volume of primary buffer B Digestion is terminated, and the digestive tissue mixture passes through. 100 μm Cell sieve filtration with pore size, continue with 10-20mL primary buffer B Rinsing the tissue to collect more tissue mass cells, 300 g enrichment centrifugation 5 min Abandoning the supernatural; use 8-10ml primary buffer B Resuspend the centrifugal precipitate (remove more impurities), 300 g enrichment centrifugation 5 min They abandoned their superiors.
If the cell pellet contains red blood cells, add 1-2 mL erythrocyte lysate 1-2 min Then, dilute to 10 mL ， 300 g enrichment centrifugation 5 min They abandoned their superiors. Organoid culture was performed directly when there was too little sedimentation or no red blood cells.
（ 4 Organoid culture
Observe the volume of cell pellets collected by centrifugation, and add 25 Double the volume of matrix glue is resuspended, forming 3D Cultivate the spatial structure to avoid bubbles during the resuspension process. The cell pellet volume is shown in the figure. 2 Add the cell pellets according to the amount shown in the figure. 300 μL 、 250 μL 、 150 μL 、 100 μL Matrix glue.
Figure 2 cell pellet volume
24 well cell culture plate according to 25μL-30μL/ Dispensing adhesive through holes The matrix adhesive is maintained throughout the 0-4 ℃ operation under conditions 。 cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix glue solidifies, 24 well cell culture plate addition per well 750μL human cervical organoid culture medium A Stand (at room temperature) 37 ℃ Incubator culture.
3 Organoid subculture (with 24 (Taking orifice plates as an example)
（ 1 Experimental materials
passage buffer G （ 4 ℃ ), organoid digestive fluid D (Room temperature or 37 ℃ ), matrix glue (in advance 24 h Put in 4 ℃ (Refrigerator melting) cervical organoid culture 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, generally after about a week of growth, microscope 10X You can see more below 20 Organoids, or size 100-200μm Organoids.
Aspirate off the medium and add an equal volume of passage buffer to each well G The pipette gently blows away the matrix glue, collecting it from... 15 mL In the centrifuge tube, each 6-8 The hole is transferred to a centrifuge tube, 4 ℃ standing 10-15 min 。
（ 3 ) Organoid digestion
The need for digestive passaging is determined based on the growth of the organoid. After centrifugation, if there is little precipitation at the bottom of the tube, no cells are found, and the matrix glue is not delaminated, it can be resuspended again to increase the centrifugal force and centrifuge again. 。
When the number of organoids is insufficient or the volume is small, 300 g centrifuge 5 min Abandon the superiors and the Qing
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 digestive fluid D After the cell pellet is blown out, it is digested at room temperature. 2-4 min Blow it every minute, each time... 20 Observe under a microscope until digestion reaches (Figure). A-B It can be stopped when in the ) state. Add 3 Passage buffer with twice the volume of organoid digestive fluid G Termination of digestion, 300 g centrifuge 5 min Abandon. 
Figure 3 Organoid passage digestion degree diagram
（ 4 ) Passage organoid culture
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. The volume of matrix rubber can be referenced. “ Organoid primary culture operation Figure 2 ” 。
24 well cell culture plate according to 25μL-30μL/ Dispensing adhesive through holes The matrix adhesive is maintained throughout the entire process. 0-4 ℃ operation under conditions 。 cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix glue solidifies, 24 well cell culture plate addition per well 750μL human cervical organoid culture medium A Stand (at room temperature) 37 ℃ Incubator culture.
4 Organoid cryopreservation (with 24 (Taking orifice plates 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 Cryopreservation of organoids
Organoids that are temporarily unused should be frozen and stored in a low-temperature environment.
Aspirate off the medium and add an equal volume of passage buffer to each well G The pipette gently blows away the matrix glue, collecting it from... 15 mL In the centrifuge tube, each 6-8 The hole is transferred to a centrifuge tube, 4 ℃ standing 10-15 min 。 300 g centrifuge 5 min Discard the supernatant and add it every three holes. 2 mL organoid cryopreservation fluid F Gently blow and mix well, then transfer to cell cryopreservation tubes. Each tube... 1 mL 。
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.
5 Organoid resuscitation culture (with 24 (Taking orifice plates as an example)
（ 1 Experimental materials
passage buffer G 、 cervical organoid culture medium A Matrix glue (in advance) 24 h Put in 4 ℃ (Refrigerator melting) 24 well cell culture plates, ice boxes, 15mL Centrifuge tube, water bath, 3mL pasteurized straw / Pipetting gun.
（ 2 Organoid resuscitation culture
The frozen organoids were removed from the low-temperature 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 mL Centrifuge tube, gently blow with 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 centrifuge tube 300 g centrifuge 5 min They abandoned the Shangqing
Add per cryopreservation tube 120μL The matrix glue is resuspended, 24 well cell culture plate according to 25μL-30μL/ When dispensing the adhesive through the holes, the matrix adhesive is maintained throughout the entire process. 0-4 ℃ Operate under these conditions. cell culture plate placement 37 ℃ Incubator 10-15 min After the matrix glue solidifies, 24 well cell culture plate addition per well 750μL human cervical organoid culture medium A Stand (at room temperature) 37 ℃ Incubator culture.
6 Use of matrix glue
In 2–8 ℃ The matrix glue was thawed overnight under ambient conditions. When using matrix glue, keep it in an ice box to prevent premature setting. Matrix glue in 37 ℃ Below 20 A gel formed within minutes.
（ 1 Characteristics of matrix rubber ：
① 4 ℃ continuous 14 Good liquidity can still be maintained today.
② Put in 37 ℃ Incubator 10-15 min It can solidify
③ It is not easily damaged during the culture process, and the culture plate is clean and does not stick when the glue is removed.
（ 2 Scope of application :
For Laboratory Research Use Only. Not For Use In Diagnostic Procedures.
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