Preparation of Acute Myocardial Tissue Slices from Biopsies of Human Neonates and Infants with Congenital Heart Disease for Physiological Measurements

Summary

Cardiac slices are a unique model for cardiovascular research and bridge the gap between single-cell and whole-heart models. This protocol describes the preparation of viable cardiac slices from myocardial tissue samples excised during surgery for congenital heart disease.

Abstract

In cardiovascular research, diverse ex vivo models are used to investigate cardiac function. These models can be categorized according to their complexity, ranging from isolated cardiomyocytes to multicellular 3-dimensional tissue preparations, such as the Langendorff-perfused heart or coronary-perfused wedges. Cardiac tissue slices bridge the gap between these models, as their relatively low thickness overcomes the need for arterial perfusion, while the native cellular alignment and extracellular matrix structure are preserved. This enables the use of tissue when coronary perfusion is not available (e.g., tissue excised during surgery for congenital heart disease). The present protocol describes the preparation of viable cardiac slices from myocardial explants from neonate and infant patients undergoing surgery for congenital heart disease. Upon extraction, the myocardial tissue is transferred to oxygenated, ice-cold, low-calcium solution and transported to the laboratory. Thereafter, the tissue is pre-cut, embedded into low-melting agarose, and sectioned with a vibratome. Tissue recovery is promoted by the stepwise increase of calcium concentration, followed by gradual rewarming to 37 °C for 1 h in the measurement solution. Afterwards, the obtained acute myocardial slices can be used for physiological experiments. Representative results for isometric force measurements and action potential recordings are provided. The importance of the solution and the vibratome parameters to the preparation of viable cardiac slices, as well as limitations regarding the control of the fiber alignment and long-term culture, are discussed.

Introduction

Ex vivo cellular studies on myocardial function rely on a spectrum of models, ranging from isolated single cells to whole-heart preparations such as the Langendorff-perfused heart.

Although isolated cardiomyocytes are the key model for many research questions, they do not entirely reflect the in vivo situation because intercellular interactions and connections to an extracellular matrix (ECM) are missing^1,2. Moreover, enzymatic digestion during the dissociation of myocardial tissue can modify the electrophysiological properties of cardiomyocytes. For instance, Yue et al.³ demonstrated that delayed rectifier potassium channel configuration was dependent upon the isolation protocol.

Multicellular preparations, such as the Langendorff-perfused heart or coronary-perfused wedge preparation, on the other hand, provide cardiomyocytes in their native cellular and extracellular environment. This allows for the investigation of phenomena that require interactions such as the development of arrhythmia. To provide proper oxygenation and to cover the metabolic demands, they require arterial perfusion due to their relatively great thickness^1,4. This restricts the use of these techniques, especially in human myocardial preparations, as explanted whole hearts or at least large tissue samples with an intact coronary artery are required.

Organotypic tissue slices have been a popular in vitro model for physiological and pathophysiological investigations for many decades. Although well-established for organs such as the brain, liver, and kidney, the use of slice preparations for functional cardiovascular research has gained more interest only recently^1,5. With a few exceptions¹, myocardial slices have extended the methodological repertoire of cardiovascular research only in the last decade. Many studies have demonstrated that viable cardiac tissue slices of high integrity can be obtained from diverse species, including mouse^6,7,8, dog⁴, guinea pig^2,9, rabbit², zebrafish¹⁰, and human^4,5,11, and at different developmental stages.

Since the precision vibratome sectioning of slices thinner than 400 µm is feasible, adequate oxygenation and nutrient supply by diffusion can be ensured for cardiac slice preparations¹. Cardiac tissue slices show a more in vivo-like profile in terms of cell composition and extracellular matrix than single cardiomyocytes or cell culture models². As arterial perfusion is not required, this preparation technique can be used for small patient biopsies.

The present protocol describes a method to prepare viable myocardial slices obtained from right ventricular tissue samples. These biopsies are an essential component during surgery on neonate and infant patients with hypoplastic left heart syndrome (HLHS) and Tetralogy of Fallot (TOF), respectively, and are discarded if not used for experimental purposes.

Protocol

This study was approved by the local ethics committee of the Medical Faculty of the University of Cologne (reference no. 07-045) and complied with the Word Medical Association Declaration of Helsinki (7th revision, Fortaleza, Brazil, 2013). Written informed consent was given by the parents of each patient.

1. Laboratory Preparation

1. Switch on the water bath that controls the temperature of a custom-made jacketed vessel (Figure 1). Set the temperature to 42 °C to maintain the temperature of melted agarose in a beaker, which will be inserted later, at 37 °C .
2. Add 500 µL of the calcium solution to 1 L of solution A (Table 1) to obtain a low-calcium solution with a Ca²⁺ concentration of 0.05 mmol/L.
3. Fill the vibratome chamber with low-calcium solution A. Oxygenate the solution in the vibratome chamber by bubbling with pure O₂. Cool the solution around the vibratome chamber with ice.
4. Insert a new steel blade according to the manufacturer's manual.
   NOTE: The steel blade must be handled carefully to prevent injury. One blade is sufficient for the whole slicing procedure on one experimental day.
5. Prepare the "slice collector" tool by carefully breaking the tip of a glass Pasteur pipette and mounting a small Peleus ball on the broken side. Use a glass cutter to make a score on one side, close to where the taper starts. Wrap the pipette in paper tissue and gently press at the point of the score to break the glass.
   NOTE: Breaking glass and handling broken glass increases the risk for injuries. Work carefully and wear protective clothes (i.e., cut-resistant gloves and goggles). Alternatively, the opening of the plastic Pasteur pipettes can be widened by cutting them with scissors. However, the tissue slices tend to stick to the inside of plastic Pasteur pipettes more frequently than to glass Pasteur pipettes.
6. Weigh 0.4 g of low-melting agarose into a beaker and add 10 mL of low-calcium solution A and a magnetic stir bar.
7. Transfer the beaker to a microwave, set the power to 750 W, and heat for 10 s. Swirl the beaker a few times and heat for an additional 5 s to completely dissolve the agarose. Cover the beaker opening with aluminum foil.
8. Transfer the beaker into the custom-made jacketed vessel, which stands on a magnetic stirring device. Stir the agarose briefly at a high speed and then at a moderate speed to avoid bubble formation in the agarose. Keep the temperature at 37 °C.
9. Fill a "collection beaker" with 40 mL of low-calcium solution A and store it on ice. Oxygenate by bubbling with pure O₂.
10. Store a 10-cm Petri dish filled with 10-15 mL of oxygenated low-calcium solution A on ice.
11. Fill a small "transport Erlenmeyer flask" with low-calcium solution A and place it in a portable container with ice. Cover the Erlenmeyer flask opening with aluminum foil or thermoplastic paraffin sealing film.

2. Transferring the Tissue from the Operating Room to the Laboratory

1. Arrive at the operating room early. Define a time trigger indicating when to leave the lab depending upon local circumstances.
   NOTE: Here, researchers leave the lab approximately at the time of the sternotomy.
2. Upon arrival to the operating room, briefly discuss the tissue transfer with the surgeon's assistant at a convenient moment, specifically concerning the proper waiting area, the transfer procedure, and the signal for tissue receipt.
   NOTE: It is crucial that the tissue pieces are transferred into low-calcium solution A immediately after excision. Arranging with the surgeon's assistant helps to minimize the time delay between the myectomy and the transfer of the tissue into the solution without interfering with the sterility of the procedure.
3. Start oxygenating the ice-cold low-calcium solution A upon arrival to the operating room using a portable oxygen cylinder.
4. Before the surgeon starts the myectomy, take the transport Erlenmeyer flask out of the ice and wipe it dry. Wait at the arranged position until the surgeon's assistant is ready to drop the tissue directly into the low-calcium solution A.
5. Return the transport Erlenmeyer flask to the portable container for cooling, restart oxygenation, and return to the lab.
   NOTE: Here, the time interval between excision and arrival in the laboratory was 17 ± 4.5 min (mean ± standard deviation, n = 21).

3. Slicing

1. Transfer the tissue (collected in step 2) to a Petri dish filled with oxygenated low-calcium solution A on ice.
2. Pre-cut the tissue into smaller blocks (approximately 3 mm x 3 mm x 3 mm) using a scalpel.
3. Transfer a single tissue block into a cylindrical steel chamber (inner: Ø 1.5 cm, 0.9 cm depth; outer: Ø 2.0 cm, 1.1 cm depth). Remove the fluid using a pipette.
4. Pour the liquid agarose solution into the steel chamber and move the tissue block into the middle using a pipette tip.
5. Immediately cool down the steel chamber on ice to solidify the agarose.
6. Carefully retrieve the agarose block with the aid of a scalpel and glue it on the vibratome specimen holder using instant adhesive, applying gentle pressure. Remove excess instant adhesive with a scalpel and insert the specimen holder into the vibratome chamber.
7. Set the cutting thickness to 300 µm. Advance the blade to the tissue. Before entering the tissue, slow down the speed at which the blade is advancing to a minimum and set the oscillation frequency to between 70 and 80 Hz.
8. Transfer the tissue slices in the collection beaker filled with oxygenated, ice-cold, low-calcium solution A using the slice collector tool.

4. Preparation of Tissue Slices for Physiological Measurements

1. After the slicing is finished, fill up the collection beaker to 90 mL with oxygenated, ice-cold, low-calcium solution A.
2. Add 405 µL of calcium solution (Table 1) to increase the Ca²⁺ concentration to 0.50 mmol/L. Incubate on ice for 15 min.
3. After 15 min, add other 405 µL of calcium solution to further increase th eCa²⁺ concentration to 0.95 mmol/L.
4. After another 15 min on ice, collect the tissue slices and transfer them to a Petri dish at 4 °C and filled with the solution suitable for the intended type of subsequent measurement.
   NOTE: Physiological buffer solutions such as Tyrode's solution (solution A) or Krebs-Henseleit buffer, as well as cell culture media, such as Iscove's modified Dulbecco's medium, can be used for subsequent measurements. Particular attention should be paid to ensuring an appropriate calcium concentration between 1.2 and 2.0 mmol/L.
5. Place the Petri dish into a humidified incubator at 37 °C to slowly rewarm the tissue slices.
   NOTE: 37 °C is typically reached after 1 h.

Results

Pictures of typical myocardial tissue slices obtained using the present protocol are shown in Figure 2. The prepared slices can be used for physiological measurements, such as force measurements or electrophysiological recordings.

For force measurements, the tissue slices were mounted onto J-shaped steel needles connected to an isometric force transducer. The slices were immersed in the measurement ...

Discussion

Cardiac slices bridge the gap between single-cell and complex multicellular models for physiological research^1,2. Here a protocol has been introduced describing the preparation of viable cardiac tissue slices from human myocardial explants obtained from neonatal and infantile patients undergoing surgery for congenital heart disease. These slices can be used for studying contractile behavior and electrophysiological properties, among other physiological parameters...

Materials

Name	Company	Catalog Number	Comments
NaCl	Carl-Roth	HN00.2	solution A: 136 mmol/L in aqua dest.
KCl	Merck	1.04936.0500	solution A: 5.4 mmol/L
NaH2PO4 * 2 H2O	Carl-Roth	T879.1	solution A: 0.33 mmol/L
MgCl2 * 6 H2O	Merck	1.05833.0250	solution A: 1 mmol/L
D(+)-glucose	Carl-Roth	HN06.3	solution A: 10 mmol/L
HEPES	Carl-Roth	HN77.3	solution A: 5 mmol/L
2,3-Butanedione monoxime	Sigma-Aldrich	B0753	solution A: 30 mmol/L
NaOH	Carl-Roth	K021.1	solution A: use to adjust pH to 7.4
CaCl2 * 2 H2O	Merck	1.02382.0250	calcium solution: 100 mmol/L in aqua dest.
Agarose Low Melt	Carl-Roth	6351.5	for slicing
Steel blades	Campden Instruments	7550-1-SS	for slicing
Vibratome	Leica	VT1000 S	for slicing
Glass pasteur pipettes	VWR	14673-010	for 'slice collector' tool
Peleus ball	VWR	612-2699	for 'Slice collector' tool
steel chamber	custom-made		for embedding tissue into agarose; cylindric shape (inner: Ø: 1.5 cm, depth 0.9 cm; outer: Ø: 2.0 cm, depth 1.1 cm)
instant adhesive	Henkel AG	PSG2C	Pattex Ultra Gel; for slicing
Jacketed vessel	custom-made		to maintain temperature of agarose in beaker at 37°C; made of steel with a plexiglass bottom
Water bath	Grant	Sub 14	for temperature control of jacketed vessel
Water pump	Aquarium Systems	Mini Jet MN404	for temperature control of jacketed vessel
IMDM	Life Technologies	31980022	for force measurements
DMEM, high glucose	Life Technologies	61965026	for microelectrode recordings
E4031 dihydrochloride	Abcam	ab120158	for microelectrode recordings