Isolating Extracellular Matrix by Liver Decellularization

INTRODUCTION Liver Fibrosis is the 12th most leading cause of the death in the United States and 45% of all deaths in the developing world are caused by a fibrotic disease. [1] With the increase rate in fibrotic disease, there is an increased need for transplants which is exceeding the number of donors available. Fibrosis in the liver remains silent until it reaches the stage of liver failure, primarily caused by alcohol abuse, hepatitis C, and nonalcoholic fatty liver. Understanding the Extracellular Matrix (ECM) of the liver is important for determining the underlying causes of fibrosis and the future of healing the damaged tissue. Collagen is the main structural protein on the extracellular matrix and is produced to aid in the strength of connective tissue that holds the liver together. When a signal triggers the ECM to synthesize collagen rapidly, the collagen crosslinking increases at a rate faster than it is degraded and in time blocks blood from getting to the liver, creating a stiff structure. [1] In time, the liver loses all primary function and is deemed to failure. The only current treatment for liver fibrosis at this stage is a transplant. [2] However, the parenchymal cells in the liverhepatocytes, which make up 75-80% of the liver, have a short survival rate after transplantation. An innovative approach in regenerative medicine involves the decellularized extracellular matrix (DLM) which proves to not only be the primary microenvironment for healthy hepatocytes, but a marker for fibrotic progression to catch the disease before it reaches to stage of Cirrhosis (liver failure). [1,3] Decellularization involves lysing the cells mechanically, chemically, or enzymatically and extracting them by a variety of methods including agitation and perfusion which involve separation of the cellular components with washing of detergents and acids that remove all remaining hepatocytes. The ECM components are ideally left intact, minimizing damage to all important proteins on the extracellular structure. [4,5] Isolating the ECM and using it as a scaffold for healthy cell growth that may have been damaged by liver fibrosis is an important rapidly growing aspect in regenerative medicine research. [5] However, an optimal decellularized method is necessary to isolate the ECM. Based on previous research, current methods of agitation reveal at most only 75% of hepatocytes have been depleted in mice liver decellularization and under 5% of the ECM material is retained. [6] To study the ECM in fibrotic tissue in comparison to healthy tissue a more optimal decellularized protocol must be established.