Vitrification: Fundamental Principles and Its Application for Cryopreservation of Human Reproductive Cells

Abstract

The fundamental understanding of cryobiology through experimentation in the 1960s, 1970s, and 1980s has led to the development of today's vitrification technology. Although human embryo and oocyte vitrification was slow to evolve, it has become an invaluable technology in the field of reproductive medicine. The aim of this chapter is to discuss some of the underlying basic principles behind forming a metastable glass phase during rapid cooling in liquid nitrogen (LN 2) and the prevention of recrystallization events upon warming. We then highlight how this understanding has led to its highly effective and reliable usage in clinical IVF. Furthermore, we describe how quality control factors (e.g., ease of use, repeatability, reliability, labeling security, and cryostorage safety) can vary between vitrification device systems, potentially influencing clinical outcomes and creating possible liability issues. An open-minded approach to continued experimentation is a necessity, especially pertaining to oocyte freeze preservation, if we are to optimize the vitrification of reproductive cells and tissue in the future.

Use of natural deep eutectic systems as new cryoprotectant agents in the vitrification of mammalian cells

“…In the first, the slow-cooling method, is given enough time for the cell to dehydrate as a result of extracellular ice formation and subsequent exosmosis during the cooling process. Then, an increase of the concentration of CPA in the cytoplasm occurs which prevent the lethal intracellular ice formation 3 , 4 .…”

Use Of Natural Deep Eutectic Systems As New Cryoprotectant Agents In The Vitrification Of Mammalian Cells

“…Then, an increase of the concentration of CPA in the cytoplasm occurs which prevent the lethal intracellular ice formation. 3,4 In the second method a high concentration of CPA is used, and the solution of cells tuns into noncrystalline or amorphous solid known as glass, reason why this method is called vitri cation. 3,5,6 During the freezing process, heterogeneous or homogeneous nucleation may occur.…”

“…The prevention of heterogeneous nucleation during the cooling process is the key factor to achieve the vitreous state. 4,7 However, when dilute solutions are being cooled, the inhibition of homogenous nucleation is more di cult to accomplish. But, an increase in the concentration of a CPA in the solution can signi cantly decrease the homogeneous nucleation temperature (T hom ) and at a certain concentration of CPA it is possible to decrease T hom below the glass transition temperature (T g ).…”

“…(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS). The amount of formazan product was measured in a microplate reader (VICTOR Nivo ™ , PerkinElmer, USA) at 490 nm, as absorbance is directly proportional to the number of viable cells in culture.…”

Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies—A Review

“…The presented material largely covers the delivery of cell-based therapies in regenerative medicine, cryopreserved in larger volumes in commercially available “cryobags.” Their processing will have used the widely published and adopted “slow cooling” cryopreservation protocols ( 15 , 33 ). Vitrification-based approaches are not included as, currently, these are only effective for small volumes of cells, typically in reproductive medicine ( 34 – 36 ). The technique can be successful for mammalian gametes and embryos, but significant technical issues are involved in scaling up to larger tissue pieces and bulk cell suspensions ( 25 , 37 , 38 ).…”