Example for process validation in biobanking: Fit for purpose testing of a cryopreservation method without isopentane

Area of Science:

• Biobanking and Sample Preservation
• Molecular Biology Techniques
• Tissue Processing

Background:

• Effective freezing is vital for preserving tissue morphology and molecular integrity in biobanking.
• Existing biobanking guidelines offer recommendations for optimal tissue freezing.
• The Vetbiobank standard protocol required validation against these guidelines for downstream application suitability.

Purpose of the Study:

• To formally validate a tissue freezing process according to ISO 20387 (2018) biobanking guidelines.
• To assess different freezing methods for optimal preservation of mouse liver and muscle tissues.
• To confirm sample suitability for downstream molecular analyses, including gene expression.

Main Methods:

• Evaluated three freezing protocols: liquid nitrogen, isopentane, and liquid nitrogen vapor.
• Assessed morphological integrity and RNA quality (RIN) of samples frozen with or without Optimal Cutting Temperature (OCT) compound.
• Validated the optimal protocol for reproducibility and robustness, including gene expression analysis of biomarkers.

Main Results:

• Freezing in liquid nitrogen yielded the best results for morphological integrity and RNA quality.
• The liquid nitrogen method demonstrated robustness across tested tissues, though OCT decreased RIN values in liver samples.
• Gene expression analyses showed comparable results across different freezing methods.

Conclusions:

• Liquid nitrogen freezing is suitable for preserving tissue samples for RNA-based investigations.
• Optimal Cutting Temperature compound negatively impacts liver tissue RNA integrity but does not affect subsequent gene expression analysis.
• Process validation confirms the suitability of the optimized freezing protocol for biobanking applications.