Effect of Ischemia Durations, Transport, Cryopreservation and Storage Conditions on Tissue Integrity, DNA, RNA, Protein Quality and Primary Cell Culture Initiation Capacity in Liver Tissues

Objectives: Standardization of pre-analytical factors is essential for ensuring the quality and reproducibility of tissue-based biomedical research. Factors such as ischemia time and freezing methods significantly impact sample integrity and the quality of downstream analyses. However, systematic evaluations of how ischemia time, transfer conditions, freezing methods, and storage parameters affect liver tissue quality remain limited. This study aimed to evaluate the effects of ischemia duration, freezing techniques, transfer conditions, and storage conditions and durations on liver tissue integrity, nucleic acid quality, protein yield, and primary cell culture initiation capacity.
Methods: Liver tissues from healthy male Wistar albino rats were subjected to varying cold ischemia durations (2h, 8h, 24h) and transfer conditions (+4°C, vacuum-sealed, culture medium) to assess tissue integrity, nucleic acid and protein quality, and cell culture initiation potential. Optimal conditions were selected for subsequent freezing techniques (controlled-rate, rapid freezing in liquid nitrogen, and freezing with isopentane) over different storage periods (3, 6, 9 months) at –80°C and –196°C. Histological assessment (HE and Masson trichrome staining) was performed to evaluate sample integrity. Nucleic acid, and protein yield and quality as well as primary cell culture initiation capacity, were tested.
Results: Tissue integrity remained stable for 0–2 hours under cold ischemia, with moderate degeneration observed after 24 hours. RNA and DNA yields were consistent across transfer conditions, with no significant differences detected. However, RNA integrity was more sensitive to ischemic conditions compared to total nucleic acid quantities. Freezing methods did not significantly differ in preserving tissue quality, and both –80 °C and –196 °C storage effectively maintained nucleic acid integrity up to 9 months. Primary cell cultures were successfully established from tissues subjected to a maximum of 8 hours of ischemia, but not from those exposed to 24 hours. Conclusion: Optimized transfer conditions and appropriate freezing and storage methods are key to preserving liver tissue quality for biobanking and downstream analyses. This study provides valuable insights for developing standardized protocols for liver tissue biobanking, which could enhance reproducibility and reliability in translational research.

Keywords: Biobanking, Cold ischemia, cryopreservation, liver tissue, pre-analytical factors, translational research