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Definition : Solidification of a solution into a glassy vitrified state from the liquid phase by an extreme elevation in viscosity while cooling at a low temperature. The water does not solidify thereby preventing ice-crystal formation.
Cooling rates : High rates in the range of -15,000ºC to -30,000ºC/min are needed along with an increase in the concentration of cryoprotectants.
Warming rates: +20,000 to +24,000 ° C/min
Advantages : Simple, fast and cheap method with no requirement of special equipment. High survival rates have been reported when used for freezing oocytes and blastocysts.
Disadvantages: Still in an early phase of development. The need for very fast cooling and storage ideally at a temperature (in the neighborhood of - 130ºC) is not easily maintained with great reliability with equipment currently available. Secondly there is a need for very fast and uniform re-warming in order to avoid "de-vitrification" (formation of ice crystals). The use of high concentrations of permeating cryoprotectants may also damage the cellular cytoskeleton.
Goals of cryopreservation : Minimize/prevent cryo-injuries from,
Overcoming hurdles : The use of ethylene glycol which has higher permeability and lower toxicity seems to overcome the cytotoxic effect. The addition of sucrose as a non penetrating cryoprotectant regulates the effects of ethylene glycol during the equilibration and vitrification procedures and also helps in removing the EG from the cytoplasm following thawing.
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The technique has to be mastered in terms of quick exposures to the media as well as the loading, sealing and plunging time. In order to minimize the damage caused by the extreme temperatures and concentrations of cryoprotectants there are a variety of loading devices available in the market which are supported by studies, for instance the cryo grid, conventional straws, open pulled straws, cryoloop and cryotips. The ideal capacity would be only 1µl so that there is minimal exposure. Not more than two oocytes or embryos need to be frozen in each tip. We are currently using the kit from Irvine Scientific with cryotips for loading.
References
1. Text book of Assisted Reproductive Techniques Laboratory and Clinical perspectives” Chapter 19 “Vitrification of human oocytes” Second Edition. Edited by David Gardner, Ariel Weissman, Colin M Howles and Zeev Shoham.
2. Juergen Liebermann, Michael, Liz Sanders and Maike Liebermann. Vitrification: Ready for Reproductive Medicine? Fertility World. Volume 2. www.ivfonline.com
3. Yasser Orief, Askan Schultze-Mosgau, Konstantinos Dafopoulos, Safaa Al-Hasani.Vitrification: will it replace the conventional gamete cryopreservation techniques? Middle East Fertility Society Journal. Vol. 10, Num. 3, 2005, pp. 171-185.
4. Yasser Orief, Askan Schultze-Mosgau, and Safaa Al-Hasani. Vitrification versus conventional cryopreservation technique.Middle East Fertility Society Journal. Vol. 10, Num. 3, 2005, pp. 207- 209.
5. Vitrification facts and prospects - Robert Ettinger - Dec.29, 2000. |
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