Tissue processing for single-cell isolation

Dissociation of human adult oral mucosa tissue samples for single-cell RNA sequencing

Ana J. Caetano¹, Paul T. Sharpe²

¹ Centre for Oral Immunobiology and Regenerative Medicine, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, United Kingdom

² Centre for Craniofacial and Regenerative Biology, Faculty of Dentistry, Oral & Craniofacial Sciences, King’s College London, United Kingdom

Summary

The human oral mucosa is composed of a wide variety of cell types; thus, digestion of pure and viable populations is necessary for downstream techniques including flow cytometry and single-cell RNA sequencing. Here, we briefly outline a protocol to isolate both epithelial and non-epithelial cells from human adult samples at high viability.

Preparation

Note: All tissues should be collected following appropriate ethical and institutional approvals.

1. Prepare all necessary buffers fresh: transport medium (1x PBS), dissociation solution (Whole Skin Dissociation Kit, human, Miltenyi, Bergisch-Gladbach, Germany), 0.04% non-acetylated BSA (UltraPure^TM BSA, ThermoFisher Scientific).
2. Ensure all dissection tools have been sterilised.
3. Ensure working surfaces, including biosafety cabinets and centrifuges, are decontaminated with 70% ethanol and RNAse AWAY decontamination reagent.
4. Perform preparation steps and buffer/stock solutions in a biosafety cabinet.
5. Use filter and wide-bore pipette tips to minimise damage to cells from shearing forces. It is critical to pipette gently and slowly.
6. Use RNAse-free 50 mL, 15 mL and 2.0 mL DNA LoBind tubes.
7. Pre-heat an incubator shaker to 37°C.

    Please refer to Key Resources Table in article for further details.

Sample collection

1. Receive gingival tissue in 10 mL of transport medium (1x PBS, Sigma) on ice, and proceed quickly with all preparation steps.

    Note: Transport of samples should be performed as quickly as possible to achieve good viability results. In this study, tissues were collected immediately after surgical resection and kept on ice for the entire process, except during dissection. Timing of the tissue preparation should aim to be completed in less than 1 hour from collection to downstream processing, with all reagents pre-cooled on ice.

Processing of tissue

    2. Transfer tissues to a petri dish containing 1-2 mL PBS. 

    3. Rinse tissues with chilled PBS to remove remaining blood. Repeat this step 3 times. 

    4. Transfer tissues to a clean petri dish or sterile cutting board and examine the gingival tissues under operating microscope. Identify anatomical landmarks for orientation to inform selection of tissue for further processing.

    Note: Tissues used for histological analysis can be separated here and fixed in 4% paraformaldehyde in PBS (PFA) overnight at 4℃.

    5. Cut tissues into <1mm³ segments using a scalpel or fine spring scissors. Use a forceps to hold the tissue while cutting. 

    Note: Tissues with a minimum size of 4-5mm were used.

    6. Move all tissue fragments to a chilled 15 mL conical tube. Tissues can be transferred using a pre-wet wide-bore 1000 pipette tip.

    Note: Some pieces may get attached to the tube plastic walls; use a forceps to push all tissues to the liquid solution.

    7. Remove excess PBS with a 200 µl or 1000 µl pipette tip. Wait for tissues to sink to the bottom of the tube before removing PBS.

    8. Add 500 µl of enzyme dissociation solution (Miltenyi, Bergisch-Gladbach, Germany) and incubate for 30 minutes at 37°C with intermittent shaking. Inspect and disturb tissues by gently pipetting every 10 minutes.

    9. Add 1mL of 0.04% BSA solution (UltraPure^TM BSA, ThermoFisher Scientific) and pipette gently to mechanically digest remaining tissues.

    Note: Instead of 0.04% BSA solution, an alternative cell culture buffer can be used, such as DMEM (Dulbecco’s Modified Eagle Medium) + 10% FBS. 

   10. Filter the resulting cell suspension through a 70-µm cell strainer into a 50 mL tube to ensure a single-cell preparation.

   Note: Wash the filter to allow maximum cell recovery. Additionally, use the rubber end of a 3 mL syringe plunger to press remaining tissues through the filter.

   11. Centrifuge 300 x g for 5 minutes.

   12. Dispose supernatant and resuspend sample in 0.04% non-acetylated BSA (UltraPure^TM BSA, ThermoFisher Scientific). 

   Note: For this study, we included a fluorescent-activated cell sorting (FACS) step prior to single-cell library preparation to ensure maximum live cell enrichment.  

   13. Transfer to a 1.5 mL microcentrifuge or FACS tube and stain with 1.5 µg of DAPI staining solution (D1306, Invitrogen). Keep samples on ice and covered in foil. Proceed immediately to cell sorting.

   Note: Samples were analysed on BD FACS Aria III fusion machine. Data analysis was performed on FlowJo v10 software. 

   14. Select live population of interest based on size using standard SSC-A and FSC-A parameters. Perform doublets exclusion using SSC-A and SSC-W parameters. Select live cells - dimly fluorescing in DAPI.

   Note: This step may require substantial optimisation to identify and select the appropriate viable cell population. In our experience, these samples contain significant cell debris, which can be minimised by adding an extra washing step, but it will yield lower cell numbers.

   15. Sort cells into chilled LoBind tubes prefilled with 0.04% BSA. Keep on ice. 

   16. Centrifuge 300 x g for 5 min and resuspend in 0.04% BSA. Calculate resuspension volume by adjusting cell concentration to a minimum of 300 cells µl^-1. 

   17. Cell viability was again manually assessed using a haemocytometer. Briefly, 10µl of cell suspension was mixed with 10µL of Trypan Blue and loaded into the haemocytometer. 

   18. Proceed immediately to single-cell library preparation.

Expected outcomes

This protocol allowed successful isolation of all major gingiva cell types, excluding neutrophils. All tissues were processed immediately after surgical resection; therefore, this protocol may provide different results if tissues are not handled in a timely manner or with the appropriate transport and preparation techniques.