Research Program
Cell Surface Interactions

Objectives
The goal of our research is to study new approaches for the creation and application of microenvironments to be used for propagation and differentiation of human stem cells. Our investigation will focus on the physical contact of stem cells with other stem cells or stromal cells in order to determine whether the number of cell contact points (lateral or vertical) affect the ability of the stem cells to self-renew or differentiate along a particular lineage. The microfabricated culture surfaces will be varied so that different ratios of stem cells and stromal cells can make physical contact in the fabricated depressions of different depths and widths.  The culture flooring also will be modified to allow the cells to be in a 3D matrix and no be restricted to setting on a flat plastic surface.  The stem cells will be surrounded be medium, cells, and/or growth factors. Comparative analyses will assist in determining the ideal in vitro environment to generate expansion of stem cells or a particular cell lineage with use of minimal amounts of support cells or growth factors due to the miniaturization of the culture system.

Methods
There will be two primary approaches to the assessment of stem cell biology. The first will utilize three dimensional (3D) microwells to investigate the effects of vertical stacking versus lateral cell contact.  Narrow microwells will be fabricated that are 10μm wide by 50μm deep to limit cell contact in the vertical direction, while wide microwells will be 20-100μm wide and 20μm deep to promote lateral contact between cells. 

To limit cell attachment and growth to the microwells, polymeric replicas will be treated with adhesion promoting factors, as well as anti-adhesion factors.  The goal of this effort will be to prevent cellular adhesion to the uppermost surface of the replicas. 

The second approach will involve direct printing of cells into tissue culture wells.

The unique combination of integrated microfluidic fluid channels, ink-versatility, high lateral resolution and alignment capability make the  “Quill Pen Lithography” (QPL) QPL technique an excellent candidate for further development to enable direct cell printing on solid substrates. QPL is a cantilever-based technology that offers a unique combination of integrated microfluidics, high spatial resolution/alignment, and compatibility with a wide range of biological materials.

Summary
Our two separate types of micromanipulations should provide significant new insight into the biology of stem cells and the influence of the physical environment on their developmental changes.  The miniaturized cultures will conserve on cells and factors.

Anticipated Accomplishments

• Advances in technology for stem cell propagation
• Understanding of the physical requirements for cell contact on proliferation versus differentiation.