Fibroblasts are dynamic cells of mesenchymal origin that regulate tissue homeostasis, extracellular matrix production, and acute wound healing. Fibroblasts respond to tissue injury and inflammation by differentiating into myofibroblasts and secreting extracellular matrix proteins. Fibroblasts are the principal mediators of the fibrotic response in all tissues and organs. Adult primary fibroblasts represent an essential tool for in vitro studies. Although they lack surface markers, fibroblasts are relatively easy to obtain and culture; primary fibroblasts are sensitive heterogeneous cell subpopulations with limited expansion potential and increased differentiation capacity. Adult primary fibroblasts fail to maintain an undifferentiated state ex vivo for long periods and quickly differentiate into myofibroblasts in culture, which necessitates the utilization of these cells either directly after isolation or after a few passages. Herein, we describe a detailed protocol for enzymatic isolation of primary cardiac fibroblasts from adult mouse hearts and their culture and expansion in a serum-containing culture medium. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol : Preparation of primary adult cardiac fibroblasts for culture and single-cell analysis

Heart failure is caused by pathological myocardial remodeling and fibrosis characterized by excessive deposition of extracellular matrix (ECM). Fibrosis is initiated as a wound repair mechanism, but the uncontrolled dynamics of this process lead to tissue scarring and organ malfunction (Senior, 2022). Fibrosis involves multiple cell types and differentiation pathways (Travers et al., 2016). In the heart, cardiac fibroblasts (CFs) are the primary source of ECM deposition and tissue fibrosis (Fan et al., 2012; Frangogiannis, 2016; Khalil et al., 2019), although other cell types are known to generate ECM components (Howard et al., 1976; Kusuma et al., 2012; Lundgren et al., 1988; Schram et al., 2010). In response to injury, CFs differentiate into activated fibroblasts, also known as myofibroblasts (MFs), which increasingly produce and secrete ECM components and acquire contractile activity through induction of genes such as Acta2 (α-smooth muscle actin [αSMA]), which allows these cells to remodel the ECM microenvironment or scar after myocardial infarction (Hinz, 2010). Thus, manipulating myofibroblast activity is an attractive therapeutic approach in adult fibrotic disease states, including heart failure (Asano, 2010; Hinz, 2010; Leask, 2010).

Although fibroblasts play a critical role in the disease, the field has lacked an in-depth understanding of this cell type differentiation stages and its activation dynamics, primarily due to the lack of reliable surface markers which allows fibroblast isolation. Recently, multiple genetic lineage-tracing mouse models were made to dissect these cells and determine their phenotypic and functional characteristics (Acharya et al., 2011; Kanisicak et al., 2016; Smith et al., 2011; Tallquist & Molkentin, 2017).

Most previous experimental approaches, such as cell lineage tracing, flow cytometry, and bulk RNA-Seq, tackled the fibroblast diversity assuming the identification of a single fibroblast-specific gene. It is important to emphasize that no exclusive single fibroblast-specific marker can currently be used for primary fibroblast isolation. Instead, a combination of markers, including periostin (Postn) (Kanisicak et al., 2016), transcription factor 21 (Tcf21) (Acharya et al., 2011), and platelet-derived growth factor alpha Pdgfrα (Ivey et al., 2019) are utilized to identify tissue-resident fibroblasts (Plikus et al., 2021). Important to consider that the fibroblast population is heterogenous given their differentiation potential. Therefore, stage-specific lineage tracing, mechanistic analysis, and mRNA expression profiling must be applied to characterize fibroblast dynamics and differentiated states in the heart after injury. Single-cell RNA-Seq technology helps explore the diversity of individual cells in a tissue and enables an unbiased definition of cardiac fibroblast subpopulations through their cell-specific transcriptomic signatures (Marín-Sedeño et al., 2021). Herein, we describe a convenient and reproducible protocol to isolate adult fibroblasts from the mouse heart. The isolated interstitial cells can be subsequently utilized for single-cell RNA-Seq (viability must be over 80%) or primary cell culture. We utilize the ventricular fibroblasts during these experiments, although atrial fibroblasts can be isolated using the same protocol. Based on our experience, due to the spontaneous differentiation, it is advisable to utilize the explanted cultured adult fibroblasts within three passages.

NOTE : The fibroblast isolation procedure is performed under a suitable biological safety hood when the cells will be utilized in culture. All solutions and dissection tools used in heart sample collection and processing must be sterile.

NOTE : Fibroblast cultures are maintained in a humidified 37°C, 5% CO₂ incubator.

NOTE : All protocols involving animals must be reviewed and approved by the appropriate Animal Care and Use Committee and must follow regulations for the care and use of laboratory animals.

In this protocol, we describe the procedure for enzymatic digestion of isolated mouse heart tissue to obtain cardiac interstitial cell populations for applications such as single-cell analysis and fibroblast culture. In addition, this protocol is utilized to obtain fibroblasts used in downstream applications such as fibroblast activation, wound healing, scratch assays, drug testing, and RNA sequencing.

Materials

• Freshly isolated mouse heart tissues

• Brilliant Violet 421 anti-mouse CD31 [390] (BioLegend, cat. no. 102424)

• PE anti-mouse CD45 Antibody Biolegend (BioLegend, cat. no. 103106)

• Calcein-AM (Biolegend, cat. no. 425201)

• Zombie NIR Fixable Viability Kit (Biolegend, cat. no. 423105)

• TruStain FcX PLUS (anti-mouse CD16/32) (Biolegend, cat. no. 156604)

• DMEM (Thermo Scientific, cat. no. 88287)

• Fetal bovine serum (FBS, Gibco, cat. no. 26140097)

• Magnesium chloride hexahydrate (Thermo Scientific, cat. no. 413415000)

• Phosphate-buffered saline (PBS, Gibco, cat. no. 20012-027)

• Penicillin–streptomycin (Fisher Scientific, cat. no. 30-002-CI)

• 2 mg/ml collagenase type IV (Worthington, 270 U/mg, cat. no. LS004188)

• 1.2 U/ml Dispase II (Sigma, 0.5 U/mg, cat. no. D4693)

• 40-µm sterile cell strainers (Corning, cat. no. 21008-949-CS)

• 5-ml Round-bottom test tubes with cell strainer (Corning, cat. no. 352235)

• 100-mm plastic cell culture dishes

• Individually wrapped sterile serological pipettes (5, 10, and 25 ml)

• 15-ml and 50-ml sterile conical tubes

• Surgical scissors and tweezers

• Micropipette and pipette tips (1000 μl)

• Humidified, 37°C, 5% CO₂ cell culture incubator

• 37°C water bath and temperature-adjustable incubating rocker.

• Rolling rocker (DLAB Scientific Inc, cat. no. SK-R30S-E/SK)

• Countess II (Thermo Fisher, AMQAX1000)

• Variable speed mini pump (VWR Scientific, cat. no. 54856-070)

• DNase I (type IIS) (Sigma-Aldrich, cat. no. D4513-VL)

Buffers

• Perfusion buffer: 1× PBS+ 2% FBS. Prepare 50 ml/mouse.
• Digestion buffer: 2 mg/mL collagenase type IV, 1.2 U/mL Dispase II in DMEM + 1% FBS (low serum) + 1% penicillin–streptomycin. Prepare 7 ml/heart.
• Washing buffer: 1× PBS + 1% FBS
• Staining Buffer/FACS buffer: 1× PBS + 2% serum + 1% BSA + 2mM EDTA
• Collection Buffer: DMEM + 2% FBS. Prepare 2 ml/sample.

Tissue collection and cleaning

1.Sacrifice the adult mice according to approved animal protocols, open the chest to expose the heart, and displace the organs to locate the vena cava.

2.Quickly perfuse the heart with ice-cold perfusion buffer by inserting a 25G needle into the left ventricle through the apex.

3.Once perfusion flow is initiated, cut the vena cava for faster perfusion.

4.Excise the heart and rinse twice in ice-cold perfusion buffer.

5.Weigh the heart (atria and ventricles), then remove the atria and weigh and isolate the ventricles (in our experiments, we usually utilize ventricular fibroblasts). Tissue collection is maintained on ice throughout the procedure. Immediately proceed to the next step.

Tissue digestion

6.Mince the ventricles into small pieces using sterile micro scissors (Fig. 1). Add 50 μl digestion buffer to facilitate mincing.

Background Information

Fibrosis is a complex cause of global morbidity since it is associated with multiple diseases such as idiopathic pulmonary fibrosis (IPF), chronic kidney disease, nonalcoholic fatty liver, cystic fibrosis, muscular dystrophy, and heart failure (Asano, 2010; Gourdie et al., 2016). Fibroblasts are mesenchymal cells within many organs, contributing to the generation and turnover of the ECM. Fibroblast activation stages range from quiescent to activated (myofibroblasts) and include yet-to-be-defined subtypes with unknown expression markers (Tallquist & Molkentin, 2017). Given their biological significance in tissue homeostasis and disease, it is essential to investigate levels of fibroblast heterogeneity intra- and inter-organ. Understanding the diversity of fibroblast subpopulations and their anatomical localization, activation, and differentiation patterns shall provide a foundation for their role in physiological and pathological processes. In addition to fibroblast heterogeneity, the complexity of fibrosis-related pathogenesis stems back to the multiple altered signaling pathways and the various cell types involved in the progression of the disease. This complication reflects the lack of current antifibrotic drug efficacy. Although multiple antifibrotic drugs that target different pathways have been developed, most of these are discontinued due to their adverse side effects (Senior, 2022).

An essential step in combating fibrosis pathogenesis and developing precision therapeutics starts with fibroblast reprogramming. Therefore, primary cardiac fibroblast cultures are crucial in developing in vitro and ex vivo disease models. Freshly isolated fibroblast subpopulations retain their activation state, which helps reveal their gene expression signatures and physiological functions. In addition, fibroblast cultures are essential to understand fibroblast differentiation stages and crucial to investigate signaling pathways amenable to manipulation and novel drug efficacy (Senior, 2022; Zhao et al., 2022).

We have previously demonstrated the ability to isolate, culture, and manipulate cardiac fibroblasts in culture. We have also investigated the gene expression signatures of fibroblast-specific knockouts of the TGFβ signaling pathway in pressure-overload-induced heart disease. We previously used primary fibroblast cultures to investigate fibroblast to myofibroblast differentiation, the secretome signature, and myofibroblast contractile activity (gel contraction assays) (Fu et al., 2018; Kanisicak et al., 2016; Khalil et al., 2017; Khalil et al., 2019).

Critical Parameters

In this protocol, we have described a simple and easily reproducible method to isolate the interstitial cardiac populations that can be utilized in different downstream applications. Our described protocol can be followed to obtain cardiac fibroblast from human heart samples. We recommend following the timeline of processing, digestion, and incubation of the tissues as described precisely for an optimal yield; refer to Table 1 for troubleshooting tips. Heart samples should be processed as soon as they are isolated. Heart perfusion should be performed with an ice-cold perfusion buffer. Tissue cleaning and mincing should be done on ice under laminar flow. 120-150 mg of heart tissues are typically digested with 2 ml of digestion buffer. If the heart weights are significantly different across the animals, the volume of the digestion buffer should be adjusted accordingly. More extended processing and digestion time leads to lower cell viability. We observed that the CD45-positive population was particularly delicate to this isolation protocol; a maximum of 70% viability rate was achieved in our hands. Time management is critical during heart collection and mincing since this step is the most time-consuming part of the protocol. Therefore, all hearts within an experiment must be cleaned, minced, and digested comparably. RBC lysis is a critical step for FAC-sorting experiments; after lysis, the interstitial cells should be completely devoid of any red color (an indication of RBC lysis). If the pellets look red, the lysis protocol should be repeated. RBCs interfere with the number of cells identified during FACS and will minimize the yield and increase the sorting time. Avoid using increased EDTA; in our hands, 2 mM is very well tolerated by the cells. We have successfully performed the tissue processing, FACS, and single-cell library preparation in one day; the whole procedure is achieved within an 8-hr duration.

Troubleshooting

A troubleshooting guide detailing the most common problems, possible causes, and solutions is provided in Table 1.

Understanding Results

This protocol isolates the total cardiac interstitial cell populations from digested heart tissues. We combined enzymatic tissue digestion and selective adhesion to obtain an enriched fibroblast population in culture. This procedure is reliable and efficient for routine primary culture experiments. The combination of enzymatic digestion and FAC sorting allows the isolation of the pure endothelial cell population (CD31+), myeloid cell population (CD45+), and CD31/CD45 double-negative cell population, which is enriched with cardiac fibroblasts. This is an unbiased approach to isolating the whole fibroblast population; however, the CD31/CD45 double-negative fraction includes nonfibroblast cells such as smooth muscle cells. In our hands, bioinformatic analysis of CD31/CD45-double negative fraction after single-cell RNAseq revealed a distinct cluster of smooth muscle cells that expresses Myh11 and Acta2 with minimal expression of known fibroblast markers.

Given that no exclusive, fibroblast-specific surface marker exists, this fibroblast isolation protocol and protocols presented by other authors encompass limitations regarding the fibroblast population purity. We highly recommended FAC sorting followed by consistent purity checking of the isolated cells of the CD31/CD45 double-negative population. Additional characterization, such as semi-quantitative real-time PCR and immunostaining, is critical to exclude and estimate the percentages of contaminating populations in every experiment. This is particularly crucial for obtaining reproducible results when utilizing the isolated fibroblast population to manipulate differentiation inhibition and drug testing experiments. In addition, it is crucial to utilize a combination of fibroblast markers to precisely characterize the fibroblast activation state. The most acceptable markers are transcription factor 21 (Tcf21), platelet-derived growth factor alpha (Pdgfra), periostin (Postn), collagen 1 (Col1a1), collagen 3 (Col3a1), Fibronectin (fn), and Tenascin (Tnc). Alternatively, lineage tracing combined with a reporter marker can be used to overcome the problem of contaminating interstitial cell populations. However, no single lineage-tracing mouse model labels all the cardiac resident and activated fibroblast subpopulations before and after heart injury.

This study strictly followed the recommendations guided by the Animal Care and Veterinary Service (ACVS) at the University of Ottawa. The University of Ottawa Institutional ACVS approved the protocol number: BMIe-3516-R2.

Time Considerations

The procedure timeline estimation is detailed below:

Heart perfusion and isolation (approx. 7-10 min per mouse)

Heart digestion (approx. 95 min)

Red blood cell removal (approx. 30 min)

Interstitial cell staining (in the dark, approx. 60 min)

Fluorescence-Activated Cell Sorting (FACS) 60 min per sample (depending on the flow cytometry apparatus utilized)

Fibroblast cell cultures (3 to 10 days)

Acknowledgments

Hadi Khalil was supported by the Swiss National Science Foundation SPARK grant (project Number: CRSK-3_190487). The Authors would like to acknowledge the unlimited support of Prof. Mona Nemer at every level throughout this work. This work was supported by grants from the CIHR to Mona Nemer (CIHR Foundation Scheme Grant Number 353388).

Author Contributions

Abir Almazloum : Data curation, writing—review and editing; Hadi Khalil : Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, resources, supervision, validation, visualization, writing—original draft, writing - review and editing.

Conflict of Interest

The authors do not disclose any conflict of interest.

Data Availability Statement

Data sharing does not apply to this article as no new data were created or analyzed in this study.

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