Epithelial Cell Culture
1) Epidermal Cell Culture
1. Material: Obtain surgical skin grafts or leftover skin pieces, preferably with a thin keratinized layer. Skin from premature abortion specimens is ideal due to its higher quality. Cut the skin into small pieces measuring approximately 0.5 to 1 square centimeter.
2. EDTA Treatment: Place the tissue in a 0.02% EDTA solution at room temperature for 5 minutes to help loosen the cells.
3. Cold Digestion: Transfer the tissue to a 0.25% trypsin solution and incubate at 4°C overnight. This step helps to gently separate the epidermis from the dermis without damaging the cells.
4. Separation: Carefully remove the skin and use forceps or a vascular clamp to separate the epidermis from the underlying dermis.
5. Warm Digestion: Take the separated epidermis, cut it into smaller fragments using scissors, and place it in a fresh 0.25% trypsin solution. Incubate at 37°C for 30 to 60 minutes to further dissociate the cells.
6. Cell Suspension: Gently pipette the mixture up and down several times to create a single-cell suspension.
7. Culture Preparation: Filter the cell suspension through an 80-mesh stainless steel Gauze, then centrifuge at low speed. Discard the supernatant and resuspend the pellet in Eagle’s medium supplemented with 20% calf serum. Adjust the cell density and inoculate the cells into culture dishes. Incubate in a CO₂ incubator at 37°C with 5% CO₂ for growth.
2) Breast Tissue Culture
Direct Culture Method (Ideal for soft tissues with minimal fibrous components):
1. Use a sharp blade to cut the breast tissue into small pieces within a container containing a small amount of culture medium or Hanks’ solution.
2. Transfer the tissue fragments along with the liquid into a centrifuge tube. Add more culture medium and gently pipette to mix. Let the tube sit for 3 to 5 minutes on a test tube rack. Aspirate the supernatant to remove non-mammary cell fractions. Repeat this process 2 to 3 times for better purification.
3. After the final treatment, add more culture solution to the tube and gently resuspend the remaining cells using a pipette. Immediately filter the suspension through 3 to 4 layers of sterile gauze into another tube, without waiting for the cells to settle.
4. Adjust the cell concentration to an appropriate level and inoculate the cells into a culture flask for further cultivation.
Collagenase Digestion (Suitable for tougher, fibrous tissues):
This method involves using collagenase to break down the extracellular matrix and release individual cells. The procedure typically includes incubating the tissue in a collagenase solution at 37°C for a specific time period, followed by mechanical disruption and filtration to obtain a viable cell suspension. This approach is particularly useful when dealing with dense or fibrous breast tissue that is difficult to dissociate using simple cutting methods alone.
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