Experiment Products Used
|
Product Name |
Cat.NO. |
|
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES |
1. Sample Preparation
Sacrifice mice at postnatal day 2-3 (P2-3) by cervical dislocation and disinfect the body surface with alcohol spray. In a sterile environment, harvest the mouse cochlea and place it in pre-chilled HBSS buffer. Use fine forceps to carefully remove connective and excess tissue attached to the cochlea.
2. Sample Digestion
Remove the cochlea from HBSS and isolate the basilar membrane tissue under a stereomicroscope. First, place it in a digestion solution and incubate at 37°C for 20 minutes to dissociate the sensory epithelial sheets. Subsequently, collect the sensory epithelial cell clusters with forceps, transfer them to TrypLE digestion solution, and continue digestion at 37°C for 45 minutes to fully dissociate them into single cells or small cell clusters.
3. Collecting Cell Suspension
After digestion, add 1 mL of DMEM/F12 complete medium to terminate the digestion reaction. Gently pipette the cell suspension approximately 20 times, then filter it through a 300-mesh (approx. 40-50 μm pore size) cell strainer to remove undigested tissue clumps. Centrifuge the filtrate at 1000 rpm for 4 minutes, discard the supernatant, and obtain the cochlear epithelial cell pellet.
4. Mixture Formation
Resuspend the cell pellet obtained in Step 3 with pre-prepared cochlear organoid complete medium containing 5% CeturegelTM Matrix. Adjust the resuspension volume based on cell counting to achieve the desired cell density.
5. Plating
Plate the mixed suspension of cells and CeturegelTM Matrix in a 96-well U-bottom ultra-low attachment culture plate, seeding approximately 3000 cells per well. The suspension volume can be optimized based on the experiment (typically 50-100 μL).
6. Culture
Incubate the plate in a 37°C, 5% CO₂ incubator. During the culture period, perform a half-medium change every two days. Typically, typical cochlear organoid structures can be observed under a microscope after 4 to 6 days of culture.
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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