Worth attention, Protocol for Organ like Operations

Tissue Culture Organ Separation:

Soak in organoid buffer containing double antibodies and gentamicin, and use ophthalmic forceps to scrape off tissue, blood, mucosa, intestinal villi, etc. Wash the culture dish three times with organoid buffer (each time the dish is replaced) and cut into approximately 1mm3 tissue blocks

1.To promote the removal of BME, the tissue was placed in a 15ml centrifuge tube, and 10 mL of ice pre cooled DMEM/F12 medium was added. The tissue was centrifuged at a speed of 200 g for 5 minutes at 4 ° C.

2. Suck out the supernatant and use TrypLE or mechanical crushing to decompose organs. The former is suitable for the head and neck, as well as tight mammary, colon, pancreas, and liver organs, while the latter is suitable for ovarian and cystic mammary, colon, pancreas, and liver organs.

Mechanical crushing method:

Resuspension the precipitate in 3 mL DMEM/F12 medium. Install P1000 suction head with filter element and (without filter element) P10 suction head on the pipette, and blow the organoid suspension up and down 30-50 times. The gun head is placed close to the bottom of the tube to generate greater pressure, which helps in the breakdown of similar organs.

TrypLE digestion method:

1. Suspend the precipitate in 3-5 mL TrypLE, blow up and down, and incubate at 37 ° C until organoid decomposition occurs. Install P1000 suction head with filter element and (without filter element) P10 suction head on the pipette, blow up and down ≥ 10 times every 3-5 minutes to promote the decomposition of similar organs. Closely monitor the digestion process and try to shorten the incubation time of TrypLE as much as possible. Different types or quantities of tissues have different digestion times, and the digestion time should not be too long. For example, the puncture sample takes 10 minutes, and small tissue fragments take 30 minutes to 1 hour.

2. Use a P1000 pipette to blow up and down 10-20 times to further promote tissue fragmentation. Add 10ml of KOSR SerumReplacer containing 10% from the top ™ Wash the cells using D/F medium and centrifuge at 200 g for 5 minutes at 4 ℃.

3. Suspend the precipitate again in a solution containing 10% KOSR SerumReplace ™ In 10 mL of culture medium, use 70-100 μ Filter with m cell filter.

4. Centrifuge the resuspended and filtered cells at 200 g for 5 minutes at 4 ° C

Attention to small details:

If the cell precipitate after screening turns red, there may be contamination of red blood cells. It is recommended to add 5 mL of red blood cell lysate and treat at 4 ° C for 5 minutes. After completion of cracking, rinse once with PBS in a 15 mL tube and centrifuge 500 g at 4 ° C for 3 minutes.

Organ like culture:

Count the digested cell suspension and calculate the required number of cells per well based on 10000 to 20000 cells. According to the calculated volume, draw the cell suspension and add it to a pre cooled 1.5 mL centrifuge tube. Fill the pre cooled culture medium to the required volume, and add the pre cooled cell suspension to an equal volume of Matrigel matrix gel. Gently mix (operate on ice throughout the entire process). Then, mix the mixture of cells and matrix gel at a rate of 40% μ Inoculate L/well (not less than 10000 cells/40ul) onto a 24 well plate. Place the inoculated culture plate in the incubator for at least 30 minutes, wait for the Matrigel to completely solidify, and then slowly add 500 pieces per well along the 24 well wall μ L pre restored to room temperature in organoid culture medium, observed under microscope after 3 days, with a particle size greater than 50 μ M means that organoids have already formed, and the culture medium is changed every 5 days for cultivation. The size of organoids is continuously observed, and most organoids are observed to be larger than 50 under the microscope μ If the size does not increase, organs can be collected for subsequent experiments.

3. Organ like passage

After the organ forms and no longer proliferates, collect organoids,

1. Add tissue digestive solution and digest at 37 ° C for 10-15 minutes.

2. Use KOSR SerimReplace ™ Termination of digestion with serum substitute or 0.2% BSA solution

3. Use 100 μ Filtering with a mesh size of m

4. Centrifuge at 1500 rpm for 3 minutes, discard the supernatant, use HBSS to resuspend and precipitate, and centrifuge again at 1500 rpm for 3 minutes; Add pre cooled organoid culture medium for resuspension, counting, and continue cultivation according to the cultivation steps.

Organ like cryopreservation: Direct cryopreservation using Qida biological tissue/organ like cryopreservation solution (product number: SD0055) is required after freezing, and liquid nitrogen is transferred the next day.