Hepatic Tissue Engineering
Diseases of the liver pose a major challenge for medicine. The spread of hepatitis C virus and the paucity of medical therapies for liver diseases mandate increased research aimed at understanding the normal and diseased liver. Existing bioartificial liver devices (dialysis-like machines that incorporate hepatocytes to perform liver functions) are in clinical trials in the United States, but results to date have been disappointing. The failure of these devices results largely from the lack of liver-specific functions supported by the devices.
All cells, including hepatocytes, interact with their local microenvironment to produce coordinated organ function. Signals arise from the extracellular matrix, cell-cell interaction, and physicochemical environment and are integrated in the cellular response. Isolation of hepatocytes from organs for use in devices requires disruption of all of these signals; therefore, our specific interest is in understanding the role of the microenvironment around isolated hepatocytes that modulates differentiated gene expression.
Until recently, the tools to control and study microenvironmental tissue structure were unavailable. With the advent of microfabrication tools in the biological realm, techniques to control and study the microenvironment have been established. These include the investigation of the role of cell-cell interactions and tissue "architecture," combinatorial cell–extracellular matrix interactions, and gradients of soluble stimuli. We are also developing strategies to extend our findings in two-dimensional model tissues to understand and build large, perfusable three-dimensional (3D) tissue constructs, with the hope of developing implantable liver "tissue" as an adjunct or alternative for whole-organ transplantation.
Conversely, we are incorporating our findings into miniaturized platforms that will serve as in vitro models of liver tissue for studying aspects of human disease for which model systems have been lacking, such as drug-induced hepatotoxicity, hepatitis C virus, and human malaria caused by Plasmodium falciparum. The sourcing of hepatic cells is problematic because of the paradoxically limited growth of primary hepatocytes in vitro. We are therefore exploring both the expansion of primary hepatocytes (a famously mitotic cell, during liver regeneration) and pluripotent stem cell sources. Our goal is to develop insight into the role of the microenvironment in order to maximize hepatocyte function, facilitate design of effective cellular therapies for liver disease, and improve our fundamental understanding of liver physiology and pathophysiology.
Miniaturization using microtechnology tools offers advantages over conventional approaches to interrogate cells but also introduces new challenges. The advantages parallel those seen in the semiconductor revolution (fast, cheap, parallel processing, microscale phenomena). The challenges arise primarily from the difficulty associated with biological constraints, such as innocuous manipulation of cells and maintenance of cellular phenotypes that mimic in vivo behavior. Toward this end, we have developed new strategies for controlling the phenotype of immobilized cells and characterized novel methods to rapidly array living cells.
Specifically, our group has explored the use of electromagnetic fields (electrophoresis [DC] or dielectrophoresis [AC]), miniaturized optical tweezers, photochemistry, patterned surface chemistries, robotic spotting, microfluidic droplet encapsulation, and microfabricated wells—all to array living cells for parallel observation. We have developed projects to utilize these tools to understand a variety of phenomena, including differentiation of pluripotent stem cells in combinatorial microenvironments, the role of 3D cell organization on tissue function, and the dynamics of cell-cell interaction using reconfigurable silicon MEMS (micro-electro-mechanical systems).
A major emphasis in our technology development has been the seamless integration with conventional biomedical platforms, with the goal of developing tools that can be easily disseminated to biomedical researchers. In recent years, we have developed tools for monitoring the status of patients with sickle cell disease, DNA damage caused by biological exposures, and tissue microenvironments that promote drug resistance. Our long-term goals are to disseminate these tools broadly to life science investigators to transform the study of living cells in much the same way microarrays have revolutionized genomics.
Our laboratory is interested in how the integration of diagnosis and therapy using multifunctional nanoparticles might transform the diagnosis and treatment of cancer. In particular, we aim to exploit nanomaterials with nanoscale properties and our knowledge of the tumor microenvironment to explore this paradigm. Our long-term vision is to develop nanoparticle bioconjugates that can be injected intravenously and that will home in on tumors, self-assemble to increase their local concentration, deliver chemotherapy locally, sense tumor activity/spread/recurrence, and allow physician intervention by remote actuation. In collaboration with Michael Sailor (University of California, San Diego), we have studied three nanoparticle cores that harness features of the nanoscale: semiconductor quantum dots that exhibit size-based optical properties, dextran-coated iron oxide particles whose assembly alters the spin-spin relaxation time of hydrogen protons on magnetic resonance imaging, and polymer-coated gold nanorods that interact resonantly with near-infrared light.
Collectively, we have explored the capabilities of these nanoparticles by studies on targeting, triggered self-assembly, remote actuation with radiofrequency fields, sensing of kinase activity, release of urinary biomarkers, and delivery of short interfering RNAs in models of ovarian, pancreatic, breast, and brain cancer (in collaboration with Phillip Sharp, Massachusetts Institute of Technology, and Bill Hahn, Dana Farber Cancer Institute).
To control the trafficking of these nanoparticle cores, we have decorated the surface with peptides in combination with polymers that prevent their nonspecific uptake in the liver and spleen. The peptides we have explored are screened in collaboration with Erkki Ruoslahti (Burnham Institute for Medical Research), using in vivo phage display whereby biological nanoparticle libraries (bacteriophages) are injected into tumor-bearing mice and purified based on their homing ability. To explore the self-assembly of these particles, we have used strategies inspired by platelets—natural microparticles that normally circulate in a latent form but can home in on sites of injury and transform to an activated state, whereby they adhere and recruit more platelets. This strategy results in assemblies of magnetic nanoparticles that may then acquire emergent properties, allowing either their enhanced visualization or remote actuation of drug delivery. We have also emulated biological systems where biological components remotely communicate via biological intermediates, such as tissue-resident macrophages that participate in the recruitment of circulating neutrophils. The resultant nanoparticle formulations then act as a "system" to produce emergent behaviors for enhancing diagnosis and therapy.
Some of this work was partially supported by grants from the National Institutes of Health, the National Science Foundation, the David and Lucile Packard Foundation, and the Bill and Melinda Gates Foundation.
As of February 25, 2013