Thompson Riley E, Meyers Melinda A, Pukazhenthi Budhan S, Hollinshead Fiona K
Colorado State University, Department of Clinical Sciences, 1601 Campus Delivery, Fort Collins, CO, 80523, USA.
Colorado State University, Department of Clinical Sciences, 1601 Campus Delivery, Fort Collins, CO, 80523, USA.
Cryobiology. 2022 Feb;104:56-62. doi: 10.1016/j.cryobiol.2021.11.003. Epub 2021 Nov 14.
Reproductive diseases in mares are a significant cause of subfertility and profound economic loss in the equine industry. Utilizing a 3D in vitro cell culture system that recapitulates the in vivo physiology will reduce time, cost, and welfare concerns associated with in vivo reproductive research in mares. If this 3D model is combined with effective cryopreservation, reproductive research on mares can occur year-round, which is not currently possible in this seasonal species. Endometrial organoids, 3D in vitro cell clusters that exhibit in vivo uterine physiology, have been established in mice, women, and mares. Here we report the first comprehensive assessment of cryopreservation of endometrial organoids in the domestic mare. Organoid growth rate was not affected by the type of freezing media. However, growth rate varied among non-cryopreserved controls, organoids cryopreserved at passage 0 (P0), and organoids cryopreserved at passage 3 (P3). Additionally, there was no difference in organoid viability among freezing media or freezing timepoint (passages). Furthermore, fresh and frozen-thawed organoids displayed positive immunohistochemical staining for ZO-1, which is a marker for intercellular tight junctions, and for periodic acid-Schiff staining as marker for organoid function through mucin production. Results demonstrate that equine endometrial organoids can be cryopreserved with 10% dimethyl sulfoxide with minimal detrimental effects while maintaining intercellular tight junctions (ZO-1) and secretory function. Availability of cryopreserved endometrial organoids may permit expanded research on uterine pathologies that negatively affect mare fertility and improve efficiency, reduce cost, and minimize animal welfare concerns associated with in vivo research in the domestic mare.
母马的生殖疾病是马匹行业中导致生育力低下和严重经济损失的重要原因。利用能够重现体内生理学的三维体外细胞培养系统，将减少与母马体内生殖研究相关的时间、成本和福利问题。如果将这种三维模型与有效的冷冻保存方法相结合，母马的生殖研究就可以全年进行，而这在这个季节性繁殖的物种中目前是无法实现的。子宫内膜类器官是一种呈现体内子宫生理学特征的三维体外细胞簇，已在小鼠、女性和母马中建立。在此，我们报告了对家养母马子宫内膜类器官冷冻保存的首次全面评估。类器官的生长速率不受冷冻培养基类型的影响。然而，非冷冻保存的对照、第0代（P0）冷冻保存的类器官和第3代（P3）冷冻保存的类器官之间的生长速率有所不同。此外，冷冻培养基或冷冻时间点（代数）之间类器官的活力没有差异。此外，新鲜的和冻融后的类器官对紧密连接标记物ZO-1进行免疫组织化学染色呈阳性，对通过粘蛋白产生作为类器官功能标记物的过碘酸希夫染色也呈阳性。结果表明，马子宫内膜类器官可以用10%二甲基亚砜进行冷冻保存，对其产生的有害影响最小，同时保持细胞间紧密连接（ZO-1）和分泌功能。冷冻保存的子宫内膜类器官的可用性可能会允许对负面影响母马生育力的子宫病理学进行更广泛的研究，并提高效率、降低成本以及将与家养母马体内研究相关的动物福利问题降至最低。
