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DMSO Vitrification Systems

There are a variety of vitrification systems to choose from.  Despite many manufacturers of media and devices, there are basically only two systems available: 1) a DMSO-based system and 2) a non-DMSO system.  Clinics have reported a variety of success rates with these products.  The bottom line is that they all can work, but you need to find what will work in your lab.  Results can vary depending on the experience of the embryologist, embryo quality, culture conditions, and patient demomographics, etc.  Although our system is non-DMSO based we offer assistance to clinics using other systems.  

Theory

DMSO systems work mainly by kinetic vitrification.  By kinetic vitrification we simply mean that there is some water around that can cause ice to form during cooling and therefore one must cool rapidly to prevent the water molecules from organizing into damaging ice crystals.  This is different than equilibrium vitrification, where the cells are more dehydrated and cooling rates do not matter; as in slow-cooling and our own I.C.E. vitrification system. 

In the DMSO systems, DMOS and Ethylene Glycol are the permeable cryoprotectants.  Initial dehydration and cryoprotectant uptake is done with a 7.5%/7.5% cryoprotectant solution.  Cells are allowed to soak for 10-15min in this dilute solution, but are not ready to be vitrified, as too much water still remains inside.  Prior to cooling in LN2, the cells are moved to the vitrification solution with a concentration of 15%/15% cryoprotectant.  This step is critical to remove most of the remaining water and fully load the cells with cryoprotectant.  There is a certain point that must be achieved for vitrification to be successful.. The cells must be dehydrated enough to prevent ice formation upon rapid cooling, but not exposed to DMSO for too long to prevent toxicity.  This is the challange of this system; long enough exposure, but not too long. 

Because there is still some water in the cells when the cooling step begins, it is necessary to cool rapidly to prevent ice formation (kinetic vitrification).  Therefore, to speed up the cooling process, small volumes of 1ul or less, and open containers that allow direct contact with LN2 are used.  If things are done properly, the system can be very successful.


Choosing a System

Media

There are many companies that sell DMSO-based vitrification solutions.  Basically, they contain the same amount of cryoprotectant (DMSO / EG).  The major differences are in the 1) base media; 2) buffer system; and 3) sugar that are used.  The base media is generally a simple salt solution similar to HTF, but can also contain amino acids and be more similar to a culture medium.  The buffer system varies and biocarbonate,  phosphate,  HEPES, or MOPS are typically used.  HEPES and MOPS tend to provide the best buffer system for media at ambient conditions.  The sugar used is either sucrose or trehalose.  Both are very effective.   

Storage Device

There are at least a dozen different storage devices on the market today.  Basically they are all similar, as they allow for a small volume of media, usually 1ul or less, to be placed on or in the device.  Most devices are open to allow a rapid cooling rate, necessary for vitrification using a DMSO-based media.  When choosing a device to use be aware of several issues:

  • Type of labeling required

  • Ease of loading

  • Ease of recovery

  • Repeatability

  • Storage requirements (space)

  • Technical problems

Among the popular devices the cryo-top, cryo-loc are open devices and the new cryo-tec, rapid-i, cryo-pette, and micro-secure are closed devices.  We have found the micro-secure to be one of the best devices, however all that are listed here can be successful if used properly.  For more information the micro-secure system click here. 

When choosing a system, please read the literature carefully and consult with others already using the system.  


Troubleshooting

There are a number of common technical problems with using the DMSO system.  Because of the variability of issues that may arise, it is difficult to troubleshoot issues in this general format.  Some advice can be found on the Help Desk.  If you have any questions or issues with the system you are using please contact us and we will be glad to help you. 

Email: jstachecki@gmail.com   or  Phone: 973-632-8635



Vitrified human oocytes - ICE Vitrification System