BioFiles Volume 5, Number 4 — Cryopreservation

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Table of Contents



Protocol for the Cryopreservation of Cell Lines



Aim

This protocol is recomended by the European Collection of Cell Cultures (ECACC) for the cryopreservation of its cell lines. This protocol employs the use of passive methods involving an electric –80 °C freezer for the cryopreservation of cell cultures. ECACC routinely uses a programmable rate controlled freezer (Planer Series Two) from Planer Products. This is the most reliable and reproducible way to freeze cells but as the cost of such equipment is beyond the majority of research laboratories the methods below are described in detail. If large numbers of cell cultures are regularly being frozen then a programmable rate controlled freezer is recommended.



Materials

  • Freeze medium (commonly 70% basal medium, 20% FBS, 10% DMSO [Product No. D2650] or glycerol, check ECACC data sheets for details)
  • 70% ethanol in water (Product No. R8382)
  • PBS without Ca2+ Mg2+ (Product No. D8537)
  • 0.25% trypsin/EDTA in HBSS, without Ca2+/Mg2+ (Product No. T4049)
  • DMSO (Product No. D2650)
  • Trypsin/EDTA (Product No. T4049)
  • HL60 (Product No. 98070106-1v1 – ECACC)


Equipment

  • Personal protective equipment (sterile gloves, laboratory coat)
  • Full-face protective mask/visor
  • Waterbath set to 37 °C
  • Microbiological safety cabinet at appropriate containment level
  • Centrifuge
  • Haemocytometer (Sigma® Bright-Line™ Product No. Z359629, Improved Neubauer-Camlab CCH.AC1)
  • Pre-labeled ampules/cryotubes
  • Cell Freezing Device (e.g. Nalgene® Mr. Frosty Product No. C1562)


Procedure

View cultures using an inverted microscope to assess the degree of cell density and confirm the absence of bacterial and fungal contaminants.

Bring adherent and semi adherent cells into suspension using trypsin/EDTA (Product No. T4049) as above and re-suspend in a volume of fresh medium at least equivalent to the volume of trypsin. Suspension cell lines can be used directly. Remove a small aliquot of cells (100–200 µL) and perform a cell count. Ideally the cell viability should be in excess of 90% in order to achieve a good recovery after freezing. Centrifuge the remaining culture at 150 g for 5 minutes.

Re-suspend cells at a concentration of 2-4x106 cells per ml in freeze medium. Pipette 1 ml aliquots of cells into cyroprotective ampules that have been labeled with the cell line name, passage number, cell concentration and date. Place ampules inside a passive freezer e.g. Nalgene Mr. Frosty (Product No. C1562). Fill freezer with isopropyl alcohol and place at –80 °C overnight. Frozen ampules should be transferred to the vapor phase of a liquid nitrogen storage vessel and the locations recorded.



Key Points

The most commonly used cryoprotectant is dimethyl sulphoxide (DMSO Product No. D2650), however, this is not appropriate for all cell lines e.g. HL60 (Product No. 98070106-1vl – ECACC) where DMSO is used to induce differentiation. In such cases an alternative such as glycerol should be used. ECACC freeze medium recommended above has been shown to be a good universal medium for most cell types. Another commonly used freeze medium formulation is 70% basal medium, 20% FBS, 10% DMSO but this may not be suitable for all cell types. Check if it works for your cells before using on a regular basis (Product No. C6164). It is essential that cultures are healthy and in the log phase of growth. This can be achieved by using pre-confluent cultures (cultures that are below their maximum cell density) and by changing the culture medium 24 hours before freezing.

The rate of cooling may vary but as a general guide a rate of between –1 °C and –3 °C per minute will prove suitable for the majority of cell cultures.

An alternative to the Mr. Frosty system is the Taylor Wharton passive freezer where ampules are held in liquid nitrogen vapor in the neck of Dewar. The system allows the ampules to be gradually lowered thereby reducing the temperature. Rate controlled freezers are also available and are particularly useful if large numbers of ampules are frozen on a regular basis. As a last resort if no other devices are available ampules may be placed inside a well insulated box (such as a polystyrene box with sides that are at least 1 cm thick) and placed at –80 °C overnight. It is important to ensure that the box remains upright throughout the freezing process. Once frozen, ampules should be transferred to the vapor phase of a liquid nitrogen storage vessel and the locations recorded.

If using a freezing method involving a –80 °C freezer it is important to have an allocated section for cell line freezing so that samples are not inadvertently removed. If this happens at a crucial part of the freezing , cell viability will be compromised.



Problem Solving Suggestions

John Ryan Ph.D
Corning Incorporated, Life Sciences
Acton, MA 01720

Viability problems associated with cryogenic storage are usually noticed soon after cultures are thawed and plated. There are four major areas where problems occur:

  1. During harvesting and processing of the cells. Problems may be caused by excessive exposure of the cells to dissociating agents; using a cryoprotective agent that is toxic; or allowing high density cell suspensions to remain too long at room temperature or at a pH that is too basic.
  2. During the cooling (freezing) process. Excessive cell damage and reduced culture viability often result from using a cooling rate that is too fast or too slow, or when the cooling process is temporarily interrupted. Not using a suitable cryoprotective agent at an appropriate concentration will also result in viability problems.
  3. During cryogenic storage. Culture viability is often reduced when vials are allowed to warm up during transfer to the freezer, or if the repository temperature is not consistently maintained at appropriate cryogenic temperatures.
  4. During thawing and recovery. Problems arise when the thawing process is too slow or the cryoprotectants are improperly removed.

These viability problems can often be corrected by using the following technique to identify the stage in the freezing process where the problem originates. Harvest enough cells to prepare at least four vials. Then remove a sample of cell suspension, equivalent in cell number to that which will be placed into the vials, and immediately place it into a culture vessel with an appropriate amount of medium and incubate. This culture will be used as a control to compare with the cultures set up in the remaining steps.

Next, add the cryoprotective agent to the remaining cells and divide among three vials. Place one vial at 4 °C for one hour. Then remove the cells from the vial, process as though they had just been thawed from the freezer, and plate in medium as above. This culture will be compared with the control culture to determine if there are any problems associated with the cryoprotective agent.

Meanwhile, process the remaining vials through the slow cooling process as usual. One vial is then immediately thawed and processed as above. This culture will be compared with the control culture to determine if there are any problems associated with the slow cooling process.

The remaining vial is then transferred to the cryogenic freezer and stored overnight before being thawed and processed as above. This culture will be compared with the control culture to determine if there are any problems associated with the cryogenic storage conditions. If additional vials of cells are available, several different recovery techniques should be used to determine if the recovery technique is the source of the problem.

By comparing all of the cultures to the original culture, it should then be possible to determine at which stage of the freezing process the problem occurred. Once this is known, the information presented in this guide and its references should be enough to eliminate the problem.

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