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. For a more detailed perspective, see Griffith, L.G. and Naughton, G., "Tissue Engineering: Current Challenges and Expanding Opportunities" Science, 295, 1009-1014 (2002).

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. A guiding example from the natural environment of the cell is the extracellular matrix molecule tenascin-C, a molecule upregulated in development and wound healing which is in structure a large hexabrachion comprising multiple adhesion and growth factor domains. We speculate that the nanoscale clustering of adhesion domains within the molecule may serve to cluster integrin receptors and influence their biological function. Using small peptide adhesion ligands, we have shown that clustering integrin ligands indeed exerts a profound influence on integrin-mediated cell adhesion and migration. Following up on the initial studies, we are currently examining the interplay of integrin ligand clustering with integrin type, ligand affinity, and mechanical context in dictating adhesion and motility behaviors. Tenascin-C contains multiple EGF-like repeats and some of these repeats can activate the epidermal growth factor receptor (EGFR). We thus speculate that another role of the tenascin-C structure is to control EGFR activation -- perhaps limiting activation to the cell surface and juxtaposing integrin and EGFR in ways that will influence the ultimate signaling properties of both. We are thus using polymeric biomaterials to control the activation of EGFR by restricting the internalization, and examining the influence on downstream cell behaviors (such as adhesion, migration and growth) as well as intracellular signaling pathways. Some of these materials and model systems are available through the NIH Cell Migration Consortium (see below). A second emphasis area is using materials that present defined adhesion and growth factor ligands to control cell behaviors for applications ranging from connective tissue progenitor cell selection and growth on bone regeneration scaffolds to control of liver cell adhesion for in vitro organogenesis of liver.

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 3DP TM 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.

Current Collaborators

Doug Lauffenburger (MIT); Anne Mayes (MIT); Alan Wells (University of Pittsburgh); Rick Horwitz (University of Virginia); George Muschler (Cleveland Clinic); Therics, Inc. (Princeton, NJ).

If you are interested in collaboration through the NIH Cell Migration Consortium: Our lab serves as part of the Biomaterials Core for the Cell Migration Consortium. Please see the Consortium web page for information on the available materials and modes of collaboration, or contact Prof. Linda Griffith directly.

Current Students, Postdocs, and Technical Staff

top

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 cm 3) 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.

Current Collaborators

Doug Lauffenburger (MIT); Harvey Lodish (MIT, Leona Samson (MIT), James Sherley (MIT), Steve Tannenbaum (MIT), Forest White (MIT), Donna Stolz (University of Pittsburgh), Alan Wells (University of Pittsburgh)

Current Students, Postdocs, and Technical Staff

top