Tonus C, Connan D, Waroux O, Vandenhove B, Wayet J, Gillet L, Desmecht D, Antoine N, Ectors F J, Grobet L
Embryology Unit, FARAH (Fundamental and Applied Research for Animals and Health) Research Center and Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
Embryology Unit, FARAH (Fundamental and Applied Research for Animals and Health) Research Center and Faculty of Veterinary Medicine, University of Liège, Liège, Belgium; Histology Unit, FARAH Research Center and Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
Theriogenology. 2017 Jan 15;88:197-206. doi: 10.1016/j.theriogenology.2016.09.022. Epub 2016 Sep 21.
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.
在本研究中，我们比较了经典的慢速冷冻（SLF）方法和无菌玻璃化（Vitrif）技术，以冷冻保存源自阿登纳鸡品种的稳定原始生殖细胞（PGC）系。解冻后的活力接近80%，两种冷冻保存方法之间没有差异。与对照组相比，两种冷冻保存方法的增殖都趋于缓慢，但仅玻璃化组的差异具有统计学意义。在对阶段特异性胚胎抗原-1表达进行流式细胞术分析以及对与PGC表型相关的几个因子进行逆转录-聚合酶链反应后，两种方法之间未发现差异。培养1周后，所有冷冻保存的细胞均达到对照组的主要形态和扩增（活力/增殖）特征。然而，慢速冷冻产生的不需要的细胞团比玻璃化更多。将PGC注射到受体胚胎中后，玻璃化的PGC显示出比慢速冷冻的PGC更明显但不显著的定殖性腺的倾向。冷冻管中的慢速冷冻仍然简单、便宜，且技术要求低于玻璃化。尽管如此，我们的无菌玻璃化方法的固有优势以及本研究表明，对于冷冻保存鸡PGC，玻璃化应被认为比经典的慢速冷冻更安全。
