Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Summary

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.

Abstract

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (Mφ/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

Introduction

Atherosclerosis is a chronic inflammatory disease associated with risk factors such as high blood pressure, hyperlipidemia, and hemodynamics¹. Carotid artery bifurcations are prone to hemodynamic changes and lead to carotid plaque formation. The clinical presentation of carotid atherosclerosis can be acute such as stroke and transient cerebral ischemia, or chronic such as recurrent transient cerebral ischemia and vascular dementia². Mechanically, carotid plaque is the outcome of the interaction between different vessel wall cells and various blood cells under pathological conditions. Therefore, revealing the single-cell atlas of carotid vessels under physiological and pathological conditions is particularly important for preventing and treating carotid plaque development.

Single-cell RNA sequencing is one of the most powerful technologies of biological research because of its ultrahigh resolution and detection of cell heterogeneity from the same cell type of organisms^3,4. Researchers have used single-cell RNA sequencing to conduct research in many fields, such as cardiovascular disease⁵ and cancer⁶. However, quickly and accurately separating tissues into single cells is still one of the main challenges. Enzymatic dissociation is a commonly used method that dominantly includes collagenase, papain, trypsin, DNase, and hyaluronidase. Specifically, collagenases are the major enzyme species for single-cell digestion, mainly hydrolyzing collagen components in connective tissues. Different collagenase types are applicable for the dissociation of different tissues, such as the mammary gland⁷, glomerulus⁸, iridocorneal angle⁹, knee joint¹⁰, aorta¹¹, and lung¹². Due to the unique physiological properties of different tissues, dissociation using the same method may cause many troubles in acquiring single cells, such as low cell viability, low cell number, and large cell debris. Therefore, the invention of digestion methods for different tissues is essential for preparing high-quality single-cell suspensions.

This protocol aims to develop a two-step cell digestion method to prepare a single-cell suspension of the carotid artery of wild-type mice. According to the characteristics of the carotid artery, we combined collagenase/DNase with trypsin to obtain a high-quality single-cell suspension of the mouse carotid artery because collagenase can hydrolyze collagen of the carotid tissues, which was further digested by trypsin into a single-cell suspension. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, four cell types, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (Mφ/DCs), were identified in normal mouse carotid arteries after digestion. The advantages of this protocol are that: 1) multiple cell types in the carotid artery can be identified, 2) cell viability is well preserved, and 3) it can be easily repeated without special equipment. This protocol is suitable for researchers who are interested in studying single-cell multiomics of the mouse carotid arteries. This protocol may also be helpful in the dissociation of other blood vessels with appropriate modifications.

Protocol

All animal procedures described below were approved by the Institutional Animal Care and Use Committee of Soochow University.

1. Reagents and materials preparation

1. Utilize 1x PBS without calcium and magnesium and 2.5 U/mL heparin sodium salt to prepare the perfusion solution. Store at 4 °C, and precool on ice when used.
2. Prepare dissociation reagent A that contains 125 CDU/mL collagenase II and 60 U/mL DNase I by diluting 1250 CDU/mL collagenase II and 3000 U/mL DNase I with HBSS. Store at 4 °C until use.
3. Prepare dissociation reagent B that contains a final concentration of 0.12% trypsin by diluting 1 mL of 0.25% EDTA-free trypsin (without phenol red) into 1 mL of 1x PBS. Store at 4 °C until use.
4. Prepare 10 mL of 1.5% FBS and 10 mL of 5% FBS in 1x PBS. Use 1.5% FBS to pool carotid arteries and keep on ice before use. Use 5% FBS to neutralize digestion and store at room temperature (RT).
5. Get the experimental materials, including 1 mL syringes, 20 mL syringes with 26-G needles, six-well cell culture plates, 1.5 mL centrifuge tubes, 40 µm sterile cell strainers, and ice containers.

2. Equipment preparation

1. Sterilize all the surgical equipment, including a stereoscopic microscope, dissecting scissors, micro-dissecting scissors, and fine-tip forceps.
2. Turn on the automated cell counter and maintain it at RT.
3. Turn on the water bath to 37 °C in advance.
4. Precool the microcentrifuge to 4 °C before use.

3. Isolation of the mouse carotid artery

1. Anesthetize the mouse using 1.25% tribromoethanol by intraperitoneal injection (0.24 mL for every 10 g of body weight) and flip the mouse to check if there is a righting reflex after 3 min. Then, euthanize the mouse by cervical dislocation and lay it on its back. Spray the mouse's skin with 75% alcohol.
2. Use sterilized dissecting scissors to open up the skin and expose the chest cavity. Cut open the diaphragm.
3. Continue cutting upward to the lower jaw and carefully remove excess connective tissues and fat, exposing the carotid artery.
4. Cut the inferior vena cava of the mouse to make blood flow out of the closed circulation.
5. Inject 20 mL of prechilled perfusion solution into the left ventricle with a syringe slowly and continuously.
   NOTE: If perfusion is successful, blood-rich organs such as the liver, spleen, and kidneys will turn gray-white.
6. Peel off all the fat and connective tissues around the carotid artery with micro-dissecting scissors under a stereoscopic microscope.
   NOTE: This step should be done slowly and carefully to prevent damage to the carotid artery.
7. Isolate the carotid artery from the mouse, wash with 1x PBS to flush the blood again, and transfer to six-well plates containing 1.5% FBS on ice.

4. Digestion of the carotid artery into single-cell suspension

1. Use micro-dissecting scissors to dissect the carotid artery longitudinally and lay flat in another 6-well cell culture plate containing 1 mL of 1.5% FBS on ice.
2. Flush the intima carefully with 1.5% FBS to remove the residual blood.
   NOTE: Flushing should be gentle and slow to avoid injuring endothelial cells.
3. Cut each carotid artery into approximately 2 mm² tissue pieces using microdissecting scissors in 1.5% FBS.
4. Transfer these tissue pieces into a 1.5 mL centrifuge tube with 1 mL wide bore tips, centrifuge at 400 x g for 5 min at 4 °C to sink the tissues to the bottom.
5. Discard the supernatant and resuspend the tissues in 500 µL of dissociation reagent A.
6. Place the tube in a 37 °C water bath for 1 h. Pipette gently with a 1 mL pipette every 10 min.
7. Add 500 µL of 5% FBS into the tube and mix well with a 1 mL pipette.
8. Filter the cell suspension through a 40 µm sterile cell strainer into a new 1.5 mL centrifuge tube and centrifuge at 400 x g for 5 min at 4 °C.
9. Remove the supernatant carefully and resuspend the cell pellet in 200 µL of dissociation reagent B.
10. Place in a 37 °C water bath and digest for another 5 min. Gently pipette every 2 min during digestion to fully dissociate into a single-cell suspension.
11. Use 200 µL of 5% FBS to terminate the digestion reaction, and centrifuge at 400 x g for 5 min at 4 °C.
12. Discard the supernatant and resuspend the cell pellet in 100 µL of 1x PBS on ice.

5. Cell suspension examination and single-cell RNA sequencing

1. Add 10 µL of AO/PI solution into 10 µL of single-cell suspension and gently mix with a 20 µL pipette.
2. Pipette 20 µL of the mixture into the chamber slide and wait for 1 min.
3. Load the slide into the automated cell counter instrument.
4. Select the AO/PI Viability assay, then wait for the quality report.
5. Use a single-cell 3ʹ reagent kit to generate single-cell gel beads in the emulsion on a single-cell controller and amplify the barcoded cDNA in a thermal cycler following the manufacturer's protocol.
   NOTE: Here, Chromium Single Cell 3ʹReagent Kits v3 was used.
6. Perform sequencing on a sequencer with a paired-end 150 bp (PE150) reading strategy. Use a software application (Cell Ranger) to convert raw base call files to fastq files using the mkfastq pipeline. Then, use the Count Pipeline of Cell Ranger for performing alignment, filtering, barcode counting, and unique molecular identifier (UMI) counting to generate feature-barcode matrices¹³.
7. Perform data dimensionality reduction and visualization with the R package Seurat¹⁴.
   1. First, the Read10X() function of Seurat reads in the output of the Cell Ranger pipeline and then uses the count matrix to create a Seurat object. Then, filter out the cells with expression of <200 or >4000 genes or a mitochondrial gene ratio that was more than 10% by the subset() function of Seurat.
   2. Use NormalizeData() and FindVariableFeatures() to normalize the data and identify 2000 highly variable features, respectively.
   3. Perform principal component analysis (PCA) on the scaled data, and cluster the cells with dims = 30 and resolution = 0.5. Finally, use the RunUMAP() function to visualize the single-cell datasets and FindAllMarkers() to find each cluster biomarker.

Representative Results

This protocol describes a two-step cell digestion method for preparing a single-cell suspension of mouse carotid arteries (Figure 1). This two-step cell digestion method combines collagenase/DNase with trypsin to effectively dissociate the mouse carotid vascular wall to obtain high-quality single-cell suspensions for single-cell sequencing. After dissociation, the cell concentration and cell viability were calculated by an automated cell counter. The bright field showed the morphology of carotid vascular cells after digestion, and AOPI dual-fluorescence staining simultaneously detected cell viability (Figure 2A,B). Notably, compared with using dissociation reagent A alone, carotid artery tissue can be more thoroughly dissociated when combined with dissociation reagent B. We further found a viable cell count of nine arteries, and the average cell diameter satisfied the requirements of single-cell sequencing after two-step cell digestion (Figure 2C). Meanwhile, a total of 176,000 single cells were collected from nine left carotids, and most vascular vessels were dissociated into single cells with high viability (Figure 2C).

After alignment, filtering, barcode counting, and UMI counting, the raw data of single-cell sequencing were analyzed with the R package Seurat. The distribution of genes per cell, UMI per cell, and mitochondrial reads per cell are shown in violin plots (Figure 3A-C). Notably, a median of ~2500 transcripts per cell was detected in each carotid artery cell. After removing the low-quality cells, 14,580 cells were detected and used for downstream single-cell RNA-seq analysis. Using 2000 variable genes with similar profiles, unsupervised Seurat-based clustering showed four main cell types, including VSMCs (74.0%), fibroblasts (16.5%), ECs (6.5%), and Mφ/DCs (3.0%), in normal mouse carotid arteries after digestion (Figure 3D-F).

Figure 1: Schematic diagram of mouse carotid artery digestion. After the mouse carotid artery was isolated and cut, 500 µL of dissociation reagent A was added to dissociate the mouse carotid artery. When the cell suspension was filtered and centrifuged, 200 µL of dissociation reagent B was added for further dissociation to obtain a single-cell suspension. Please click here to view a larger version of this figure.

Figure 2: Single-cell suspension preparation from mouse carotid artery. (A) The cell morphology and cell viability of carotid artery cells after 1 h digestion with dissociation reagent A are shown in the bright field view and AOPI staining. Nucleated cells stained with green fluorescence are living cells, while those stained with red fluorescence are dead cells. Scale bar = 100 µm. (B) The cell morphology and cell viability of carotid artery cells after two-step cell digestion are shown in the bright field view and AOPI staining. Nucleated cells stained with green fluorescence are living cells, while those stained with red fluorescence are dead cells. Scale bar = 100 µm. (C) The cell viability, total cell count, viable cell count, and average cell diameter were measured after two-step cell digestion. Please click here to view a larger version of this figure.

Figure 3: Standard quality control (QC) metrics for scRNA-seq and the cell landscape of the carotid artery. (A) Violin plots showing the distribution of genes per cell (nFeature RNA), (B) UMI per cell (nCount_RNA), and (C) mitochondrial reads per cell (percent mt) for the scRNA-seq data. (D) Uniform manifold approximation and projection (UMAP) plot showing the four identified cell types of the carotid artery. (E) Dot plot showing the markers defining each type of cell cluster in panel D. The size of each circle denotes the cell proportion within the group expressing each transcript. The blue dots indicate highly expressed genes and the gray dots indicate genes with low expression. (F) Stacked bar plot displaying the relative abundance of the four cell types detected by scRNA-seq of wild-type mice (WT). Please click here to view a larger version of this figure.

Discussion

Here we provide a detailed protocol for the preparation of a high-quality single-cell suspension from the carotid artery of wild-type mice, in which a two-step digestion method integrating the digestion process of collagenase/DNase and trypsin was constructed. After the quality check of the single-cell suspension, we found that it satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. Moreover, a variety of cell types in the carotid artery, including VSMCs, fibroblasts, ECs, and Mφ/DCs, were successfully detected after single-cell data processing.

Current methods for preparing carotid single-cell suspensions are still scarce. Depuydt et al. obtained a single-cell suspension from human carotid plaques by combining collagenase, DNase, Human Albumin Fraction V, and Flavopiridol at 37 °C for 30 min¹⁵. After fluorescence-activated cell sorting, they identified 14 distinct cell populations, including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells¹⁵. Furthermore, researchers digested rat common carotid arteries using 0.25% trypsin-EDTA and 0.1% collagenase at 37 °C and successfully performed single-cell RNA sequencing¹⁶. Five cell types, including VSMCs, fibroblasts, ECs, transitional cells, and macrophages, were identified in both normal and experimental carotid arteries¹⁶. In addition, a protocol for obtaining EC-enriched single-cell preparations of the carotid artery has also been established¹⁷. After completing luminal enzymatic digestion, carotid artery flushing will obtain enough single-cell pellets for scRNAseq and scATACseq¹⁷. To flexibly control the digestion time, we developed a two-step digestion method for mouse carotid arteries, separating the steps of tissue dissociation and single-cell suspension preparation. The process starts with 125 CDU/mL collagenase II and 60 U/mL DNase I for 1 h, followed by EDTA-free trypsin for 5 min. Significantly, we can appropriately modify any step of the method for the digestion of other blood vessel tissues based on the characteristics of blood vessels.

There are some limitations of the described method. First, we do not know whether this method can be used for primary cell culture in vitro. Since the carotid artery wall is thinner and has fewer cells, how to culture and expand in vitro is challenging. Second, unlike other digestion methods, this protocol requires gentle pipetting to dissociate cells during digestion. A shaking water bath may also result in a good-quality single-cell suspension. Third, since our method is applicable to normal carotid arteries, whether it is suitable for the digestion of carotid plaque remains to be studied.

In conclusion, we designed a two-step digestion method to obtain a high-quality single-cell suspension from the carotid artery of wild-type mice. This protocol may be used to prepare single-cell suspensions for other vessels with minor modifications, making it very helpful in cardiovascular research.

Materials

Name	Company	Catalog Number	Comments
0.25% EDTA-free trypsin	Beyotime	C0205	Dilute 1 mL of 0.25% EDTA-free trypsin into 1 mL of 1x PBS.
0.9% NaCl saline solution	Beyotime	ST341	Dilute the heparin sodium solution into a final concentration of 10 mg/mL
1 mL syringes	SKJYLEAN	sk-r009	To perform cardiac perfusion
1.5 mL centrifuge tubes	KIRGEN	KG2211W	To centrifuge the tissue piece and cell suspension
20 mL syringes	SKJYLEAN	sk-r013	To perform cardiac perfusion
40 µm cell strainer	JETBIOFIL	css010040	To filter undigested tissue fragments
AO/PI kit	Hengrui Biological	RE010212	To identify whether the cell is alive or dead
Automated cell counter	Countstar	Mira FL	To analyze the cell morphology and cell viability of digested carotid vascular cells
Cell Ranger software	10× Genomics	3.0.2	To process Chromium single-cell RNA-seq output and perform clustering and gene expression analysis
Chromium Single Cell 3'Reagent Kits v3	10× Genomics	1000075	To prepare single-cell RNA-seq libraries of single-cell suspension
Collagenase II	Sigma-Aldrich	C6885	Dilute with HBSS to a final concentration of 125 CDU/mL
Deoxyribonuclease I	Worthington	LS002140	Dilute with HBSS to a final concentration of 60 U/mL
Fetal bovine serum	HyClone	SH30088.03	Termination of the digestion reaction
Hank's balanced salt solution	Gibco	14175095	Store at the room temperture
Heparin sodium salt	Solarbio Life Science	H8060	Dilute with 0.9% NaCl to a final concentration of 10 mg/mL
Microcentrifuge	Thermo Fisher Scientific	75002560	Applied for spining down the tissues and cell pellets
NovaSeq 6000	Illumina	N/A	Sequencer
Phosphate-buffered saline	Solarbio Life Science	P1000	Used for cardiac perfusion and resuspension of cells
Seurat package- R	Satija Lab	3.1.2	To performed dimensionality reduction, visualization, and analysis of scRNA-sequencing data
Six-well cell culture plates	NEST	703002	Place the vascular tissue
Water bath	Jinghong	DK-S22	Keep the digestion temperature at 37 °C