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

Summary

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.

Abstract

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.

Introduction

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 rejection^1,2. 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....

Protocol

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 clamped perfusion line. Perforate the inferior vena cava (IVC) to vent the right heart. Cut either the left superior pulmonary vein or the left atria....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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