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Principles and Theory Of Controlled Rate Freezing

Contract No. QLRI-CT-2001-01645

Protocol Validation Information Pack

Work Pack 4

Evaluation Of Preservation And Genetic Stability Test Protocols

Validation of Cryopreservation Protocols

Part B (I)

Traditional Controlled Rate Freezing

Programmable Freezer


Validation Contact Details

Dr JG Day

Curator CCAP

Culture Collection of Algae and Protozoa

Scottish Association for Marine Science

Dunstaffnage Marine Laboratory,

Dunbeg, Argyll, PA37 1QA, UK

John.Day-SAMS@sams.ac.uk

Tel: 0044 (0) 1631 559000

Direct dial: 0044 (0) 1631 559349

Fax: 0044 (0) 1631 559001

www.ccap.ac.uk

www.cobra.ac.uk

www.ukncc.co.uk


Introduction

 

Cryopreservation protocols are either based on: (1) traditional 2-step controlled rate freezing methodologies that apply colligative chemical cryoprotectants and require the precise control of cooling rates, terminal transfer temperatures and extracellular ice nucleation or (2) vitrification methods that depend upon cryoprotective strategies that increase cellular viscosity to a critical point at which water forms an amorphous, non-crystalline glass on exposure to liquid nitrogen (at -196oC). Traditional cryopreservation requires the formation of extracellular ice.

 

Why are we using the traditional controlled rated freezing protocol in the COBRA Project?

The majority of cryopreserved algal strains cryogenically stored to date (Day, 1998 a,b, 1999, Day et al., 1997, Morris 1978, 1980, Taylor and Fletcher, 1999) have been processed using traditional colligative chemical cryoprotectants (dimethyl sulphoxide {DMSO}, glycerol and methanol) in association with two-step protocols involving cooling to a subzero holding temperature (-40/-60°C) prior to plunging into liquid nitrogen (Morris, 1978; 1980; Morris & Canning, 1978; Morris & McGrath, 1981; Day, 1999). Cooling rates are controlled by either computer controlled Programmable Freezers or a simplified, solvent cooled temperature controlled tank called a “Mr Frosty”.

 

Principles and Theory Of Controlled Rate Freezing

 

The traditional approach to cryopreservation involves exposure of cells to subzero temperatures using gradual (slow), and controlled rates of temperature reduction. The success of the method is dependent upon minimising the effects of two potentially injurious factors:

 

(1) Ice

(2) Dehydration

The “two-factor hypothesis” of freezing injury was originally proposed by Mazur et al. (1972) who demonstrated that the rate of change of temperature at which cells were exposed to freezing was critically important because it controlled the rate at which water moves across cell membranes and hence determines cell solute concentration. Ice damage is a critical factor in algal cryoinjury (Day et al., 1998a,b, Morris & McGrath 1981; Fleck et al. 1997,1999) and changes in water movement and water status (as a solid {ice} or a liquid) during freezing influences survival after cryogenic storage. When water moves from intra-cellular to extra-cellular locations during slow freezing the colligative properties change and cell solutes become concentrated. An algal cell cryoprotected with traditional, penetrating colligative chemical cryoprotectants (e.g. dimethyl sulphoxide, glycerol, methanol) and exposed to controlled rate, slow freezing (-10 to 0.5oC) will experience the following cryogenic effects:



 

1. Ice initially forms extracellularly, by the process of “seeding” or ice nucleation. This occurs outside the cell as there are few ice nucleating agents inside cells. These are surfaces and structures (dust particles, ice nucleating proteins, bacteria) that act as templates for crystal growth.

 

2. Ice grows outside the algal cell, a water vapour deficit is formed between the inside and outside and, intracellular water moves to the outside of the cell where it then freezes. This process may be considered cryo-dehydration and the cell shrinks as more water moves outside.

 

3. As (2) progresses cell solute concentration increases and as a result the freezing point is depressed by the process of “supercooling”or freezing point depression.

 

4. Controlled rate cooling progresses to a “terminal transfer temperature” at this point, the cells in combination with their cryoprotectants are then transferred to LN2.

 

At the point of transfer survival will be dependent upon reducing or circumventing the intracellular ice damage and mitigating the effects of solute concentration though the application of colligative cryoprotectants. This is achieved by either/and/or:

· Attaining water content so low (enhanced by the uptake of colligative cryoprotectants and cryo-dehydration) that there is not sufficient water to form large ice crystals, those that are formed are so small that their injurious effects are limited.

· Manipulating cell solute concentration via the process of cryo-dehydration such that cellular viscosity reaches a point at which exposure to ultra low temperatures causes the remaining intracellular water to become vitrified.

 

 

By using (penetrating) colligative cryoprotectants to protect the cells against damaging solution effects and computerized programmable freezers to control cryogenic parameters it is possible to develop a cryopreservation protocols that allow survival after liquid nitrogen storage.

 


Date: 2015-12-17; view: 999


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