Experiment Products Used
|
Product Name |
Cat.NO. |
|
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES |
1. Sample Preparation
Obtain endometrial biopsy samples under aseptic conditions.
2. Sample Washing
Rinse the biopsy tissue with pre-chilled PBS buffer until the rinse solution becomes clear. The sample weight should be approximately 1g.
3. Sample Processing
Place the washed endometrial tissue in a culture dish and mince it into approximately 1 mm³ tissue fragments using sterile surgical scissors.
4. Sample Digestion
Add an appropriate amount of collagenase digestion solution to the tissue fragments. Incubate in a 37°C constant-temperature water bath shaker or incubator for 15-30 minutes.
5. Washing
After digestion, add DMEM/F12 complete medium containing 1% FBS to terminate the digestion. Centrifuge the digestion mixture (specific speed and time can be set according to standard cell centrifugation conditions, e.g., 300-500 g for 5 minutes). Discard the supernatant to remove collagenase and the termination solution.
6. Collecting Cell Suspension
Resuspend the cell pellet in an appropriate amount of medium. Filter the suspension sequentially through 100 μm and 40 μm cell strainers. First, filter through the 100 μm strainer to remove undigested tissue blocks, then filter the filtrate through the 40 μm strainer. The cell clusters retained on the 40 μm strainer are the desired glandular epithelial cell clusters.
7. Washing
Carefully rinse the back of the 40 μm strainer with an appropriate amount of organoid basal medium to collect the glandular epithelial cell clusters. Centrifuge the collected cell suspension (e.g., 300-500 g for 5 minutes) and discard the supernatant.
8. Mixture Formation
Resuspend the glandular epithelial cell pellet in pre-chilled medium containing 5% CeturegelTM Matrix. Based on the volume of the cell pellet, prepare the mixed suspension at a 1:3 ratio (pellet volume : matrix-medium volume) and keep on ice.
9. Plating
Plate the mixed suspension in the center of the bottom of a 24-well plate, adding 30 μL per well. Avoid letting the suspension touch the side walls of the well. Place the plate in a 37°C, 5% CO₂ incubator for approximately 30 minutes to allow the matrix to solidify completely.
10. Culture
After the CeturegelTM matrix has solidified, slowly add 800 μL of prepared endometrial organoid-specific growth medium along the side wall of each well, ensuring the matrix droplet is completely submerged. Return the plate to the 37°C, 5% CO₂ incubator. Change the medium with fresh growth medium every 2 days and observe the growth status of the organoids. Typically, endometrial organoids will form within 3-5 days.
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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