Epithelial Cell Culture
1) Epidermal Cell Culture
1. Materials: Obtain surgical skin grafts or leftover skin pieces from surgery, preferably with a thin keratinized layer. Skin from premature infants is often 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 0.02% EDTA solution at room temperature for 5 minutes. This helps to loosen the cell junctions and make the separation process easier.
3. Cold Digestion: Transfer the tissue to 0.25% trypsin solution and incubate at 4°C overnight. The cold digestion allows for gentle dissociation of 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 dermis. Be gentle to avoid tearing the delicate epidermal layer.
5. Warm Digestion: Take the separated epidermis, cut it into smaller fragments with scissors, and place them in a fresh 0.25% trypsin solution. Incubate at 37°C for 30 to 60 minutes to further digest the tissue and release individual cells.
6. Cell Suspension: Use a pipette to gently and repeatedly aspirate and expel the mixture, ensuring a uniform cell suspension without excessive shearing.
7. Culture Preparation: Filter the cell suspension through an 80-mesh stainless steel sieve to remove any large debris. Centrifuge at low speed, discard the supernatant, and resuspend the pellet in Eagle’s medium supplemented with 20% calf serum. Inoculate the cells into a culture dish and incubate in a CO₂ incubator at 37°C with 5% CO₂ for optimal growth.
2) Breast Tissue Culture
Direct Culture Method: (Ideal for soft tissues with minimal fibrous components)
1. Using a sharp blade, cut the tissue into small pieces within a container that contains a small amount of culture medium or Hanks’ solution. This helps keep the tissue viable during preparation.
2. Transfer the tissue fragments along with the liquid into a centrifuge tube. Add a bit more culture medium, and gently pipette the mixture for a few minutes. Let the tube sit on a rack for 3 to 5 minutes to allow heavier cells to settle. Aspirate the supernatant to remove non-mammary cell fractions. Repeat this process 2 to 3 times to ensure purity.
3. After the final step, add more culture medium to the tube and gently resuspend the sediment using a pipette. Immediately filter the mixture through 3 to 4 layers of sterile Gauze into another tube. Do not wait for the cells to settle—filter immediately to maintain cell viability.
4. Adjust the cell density as needed and inoculate the cells into a culture flask for further cultivation.
Collagenase Digestion: (Best suited for harder tissues with high fibrous content)
This method involves using collagenase to break down the extracellular matrix, making it easier to isolate epithelial cells from dense or fibrous breast tissue. The process typically includes enzymatic digestion at specific concentrations and incubation times, followed by filtration and cell collection similar to the direct culture method. This approach is particularly useful when working with tissues that are difficult to dissociate using trypsin alone.
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