Trypsin-EDTA (0.05%), phenol red

Dissociation of cells from Primary Tissue

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 supernatant. Repeat the wash 2 or 3 times.
2. Place the container with the tissue pieces on ice, and remove any remaining supernatant. 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.

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 disaggregation 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.

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 disaggregation is required.
5. Wash suspension several times by centrifugation in the balanced salt solution.
6. Resuspend the pellet in culture medium. Count and seed the cells for culture.

REFERENCE:
Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.

The basal Diploid Growth Serum-Reduced Medium already contains 6 mM L-glutamine.

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We recommend evaluating performance with Diploid Production Serum-Free Medium using current conditions, however, multiplicity of infection, time of infection, and time of harvest may be different than with conventional media. Closely monitor cells for cytopathic effect and evaluate viral titers at multiple time points.

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Diploid Growth Serum-Reduced Medium can be supplemented with Fetal Bovine Serum, Newborn Calf Serum, Donor Bovine Serum with Iron, or Bovine Serum.

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Diploid Growth Serum-Reduced Medium has been evaluated with fibroblast cell lines, including MRC-5, WI-38, IMR-90, BS-2, and CEF cells.

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