Cryopreservation of chicken primordial germ cells by vitrification and slow freezing: A comparative study.

In the present study, we compare a classical slow freezing (SLF) method and an aseptic vitrification (Vitrif) technique to cryopreserve a stable primordial germ cell (PGCs) line issued from the Ardennaise chicken breed. Viability immediately after warming was close to 80% and did not differ between the two cryopreservation methods. Proliferation tended to be slower for both cryopreservation methods compared with controls, but the difference was significant only for Vitrif. No difference was found between the two methods after flow cytometry analysis of stage-specific embryonic antigen-1 expression and reverse transcription-polymerase chain reaction on several factors related to PGC phenotype. After 1 week in culture, all cryopreserved cells reached controls' main morphologic and expanding (viability/proliferation) features. However, SLF generated more unwanted cells clusters than Vitrif. After injection of the PGCs into recipient embryos, vitrified PGCs reported a clear, yet not significant, tendency to colonize the gonad at a higher rate than slow frozen PGCs. SLF in cryovials remains simple, inexpensive, and less technically demanding than Vitrif. Nevertheless, the intrinsic advantages of our aseptic Vitrif method and the present study suggest that this should be considered as safer than classical SLF for cryopreserving chicken PGCs.

Lower intracellular concentration of cryoprotectants after vitrification than after slow freezing despite exposure to higher concentration of cryoprotectant solutions.

STUDY QUESTION: What is the intracellular concentration of cryoprotectant (ICCP) in mouse zygotes during vitrification (VIT) and slow-freezing (SLF) cryopreservation procedures? SUMMARY ANSWER: Contrary to common beliefs, it was observed that the ICCP in vitrified zygotes is lower than after SLF, although the solutions used in VIT contain higher concentrations of cryoprotectants (CPs). WHAT IS KNOWN ALREADY: To reduce the likelihood of intracellular ice crystal formation, which has detrimental effects on cell organelles and membranes, VIT was introduced as an alternative to SLF to cryopreserve embryos and gametes. Combined with high cooling and warming rates, the use of high concentrations of CPs favours an intracellular environment that supports and maintains the transition from a liquid to a solid glass-like state devoid of crystals. Although the up-to-date publications are reassuring in terms of obstetric and perinatal outcomes after VIT, a fear about exposing gametes and embryos to high amounts of CPs that exceed 3-4-fold those found in SLF was central to a debate initiated by advocates of SLF procedures. STUDY DESIGN, SIZE, DURATION: Two experimental set-ups were applied. The objective of a first study was to determine the ICCP at the end of the exposure steps to the CP solutions with our VIT protocol (n = 31). The goal of the second investigation was to compare the ICCP between VIT (n = 30) and SLF (n = 30). All experiments were performed in triplicates using mouse zygotes. The study took place at the GIGA-Research Institute of the University of Liège. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cell volume is modified by changes in extracellular osmolarity. Hence, we estimated the final ICCP after the incubation steps in the VIT solutions by exposing the cells to sucrose (SUC) solutions with defined molarities. The ICCP was calculated from the SUC concentration that produced no change in cell volume, i.e. when intra- and extracellular osmolarities were equivalent. Cell volume was monitored by microscopic cinematography. ICCP was compared between SLF and VIT based on the principle that a high ICCP lowers the probability of (re)crystallization during warming but increases the probability of over-swelling of the cell due to fast inflow of water. The survival rates of mouse zygotes after SLF or VIT were compared using either (i) various warming rates or (ii) various concentrations of SUC in the warming dilution medium. MAIN RESULTS AND THE ROLE OF CHANCE: The ICCP in mouse zygotes during the VIT procedure prior to plunging them in liquid nitrogen was ∼2.14 M, i.e. one-third of the concentration in the VIT solution. After SLF, the warming rate did not affect the zygote survival rate. In contrast, only 3/30 vitrified zygotes survived when warmed slowly but as many as 30/30 zygotes survived when warming was fast (>20 000°C/min). Vitrified zygotes showed significantly higher survival rates than slow-frozen zygotes when they were placed directly in the culture medium or in solutions containing low concentrations of SUC (P < 0.01). These two experiments demonstrate a lower ICCP after VIT than after SLF. LIMITATIONS, REASONS FOR CAUTION: The results should not be directly extrapolated to other stages of development or to other species due to possible differences in membrane permeability to water and CPs. WIDER IMPLICATIONS OF THE FINDINGS: The low ICCP we observed after VIT removes the concern about high ICCP after VIT, at least in murine zygotes and helps to explain the observed efficiency and lack of toxicity of VIT. STUDY FUNDING / COMPETING INTEREST(S): The study was funded by the FNRS (National Funds for Scientific Research). The authors declare that they have no competing interests.