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. These interactions are important in normal physiology of many organ systems, in embryogenesis where differentiation cues are determined by the local cellular microenvironment, and implicated in the pathophysiology of certain diseases. The ability to spatially control cells at the single cell level using micropatterning would allow the precise manipulation of cell-cell interactions of interest.

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. We have used photolithography to pattern biomolecules (collagen 1) on glass which mediates cell adhesion of the first cell type, hepatocytes, followed by non specific, serum-mediated attachment of fibroblasts to the remaining unmodified areas. This co-culture technique allowed the manipulation of the initial cellular microenvironment without variation of cell number. 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. In particular, 2 to 3-fold variations in steady-state levels of representative hepatocellular functions were achieved from identical numbers of cells. Furthermore, our results indicated that the use of microfabrication to control cell-cell interactions may allow modulation over the kinetics of functional up-regulation;​ in fact, micropatterned co-cultures displayed increased levels urea synthesis up to 1 week earlier than randomly distributed, unpatterned co-cultures with the same cellular constituents. 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. We found that hepatocytes adjacent to the heterotypic expressed increased levels of intracellular albumin (a marker of hepatic synthetic function);​ whereas, hepatocytes far from the heterotypic interface contained undetectable levels of albumin. 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. We determined that fibroblast number could be reduced by twelve-fold with only a modest reduction in hepatic tissue function. 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.