Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Summary

Brush cells are rare cholinergic chemosensory epithelial cells found in the naïve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

Abstract

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Introduction

Brush cells belong to a class of chemosensory epithelial cells characterized by the expression of bitter taste receptors and the taste receptor transduction machinery found in taste bud cells. Unlike taste bud cells, chemosensory epithelial cells are scattered in epithelial surfaces and are referred to as solitary chemosensory cells (SCCs) and microvillous cells in the nasal epithelium^1,2, brush cells in the trachea^3,4, and tuft cells in the intestine^5,6. Epithelial cells expressing bitter taste receptors and the bitter taste transduction machinery are also found in the urethra^7,8 and the auditory tube⁹. Airway brush cells have unique functions in neurogenic and immune airway responses. They are acetylcholine-producing chemosensory cells that evoke protective respiratory reflexes upon activation with bitter compounds and bacterial metabolites like quorum-sensing substances¹⁰. Airway brush cells are also the dominant airway epithelial source of IL-25, which regulates aeroallergen-elicited type 2 inflammation in the airways³.

Characterization of the full transcriptome of lower airway brush cells and their response to environmental stimuli has been limited by their low numbers in the tracheal epithelium and very limited numbers beyond the large bronchi¹⁰. Techniques used for the isolation of chemosensory cells from the intestinal epithelium have not yielded proportionally high numbers from the trachea, possibly because of the intimate contacts of tracheal brush cells with nerve endings¹⁰ or other tissue-specific factors in the respiratory mucosa such as the composition of adherens and tight junction proteins. Recent reports of successful isolation of tracheal brush cells in higher numbers for single cell RNA sequencing analysis employed either a 2 h incubation with papain or an 18 h incubation with pronase^11,12. Since longer incubations with digestive enzymes can decrease cell viability and alter the transcriptional profile of cells from digested tissues¹³, this could bias comparative analysis with other chemosensory epithelial populations.

Here, we report a method for the isolation of tracheal brush cells for RNA sequencing³. Treatment of the trachea with high-dose dispase separates the epithelium from the submucosa. Subsequent digestion of the epithelial sheet with papain allows for excellent recovery of this structural cell.

Protocol

Before conducting the following experiments, ensure that all animal care use and protocols are approved by the Institutional Animal Care and Use Committee (IACUC) and performed in accord with the National Research Council's "Guide for the Care and Use of Laboratory Animals" (8^th Edition, 2011) and the ARRIVE guidelines. All procedures described below have been reviewed and approved by the Institutional Animal Care and Use Committee at the Brigham and Women's Hospital.

1. Preparation of Reagents

1. Prepare Dispase digestion solution, which is a PBS solution containing 16 U/mL dispase and 20 µg/mL DNase I. Ensure that the dispase powder is fully dissolved before warming up the solution in a water bath at 37 °C.
2. Add 5% heat-inactivated fetal bovine serum (FBS) to Dulbecco Modified Eagle Medium (DMEM) to make a stopping solution.
3. Prepare Tyrode I buffer: add 26 U/mL papain (20 µL/mL of 48 U/mg papain solution) and 10 µL/mL L-cysteine to HEPES-Tyrode's buffer without calcium.
4. Prepare Tyrode II buffer: add 2 μL/mL leupeptin (5 mg/mL) to HEPES-Tyrode's buffer with calcium.
5. Prepare FACS buffer: use Hanks' Balanced Salt Solution (HBSS) without calcium, magnesium & phenol red, supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA) and add 2% FBS.

2. Dissection of Mouse Trachea

NOTE: Mice used in this protocol are ChAT^(BAC)-eGFP (B6.Cg-Tg(RP23- 268L19-EGFP)2Mik/J), 3-6 months of age of both sexes. Minimize the exposure of the tissue to direct light to reduce photobleaching of eGFP.

1. Euthanize the mouse with 100 mg/kg pentobarbital euthanasia solution injected intraperitoneally or using standard protocols approved by the IACUC.
2. Fix the mouse on a surgical board in the supine position with 21 G needles with extended upper and lower extremities. Spray the fur with 70% EtOH to sanitize the area.
3. Using straight forceps, lift the skin and fur of the abdomen and make an incision in the center with dissecting scissors (straight scissors, 3 cm). Using the scissors, separate the skin from the subcutaneous tissue from the abdomen to the mandibula. While holding the subcutaneous tissue up with the forceps, make a small incision with the scissors in the center of the abdominal wall.
4. Open the peritoneum with a V-shaped incision. Using the forceps, gently move the small intestine to the side, locate the abdominal aorta and vena cava and make an incision with the dissecting scissors to allow for rapid exsanguination.
5. Locate the diaphragm. Using an 18 G needle, make an opening in the diaphragm just below the sternum to deflate the lungs. Carefully separate the diaphragm from the rib cage using sharp-pointed straight dissecting scissors to cut along the base of the ribs.
6. Using the forceps, lift the exposed end of the sternum and cut the sternum longitudinally from the base of the rib cage to the neck. Make a central cervical incision with short straight scissors (2 cm) and separate the two lobes of the submandibular gland.
7. Carefully remove the surrounding connective tissue and the thymus overlying the carina with a pair of fine point high precision forceps.
8. Dissect the trachea free first by separating the proximal end at the level of the epiglottis and then by dissecting the distal end at the level of the bifurcation of the trachea.
9. Locate the epiglottis and cut the trachea longitudinally from the epiglottis to the carina.

3. Tracheal Epithelial Digestion

1. Place the trachea into a 1.5 mL tube containing 750 µL of pre-warmed (to 37 °C) dispase digestion solution. Incubate on a shaker at 200 rpm for 40 min at room temperature. Cover the tube with aluminum foil to reduce the exposure to direct light.
2. Add 750 µL of cold DMEM with 5% FBS to stop the reaction. Place on ice.
3. Transfer the trachea to a Petri dish (100 mm x 15 mm) and place under a dissecting microscope. Orient the trachea with the epithelial side facing up. The longitudinally dissected trachea has a semi-cylindrical shape maintained by the cartilaginous rings. The epithelium is on the concave surface.
4. Tether the epiglottis area of the trachea with straight forceps to the Petri dish and using a size 22 disposable scalpel, scrape the epithelium off the trachea. The epithelial layer separates as a translucent sheet.
5. Mince the epithelium with the scalpel. Transfer the epithelial layer to a 2 mL tube.
6. Rinse the Petri dish with 750 µL of Tyrode I buffer and transfer to the 2 mL tube containing the epithelial layer.
7. Incubate the tracheal epithelial layer in Tyrode I buffer for 30 min at 37 °C on a shaker at 200 rpm. Cover the tube with aluminum foil to reduce the exposure to direct light.
8. Add 750 µL of cold Tyrode II buffer. Vortex the digested tissue vigorously for 20-30 s. Triturate the homogenate with a syringe attached to an 18 G needle 10 times.  Switch to a 21 G gauge needle and triturate 10-20 more times.
9. Filter the cells through a 100 µm strainer into a 50 mL conical tube. Add 30:1 vol/vol of cold FACS buffer.
10. Spin at 350 x g for 10 min at 4 °C and discard the supernatant. Resuspend the pellet in cold FACS buffer and transfer the suspension to a 12 mm x 75 mm (5 mL) polystyrene tube. Spin again at 350 x g for 10 min at 4 °C and discard the supernatant.
11. Re-suspend the pellet in 100 µL of FACS buffer.
12. Add 1 µL of anti-mouse CD16/32 blocking antibody to block non-specific binding and incubate for 15 min on ice. Do not wash.
13. Add the following antibodies and the respective isotype controls: pacific blue anti-mouse CD45 or rat IgG2a, k (0.25 µg/10⁶ cells in 100 µL volume) and allophycocyanin (APC) anti-mouse EpCAM or rat IgG2b, k (0.5 µg/10⁶ cells in 100 µL volume) monoclonal antibodies. Incubate for 45 min on ice protected from direct light. Add 4.5 mL of cold FACS buffer, mix and spin at 350 x g for 10 min at 4 °C. Discard the FACS buffer and re-suspend the pellet in 300 µL of cold FACS buffer.
14. Add propidium iodide (PI) (5 µg/mL) immediately before flow cytometric sorting.
    NOTE: Brush cells have an irregular shape with several processes that could potentially increase their adherence to filters (Figure 3C). We compared 30 μm to 70 μm and 100 μm filters and found that larger pore filters ensured better yields. In addition, thorough trituration of the cell suspension after papain digestion significantly improves the brush cell yields. We recommend using an 18G needle for initial trituration followed by a smaller bore (21 or 23 G needle) for finer dissociation of cells.

4. Flow Cytometry Gating Strategy

1. Identify cells from debris by forward and side scatter angle. Exclude the doublets using forward scatter height and width and side scatter height and width. The doublets would be the cells that have high width values.
2. Within the single cells, identify the live cells as the population that is PI negative.
3. Within the live single cells, identify the CD45 low to negative cells based on the isotype control.
4. Within the CD45 low/negative cells, identify the EpCAM positive cells that are also eGFP positive (in the FITC channel). This is the population of brush cells (Figure 2A).

Results

This procedure has been successfully implemented to isolate tracheal brush cells for RNA sequencing³. After isolation of the trachea and digestion of the tissue with a 2-step protocol (Figure 1), cells were collected and stained with fluorescently-labeled CD45 and EpCAM after exclusion of dead cells with PI. After gating out doublets based on forward and side scatter characteristics, we defined brush cells as low/negative for CD45, positive for EpCAM and positive for ...

Discussion

We found that a combination of high-dose dispase treatment for 40 min followed by a short papain treatment (30 min) provides an optimal protocol for tracheal digestion and brush cell isolation. This combination both avoids extensive digestion and produces the highest yield of brush cells, compared to alternate protocols.

While lung digestion to extract hematopoietic cells has classically relied on mild digestive enzymes like collagenase IV¹⁵, isolation of epithelial cel...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibodies
Anti-GFP (Polyclonal goat Ig)	Abcam	cat# ab5450
APC anti-mouse CD326 (EpCAM)  (G8.8)	Biolegend	cat#118214
APC Rat IgG2a, k isotype control	Biolegend	cat#400511
DAPI	Biolegend	cat#422801
Donkey anti-goat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Life Technologies/Molecular Probes	cat#A-11055
Normal Goat IgG	R&D Systems	cat#AB-108-C
Pacific Blue anti-mouse CD45 (30F-11)	Biolegend	cat#103126
Pacific Blue Rat IgG2b, k isotype control	Biolegend	cat#400627
TruStain FcX (anti-mouse CD16/32) Antibody	Biolegend	cat#101320
Chemicals, Peptides, and Recombinant Proteins
Dispase	Gibco	cat# 17105041
DNase I	Sigma	cat# 10104159001
HEPES-Tyrode’s Buffer Without Calcium (10 mM HEPES, 135 mM NaCl, 2.8 mM KCl, 1 mM MgCl2, 12 mM NaHCO3, 0.4 mM NaH2PO4, 0.25% BSA, 5.5 mM Glucose. Prepared in 18.2 megohms water and filtered through 0.22 µm filter	Boston BioProducts	cat# PY-912
Tyrode’s Solution (HEPES-Buffered) 140 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2 and 10 mM glucose. Prepared in 18.2 megohms water and filtered through 0.22 µm filter. )	Boston BioProducts	cat# BSS-355
L-Cysteine	Sigma	cat# C7352
Leupeptin trifluoroacetate salt	Sigma	cat# L2023
Papain from papaya latex	Sigma	cat# P3125
Propidium iodide	Sigma	cat# P4170
Experimental Models: Organisms/Strains
ChATBAC-eGFP (B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J)	The Jackson Laboratory	7902
Equipment
LSM 800 with Airyscan confocal system on a Zeiss Axio Observer Z1 Inverted Microscope	Zeiss
LSRFortessa	BD	647465
Disposable equipment
1.5 mL sterile tubes	Thomas Scientific	1157C86
5 mL Poysterene Round-bottom Tube, 12 mm x 75 mm style	Falcon	14-959-1A
50 mL Polypropylene conical tube, 30 mm x 115 mm style	Falcon	352098
Feather Disposable Scalpel no.12	Fisher Scientific	NC9999403
Petri dish, 100 mm x 15 mm Style	Falcon	351029
Sterile cell strainer, 100 μm	Fisherbrand	cat#22363549