Experiment Products Used
|
Product Name |
Cat.NO. |
|
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES |
1. Sample Preparation
Sacrifice 8-week-old C57 male mice by cervical dislocation and immerse in alcohol for 5 minutes for disinfection. In a sterile environment, harvest approximately 8 cm of small intestine tissue and place it in pre-chilled 4°C DPBS solution.
2. Sample Washing
Wash the small intestine tissue twice with 10 mL DPBS solution.
3. Sample Processing
Use surgical scissors to longitudinally dissect the intestinal tissue. Use a sterile scalpel to gently scrape the intestinal villi from the luminal surface; repeat this step 3 times. Subsequently, wash the tissue 3 times with 10 mL DPBS. Next, mince the tissue into approximately 2 mm wide fragments and wash again with 10 mL DPBS 3 times.
4. Sample Digestion
Add 10 mL DPBS and 100 μL of 0.5 M EDTA solution to the tissue fragments and mix gently. Incubate on a shaker at 4°C and 90 rpm for 30 minutes.
5. Washing
After digestion, discard the supernatant containing EDTA. Add 10 mL of 0.1% BSA solution to wash the tissue pellet; repeat this washing step 2 times.
6. Collecting Cell Suspension
Add 10 mL of 0.1% BSA solution to the washed tissue and pipette repeatedly. Examine a small aliquot of the suspension under a microscope to observe the release of organoid-like structures. Filter the entire tissue suspension through a 70 μm cell strainer to collect cell clusters. Repeat this pipetting and filtration step 2 times.
7. Washing
Centrifuge the filtrate collected in Step 6 at 1000 rpm, 4°C for 3 minutes. Discard the supernatant to obtain the crypt/organoid-like structure pellet.
8. Mixture Formation
Resuspend the small intestine crypt pellet obtained in Step 7 with a mixture of 180 μL CeturegelTM Matrix and 60 μL basal medium (total volume 240 μL, matrix-to-medium ratio 3:1). Pipette gently to mix and keep on ice.
9. Plating
Plate the mixed suspension in the center of the bottom of each well in a 24-well plate, seeding 30 μL per well. Place the plate in a 37°C, 5% CO₂ incubator and incubate for approximately 30 minutes to allow the matrix to solidify completely.
10. Culture
After the matrix has solidified, slowly add 500 μL of pre-warmed small intestine organoid complete medium along the side wall of each well. Return the plate to the 37°C, 5% CO₂ incubator. Change the medium with fresh complete medium every 3 days and observe the growth and morphology of the organoids.
Organoid Construction Experiment Tips
1.Sample Processing: Maintain sterile operation, quickly remove necrotic tissue, keep samples at low temperature, and ensure initial cell viability.
2.Digestion Process: Strictly control digestion solution concentration, temperature, and time; terminate via microscopic inspection to avoid over-digestion.
3.Matrix Operation: Perform all steps on ice, resuspend and plate quickly to prevent gel solidification, and ensure centered gel spotting.
4.Culture Maintenance: Use fresh medium, change medium gently and regularly, maintain a stable culture environment, and frequently inspect morphology microscopically.
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|
41423ES |
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|
41424ES |
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|
41451ES |
CebraryTM Organoid Rinsing Solution |
|
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|
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