Experiment Products Used
|
Product Name |
Cat.NO. |
|
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES |
1. Digestion Enzyme Preparation
Prepare the HRA composite digestion enzyme working solution: Take 4.7 mL of serum-free basal medium and sequentially add 200 μL of Solution H, 100 μL of Solution R, and 30 μL of Solution A. Mix thoroughly. Label the tube with the name and date.
2. Sample Pretreatment
Place the tumor tissue in a sterile petri dish. Use surgical scissors and forceps to carefully remove macroscopically visible necrotic (black) areas and major blood vessels (red) areas. Subsequently, wash the tissue blocks several times with a sufficient amount of serum-free basal medium.
3. Sample Processing
Add a small amount of serum-free basal medium to the washed tissue and mince it thoroughly into a paste using surgical scissors or tissue scissors.
4. First Digestion
Add the pre-prepared HRA digestion enzyme working solution to the minced tissue paste, gently pipette to mix, and transfer to a centrifuge tube. Place the tube in a 37°C shaker and digest at 440 rpm for 15 minutes. During digestion, take samples for microscopic examination at any time. When a large number of single cells or cell clusters of ideal size are observed, immediately add serum-containing complete medium (1.5 times the volume of the digestion solution) to terminate the digestion.
5. Cell Suspension Collection
Filter the mixture after digestion termination through a cell strainer into a 50 mL centrifuge tube to collect the filtrate containing single cells/cell clusters. Aliquot the filtrate into 15 mL centrifuge tubes and centrifuge at 350 g for 4 minutes to obtain the cell pellet. Retain the remaining undigested tissue blocks after filtration for secondary digestion.
6. Second Digestion
Add fresh serum-free basal medium to the retained tissue blocks and repeat the digestion and centrifugation process from Steps 4 and 5 (i.e., add HRA digestion, terminate, filter, centrifuge) to obtain more cell pellets. Combine the cell pellets obtained from both digestions.
7. Mixture Formation
Discard the supernatant after centrifugation and resuspend the final cell pellet with pre-chilled CeturegelTM Matrix. It is recommended to resuspend to a cell density of approximately 10⁶ cells per 50 μL of matrix. The resuspended mixture must be kept on ice at all times, and subsequent operations should be performed as quickly as possible to prevent the matrix from solidifying.
8. Plating
Plate the matrix-cell mixed suspension in the center of the bottom of each well in a 24-well plate, adding 30-50 μL per well. Be careful to avoid the suspension touching the well walls. Place the plate in a 37°C, 5% CO₂ incubator and incubate statically for approximately 30 minutes to allow the matrix to solidify completely.
9. Culture
After the matrix has solidified, slowly add 800 μL of pre-warmed organoid-specific complete growth medium along the side wall of each well, ensuring the matrix droplet is completely submerged. Return the plate to the incubator for routine culture. Change the medium with fresh complete medium every 3 days and regularly observe the formation and growth status of organoids under a microscope.
Experimental Tips for Organoid Construction
- Sample Handling: Maintain sterile technique, quickly remove necrotic tissue, and keep the sample cold to ensure initial cell viability.
- Digestion Process: Strictly control the concentration, temperature, and duration of the digestion solution. Terminate digestion promptly based on microscopic examination to avoid over-digestion.
- Matrix Handling: Perform all steps on ice, resuspend and plate quickly to prevent the matrix from solidifying prematurely, and ensure the droplet is centered.
- Culture Maintenance: Use fresh medium, change it gently and regularly, maintain a stable culture environment, and frequently observe morphology under a microscope.
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