Protocol for simultaneous isolation of high-quality and high-quantity cardiomyocytes and non-myocyte cells from adult rat hearts

Summary

Isolating high-quality different cell types is a powerful approach for understanding cellular compositions and features in the heart, but it is challenging. The available protocols typically focus on isolating one or two cell types. Here, we present a protocol to simultaneously isolate high-quality and high-quantity cardiomyocytes and non-myocyte cells, including immune cells, from adult rat hearts. We describe steps for purifying cells using bovine serum albumin. We also detail procedures for viability analysis and cell type identification using fluorescence-activated cell sorting.

For complete details on the use and execution of this protocol, please refer to Zhang et al.,¹ Valkov et al.,² Vang et al.,³ and Li et al.⁴

Graphical abstract

Highlights

• •Protocol to isolate cardiac cells using Langendorff perfusion and enzymatic digestion
• •Steps to purify cardiomyocytes and non-myocyte cells using bovine serum albumin
• •Steps for medium preparation and culturing high-quality cardiomyocytes and fibroblasts
• •Characterization of non-myocyte cells using fluorescence-activated cell sorting

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Isolating high-quality different cell types is a powerful approach for understanding cellular compositions and features in the heart, but it is challenging. The available protocols typically focus on isolating one or two cell types. Here, we present a protocol to simultaneously isolate high-quality and high-quantity cardiomyocytes and non-myocyte cells, including immune cells, from adult rat hearts. We describe steps for purifying cells using bovine serum albumin. We also detail procedures for viability analysis and cell type identification using fluorescence-activated cell sorting.

Before you begin

The protocol below describes the specific steps for simultaneously isolating high-quality and high-quantity cardiomyocytes and non-myocyte cells from adult rat hearts. Before you begin, please be sure the following steps are completed. Secure an approval of an animal protocol from the Institutional Animal Care and Use Committee before any animal experiments. Collect all required materials and equipment. Refer to key resources table and materials and equipment sections for a complete list of materials and equipment. Prepare stock solutions, working solutions, and culture media ahead of time. Prepare the enzymatic digestion solutions freshly on the isolation day.

Institutional permissions

All animal work described here was approved by the Institutional Animal Care and Use Committee at the Providence VA Medical Center and conformed with the Health Research Extension Act, US Public Health Service, and US National Institutes of Health policy.

Background

The myocardium consists of different cell types, which interact in a dynamic fashion to maintain the physiological function in the normal heart and to respond to alterations in pathological stimuli. It has been long viewed that cardiac myocytes, fibroblasts, endothelial cells, and vascular smooth muscle cells are the major cellular constituents of the heart and together they maintain the electrical, chemical, and biomechanical functions of the heart.^5,6 In addition to those cell types, immune cells are increasingly appreciated as another important cellular constituent in the normal and diseased hearts.^{7,8,9,10,11,12} They store and release a variety of biologically active mediators including various cytokines that can critically regulate myocardial homeostasis and function via autocrine, paracrine, and cell-cell interactions.^13,14,15,16

Preclinical animal models are widely used to study the underlying mechanisms of cardiac diseases.¹⁷ Isolation of different cell types from the heart is a powerful approach for understanding cellular compositions in normal hearts and their alterations during the progression of cardiac diseases. Rat is a standard model organism to study the heart and has some unique features: there are specific physiological differences in the heart between rat and mouse (e.g., heart rate, size, and amount of collagen); cardiac phenotypic changes in certain disease models such as pulmonary hypertension are more pronounced in rats than mice; because of the larger size of the rat hearts, more cells can be isolated from adult rat hearts for in vitro mechanistic studies, such as those involving gene manipulation and drug treatments. However, due to the large rat myocardium (10–12-fold more than mouse myocardium), isolation of high-quality and high-quantity cells from adult rat hearts is generally challenging. This is particularly true for isolation of the less abundant cell types such as immune cells.

Here, we present a modified isolation protocol utilizing Langendorff perfusion and subsequent enzymatic digestion^1,2,3,4 to isolate cardiomyocytes and non-myocytes cells including immune cells with high quality and high quantity. In addition, we present a detailed protocol using fluorescence-activated cell sorting (FACS) for identification of live non-myocyte cell types. Advantages from our isolation approach include i) Isolation of immune cells together with all other cardiac cells from the same heart, which allows for investigations of multiple cell types simultaneously. This approach will also reduce the animal number to be used if two or more cell types are analyzed; ii) Establishment of a standardized protocol with consistent enzyme composition and concentration, which will allow the comparison of data from different laboratories; iii) Cardiomyocytes and non-myocyte cells are purified using bovine serum albumin (BSA) solutions that are easy to prepare and have minimal effects on cell viability (compared to Percoll gradient); and iv) Isolated cells can be used for further experiments such as single cell RNA sequencing, gene expression profiling, functional and biochemical assays, or can be cultured for various in vitro experiments. In this protocol, we also discuss potential problems and the solutions to help users establish this technique in their laboratories successfully. Together, we describe a standard protocol that can be adapted to each laboratory and is valuable for cardiac studies requiring analysis of single cells of different cell types isolated from adult rat hearts.

Before you begin, please find a list of specific reagents, labware, and equipment used in this protocol in the key resources table.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
CD45 (1:50) – FITC	Thermo Fisher Scientific	Cat# 11-0461-82
CD31 (1:50) – PE	BD Biosciences	Cat# 555027
Vimentin (1:50) – FITC	Santa Cruz	Cat# 6260
Chemicals, peptides, and recombinant proteins
Sodium chloride (NaCl)	Sigma	Cat# 746398
Potassium chloride (KCl)	Sigma	Cat# 746436
Magnesium sulfate anhydrous (MgSO4)	Sigma	Cat# M2643
Potassium phosphate monobasic (KH2PO4)	Fisher	Cat# P380
Sodium bicarbonate (NaHCO3)	Sigma	Cat# S6014
Dextrose	Sigma	Cat# G5767
HEPES	Sigma	Cat# H4034
Calcium chloride (CaCl2)	Sigma	Cat# C3306
Collagenase II	Worthington	Cat# 4176
Hyaluronidase	Sigma	Cat# H2126
Trypsin IX	Sigma	Cat# T0303
DNase type 1	Worthington	Cat# L5002140
Dulbecco’s modified Eagle’s medium (DMEM), low glucose, pyruvate, no glutamine, no phenol red	Gibco	Cat# 11054-001
Penicillin-Streptomycin (PS)	Sigma	Cat# P0781
Bovine serum albumin (fatty acid free)	Gold Biotechnology	Cat# A-420-1
Laminin	BD Biosciences	Cat# 354323
Albumin	Sigma	Cat# A7906
L-carnitine	Sigma	Cat# C0283
Creatine	Sigma	Cat# C3630
Taurine	Sigma	Cat# T8691
Insulin solution (10 mg/mL)	Sigma	Cat# I0516
Medium 199	Sigma	Cat# M7528
Dulbecco’s modified Eagle’s medium (DMEM), high glucose (HG)	Gibco	Cat# 12800-017
Fetal bovine serum (FBS)	Sigma	Cat# F6182
Triton X-100	Sigma	Cat# SLBZ4187
Goat serum	Sigma	Cat# G9023
Software and algorithms
NanoCellect Volf Viewer 3.2.3603	NanoCellect	Version 3.2.3603
Experimental models: Organisms/strains
Rats (Sprague Dawley)	Charles River Laboratories	Strain Code: 001
Other
LIVE/DEAD Fixable Orange (602) Viability Kit, for 561 nm excitation	Thermo Fisher Scientific	Cat# L34983
Heating coil (for perfusion)	Radnoti	Cat# 158831
Peristaltic pump drive	Masterflex	Cat# 170100A
Shaking water bath	Edvotek	Model# SHWB10
Circulating water bath	Cole-Parmer	1B1190564
Narrow pattern forceps	Fine Science Tools	FST 11003-12
Mayo scissors (large scissor for surgery)	Fine Science Tools	Cat# 14512-15
Weight boat large	Heathrow Scientific HD	Cat# 14251C
Nylon mesh 200 μm	ELKO Filtering	Cat# 03-200/45
Cell strainer 70 μm	CELLTREAT Scientific Products	Cat# 229483
5 mL polystyrene round-bottom tube with cell strainer cap	Falcon	Cat# 352235
Wolf microfluid sorting cartridge, bulk	NanoCellect	Cat# 150410
Calibration beads w/ 0.01% Tween 20, 0.02% NaN3	NanoCellect	Cat# 170111

Materials and equipment

Ca²⁺ free Krebs-Henseleit (KH) Buffer (20x Stock solution)

Concentration in 20x	Chemical name	Molecular weight	1 L	0.5 L
2.36 M	NaCl	58.44	137.9 g	68.95 g
93.9 mM	KCl	74.55	7.0 g	3.50 g
24.0 mM	MgSO4	120.4	2.89 g	1.45 g
24.0 mM	KH2PO4	136.09	3.27 g	1.64 g
0.5 M	NaHCO3	84.01	42.0 g	21.0 g
-	Milli-Q H2O	-	Bring up to 1 L	Bring up to 0.5 L

Ca²⁺ free Krebs-Henseleit (KH) Buffer (1x Working solution)

Concentration in 1x	Reagent	Amount of 20x Stock solution	Molecular weight	Amount in weight or volume
118.0 mM	NaCl	100 mL	-	-
4.7 mM	KCl	100 mL	-	-
1.2 mM	MgSO4	100 mL	-	-
1.2 mM	KH2PO4	100 mL	-	-
25.0 mM	NaHCO3	100 mL	-	-
11.0 mM	Dextrose	-	180.2	4.0 g
8.4 mM	HEPES	-	238.8	4.0 g
-	Milli-Q H2O	-	-	1500 mL

DMEM/PS medium for cardiomyocytes

Reagent	Amount
HEPES	5.94 g
DMEM (Gibco 11054–001)	1 L
PS	10 mL

Culture medium for fibroblasts

Reagent	Amount
DMEM (Gibco 12800–017) supplemented with 3.7 g/L NaHCO3	890 mL
FBS	100 mL
PS	10 mL

ACCTI medium (also known as adult cardiomyocyte culture medium)

Reagent	MW or conc.	In 500 mL of medium 199	Final concentration
Albumin	-	1 g	2 g/L
L-Carnitine	197.9	0.2 g	2 mM
Creatine	149.15	0.375 g	5 mM
Taurine	125.1	0.31 g	5 mM
Insulin Solution	10 mg/mL	28.67 μL	10-7 M
PS	100x	5 mL	1x

Enzyme I Isolation Solution

Reagent	Amount	Concentration in solution
Collagenase II	30 mg	0.3 mg/mL
Hyaluronidase	30 mg	0.3 mg/mL
CaCl2 (1 M)	5 μL	50 μM
1x KH Buffer	100 mL	-

Enzyme II Isolation Solution

Reagent	Amount	Concentration in solution
Enzyme I Solution	28 mL	-
Trypsin IX (0.6 mg/mL)	1 mL	0.02 mg/mL
DNase I (0.6 mg/mL)	1 mL	0.02 mg/mL
CaCl2 (1 M)	15 μL	0.5 mM

Washing solution for cardiomyocytes

Reagent	Volume
1x KH Buffer	15 mL
DMEM/PS medium for cardiomyocytes	15 mL

Composition of perfusion system

The Langendorff perfusion system consists of two parts: 1) Circulating water bath that is used to circulate the warmed water to the heating coil to keep the digestion solution at 37°C (Figures 1A) Peristaltic pump that circulates the digestion solution to retrogradely perfuse the heart via aorta (Figure 1B). In addition, a shaking water bath with controlled temperature at 37°C is used for further tissue digestion in the enzyme solution II after perfusion (Figure 1C).

Figure 1.

Langendorff perfusion system

Illustration of key components.

(A) Circulating water bath with temperature control.

(B) Peristaltic pump for circulating Enzyme I solution.

(C) Shaking water bath for tissue digestion with Enzyme II solution.

Prepare perfusion system

Timing: 15 min

Prepare the Langendorff system and the surgical area

• •
  Prepare the Langendorff system:

  • ○
    Turn on the circulating water bath, set the temperature to ∼38°C (Figure 1A).
  • ○
    Turn on the shaking water bath (Figure 1C).
  • ○
    Place the perfusion tubing into proper channel(s) of the perfusion pump and turn on the perfusion pump (Figure 1B).
  • ○
    Rinse the perfusion system with ddH₂O for 5 min and then 70% ethanol for 5 min.
  • ○
    Measure and adjust the temperature of the solution exiting out of the cannula to 37°C.

    Note: Change the temperature setting of the circulating water bath as needed.

  • ○
    Measure and adjust the flow rate to ∼ 8 mL/min.
  • ○
    Measure the time of the solution from getting into the perfusion tubing to exiting from the cannula.
  • ○
    Fill up the system with Ca²⁺-free, 1x KH buffer, and circulate for a few minutes.
  • ○
    Inspect and get rid of any air bubbles in the system.
  • ○
    Keep the clip and suture nearby for heart cannulation via aorta.

    Note: Now the perfusion system is ready to be used.

• •
  Equipment and materials for heart harvesting:

  • ○
    Set up the surgical area.
  • ○
    Place all the surgical tools (scissors and forceps) in a beaker filled with 70% ethanol for at least 15 min.
  • ○
    Fill two 6-cm culture dishes with ice cold Ca²⁺-free, 1x KH buffer and leave them on ice.
  • ○
    Have the isoflurane anesthetic vaporizer ready.
  • ○
    Have a 1 mL syringe filled with heparin.

Prepare reagents for isolation

Timing: 30 min

CRITICAL: Use filter-sterilized medium and sterilized nylon mesh and glassware. Set up experimental conditions in the cell culture room for cell isolation.

• •
  Prepare solutions and culture media ahead of time:

  • ○
    Prepare 20x Ca²⁺ free, KH Buffer (Stock Solution).

    Note: Dissolve all chemicals in Milli-Q H₂O, sterilize using 0.22 μm filter in a class II biological safety cabinet, and store the stock solution at 22°C–25°C in a closed container for up to 6 months.

  • ○
    Prepare 1x Ca²⁺ free, KH buffer (Working Solution).

    Note: Mixing all components in Milli-Q H₂O, sterilize using 0.22 μm filter in a class II biological safety cabinet, and store the working solution in a closed container at 4°C for up to 3 months.

  • ○
    Prepare DMEM/PS medium.

    Note: The final concentration of HEPES is 25 mM. Adjust pH to 7.3 and then sterilize using 0.22 μm filter in a class II biological safety cabinet. Store at 4°C for up to 3 months.

  • ○
    Prepare ACCTI medium (A.k.a. adult cardiomyocyte culture media).

    Note: Weigh out Albumin, L-Carnitine, Creatine, Taurine and put them into a sterile glass bottle. Add 100 mL of Medium 199 (from a 500 mL medium bottle) inside a class II biological safety cabinet and mix until the chemicals are fully dissolved. Add this 100 mL Medium 199 supplemented with the chemicals back to the same 500 mL medium bottle, add insulin and PS, and sterilize using 0.22 μm filter. The complete medium can be stored at 4°C for up to 3 months.

• •
  Prepare reagents and solutions in the isolation day for heart digestion (enough for 1–2 isolations):

  • ○
    Have the following materials ready and use in a class II biological safety cabinet.

    • -
      Autoclaved nylon mesh filters (2 pieces).
    • -
      Sterile 70-μm filters (2 pieces).
    • -
      5 mL, 10 mL, and 25 mL serological pipets.
    • -
      15 mL and 50 mL conical tubes.
  • ○
    Prepare Enzyme I Solution and keep at 22°C–25°C.

    Note: Prepare the solution freshly on the day of isolation. Accurately measure enzymes and put them into a sterile glass jar. In a class II biological safety cabinet, add the amount of 1x KH buffer and CaCl₂. Mix well until complete dissolution is achieved.

    CRITICAL: There is batch difference in collagenase II. It is critical to test collagenase II from different batches before ordering. For long-term storage, keep collagenase II at −20°C or below.

  • ○
    Prepare Enzyme II Solution and keep at 37°C.

    Note: Prepare in a separate sterile glass jar using the Enzyme I solution that is freshly prepared. All steps are performed inside a class II biological safety cabinet. Mix thoroughly.

    Note: Trypsin IX and DNase I solutions are prepared ahead of time to the indicated concentration using 1x KH Buffer, aliquoted, and stored at −20°C for usage.

  • ○
    Prepare the Washing Solution for cardiomyocytes and keep it at 37°C.
  • ○
    Aliquot 50 mL of DMEM/PS medium and keep it at 37°C.
  • ○
    Prepare 6.5% BSA solution for cardiomyocyte purification by dissolving 0.65 g of BSA in 10 mL of washing solution. Prepare fresh and keep at 22°C–25°C.
  • ○
    For cardiomyocyte plating and/or culture:

    • -
      Coat glass bottom dishes or plates using laminin (10–20 μg/mL in DMEM/PS) and allow it to sit for at least 30 min at 22°C–25°C before use.

      Note: Volume of laminin added should be enough to cover the dish/plate (e.g., 1 mL per well of a 6-well plate. Unused laminin-coated dishes or plates (with laminin solution in) can be kept inside the biological safety cabinet or incubator for 3–5 days.

    • -
      Aliquot sufficient ACCTI medium for cardiomyocyte culture.
  • ○
    Prepare culture medium for fibroblasts by adding 10% FBS and 1% PS in DMEM (HG) media. Aliquot and warm at 37°C.
  • ○
    Aliquot and warm DMEM (HG) media at 37°C.
  • ○
    Prepare 1% BSA solution by dissolving 400 mg of BSA in 40 mL of 1x KH buffer. Add 8 mL of this solution per 15 mL conical tube. Keep at 22°C–25°C.

Prepare flow cytometer

Timing: 30 min

CRITICAL: Ensure that all solutions intended for use in the flow cytometer instrument undergo filtration using a 0.22 μm filter.

• •
  Set up WOLF G2 Cell Sorter:

  • ○
    Gather a cartridge and calibration beads.
  • ○
    Follow manufacture settings to turn on the instrument and open the software.
  • ○
    Prepare the instrument with the following steps: cartridge insertion, tubing connection, cartridge priming and chip alignment, chip calibration and flush the cartridge with filtered 1x PBS.

Step-by-step method details

This protocol is presented in four subsections below starting with the heart harvest and digestion through cell analysis by FACS.

Heart harvesting and digestion

Timing: 45 min

• 1.
  Heart collection and cannulation:

  • a.
    Sacrifice a rat for heart collection.

    • i.
      Heparinize a rat with sodium heparin intraperitoneally injected at 1000 U/kg and wait for 15 min.

      Note: This step is to prevent blood coagulation and embolism in coronary circulation.

    • ii.
      Put the rat into the induction chamber and fill the chamber with 3%–5% isoflurane.
    • iii.
      When the rat is anesthetized, quickly transfer it to the surgical area, lay it down on its back, and continue anesthesia with isoflurane via nose cone.
    • iv.
      Confirm the rat is in deep anesthesia with no response to toe pinch.
    • v.
      Spray chest and upper abdomen with 70% ethanol.
    • vi.
      Perform a laparotomy and then bilateral thoracotomy to expose the heart and lungs.
    • vii.
      Euthanize the rat via exsanguination by cutting the inferior vena cava.
  • b.
    Quickly dissect the aorta for cannulation.

    • i.
      Quickly cut off the heart at the aortic arch level or above.
    • ii.
      Immediately put the heart into one of the 6-cm culture dishes filled with ice-cold Ca²⁺-free, 1x KH buffer.

      Note: The heart stops contracting within a few seconds.

    • iii.
      Quickly transfer the heart to the 2^nd 6-cm culture dishes filled with ice-cold Ca²⁺-free, 1x KH buffer.
    • iv.
      Quickly dissect the ascending aorta by removing surrounding tissues.

      CRITICAL: Be sure to keep the ascending aorta as long as possible for easy cannulation.

  • c.
    Quickly cannulate heart to the Langendorff perfusion system via the aorta (Figure 2).

    • i.
      Turn on the perfusion pump to circulate the Ca²⁺-free, 1x KH buffer.
    • ii.
      Put the aorta on the cannula connected to the Langendorff system.

      CRITICAL: The cannula needs to be inserted into the ascending aorta but does not pass the aortic valve. See Figure 2B and refer to problem 1 in the troubleshooting section.

    • iii.
      Hold the heart in place using a clip.
    • iv.
      Tie the aorta to the cannula using a suture underneath the clip.

      CRITICAL: Make sure there is no leak from the tie. Refer to problem 1 in the troubleshooting section for further information.

  • d.
    If the cannulation is successful, the blood in the coronary circulation will be quickly rinsed out and the coronary vessels will become clear.

    Note: The myocardium will show light pink color during the entire perfusion period.

    CRITICAL: Quickly dissecting the ascending aorta and cannulating the heart is the most crucial step for this isolation protocol, which directly affects the quality and quantity of the isolated cells. This step from removing the heart to cannulating the heart to the Langendorff system typically takes ∼1-2 min and will need some practice for new users.

• 2.
  Perfuse the heart with Enzyme I solution:

  • a.
    Continue to perfuse the heart with Ca²⁺-free, 1x KH buffer for 2 min to rinse all the residual blood in the coronary circulation.
  • b.
    Quickly switch the perfusion solution from KH buffer to digestion enzyme I solution.

    CRITICAL: Do not introduce air bubbles to the perfusion system during the switch. If the heating coil does not have a physical bubble trap, the perfusion pump has to be turned off during the switch and then turned on. Refer to problem 2 in the troubleshooting section.

  • c.
    Use the time measured from the prepare perfusion system section to estimate the time that the enzyme I solution reaches the heart and start to recycle the enzyme solution.
  • d.
    Start the timer when the enzyme I solution reaches the heart and continue to perfuse the heart for approximately 10 min.
  • e.
    Use forceps with flat tips to check the softness of the heart when it is almost 10 min.
  • f.
    Stop the perfusion when the heart is very soft with little resistance when squeezing it with the forceps.

    CRITICAL: Stopping the perfusion at the right time is critical. Stopping earlier will lead to under-digestion, while stopping later will cause over-digestion. In our experience with the described flow rate and the peristaltic pump we used, it will take about 10 min. The perfusion time is expected to be longer for ageing hearts or diseased hearts with cardiac remodeling. Refer to problems 3, 4, 5 in the troubleshooting section for further information.

• 3.
  Digestion with Enzyme II solution:

  • a.
    Remove the heart from the cannula and transfer to a 6-cm dish filled with ∼10-15 mL digestion enzyme II.
  • b.
    Trim off the aorta and the left and right atria.

    Note: If isolating cells from each ventricle is preferred, this is the time to separate the left and right ventricles and process them separately from this time point.

  • c.
    Cut the ventricular tissues using a fine scissor into small pieces within 1 min.
  • d.
    Pool the minced tissue back to the original glass bottle with the enzyme II solution in total 30 mL.
  • e.
    Incubate at 37°C in a shaking water bath setting at 125 rpm for 10 min.
  • f.
    After time is up, take the bottle from the shaking water bath and proceed to cell dissociation in the biosafety cabinet.

Figure 2.

Cannulation of an adult rat heart

(A) Adult rat heart cannulated for perfusion.

(B) Schematic view of the position of the cannula inside the aorta relative to the aortic valve and the suture tie.

Cell dissociation and preparation for culture/analysis

Timing: 30 min

Figure 3 illustrates the steps for cell dissociation and preparation for culture/analysis.

CRITICAL: All procedures for cell suspension preparation must be conducted in a class II biosafety cabinet to avoid contamination.

CRITICAL: Cardiomyocytes and non-myocyte cells can be isolated and collected simultaneously during cell dissociation.

• 4.
  Cell dissociation and preparation (Figures 3A–3C):

  • a.
    Spray the outside of the glass bottle containing the digested heart tissue with 70% ethanol before placing it inside the biosafety cabinet.
  • b.
    Gently pipette the tissue 5–10 times in the glass bottle using a 25 mL serological pipette.

    Note: Gently pipette to avoid cell damage. The digested tissue solution should be able to go through the tip of a 25 mL serological pipette easily (if not, it indicates under digestion).

  • c.
    Filter the digested tissue solution through a 200-μm nylon mesh into a 50 mL conical tube. The total volume should be around 30 mL.
  • d.
    Centrifuge at 17 × g for 3 min at 22°C–25°C.
  • e.
    Supernatant contains the non-myocyte cells, while the pellet contains cardiomyocytes (Figure 3C).
  • f.
    Carefully take out the supernatant and put it into a 50 mL conical tube filled with 10 mL fibroblast culture medium for non-myocyte cell preparation (see step 6).

    CRITICAL: Step 5 below is for cardiomyocyte purification and culture. Step 6 is for preparation of non-myocyte cells.

• 5.
  Cardiomyocyte purification and culture (Figure 3D):

  • a.
    Resuspend the cardiomyocyte pellet in a 10 mL washing solution and let it sit for up to 5 min, during which the myocytes will settle down to the bottle by gravity.
  • b.
    Carefully remove the top 5 mL of the supernatant without interrupting the loose cardiomyocyte pellet.
  • c.
    Gently mix the remaining 5 mL solution containing cardiomyocytes and gently load it onto the top of the 6.5% BSA prepared in a 50 mL conical tube.

    CRITICAL: Be slow. After loading, having a clear two-phase separate is key.

  • d.
    Let it sit for ∼13 min at 22°C–25°C, during which the rod-shaped healthy cardiomyocytes will settle down to the bottom of the tube by gravity.

    Note: Pay attention to the time. Do not let it sit longer than 18 min; otherwise, the rounded, non-healthy cardiomyocytes will also reach the bottom.

  • e.
    Aspirate the supernatant and resuspend the purified cardiomyocyte pellet in 10 mL DMEM/PS medium.
  • f.
    Check the quality and assess the cell number under a microscope.

    CRITICAL: At this step, cardiomyocytes can be used for various analyses such as electrophysiology, contractility measurement, and acute treatment.

    Note: Typical yield of rod-shaped healthy cardiomyocytes (Figures 4A and 4B) from entire ventricles of an adult rat heart is around 4–5 × 10⁶. Cell density will depend on the volume of DMEM/PS medium used for resuspension in the previous step. Refer to problems 4 and 5 in the troubleshooting section for further information.

  • g.
    To culture the cardiomyocytes, plate them in the desired density to laminin-coated culture dishes or plates.

    Note: Before plating, completely aspirate all the laminin solution in each dish or well. Any remaining laminin solution may affect cardiomyocyte viability.

  • h.
    Put cells into the incubator and let them sit for 1 hr to attach.
  • i.
    After that, change the medium to the ACCTI media for culture.

    Note: Cardiomyocytes are sensitive to sudden temperature changes. It is recommended to not put the dishes directly on the steel surface of the biosafety cabinet during the handling; instead, put them onto a polystyrene foam.

• 6.
  Preparation of non-myocyte cells (Figures 3E and 3F):

  • a.
    Centrifuge the collected supernatant containing non-myocyte cells at 738 xg for 5 min at 22°C–25°C.
  • b.
    Aspirate the supernatant, the pellet is ready to be used for purification of non-myocyte cells and preparation for FACS (Figure 3E).

    Note: The size of the cell pellet reflects the overall yield of the non-myocyte cells. Refer to problems 4 and 5 in the troubleshooting section for further information.

    Alternative: As illustrated in Figure 3F, if a user just wants to get isolated cardiac fibroblasts for cell culture and subsequent assays, a simple “pre-plating” approach can be done, for which the cell pellet is resuspended in fibroblast culture medium, plated into a regular cell culture dish (no special coating required), and put into incubator at 37°C. After 2 h, wash the cells with 37°C 1x PBS for 3 times and then change to fibroblast culture medium (DMEM/high glucose supplemented with 10% FBS and 1% PS). This step critically removes unattached or loosely attached cells, including vascular smooth muscle cells, endothelial cells, and a few remaining myocytes. The high purity of the fibroblast preparations using this approach has been confirmed by positive staining for vimentin and the absence of staining for endothelial cells and vascular smooth muscle cell markers by our laboratory.¹

  • c.
    Resuspend pellet in 12 mL of fibroblast culture medium (DMEM/high glucose supplemented with 10% FBS and 1% PS).
  • d.
    Filter twice using two 70 μm filters to remove myocytes, large cell clusters, and debris.

    CRITICAL: These will clog the flow cytometer.

  • e.
    Slowly load all 12 mL cells into 4 of 15 mL conical tubes containing 8 mL of 1% BSA solution (Figure 3E) to further get rid of debris.

    CRITICAL: Be slow. After loading, having a clear two-phase separate is key.

  • f.
    Centrifuge at 738 xg for 5 min at 22°C–25°C.

    Note: A pellet will form, and supernatants will have a light pink color.

  • g.
    Aspirate supernatant as waste and add 12 mL of warm fibroblast culture medium to each pellet for washing; gently mix.
  • h.
    Centrifuge at 738 xg for 5 min at 22°C–25°C. Aspirate supernatant as waste.
  • i.
    Resuspend the pellet according to the size of the pellet (typically 6 mL for the whole heart).
  • j.
    The non-myocyte suspension is now ready for staining and FACS.

Figure 3.

Cell dissociation and preparation

Illustration of key steps after enzyme digestion for cell dissociation and preparation for culture or analysis.

(A) Filtering the digested tissue using a 200-μm filter.

(B) Centrifugation.

(C) Separation of cardiomyocyte and non-myocyte cell portions after low-speed centrifugation.

(D) Cardiomyocyte purification and plating.

(E) Non-myocyte cell purification for FACS.

(F) “Pre-plating” approach for fibroblast culture only.

Figure 4.

High quality viable adult rat cardiomyocytes from the isolation day and 6 days post-isolation

(A and B) Representative images of cardiomyocytes from isolation day at 4x (A) and 40x (B).

(C and D) Representative images of cardiomyocytes 6 days post-isolation at 4x (C) and 40x (D).

Cell Staining for FACS

Timing: 50–120 min

Staining protocol for non-myocyte population. All steps are performed at 22°C–25°C.

• 7.
  Superficial marker labeling (e.g., Live/Dead dye, CD45, CD31):

  • a.
    Take proper amount of non-myocyte cell suspension and add the following markers.

    Note: use fluorescent conjugated primary antibodies if possible.

    • i.
      Stain with Live/Dead dye (1:1000 dilution).
    • ii.
      Stain with anti-CD45 antibody (immune cell marker; 1:50 dilution).
    • iii.
      Stain with anti-CD31 antibody (endothelial cells marker; 1:50 dilution).
  • b.
    Allow samples to sit in the dark for 30 min.
  • c.
    Wash with 1x PBS.
  • d.
    Centrifuge the samples at 416 xg for 5 min.
  • e.
    Carefully remove the supernatant as waste.
  • f.
    Resuspend the pellet in 1 mL of filtered 1x PBS.
  • g.
    The samples are now ready for FACS analysis/sorting.

    CRITICAL: Prolonged labeling time and waiting time before FACS will reduce the cell viability. Refer to problem 5 in the troubleshooting section for further information.

• 8.
  Intracellular marker labeling (e.g., Vimentin):

  • a.
    Fix cells with 4% PFA for 15 min.
  • b.
    Wash with 1x PBS and centrifuge at 900 xg for 5 min.
  • c.
    Remove supernatant.

    CRITICAL: Fixed cells are lighter and become transparent. In case of no visible pellet, opt for leave ∼100 μL to avoid discarding the pellet by mistake.

  • d.
    Resuspend pellet in 0.1% Triton x-100 for 10 min.
  • e.
    Wash with 1x PBS.
  • f.
    Centrifuge at 900 xg for 5 min. Remove supernatant.

    CRITICAL: The pellet is transparent. Be careful not to discard it together with supernatant.

  • g.
    Add 100 or 200 μL of blocking solution (5% goat serum diluted in 1x PBS) and incubate for 30 min.
  • h.
    Add primary antibodies. In this case, add anti-Vimentin antibody (fibroblast cells marker; 1:50 dilution) and incubate for 60 min in dark.
  • i.
    Wash with 1x PBS.
  • j.
    Centrifuge at 900 xg for 5 min. Remove supernatant.

    CRITICAL: The pellet is transparent. Be careful not to discard it together with supernatant.

  • k.
    Resuspend the pellet using 1 mL of 1x PBS and the sample is ready for FACS.

FACS analysis using WOLF cell sorter

Timing: 20 min

We used the WOLF Cell Sorter (NanoCellect) for the FACS analysis. WOLF Cell Sorter is a microfluidic-based cell sorter, which has low pressure that does not exert stress on the cells or create hazardous aerosols. This is especially important for sorting fragile cell types and maintaining high cell viability for downstream live cell applications.

For analysis only, any flow cytometers equipped with a proper laser and filters should work. It is necessary to check whether a flow cytometer meets the requirements of a specific application before any experiments.

CRITICAL: Ensure that all solutions used with the WOLF system are filtered twice through a 0.22 μm filter.

• 9.
  Loading sample:

  • a.
    After filtering, place the sample in the sample channel.
  • b.
    Press the “New sample” and “Analyze” buttons.
  • c.
    In the resulting “Name for new sample” pop-up window, type a name for the sample, and select the “New sample” button to trigger the uptake of the sample.
  • d.
    Run sample for a few seconds.
  • e.
    Check the average “events/μL”.

    Note: If it exceeds the expected value (>1000 events/μL), dilute the sample to achieve the desired cell concentration.

  • f.
    Adjust the threshold and voltage as needed.
  • g.
    When counts reach 5000 events, reset counts, and then start the analysis.
• 10.
  Gating.

  • a.
    Gate “Cells”.

    • i.
      Select the cells by using a polygon gate.
    • ii.
      Select cells based on size (FSC) and complexity (BSC) in bivariate plots and log scales.
    • iii.
      Name the gate as “Cells”.
  • b.
    Gate “Singular Cells”.

    • i.
      Double-click on “Cells” gate to pull out a new plot.
    • ii.
      In this plot, select FSC-width against FSC-H to visualize cluster of single cells.
    • iii.
      Draw a rectangular gate to select single cells and name as “Singular”.
  • c.
    Analyzing the samples that serve as negative control (e.g., staining without adding primary antibody).

    • i.
      Load a sample.
    • ii.
      Select a new 1D plot.
    • iii.
      Choose the channel based on the excitation/emission wavelengths of fluorescein.
    • iv.
      Determine the fluorescence range using the negative control (Figures 5 and 6 - Top Panels).
  • d.
    Analyzing the cell samples labeled by primary antibody and fluorescence probes.

    • i.
      Load a sample.
    • ii.
      Select a new 1D plot.
    • iii.
      Using the gating for negative control from step c above to determine the positive signals/peak (Figures 5 and 6 – Bottom Panels).

Note: Refer to problem 6 in the troubleshooting section for further information.

CRITICAL: Negative control is required for each fluorescence probe with different excitation/emission wavelengths.

Figure 5.

Viability assessment using FACS and live/dead dye staining

(Top) Representative FACS data from negative control cells (without dye staining). (Bottom) Representative FACS data from cells stained with LIVE/DEAD Dye. Live cells show negative fluorescence, whereas dead cells show positive fluorescence. Percentages of cells with negative or positive fluorescence are also included to show the percentages of live and dead cells, respectively.

Figure 6.

FACS of non-myocyte cells with cell type specific markers

(Top Panel) Representative FACS data showing negative controls for the indicated markers. (Bottom Panel) Representative FACS data showing cells stained CD45 (left), Vimentin (Vim, middle), or CD31 (right). Percentages of positively and negatively stained cells for each marker are included in the respective panel.

Expected outcomes

We describe here a highly practical protocol for simultaneous isolation of high-quality and high-quantity cardiomyocytes and other non-myocyte cells including fibroblasts, endothelial cells, and immune cells from adult rat hearts. We also describe the detailed procedure to use FACS to identify/sort out different viable non-myocyte cell types.

Cardiomyocytes are the major cell type in the heart directly determining cardiac contractility. Our protocol can isolate high-yield viable cardiomyocytes from adult rat hearts (4–5 × 10⁶ per adult heart) that can be easily identified by their rod shape (Figure 4). The Purity of rod-shaped cardiomyocytes from our protocol is typically ∼95% (Figure 4A). The rod-shaped cardiomyocytes showing a clear striation pattern with an absence of membrane blebbing suggest the high quality of these cardiomyocytes (Figure 4B). These cardiomyocytes can be cultured for 6-days post isolation (Figures 4C and 4D), which provides sufficient time for in vitro manipulation and treatments. Based on our experience, using oxygenated enzyme solutions do not further increase the yield or quality of cardiomyocytes. We also found that 2,3-butanedione monoxime (BDM) is not required, particularly it can significantly interfere with the contractility measurements of the isolated cardiomyocytes. Different from mouse cardiomyocytes, cardiomyocytes from adult rats do not require steps for slow calcium recovery.

Unlike cardiomyocytes that can be easily identified under a microscope, the non-myocyte cells, which are a heterogeneous mix of cells, need to be assessed based on their expression of specific molecular markers. Specifically, we employed FACS to determine the viability, cell types, and yields by labeling them with live/dead dye and the established specific markers. Figure 5 shows the representative FACS determining cell viability of isolated non-myocyte cells that were stained with live/dead dye (without live/dead dye as negative controls). From our cell isolations, we can consistently get high cell viability at 91 ± 5% (mean ± SD, n = 4 independent isolations). Given the increased demands in the field to analyze cells from left and right ventricles separately, we also separated left and right ventricles after Langendorff perfusion to isolate different cell types from each ventricular free wall. To identify each cell type and yield, we labeled cardiac fibroblasts using vimentin, endothelial cells using CD31, and immune cells using CD45. Please note that we used CD45 as a pan-marker for immune cells to demonstrate the feasibility of our protocol in isolation of high quantity viable immune cells in general. It will be up to the users to further characterize diverse immune cell populations, including neutrophils, macrophages, and lymphocytes with their specific subpopulation markers. Figure 6 shows the representative FACS results. Our data in Table 1 show that, among the non-myocyte cells in the left ventricle, 31 ± 4% is vimentin positive cells, 35 ± 2% is CD31 positive cells, and 17 ± 2% is CD45 positive cells. A similar cell composition is found in the right ventricle. Consistent with the fact that the left ventricular mass is much bigger than the right ventricular mass in the normal heart, the estimated numbers of each cell type in the left ventricle are higher than that in the right ventricle (Table 1). Those cells then can be used for phenotypic analysis or used in cell culture for mechanistic studies.

Table 1.

Characterization of non-myocyte cells through FACS

Cell marker	Left ventricle (free wall)		Right ventricle (free wall)
	Percentage	Cell number (x106)	%	Cell number (x106)
Vimentin +	31 ± 4	0.94 ± 0.35	32 ± 5	0.17 ± 0.09
CD31 +	35 ± 2	0.94 ± 0.18	34 ± 7	0.19 ± 0.07
CD45 +	17 ± 2	0.37 ± 0.17	17 ± 3	0.10 ± 0.05

Mean ± SD; n = 3–4 independent isolations. Rat age ∼10–15 wks.

In addition, we would like to add the following notes: 1) While we used Sprague-Dawley rats for the data presented, the protocol can be used for different strains of rats in both male and female. 2) The protocol can be used for all ages of adult rats and both normal and diseased hearts. 3) For the isolation using ageing animals or disease hearts with significant remodeling and fibrosis development, it is necessary to increase the time for the Langendorff perfusion with the digestion enzymes. We suggest not changing other conditions unless it is absolutely necessary. 4) Collagenase II is critical for the isolation and needs to be tested before a new batch is to be used.

Limitations

We present here a highly practical isolation protocol with detailed steps. The most challenging part would be the step to quickly cannulate the heart via the aorta to the Langendorff perfusion system (Figure 2). However, based on our >15 years’ experience, this needs practice. With practice, all different levels of our trainees (research assistants, graduate students, and postdoctoral researchers) mastered the skill successfully.

Troubleshooting

Problem 1

Heart is cannulated but not digested properly (Step 1.c under heart harvesting and digestion section).

Potential solution

The problem could come from improper cannulation, in which the cannula is inserted into the left ventricular chamber that causes inefficient coronary perfusion and poor digestion of the heart. Leak from the tie can also cause a similar problem. Thus, it is critical during cannulation to be sure that the end of the cannula does not pass the aortic valve and there is no leak from the tie that holds the aorta to the cannula (Figure 2). In addition, heart showing dark red color is also a sign for improper cannulation and poor coronary perfusion.

Problem 2

Heart shows very pale or white color during perfusion (Step 2b under heart harvesting and digestion section).

Potential solution

This is typically caused by introducing air bubbles into the coronary circulation, which form air embolism and subsequently induce myocardial ischemia/death. It is important to remove any air bubbles in the perfusion system before heart cannulation and closely monitor any potential air bubble formed during the perfusion. To have a heating coil with an air bubble trap is very helpful.

Problem 3

Heart is cannulated properly, but myocardial tissue is not fully digested (Step 2f under heart harvesting and digestion section).

Potential solution

This could be caused by the short digestion time, so the tissue is only partially digested. It is critical to check the heart softness frequently at the end of perfusion to make sure the heart is digested properly.

Problem 4

No cells or very low yield of cells after isolation (Step 2f under heart harvesting and digestion section and Steps 5f and 6b under cell dissociation and preparation for culture/analysis section).

Potential problem and solution

This may be due to over-digestion or under-digestion of the cardiac tissue. Using the softness of the perfused heart to determine the proper digestion time. Stopping perfusion at the right time is important in this matter. For isolation of cells from ageing animals or disease hearts with significant remodeling and fibrosis development, it is necessary to increase the time for the Langendorff perfusion with the digestion enzymes in comparison to the cell isolation using normal young hearts.

Problem 5

Cell yield is good, but viability is low (Step 2f under heart harvesting and digestion section, Steps 5f and 6b under cell dissociation and preparation for culture/analysis section, and Step 7g under cell staining for FACS section).

Potential solution

Over-digestion of heat tissue could cause this phenomenon. This can be solved with proper digestion. Prolonged labeling time and waiting time before FACS could be another reason, for which it is important to plan the experiments efficiently to reduce the lag time between each step.

Problem 6

No positive peak in FACS analysis with cell type-specific markers (Step 10d under FACS analysis using WOLF cell sorter section).

Potential solution

If the isolated cells are viable and healthy, the problem is highly likely from the primary antibodies and the labeling process. It is necessary to validate that an antibody can be used for FACS and is specific to the protein to be labeled. For cell surface markers, be sure that the epitope to be detected by the antibody is on the cell surface; otherwise, fixing and permeabilizing cells are required for proper labels. Negative controls have to be added to all conditions for properly gating in FACS. Optimization for any new antibodies is critical.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Peng Zhang (peng_zhang@brown.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to the technical contact, Peng Zhang (peng_zhang@brown.edu) or Alexsandra Zimmer (alexsandra_zimmer@brown.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any unique datasets or code.