Dissociation of Cells from Primary Tissue
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.
1. Collegenase
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).
Add Collegenase (50 to 200 units/ml in HBSS).
Incubate at 37°C for 4 to 18 h. Addition of 3 mM CaC12 increases the efficiency of dissociation.
Filter the cell suspension through a sterile stainless steel mesh to separate the dispersed cells and tissue fragments larger pieces. Fresh Collegenase can be added to the fragments if further disaggregation is required
Wash suspension several times by centrifugation in HBSS.
Resuspend the pellet in culture medium. Count and seed for culture.
2. Trypsin
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 resuspend 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, 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),
Incubate at 4°C for 6 to 18 h to maximize penetration of the enzyme with little trypsin activity.
Decant and discard the trypsin from the tissue pieces. Incubate the tissue pieces with residual trypsin at 37°C for 20 to 30 min.
Add warm complete medium to the tissue pieces and gently disperse the tissue by pipetting. If using a serum-free medium, also add a soybean trypsin inhibitor,
Filter the cell suspension through sterile stainless steel mesh (100 to 200 um) to completely disperse any remaining tissue. Count and seed the cells for culture.
3. Dispase
Mince tissue into 3- to 4-mm pieces with a sterile scalpel or Wash the tissue pieces several times in a calcium and mal free balanced salt solution
Add dispase (0.6 to 2.4 units/ml in calcium and magnesium balanced salt solution).
Incubate at 37°C for 20 min to several hours
Filter the cell suspension through a sterile stainless steel mesh to separate the dispersed cells and tissue fragments larger pieces. Fresh dispase can be added to the fragments disaggregation is required.
Wash suspension several times by centrifugation in the balanced salt solution.
Resuspend the pellet in culture medium. Count and seed for culture.
Reference:
Culture of Animal Cells: A Manual of Basic Technique, Freshney, R. (1987), Alan R. Liss, Inc.
New York.
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