Human Liver Tissue Storage Methods For Multiomic Applications
Catia Perciani, Xue-Zhong Ma, Justin Manuel, Sai Chung, Ian McGilvray, Sonya Macparland, Diana Nakib
Abstract
Sampling different areas of the diseased or healthy liver and storing the tissue in a variety of mediums to allow for an in-depth characterization of heterogeneity and the identification of unique cell populations through the employment of multiomic technologies, such as single-cell RNA-sequencing, single-nucleus RNA-sequencing, flow cytometry, and spatial transcriptomics.
Steps
Sample Collection
Collect explanted tissue section in 30mL of HBSS 1x with Ca2+ and Mg2+ in a 50mL conical tube and keep on ice, ideally to process within 30 minutes of removal from patient or removal from the flushed donor organ in preservation solution.
Caudate will be perfused and dissociated as described in Human Liver Caudate Lobe Dissociation for ScRNA-seqV.2
Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
For the purpose of spatial transcriptomics , OCT-embed for the viable freezing and storing for future slicing of tissue for spatial analyses. * O.C.T. (Optimal Cutting Temperature) Compound (Fisher Health Care 4585)
- Additional approach: Tissue snap frozen in liquid nitrogen can be embedded in OCT after LN storage and then processed for spatial transcriptomics.
Tissue should remain on ice in HBSS 1X with Ca2+ and Mg2+ until ready to be sectioned.
Transfer tissue to a petri dish placed on top of a flat ice pack to begin sectioning with sterile scalpels and sterile forceps.
Section tissue for OCT-embedding, it should be equal to or smaller than 1cm x 1cm x 0.3 cm (W x L x H).
Remove excess liquid around the tissue (e.g. by using a dry petri-dish surface).
Cover the base of the mold with a flat layer of OCT and place the tissue on top (OCT blocks must be pre-labeled).
Add OCT around and on top of the tissue (the tissue must be completely submerged in OCT).
Place the OCT blocks on top of a flat layer dry ice.
When the block looks completely white, wrap them with plastic microfilm and secure the plastic, make sure to re-label on top of the plastic microfilm.
Store the blocks in -80ºC Fridge until future usage.
-80°C
Formalin-Fixing Paraffin-Embedding
For the purpose of histology and immunohistochemistry, formalin-fixing and paraffin-embedding (FFPE) tissue allows for the long-term storage of tissue.
Fill and label a 15mL conical tube with 10mL of 10% Formalin .
Ensure to keep the tissue in the petri dish on the ice pack while sectioning, aim for a size of 1cm x 1cm x 1cm (W x L x H).
Place the tissue section in the tube, fully submerged in 10% Formalin and keep it in the fridge until you are ready to submit it for paraffin-embedding and sectioning for stains.
To ensure long-term storage and maintenance of RNA quality, ensure that the FFPE blocks are placed in 4ºC fridge.
4°C
Snap-Freezing Tissue Sections
For the purpose of single-nucleus RNA-sequencing snap-freeze (dry) for the maintenance and storage of nuclei.
Fill a canister with 1L of liquid nitrogen.
Label 1.5ml or 2ml cryovials with study ID, date, lab name, and "Snap-Frozen."
Section tissue for snap-freezing, it should be approximately 1cm x 1cm x 0.3 cm (W x L x H) in size.
Remove as much excess liquid as possible by placing it on a dry petri dish surface, then transfer to cryovial, close it shut and drop it in the liquid nitrogen container.
Once frozen, use forceps to remove them from the container, and place them a cardboard box in a liquid nitrogen storage tank until future usage.