Nov 17, 2022
Human Liver Tissue Storage Methods For Multiomic Applications
- Diana Nakib1,
- Catia Perciani1,
- Sai Chung1,
- Xue-Zhong Ma1,
- Justin Manuel1,
- Ian McGilvray1,
- Sonya Macparland1
- 1University of Toronto
- Human Cell Atlas Method Development Community
Protocol Citation: Diana Nakib, Catia Perciani, Sai Chung, Xue-Zhong Ma, Justin Manuel, Ian McGilvray, Sonya Macparland 2022. Human Liver Tissue Storage Methods For Multiomic Applications. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge34qdl47/v1
Manuscript citation:
Tallulah S Andrews, Jawairia Atif, Jeff C Liu, Catia T Perciani, Xue‐Zhong Ma, Cornelia Thoeni, Michal Slyper, Gökcen Eraslan, Asa Segerstolpe, Justin Manuel, Sai Chung, Erin Winter, Iulia Cirlan, Nicholas Khuu, Sandra Fischer, Orit Rozenblatt‐Rosen, Aviv Regev, Ian D McGilvray, Gary D Bader, Sonya A MacParland (2022) Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatology communications Volume 6 Issue 4
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2022
Last Modified: November 17, 2022
Protocol Integer ID: 71406
Keywords: Multiomics, Liver, Tissue Storage, Transcriptomics, human cell atlas methods development community, human liver tissue storage methods for multiomic application, human liver tissue storage method, spatial transcriptomic, multiomic application, identification of unique cell population, multiomic technology, unique cell population, healthy liver, cell rna, employment of multiomic technology, cell population, flow cytometry, nucleus rna, sequencing, rna, storage, tissue
Funders Acknowledgements:
Chan Zuckerberg Initiative
Grant ID: CZF2019-002429
CIHR
Grant ID: PJT 162098
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.
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.
Materials
General Materials:
- 1 x ice bucket
- 2 x sterile scalpels
- 1 x sterile spring scissor (14 mm cutting edge, 0.275 mm tip diameter, 12 cm length, Fine Science Tools)
- 2 sterile forceps
- Flat ice pack
- Petri dishes
- HBSS 1X with Ca2+ and Mg2+ (Gibco A14025-092, Bleiswijk, The Netherlands)
- 50 ml conical tube
OCT-Embedding Materials:
- 1 x dry-ice bucket
- Pre-labeled base mold (15 x 15 x 5 mm, Ref 22363553, Fisher Brand)
- O.C.T. (Optimal Cutting Temperature) Compound (Fisher Health Care 4585)
- Plastic Film (to wrap the OCT blocks)
Formalin-Fixing Materials:
- Formalin solution, neutral buffered, 10% (Sigma, HT501128)
- 1x 50mL conical tube
Snap-Freezing Materials:
- 1x liquid nitrogen canister
- 1L liquid nitrogen
- 1.5mL cryovials
Troubleshooting
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).
Note
To ensure complete submersion in OCT, avoid the presence of bubbles above or surrounding the tissue.
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
5m
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.
Note
Ensure label contains sample ID, date, lab name, and solution (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
15m
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.