1. Isolation of cells from primary tissues: There are several techniques available for dissociating tissue fragments into single-cell suspensions. The most commonly used methods include mechanical dissociation, enzymatic dissociation, and chelating agent-based dissociation. Among these, enzymatic digestion is the most widely used method for obtaining high-quality cell suspensions. It is essential to minimize the exposure time of cells to enzymes in order to preserve their viability and function. The following procedures can be followed to efficiently break down the tissue and obtain a high yield of active cells.
(1) Trypsin
• After removing any non-target tissue, use sterile scissors and a scalpel to cut the remaining tissue into small pieces (3–4 mm). Wash the tissue in a calcium- and magnesium-free balanced salt solution. Allow the tissue to settle and remove the supernatant. Repeat this washing process 2–3 times to ensure thorough cleaning.
• Place the tissue on ice and add trypsin (1 ml per 100 mg of tissue), which has been dissolved in a 0.25% calcium-free balanced salt solution.
• Incubate at 4°C for 6–18 hours to allow the enzyme to penetrate the tissue slowly and gently.
• After incubation, remove the trypsin and incubate the tissue at 37°C for 20–30 minutes to complete the digestion.
• Add the tissue to pre-warmed complete medium and gently pipette to disperse the tissue. If using serum-free medium, add soybean trypsin inhibitor to neutralize the enzyme.
• Pass the suspension through a sterile stainless steel mesh (100–200 μm) to separate individual cells from larger debris. Count the cells and seed them for further culture.
(2) Collagenase
• Cut the tissue into 3–4 mm pieces and wash it several times with Hanks’ Balanced Salt Solution (HBSS).
• Dissolve collagenase (50–200 units/ml) in HBSS and add it to the tissue.
• Incubate the tissue at 37°C for 4–18 hours. Adding 3 mM CaCl₂ can enhance the efficiency of the digestion.
• Filter the cell suspension through a sterile stainless steel or nylon mesh to remove large particles and undigested tissue.
• Centrifuge the suspension and wash it with HBSS to remove residual enzymes.
• Resuspend the cells in culture medium, count them, and proceed with seeding and culturing.
(3) Dispase
• Cut the tissue into 3–4 mm pieces and wash it several times with a calcium- and magnesium-free balanced salt solution.
• Add Dispase (0.6–2.4 units/ml) dissolved in the same balanced salt solution.
• Incubate the tissue at 37°C for 20 minutes to several hours, depending on the tissue type.
• Filter the cell suspension through a sterile mesh to separate individual cells from debris. If needed, add fresh Dispase to the remaining tissue for further digestion.
• Centrifuge the suspension and wash it with the balanced salt solution.
• Resuspend the cells in medium, count, and seed them for culture.
2. Isolation of cells from original culture vessels: This procedure is commonly used to detach cells from the culture surface while maintaining their integrity. However, the optimal conditions may vary between cell lines, so it is important to test and adjust the protocol accordingly.
• Check cell viability before reseeding. Ideally, viability should be above 90%.
• For serum-free media, reduce the amount of trypsin used to avoid over-digestion.
(1) Remove the spent culture medium.
(2) Wash the cells with a calcium- and magnesium-free balanced salt solution or a solution containing EDTA. Add the washing solution to the side of the flask facing the cells, swirl for 1–2 minutes, and then remove the solution.
(3) Add 2–3 ml of the selected dissociation solution (as specified in the table) to the side of the flask containing the cells. Ensure the solution covers the cell layer. Incubate at 37°C and gently shake the flask. Cells typically detach within 5–15 minutes, but the exact time depends on the cell line. Monitor closely to prevent over-digestion. For stubborn cells, a gentle tap can help speed up the process.
(4) Once all cells have detached, place the flask upright to let the cells collect at the bottom. Add complete medium and pipette gently across the monolayer to disperse the cells.
(5) For serum-free cultures, add soybean trypsin inhibitor. A common ratio is 1:1 (v:v) of 0.25 mg/ml trypsin inhibitor to trypsin to effectively neutralize the enzyme activity.
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