Improved low-CPA vitrification of mouse oocytes using quartz microcapillary

Abstract

Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management.

Microfluidics for cryopreservation

“…Its extension form was further successfully used for the design of CPA equilibration for adherent endothelial cells (Davidson et al, 2015). Compared with programmed slow freezing, vitrification/low-CPA vitrification requires comparatively more CPA; therefore CPA addition and removal processes are more time consuming and costly (Choi et al, 2015a; Choi et al, 2015b; Huang et al, 2015; Wang et al, 2016). …”

“…More importantly, this technique provides an urgently needed method for preserving very limited volumes of biological samples, as often occurs with assisted reproductive technologies and stem cell research (Araki et al, 2015; Cohen and Garrisi, 1997; Cohen et al, 1997; Montag et al, 1998; Rao et al, 2015; Wang et al, 2016). Microfluidics can further be used to produce biological agents and cells in micro-scale volumes, enabling low-CPA droplet or encapsulation vitrification (Choi et al, 2015a; He et al, 2008; Huang et al, 2015; Risco et al, 2007; Tasoglu et al, 2013). …”

“…Trillions of cells are biopreserved globally for daily clinical use. For example, cryopreservation of human oocytes preserves future fertility of young females who may experience future infertility due to exposure to environmental/occupational hazards or aggressive medical treatments and such cryopreservation avoids moral, ethical, and religious issues associated with human embryo preservation (Choi et al, 2015a; Nagashima et al, 1995; Palasz and Mapletoft, 1996; Rall and Fahy, 1985; Redmond et al, 1988; Smorag and Gajda, 1994; Steponkus et al, 1990; Trounson and Mohr, 1983). Similarly, due to the significant decline in sperm quality in global males (Merzenich et al, 2010), increased incidence of azoospermia and oligospermia, and rapid increases in testicular cancer in young males, spermatozoa and spermatogonia stem cell preservation can be undertaken (Zou et al, 2013) and cryopreservation of sperm, ova, and fertilized eggs is currently in clinical practice worldwide.…”

“…Due to numerous intrinsic features, such as ease of mass production and specific designs and changes, ease of component integration, disposability, low cost, and requirement of smaller reagents or analyte volumes, as well as ease of rapid implementation of a high-efficiency mixture, microfluidics techniques emerged in early 1980 have been introduced for cryopreservation approaches. These fields include controlled loading/unloading of CPAs into the cells with stepwise, linear and complex CPA profiles (Heo et al, 2011; Park et al, 2011; Song et al, 2009), programmable freeze-thawing cycles (Afrimzon et al, 2010; Deutsch et al, 2010; Li et al, 2010), and low-CPA vitrification by ultra-rapid freezing (Choi et al, 2015a; He et al, 2008; Zou et al, 2013) using microfluidics. Such innovation offers a tremendous potential to revolutionize cryopreservation.…”

Modeling and experimental studies of enhanced cooling by medical gauze for cell cryopreservation by vitrification

“…On the other hand, vitrification totally avoids the formation of intracellular ice formation (IIF) which is considered to be fundamental to all cryoinjuries. Vitrification experiments for cryopreservation are usually conducted by plunging vitrification devices with cell suspensions into liquid nitrogen (LN 2 ) [6]. Owing to the large temperature difference between sample and LN 2 , this procedure is commonly accompanied by film boiling of LN 2 on device surface [7, 8], which consequently blocks heat transfer surrounding the sample and leads to incomplete vitrification.…”