Cryopreservation Protocol

Materials and Reagents

• 1–2 mL cryovials
• CoolCell® freezing unit or programmable cooler
• 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

Labeling and Documentation

• Label cryovials with the date, researcher's name, cell number, passage number, cell type, and any additional information like genetic modifications.

Freezing Medium Preparation

• Prepare cryoprotective freezing medium according to the cell type:
• For FBS-containing media cultures: 90% FBS + 10% DMSO.
• For serum-free media cultures: 90% conditioned media + 10% DMSO.
• For cells requiring glycerol: 90% FBS + 10% glycerol.
• For a convenient and tailored freezing solution, consider acquiring our pre-formulated freeze medium CM-1 or CM-ACF. Opt for our CM-1 freeze medium if your protocol requires a medium containing Fetal Bovine Serum (FBS), or choose our CM-ACF medium for an FBS-free alternative.

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 rpm 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.
• Aliquot 1.0 mL (or more as required) of the cell suspension into labeled cryovials.
• Place vials in a 4°C refrigerator for 20 minutes.
• Transfer the vials to a programmable cooler or an insulated box in a -70°C to -90°C freezer to freeze cells at a controlled rate of 1°C per minute.
• After the initial freezing, move the vials to liquid nitrogen or electrical freezer storage at below -150°C for long-term preservation.

Aseptic Technique

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