Homo sapiens (Primary cells)
This simple protocol is for the cryopreservation of disaggregated primary cells from human tissue samples. It is adapted from Current Protocols in Cytometry. The protocol stores the cells at -70°C, and notes that cells can be stored at this temperature for 4 – 6 months. It makes no mention of longer-term storage at cryogenic temperatures.
• Whole primary cell suspension, unfixed
• Growth medium (e.g., RPMI, MEM, Medium 199) containing 5% (v/v) heat-inactivated FBS
• Cryopreservation solution: 1.765 g NaCl (0.3 M final) 15 ml dimethyl sulfoxide (DMSO; 15% final) H2O to 100 ml, stored at 4°C
• 1.25-ml cryovials
• 12 × 75–mm polystyrene tubes
• Beckman RJ-6 centrifuge and TH-4 rotor
1. Transfer 1.0 ml whole-cell suspension at a concentration of 3 to 5 × 106 cells/ml in growth medium containing 5% FBS into 12 × 75–mm polypropylene tube.
2. Centrifuge 5 min at 150 × g (1200 rpm in TH-4 rotor), 4°C.
3. Decant supernatant and quickly resuspend cells in 1.0 ml cryopreservation solution.
4. Immediately transfer contents of tube into a labeled 1.25-ml cryovial and place in −70°C freezer.
Dressler, L. G. and Visscher, D. 2001. Handling, Storage, and Preparation of Human Tissues. Current Protocols in Cytometry. 5.2.1–5.2.15.
Added by: Carlton Hoyt on July 25, 2012
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