Experiment Products Used
|
Product Name |
Cat.NO. |
|
Ceturegel™ Matrix for Organoid culture, Phenol Red-Free, LDEV-Free |
40192ES |
1. Sample Preparation
Obtain ovarian cancer surgical specimens under sterile conditions. Rinse the specimen surface with saline. Excise tissue blocks of approximately 111 cm, carefully remove visible necrotic tissue and major vessels using forceps, and place them in 4℃ pre-chilled DPBS solution containing 1% antibiotics.
2. Sample Washing
Gently rinse the specimen 2-3 times using saline containing 1% triple antibiotics with a syringe.
3. Sample Processing
Place the washed ovarian tumor tissue in a sterile petri dish and mince it into approximately 1 mm³ fragments using surgical scissors, then transfer to a new 50 mL centrifuge tube. Add pre-chilled DPBS and gently wash the tissue fragments 3-5 times.
4. Sample Digestion
Add 10-15 mL of pre-chilled DPBS digestion solution containing 3-5 mM EDTA to the washed tissue fragments. Incubate at 4℃ for approximately 30 minutes, gently shaking the centrifuge tube once every 10 minutes.
5. Washing
After digestion, carefully discard the supernatant containing EDTA. Add new pre-chilled DPBS buffer and gently rinse the tissue pellet 2-3 times to thoroughly remove residual EDTA.
6. Collecting Cell Suspension
Add 10-15 mL of pre-chilled DPBS solution containing 0.1% BSA to the washed ovarian tumor tissue fragments. Use a pipette to repeatedly blow and resuspend, separating crypt-like structures from the matrix. Observe a small amount of suspension under a microscope; stop pipetting when sufficient crypt-like structures are found. Filter the entire tissue suspension through a 70 μm cell strainer to collect the filtrate.
7. Washing
Centrifuge the collected cell suspension at 1500 rpm, 4℃ for 3 minutes and discard the supernatant. Repeat steps 5 (DPBS washing) and 6 (pipetting and filtering) twice. Finally, centrifuge the collected suspension under the same conditions to obtain a relatively pure crypt-like structure pellet.
8. Mixture Formation
Resuspend the tissue pellet obtained in step 7 with pre-chilled CeturegelTM Matrix. Adjust the resuspension concentration so that approximately 200-600 crypt-like structures are contained in every 10 μL of matrix suspension. The resuspended mixture must always be kept on ice, and subsequent operations should be performed as quickly as possible.
9. Plating
Seed the matrix-tissue mixture suspension at the center bottom of each well in a 24-well plate, 30-50 μL per well, ensuring the suspension does not contact the well sidewalls. Place the culture plate in a 37℃, 5% CO₂ incubator and incubate statically for approximately 30 minutes to allow the matrix to solidify completely.
10. Culture
After the matrix has completely solidified, slowly add prepared ovarian cancer-specific organoid growth medium along the well wall, 800 μL per well, ensuring the gel droplets are completely submerged. Place the culture plate in a 37℃, 5% CO₂ incubator. Change to fresh medium every 3 days and regularly observe organoid growth status and morphology under a microscope. Typically, ovarian cancer organoids form within 5-7 days of culture.
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.
Related Products
|
Cat.NO. |
Product Name |
|
41423ES |
CebraryTM Tissue Digestion Solution |
|
41424ES |
CebraryTM Tissue Storage Solution |
|
41451ES |
CebraryTM Organoid Rinsing Solution |
|
40613ES |
Antimicrobial Agent for Primary Cells |
|
41433ES |
CebraryTM Ovarian Cancer Organoid Growth Medium (Human) |