Medical School of Chinese PLA, Chinese PLA General Hospital, Beijing, People's Republic of China.
Department of Orthopedics, Chinese PLA General Hospital, Beijing, People's Republic of China.
Biopreserv Biobank. 2024 Jun;22(3):225-234. doi: 10.1089/bio.2023.0005. Epub 2023 Aug 18.
Different experiments require different sample storage methods. The commonly used preservation methods in biobank practice cannot fully meet the multifarious requirements of experimental techniques. Programmable controlled slow freezing (PCSF) can maintain the viability of tissue. In this study, we hypothesized that PCSF-preserved samples have potential advantages in matching subsequent experiments compared with existing methods. We compared the differences on skeletal muscle tissue RNA integrity, protein integrity, microstructure integrity, and cell viability between four existing cryopreservation methods: liquid nitrogen (LN) snap-freezing, LN-cooled isopentane snap-freezing, RNAlater-based freezing, and PCSF. RNA integrity was evaluated using agarose gel electrophoresis and RNA integrity number. Freezing-related microstructural damage in the muscle tissue was evaluated using ice crystal diameter and muscle fiber cross-sectional area. Protein integrity was evaluated using immunofluorescence staining. Cell viability was evaluated using trypan blue staining after primary muscle cell isolation. PCSF preserved RNA integrity better than LN and isopentane, with a statistically significant difference. RNAlater preserved RNA integrity best. PCSF best controlled ice crystal size in myofibers, with a significant difference compared with LN. The PCSF method best preserved the integrity of protein epitopes according to the mean fluorescence intensity results, with a significant difference. Cell viability was best preserved in the PCSF method compared with the other three methods, with a significant difference. PCSF protected the RNA integrity, microstructural integrity, protein integrity, and cell viability of skeletal muscle tissue. The application of PCSF in biobank practice is recommended as a multi-experiment-compatible cryopreservation method.
不同的实验需要不同的样本储存方法。生物库实践中常用的保存方法不能完全满足实验技术的多样化要求。程控慢冻（PCSF）可以保持组织的活力。在这项研究中，我们假设与现有方法相比，PCSF 保存的样本在匹配后续实验方面具有潜在优势。我们比较了四种现有的冷冻保存方法（液氮（LN）快速冷冻、LN 冷却异戊烷快速冷冻、Rnalater 基础冷冻和 PCSF）对骨骼肌组织 RNA 完整性、蛋白质完整性、微观结构完整性和细胞活力的差异。使用琼脂糖凝胶电泳和 RNA 完整性数评估 RNA 完整性。使用冰晶直径和肌纤维横截面积评估冷冻相关的肌肉组织微观结构损伤。使用免疫荧光染色评估蛋白质完整性。通过原代肌细胞分离后使用台盼蓝染色评估细胞活力。PCSF 比 LN 和异戊烷更能保存 RNA 完整性，差异具有统计学意义。Rnalater 保存 RNA 完整性最好。PCSF 控制肌纤维中的冰晶大小最好，与 LN 相比差异显著。根据平均荧光强度结果，PCSF 方法保存蛋白质表位的完整性最好，差异显著。与其他三种方法相比，PCSF 方法保存细胞活力最好，差异显著。PCSF 保护骨骼肌组织的 RNA 完整性、微观结构完整性、蛋白质完整性和细胞活力。建议在生物库实践中应用 PCSF 作为一种多实验兼容的冷冻保存方法。
