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How to handle cell cultures on receipt

Frozen cell lines

Upon receiving a shipment of frozen cells it is important that the end user gives the shipment attention without delay. The technical advice accompanying the cell lines should be consulted before removing ampoules from the dry ice. Correct handling immediately upon receipt is critical to successfully establishing the cell line in the end user’s laboratory.

At the time a cell line is ordered from ECACC, end users should also consider the culture conditions for the new cell line and make sure the appropriate medium will be available when the cells arrive.

Upon receipt, frozen ampoules should be transferred directly to gaseous phase liquid nitrogen without delay, unless they are to be used straight away. DO NOT use a -80°C freezer as an alternative; this will result in loss of viability.

For the majority of cell lines there are approximately 2- 4 x 106 cells per frozen vial (1 ml per vial). Immediately post resuscitation of cell cultures it is important to perform a viable cell count to use when seeding the cells.  Do not simply rely on the viable cell count figure provided.  This will enable you to seed at the correct viable cell density. 

 

Growing cell lines 

Growing cultures can be provided for most cell lines and are particularly valuable for people with less experience of growing cells or for cell lines that are known to be difficult to resuscitate. They are provided in plastic flasks in transport medium that does not contain any antibiotics. 

Growing cell cultures should be checked on receipt using an inverted microscope. If you have any problems or queries email  or phone us +44 (0)1980 612684.

Check the cell density. Immediately check the cells upon arrival for any obvious defects by using an inverted microscope. Lay the flask flat in an incubator under the conditions described in the cell line data and incubate overnight. Alternatively, if the cell density is too high (more than 80% confluent) subculture the cells (harvest and reseed) immediately.   Follow the guidance for adherent or suspension cells below:

Adherent cell lines
  1. After overnight incubation, remove most of the transport medium – leaving sufficient to just cover the cells. Incubate under the correct conditions as described in the cell line data until the required degree of confluence is achieved
  2. Once 80% confluence is achieved, unless otherwise specified, carefully remove the culture medium, wash the monolayer twice with phosphate buffered saline (PBS) then add 1-2ml of 0.25%Trypsin/EDTA solution ensuring the cells are covered – decant excess Trypsin/EDTA immediately. Warning: some cell lines are damaged by trypsin
  3. Incubate at the temperature specified until the cells start to detach from the flask – normally 2-10 minutes
  4. Pre-warm fresh medium to the correct incubation temperature for the cells then add 5ml to re-suspend the cells and inactivate the trypsin
  5. Count the viable cells. This can be done microscopically using a haemocytometer and trypan blue stain (the non-viable cells will be stained blue) although other methods are available. Calculate the amount of medium required and flask size necessary to achieve the required cell density. For examples of calculations and methods visit our cell counting page.
Suspension cell lines
  1. Following overnight incubation, determine the viable cell density.  This can be done microscopically using a haemocytometer and trypan blue stain (the non-viable cells will be stained blue) although other methods are available - see link above for our cell counting page.
  2. Please refer to the cell line data sheet for the correct subculture protocol

 

 

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