Homo sapiens (Primary cells)

(Cryopreservation of Primary Cells from Human Tissue)

Intro/Description:

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.

Materials:

• 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

Protocol:

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.

References:

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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