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Here we describe protocols to culture adult zebrafish hearts as explants, and to study regeneration of epicardial tissue ex vivo. Uninjured or injured adult hearts are excised, washed, and cultured in an incubator with gentle agitation. Preparation of heart explants is accomplished within 2 hours, and explants can be maintained in culture for 30 days or longer. Dynamic behaviors of epicardial cells are monitored through live imaging of fluorescent transgenes in the explant, using stereofluorescence microscopy. We also describe ex vivo procedures for genetic ablation of the epicardium, cell proliferation assays, tissue grafts, and bead grafts. Basic cell culture and surgical skills are required to carry out these protocols. As opposed to existing protocols for culturing isolated zebrafish epicardial cells on matrices, procedures described here maintain epicardial cells on an intact cardiac surface, better enabling in vivo cell behaviors. Our protocols complement and extend in vivo studies of heart regeneration.
Zebrafish provide a valuable model system to study regeneration of cardiac tissue, as injured hearts can regenerate large portions of lost muscle through the proliferation of spared cardiomyocytes. Non-muscle supporting cells like epicardium, endocardium, vasculature, nerves, and inflammatory cells play critical roles during heart regeneration1-6. The epicardium, a mesothelial layer covering the surface of all vertebrate hearts, is required for normal muscle regeneration after cardiac injury in zebrafish4, 5, 7-10. Studies in zebrafish and mammals have led to many proposed roles for the epicardium: in vascularization of new muscle, as a source of mitogens, as a contributor to muscle survival, and as a cellular target for direct reprogramming strategies11-17.
Tissue regeneration is by definition an in vivo phenomenon. However, regeneration studies in adult zebrafish suffer from their long animal generation times (~3 months) and the dearth of molecular genetic tools for inducibly manipulating adult regenerative events. Moreover, the heart as an internal organ is technically challenging to access for live imaging of regenerative events. To address these challenges, we have developed an explant system that enables culturing of adult hearts for several weeks, while maintaining much of the morphology and contractility of the heart7. With this method and molecular genetic tools for genetic ablation and live monitoring of epicardial cells, we defined and dissected regenerative capacity of adult zebrafish epicardial tissue in a recent research report7. Additionally, this explant culture system can be adapted for tissue grafting and chemical screening approaches. Here, we describe in detail this protocol for explant culture, and important applications.
Our original methods for culturing heart explants incubated extracted hearts for 7 days with Liebovit's L-15 medium supplemented with 10% fetal calf serum and antibiotics10. With this method, heart explants tended to attach to the dish bottom, altering ventricular shape and challenging live visualization of epicardial cell behaviors on the cardiac surface. Spontaneous internal infarcts were formed within ~7 days inside the explants, suggesting poorly maintained cardiac muscles. An additional protocol exists to culture isolated epicardial cells from adult zebrafish on a fibrin matrix18. With this method, epicardial cells migrate into the fibrin gel from explanted chunks of ventricle apex. This method is advantageous for cellular and molecular analysis; however it is a more artificial environment than the cardiac surface. Several other methods exist for culturing mammalian cardiac slices for weeks while maintaining contractile activity19-22; yet, these systems are not applicable for studies of the epicardium.
To visualize and manipulate epicardial cells on an intact cardiac surface for extended imaging experiments, we refined our explant culture protocol with the following specific modifications. a) Instead of non-carbonate buffered L-15 medium, we used carbonate buffered DMEM medium, which is ideal for a CO2 incubator. We also tested DMEM/F12 and IMDM medium without noticing apparent differences. We ultimately chose to use DMEM medium, as it is inexpensive and sufficient for the experiments described here. b) We added 1% non-essential amino acids (NEAA) and 0.1% 2-mercaptoethanol, used previously for long-term heart slice cultures19. NEAA increases cell growth and viability, while 2-mercaptoethanol is a reducing agent that than can limit oxygen radical levels. c) We used a cocktail of antibiotics - Primocin - for the first few days of primary culture to reduce the incidence of contamination18. We also tested a combination of penicillin-streptomycin and fungizone, which was inadequate in reducing contamination. d) Most importantly, we agitated the culture plate at 150 rpm in an orbital shaker while culturing, which allows the explant to remain suspended in the culture and prevents ventricular adherence and deformation. Agitating manually every 12 h without a shaker was typically not sufficient to prevent attachment of explants to the dish bottom. These alterations in methodology allow the heart surface to remain largely intact during culture. With the refined protocol, we were able to longitudinally monitor epicardial cell migration and proliferation on the surface of a beating heart for at least one month. This protocol is suitable for studies that focus on the epicardium and epicardial regeneration. We have not tested pacing of the explanted hearts, different energy substrates and application of hyperoxygenation, which have the potential to further optimize the protocol.
The overall experimental scheme is shown in Fig. 1. In this protocol, we will describe explant culture of intact hearts and ex vivo epicardial ablation. To ablate epicardial cells, we generated a BAC line by fusing a bacterial Nitroreductase (NTR) to an mCherry fluorescent reporter gene cassette, driven by tcf21 regulatory sequences, which are epicardial and epicardial-derived-cell-specific in the heart (tcf21:mCherry-NTR)5, 7. After treatment with metronidazole (Mtz), which is processed by NTR into a cytotoxin, this line induced cell death specifically of epicardial and epicardial-derived cells23, 24. We typically use a tcf21:nucEGFP BAC reporter strain to mark epicardial cell nuclei5. In optional procedures (Box 1-3), we describe an assay for proliferation by epicardial cells in culture by incorporation of the nucleotide analog 5-ethynyl-2’-deoxyuridine (EdU) (Box 1); the engraftment of a freshly collected outflow tract to the base of an epicardially-ablated ventricle (Box 2); and embedding of beads with Hh proteins onto the ventricular base (Box 3).
Our original ex vivo explantation method was developed to study epicardial and endocardial cell gene expression after the formation of spontaneous internal infarcts10. This method is suitable only for short-term culture (7 days). Also, as heart explants attach to the culture dish and cardiac muscle undergoes rapid necrosis, there is considerable opportunity for artifacts. In vitro primary cell culture of isolated epicardial cells is effective to characterize epicardial cell-extracellular matrix interactions, and can be expanded to tests of cell-cell interactions by co-culture18. Although a more artificial environment than the cardiac surface, dissociated cell culture has certain advantages for cellular and molecular studies. including the capacities for gene transfection and live imaging. The refined protocol we describe here combines the advantages of these two systems. It is suitable for cellular and molecular analyses while largely maintaining the cardiac surface environment (described in the next section). Other methods for culturing cardiac slices are sufficient for testing cardiac muscle behaviors but are not applicable for studies of the epicardium19-22.
Recently, by generating transgenic tools to ablate the epicardium in zebrafish, we found that the epicardium is required for normal regeneration of heart muscle after injury. Moreover, with the aid of this ex vivo explant culture system, we identified unexpected features by which the epicardium itself regenerates after its depletion. The procedures we describe have considerable advantages for investigating the cellular and molecular mechanisms of epicardial cell gene regulation, proliferation, migration, and overall regeneration in real time while largely recapitulating in vivo environment. The heart explants are also suitable for assays of gene expression by in situ hybridization. We include procedures here that enable the study of tissue-tissue interactions, including tissue and bead grafting, which can potentially be expanded to include other tissues. A clear application for these methods is chemical screening; for instance, one can test hundreds or thousands of compounds for influences on epicardial cell behaviors while on the cardiac surface. Those factors that induce epicardial cell proliferation or the activation of markers of tissue regeneration have the to potential to influence the efficacy of heart regeneration.
The protocol described here has been successfully used in studying epicardial cell behaviors, but it is not suitable for detailed studies of cardiomyocytes. We detect very limited proliferation of cardiomyocytes during culture, possibly due to missing ingredients or suboptimal environmental conditions. Rates of contraction decrease during culture, and hearts occasionally show internal infarcts. Further development of the protocol is required to improve its applicability for ex vivo studies of cardiac muscle. Most applications can be completed in 2 weeks, during which the hearts appear grossly normal in morphology and beating. For long-term culture of more than 2 weeks, the heart shape becomes slightly altered. Shrinking is observable, and the pyramidal ventricle appears more rounded, likely secondary to the shaking of the culture. After grafting of cardiac chambers, the ventricular shape may change slightly due to pressure exerted from the agarose used in the procedure. However, no apparent difference in epicardial cell behavior (such as proliferation and migration) was observed in ventricles of different shapes.
Depending on the experimental purpose, partial resection can be performed by removing 10-20% of the ventricular apex as described previously, before explant culture1. Epicardial ablation can be performed before or during explant culture. Hearts can be collected at any time following in vivo manipulations. Examples can be found in our recent publication7. Hearts after resection or ablation injury will be adhesive and may be difficult to separate from pericardial material. In this report, we will describe explant culture of intact hearts and those undergoing ex vivo epicardial ablation. As heart sizes, surgery size and positions, and ablation effects are variable among different fish and clutches, at least 10 explants per treatment are required for each experiment. Five sham-treated (uninjured or unablated) explants should be included each time as a system control for culture procedures. At least two independent replicates are required for conclusions. If treatments will be applied during explant culture, such as chemical treatment and bead embedding, uninjured explants and/or explants with surgery or ablation should be randomized into different groups for each experiment. Finally, only clutchmates (or hearts collected from clutchmates) will be used for each experiment.
While shaking, explants will gather in the middle of the culture dish or well. If epicardial cells are ablated or heart surgeries are performed prior to culturing, explants are adhesive and will become attached to each other when cultured as multiples. In this situation, one heart per well is recommended and multi-well plates are economical. On the other hand, small wells of 24- or 48- well plate are not conducive for live imaging. As the samples must remain covered while imaging, only the center of these small wells can provide an undistorted view. The 12-well plate is the best choice for most experiments. Uninjured hearts can be cultured together in larger wells or petri dishes.
There are various ways to assay indices of proliferation in cells on the cardiac cell surface. To mark cells undergoing DNA synthesis, we use the nucleotide analog EdU. A 1-hour period of EdU incorporation at 25 μM is sufficient to label a large number of cells. Phosphorylated Histone H3 is a marker of cells in G2/M phase, recognized by several commercially available antibodies. We choose EdU as an example here. Epicardial cell nuclei are best identified by the tcf21:nucEGFP strain5. Two independent replicates and 10 explants per treatment are required for quantification of proliferation indices.
Small molecule compounds can be included in the incubation protocols to examine effects on epicardial proliferation and regeneration7. It is important to maintain sterile medium, accomplished either by filter sterilization of the medium with diluted drug, or by keeping the drug stocks sterile. For each chemical compound, two independent replicates with at least 10 explants of uninjured or epicardially ablated hearts per replicate are treated, and 10 more vehicle treated explants of hearts from the same clutch of zebrafish served as a control. Medium should be changed every other day with fresh chemicals. For uninjured hearts, effects on epicaridial proliferation are assessed by quantification of cell numbers using the tcf21:nucEGFP reporter at 2-6 days of culture. For effects on epicardial regeneration after ablation, chemicals are added at 2 days post Mtz treatment, and explants from the tcf21:nucEGFP strain are imaged every day for up to 14 days to assess epicardial proliferation and migration.
We have successfully engrafted the bulbous arteriosus (cardiac outflow tract) or ventricular apex to the base or apex of ventricles that have had their epicardium genetically depleted7. Here we describe the engraftment of freshly collected outflow tract to the base of an epicardially-ablated ventricle. In this experiment, about 50% of the explants will be tightly grafted together after 24 h contact in agarose. For 10 successful grafts, at least 20 attempts are required. As the grafted positions vary across grafts, we prefer to make conclusions based on 3 independent replicate experiments. The protocol should also be applicable to other tissue combinations.
Agarose beads can be used to test factor function by controlled protein release. Here we will embed beads soaked with Hh proteins onto the ventricular base as an example. Beads soaked with BSA protein serve as a control. At least 50% of ventricles successfully engraft and maintain beads during culture. Three independent replicates with at least 20 attempts per replicate are required for each experiment. Heparin acrylic beads can also be used25.
Adult zebrafish of the Ekkwill and Ekkwill/AB strains were used, maintained as described1. Animals between 4 and 12 months of both sexes are generally used. Transgenic lines used in this study were Tg(tcf21:mCherry-NTR)pd108 (ref. 7), Tg(tcf21:nucEGFP)pd41 (ref. 26), Tg(cmlc2:actin3-EGFP)sd10 (ref. 27) and Tg(fli1a:EGFP)y1 (ref. 28). All transgenic strains were analyzed as hemizygotes. Animal procedures were approved by the Institutional Animal Care & Use Committee at Duke.
Fish water (aquarium water from the zebrafish facility)
Tricaine (Sigma, cat. no. A5040) CAUTION Avoid inhalation and direct contact with eyes and skin.
DMEM cell culture medium (Invitrogen, cat. no. 11965)
FBS (Thermo Scientific, cat. no. SH30071.03)
Primocin (InvivoGen, cat. no. ant-pm-2)
PBS (Corning, cat. no. 46-013-CM)
Ethanol (VWR, cat. no. 89125-172)
Metronidazole (Mtz, Sigma, cat. no. M1547) CAUTION Mtz is a carcinogen. Avoid contact with skin and eyes. Avoid inhalation. Wear gloves and eye protection.
L-Glutamine (Invitrogen, cat. no. 25030-081)
MEM-NEAA (Invitrogen, cat. no. 11140-050)
Penicillin-streptomycin (Invitrogen, cat. no. 15140-122)
Low-melting agarose (Fisher Scientific, cat. no. BP1360-100)
Affi-Gel Blue beads (Bio-Rad, cat. no. 1537301)
5-ethynyl-2’-deoxyuridine (EdU, Life Technologies, cat. no. A10044) CAUTION Avoid contact with skin and eyes. Avoid inhalation. Wear gloves and eye protection.
Recombinant mouse Sonic Hedgehog (C25II), N-terminus protein (R & D Systems, cat. no. 464-SH-025)
Bovine serum albumin (BSA, VWR, cat. no. 97061-420)
Paraformaldehyde (Fisher Scientific, cat. no. O4042-500) CAUTION Avoid contact with skin and eyes. Avoid inhalation. Wear gloves and eye protection.
Fluoromount G mounting medium (Fisher Scientific, cat. no. 0100-01)
Finger bowls (90 × 50 mm; VWR, cat. no. 89000-288)
1.7 ml Eppendorf tubes (EP tubes, Genesee Scientific, cat. no. 24-281)
12-well plates (Corning, cat. no. 3737)
60 mm Petri dishes (Corning, cat. no. 351007)
Millex-GP Filter units (0.22μm, Millipore, cat. no. SLGP033RB)
Syringes (BD, cat. no. 302831)
Sterile pipette tips (Corning, cat. no. 4135, 4139 and 4140)
Sterile disposable plastic transfer pipettes (VWR, cat. no. 414004-016)
Cover slips (Fisher Scientific, cat. no. 12-544-E)
Stainless steel microforceps (Dumont, cat. no. 5-inox-H)
Stainless steel microscissors (World Precision Instruments, cat no. 14124)
Sponges (1.5 × 5 × 3 cm) with a single center groove cut using scissors (0.5 × 2.5 cm)
Plastic spoons, heavy duty (Staples)
Falcon tubes (15 ml, 50 ml, Corning, cat. no. 430790, 430828)
Dissecting microscope (Leica)
Water bath (Fisher Scientific, ISOTEMP 150)
125 ml flasks (Corning)
Biological safety cabinet with laminar flow and UV light (Labconco, Class II, type A2)
Cell culture incubator (28°C, 5% CO2, humidified; Fisher Scientific, Model 3530)
Orbital shaker (Troemner, cat. no. 12620-938)
GeneMate GyroMixer, Variable Speed (BioExpress, cat. no. R-3200-1XL)
Leica MZ05FA stereofluorescence microscope with camera
The stock solution contains 15 mM ethyl 3-aminobenzoate methanesulfonate (tricaine) and 20 mM Tris-HCl, pH 7.4. Store this stock solution at 4°C for as long as one month, and at −20°C for longer term storage. To anaesthetize adult zebrafish, dilute 4.2 ml of the stock solution into 100 ml of fish water.
Culture medium consists of DMEM plus 10% fetal bovine serum, 1% MEM-NEAA (non-essential amino acids), 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-mercaptoethanol. Primocin™ was added just before use to prohibit microbial contaminants during the first few days primary culture. Sterile medium can be stored at 4°C for up to one month without effects on culture results. CAUTION Medium and PBS must be sterile filtered.
For epicardial ablation, freshly prepare 1 mM Mtz solution in culture medium for use. First make 10-20 ml of a 10 mM Mtz solution in a 50 ml Falcon tube by vortexing and pipetting. Then dilute 1:10 to the desired volume, and filter sterilize with a 0.22 μm filter unit.
CAUTION As Mtz is difficult to dissolve, ensure it is fully dissolved before use.
Keep a separate bottle of agarose powder for culture use only, to reduce chance of contamination. To make 30 ml 1% low-melting agarose, weigh 0.3 g of powder and put into autoclaved 125 ml flask and add 30 ml sterile PBS. Microwave for several minutes until agarose is fully dissolved. Store the agarose in a 37°C water bath until use.
CRITICAL Use sterile PBS and flask.
CAUTION A sponge that is used repeatedly could accumulate contaminating microorganisms. Washing with EtOH will reduce the chance of contamination.
CRITICAL STEP Steps 2–7 must be performed on a clean lab bench using sterile tools and filter pipette tips.
Extracting hearts TIMING 2-5 min per heart
CRITICAL STEP Animal procedures must be performed in accordance with institutional guidelines.
CRITICAL STEP If more then one heart is processed, store hearts in a Petri dish with medium at room temperature. Keep the lid of the culture dish closed.
CRITICAL STEP Steps 6-10: Be careful not to damage the epicardium, which may influence experiments.
CRITICAL STEP All explant transfer procedures should be performed in this way.
CRITICAL STEP Steps 8–12 must be performed in a biological safety hood using sterile pipette tips and PBS.
Heart culture and epicardial ablation TIMING 5 min
CRITICAL STEP Medium must be warmed before use. Ablation medium must be freshly prepared just before dissecting.
CRITICAL STEP Ensure the shaker is working properly throughout the culture period.
Box 1. Proliferation assay
CAUTION Follow institutional guidelines to dispose of medium with Mtz.
Box 2. Chamber grafts
Box 3. Bead grafts
CRITICAL While taking images, keep the lids of culture plates closed.
|11-13, b5, c6||Contamination with bacteria||Bench is not clean||Clean bench with 70% ethanol or perform procedures in a cell culture hood|
|Tools are not sterile||Sterilize tools with 70% ethanol, use sterile pipette tips or filter tips|
|Reagents are not sterile||Filter sterilize reagents|
|Plate lid is open during culture or imaging||Keep lid closed while culturing and imaging|
|Primocin is not added||Add Primocin for the first few days of culture|
|11||Heart explant is attached to dish bottom||Culture plate is not shaking properly||Make sure the shaker is always working at 150 rpm during culture|
|13||Absent or weak epicardial ablation effect||Mtz is not fully dissolved||Ensure Mtz is fully dissolved when preparing 10 mM solution|
|b5||Chamber engraft fails||Chambers are not contacted closely||Use smaller hole to hold chambers tightly|
|c6||Beads floating||Too much medium||Add less medium|
Steps 1-3, Preparation: 1 h
Steps 4-5, Extracting hearts: 2-5 min per heart
Steps 6-8, Clean hearts: 5 min
Steps 9-11, Heart culture and epicardial ablation: 5 min
Step 12, Release Mtz treatment: 30 min
Step 13, Imaging: up to 30 days
Optional Steps a1-a3: Proliferation assay: 5-8 h
Optional Steps b1-b5: Chamber engraftment: 25-50 h
Optional Steps c1-c5: Beads embedding: about 26 h
During culture, cardiac contraction will gradually slow, although explants can continue beating for more than one month (ref. 7, Supplementary video 1)7. Sarcomeric structure, visualized using a transgenic cmlc2:actinin3-EGFP line, is largely maintained during the first 2 weeks of culture, and muscle necrosis is rare (Fig. 4). The heart shape will also change gradually, shrink slightly and become rounded. Despite these minor changes, epicardial proliferation and migration are not affected. Epicardial cells will proliferate spontaneously without any treatment, with cell density increasing during the first 2 days, as assessed by staining with an antibody against Raldh2, and EdU incorporation and staining (Fig. 4; Fig. 5 and ref. 7)7. Proliferating endocardial cells were very rare, and were not detected in most hearts after 24h EdU incorporation (day 2-3) and assessment at day 3 by using a fli1a:EGFP reporter line (Fig. 5b). Some EGFP+/EdU+ endothelial cells were detected, but a nuclear marker of endothelial cells is required for further confirmation.
The cultured explants are suitable for in situ hybridization (Fig. 6), and we detected epicardial induction of raldh2, tbx18 and wt1 in uninjured hearts during explant culture. For most explants, raldh2 was only detected in epicardial cells, while some explants displayed endocardial expression.
With Mtz treatment, epicardial cell death can be observed from day 2 (1 day post Mtz incubation, dpi) with debris protruding from the heart surface. Full ablation can be achieved at day 3 (2 dpi) with a significant portion of the ventral surface demonstrating absent tcf21:nucEGFP fluorescent. Epicardial regeneration is visualized by daily imaging; epicardial cells located at the ventricular base proliferate and migrate toward the ventricular apex (Fig. 7a). It takes approximately 2 weeks for epicardial cells to completely repopulate the ventricular surface (Fig. 7a and ref. 7, Fig. 2). In chamber engraftment experiments, about 50% of the explants will be tightly connected after 24 h contact in agarose (Fig. 7b, c). The ratio will be higher after 48 h contact in agarose. The ventricular epicardium loses regenerative capacity after removal of the outflow tract; however, a fresh donor outflow grafted to the ventricular base can rescue the defect (Fig. 7c and ref. 7, Fig. 3). In bead-grafting experiments, some ventricles lose the beads after release from agarose; these samples must be discarded. Ventricles also lose beads during 1-week culture and also must be discarded. At least 50% of ventricles successfully engraft and maintain beads during culture. Beads soaked with Hh protein can induce epicardial cell regeneration within the host ventricle after outflow tract removal (ref. 7, Fig. 4), implicating this factor in mechanisms of regeneration.
In summary, we describe in detail an explant culture system that maintains aspects of cardiac physiology and recapitulates in vivo observations of epicardial regeneration, while better representing such observations than cultured primary cells7. At the same time, cellular and molecular studies are convenient and efficient.
Following Steps 1-11. To detect proliferation, EdU should be added to the culture medium at a concentration of 25 μM.
CRITICAL STEP PBS washes are important; otherwise unincorporated EdU will produce high background.
Following Steps 1-12. Here, we graft freshly collected outflow tracts to an epicardially ablated ventricle as an example. The ventricle was treated with 1 mM Mtz for 24 h and cultured for 2 more days before engraftment.
CRITICAL STEP Steps b1-b5 should be performed in a biological hood with sterile tools.
CRITICAL STEP Several holes with different sizes can be made in case heart size varies among your animals.
CRITICAL STEP Ensure the size of the hole is appropriate - not too small to squeeze chambers.
CRITICAL STEP Gently place the dish in the incubator, and prevent agitation until explants are released from the agarose in subsequent steps.
Following Steps 1-12. Here we graft beads soaked with Hh proteins onto the ventricular base as an example.
CRITICAL STEP Steps c1-c6 can be performed in a biological hood using sterile tools.
CRITICAL STEP Beads must be washed thoroughly to remove preservative.
CRITICAL STEP Two to three beads can be applied to one site. Not all beads will be successfully grafted.
CRITICAL STEP Do not add too much medium, as this may cause the beads to float.
CRITICAL STEP Gently place the dish in the incubator, and keep still until the release of explants from the agarose.
We thank J. Wang and S. Singh for generating transgenic fish; J. Burris, N. Lee, A. Dunlap, S. Davies, T. Thoren, and N. Benkaci for fish care; A.L. Dickson for artwork; and A. Shoffner for comments on the manuscript. This work was funded by a postdoctoral fellowship from the American Heart Association to J.C. and grants from NIH (R01 HL081674 and R01 HL131319) to K.D.P.
Wang, J., Cao, J., Dickson, A.L. & Poss, K.D. Epicardial regeneration is guided by cardiac outflow tract and Hedgehog signalling. Nature 522, 226-230 (2015).
J.C. and K.D.P. designed the study and wrote the manuscript. J.C. performed all experiments.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.