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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.
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.
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.
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:
1.
Culture of Animal Cells: A Manual of Basic Technique, Freshney, R. (1987), Alan R. Liss, Inc.,
New York.
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