Generation of Porcine Ventricular Slices: A Vibratome-based Technique to Prepare Ultra-thin Slices of Pig Heart Tissue

Overview

This video demonstrates a vibratome-based procedure to generate viable, ultra-thin sections of pig heart. The prepared tissue slices can be cultured and used as model systems for pharmacological testing.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Pig Heart Tissue Slicing

1. Set up the tissue bath on the vibratome, add ice to the tissue bath cooling jacket, then add Tyrode's solution into the tissue bath. Set up 1 L plastic jar to collect the disposal of melted ice from the tissue bath cooling jacket. For 1 L of the slicing Tyrode's solution, mix 3 g/L 2,3-butanedione monoxime (BDM), 140 mM NaCl (8.18 g), 6 mM KCl (0.447 g), 10 mM D-glucose (1.86 g), 10 mM HEPES (2.38 g), 1 mM MgCl₂ (1 mL of 1 M solution), 1.8 mM CaCl₂ (1.8 mL of 1 M solution), up to 1 L of ddH₂O. Adjust the pH to 7.40 using NaOH. Then, filter the solution using a 1,000 mL, 0.22 µm, vacuum filter/storage system and store at 4 °C overnight.
2. Under the biosafety cabinet, transfer the pig heart to a tray containing 1 L of fresh cold cardioplegic solution. For 1 L of the cardioplegic solution, mix 110 mM NaCl (6.43 g), 1.2 mM CaCl₂ (1.2 mL of 1 M solution), 16 mM KCl (1.19 g), 16 mM MgCl₂ (3.25 g), 10 mM NaHCO₃ (0.84 g), 1 U/mL heparin, up to 1 L of ddH₂O. Adjust the pH to 7.40 using NaOH. Then, filter the solution using a 1,000 mL, 0.22 µm, vacuum filter/storage system and store at 4 °C overnight.
   NOTE: Add 1,000 U/L heparin in cardioplegia solution the day of slicing. 
3. Dissect the heart to isolate the left ventricle using retractable sterile scalpels. Then, cut the left ventricle into blocks of 1−2 cm³ each using razor rectangle blades. Use one piece for slicing and keep the remaining pieces in a 50 mL tube in cold Tyrode's solution on ice for cutting later, if needed.
   NOTE: Massage the tissue before slicing with hands, especially if this is the second block. Massaging helps to restore the correct muscle fiber configuration and prevents any stiffness within the heart block.
4. Add 1−2 drops of tissue glue (Table of Materials) to the metal sample holder and stick a piece of the 4% agar block with a surface area of approximately 1 cm².
5. Add 1−2 drops of tissue glue on the agar.
6. Stick the heart block to the agar with the cardiac epicardium side facing down on the tissue glue and make sure it is as flat as possible. Then, transfer the tissue holder with the heart block to its position in the slicing bath of the vibratome.
7. Attach the oxygen tube to the slicing bath and the metal tray filled with Tyrode's solution for collecting the slices (oxygenated Tyrode's bath). Then, add 40 µm cell strainers in the metal tray to collect the slices after cutting.
8. Adjusting the slicing position
   1. Using the vibratome operating software and the dashboard, adjust the height of the blade/sample. Select the height button and follow the on-screen instructions for adjusting the height. Ideally, have the blade close to the top of the tissue but below the papillary muscles and make sure there is liquid covering the tissue and the blade before slicing.
   2. Adjust where to begin slicing the tissue by selecting the advance button. Press the slice button and increase the speed using the knob to move the blade towards the edge of the tissue. Then, press slice again to stop, and press the advance button to inactivate the process.
   3. Adjust the cutting parameters: advance speed = 0.03 mm/s, vibration frequency = 80 Hz, and horizontal vibration amplitude = 2 mm. Then, start slicing by selecting the slice button.
   4. Let the vibratome slice until it reaches the end of the tissue but before it hits the back end of the specimen holder. At this point, press slice again to stop. Hit the Return button to go back to the start position.
   5. Select auto repeat (clicking it twice) which will allow the vibrating microtome to auto repeat the slicing process for up to 99 times.
9. Collecting slices
   1. Wait until slices are full length and appear good (after getting past the papillary muscle layers), then start collecting the slices.
   2. Collect the slices using a plastic Pasteur pipette filled with cold Tyrode's solution to gently grab the tissue from the bath. If necessary, use forceps and spring scissors to dissociate the slice from the heart block if the tissue is still attached.
   3. Transfer the slice to one cell strainer in the oxygenated Tyrode's bath (see step 1.7) and use the liquid in the plastic Pasteur pipette to get the tissue to lie flat on one of the cell strainers, then add a metal washer on the top to hold the tissue down.

Materials

Name	Company	Catalog Number	Comments
Agarose	Bioline USA	BIO-41025
2,3-Butanedione monoxime (BDM)	Fisher	AC150375000
Calcium Chloride (CaCl2)	Fisher	C79-500
Ceramic Blades for Vibrating Microtome	Campden Instruments	7550-1-C
D-Glucose	Fisher	D16-1
Disposable Scalpel #20	Biologyproducts.com	DS20X
Falcon Cell Strainers, Sterile, Corning	VWR	21008-952
Heparin sodium salt	Sigma-Aldrich	H3149-50KU
HEPES	Fisher	BP310-1
Histoacryl BLUE Tissue glue	Amazon		https://www.amazon.com/HISTOACRYL-FLEXIBLE-1051260P-Aesculap-Adhesive/dp/B074WB5185/
Iris Spring scissors	Fisher	NC9019530
Iris Straight Scissors	Fisher	731210
Magnesium Chloride (MgCl2)	Fisher	M33-500
Oxygen regulator	Praxair
Oxygen tanks	Praxair
Plastic Pasteur pipettes	Fisher	13-711-48
Potassium Chloride (KCl)	Fisher	AC193780010
Razor rectangle blades	Fisher	12-640
Retractable scalpels	Fisher	22-079-716
Sodium Bicarbonate (NaHCO3)	Fisher	AC217125000
Sodium Chloride (NaCl)	Fisher	AC327300010
Vibrating Microtome	Campden Instruments	7000 SMZ-2