Protocol

Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples

This protocol was adapted from "Growth and Manipulation of Cells in Culture," Chapter 2, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This three-volume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman.

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INTRODUCTION

This protocol provides methods for cryofreezing and subsequent thawing of mammalian cells. Pre-confluent cells are trypsinized, pelleted, resuspended in freezing medium, and gradually frozen. When needed, frozen cells are thawed quickly under running tap water and transferred to growth medium.

MATERIALS

Reagents

Ethanol, 70%

Freezing medium

The optimal freezing medium components can be determined by comparing levels of FBS (10-20%) and either DMSO (5-20%) or glycerol (10-15%). Measure recovery after a few days in low-temperature storage.

Growth medium

Pre-confluent cells

Trypsin working solution

Equipment

Cryofreezer (Nalgene) or controlled-rate freezing device

Flasks (e.g., T-25)

Incubator, preset to 37°C

Liquid nitrogen chamber

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