Research Program
Cellular Microdynamics

Project #
CM10

Participating Faculty:	Abraham Stroock, Larry Bonassar, Thomas Sato, Michael Shuler, Tracy Stokol, Mingming Wu.
NBTC Students/Postdocs:	Brittany Held, Jong Sung, James Camp, Valerie Cross.
Other Students/Postdocs:	Kevin Wong, Dr. Eugene Kalinin, Karla Comacho, Ankur Chaudhury, Rosa Rosales, Dr. Daniel Rhodes, Christopher Lee, Inna Lipchina.

Objectives

Tools:  1) Develop microfluidic and micromechanical methods to study and control single cell and multicellular dynamics in three-dimensional (3-D) cultures in vitro.  2) Develop optical and biochemical techniques to characterize cellular dynamics with sub-cellular resolution within 3-D cultures. 

Studies:  1) Elucidate drivers and dynamics of vasculogenesis and angiogenesis.  2) Develop an accurate model of the endothelial barrier as it serves to mediate metastasis and pharmacokinetics.  3) Engineer functional vascular structure within 3-D scaffolds for tissues in the context of regenerative medicine.

Methods
1) Living lithography (Stroock, Bonassar).  We have adapted soft lithographic methods to transfer photolithographically defined features into cell compatible hydrogels such as calcium alginate, agarose, and collagen with cells in the bulk and on surfaces.  Living cells can be present in the bulk of the material and plated onto the surfaces. 
2) Endothelializable microfluidic networks (Stokol, Shuler). We have designed and fabricated multi-scale networks of semi-cylindrical microchannels in silicon (xenon fluoride etch).  These structures have been transferred into polystyrene for casting into PDMS. We have performed cultures of endothelial cells in these channels. 
3) Microfluidic migration assay mammalian cell micrgration assays (Wu).  We have developed a microfluidic platform in agarose that allows  for imposition of gradients of multiple soluble factors on mammalian cells.  The fluid in the cell chamber can be either stationary or flowing.  

Summary
Our team has made significant advances toward uniting microfabrication and microfluidics with complex, 3-D cell culture.  We have used hydrogel-based microfluidic devices to allow for the control of the soluble chemistry in the microenvironment of cells via convective mass transfer.  As illustrated in Fig. 1, channels embedded with a hydrogel act as a vascular system that can control the chemical state of the bulk of a 3-D culture both temporally (Fig. 1a) and spatially (Fig. 1b and 1c).  In order to form physiologically relevant templates for vascular structure, we have developed new fabrication processes in silicon to create multi-generational networks of channels with round cross-sections (Fig. 2).  In an important step toward understanding the physical cues that control the initial stages of vascular development, we have mapped the effects of geometrical and mechanical characteristics of a 3-D culture on the behavior of vascular endothelial cells (Fig. 3).  For example, we find that mechanical confinement and cell-seeding density have dramatic impacts on cellular self organization.  These studies have allowed us to identify conditions that lead to the formation of extensive vascular networks in vitro (Fig. 3c).  These advances represent exciting steps for advanced cell culture and form basis engineering microstructure with which to study and control the development of microphysiological structure in vitro.

Accomplishments

• Demonstration of microfluidic scaffolds for spatial and temporal control of soluble chemistry in 3-D tissue cultures.
• Map of effects of geometrical parameters on vasculogenesis in vitro.
• Endothelialized networks of microchannels for studies of cancer metastasis.