Organoid culture medium kit storage method:  

1. Organoid culture medium can be stored at 4°C for 3 months. After receipt, store at 4°C and use within 1 month. For long-term storage, it is recommended to store at -20°C, avoiding more than 2 freeze-thaw cycles.  

2. Tissue preservation solution and primary tissue digestion solution contain nutrients to maintain cell viability. To preserve the activity of the reagent components, store at -20°C for long-term storage, avoiding more than 2 freeze-thaw cycles.

Operation steps and methods:

1. Organize the pretreatment

1.1 Experimental materials

Primary tissue culture buffer (4℃ pre-cooled), tissue preservation fluid, sampling tube, tissue transport box and ice bag should be prepared in advance.

1.2 Acquisition and transportation of the organization

The sampling and transportation of tissues is the first and most easily overlooked step in the successful construction of organoids. If the tissue is not properly preserved in the early stage, it will lead to poor cell activity, contamination, few normal cells and other problems, which will reduce the success rate of organoids construction.

The tissue should be put into the tissue preservation solution within 30 minutes after being isolated. The tissue was washed 3-5 times with Primary tissue culture buffer, and the blood on the surface of the tissue was flushed clean. The tissue was put into the tissue preservation solution, and the sampling tube was transported in 4℃ low temperature storage (within 72 hours).

2 Primary culture of organoids (using 24-well plate as an example)

2.1 Experimental materials

Prepare the following materials in advance: primary culture buffer (4℃), primary tissue digestion solution (37℃), matrix gel (pre-melted in a 4℃ freezer 24 hours prior), organoid medium (room temperature or 37℃), forceps (10㎝), sharp ophthalmic scissors/surgical blades, disposable 60 mm culture dishes, 15 mL/50 mL centrifuge tubes, 1.5 mL EP tubes, 100㎛ cell strainers, 3 mL Bunsbach pipettes/1000 μL pipettes, 24-well cell culture plates, and metal ice packs.

2.2 Organoid construction

2.2.1, Organization processing

After taking the samples, the organization suggested that they should be stored and transported under 2-8℃ conditions, and quickly transferred to a clean laboratory for organoid construction. The tissues were photographed and detailed information was registered.

2.2.1.1 Cleaning of the organization

After disinfection of the sampling tube, the tissue was taken out in the laminar flow unit and put into a petri dish. Primary tissue culture buffer was added, and the tissue was blown and cleaned with 3 mL pasteur pipette or 1000 uL pipette, and the cleaning operation was repeated for more than three times.

2.2.1.2 Disintegration and digestion of tissues

Using ophthalmic scissors or surgical blades to remove tissue debris, the samples are transferred with forceps into a 1.5 mL EP tube. The tissue is mechanically dissociated into 1-3 mm³ tissue blocks using ophthalmic scissors, then transferred to a 15 mL centrifuge tube. Add 5-8 mL primary tissue digestion solution and vortex-blade digest for 15-25 minutes at 37°C. Observe the digestion process under a microscope every 10 minutes. Take a small amount of digestion solution for microscopic examination. When numerous cell clusters or individual cells smaller than 70 um are observed, proceed to the next step. The extent of tissue digestion is shown in Figure 1.

If the tissue quantity is too small, the biopsy tissue is digested in 1 mL primary tissue digestion solution in 1.5 mL EP tube.

Figure 1: The cancerous tissue is digested into more cell clusters or more single cells.

2.2.1.3 Organization filtering

The digested tissue is filtered through a 100 μm cell sieve. The filtrate is collected and washed with three times the volume of Primary tissue culture buffer to terminate digestion. After 300 g enrichment centrifugation for 5 minutes, the supernatant is discarded. If red blood cells are present in the cell sediment, add 1-2 mL of red blood cell lysing solution and incubate for 1-2 minutes. Dilute to 10 mL and perform 300 g enrichment centrifugation for 5 minutes before discarding the supernatant. When minimal or no red blood cells are present in the sediment, proceed directly to organoid culture.

2.2.1.4 Organoid culture

The volume of the centrifuged cell precipitate was observed. A 25-fold volume of matrix gel was added to resuspend the cells, forming a 3D culture spatial structure while avoiding bubble generation during suspension. As shown in Figure 2, the cell precipitate volumes were divided into four groups: 200 ul, 150 ul, 100 ul, and 50 ul matrix gel according to the cell precipitate volumes specified in the Figure2.

       Figure 2: Cell pellet volume

For the 24-well cell culture plate, apply a matrix adhesive solution of 25-30 μL per well, maintaining the adhesive concentration at 0-4℃ throughout the process. Place the plate in a 37℃ incubator for 10-15 minutes until the matrix gelatinizes. After solidification, add 500-750 μL organoid medium (preheated at 37℃) to each well for cultivation.

3. Generation of organoids (using 24-well plate as an example)

3.1 Experimental materials

Generation buffer (4℃), organoid generation digestion buffer (room temperature or 37℃), matrix gel (placed in 4℃ refrigerator for 24 h to melt), organoid medium (room temperature or 37℃), 1.5 mL/15 mL centrifuge tube, 24-well cell culture plate, and ice box.

3.2 Organoid propagation

Select appropriate organoids for subculture. Generally, after about one week of growth, more than 20 organoids or organoids with a size of 100-200um can be seen under the microscope at 4X.

After aspirating the culture medium, add an equal volume of generation buffer to each well, gently blow the matrix glue with a pipette, collect it in a 15 mL centrifuge tube, and transfer it to a centrifuge tube for 6-8 holes, then 4℃ stand at 10-15 min or-20℃ stand at 4-8 min.

3.2.1 Organoid digestion

The need for digestion and subculture should be determined according to the growth of the organoids. If there is little precipitate at the bottom of the tube, no cells are seen, and the matrix gel is not stratified after centrifugation, the tube can be resuspended again and centrifuged again with increased centrifugal force.

When the number of organoids is insufficient or the volume is small, centrifuge at 300g for 5 min and discard the supernatant.

When the number of organoids is large or the volume is large, 300g centrifuge for 5min and discard the supernatant. Digestion solution digestion or mechanical digestion can be selected.

Digestion with digestion buffer: Add 1-2 mL of organoid generation digestion buffer. After dispersing the cell precipitate, digest at room temperature for 1-3 minutes. Perform hemolysis every minute and observe under a microscope until digestion progresses from (Figure A) to (Figure B). Terminate digestion by adding three times the volume of organoid digestion buffer in the generation buffer, then centrifuge at 300 g for 5 minutes and discard.

Mechanical digestion: Add 1 mL of transfection buffer and blow 1 mL of gun head 20-30 times until the state from (Figure A) to (Figure B) is stopped. Add 10 mL of transfection buffer and centrifuge at 300g for 5 minutes.

Figure 3: Organoid Generation Digestion Degree Diagram

3.2.2 Transgenerated organ culture

Monitor the volume of organoid precipitate collected by centrifugation. If the precipitation is minimal, reserve an equal volume of supernatant for dispensing. For substantial precipitates, completely aspirate the supernatant and resuspend the organoids using 25 times the volume of matrix gel compared to the precipitate. The appropriate volume of matrix gel can be referenced from "Protocol Figure 2: Primary Culture of Organoids".

For the 24-well cell culture plate, apply a matrix adhesive solution of 25-30 μl per well under continuous 0-4℃ temperature control. Place the plate in a 37℃ incubator for 10-15 minutes until the adhesive solidifies. Then add 500-750 ul of organoid medium (preheated at 37℃) to each well for cultivation.

4. Cryopreservation of organoids (using 24-well plate as an example)

4.1 Experimental materials

Transient buffer (4℃), organoid cryopreservation solution (4℃), 15 mL centrifuge tube, cell cryovial, programmed cooling box, pipette.

4.2 Organoid cryopreservation

Organoids that are not in use should be cryopreserved and stored at low temperatures.

Aspirate the culture medium and add an equal volume of generation buffer to each well. Gently disperse the matrix gel using a pipette and transfer it to 15 mL centrifuge tubes. Transfer 6-8 wells to a single centrifuge tube and let it stand at 4℃ for 10-15 minutes. Centrifuge at 300 g for 5 minutes, then discard the supernatant. Add 2 mL of organoid cryopreservation solution to every three wells, gently aspirate and mix thoroughly. Transfer the mixture into cell cryovials at 1 mL per tube.

Prepare the labeled information, place it in the cooling box, transfer to the-80℃ freezer. After 48 hours, store in a liquid nitrogen tank. Alternatively, place in a 4℃ freezer for 40 minutes, then transfer to the-20℃ freezer for 2 hours, and finally move to the-80℃ freezer. After 48 hours, store in a liquid nitrogen tank.

5. Organoid recovery culture (using 24-well plate as an example)

5.1 Experimental materials

Transferred buffer, organoid medium, matrix gel (placed in 4℃ refrigerator for 24 hours in advance to melt), 24-cell culture plate, ice box, 15mL centrifuge tube, water bath, 3mL pasteur pipette/transfer gun.

5.2 Organoid recovery culture

After thawing cryopreserved organoids from low-temperature conditions, immediately place them in a 37°C water bath to ensure complete dissolution. Gently shake the storage tube during thawing to allow rapid liquefaction. Transfer the thawed organoids into a 15 mL centrifuge tube and gently pipette 6-8 times with a pipette. Centrifuge at 300 g for 5 minutes, then discard the supernatant. Resuspend the cells using an appropriate amount of generation medium, then transfer to a 1.5 mL centrifuge tube and centrifuge at 300 g for 5 minutes. Finally, discard the supernatant.

Add 100 μL of matrix gel to each tube and resuspend. For the 24-well cell culture plate, apply 25-30ul per well using a pipette, maintaining the matrix gel at 0-4℃ throughout the process. Place the plate in a 37℃ incubator for 10-15 minutes until the matrix gel solidifies. Then add 500-750ul of organoid medium (preheated at 37℃) per well for cultivation.

6. Use of matrix gel

Under 2-8℃ conditions, the matrix gel is thawed overnight. When using the matrix gel, keep it in an ice tray to prevent premature solidification. The matrix gel forms a gel within 20 minutes at 37℃.

6.1 Characteristics of matrix gel:

Ø 4℃ It can still maintain good liquidity for 14 consecutive days

Ø Place in a 37℃ incubator for 10-15 min to solidify

Ø During the cultivation process, it is not easy to break, and the adhesive is removed cleanly without sticking to the culture plate