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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

Published: November 26th, 2018

DOI:

10.3791/58123

1Regenerative Medicine Research, Texas Heart Institute, 2Lifegift Organ Donation Center, 3Biomedical Engineering, Texas A&M University

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing "cellularization" at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more "physiological" method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

Heart failure leads to high mortality in patients. The ultimate treatment option for end-stage heart failure is allo-transplantation. However, there is a long wait-list for transplantation due to the shortage of donor organs, and patients face post-transplantation hurdles that range from life-long immunosuppression to chronic organ rejection1,2. Bioengineering functional hearts by repopulating decellularized human-sized hearts with a patient's own cells could circumvent these hurdles3.

A major step in "engineering" a heart is the creation of a sca....

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All experiments adhered to the ethics committee guidelines from the Texas Heart Institute.

1. Organ Preparation

NOTE: In collaboration with LifeGift, a nonprofit organ procurement organization in Texas (http://www.lifegift.org), donated human hearts not suitable for transplant were used for research with approved consent.

  1. To procure hearts, intravenously infuse 30,000 U heparin to the hearts. Securely suture cardioplegia cannula in the aorta and attach a.......

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After a 7-day decellularization with antegrade aortic perfusion under constant pressure of 120 mmHg, the human heart turned translucent (Figure 6B). The heart was grossly dissected into 19 sections for biochemical (DNA, GAG and SDS) analysis (Figure 6C) to evaluate the final decellularized product.

Throughout the decellularization process, infusion flow rate of differen.......

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To our knowledge, this is the first study to report inverted decellularization of human hearts inside a pressurized pouch with time-lapse monitoring of flow rate and cell debris removal. The pericardium-like pouch keeps the orientation of the heart stable throughout the decellularization procedure. Submerging and inverting the whole hearts inside a pouch prevents dehydration and minimizes excessive strain on the aorta (from heart weight) when compared to the conventional upright Langendorff perfusion decellularization me.......

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This research was supported by the Houston Endowment grant and the Texas Emerging Technology Fund. The authors acknowledge the organ procurement agency LifeGift, Inc. and the donor's families for making this study possible.

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Name Company Catalog Number Comments
2-0 silk suture Ethicon SA85H Suture used to ligate superior and inferior vena cava
1/4" x 3/8" connector with luer NovoSci 332023-000 Connect aorta and pulmonary artery
Masterflex platinum-cured silicone tubing Cole-Parmer HV-96410-16 Tubing to connect heart chambers/veins
infusion and outflow line Smiths Medical MX452FL For flowing solutions through the vasculature
Polyester pouch (Ampak 400 Series SealPAK Pouches) Fisher scientific 01-812-17 Pericardium-like pouch for containing heart during decellularization
Snapware Square-Grip Canister Snapware 1022 1-liter Container used for perfusing heart
Black rubber stoppers VWR 59586-162 To seal the perfusion container
Peristaltic pump Harvard Apparatus 881003 To pump fluid through the inflow lines and to drain fluids
2 L aspirator bottle with bottom sidearm VWR 89001-532 For holding solutions/perfusate
Quant-iT PicoGreen dsDNA Assay kit Life Technologies P7589 For quantifying dsDNA
Calf thymus standard Sigma D4522 DNA standard
Blyscan Glycosaminoglycan Assay Kit Biocolor Ltd Blyscan #B1000 GAG assay kit
Plate reader Tecan Infinite M200 Pro For analytical assays
GE fluoroscopy General Electric OEC 9900 Elite Angiogram
Visipaque GE 13233575 Contrast agent

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