Cryogenic Preservation of Biopsy Tissue

The Clinical Need

  • Biopsy tissue samples are preserved in formalin or in ice, each has its merits and pitfalls:
    • Formalin retains tissue ultrastructure but degrades proteins DNA, RNA, which causes insufficient yield for biomarker analysis
    • Freezing causes histology artifacts and degrades ultrafine structures, which limits histopathology diagnosis (e.g. tumor invasiveness)
  • Tissue handling becomes critical when biomarkers are used to guide therapy (e.g. process standardization, effect of ischemia on level of sensitive biomarkers)

Technology Innovation

Novel cryogenic devices employ proprietary Differential Freezing Profile (DFP) to preserve cell viability, in-vivo biochemical profile, and ultrastructure in biopsy samples
  • CryoBxTM: in-situ cryobiopsy device
  • CryoTrayTM: chemo-cryo preservation cartridge
  • Proprietary Differential Freezing Profile:
    • frozen part maintains cellular viability and in-vivo biomolecular profile
    • non-frozen part maintains cellular ultrastructure for histology and IHC

Proof of Concept

Effect of sample ischemia on biomarker level

Collaborator: Dr. Akhilesh Pandey, Johns Hopkins Medical Institutions
Mass-spectrometry analysis of NCI-N87 gastric cancer cell line:
  • Ratio of phosphorylation level in conventional versus cryobiopsy samples in ~650 peptides analyzed.
  • Tissue ischemia changes phosphorylation level in many peptides.
  • pHSP27-S82 shows ~2.5 fold increase in conventional sample compared with cryobiopsy sample following 2-minute incubation in room temperature.
Western blot analysis in two tumor cell line:
  • Total HSP27 is similar in conventional and cryogenic biopsy samples.
  • Phosphorylated HSP27-S82 is higher in the conventional biopsy sample compared to the cryogenic sample
Cryobiopsy needle used in "proof of concept" studies
Biopsy sample has frozen part and non-frozen part through the use of Differential Freezing Profile

Effect of sample freezing on histopathology

Collaborator: Dr. Deborah Dillon, Brigham and Women's Hospital, Boston
Effect of tissue freezing on histology and IHC
  • Non-frozen part of samples maintains tissue ultrastructure which enables high quality histology (e.g. H&E staining) and IHC (e.g. cytoplasmic pS6).
  • Frozen part of samples shows structural damage due to ice formation, but is ideal for biomarkers (enzymes, DNA, RNA)

This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 761525.