Wieser Monika, Burger Stefanie, Ertl Reinhard, Kummer Stefan, Stargardt Melanie, Walter Ingrid
VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria.
Front Mol Biosci. 2022 Sep 30;9:876670. doi: 10.3389/fmolb.2022.876670. eCollection 2022.
The freezing process of tissue samples is crucial for the preservation of morphological and molecular features. Several biobanking guidelines describe freezing techniques for optimal outcomes. As the Vetbiobank standard freezing protocol does not comply with those recommendations in detail, a process validation was performed to demonstrate that samples are suitable for downstream applications. Here we give a formal example of a process validation in the biobanking setting, as required by the biobanking guideline ISO 20387 (2018). Three different freezing protocols, freezing in liquid nitrogen, freezing isopentane precooled on dry ice and freezing liquid nitrogen vapor, were assessed based on morphological integrity of mouse liver and muscle tissue samples. Samples were either frozen in cryotubes (without Optimal Cutting Temperature compound, OCT) or in cryomolds (with OCT). The protocol providing the best results was validated for reproducibility and robustness in terms of defined acceptance criteria for morphological evaluability, A260/A280 ratio, and RNA integrity number values (RIN). In addition, performance tests were run by gene expression analyzes of selected, tissue specific biomarkers to confirm that processed samples are fit for purpose. From the three applied freezing protocols, freezing in liquid nitrogen generated best results. Reproducibility acceptance criteria were met for both, morphological integrity and RNA quality. The freezing method was robust for the tested tissue types and the application of OCT, with exception of liver tissue, where it led to a significant decrease of the RIN value. Gene expression analyzes showed good comparability of results regardless of the applied freezing method. Freezing of tissue samples in liquid nitrogen provides samples of adequate quality for subsequent RNA investigations. A negative impact of OCT on the RIN value of liver samples was observed, which was independent from the applied freezing protocol and showed no impact on subsequent gene expression analysis.
组织样本的冷冻过程对于形态和分子特征的保存至关重要。一些生物样本库指南描述了实现最佳结果的冷冻技术。由于兽医生物样本库标准冷冻方案未详细符合这些建议，因此进行了过程验证以证明样本适用于下游应用。在此，我们按照生物样本库指南ISO 20387（2018）的要求，给出生物样本库环境下过程验证的一个正式示例。基于小鼠肝脏和肌肉组织样本的形态完整性，评估了三种不同的冷冻方案：在液氮中冷冻、在干冰预冷的异戊烷中冷冻以及在液氮蒸汽中冷冻。样本要么在冻存管（不含最佳切割温度复合物，OCT）中冷冻，要么在冻模（含OCT）中冷冻。根据形态可评估性、A260/A280比值和RNA完整性数值（RIN）的既定验收标准，对提供最佳结果的方案进行了可重复性和稳健性验证。此外，通过对选定的组织特异性生物标志物进行基因表达分析来进行性能测试，以确认处理后的样本符合用途要求。在所应用的三种冷冻方案中，在液氮中冷冻产生了最佳结果。形态完整性和RNA质量均符合可重复性验收标准。除肝脏组织外，该冷冻方法对于所测试的组织类型和OCT的应用具有稳健性，在肝脏组织中它导致RIN值显著下降。基因表达分析表明，无论应用何种冷冻方法，结果都具有良好的可比性。在液氮中冷冻组织样本可为后续的RNA研究提供质量足够的样本。观察到OCT对肝脏样本RIN值有负面影响，但这与所应用的冷冻方案无关且对后续基因表达分析无影响。
