Chicken intestine Organid Culture

Figure 1 Tissue digestion into multiple crypts

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 uL 、 250 uL 、 150 uL 、 100uL Matrix glue.  
                                                                                                      Figure 2 cell pellet volume

24 well cell culture plate according to 25 uL-30 uL/ When dispensing the adhesive through the holes, the matrix adhesive is 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 chicken small intestine organoid culture medium A (Preheat) for incubation.
III. Organoid subculture (with 24 (Taking orifice plates as an example)
1. Experimental Materials:
passage buffer G （ 4 ℃ ), organoid passage digestive fluid D (Room temperature or 37 ℃ ), matrix glue (in advance 24 h Put in 4 ℃ (Refrigerator melting) chicken small intestine 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 100-200㎛ 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 。
2.1 organoid digestion
The need for digestive passaging is determined based on the growth of the organoid. 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.
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 passage digestive fluid D After the cell pellet is blown out, it is digested at room temperature. 1min Blow every minute and observe under a microscope until digested (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.

                                                                         A                                                                                     B
                                                                       

                                                                                            Figure 3 Organoid passage digestion degree diagram

2.2 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 25 uL-30 uL/ 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 gum has set, add to each well. 500-750 uL chicken small intestine organoid culture medium A (Preheat) for incubation.
four 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 Organoid cryopreservation:
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.
V. Organoid resuscitation culture (with 24 (Taking orifice plates as an example)
1 Experimental materials:
passage buffer G 、 chicken small intestine 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 100 uL The matrix glue is resuspended, 24 well cell culture plate according to 25 uL-30 uL/ 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 39 ℃ Incubator 10-15 min After the matrix gum has set, add to each well. 500-750 uL chicken small intestine organoid culture medium A (Preheat) for incubation.
VI. Use of matrix adhesive
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. Matrix adhesive characteristics:
（ 1 ） 4 ℃ continuous 14 Good liquidity can still be maintained today.
（ 2 ) Put in 37 ℃ Incubator 10-15 min It can solidify
（ 3 The culture plate is not easily damaged during the cultivation process, and the glue is clean and does not stick to it.