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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Protoc. Author manuscript; available in PMC 2013 February 1.
Published in final edited form as:
PMCID: PMC3541820
NIHMSID: NIHMS428237

In vitro culture of epicardial cells from adult zebrafish heart on a fibrin matrix

Abstract

We describe here a protocol for culturing epicardial cells from adult zebrafish hearts, which have a unique regenerative capacity after injury. Briefly, zebrafish hearts first undergo ventricular amputation or sham operation. Next, the hearts are excised and explanted onto fibrin gels prepared in advance in a multiwell tissue culture plate. The procedure allows the epicardial cells to outgrow from the ventricle onto a fibrin matrix in vitro. This protocol differs from those used in other organisms by using a fibrin gel to mimic blood clots that normally form after injury and that are essential for proper cell migration. The culture procedure can be accomplished within 5 h; epicardial cells can be obtained within 24-48 h and can be maintained in culture for 5-6 d. This protocol can be used to investigate the mechanisms underlying epicardial cell migration, proliferation and epithelial-to-mesenchymal transition during heart regeneration, homeostatic cardiac growth or other physiological processes.

Keywords: epicardial cell, fibrin matrix, primary culture, zebrafish, adult heart regeneration

INTRODUCTION

Hearts are composed of multiple tissue types, including endocardium, epicardium and myocardium1. The epicardium is a mesoepithelial cell layer that originates from the proepicardium and has an essential role in the heart development and morphogenesis2,3. The study of the epicardium in adult zebrafish hearts is of particular interest because of its potential role in regeneration4-7 and homeostatic cardiac growth8. As in vivo imaging of adult zebrafish hearts is technically difficult, and epicardium-specific gene expression is not always feasible for in vivo studies in adult zebrafish, we established in vitro zebrafish primary epicardial cell cultures from adult zebrafish hearts on fibrin matrices to circumvent these obstacles9. By adjusting the culture conditions to control each component in a defined way, the explant culture system can complement in vivo observations and provide an alternative approach to investigate the detailed mechanisms underlying cell and growth factor functions, as well as the role of extracellular matrix (ECM) components during regeneration. Coupled with techniques such as time-lapse microscopy, the in vitro cell culture system can also be a valuable approach for visualizing and recording morphological features of cell migration10,11. We have analyzed cell proliferation and epithelial-to-mesenchymal (EMT) transition of adult zebrafish epicardial cells using the protocol described herein9.

Development of the protocol using fibrin gels

This is the first protocol described, to our knowledge, for culturing primary epicardial cells from adult zebrafish hearts. Other protocols for the culture of embryonic epicardial cells have been developed for chicken and mouse, and they have been important tools in the study of the detailed molecular and cellular mechanisms of heart development12-15. The difference between our culture protocol and others is that the heart explant is cultured on a fibrin gel, whereas previous protocols used collagen or gelatin12-15. Fibrin gels are prepared from fibrinogen (Fg) treated with thrombin16,17. The working concentration of Fg at 2 mg/ml was used to reflect its physiological concentration in the blood of vertebrates (2-3 mg/ml of plasma)18. As an aqueous gel, fibrin is ideal for the adherence of the explanted heart tissues. On the basis of the observations in our laboratory, adult zebrafish epicardial cells migrated out poorly when heart explants were placed on culture dishes coated with other types of ECM proteins such as collagen or gelatin. This is consistent with the in vivo amputation model of heart regeneration, wherein a fibrin clot is formed at the injured site followed by epicardial cell invasion into the fibrin clot6,9.

Application of the methods

Establishing in vitro primary cell cultures from adult zebrafish is one approach to expand the utility of this organism for mechanistic studies of injury repair at the cellular level. Fg/fibrin has been widely used for in vitro cell culture systems and for tissue engineering because of its physiological relevance, pliability and its ability to be mixed with other ECM components19-21. One potential future application is the characterization of epicardial cell-ECM interactions by mixing fibrin with other ECM components, such as collagen or hyaluronic acid22,23. In vitro culture is also useful for the study of the function of growth factors and downstream signaling pathways regulating migration, proliferation and differentiation of zebrafish epicardial cells. The approach can be expanded to include other cell types in coculture settings to analyze the interactions between epicardial cells and cardiomyocytes or endothelial cells for their cooperative roles in injury repair and regeneration. In coculture experiments, different cell types can be marked with transgenic lines such as Tg(wt1b:GFP)24 (epicardial cells), Tg(cmlc2:GFP)25 (cardiomyocytes) and Tg(fli1a-EGFP)26 (endothelial cells). It is expected that our in vitro culture system will facilitate studies on the mechanisms of zebrafish heart regeneration.

Experimental Design

The overall experimental scheme is shown in Figure 1. The following points should be carefully considered before starting the experiment.

Figure 1
Flowchart outlining the timeline of the described experimental procedures.

Sham operation or heart surgery

Depending on the purpose of the study, either sham-operated (mock surgery with un-injured hearts) or regenerating hearts (after amputation or other types of injuries) can be used. The details of zebrafish care and usage are described in REAGENT SETUP. The protocol we describe herein is an example in which the hearts are explanted 4-14 days after ventricular amputation or sham operation. However, the hearts can be collected at any time point after the initial surgical procedure for explant, depending on the aim of the studies and the experimental design. In this protocol, we use the apex of the heart (one-third of the ventricle), which contains the regenerating area. Using one-third of the ventricle instead of the whole ventricle can maximize the contact area between the regenerating tissue and the fibrin gel. However, depending on the research purpose (e.g., obtaining epicardial cells from sham-operated hearts), the whole ventricle can also be placed onto the gel. The procedures of heart amputation and sham operation are performed as described elsewhere 4,9.

Use of a glass coverslip for immunostaining

If immunostaining of the epicardial cells is planned, we recommend placing a glass coverslip onto the bottom of the multiwell plate before preparing the fibrin gels. This will make handling of the stained cells easier because it will be possible to place the entire fibrin gel layer onto the slide by lifting the coverslip. This procedure is described in Box 1. This step is not necessary if the cells will be used for other purposes (e.g., RNA extraction).

Box 1

Sterilization of a glass coverslip before fibrin gel preparation TIMING ~1h

This procedure is optional and should be carried out only if immunostaining is required (Steps 22-29). Carry out the entire procedure in a tissue culture hood.

  1. Place a standard 12-mm-diameter round glass coverslip at the bottom of the well of a 24-well cell culture plate (Fig. 3b). Alternatively, 6- or 12-well cell culture plates may also be used for easier handling and processing of the fibrin gel specimen for immunocytochemistry.
  2. Sterilize the coverslip by rinsing the well with 70% (vol/vol) ethanol three times for 5 min each, and do a final rinse with 100% ethanol.
  3. After removing the ethanol, dry the plate in the hood with the lid open for at least 1 h.

Choice of multiwell plate

Depending on the purpose of the experiment, 300 μl of Fg-thrombin mixture is used per well of a 24-well plate; this should form a fibrin gel of approximately 1.5 mm in thickness. However, because of the viscosity of the mixture and the capillary effect, the thickness of the center part of the gel is around 1 mm and gradually increases around the edges of the well. This phenomenon will cause difficulty during imaging because the cells will be on different focal planes. To prevent this, larger multiwell plates (e.g., 6-or 12-well plates) can be used with the same ratio of Fg to thrombin, with the total volume adjusted accordingly on the basis of the surface area of the well. For immunocytochemistry and cell imaging, larger multiwell plates (e.g., 6-or 12-well plates) can be used with adjusted Fg-thrombin volume, i.e., 600 μl per well for 12-well plates and 1,500 μl per well for 6-well plates.

Use of pharmacological agents

We have been successful in studying different signaling pathways with various pharmacological agonists and inhibitors using this epicardial cell culture9. As with any in vitro cell culture study, care must be taken when interpreting the results. When performing this type of assay, it is important to determine dose-dependent effects and use proper controls. In general, we use the solvent that is used to dissolve the pharmacological agonists and inhibitors as a negative control. Depending on the research design, epicardial cell outgrowth, proliferation and EMT can be determined and quantified with image analysis software.

MATERIALS

REAGENTS

Tricaine solution (4 mg/ml; Sigma, cat. no. A5040)

! CAUTION Avoid inhalation and contact with eyes.

Fish water27

Ethanol (70% (vol/vol); Sigma, cat. no. 459844)

Ethanol (100%; Sigma, cat. no. 459844)

Fibrinogen (Fg; Calbiochem, cat. no. 341573)

Thrombin (Sigma-Aldrich, cat. no. T9549)

DMEM cell culture medium (Cellgro, cat. no. 10-013)

HEPES (Sigma, cat. no. H3375)

DMEM powder (Sigma, cat. no. D5648)

FBS (Cellgro, cat. no. 35-015-CV)

Primocin (antibiotic, Invivo Gen, cat. no. ant-pm-1)

Paraformaldehyde (Electron Microscopy Sciences, cat. no. 15710)

! CAUTION Paraformaldehyde is a carcinogen. Wear gloves and eye protection. Avoid inhalation.

PBS (Cellgro, cat. no. 21-030-CV)

Triton X-100 (Sigma, cat. no. T-8787)

5-Bromo-2′-deoxyuridine (BrdU; Sigma, cat. no. B5002)

! CAUTION BrdU is a carcinogen. Wear gloves and eye protection. Avoid inhalation.

Hank’s balanced salt solution (Hank’s solution; Cellgro, cat. no. 21-020)

ZO-1 mouse monoclonal antibody (Invitrogen, cat. no. 339100)

Tcf21 rabbit polyclonal antibody (AnaSpec, cat. no. 55867)

Phospho-histone-3 antibody (Upstate/Millipore, cat. no. 06-570)

BrdU rat monoclonal antibody (Abcam, cat. no. ab6326)

Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, cat. no. A11029)

! CAUTION This reagent is light sensitive. Avoid exposure to light by wrapping thetube with thin foil.

Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes, cat. no. A11012)

! CAUTION This reagent is light sensitive. Avoid exposure to light by wrapping thetube with thin foil.

Alexa Fluor 594 goat anti-rat IgG (Molecular Probes, cat. no. A11007)

! CAUTION This reagent is light sensitive. Avoid exposure to light by wrapping thetube with tin foil.

Vectashield with DAPI (Vector, cat. no. H-1200)

Sodium hydroxide

EQUIPMENT

Microtubes (0.5ml; Denville, cat. no. C18001-GD)

Microtubes (1.5 ml; Denville, cat. no. C19001B)

Glass bowl (90 × 50 mm; Kimax)

Dissecting microscope (Jenco)

Sponge (1.5 × 5 × 3 cm) with groove (0.5 × 2.5 to 3 cm) (available at any grocery store)

Filter unit (0.2 μm; Nalgene Labware, cat. no. 156-4020)

Stainless steel fine microforceps (Sigma, cat. no. T-4537)

Stainless steel microscissors (Fine Science Tools, cat. no. 15000-00)

Surgical blades (BD Bard-Parker, cat. no. 371215)

Plates, 6, 12 and 24 wells (Greiner Bio-One, cat. no. 567165, 665180, 662940)

Dish (35 * 10 mm; Fisher, cat. no. 08-772A)

Pipet-Aid (Drummond, cat. no. 4-000-100)

Sterile micropipettes (Denvill)

Kimwipes (Kimberly-Clark)

Cell culture incubator (28 °C, 5% CO2, humidified; Thermo Scientific)

Biological cell culture hood with laminar flow and UV light (Baker)

Round coverslips (VWR, cat. no. 48300-560)

Slide glass (Fisher Scientific)

Suction flask with vacuum (Pyrex)

Fluorescence and phase-contrast microscope with camera (Leica, model no. DMI6000 B)

Phase-contrast microscope with camera (AMG, EVOS xl microscope)

Tweezer

REAGENT SET UP

Zebrafish care and use Wild-type (AB or EKK strain) or transgenic zebrafish are housed and cared for in a standard zebrafish facility as described27, and are in accordance to Institutional animal care and use committee guidelines. Eight months to 1.5-year-old adult fish, either male or female, can be used for the sham operation or heart amputation. Depending on the research purpose, statistical and analytical methods to be used, either wild-type or transgenic fish will be used and the number of fish should be calculated in advance. When calculating the numbers of fish to be used for experiments, a few aspects will be considered. In general, we put one fish heart in each well and only one treatment is done per well. However, for collecting RNA and protein or cocultures, more hearts can be cultured in one well. The number of fish hearts needed depends on how many treatments are required. As there is a 10-20% lethality rate within 24 h for the fish after heart amputation, we suggest that 10% more fish should be used for amputation than for the sham operation in order to get the same number of samples in the end.

Tricane Solution: Tricaine contains 15mM M ethyl 3-aminobenzoate methanesulfonate and 20mM M Tris-HCl, pH 7.4. Dissolve 4 g of ethyl 3-aminobenzoate methanesulfonate in 979 ml of deionized water, and add 21 ml of 1 M Tris-HCl, adjusting the pH to 7.4. Store this stock solution at 4 °C for 1 month. To anesthetize adult zebrafish, dilute 4.2 ml of the stock solution into 100 ml of fish water27 (i.e., water from the fish aquarium system).

HDMEM (Hepes - treated DMEM): HDMEM contains 23 mM HEPES and 15 mM NaCl. To prepare HDMEM, Dissolve 1 pack of DMEM powder into 900 ml of deionized water. Add 5.49 g of HEPES, 880 mg of NaCl and adjust pH to 7.4 by adding 10 N NaOH and the volume to 1 liter. Sterilize the HDMEM by filtering the solution using a Nalgene filter unit with a 0.2-μm pore size. Sterile HDMEM can be stored at 4 °C for up to 3 months.

Fibrinogen Stock Solution (20 mg/ml): Reconstitute the lyophilized fibrinogen (Fg) under sterile conditions by adding PBS to Fg to reach a stock concentration of 20 mg/ml. Incubate the reconstituted Fg in a 37°C waterbath with occasional shaking for 30 min. This is to ensure that Fg is completely dissolved. Make 500 μl aliquots of the Fg stock solution in 1.5 ml sterile microtubes and the stock can be stored at −20°C for at least 1 year.

Δ CRITICAL The ability of each batch of Fg to form clots should be determined by titration for quality control17 (Box 2). Stocks that have a clottability of less than 95% should be discarded. Avoid repeated freezing and thawing.

Box 2

Determining clottability of Fg stock solution • TIMING ~ 1h

Clottability is defined as % (polymerized Fg)/(total Fg). Briefly, this is measured as described below.

  1. Mix 1 ml of Fg in serially titrated concentrations (i.e., 1.25, 2.5, 5 and 10 mg/ml) with 5 U of thrombin.
  2. Incubate for 30 min at 37 °C.
  3. Centrifuge the reactions at 25°C, 12,000g for 15 min.
  4. Determine the protein concentration of nonpolymerized Fg in the supernatant by measuring the optical density at 280 nm (OD280).

Δ CRITICAL Fg precipitates at below room temperatures (25°C) and may not completely dissolve at room temperature when reconstituted from the lyophilized powder. Therefore, it is important to place the reconstituted Fg in the 37 °C water bath for 30 min with occasional shaking to ensure that Fg completely dissolves and that the concentration of Fg is consistent from preparation to preparation. The Fg concentration in the stock solution may be checked using conventional protein assays.

Thrombin Stock Solution (50 U/ml): Sterile procedure should be used for reconstituting thrombin. Thrombin is supplied either as a liquid or a lyophilized powder from commercial vendors. To reconstitute the lyophilized thrombin, add de-ionized water to the uncapped bottle to achieve a stock concentration of 50 U/ml. Make 25 μl aliquots of the stock solution using 0.5 ml sterile microtubes. Since repeated freezing and thawing can inactivate its enzymatic activity, it is necessary to aliquot the liquid stock solution and freeze the aliquots at −20°C. The stock solution can be stored for 6 months.

BrdU Stock Solution (2.5 mg/ml): Dissolve BrdU in Hank’s solution or PBS to a final concentration of 2.5 mg/ml. The BrdU stock solution can be stored at −20°C for 6 months.

! CAUTION BrdU is a carcinogen and needs to be prepared in the chemical hood with proper protection.

PROCEDURE

Preparation of the fibrin gels • TIMING 1.5 h

1 Thaw a 500 μl aliquot of Fg stock solution (20 mg/ml) in a 37 °C water bath for ~10 min with occasional gentle shaking.

Δ CRITICAL STEP Once it is thawed, the Fg stock solution should be kept at room temperature. Δ CRITICAL STEP It is important to completely dissolve Fg in the stock solution in the 37 °C water bath for 10-30 min. Check for spontaneous clotting due to freezing and thawing of the stock solution by inverting the vial several times. A completely thawed stock solution should be translucent and homogenous. Spontaneously formed clots usually attach to the wall of the tube and appear as uneven semitranslucent lumps. When this happens, discard the vial and thaw a new vial of Fg stock solution. Incomplete thawing and/or spontaneous clotting decreases Fg concentration in the stock solution and thereby alters the consistency and quality of the fibrin gels.

? TROUBLESHOOTING

2 Thaw an aliquot of thrombin stock solution (50 U/ml, 25 μl) quickly by holding the tube and warming it in your hand or by placing it in a 37 °C water bath.

Δ CRITICAL STEP Once thawed, keep the thrombin stock solution on ice until needed.

3 Warm DMEM culture medium to room temperature.

4 Dilute the Fg stock solution (20 mg/ml) with prewarmed DMEM culture medium (from Step 3) to reach a final concentration of 2 mg/ml (1:10 dilution) in a tissue culture hood. Mix the Fg working solution by inverting the tube several times.

Δ CRITICAL STEP Use sterile technique for Steps 4-8.

Δ CRITICAL STEP Always prepare an excess of Fg working solution for incidentals. Keep the Fg working solution at room temperature.

5 Add thrombin (50 U/ml) to the Fg working solution to reach a final concentration of 0.2 U/ml. Quickly mix the thrombin and Fg by inverting the tube several times. Proceed immediately with Step 6.

Δ CRITICAL STEP Thrombin converts soluble Fg into fibrin by cleaving the end peptides of Fg, which then forms end-to-end and side-by-side polymers16. The speed of conversion and the quality of the clot depend on the concentration of thrombin and the reaction temperature. The final thrombin concentration of 0.2 U/ml was chosen to allow fibrin gel to form in a reasonable time frame, i.e., 15-20 min during gel preparation. For example, we add 20 μl of the thrombin stock solution for every 5 ml of Fg working solution. This concentration of thrombin allows the fibrin gel to form in 20 min at 28 °C.

6 Pipette 300 μl of the Fg-thrombin mixture (from Step 5) into the well of a 24-well cell culture plate. Carry out the procedure relatively quickly to prevent clotting before finishing the procedure; make sure that the mixture completely covers the bottom of the well.

7 Incubate the plate for 30 min in the tissue culture hood without disturbance.

8 After 30 min, check if the fibrin gel has formed by tilting the plate.

Δ CRITICAL STEP Monitor the formation of the gels to ensure that they form in the appropriate time and manner to maintain consistency. Discard the fibrin gel if an extended time period (more than 60 min) is required to form the gels and start again from Step 1. If problems persist, use a new thrombin stock solution. A fully formed fibrin gel should maintain its shape and appear slightly opaque.

Δ CRITICAL STEP Fibrin gels should form in about 20 min under the conditions described here. The consistency of the fibrin gel is crucial.

Δ PAUSE POINT The plate containing the fibrin gels can be kept in the 28 °C incubator for longer periods of time (up to 2 h) while you wait for the next steps (obtaining ventricular tissue) to be completed.

? TROUBLESHOOTING

Obtaining adult zebrafish hearts and isolating the ventricular apex • TIMING 5-7 min per fish

9 Euthanize the fish with Tricaine solution (4.2 ml of Tricaine stock in 100 ml of fish water, 15 min or longer) and position the fish, ventral side up, in the groove of a moistened sponge under a dissecting microscope (Fig. 2a).

Figure 2
Dissection of the heart from adult zebrafish. (a) An euthanized adult zebrafish (ventral side up) positioned in the groove of a soft sponge. (b) View of the heart exposed in the chest cavity. After removal of the scales at the chest area, a longitudinal ...

Δ CRITICAL STEP All zebrafish handling should follow institutional guidelines and should be approved by institutional animal care and use committees.

Δ CRITICAL STEP Steps 9-13 can be performed on the bench; however, sterile surgical instruments should be used.

10 Open the chest wall of the fish by a longitudinal cut between the gills (Fig. 2b and Supplementary Video 1). While holding the bulbus arteriosus with a pair of tweezers, gently pull the heart out of the chest cavity and separate it from the fish by severing the ventral aorta and sinus venosus with microscissors1 (Fig. 2c and Supplementary Video 1).

11 Place the heart into a 3.5 × 10 mm dish and rinse it with HDMEM three times to remove the blood and tissue debris. Dispose of the fish carcass appropriately. Δ CRITICAL STEP If more than one heart is being processed, the dissected whole hearts can be stored in HDMEM at room temperature during the next heart dissection. We do not recommend leaving the heart in HDMEM for more than 1 h. When all of the hearts are collected, isolate the apex of each (Steps 12 and 13).

12 Under a dissecting microscope, remove the atrium and the bulbus arteriosus using microscissors, isolate the apex of the heart (posterior third of the ventricle) by cutting and separating it from the rest of the heart with a scalpel, and place it into a new 3.5 × 10 mm dish containing HDMEM.

13 Remove the blood in the ventricular cavity by gently pressing on the ventricle while rinsing it with a large amount of HDMEM.

Δ CRITICAL STEP The heart ventricle needs to be placed into the explant culture within 1 h (Step 14).

Placement of the tissue onto the fibrin gel • TIMING 5 min

14 Take the 24-well plate containing the fibrin gels (from Step 6) out of the incubator, open the lid, and gently place the isolated ventricular tissue (from Step 13) on the central surface of the fibrin gel.

Δ CRITICAL STEP The isolated heart apex should be correctly positioned with the apex facing down to maximize the contact area of the epicardial cells of the regenerating area and to prevent the contact of the exposed myocardium and endocardium onto the fibrin gel.

15 Gently press down on the tissue with tweezers to aid tissue attachment to the fibrin gel.

16 Carefully remove the excess HDMEM surrounding the ventricle by gentle suction, using a micropipette or a capillary tube (Fig. 3a,b).

Figure 3
Explant culture of adult zebrafish hearts on fibrin gels. (a) A top view of the heart apex (1/3 of the ventricle) explanted on a fibrin gel. (b) An illustration of the culture set up with epicardial cell outgrowth onto the fibrin gel. White asterisk: ...

Δ CRITICAL STEP Excess HDMEM surrounding the tissue explant can cause the tissue to detach when medium is added (Step 18). As the outgrowth and migration of epicardial cells from the ventricle onto the fibrin matrix requires close contact between the ventricle and fibrin gel, it is important to remove as much of the HDMEM surrounding the ventricle as possible without subjecting the tissue to desiccation. Because of the aqueous nature of the fibrin gel, it will be impossible and not necessary to remove all of the HDMEM.

Attachment of heart tissues • TIMING 30-60 min

17 Return the plate to the 28°C tissue culture incubator for an additional 30-60 min to allow attachment of the ventricular tissue to the fibrin gel.

Δ CRITICAL STEP During this time, remove the plate from the tissue culture incubator (28 °C) every 15 min and check the tissue under a microscope to ensure that the tissue has not dried out too much. The volume of HDMEM surrounding the tissue should decrease as a result of evaporation and appear as a thin meniscus of liquid around the tissue. If the meniscus is not visible and the tissue has a desiccated appearance, this may be an indication of overdrying. If the tissue has dried out, discard the tissue. Leaving the heart explant on the fibrin gel without cell culture medium for more than 60 min is not recommended.

Addition of cell culture medium • TIMING 2 min

18 Add 300 μl of DMEM containing primocin and 0.5% (vol/vol) FBS to the well.

Δ CRITICAL STEP Take precautions not to disturb the attachment of the ventricle to the fibrin gel. The medium should be added very slowly along the side of the well. Any disturbance of the ventricle attachment to the fibrin gel may cause the tissue to float or dislodge, which will prevent epicardial cell outgrowth.

? TROUBLESHOOTING

Cell migration and proliferation • TIMING ~1d

19 Incubate the plate in a tissue culture incubator at 28 °C, supplemented with 5% CO2 and 100% humidity.

20 After 24 h, check the fibrin gel for epicardial cell migration under an inverted phase contrast microscope. No change of medium is necessary during the first 2-d culture period, unless required by the research design (e.g., treatment with pharmacological compounds, growth factors or BrdU).

? TROUBLESHOOTING

21 Change the medium every other day. Make sure that disruption of the ventricle does not occur during medium changes by adding the culture medium very gently.

Δ CRITICAL STEP To detect DNA synthesis and cell proliferation, BrdU can be added at a concentration of 10 mg/ml in the feeding medium for 12 h after the initial 3-d culture period. The epicardial cells with regenerating (amputated) heart culture can be used as the positive control for the BrdU incorporation assay, as these cells actively undergo proliferation.

Immunocytochemistry to confirm epicardial cell identity • TIMING 5-8 h

22 Carefully remove the medium from the well containing the fibrin gel and the epicardial cells.

23 Gently add 500 μl of 4% (wt/vol) paraformaldehyde to the well and fix the epicardial cells for 10 min at room temperature.

Δ CRITICAL STEP The ventricle is likely to be still attached to the fibrin gel at this time. Do not remove the ventricle because manual removal before or during the fixation step will disrupt the epicardial cell monolayer and cause it to dislodge.

24 Remove the paraformaldehyde after 10 min and rinse the well with PBS three times for 5 min each time.

25 Permeabilize the cells with 300 μl of 1% (vol/vol) Triton X-100 (plus 2 N HCl, optional for BrdU) in PBS for 60 min and rinse with PBS three times for 5 min each.

26 Treat the fixed and permeablized cells with antibody blocking solution before the antibody staining step according to the manufacture’s recommendations. Please refer to Kim et al.9 for conditions for epicardial cell-specific nuclear Tcf21 and BrdU staining. The entire procedure takes place in the well of the 24-well tissue culture plate.

Δ CRITICAL STEP During this procedure, it is likely that the heart explant will detach from the fibrin gel. If the explant is still attached to fibrin gel, it is not necessary to remove the tissue, because the stained cells can still be mounted even if the tissue is still attached. Do not remove the tissue manually. For the negative control, the cells can be stained without primary antibody or species-specific IgG antibody, which should result in no specific staining of the cells.

27 Add 2 drops (around 30 μl) of Vectashield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) to the center of a glass microscope slide and set the slide aside.

28 Carefully remove the epicardial cells (with the underlying fibrin gel still attached to the 12-mm glass coverslip) from the 24-well cell culture plate; use forceps to reach and lift the coverslip from the bottom of the well (Box 1). The entire specimen should be easily removed from the well. Mount upside down in the mounting medium on the glass microscope slide prepared in Step 27.

29 Press the glass coverslip gently to compress the fibrin gel and to remove trapped bubbles. At this point, the specimen is ready to be examined.

Δ PAUSE POINT Prevent desiccation of the specimen and quenching of the chromophores by storing the specimen in a humidified box at 4 °C away from light. Images should be taken within 2-3 d.

? TROUBLESHOOTING

Troubleshooting advice can be found in Table 1.

Table 1
Troubleshooting table

TIMING

Steps 1-8, Preparation of the fibrin gels: 1.5 h

Steps 9-13, Dissection of zebrafish hearts and apex of ventricles: 5-7 min per fish

Steps 14-16, Placement of the tissue onto the fibrin gel: 5 min

Step 17, Attachment and cell culture: ~1 h

Step 18, Addition of cell culture medium: 2 min

Steps 19-21, Cell migration and proliferation: ~1 day

Steps 22-29, Assays, including immunocytochemistry and BrdU assay: 5-8 h

ANTICIPATED RESULTS

Epicardial cells can be seen migrating out of the heart explant onto the surface of fibrin matrix within 24 h of the procedure. When sham-operated hearts are used, the initial epicardial cell migration occurs approximately 48-72 h after the hearts were placed in culture. Sufficient outgrowth of the epicardial cell monolayer for further analysis generally occurs around 4-5 d. The outgrowth of the epicardial cells can be measured and quantified by the distance from the edge of the heart tissue to the periphery of the monolayer (Fig. 3c). Epicardial cells cultured from regenerating hearts (7 d.p.a. (days post amputation)) have significantly more outgrowth than cells from sham-operated hearts (Fig. 3d). Under the light microscope, the epicardial cells exhibit a cobblestone-like shape (Figs. 3 and and4),4), which is typical of epithelial cells. In addition, these cells show nuclear staining for the epicardial marker nuclear Tcf21 (also called epicardin), which confirms their epicardial identity (Fig. 4a,b). Staining for the tight junction marker ZO-1 showed close contacts between cells, further confirming their epithelial nature (Fig. 4b). The epicardial identity of these cells can be further confirmed using several transgenic lines expressing epicardial reporters, such as Tg(wt1b:GFP)24, Tg(tcf21:DsRed2)28 and the enhancer trap line ET27 29. Using hearts from Tg(cmlc2-GFP) fish to prepare the culture, in which the myocardium is marked by GFP, we confirmed that there are no cardiomyocytes in the monolayer (Fig. 4c,). When epicardial cells are cultured from regenerating hearts 4 d.p.a., they readily undergo mitosis and DNA synthesis in vitro (Fig. 5), consistent with in vivo findings9. We do not culture these epicardial cells beyond 1 week, because the cells at the periphery of the monolayer start changing morphology and do not survive well for long-term culture. Although we observed that epicardial cells lose cell-cell contact, a characteristic of EMT, when the cells were treated with platelet-derived growth factor (PDGF)-BB9, we have not observed invasion of the epicardial cell into the fibrin gel within 1 week in culture. This is different from what was reported for embryonic chick hearts12 and could be further investigated.

Figure 4
Epicardial cell outgrowth on fibrin gels. (a) Tcf21 antibody staining (red) for epicardial cells. DAPI staining is shown in blue. b) Double staining the epicardial cells with Tcf21 (red) and ZO1 (green) antibodies. (c) and (d): Epicardial culture of hearts ...
Figure 5
Proliferation of epicardial cells on fibrin gels. Epicardial cells from a regenerating heart (4 dpa) culture were stained with a) an antibody against phospho-histone-3 (green), a marker for mitosis; and b) BrdU antibody (red) to determine DNA synthesis. ...

Supplementary Material

Supp. video 1

ACKNOWLEDGMENTS

This work was supported by the American Heart Association (0730214N to C.-L.L.), National Heart, Lung, and Blood Institute Grants (R01HL096121 to C.-L.L. and R01HL096121S1 to C.-L.L. for N.R.), the Wright Foundation (to C.-L.L.), a Research Career Development Award from the Saban Research Institute (C.-L.L.), a National Institute of General Medical Sciences Grant (R01GM055081 to T.-L.T.) and California Institute for Regenerative Medicine (CIRM) postdoctoral fellowships (J.K.). We thank Q. Wu for excellent technical support, E. Fernandez for helping with taking the video and M. Chao for critically reviewing the manuscript prior to submission.

Footnotes

Supplementary Information

Supplementary Video: Dissection of zebrafish to collect the heart for culture.

AUTHOR CONTRIBUTIONS

J.K., T.-L.T. and C.-L.L. designed the study; J.K., N.R. and Y.H. carried out the experiments; and J.K., N.R., T.-L.T. and C.-L.L. wrote the manuscript.

COMPETING FINANCIAL INTERSTS STATEMENT

The authors declare that they have no competing financial interests.

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