Published: November 26th, 2018
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....
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.
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.......
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.......
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.....
|2-0 silk suture
|Suture used to ligate superior and inferior vena cava
|1/4" x 3/8" connector with luer
|Connect aorta and pulmonary artery
|Masterflex platinum-cured silicone tubing
|Tubing to connect heart chambers/veins
|infusion and outflow line
|For flowing solutions through the vasculature
|Polyester pouch (Ampak 400 Series SealPAK Pouches)
|Pericardium-like pouch for containing heart during decellularization
|Snapware Square-Grip Canister
|1-liter Container used for perfusing heart
|Black rubber stoppers
|To seal the perfusion container
|To pump fluid through the inflow lines and to drain fluids
|2 L aspirator bottle with bottom sidearm
|For holding solutions/perfusate
|Quant-iT PicoGreen dsDNA Assay kit
|For quantifying dsDNA
|Calf thymus standard
|Blyscan Glycosaminoglycan Assay Kit
|GAG assay kit
|Infinite M200 Pro
|For analytical assays
|OEC 9900 Elite
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