Karel Domansky

Design, modeling and fabrication of a constant flow pneumatic micropump

This paper characterizes a bi-directional pneumatic diaphragm micropump and presents a model for performance of an integrated fluidic capacitor. The fluidic capacitor is used to convert pulsatile flow into a nearly continuous flow stream. The pump was fabricated in acrylic using a CNC mill. The stroke volume of the pump is ~1 µL. The pump is self-priming, bubble tolerant and insensitive to changes in head pressure and pneumatic pressure within its operating range. The pump achieves a maximum flow rate of 5 mL min−1 against zero head pressure. With pneumatic pressure set to 40 kPa, the pump can provide flow at 2.6 mL min−1 against a head pressure of 25 kPa. A nonlinear model for the capacitor was developed and compared with experimental results. The ratio of the time constant of the capacitor to the cycle time of the pump is shown to be an accurate indicator of capacitor performance and a useful design tool.

Design of Solid State Array for Simultaneous Potentiometric and Impedance Sensing in Gas Phase

A general sensing platform consisting of eight modules has been designed and fabricated in silicon. The operating function of this platform has been divided between a chemical sensing chip (CSC) and an electronic service chip (ESC). The CSC uses a gold metallization and a high temperature silicon nitride as the passivation layer. The smallest feature size on the CSC is 20 µm. It houses eight sensing modules each consisting of a pair of gold electrodes for measurement of impedance of the conducting polymer and a field-effect transistor for measurement of chemical modulation of the work function of the same polymer. The modules are separated from each other by a patterned 25 µm thick polyimide. The ESC was fabricated by a standard CMOS technology with 1.25 µm feature size. The CSC and ESC chips are connected by flip-chip bonding which greatly simplifies the packaging. The operation of the combined impedance and work function sensing has been verified by exposure to ammonia in ppm range.

Perfused Microreactors for Liver Tissue Engineering

We developed scalable microreactors that foster the development of 3D microscopic pieces of tissue. By integrating microreactors, reservoirs, and pumps in the multiwell cell culture plate format, we created a high throughput cell culture system. However, in contrast to commonly used 2D static cell culture in multiwell plates, our new system allows 3D perfused cell culture. The system provides a means to conduct assays for toxicology and metabolism and can be used as a model for human diseases such as hepatic diseases, exposure-related pathologies, and cancer

BioMEMS applied to the development of cell-based bioassay systems

Biological applications of MEMS technology (bioMEMS) is of increasing interest in the development of miniature and portable instrumentation for cell-based microassays and sensor applications. A major bioMEMS challenge is the physical incorporation of living cells into sensors and diagnostic devices and creation of the environmental conditions conducive for organization of differentiated cells into tissue-like structures. Our work towards these goals is illustrated by a tissue-based bioassay system we are developing based on a miniature cross-flow bioreactor constructed from of an array of cell-filled microchannels integrated into an environmentally-controlled polymer microfluidics manifold. We describe our microchannel array and manifold manufacturing methods and report on the in vitro culture of cell populations in the bioreactor.

Multiwell cell culture plate format with integrated microfluidic perfusion system

A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

850. Quantitative Analysis of Non-Viral Gene Therapy in a Three-Dimensional Liver Tissue Construct

Successful delivery of DNA lies at the heart of gene therapy, and its feasibility in treating a number of diseases depends on the continued development of more effective gene delivery vectors. While vectors based upon recombinant viruses have shown high transfection efficiencies, they may also pose certain health risks to patients and can be difficult to target to individual cell or tissue types of interest. Non-viral vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type, tissue, or pathology, but these vectors are still plagued with low transfection levels. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus. Current evaluations of non-viral gene delivery treatments in more clinical settings often focus on a single barrier at a time, and as a result, may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro experiments may not correlate well with in vivo data. A scaled up and improved three-dimensional, perfused bioreactor has been designed and built that allows for the long-term culture of primary hepatocytes. Within the microfabricated flow channels of this reactor, cells self assemble over time into tissue structures that more closely mimic hepatic morphology and phenotype than conventional two-dimensional culture systems. By studying non-viral gene delivery in this system, quantitative experiments and experimentally-driven computational models can be developed that may better describe how a vector will perform in vivo. Methodologies in density gradient electrophoresis (DGE) have been adapted to obtain greater resolution in subcellular fractionation. An experimental scheme has been developed which utilizes a newly constructed DGE device that has demonstrated proof of principle for the separation and collection of the vesicular organelles that play an important role in gene delivery. Combined with quantitative downstream assays for both the DNA plasmid and the polymer carrier, vector dynamics can now potentially be tracked at cell entry, progressive stages of vesicular trafficking and escape, and nuclear import, providing data sets which may in turn lead to more accurate and predictive mathematical models. Through a systematic iteration of quantitative experiments and computational simulations, these models will be fine-tuned for different polymer carriers administered to the hepatic tissue constructs, potentially allowing for optimization of specific vector properties and increased success of non-viral approaches.

Tissue-Engineered Liver Microreactor as an in vitro surrogate assay for gene delivery (Ad.CMV.EGFP delivery)

The isolation and culture of cells in vitro alters the structural and functional properties that they exhibit as part of an organized tissue, and could be one of the factors that lead to the lack of correlation between in vitro and in vivo gene delivery. This issue is of particular importance in the case of liver. Hepatocytes undergo a rapid loss of liver-specific function, cell viability and cyto-architecture when maintained under conventional culture conditions. This problem indicates the necessity for an organized in vitro tissue culture system, in order to improve the predictive value of in vitro assays for the performance of gene delivery vectors in vivo. The Tissue-Engineered Liver Microreactor developed in our lab provides a perfused 3D liver culture system. We are pursuing this system as a potential in vitro assay for gene delivery studies. Using an adenoviral vector and enhanced Green Fluorescent Protein as a reporter, we can detect and quantify gene delivery efficiency and levels of gene expression in this 3D system, by the application of techniques such as two-photon microscopy and spectrometry.