A Chemically Defined Medium for Rabbit Embryo Cryopreservation

Abstract

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbecco's phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v−1) of CRYO3 or 18% (v.v−1) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0.066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions.

Insights into Species Preservation: Cryobanking of Rabbit Somatic and Pluripotent Stem Cells

“…This synthetic medium induced acceptable viability rates, growth recoveries and pluripotency gene expression when supplemented with 4–5% cryoprotectant. These data are consistent with our previous studies on embryo cryopreservation [ 38 , 39 , 44 ] and with other studies which showed that FBS could be replaced by a commercial synthetic media containing a high molecular weight polymer when freezing human or mouse PSCs [ 52 , 53 , 54 , 55 ]. In our study, we compared CRYO3, a synthetic commercial product accredited for clinical purposes, with CryoStor ® CS10, a widely used stem cell freezing medium of unknown composition.…”

“…We further observed that the CRYO3-based medium exhibited a more variable ∆H than CryoStor ® CS10, suggesting that the former was less thermodynamically stable. However, this instability of CRYO3 was not previously observed in studies of embryo freezing media [ 38 , 44 ]. Except for CryoStor ® CS10, all freezing media exhibited similar thermodynamic patterns that corresponded to reduced viability in the thawed cells.…”

“…To avoid the risks associated with serum, home-made and commercial freezing media has been supplemented with natural macromolecules, such as soybean lecithin [ 33 ] and silk sericin [ 34 ], or synthetic macromolecules, such as liposomes [ 35 ], polysaccharides [ 36 ], and polyvinylpyrrolidones [ 37 ], with varying degrees of success depending on the cryopreserved cells. Previous studies have already demonstrated the effectiveness of one type of commercial chemically defined media, STEM ALPHA.CRYO3 (hereinafter referred to as CRYO3), in the cryopreservation of rabbit embryos [ 38 , 39 ]. CRYO3 is a patented medium that lacks serum, protein, and dextran and is manufactured according to good manufacturing practice, in accordance with directive 2001/83/EC.…”

Use of polyethyleneoxide and hydroxyethylstarch as blood plasma substitutes in the cryopreservation of testis interstitium cells in mice

“…Існують роботи, в яких 6-7% ГЕК розглянуто як замінник протеїнів сироватки крові для кріоконсервування еритроцитів [28,29]. Нами також використаний цей підхід при обранні концентрацій полімерних сполук -ПЕО та ГЕК, які потенційно можуть заміщувати дію протеїнів сироватки під час кріоконсервування [30], але ПЕО за даних концентрацій виявився неефективним.…”

Vitrification of Biological Cells Using a Cryogenic Fine Solid Particulate Spray

“…Although a great deal of effort has been expended in improving cell cryopreservation [1,2], a considerable amount of specially designed cryopreservation solution [3][4][5][6][7] is still required and low cell viability at the time of thawing remains a serious problem. Accordingly, availing of the newly developed high-speed vitrification cryopreservation system, we propose an ultra-high heat flux vitrification (quenching) method that uses a micro-nano fine solid nitrogen (SN 2 ) particulate spray [8,9].…”