Controlling cell-cell interactions in hepatic tissue engineering using microfabrication

The repair or replacement of damaged tissues using in vitro strategies has focused on manipulation of the cell environment by modulation of cell-extracellular matrix interactions, cell-cell interactions, or soluble stimuli. Development of functional tissue substitutes through 'tissue engineering' has been facilitated by the ability to control each of these environmental influences; however, in co-culture systems with two or more cell types, cell-cell interactions have been difficult to manipulate precisely.

We have developed an adaptable method for generating two-dimensional, anisotropic model surfaces capable of organizing two different cell types in discrete spatial locations. We have chosen a primary rat hepatocyte/3T3 fibroblast cell system due to its potential clinical significance in bioartificial liver design and also based on widely reported interactions observed in this co-culture model. Specifically, we were able to control the level of homotypic and heterotypic interactions in co-cultures over a wide range.

Modulation of initial cell-cell interactions was found to have significant effects on liver-specific markers of metabolic, synthetic, and excretory function. Our data indicate that control over cell-cell interactions will allow the control of bulk tissue function based on the local microenvironments. The mechanisms by which hepatocytes and fibroblasts interact to produce a differentiated hepatocyte phenotype were also investigated. Variations in bulk tissue function were due to spatial heterogeneity in the pattern of induction of hepatocyte differentiation within a hepatocyte population due to interaction with mesenchymal cells. Although the actual molecular basis of this signaling was not identified, our experimental results indicated that the source of the observed induction pattern was a tightly cell-associated fibroblast product.

Clinical implementation of a co-culture based, bioartificial liver requires optimization of hepatic function based on fibroblast number and various bioreactor design constraints. To this end, we utilized microfabrication to achieve a reduction in fibroblast number while preserving the heterotypic interface. These data were combined with a simple model of oxygen transport and viscous energy losses in a hypothetical multi-unit bioreactor, to determine design criteria for a microfabricated, co-culture based bioartificial liver. This general approach has potential applications in many areas of tissue engineering, implantation biology, and developmental biology, both in the arena of basic science and in the development of cellular therapeutics. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)