Cell Culture Protocols

Procedure for handling frozen ampoules upon receipt

Upon receipt, frozen ampoules should be transferred directly to gaseous phase liquid nitrogen without delay if not to be used straightaway. DO NOT use a -80^oC freezer as an alternative. Please contact CellBank Australia within 24 hours if there appears to be a problem.

Resuscitation of frozen cells

Safety Notice: It is important to handle the ampoules with care and wear a laboratory coat, full protective face mask and gloves. On rare occasions, an ampoule may explode on warming due to the expansion of trapped residual liquid nitrogen.

Note: Do not totally immerse the ampoule as this may increase the risk of contamination.

1. In a microbiological safety cabinet, hold a tissue soaked in 70% alcohol around the cap of the frozen ampoule and turn the cap a quarter turn to release any residual liquid nitrogen that may be trapped. Re-tighten the cap. Quickly transfer the ampoule to a 37^oC waterbath until only one or two small ice crystals, if any, remain (1-2 min). It is important to thaw rapidly to minimize any damage to the cell membranes.

2. Wipe ampoule with a tissue soaked in 70% alcohol prior to opening.

3. Slowly pipette the whole content of the ampoule into 5ml pre-warmed medium that has already been supplemented with the appropriate constituents. Determine the viable cell density using trypan blue stain, a haemocytometer and an inverted microscope to count the cells. For adherent cell lines: Adjust the volume of the medium, and if necessary the flask size, to achieve the cell seeding density recommended on the cell line data sheet. A pre-centrifugation step to remove cryoprotectant is not normally necessary as the first media change will remove residual cryoprotectant. If it is, then this will be specified on the data sheet. If the cells are to be used immediately (e.g. for a cell based assay), rather than subcultured, it may be advisable to perform a pre-centrifugation step to remove cryoprotectant. For suspension cell lines: A pre-centrifugation step to remove cryoprotectant is recommended i.e. pellet the cells by centrifugation at 150 x g for 5 minutes and resuspend the cell pellet in fresh medium using the appropriate volume to achieve the correct seeding density.

4. Incubate at the temperature and CO₂ level recommended on the data sheet. If a CO₂ fed incubator is used, the flask should have a vented cap to allow gaseous exchange.

Procedure for handling growing cultures upon receipt

Immediately check the cells upon arrival for any obvious defects by using an inverted microscope and then lay the flask flat in an incubator, set at the temperature specified in the cell line data sheet, overnight. If the cell density is too high, follow the procedure below and as described in the cell line data sheet. Please contact CellBank Australia within 24 hours if there appears to be a problem.

Note: The transport medium does not include antibiotics.

Adherent Cell Lines

1. Following overnight incubation, remove most of the transport medium leaving sufficient medium to cover the cells (5-8ml for a 25cm² flask). Incubate at the temperature and CO₂ level recommended on the cell line data sheet until the required degree of cell confluence is achieved.

2. At 80% confluence, unless specified otherwise on the cell line data sheet, carefully remove the culture medium and wash the monolayer of cells twice with PBS. Add 1-2ml of ^#0.25% Trypsin/ 0.2% EDTA solution to the 25cm² flask ensuring that all cells are covered. Decant the excess Trypsin/EDTA immediately.

3. Incubate (unless specified otherwise on data sheet) until the cells start detaching from the flask – usually takes 2-10 minutes.

4. Add 5ml fresh pre-warmed (to incubation temperature specified on data sheet) medium to resuspend the cells and quench the trypsin. Determine the viable cell density using trypan blue stain, a haemocytometer and an inverted microscope to count the cells. Seed into a fresh flask(s), adjusting the volume of the medium, and if necessary the flask size, to achieve the cell seeding density recommended on the cell line data sheet.

#Note: Trypsin can cause damage to some cell lines. Before commencing any work, please check the cell line datasheet to determine if trypsin is appropriate and for specific procedures involved when using serum free media.

Suspension Cell Lines

1. Following overnight incubation, determine the viable cell density using trypan blue stain, a haemocytometer and an inverted microscope to count the cells. Seed at the cell seeding density recommended in the data sheet by adjusting the volume using fresh pre-warmed medium in a larger flask if necessary.

Procedure for viability testing

Counting of cells (haemocytometer)

1. Prepare an alcohol cleaned cover-slip, haemocytometer and cell supension.

2. Moisten and affix cover-slip to the haemocytometer and ensure adherence by looking for "Newton's Rings" - rainbow-like rings under the cover-slip.

3. Mix equal volumes of 0.4% trypan blue stain and a homogenous cell suspension e.g. mix 100µl trypan blue stain with 100 µl cell suspension.

4. Pipette trypan blue/cell mix (approximately 10µl) at the edge of the cover-slip and allow to run under the cover slip.

5. Focus down a phase contrast microscope on the haemocytometer grid and count viable (live) and non-viable (dead) cells in one or more large corner squares  - recording cell counts. 
    - Trypan Blue is taken up in non-viable cells and excluded from viable live cells.
    - Live cells appear colourless and bright (refractile) under phase contrast while dead cells stain blue and non-refractile. 
    - Cell suspension may require concentration or dilution to ensure accurate counting.

6. Count around 40 to 70 cells to obtain an accurate cell count.

7. To calculate cell concentration per ml:

Take the average number of cells in one large square x reciprocial dilution factor x 10⁴ 
  - reciprocal dilution factor is usually 2 (1:1 dilution with trypan blue). 
  - 10⁴ = conversion factor to convert 10^-4ml to 1ml
(Vol. of 1 large square = 1mm² x 0.1mm = 0.1cm x 0.1cm x 0.01cm = 10^-4cm³ or 10^-4ml). 

Calculation of Cell Viability:

1. No. of viable cells counted divided by total number of cell counted (viable + dead) x 100 = % viable cells

2. Percentage of non viable cells = 100% - % viable cells

Procedure for freezing cells

CellBank Australia recommends freezing a stock of the cell line(s) soon after receipt (2-4 x 10⁶ cells/ml) as a precaution. The following guide is offered for the preparation and freezing of cell lines.

1. Harvest the cells in the log phase of growth in the same manner used for routine subculture. For adherent cell lines harvest as close to 80-90% confluency as possible.

2. The standard procedure is to use 90% serum + 10% cryoprotectant for all cell lines unless otherwise specified on the data sheet. Allow 1ml for each ampoule. Most cell lines can be frozen in the appropriate culture medium supplemented with 20% serum and 10% cryoprotectant. The cryoprotectant is usually DMSO but in certain instances glycerol is recommended. If DMSO is not suitable an alternative will be specified on the cell line data sheet. 

3. Pellet cells by centrifugation e.g. 150 x g for 5 minutes. Resuspend the cell pellets in the appropriate freeze medium to give a final cell concentration of 2-4 x 10⁶ cells/ml and pipette 1ml into each ampoule.

4. Freeze the cells at a cooling rate between 1-3^oC/min using a programmable rate controlled freezer or suitable alternative^#. When the temperature reaches at least -130^oC, transfer the ampoule to a gas phase liquid nitrogen storage vessel. It is advisable to test cell viability by thawing one ampoule after short term storage in gas phase nitrogen.

^#A polystyrene box or alcohol bath (e.g. a Nalgene 5100 ‘Mr Frosty’ box Sigma cat no. C1562) can be used in a -80^oC freezer for up to 24 hours prior to transfer to gaseous phase liquid nitrogen.

Reference.

Cryopreservation of Animal Cells in Advances in Biotechnology Processes (1988), 7, A.R. Liss