Department of Obstetrics and Gynecology, Medical Faculty, Cologne University, 50931 Cologne, Germany.
Institute of Biology and Immunology of Reproduction of Bulgarian Academy of Sciences (BAS), 1113 Sofia, Bulgaria.
Int J Mol Sci. 2023 Dec 22;25(1):214. doi: 10.3390/ijms25010214.
Cryopreservation of human testicular tissue, as a key element of anticancer therapy, includes the following stages: saturation with cryoprotectants, freezing, thawing, and removal of cryoprotectants. According to the point of view existing in "classical" cryobiology, the thawing mode is the most important consideration in the entire process of cryopreservation of any type of cells, including cells of testicular tissue. The existing postulate in cryobiology states that any frozen types of cells must be thawed as quickly as possible. The technologically maximum possible thawing temperature is 100 °C, which is used in our technology for the cryopreservation of testicular tissue. However, there are other points of view on the rate of cell thawing, according to how thawing should be carried out at physiological temperatures. In fact, there are morphological and functional differences between immature (from prepubertal patients) and mature testicular tissue. Accordingly, the question of the influence of thawing temperature on both types of tissues is relevant. The purpose of this study is to explore the transcriptomic differences of cryopreserved mature and immature testicular tissue subjected to different thawing methods by RNA sequencing. Collected and frozen testicular tissue samples were divided into four groups: quickly (in boiling water at 100 °C) thawed cryopreserved mature testicular tissue (group 1), slowly (by a physiological temperature of 37 °C) thawed mature testicular tissue (group 2), quickly thawed immature testicular tissue (group 3), and slowly thawed immature testicular tissue (group 4). Transcriptomic differences were assessed using differentially expressed genes (DEG), the Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and protein-protein interaction (PPI) analyses. No fundamental differences in the quality of cells of mature and immature testicular tissue after cryopreservation were found. Generally, thawing of mature and immature testicular tissue was more effective at 100 °C. The greatest difference in the intensity of gene expression was observed in ribosomes of cells thawed at 100 °C in comparison with cells thawed at 37 °C. In conclusion, an elevated speed of thawing is beneficial for frozen testicular tissue.
人类睾丸组织的冷冻保存是癌症治疗的关键要素，包括以下阶段：用冷冻保护剂饱和、冷冻、解冻和去除冷冻保护剂。根据“经典”低温生物学中的观点，解冻模式是冷冻保存任何类型细胞（包括睾丸组织细胞）整个过程中最重要的考虑因素。低温生物学中的现有假设指出，任何冷冻类型的细胞都必须尽快解冻。技术上最大可能的解冻温度为 100°C，我们的睾丸组织冷冻保存技术中就使用了这个温度。然而，对于细胞解冻的速度，还有其他观点，即应该如何在生理温度下进行解冻。事实上，未成熟（来自青春期前患者）和成熟的睾丸组织之间存在形态和功能上的差异。因此，关于解冻温度对这两种组织的影响的问题是相关的。本研究的目的是通过 RNA 测序探索不同解冻方法对冷冻保存的成熟和未成熟睾丸组织的转录组差异。收集和冷冻的睾丸组织样本分为四组：快速（在 100°C 的沸水中）解冻的冷冻成熟睾丸组织（第 1 组）、缓慢（通过 37°C 的生理温度）解冻的成熟睾丸组织（第 2 组）、快速解冻的未成熟睾丸组织（第 3 组）和缓慢解冻的未成熟睾丸组织（第 4 组）。使用差异表达基因（DEG）、京都基因与基因组百科全书（KEGG）、基因本体论（GO）和蛋白质-蛋白质相互作用（PPI）分析评估转录组差异。在冷冻保存后，成熟和未成熟睾丸组织的细胞质量没有发现根本差异。通常，成熟和未成熟睾丸组织在 100°C 时解冻效果更好。在 100°C 下解冻的细胞与在 37°C 下解冻的细胞相比，核糖体的基因表达强度差异最大。总之，提高解冻速度有利于冷冻睾丸组织。
