Experiment Products Used
|
Product Name |
Cat.NO. |
|
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES |
1. Sample Preparation
Under sterile conditions, harvest mouse liver tissue via microsurgery.
2. Sample Digestion
Transfer the liver tissue to a centrifuge tube and add an appropriate amount of pre-warmed primary mouse liver tissue digestion solution. The volume of the digestion solution should be 3-5 times the volume of the tissue. Subsequently, place the centrifuge tube at 37°C and digest for 10-15 minutes until the tissue is dissociated into a single-cell suspension.
3. Sample Washing
Filter the digested cell suspension through a cell strainer of appropriate pore size to remove undigested tissue fragments. Then, wash the filtered cell suspension with pre-chilled organoid-specific washing solution (or basal medium). After centrifugation, discard the supernatant. Repeat this process several times to obtain a high-purity, high-viability single-cell pellet.
4. Mixture Formation
Perform this step on ice. Estimate and record the approximate volume of the cell pellet obtained in Step 3. Calculate and add pre-chilled CeturegelTM Matrix at 25 times the volume of the cell pellet. Gently pipette to resuspend the cells, forming a homogeneous cell-matrix mixed suspension.
5. Plating
Plate the mixed suspension in the center of the bottom of each well in a 24-well plate, adding 25 μL per well. Place the plate in a 37°C, 5% CO₂ incubator and incubate for 10-15 minutes to allow the matrix to solidify completely.
6. Culture
After the matrix has solidified, slowly add 500-750 μL of mouse normal liver organoid medium (restored to room temperature) along the side wall of each well, ensuring the matrix droplet is completely submerged. Return the plate to the incubator to begin culture.
7. Subsequent Maintenance
According to the medium instructions or routine organoid culture experience, regularly change the fresh medium and observe the formation (typically requiring 3-7 days) 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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