Experiment Products Used
|
Product Name |
Cat.NO. |
|
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES |
1. Sample Preparation
Sacrifice the mouse by cervical dislocation and spray the surface with alcohol for sterilization. In a sterile environment, harvest 3-15 cm of colonic tissue from the proximal anal end. Carefully remove the mesentery and fat from the exterior of the intestine using forceps, then place the tissue in pre-chilled DPBS solution containing 1% antibiotics (penicillin/streptomycin) at 4°C.
2. Sample Washing
Flush the intestinal lumen 2-3 times using a syringe. Use surgical scissors to carefully cut the intestine longitudinally, with the lumen facing up. Use a scalpel to gently scrape the intestinal villi from the luminal surface. Once the villi are removed (the tissue appears translucent), place the intestinal tissue in a new petri dish with DPBS and wash 2-3 times.
3. Sample Processing
Mince the washed colonic tissue into 2 mm fragments and transfer them to a new 50 mL centrifuge tube. Gently wash with DPBS 3-5 times to remove villi cells and floating fat tissue.
4. Sample Digestion
Add 10-15 mL of pre-chilled DPBS containing 3-5 mM EDTA to the colonic tissue fragments for digestion. Incubate at 4°C for approximately 30 minutes, gently shaking the centrifuge tube every 10 minutes.
5. Washing
After digestion is complete, discard the EDTA digestion supernatant. Gently rinse the tissue pellet 2-3 times with fresh DPBS buffer to remove residual EDTA.
6. Cell Suspension Collection (First Round)
Add 10-15 mL of pre-chilled DPBS containing 0.1% BSA to the colonic tissue fragments. Pipette repeatedly to resuspend the tissue fragments, allowing the crypts and basal layers to separate. Examine a small aliquot under a microscope; stop pipetting once crypt-like structures are observed. Filter the suspension through a 70 μm strainer to collect the crypt suspension that passes through.
7. Washing and Centrifugation
Centrifuge the collected suspension at 1500 rpm, 4°C for 3 minutes. Discard the supernatant.
8. Repeat Collection and Centrifugation
Add 10-15 mL of pre-chilled DPBS containing 0.1% BSA to the tissue pellet in the centrifuge tube again. Repeat the pipetting and filtration steps from Step 6, collect the filtrate, and centrifuge under the same conditions. Perform this twice in total and combine the crypt pellets obtained from both centrifugations.
9. Mixture Formation
Resuspend the crypt tissue pellet with CeturegelTM Matrix. Adjust the concentration so that every 10 μL of matrix suspension contains 200-600 crypts. Keep the resuspended mixture on ice and proceed as quickly as possible to prevent the matrix from gelling.
10. Plating
Plate the mixed suspension in the center of the bottom of each well in a 24-well plate, adding 30-50 μL per well. Avoid letting the suspension touch the side walls of the plate. Place the plated culture dish in a 37°C CO₂ incubator and incubate for approximately 30 minutes to allow the Ceturegel® Matrix to solidify.
11. Culture
Once the matrix has completely solidified, slowly add 800 μL of prepared mouse colonic organoid growth medium along the wall of each well until the mixture is completely submerged. Place the 24-well plate in a 37°C CO₂ incubator. Change the medium with fresh medium every 3 days and observe the growth status of the organoids. Generally, mouse colonic organoids will form within 5-7 days.
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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|
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|
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|
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|
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