Single Nuclei Isolation from Coronary Endarterectomy Tissue of Coronary Artery Bypass Graft Patients

Summary

Coronary endarterectomy tissue samples were processed to isolate single nuclei using an optimized dissociation protocol. The integrity of the isolated nuclei was validated through three complementary approaches: trypan blue exclusion staining to assess membrane integrity, DAPI-based confocal microscopy to confirm nuclear morphology, and flow cytometry to quantify nuclear yield and purity.

Abstract

Atherosclerosis is a chronic, progressive inflammatory condition characterized by the accumulation of plaque in the walls of arteries. During the progression of plaque formation, certain lesions become unstable, referred to as high-risk plaques, and have the potential to cause myocardial infarction (MI). An atherosclerotic plaque comprises smooth muscle cells, endothelial cells, immune cells, fat cells, and necrotic cells, any of which can cause plaque rupture. It is crucial to study cellular heterogeneity within atherosclerotic plaques to better understand the mechanisms underlying plaque instability. We present an easily reproducible protocol for isolating and validating nuclei from human coronary endarterectomy tissue obtained from patients undergoing coronary artery bypass graft (CABG) surgery. All tissues are kept on wet ice in Roswell Park Memorial Institute (RPMI) medium to preserve cellular integrity before harvesting. The tissue samples are finely minced and then gently disrupted with a micro pestle in lysis buffer to lyse cells and release nuclei. The isolated nuclei are then assessed for integrity and yield using the trypan blue exclusion assay in a compound inverted microscope. DAPI staining enables visualization of intact nuclei by confocal microscopy, and flow cytometry provides fluorescence-based quantification to assess nuclear isolation yield and validate the nuclear population. In contrast to traditional methods of isolation via single-cell dissociation, nuclear isolation reduces cellular stress and maintains the native transcriptomic environment, thereby preventing changes in gene expression. The high-quality nuclear preparation obtained through this method is compatible with downstream applications such as single-nuclei RNA sequencing (snRNA-seq).

Introduction

Atherosclerotic cardiovascular disease (ASCVD) is the world's leading cause of death and disability, and despite major therapeutic advances, its burden remains undiminished¹. Atherosclerosis is a chronic inflammatory disease of the arterial wall, driven by complex and dynamic interactions among diverse cell populations that collectively orchestrate the silent progression from early subclinical disease to life-altering cardiovascular (CV) events².

In medium and large arteries, the buildup of cholesterol deposits within macrophages is a hallmark of atherosclerosis³. The atherosclerotic plaque consists of fatty acids, cholesterol, inflammatory cells, calcium, fibrin, smooth muscle cells, endothelial cells, and cellular debris within the subendothelium⁴. Atherosclerotic plaques are classified into two types: low-risk and high-risk. High-risk plaques are identified by their tendency to trigger a subsequent cardiovascular event, typically through mechanisms such as plaque erosion or rupture⁵.

The characteristics of high-risk plaques include large lipid cores, several inflammatory cells, fewer smooth muscle cells, a thin fibrous cap, microcalcification, intraplaque haemorrhage, neo vessels, and a necrotic core⁶. The detection of high-risk plaques is clinically performed using non-invasive and invasive imaging modalities, namely, coronary computed tomography angiography (CCTA) and optical coherence tomography (OCT), respectively^7,8. Despite numerous advancements in cardiovascular imaging, it is still challenging to predict the rupture of atherosclerotic plaque that causes myocardial infarction (MI), and it is not always useful in clinical settings⁹.

Despite decades of research, we still lack reliable circulating biomarkers that can specifically identify high-risk plaques phenotypes, leaving a critical gap in our clinical management of atherosclerosis. This underscores the need for molecular strategies that can characterize these plaques at a more fundamental biological level^7,10. In this context, RNA-based markers have gained considerable attention, as they can capture the intricate transcriptional activity occurring within the plaque microenvironment¹¹. Recent developments in single-cell RNA sequencing (scRNA-seq) have proven particularly powerful, offering an unprecedented resolution to profile individual cells within atherosclerotic plaques and uncover distinct cell populations, gene expression patterns, and molecular pathways that underlie plaque vulnerability^12,13. Despite these developments, little knowledge exists on the regulatory processes, interactions, and functional importance of the various cell types implicated in atherosclerosis development¹⁴.

When atherosclerotic burden is severe and diffuse, surgical revascularization becomes the definitive treatment strategy. Coronary endarterectomy (CE) is a surgical adjunct to coronary artery bypass graft (CABG) surgery in which atherosclerotic plaque is mechanically removed from diffusely diseased coronary arteries to enable bypass grafting and achieve complete myocardial revascularization^15,16. Atherosclerotic plaques and endarterectomy specimens are particularly challenging to work with because they contain abundant necrotic tissue, lipid deposits, fibrin, and calcifications. When these tissues are broken down for analysis, they generate substantial debris that clogs filters and microfluidic systems, making the isolation of viable single cells extremely difficult^17,18. In fact, one carotid plaque study found that viable cells represented less than 1% of all particles after tissue processing; the rest was simply plaque debris¹⁸.

Further compounding this challenge, atherosclerotic plaques exist in a state of chronic metabolic and oxidative stress, where cells undergoing apoptosis, senescence, and other degenerative processes render cell membranes increasingly fragile and susceptible to disruption, making the isolation of viable single cells extremely difficult^18,19,20. The mechanical and enzymatic digestion required to prepare samples for scRNA-seq often damages these already compromised cells, further reducing viability and resulting in poor cell capture rates. This process also tends to selectively lose fragile or rare cell populations, which introduces significant bias into both the transcriptomic data and the cell types represented in the final results. This problem becomes even more pronounced in calcified or heavily diseased plaques, where the harsh processing conditions needed to break down the tissue cause additional damage to cell viability^18,21,22.

In contrast, the nucleus remains structurally intact even under these adverse conditions²³. This is primarily due to the nuclear envelope, a double lipid bilayer reinforced by nuclear lamins (Lamin A/C and Lamin B), which provides mechanical rigidity and resistance to the osmotic, enzymatic, and physical stresses that readily disrupt the plasma membrane^24,25. The nuclear lamina also preserves nuclear morphology even when the surrounding cytoplasm is severely compromised²⁶. This makes single-nucleus RNA sequencing (snRNA-seq) a more practical and reliable alternative to single-cell RNA sequencing (scRNA-seq) for profiling atherosclerotic tissue²⁷. By capturing transcriptional information from intact nuclei rather than whole cells, snRNA-seq circumvents the limitations imposed by poor cell viability and high debris content, enabling more accurate and comprehensive transcriptomic profiling of the plaque microenvironment¹⁸.

Therefore, in this study, we present an optimized nuclei isolation protocol specifically developed for coronary endarterectomy tissue, incorporating non-ionic detergent-based lysis and empirically adjusted lysis duration based on the degree of tissue calcification, to consistently yield high-quality nuclei with preserved structural and RNA integrity suitable for downstream snRNA-seq analysis.

Protocol

The present study was performed with approval and in accordance with the guidelines of the Human Ethical Committee of U. N. Mehta Institute of Cardiology and Research Centre (EC/Approval/26/Cardio/14/12/2024) and Gujarat Biotechnology University (GBU_IHEC/2023/006). Informed consent was obtained from all participants prior to sample collection. The patients undergoing coronary artery bypass graft surgery with coronary endarterectomy were selected for the current study. Following surgical removal, the endarterectomy tissue was immediately placed into serum-free RPMI medium, maintained at 4 °C, and transported to the laboratory within approximately 12 h in a cryovial. See the Table of Materials for details on all materials used in the following protocol.

1. Nuclei isolation

NOTE: Perform all the steps on ice and use sterile materials. Prepare all buffers freshly.

1. Wash the tissue once or twice with serum-free RPMI medium to remove blood stains and fragments. Using sterile forceps, transfer the tissue onto a sterile petri dish and record the weight.
2. Using a surgical blade (no. 10), mince the tissue into small pieces ∼1 mm³ in size.
3. Prepare 1 mL lysis buffer by combining 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 (using stock solutions of 1 M for each component), 0.1% (v/v) Nonidet P-40 in nuclease-free water. Add the components sequentially in the following order: nuclease-free water first, then Tris-HCl, NaCl, MgCl₂, and NP-40 last, to avoid foaming.
   NOTE: The lysis buffer should be prepared fresh on the day of use and kept chilled on ice at all times. Do not freeze.
4. Add chilled 1 mL lysis buffer to the tissue in a 5 mL microcentrifuge tube.
5. Lyse the tissue on ice using a micro pestle by applying a gentle, consistent force and swirling the micro pestle clockwise and anticlockwise alternately. Continue the lysis for approximately 30 min, ensuring the sample remains cold throughout.
   NOTE: Lyse the tissue until it turns turbid. Use a 1000 µL tip to mix gently by pipetting.
6. Filter the solution using a 100 µm pore-size strainer into a 15 mL centrifuge tube to remove debris and tissue chunks.
   NOTE: The filtrate should appear less turbid with visible debris retained on the strainer.
7. Centrifuge the nuclei at 500 x g for 5 min at 4 ˚C using a swinging bucket rotor.
   NOTE: A visible pellet should form at the bottom of the tube.
8. Remove the supernatant using a regular-bore pipette tip without disturbing the pellet.
9. Prepare nuclei wash and resuspension buffer by combining 1% bovine serum albumin (BSA) (using 10% of stock solution), 0.2 U/µL RNase inhibitor (using 20 U/µL stock solution) in 1X Dulbecco's phosphate buffer saline (DPBS). Add the components sequentially in the following order: DPBS first, then BSA, and finally RNase inhibitor.
   NOTE: The buffer should be prepared fresh on the day of use and kept chilled on ice at all times. Do not freeze.
10. Resuspend the pellet in nuclei wash and resuspension buffer. Chill the buffer on ice before use.
11. Assess lysis efficiency and cell viability by staining with 5 µL trypan blue and counting in a hemocytometer.
    NOTE: If viable cells are still present, centrifuge at 500 x g for 5 min at 4 ˚C, then repeat sections 3-10.
12. Filter the solution using a 30 µm pore-size strainer into a 15 mL centrifuge tube to remove debris and clumps.
13. Centrifuge the nuclei at 500 x g for 5 min at 4 ˚C using a swinging bucket rotor.
14. Remove the supernatant using a regular-bore pipette tip, then resuspend the pellet in 1 mL of nuclei wash and resuspension buffer.

2. Characterization of isolated nuclei

1. Trypan blue staining for assessing the integrity of nuclei.
   1. Take 15 µL of the isolated nuclei suspension in a 1.5 mL microcentrifuge tube.
   2. Add 5 µL of trypan blue dye (0.4% w/v) and mix gently by pipetting.
   3. Incubate at room temperature for 5 min to allow proper staining.
   4. Load 10 µL of the stained mixture onto a hemocytometer.
   5. Observe under a compound inverted microscope to examine morphology and count the number of nuclei.
2. Flow cytometry analysis of single nuclei suspension
   1. Take 1 mL of isolated nuclei suspension and centrifuge the nuclei suspension at 500 x g for 5 min at 4 ˚C using a swing bucket rotor.
   2. Remove the supernatant using a regular-bore pipette tip and resuspend the pellet in 200 µL FACS Buffer (1X PBS and 1% BSA).
   3. Divide 200 µL of resuspended nuclei into two aliquots: keep 100 µL as the unstained control and use 100 µL as the stained sample.
   4. Add 1 µL 4', 6-diamidino-2-phenylindole (DAPI) (1 mg/mL stock) to the stained sample to achieve a final concentration of 10 µg/mL. Mix gently and incubate at room temperature in the dark for 5 min prior to flow cytometric analysis.
   5. In the flow cytometer used for this study, use the violet laser at 405 nm, and detect using 448/45 nm. Run the unstained control sample first and acquire a minimum of 10,000 events of the total population.
   6. Set the voltage at 183 V for forward scatter (FSC), 300 V for side scatter (SSC), and 400 V for V450 (DAPI Laser). Set the threshold at 10,000 for FSC.
      NOTE: All instrument voltage and gain settings are constant across samples to maintain consistency.
   7. Plot forward scatter area (FSC-A) versus forward scatter height (FSC-H) to distinguish singlets from doublets and aggregates. Draw a gate around events displaying a linear relationship to retain only singlet nuclei.
   8. Generate a bivariate plot of FSC-A versus side scatter area (SSC-A). Apply a broad gate encompassing the entire scatter population, as isolated nuclei typically exhibit a heterogeneous scatter profile without distinct subpopulations.
   9. Create a 2D density (pseudo color) plot of SSC-A versus Violet 450 nm-area (V450-A). Use the unstained control to define background fluorescence and guide precise gate placement. Identify and gate the distinct DAPI⁺ population corresponding to intact nuclei.
      NOTE: As a single fluorochrome (DAPI) is used, fluorescence compensation is not required.
3. Imaging the isolated nuclei using confocal microscopy
   1. Take 50 µL of nuclei suspension and add 1 µL DAPI (1 mg/mL) to the stained sample to achieve a final concentration of 10 µg/mL, mix gently, and incubate at room temperature for 5 min in the dark prior to confocal imaging.
   2. Mount 5 µL of stained nuclei suspension onto a clean glass slide and cover it with a coverslip.
      NOTE: Avoid fixation to preserve nuclear morphology.
   3. Visualize the nuclei using an inverted confocal microscope. Open the confocal microscope software and observe first in 10X bright field, then switch to 40X bright field.
   4. Acquire confocal images using a laser scanning confocal microscope equipped with a spectral detector and objectives PLAN APO λD 40X (NA 0.95, RI 1.0). Excite DAPI using a 405 nm laser and collect emission between 429-474 nm.
   5. Imaging parameters were as follows:
      1. Detector mode: Multi-channel (GaAsP)
      2. Scanner: Galvano unidirectional
      3. Scan mode: Band
      4. Line averaging: 2X
      5. Resolution: 1024 × 1024 pixels
      6. Dwell time: 2.0 µs
      7. Pinhole size: 47.7 µm
      8. Laser power: 0.1-9.3% (depending on field intensity)
      9. Detector gain: 37.8-45.9%
      10. Zoom: 1.0X
      11. Calibration: 0.43-0.58 µm/pixel
   6. Acquire all images using the AX camera with minimal crosstalk mode under identical optical configurations, and the perfect focus system (PFS) should be active during imaging to maintain z-plane stability.

Results

The human coronary endarterectomy tissue was collected from patients undergoing CABG surgery. The tissue was weighed and processed for nuclei isolation, and further isolated nuclei were confirmed by flow cytometry and confocal microscopy (Figure 1).

Trypan blue assessment of nuclei isolation quality
The tissue kept in RPMI medium was weighed, and it measured 247.8 mg. The tissue was minced, then homogenized in lysis buffer using a micropestle ...

Discussion

An optimized workflow for the isolation of nuclei from human coronary endarterectomy tissue is described in the current protocol. The nuclear isolation protocol described here was specifically designed to overcome the critical limitations of obtaining viable, intact cells from advanced coronary atherosclerotic plaques. Unlike conventional scRNA-seq approaches that rely on harsh enzymatic digestion and mechanical dissociation^21,30, our optimized protocol utilizes ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 μm strainer	Miltenyi Biotech	130-098-463
30 μm strainer	Miltenyi Biotech	130-098-458
Bovine Serum Albumin	HiMedia	TC546-25G
Centrifuge	MPW	NA	https://mpw.pl/en/offer/mpw-352r
Compound Microscope	Nikon	NA	Nikon Eclipse Ts2 inverted microscope (https://www.microscope.healthcare.nikon.com/en_AOM/products/inverted-microscopes/eclipse-ts2)
Confocal Microscope	Nikon	NA	Nikon Ti2-E inverted confocal microscope (https://www.microscope.healthcare.nikon.com/en_AOM/products/inverted-microscopes/eclipse-ti2-series)
Coverslips	HiMedia	BG011C
CRYOCHIL Wide Mouth Specimen Vial	Tarsons	883191
DAPI	Sigma	D9542-5MG
DPBS	Gibco (Thermofischer)	10010-023
Centrifuge Tube (15 mL)	Tarsons	546021
Flow cytometer	BD Biosciences	663029	BD FACSLyric (3L12C Instrument CEIVD)
Hemocytometer	Axibio	CCB 100
Microcentrifuge Tube (1.5 mL)	Tarsons	500010C
Microcentrifuge Tube (5 mL)	Tarsons	500050
MgCl2	Sigma	M1028-100ML
Micro pestle	Tarsons	160020
NaCl	Sigma	S9888-500G
NP-40	HiMedia	MB143-100ML
Nuclease free water	Gibco (Thermofischer)	W4502-1L
RNase inhibitor	Sigma	333540201
RPMI 1640	Gibco (Thermofischer)	11875093
Slide	Riviera	72910135
Tris-HCl	Sigma	T2194-100ML
Trypan blue	Sigma	302643-25G