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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Published: November 5th, 2016

DOI:

10.3791/54459

1Lillehei Heart Institute, University of Minnesota

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

Heart failure is common and deadly. It is a progressive disease that results in decreased contractility of the heart, which impairs blood flow to organs and leaves the metabolic demands of the body unmet. It is estimated that 5.7 million Americans have heart failure and it is the primary cause of hospitalization in the United States9. The collective cost of treating patients in heart failure in the United States exceeds $300 billion dollars per year 9-10. The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation....

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All mouse experiments were performed in accordance with US Animal Welfare Act and were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.

1. Method for Mouse Heart Isolation

  1. Euthanize a neonatal mouse by decapitation with a single use blade.
  2. Swab the thorax with 70% ethanol.
  3. Dissect the skin from the chest by cutting it away from the chest wall with standard scissors while pulling the skin laterally with a pair of #5 forceps.
  4. Perfor.......

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Decellularization
On average, the time to decellularization of a P3 heart using this protocol is approximately 14 hr. given an average heart weight of 23 mg for the P3 neonate.

Acellularity
Figure 3a
demonstrates a fully intact P3 neonatal heart (whole mount). Figure 3b shows the same heart following decellularization. Figures 4a and 4b show the hema.......

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The dependence of this technique on repeated perfusions of the heart makes the avoidance of an embolism a critical component of a successful outcome. From the initial catheterization of the heart in Steps 2.2-2.6, to the changes of solution between Steps 2.8-2.14, there are manipulations that can allow introduction of air bubbles which compromise the flow of perfusate into the myocardium. Due to the diminutive size of the neonatal heart, even minute bubbles in the vasculature can cause a technical infarct, thus rendering.......

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The authors gratefully acknowledge Ms. Cynthia DeKay for the preparation of the figures.

....

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Name Company Catalog Number Comments
1. Materials For Mouse Heart Isolation
P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J) Jackson Laboratories 21577 or equivalent
60 mm Culture dish BD Falcon 353004 or equivalent
Phosphate buffered saline pH 7.4 (sterile) Hyclone SH30256.01 or equivalent
Single Use Blade Stanley 28-510 or equivalent
Standard Scissors Moria Bonn (Fine Science Tools) 14381-43 or equivalent
Spring Scissors 10 cm Fine Science Tools 15024-10 or equivalent
Vannas Spring Scissors - 3mm Cutting Edge Fine Science Tools 15000-00 or equivalent
#5 Forceps Dumnot (Fine Science Tools) 11295-00 or equivalent
2. Materials For Decellularization
Inlet adaptor Chemglass CG-1013 autoclavable
Septum Chemglass CG-3022-99 autoclavable
1/8 in. ID x 3/8 OD C-Flex tubing Cole-Parmer  EW-06422-10 autoclavable
Male luer to 1/8" hose barb adaptor McMaster-Carr 51525K33 autoclavable
Female luer to 1/8" hose barb adaptor McMaster-Carr 51525K26 autoclavable
Prolene 7-0 surgical suture  Ethicon 8648G or equivalent
Ring stand Fisher Scientific S47807 or equivalent
Clamp Fisher Scientific 05-769-6Q or equivalent
Clamp regular holder Fisher Scientific 05-754Q or equivalent
60 cc syringe barrel  Coviden 1186000777T or equivalent
Beaker Kimble 14000250 or equivalent
22g x 1 Syringe Needle  BD 305155 or equivalent
12 cc syringe  Coviden 8881512878 or equivalent
3-way stop cock   Smith Medical  MX5311L or equivalent
22 x 1 g needle  BD 305155 or equivalent
PE50 tubing  BD Clay Adams Intramedic 427411 Must be formable by heat. Polyethylene recommended
1% SDS  Invitrogen 15525-017 Ultrapure grade recommended. Make up fresh solution and filter sterilize before use. 
1% Triton X-100  Sigma-Aldrich T8787 Make up fresh solution from a 10% stock and filter sterilize before use. 
Sterile dH2O Hyclone SH30538.02 Or MilliQ system purified water.
1X Pen/Strep  Corning CellGro 30-001-Cl or equivalent
3. Materials For DNA Quantitation
Proteinase K  Fisher BP1700 >30U/mg activity
KCl Sigma-Aldrich P9333 or equivalent
MgCl*6H2O Mallinckrodt 5958-04 or equivalent
Tween 20  Sigma-Aldrich P1379 or equivalent
Tris base/hydrochloride Sigma-Aldrich T1503/T5941 or equivalent
Pico-Green dsDNA assay kit Life Technologies  P7589 requires fluorimeter to read
4. Method for fixation and sectioning of tissue. 
Paraformaldehyde Sigma-Aldrich P6148 or equivalent
Gelatin Type A from porcine skin Sigma-Aldrich G2500 must be 300 bloom or greater
5. Method for tissue histology
Cryomolds 10 x 10 x 5mm Tissue-Tek 4565 or equivalent
Cryostat Hacker/Bright Model OTF or equivalent
Microscope Slides  25 x 75 x 1 mm Fisher Scientific 12-550-19 or equivalent
Hematoxylin 560  Surgipath/Leica Selectech  3801570 or equivalent
Ethanol Decon Laboratories 2701 or equivalent
Define Surgipath/Leica Selectech  3803590 or equivalent
Blue buffer  Surgipath/Leica Selectech  3802915 or equivalent
Alcoholic Eosin Y 515  Surgipath/Leica Selectech  3801615 or equivalent
Formula 83 Xylene substitute  CBG Biotech  CH0104B or equivalent
Permount Mounting Medium  Fisher Chemical  SP15-500 or equivalent
Collagen IV Antibody Rockland 600-401-106.1 or equivalent
α-Actinin Antibody Abcam AB9465 or equivalent
mCherry Antibody Abcam AB205402 or equivalent
NKX2.5 Antibody Santa Cruz Biotechnology SC-8697 or equivalent
Donkey anti-mouse AF488 Antibody Life Technology  A21202  or equivalent
Donkey anti-chicken AF594 Antibody Jackson Immunoresearch  703-585-155  or equivalent
Donkey anti-goat CY5 Antibody Jackson Immunoresearch  705-175-147 or equivalent
Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594 Jackson Immunoresearch  111-587-003  or equivalent
Prolong Gold Antifade Mountant with DAPI ThermoFisher P36930 or equivalent
6. Isolation of neonatal ventricular cardiomyocytes using pre-plating.
HBSS (Ca, Mg Free) Hyclone SH30031.02 or equivalent
HEPES (1M) Corning CellGro 25-060-Cl or equivalent
Cell Strainer BD Falcon 352340 or equivalent
50 mL tube BD Falcon 352070 or equivalent
Primeria 100 mm plates Corning 353803 Primeria surface enhances fibroblast attachment promoting a higher myocyte purity
Trypsin Difco 215240 or equivalent
DNase II Sigma-Aldrich D8764 or equivalent
DMEM (Delbecco's Minimal Essential Media) Hyclone SH30022.01 or equivalent
Vitamin B12  Sigma-Aldrich V6629 or equivalent
Fibronectin coated plates  BD Bioscience 354501 or equivalent
Fetal bovine serum  Hyclone SH30910.03 or equivalent
Heart bioreactor glassware Radnoti Glass Technology 120101BEZ Must be sterilizable by autoclaving or gas.

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