Comprehensive Guide to Freezing Cells: Ensuring Viability and Sterility

Freezing cells is a critical process in cell culture management, ensuring long-term storage and preservation of valuable cell lines. Whether working with human or animal cells, following precise protocols and maintaining strict aseptic techniques are paramount to achieving successful cryopreservation.

This guide provides detailed instructions on the necessary materials, reagents, and step-by-step procedures to effectively freeze cells while preserving their viability and sterility. By adhering to these guidelines, researchers can confidently store and later revive cell cultures with minimal loss in cell viability, maintaining the integrity and reliability of their experimental results.

To begin, it is essential to establish a sterile working environment. Proper aseptic techniques play a crucial role in preventing contamination and ensuring the sterility of the cultures.

Aseptic Technique

Follow aseptic techniques throughout the process to ensure the sterility of the cultures.

Comprehensive Cell Freezing Process

This flowchart provides a detailed visual guide to the essential steps involved in the proper freezing of cells. Follow these procedures to ensure high cell viability and integrity during the cryopreservation process.

Freezing Medium Preparation

Prepare cryoprotective freezing medium according to the cell type:

FBS-containing media cultures: 90% FBS + 10% DMSO or 70% growth medium, 20% FBS and 10% DMSO
Cells requiring glycerol: 90% FBS + 10% glycerol

Consider acquiring our pre-formulated freeze medium:

Freeze Medium CM-1: For protocols requiring a medium containing Fetal Bovine Serum (FBS)
Freeze medium CM-ACF - serum free: For an FBS-free alternative

Labeling and Documentation

Label cryovials with the following details:

Date
Researcher's name
Cell number
Passage number
Cell type
Any additional information (e.g., genetic modifications)

Cell Preparation

For Adherent Cells:

Monitor cells until they reach 85–95% confluence
Aspirate the old culture medium
Rinse the cell monolayer with calcium- and magnesium-free PBS
Detach cells with Accutase, trypsin, or trypsin-EDTA and neutralize with culture media if required
Transfer the cell suspension into a 50 mL Falcon tube

For Suspension Cells:

Check cell density and health under a microscope
Transfer the cell suspension directly into a 50 mL Falcon tube

Cell Counting and Centrifugation

Count cells using a hemocytometer or automated cell counter
Centrifuge at 300g for 3-5 minutes at room temperature to pellet cells
Aspirate the supernatant carefully, avoiding disturbance of the cell pellet
Warm the prepared freezing media to 37°C before use

Cryopreservation

Resuspend cell pellet in freezing media to a density of 2.0 million cells/mL for adherent, and 5 million cells/mL for suspension cells, or your desired concentration
Aliquot 1.0 mL (or more as required) of the cell suspension into labeled cryovials
Use a slow-freezing method before transferring the cells to long-term storage

? Method	? Steps
❄️ Manual Freezing	1️⃣ Place cells in freeze medium in a 4°C freezer for 40 minutes. 2️⃣ Transfer to a -80°C freezer for 24 hours. 3️⃣ Store cells in liquid nitrogen for long-term preservation.
? Using Mr. Frosty	1️⃣ Prepare cells in cryovials with freeze medium. 2️⃣ Place cryovials in the Mr. Frosty container. 3️⃣ Store at -80°C for 24 hours before transferring to liquid nitrogen.
? Controlled-Rate Freezer	1️⃣ Program the device to gradually decrease the temperature. 2️⃣ Place prepared cells in the freezer. 3️⃣ After the freezing cycle, transfer cells to liquid nitrogen.

Store the cryovials at temperatures below -130°C or in liquid nitrogen for long-term preservation.

Materials and Reagents

1–2 mL cryovials
CoolCell® freezing unit or controlled-rate freezer
Cryoprotective freezing medium (e.g., DIY cryoprotectant media, CM-1, CM-ACF, or specialized media)
Fetal Bovine Serum (FBS) or conditioned media
DMSO or glycerol
Calcium- and magnesium-free PBS
Accutase, Trypsin, or Trypsin-EDTA (for adherent cells)
15 mL or 50 mL Falcon tubes
Hemocytometer or automated cell counter
Centrifuge