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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.


  • 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.
Note: The exact protocols may vary based on the type of cells, their sensitivity to cryoprotectants like DMSO or glycerol, and the specific equipment and materials available. Always consult the manufacturers' instructions for the reagents and equipment being used for detailed guidance.

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