TITLE: Human Liver Tissue Storage Methods For Multiomic Applications
AUTHORS: Diana Nakib, Catia Perciani, Sai Chung, Xue-Zhong Ma, Justin Manuel, Ian McGilvray, Sonya Macparland

[DESCRIPTION]
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.

[GUIDELINES]
All biological samples should be handled and processed according to Biosafety Guidelines. This procedure should be performed in accordance with all applicable safety procedures and using aseptic technique.

[STEPS]
SECTION: Sample Collection
1. 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.
SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3. 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.
SECTION: Snap-Freezing Tissue Sections
5. For the purpose of single-nucleus RNA-sequencing snap-freeze (dry) for the maintenance and storage of nuclei.
SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.1. Tissue should remain on ice in HBSS 1X with Ca2+ and Mg2+ until ready to be sectioned.
SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.2. Transfer tissue to a petri dish placed on top of a flat ice pack to begin sectioning with sterile scalpels and sterile forceps.


SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.3. 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).
SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.4. Remove excess liquid around the tissue (e.g. by using a dry petri-dish surface).
SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.5. Cover the base of the mold with a flat layer of OCT and place the tissue on top (OCT blocks must be pre-labeled).


SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.6. Add OCT around and on top of the tissue (the tissue must be completely submerged in OCT).

 

SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.7. Place the OCT blocks on top of a flat layer dry ice.
SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.8. 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.
SECTION: Optimal Cutting Temperature (OCT)-Embedding Tissue Sections
3.9. Store the blocks in -80ºC Fridge until future usage.

-80 °C 

SECTION: Formalin-Fixing Paraffin-Embedding
4.1. Fill and label a 15mL conical tube with 10mL of 10% Formalin.


 

SECTION: Formalin-Fixing Paraffin-Embedding
4. For the purpose of histology and immunohistochemistry, formalin-fixing and paraffin-embedding (FFPE) tissue allows for the long-term storage of tissue.
SECTION: Snap-Freezing Tissue Sections
5.1. Fill a canister with 1L of liquid nitrogen.
SECTION: Formalin-Fixing Paraffin-Embedding
4.2. 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).
SECTION: Snap-Freezing Tissue Sections
5.3. Section tissue for snap-freezing, it should be approximately 1cm x 1cm x 0.3 cm (W x L x H) in size.


SECTION: Snap-Freezing Tissue Sections
5.5. 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.


SECTION: Formalin-Fixing Paraffin-Embedding
4.3. 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. 
SECTION: Formalin-Fixing Paraffin-Embedding
4.4. To ensure long-term storage and maintenance of RNA quality, ensure that the FFPE blocks are placed in 4ºC fridge.

4 °C 

SECTION: Snap-Freezing Tissue Sections
5.2. Label 1.5ml or 2ml cryovials with study ID, date, lab name, and "Snap-Frozen."
SECTION: Snap-Freezing Tissue Sections
5.4. 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.
SECTION: Sample Collection
2. Caudate will be perfused and dissociated as described in Human Liver Caudate Lobe Dissociation for ScRNA-seqV.2