實驗概要
A common method to obtain single cell suspensions from primary tissue is enzymatic disaggregation. Expose the cells to enzymes for a minimal amount of time to preserve maximum viability. The following procedures disaggregate whole tissue to obtain a high yield of viable cells.
實驗步驟
1. TrypLE? Products
TrypLE? products are formulated to allow direct substitution into your existing protocols. The following general procedure can be used to remove various cell lines from cultureware while maintaining cellular integrity. Optimal conditions and concentrations employed for individual systems should be determined empirically.
1) Decant media from flask. Rinse flask with 5 ml of Dulbecco’s Phosphate Buffered Saline (D-PBS) without calcium and without magnesium (GIBCO? cat. number 14190). Decant D-PBS.
2) Add an appropriate volume (i.e. 2 ml in a 75 cm2 flask) of prewarmed TrypLE? to flask. Rock vessel to coat cell sheet completely.
3) Incubate at 37°C until cells have detached (observe at 5 minute intervals). Gently rap vessel to dislodge cells.
4) Dilute in 2 to 5 ml of cell culture growth media and transfer cell suspension to a 15 ml centrifuge tube.
5) Centrifuge for 5 to 10 minutes at 100 × g. Discard supernatant and suspend cell pellet with 2 to 5 ml of fresh growth medium.
2. Trypsin
1) After dissecting off unusable tissue, mince the remaining tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces by resuspending in a balanced salt solution without calcium and magnesium. Allow the tissue pieces to settle, and remove the supernate. Repeat the wash 2 or 3 times.
2) Place the container with the tissue pieces on ice, and remove any remaining supernate. Add 0.25% trypsin in a balanced salt solution without calcium or magnesium (1 ml of trypsin for every 100 mg of tissue).
3) Incubate at 4°C for 6 to 18 h to maximize penetration of the enzyme with little trypsin activity.
4) Decant and discard the trypsin from the tissue pieces. Incubate the tissue pieces with residual trypsin at 37°C for 20 to 30 min.
5) Add warm, complete media to the tissue pieces and gently disperse the tissue by pipetting. If using a serum-free medium, also add soybean trypsin inhibitor.
6) Filter the cell suspension through sterile, stainless steel mesh (100 to 200 μM) to completely disperse any remaining tissue. Count and seed the cells for culture.
3. Collagenase
1) Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces several times with Hanks' Balanced Salt Solution (HBSS).
2) Add collagenase (50 to 200 U/ml in HBSS).
3) Incubate at 37°C for 4 to 18 h. Addition of 3 mM CaCl2 increases the efficiency of dissociation.
4) Filter the cell suspension through a sterile stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh collagenase can be added to the fragments if further disagregation is required.
5) Wash suspension several times by centrifugation in HBSS.
6) Resuspend the pellet in culture medium. Count and seed the cells for culture.
4. Dispase
1) Mince tissue into 3 to 4 mm pieces with a sterile scalpel or scissors. Wash the tissue pieces several times in a calcium and magnesium-free balanced salt solution.
2) Add dispase (0.6 to 2.4 U/ml in calcium and magnesium-free balanced salt solution).
3) Incubate at 37°C for 20 min to several hours.
4) Filter the cell suspension through a sterile, stainless steel or nylon mesh to separate the dispersed cells and tissue fragments from the larger pieces. Fresh dispase can be added to the fragments if further disaggegation is required.
5) Wash suspension several times by centrifugation in the balanced salt solution.
