A magnetically-stabilized, continuous-flow bioreactor was designed and applied for the controlled growth of rat aortic smooth muscle cells (RASMC) in a pre-determined shape in a three-dimensional environment. The cells were immobilized on magnetic agarose beads (MABs) and grown into a tube-shaped tissue. By adjusting the experimental parameters, the size of the MABs were controlled. The surfaces of the MABs were biochemically modified and RASMC cell growth on the modified MABs was tested. Initial RASMC tissue rings with MABs grew in the magnetic field inside the continuous flow of culture medium in the first few days. The RASMC tissue tube was formed in a week, and allowed to mature up to about a month before removing from the bioreactor to characterize it. Histological staining of RASMC tissue tube showed that RASMC were circumferentially aligned perpendicular to the direction of the flow of culture medium. The majority of the cells in the RASMC tissue tube grown out of the MABs stabilized in the magnetic field in the continuous flow were healthy and highly proliferating. The system has applications in the fields of tissue regeneration, pharmaceutical production, stem cell amplification and biofuel production.

Antibiotic resistance is a growing and widely recognized public health issue. Today, more than 70% of bacteria are resistant to at least one of the most commonly used antibiotics. Bacteria evolve with increasing antibiotic resistance due to the selective pressure that administration of conventional antibiotics creates on cell viability, wherein those bacteria that survive antibiotics become dominant. The emergence of “super” bacteria that carry multiple resistant genes calls for the development of novel antimicrobial strategies that place less selective pressure on the target bacteria. Rather than killing bacteria with antibiotics, interruption of bacterial communication networks - or quorum sensing (QS) - might delay the population-scale behaviors of target bacteria in gene regulation and buy time for the host immune system to fight back. Microfluidic environments provide a controlled and attractive opportunity to study bacterial QS and to explore these strategies.

Here we report in vitro signaling between localized, spatially distinct cell populations in controlled 3D fluidic microenvironments. First, a freestanding chitosan membrane was fabricated by using pH gradients generated at the flow interface of two converging flows. Next, alginate membranes were fabricated by cross-linking alginate sequentially on both sides of the chitosan membrane using diffusion of calcium ions through the semi-permeable chitosan membrane. Finally, cell assembly was achieved by suspending cells in the alginate solution to embed the target cells into the alginate scaffolds, realized as a micro-sandwich structure of cells in alginate on both sides of the chitosan membrane. Signal molecules transmitted in situ from one cell population were transported either by diffusion to (1) surrounding cells and (2) nearby segregated cell population, or by convection to (3) cell populations that are relatively far away in a separated microchannel. Induced quorum sensing responses, the production of fluorescence proteins functionally linked to QS genes, were observed for all three configurations. Importantly, these membrane-based 3D scaffolds offer convenient top-down visualization and easy access to both sides of the scaffolds. These approaches provide a versatile and powerful platform to understand and modulate collective and interruptive cellular responses in bacterial quorum sensing.

We seek to understand the response of the human body to various pharmaceuticals. Our platform technology is an in vitro system that combines microfabrication and cell cultures and is guided by a computer model of the body. We called this in vitro system a micro cell culture analog (microCCA) or a “Body-on-a-Chip”. A microCCA device contains mammalian cells cultured in interconnected micro-chambers to represent key body organs linked through the circulatory system and is a physical representation of a physiologically based pharmacokinetic model. ^{(1, 2)} MicroCCAs can reveal toxic effects that result from interactions between organs as well as provide realistic, inexpensive, accurate, rapid throughput toxicological studies that do not require animals. The advantages of operating on a microscale include the ability to mimic physiological relationships more accurately as the natural length scale is order of 10 to 100 microns.

We have used a microCCA to test potential combination therapies (Tegafur and uracil) for colon cancer. ⁽³⁾ Tegafur is a prodrug for 5-FU and uracil an inhibitor of DPD, an enzyme which deactivates 5-FU. Simple microwell plates cannot probe this system, but the microCCA predicts the types of responses observed experimentally. A “pumpless” system that would be easy to utilize has been demonstrated with Tegafur also.⁽⁴⁾ We have coupled these body modules with a micro model of the GI tract to examine the response to oral exposure of drugs, chemicals, or nanoparticles. ⁽⁵⁾

Overall, we believe that in vitro, microfabricated devices with cell cultures provide a viable alternative to animal models to predict toxicity and efficacy in response to pharmaceuticals.

References

1. Sin, A., K.C. Chin, M.F. Jamil, Y. Kostov, G. Rao, and M.L. Shuler. The Design and Fabrication of Three-Chamber Microscale Cell Culture Analog Devices with Integrated Dissolved Oxygen Sensors. Biotechnol. Prog. (2004), 20:338-345.

2. Khamsi, R. Meet the Stripped Down Rat. Nature (2005), 435(5 May):12-13.

3. Sung, J.H. and M.L. Shuler. A Micro Cell Culture Analog (microCCA) with 3-D Hydrogel Culture of Multiple Cell Lines to Assess Metabolism-Dependent Cytotoxicity of Anti-Cancer Drugs.

Lab Chip (2009), 9:1385-1394.

4. Sung, J.H., C. Kam, and M.L. Shuler. A microfluidic device for pharmacokinetic-pharmacodynamic (PK-PD) model on a chip. Lab Chip (2010) 10: 446-455.

5. Mahler, G.J., M.B. Esch, R.P. Glahn, and M.L. Shuler. Characterization of a gastrointestinal tract microscale cell culture analog used to predict drug toxicity. Biotechnol. Bioeng. (2009) 104:193-205.

Transformation, the process by which a bacterium takes up and incorporates extracellular DNA, is one of the primary enabling technologies in the biotechnology field. This allows a researcher to program bacteria, equipping them with a complement of genes to accomplish a task, such as producing a molecule of interest or acting as a sensor. We describe the simultaneous transformation and localization of Escherichia coli bacteria in response to an electric signal within a microfluidic device. We demonstrate that these transformed bacteria can act as fluorescent sensors of isopropyl β-D-1-thiogalactopyranoside (IPTG), a chemical stimulus, or low dissolved oxygen levels, an environmental stimulus.

This method focuses on bacterial transformation with the added benefit of simultaneous entrapment within an alginate hydrogel at a desired electrode address. This offers the ability to create microfluidic cell-based sensors in a single, simple step. To transform and deposit bacteria, the device was filled with a mixture of electrocompetent cells, 200ng plasmid, 0.5% alginate, and 0.125% CaCO₃ and subjected to a 30V/cm DC electric field for 3 minutes on ice. The cells were allowed to recover at 37ºC for an hour, cultured for 16 hours, and induced with a chemical signal, IPTG, for 4 hours. This resulted in increased expression of DsRed, a red fluorescent protein.

Dissolved oxygen is an important parameter for many cell culture experiments. To create a dissolved oxygen sensor, E. coli were transformed with a plasmid that causes production of green fluorescent protein (GFP) in response to decreased dissolved oxygen concentration in the surrounding medium. Following the above transformation and culturing method, the cells were induced with media that had been deoxygenated in a vacuum chamber, resulting in an increase in GFP expression.

This method is versatile in terms of creating microfluidic cell-based sensors. We envision many exciting applications of this work, including the development of dynamically reconfigurable microfluidic biosensors and high-throughput screening methods for plasmid libraries generated by protein engineering and directed evolution experiments.