Microscale Tissue Engineering: A 3D Perfused Liver Bioreactor

Our research is in the field of tissue engineering. Broadly defined, tissue engineering is the process of creating living, physiological, 3D tissues and organs. The process starts with a source of cells derived from a patient or from a donor. The cells may be immature cells, in the stem cell stage, or cells that are already capable of carrying out tissue functions; often, a mixture of cell types (e.g., liver cells and blood vessel cells) and cell maturity levels are needed. Coaxing cells to form tissue is inherently an engineering process, as they need physical support (typically in the form of some sort of 3D scaffold) as well as chemical and mechanical signals provided at appropriate times and places to form the intricate hierarchical structures that characterize native tissue.

The process of forming tissues from cells is a highly orchestrated set of events that occur over time scales ranging from seconds to weeks and dimensions ranging from 0.0001 cm - 10 cm. Research projects in the lab address problems across this spectrum. At one end, we study basic biological and biophysical processes at the molecular and cellular level. This helps us understand what processes the cells need help with, and what events they can accomplish themselves. Our work at this end of the spectrum has led to the development of new tools for biologists to use in fundamental studies of cell behavior. At the other end of the spectrum, we develop new materials and devices that are needed to direct the process of tissue formation, under the classical engineering constraints of cost, reliability, government regulation, and societal acceptance. We are also developing new integrated micro-bioreactor systems to grow 3D tissues for use in drug development, and as physiological models of human diseases such as cancer metastasis. Research and development in this area includes integration of materials and scaffold engineering with computation models of fluid flow and nutrient metabolism.

Biomaterials and Scaffolds

At the molecular level, our lab focuses on developing polymeric materials that control receptor-mediated cell behaviors. One emphasis area is on developing a mechanistic understanding of how the physical context of ligand presentation controls processes such as cell adhesion, migration, and growth. For example, using small peptide adhesion ligands, we have shown that clustering integrin ligands indeed exerts a profound influence on integrin-mediated cell adhesion and migration. A second emphasis area is controlling the activation of receptors in the epidermal growth factor receptor (EGFR) family, which comprises EGFR along with Her2, Her3, and Her4. These receptors play essential roles in development, wound healing, and cancer. We use polymeric biomaterials to control the spatial and temporal activation of growth factors and have shown that tethering factors to the culture substrate to inhibit internalization dramatically influences signaling responses. In addition to using materials and protein engineering approaches to understand biology of EGFR signaling, we are translating these discoveries into practical application in regenerative medicine, particularly in bone tissue engineering.

PCL Scaffold used in Cleveland Clinic dog studies: Scaffold Macro Architecture

At the level of tissue-scale devices, we seek to fabricate scaffolds with defined architectures over length scales ranging from a few tens of microns to centimeters, and to do so with a range of materials important for tissue engineering. We are thus implementing a solid free-form fabrication technique, the 3DPTM printing process, in which devices are built as a series of thin two-dimensional layers beginning with a computer design of the desired architecture. This process was invented at MIT and has been commercialized for tissue engineering applications by Therics, Inc. The main application we are currently pursuing is generation of scaffolds for use in large segmental defects in bone, and are integrating surface modification with growth factors to enhance selection and survival of connective tissue progenitor cells in human bone marrow.

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Microscale Liver and Bone Marrow Tissue Engineering

Scalable Liver Bioreactor Technologies for High-Throughput Drug Toxicity & Metabolism Studies

Many therapeutic applications of tissue engineering involve disease processes that might be prevented or treated if better drugs were available or if the processes could be better understood. Animal models, although they provide great insight into human disease, sometimes fall short of capturing the full spectrum of human pathologies and responses to therapy and are not readily adaptable to high throughput studies. Cell culture models, although high throughput, often fail to replicate physiological processes adequately. For example, liver cells lose their susceptibility to hepatitis infection and many aspects of drug metabolism when they are taken from the body and placed in culture. We are thus developing microscale 3D tissues in order to capture higher order physiological behavior of human tissues in vitro in a reasonably high throughput format.

One application area is development of physiological models of liver. The in vivo microenvironment of hepatocytes includes signaling mechanisms mediated by cell-cell and cell-matrix interactions, soluble factors, and mechanical forces. In an attempt to mimic key facets of the in vivo microenvironment, we have developed a microfabricated bioreactor system that fosters three dimensional tissue morphogenesis under continuous perfusion conditions. A key feature of the bioreactor is the distribution of cells into many tiny (~0.001 cm3) tissue units that are relatively uniformly perfused with culture medium. The total mass of tissue in the system is readily adjusted for applications requiring only a few thousand cells to those requiring over a million cells by keeping the microenvironment the same and scaling the total number of tissue units. We are currently conducting fundamental studies characterizing cell dynamics and liver-specific gene expression as a function of several system parameters, and using and modifying the system for a range of applications including prediction of drug toxicity, evaluation of liver responses to environmental toxins, and models of cancer metastasis.

A second application area is development of in vitro models for assessment of toxicity in the hematopoietic system. Here, we are employing an in vitro erythropoiesis culture system developed by the Lodish lab, and attempting to build a quantitative model of responses to agents that damage DNA, such as chemotherapeutic drugs.

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