A241 Cell Culture

Problem 6 Until cells freeze over

Republic Polytechnic Singapore

Activity Owner : Jasmine Heng (Dr) Inputs : Ritu Bhalla (Dr) Approved By : Ventris Dsouza (Dr) Module Chair : Ritu Bhalla (Dr)
Copyright 2011 by Republic Polytechnic, Singapore

The Problem


You are shown 2 animals which survive the extreme cold You are also presented with the mechanism of how one of these animals cope with its freezing environment Your task is to freeze-store the cell line which is not required for use in the lab till next year

Copyright 2011 by Republic Polytechnic, Singapore

What do you recognise?



Glucose, converted from glycogen in the liver of wood frogs, lowers the temperature at which ice crystals form, protecting its cells from the ice when freezing does occur There is damage to cells from crystals of ice formed when cells are frozen Cell lines that are not required for long periods of time can be stored frozen There is a need to protect the cells from harmful effects of freezing
Copyright 2011 by Republic Polytechnic, Singapore

The Approach
      

What is the strategy used by Wood Frog? Why freeze cells in the lab? What can happen to frozen cells? How to prevent damage from freezing? Stages of cryopreservation What are the materials required for the procedure? How to document storage records?
Copyright 2011 by Republic Polytechnic, Singapore

What happens inside the wood frogs liver?

Glycogen

Glucose

Prevents ice crystals formation through freezing point depression

Copyright 2011 by Republic Polytechnic, Singapore

Why freeze-store cells in the lab?

Prone to genetic variation

Contamination/ Cross-contamination

Stable supply of cells

Maintain backups/ reserves

Save time/ materials
Copyright 2011 by Republic Polytechnic, Singapore

Storage and transportation

What happens when cells are being frozen?

Ice crystals formation

extracellular solute concentration

Deformed/ ripped cell

Copyright 2011 by Republic Polytechnic, Singapore

Factors to consider to prevent freezing damage

Prevent Ice formation !!!
Slow

freezing Cryoprotective agent Storage temperature

Copyright 2011 by Republic Polytechnic, Singapore

Stages in Cryopreservation
* Control size & rate of ice crystal formation
* Water

ice

From -2 to -5 degrees; there is a release of energy as heat

Cooling rate is proportional to the difference in temperature between cryo vials and ambient air

Copyright 2011 by Republic Polytechnic, Singapore

Cryoprotective agent


Helps to protect the biological tissue against damage caused due to freezing. Eg DMSO Protects cells against harm caused by freezing leading to water crystals and dehydration
 

Reduce likelihood of ice nuclei formation Allowing more time for cell shrinkage

 

DMSO- precautions !! Kill cells with overexposure at non-freezing temperatures Toxic; penetrates natural & synthetic membranes (skin & gloves) Handle with extreme care
Copyright 2011 by Republic Polytechnic, Singapore

What materials are required for the procedure?

Freezing Media (Media with 10% FBS and 10% DMSO)

Freezing container, Mr Frosty

Cryovials

Liquid nitrogen storage tanks

Copyright 2011 by Republic Polytechnic, Singapore

Maintaining Records


Proper labelling on cryo vials (Name of Cell Line & User, Date stored, Passage Number) Proper records of contents in liquid nitrogen storage  Details of exact location of vial - tank, box, rack, cannister, cane, position  Name of Cell Line & User, Date stored, Passage Number

WHY?
Copyright 2011 by Republic Polytechnic, Singapore

You have learnt

The

rationale for freezing cells for freezing cells for storage in Liquid Nitrogen

Procedure Use

of DMSO as a freezing agent required for freezing cells for storage of maintaining cryopreservation records

Materials

Importance

End of Activity 6
Copyright 2011 by Republic Polytechnic, Singapore