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SOP-003: Cryopreservation

1. Purpose

To preserve viable mammalian cells for long-term storage while minimizing cellular damage during freezing and maximizing post-thaw recovery.

2. Scope

This SOP applies to the cryopreservation of adherent and suspension mammalian cell lines intended for long-term storage in liquid nitrogen.

3. Principle

During freezing, intracellular water can form ice crystals that damage cellular structures. Cryoprotective agents such as dimethyl sulfoxide (DMSO) reduce ice crystal formation, while controlled-rate freezing minimizes osmotic stress and improves long-term cell survival.

4. Materials and Equipment

Materials​

  • Healthy mammalian cell culture
  • Complete growth medium
  • DMSO (Dimethyl Sulfoxide)
  • Cryovials
  • Sterile centrifuge tubes

Equipment​

  • Class II Biosafety Cabinet
  • Centrifuge
  • Controlled-rate freezing container
  • −80°C freezer
  • Liquid nitrogen storage system
  • Pipettes and sterile pipette tips

5. Safety Considerations

  • Wear appropriate personal protective equipment (lab coat, gloves and eye protection).
  • Handle DMSO carefully and avoid skin contact.
  • Handle liquid nitrogen using cryogenic gloves and face protection.
  • Treat all cell cultures as potentially biohazardous.
  • Follow institutional biosafety and cryogenic safety guidelines.

6. Procedure

6.1 Evaluate Cell Health​

  1. Examine the culture under an inverted microscope.

  2. Freeze only healthy, actively growing cultures.

💡 Why? Healthy cells tolerate freezing stress much better and exhibit higher post-thaw viability.

⚠ Important: Do not freeze contaminated, unhealthy or overconfluent cultures.

🔍 Check: Cells should display normal morphology, good viability and no signs of contamination.

6.2 Determine Cell Number​

  1. Count cells and determine viability.

💡 Why? Consistent cell numbers improve reproducibility and ensure similar recovery across cryovials.

⚠ Important: Record both total cell number and viability before freezing.

🔍 Check: Cell concentration and viability have been documented.

6.3 Harvest Cells​

  1. Detach adherent cells using Trypsin-EDTA or collect suspension cells directly.

  2. Neutralize trypsin with complete growth medium.

  3. Transfer the cell suspension into a sterile centrifuge tube.

💡 Why? Cells must be in a healthy single-cell suspension before cryopreservation.

⚠ Important: Avoid prolonged trypsin exposure.

🔍 Check: The suspension should contain minimal cell clumps.

6.4 Centrifuge Cells​

  1. Centrifuge the cells using conditions appropriate for the cell type.

  2. Carefully remove the supernatant.

💡 Why? Centrifugation concentrates the cells and removes residual enzymes and waste products.

⚠ Important: Excessive centrifugation force may damage cells.

🔍 Check: An intact cell pellet should be visible.

6.5 Prepare Freezing Medium​

  1. Prepare fresh freezing medium immediately before use.

A commonly used formulation is:

ComponentFinal Concentration
Complete growth medium90%
DMSO10%

💡 Why? DMSO acts as a cryoprotectant by reducing intracellular ice crystal formation during freezing.

⚠ Important: Prepare freezing medium fresh to maintain sterility and cryoprotectant effectiveness.

6.6 Resuspend Cells in Freezing Medium​

  1. Gently resuspend the cell pellet in cold freezing medium.

💡 Why? Uniform exposure to the cryoprotectant provides consistent protection during freezing.

⚠ Important: Mix gently to avoid mechanical damage.

🔍 Check: The suspension should appear homogeneous.

6.7 Aliquot into Cryovials​

  1. Dispense the desired volume into sterile cryovials.

  2. Label each cryovial with:

  • Cell line
  • Passage number
  • Date
  • Operator initials

💡 Why? Proper labeling ensures traceability and prevents sample mix-ups.

⚠ Important: Verify labels before freezing.

🔍 Check: All cryovials are clearly and permanently labeled.

6.8 Controlled-Rate Freezing​

  1. Place cryovials into a controlled-rate freezing container.

  2. Store at −80°C overnight.

💡 Why? Cooling at approximately 1°C per minute allows water to leave cells gradually, reducing intracellular ice formation.

⚠ Important: Do not place freshly prepared cryovials directly into liquid nitrogen.

🔍 Check: Cryovials remain upright within the freezing container.

6.9 Transfer to Long-Term Storage​

  1. Transfer cryovials from the −80°C freezer into liquid nitrogen storage.

💡 Why? Cryogenic temperatures effectively halt cellular metabolism, allowing long-term preservation.

⚠ Important: Minimize the time cryovials remain at room temperature during transfer.

🔍 Check: Cryovials are stored in the correct liquid nitrogen rack and location has been recorded.

7. Quality Control

Record and verify:

  • Cell line
  • Passage number
  • Cell viability before freezing
  • Number of cells per vial
  • Freezer location
  • Liquid nitrogen storage location

8. Troubleshooting

ProblemPossible CauseCorrective Action
Poor post-thaw viabilityUnhealthy starting cultureFreeze only healthy cultures
Low recoveryRapid freezingUse controlled-rate cooling
Excessive cell deathIncorrect DMSO concentrationPrepare fresh freezing medium
Sample mix-upPoor labelingUse standardized labeling procedures

9. Documentation

Record the following information:

  • Cell line name
  • Passage number
  • Cell concentration
  • Cell viability
  • Freezing medium composition
  • Number of cryovials prepared
  • Storage location
  • Operator name
  • Date of cryopreservation

10. Common Beginner Mistakes

  • Freezing unhealthy or overconfluent cultures.
  • Skipping cell counting before freezing.
  • Preparing freezing medium long before use.
  • Mixing cells vigorously after adding DMSO.
  • Placing cryovials directly into liquid nitrogen.
  • Incomplete cryovial labeling.
  • Leaving cryovials at room temperature for extended periods.

11. References

  • Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications.
  • ATCC. Cryopreservation Guidelines.
  • ECACC. Cell Banking Procedures.