Dissociation of Cells from Primary Tissue

实验概要

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.

实验步骤

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.