CryopreservationofCellLines

Aim
The protocol below describes the use of passive methods involving an electric -80ºC freezer for the cryopreservation of cell cultures. ECACC routinely use 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% FCS, 10% DMSO (Prod. No. D2650) or glycerol, check ECACC data sheets for details).

• 70% ethanol in water (Prod. No. R8382)

• PBS without Ca₂₊ Mg₂₊(Prod. No. D8537)

• 0.25% trypsin/EDTA in HBSS, without Ca₂₊/Mg₂₊(Prod. No. T4049)

• DMSO (Prod. No. D2650)

• Trypsin/EDTA (Prod. No. T4049)

• HL60 (Prod. No. 98070106-1v1)

Equipment

• Personal protective equipment (sterile gloves, Laboratory coat)

• Full-face protective mask/visor

• Waterbath set to 37ºC

• Microbiological safety cabinet at appropriate containment level

• Centrifuge

• Haemocytometer (Sigma Bright- line Prod. No. Z359629, Improved Neubauer – Camlab CCH.AC1)

• Pre labeled ampules/cryotubes

• Cell Freezing Device (e.g. Nalgene Mr. Frosty Prod. No. C1562)

Procedure

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

2. Bring adherent and semi adherent cells into suspension using trypsin/EDTA (Prod. No. T4049) as above (Protocol 3 and 4 – Subculture of adherent/attached and semi-adherent cell lines) and re-suspend in a volume of fresh medium at least equivalent to the volume of trypsin. Suspension cell lines can be used directly.

3. Remove a small aliquot of cells (100-200uL) and perform a cell count (Protocol 6 – Cell Quantification). Ideally the cell viability should be in excess of 90% in order to achieve a good recovery after freezing.

4. Centrifuge the remaining culture at 150g for 5 minutes.

5. Re-suspend cells at a concentration of 2-4x106 cells per ml in freeze medium.

6. Pipette 1ml aliquots of cells into cyroprotective ampules that have been labeled with the cell line name, passage number, cell concentration and date.

7. Place ampules inside a passive freezer e.g. Nalgene Mr. Frosty (Prod. No. C1562). Fill freezer with isopropyl alcohol and place at –80ºC overnight.

8. Frozen ampules should be transferred to the vapor phase of a liquid nitrogen storage vessel and the locations recorded.

Key Points

1. The most commonly used cryoprotectant is dimethyl sulphoxide (DMSO Prod. No. D2650), however, this is not appropriate for all cell lines e.g. HL60 (Prod. No. 98070106-1v1) where DMSO is used to induce differentiation. In such cases an alternative such as glycerol should be used (refer to ECACC data sheet for details of the correct cryoprotectant).

2. 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% FCS, 10% DMSO but this may not be suitable for all cell types. Check it works for your cells before using on a regular basis (Prod. No. C6164).

3. 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.

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

5. 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.

6. 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 1cm thick) and placed at –80ºC 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.

7. If using a freezing method involving a -80ºC 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 process then viability and recovery rates will be adversely affected.