Published: November 5th, 2016
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....
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
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.
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.......
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.......
|1. Materials For Mouse Heart Isolation
|P1 mouse pups (as shown; B6;D2-Tg(Myh6*-mCherry)2Mik/J)
|60 mm Culture dish
|Phosphate buffered saline pH 7.4 (sterile)
|Single Use Blade
|Moria Bonn (Fine Science Tools)
|Spring Scissors 10 cm
|Fine Science Tools
|Vannas Spring Scissors - 3mm Cutting Edge
|Fine Science Tools
|Dumnot (Fine Science Tools)
|2. Materials For Decellularization
|1/8 in. ID x 3/8 OD C-Flex tubing
|Male luer to 1/8" hose barb adaptor
|Female luer to 1/8" hose barb adaptor
|Prolene 7-0 surgical suture
|Clamp regular holder
|60 cc syringe barrel
|22g x 1 Syringe Needle
|12 cc syringe
|3-way stop cock
|22 x 1 g needle
|BD Clay Adams Intramedic
|Must be formable by heat. Polyethylene recommended
|Ultrapure grade recommended. Make up fresh solution and filter sterilize before use.
|1% Triton X-100
|Make up fresh solution from a 10% stock and filter sterilize before use.
|Or MilliQ system purified water.
|3. Materials For DNA Quantitation
|Pico-Green dsDNA assay kit
|requires fluorimeter to read
|4. Method for fixation and sectioning of tissue.
|Gelatin Type A from porcine skin
|must be 300 bloom or greater
|5. Method for tissue histology
|Cryomolds 10 x 10 x 5mm
|Microscope Slides 25 x 75 x 1 mm
|Alcoholic Eosin Y 515
|Formula 83 Xylene substitute
|Permount Mounting Medium
|Collagen IV Antibody
|Santa Cruz Biotechnology
|Donkey anti-mouse AF488 Antibody
|Donkey anti-chicken AF594 Antibody
|Donkey anti-goat CY5 Antibody
|Fab Fragment Goat Anti-Rabbit IgG (H+L) AF594
|Prolong Gold Antifade Mountant with DAPI
|6. Isolation of neonatal ventricular cardiomyocytes using pre-plating.
|HBSS (Ca, Mg Free)
|50 mL tube
|Primeria 100 mm plates
|Primeria surface enhances fibroblast attachment promoting a higher myocyte purity
|DMEM (Delbecco's Minimal Essential Media)
|Fibronectin coated plates
|Fetal bovine serum
|Heart bioreactor glassware
|Radnoti Glass Technology
|Must be sterilizable by autoclaving or gas.
Copyright © 2024 MyJoVE Corporation. All rights reserved