Hepatocyte culture on collagen grids in the presence and absence of PEG grids

We are developing a novel, microfabricated in vitro system that mimics the in vivo organization of the liver acinus to study the dynamic sequence of expression of pro- and anti-inflammatory mediators induced after ischemia-reperfusion injury. Elements of this model include the Living Cell Array (LCA) and the development of liver sinusoid models using novel microfabrication techniques for controlling cell-cell interactions, and biocompatible polyelectrolytes for building multilayered cellular architectures.

Several stimuli in different concentrations can be prepared and delivered through microfluidic networks to an array of cell culture chambers [click on image for a larger view]

The Living Cell Array (LCA) consists of an arrangement of 500x800mm cell culture chambers and microfluidic channels that deliver nutrients, stimulate the cells and remove the waste. Through proper design of the microfluidic network several concentrations of the stimulus are generated by continuous-flow diffusive mixing of adjacent laminar flow streams and delivered to the cells. Gene expression dynamics are monitored in the LCA device by profiling the induction of NF-kB in HeLa-NF reporter cells and comparing to standard tissue culture formats. We are now extending this platform to accommodate many reporters on a single device to profile the dynamics of multiple pathways in hepatocytes during inflammatory responses.

Microscale liver tissue in a PDMS flow-through device [click on image for a larger view]

We are exploring novel approaches to enhance the control of homotypic and heterotypic cell-cell interactions inside the LCA. Thus, we are combining robotic protein printing and PEG photolithography to superimpose an array of PEG microwells over robotically printed collagen islands. Primary hepatocytes were found to preferentially adhere to underlying collagen regions while becoming confined within PEG microwells. Cells residing on a collagen spot are permitted to form intercellular contacts, while cells in microwells are not allowed to form cell-cell contacts.

We are also working towards assembling a microscale liver tissue on chip, using novel fabrication and tissue culture techniques. This ‘liver microchip’ design could be used to rapidly screen new drugs for liver toxicity, accelerating their development and introduction to the market. The microscale liver tissue will also be used to study emerging pathways in hepatitis C viral infection.

We are developing a novel approach for tissue engineering employing nano-scaffolds in the form of ultra-thin biocompatible polyelectrolyte films to build multilayered cellular architectures. Alternatively charged layers of chitosan and DNA are assembled on hepatocytes monolayers and used as scaffolds for a second layer of endothelial cells on top of the first. Enhanced hepatocytes function (albumin secretion) was reported.

Computer simulations show that hepatocytes can be protected from shear stress by microfabricated structures [click on image for a larger view]

Using numerical simulation tools we optimized the design of the microfluidic system to satisfy the increased metabolic needs of hepatocytes in the new liver sinusoid models. We incorporated microfabricated features into the design of the cell culture chambers to optimize the delivery of oxygen and nutrients, and simultaneously protect the cells from the shear stresses.