Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090, Belgium.
Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, 1090, Belgium.
Reprod Biomed Online. 2018 Jul;37(1):6-17. doi: 10.1016/j.rbmo.2018.04.045. Epub 2018 Apr 27.
From a clinical perspective, which parameters grant optimal cryopreservation of mouse testicular cell suspensions?
We studied the effect of different cryopreservation rates, the addition of sugars, different vessels and the addition of an apoptotic inhibitor on the efficiency of testicular cell suspension cryopreservation. After thawing and warming, testicular cell suspensions were transplanted to recipient mice for further functional assay. After selecting the optimal cryopreservation procedure, a second experiment compared the transplantation efficiency between the selected freezing protocol and fresh testicular cell suspensions.
Multiple- and single-step freezing did not differ significantly in terms of recovered viable cells (RVC) (33 ± 28% and 38 ± 25%). The addition of sucrose did not result in a higher RVC (33 ± 20%). Cells frozen in vials recovered better than those frozen in straws (52 ± 20% versus 33 ± 20%; P = 0.0049). The inclusion of an apoptosis inhibitor (z-VAD[Oe]-FMK) significantly increased the RVC after thawing (61 ± 18% versus 50 ± 17%; P = 0.0480). When comparing the optimal cryopreservation procedure with fresh testicular cell suspensions, a lower RVC (63 ± 11% versus 92 ± 4%; P < 0.0001) and number of donor-derived spermatogonial stem cell colonies per testis (34.04 ± 2.34 versus 16.78 ± 7.76; P = 0.0051) were observed.
Upon freeze-thawing or vitrification-warming, and assessment of donor-derived spermatogenesis after transplantation, Dulbecco's modified Eagle's medium supplemented with 1.5M dimethyl-sulphoxide, 10% fetal calf serum and 60 µM of Z-VAD-(OMe)-FMK in vials at a freezing rate of -1°C/min was optimal.
从临床角度来看，哪些参数能使小鼠睾丸细胞悬液的冷冻保存达到最佳效果？
我们研究了不同的冷冻保存率、添加糖、不同的容器以及添加凋亡抑制剂对睾丸细胞悬液冷冻保存效率的影响。解冻和复温后，将睾丸细胞悬液移植到受体小鼠中进行进一步的功能检测。在选择最佳的冷冻程序后，第二项实验比较了选定的冷冻方案与新鲜睾丸细胞悬液的移植效率。
多步和单步冷冻在回收的有活力的细胞（RVC）方面没有显著差异（分别为 33 ± 28%和 38 ± 25%）。添加蔗糖并没有导致更高的 RVC（33 ± 20%）。在小瓶中冷冻的细胞比在 straw 中冷冻的细胞恢复得更好（52 ± 20%对 33 ± 20%；P = 0.0049）。添加凋亡抑制剂（z-VAD[Oe]-FMK）显著增加了解冻后的 RVC（61 ± 18%对 50 ± 17%；P = 0.0480）。将最佳冷冻程序与新鲜睾丸细胞悬液进行比较时，观察到 RVC 较低（63 ± 11%对 92 ± 4%；P < 0.0001）和每个睾丸中供体源性精原干细胞集落的数量较少（34.04 ± 2.34 对 16.78 ± 7.76；P = 0.0051）。
在冻融或玻璃化-复温后，通过评估移植后供体来源的精子发生情况，在小瓶中以 -1°C/min 的冷冻速率，使用添加了 1.5M 二甲基亚砜、10%胎牛血清和 60µM z-VAD-(OMe)-FMK 的 Dulbecco 改良 Eagle 培养基是最佳的。
