November 2005, Issue 5

Exploring Human Tissues with 3D Tissue Cultures

Jonathan A. Garlick, DDS, PhD, joined the Tufts University School of Dental Medicine (TUSDM) in September 2004 as a professor in the Department of Oral and Maxillofacial Pathology and director of the Division of Cancer Biology and Tissue Engineering. He also has appointments in the Program for Cell, Molecular & Developmental Biology in the Sackler School of Graduate Biomedical Sciences and in the Department of Biomedical Engineering. He came to Tufts from the School of Dental Medicine at Stony Brook University, where he was head of oral pathology. Garlick has three current research goals: using three-dimensional (3D) human tissue cultures to explore the very early stages of oral and cutaneous cancers and understand factors regulating stem cell behavior in 3D tissues; setting up the Center for Integrated Tissue Engineering (CITE) at TUSDM to expand scientific exploration using bioengineered human tissues; and developing 3D tissue culture into a platform technology for the discovery and testing of drugs and the development of imaging and diagnostic tools.

Garlick’s research group works primarily with human cutaneous and oral lining tissues, but their research is relevant to any of the body’s epithelial lining tissues, including those of the cervix and the esophagus. For some applications, the 3D tissue culture system that Garlick helped pioneer is a major advance over traditional single-layer tissue cultures. Through a technique known as "raft" culturing, the 3D system allows tissues that mimic the architecture of the body to be grown in the laboratory. A clear polycarbonate box with a central pedestal supports the raft upon which the cells are grown. Cells are fed from the culture medium below and are open to the air above. This air-liquid interface more closely resembles the natural environment of the lining cells.

To grow artificial human skin, a single layer of collagen populated with dermal fibroblasts is laid down on the pedestal to establish the connective tissue upon which epithelial cells grow. The collagen is allowed to contract in medium for a week before a single layer of epithelial cells is laid down on it. After the skin cells have been immersed in medium for a few days, the level of medium is lowered so that the cells may grow in an air-liquid interface. A stratified tissue develops, which consists of a lower connective tissue layer with an upper epithelial tissue layer and the epithelium’s basement membrane in between. The 3D tissue does not overgrow the culture dish but instead reaches and maintains equilibrium between cell growth and programmed cell death. As new cells develop in the lower layer, old cells are sloughed off from the top layer, as happens normally in the body.

“Having both cell types present in their in vivo environment allows the cells to nurture each another,” says Garlick. “The connective tissue fibroblasts make growth factors that support the growth and differentiation of the surface epithelial cells, and the surface epithelial cells secrete factors that support the growth of fibroblasts. This 3D system allows us to take advantage of the cross talk between the epithelium and the mesenchyme [the connective tissue], which in some ways recapitulates normal tissue development.”

Garlick’s research group has used these 3D tissue cultures to further our understanding of early epithelial cancer development. To adapt normal skin cells to the study of precancer, Garlick’s group cultures normal epithelial cells with potentially cancerous cells that carry a beta galactosidase marker. “We study the effect of the tumor microenvironment, which is a dominant regulator of cancer progression,” says Garlick. “If the environment in which the cells are present is normal and not perturbed, then you have normal development of that tissue. If you have a small number of abnormal cells, the normal cells can still be dominant because they exert a suppressive effect on the potential tumor cells and prevent those cells from becoming malignant. In skin and other lining tissues, the abnormal cells are actually lost from the tissue as the cells move to the surface and are sloughed off. This is an intrinsic self-cleansing mechanism that is not dependent on the immune system or other outside factors. However, if you put in a critical threshold of potentially cancerous cells, the environment is no longer able to regulate them and the cells manifest their biological potential and become cancerous.”

The research group also reported that the dormant state of epithelial precancer cells can be overcome and cancer progression enabled by treating the tissue with tumor promoters such as 12-O-tetradecanoylphorbol- 13-acetate (TPA) or UV irradiation, which disrupts normal protein function. For example, a reduction in the function of the cell adhesion molecule E-cadherin (a transmembrane protein that binds to several transcription regulating molecules within cells) results in degradation of the basement membrane and invasion of tumor cells into the connective tissue. “The loss of E-cadherin activates a program that initiates the process of [tumor] cell invasion,” says Garlick. “You increase the production of enzymes called MMPs (matrix metalloproteinases) that can break down the basement membrane barrier so the cells can migrate more efficiently.” Once the precancer cells cross the basement membrane and invade the connective tissue, they can enter the circulatory or lymph system and travel to other parts of the body (metastasis). Garlick’s laboratory continues to use 3D tissue models to study cell-cell and cell-matrix interactions during early cancer development and molecular changes that accompany these initial events.

“It’s very exciting," says Garlick. “As pathologists, we looked at a tissue and saw changes in tissue structure that we thought were the outcome of the disease process. But now we’re understanding that the changes in tissue structure are actually driving the disease process.”

Garlick has spearheaded the establishment of the Center for Integrated Tissue Engineering (CITE) at TUSDM. The center will give advice on 3D tissue experiments and train those interested in generating these tissues. The CITE will also generate 3D tissues, process the tissues, and provide imaging analysis for academic and industrial laboratories. Look to a future issue of Research News @ Tufts for a feature on the CITE.

Garlick also foresees the use of bioengineered human tissues fabricated in the CITE in drug development and development of imaging and diagnostic tools. “These in vitro assays can be scaled up and used as a cost-effective system to screen drug candidates. This will take drug screening to an environment in which we can more closely mimic and simulate human tissues.”

“The collaborative environment at Tufts has really been amazing,” commented Garlick. “The network already in place here is very strong in my areas of specialty: tissue engineering, stem cell biology, and cancer biology.” Garlick is actively involved in collaborations with Philip Tsichlis, director of the Molecular Oncology Research Institute at Tufts–NEMC and professor of medicine at Tufts University School of Medicine; David Kaplan, chair of the Department of Biomedical Engineering at Tufts School of Engineering; and Ira Herman, professor of physiology at Tufts University School of Medicine.

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