1 - 2:00 - Kim Horger , Michael Mayer

Dried Films of Hydrogels Enable Formation of Giant Liposomes in Phosphate Buffered Saline

Abstract:
Giant liposomes (GL) are cell-sized, spherical shells of lipid bilayers that are useful models for studying properties of cell membranes; however, some of these properties (e.g. ion channel activity) can be affected by the ionic strength of the surrounding solution. Hence, preparation of GLs in solutions of physiological ionic strength, such as phosphate buffered saline (PBS), is desirable but is challenging with traditional methods of generating GLs. Here, we present a simple method of forming GLs in ionic solutions by applying the established method of electroformation to a surface that has been pre-treated with a dried film of hydrogel supporting a film of lipids. This technique generated large numbers of GLs with various lipid compositions (zwitterionic, PEGylated, or negatively charged) in PBS and in deionized water, even without applying an electric field. The results of these experiments provide insight into the fundamental mechanisms that facilitate the formation of GLs.

2 - 2:15 - Daniel Estes , Michael Mayer

High-throughput screening of ion channels in primary human lymphocytes

Abstract:
We present a novel method to quantify functional activity of ion channels in human lymphocytes. An effective immune response to many pathogens requires the activation and proliferation of lymphocytes, and ion channels play important roles in these processes. A significant challenge, however, in studying these ion channels is the lack of an accessible technique to measure functional activity. Here, we present an automated, high-throughput assay capable of measuring functional ion channel activity in over 200 individual T lymphocytes within one hour (~20-fold higher throughput than traditional techniques). We used this assay to study the changes in the voltage-gated potassium channel, Kv1.3, after activation of T cells, as well as to profile Kv1.3 activity in a variety of different cells of the immune system. This assay represents a general and accessible method to study ion channels in primary cells, and may be useful for studying the role of ion channels in human autoimmune disease.

4 - 3:00 - Geeta Mehta , Shuichi Takayama , Jennifer Linderman

Microfluidic Technologies towards Creating In Vitro Niches for Hematopoietic Stem Cells

Abstract:
The goal of this research work is to create in vitro microenvironments for long term culture of hematopoeitic stem cell (HSC) in microfluidic bioreactors and to study the role of HSCs in the bone marrow niche. In vivo, HSCs reside in the bone marrow osteoblastic and vascular niches in adult mammals. Some of the defining features of their in vivo niche comprise of: very small number of HSCs, heterogeneous population of bone marrow cells that support HSCs, and very low oxygen tension. In vivo studies with HSCs are often tedious and time consuming, while the conventional in vitro cultures do not capture the microenvironment found in vivo. We are using microfluidic tools to study and re-create the microenvironmental hematopoeitic stem cell (HSC) niches in vitro. We engineer these niche elements in microfluidic bioreactors by: modulation of oxygen tension in the microbioreactors, optimal attachment and growth of HSC supporting bone marrow stromal cells, and also by culturing small numbers.

5 - 3:15 - Khamir Mehta , Jennifer Linderman

Towards Efficient Design and Quantitative Characterization of Microfluidic Cell Culture Systems for Tissue Engineering

Abstract:
The ability to manipulate and quantitatively measure the cellular behavior inside novel microfludic bioreactors is of fundamental interest to various applications including tissue engineering. Here, we demonstrate the application of mathematical model describing the spatio-temporal distribution of nutrient and growth factor concentrations in design optimization of such bioreactors and extend it to infer cellular parameters from experimental measurements. We show that the reactor design and operating conditions offer a handle to control the spatial gradients in nutrient concentrations inside the bioreactor. We use experimental measurements of oxygen concentrations in a poly(dimethylsiloxane) (PDMS) microreactor culturing C2C12 cells to infer quantitative information on cellular uptake rates, and hence demonstrate a methodology to quantify cellular responses. Our results are significant for the development, design, and optimization of novel in vitro systems for tissue engineering.

10 - 4:45 - Yusuf Murgha

Amplification Methods to Probe Single Cell Genome

Abstract:
Microarray technology has enabled biologists to monitor the whole genome on a single device, thereby giving a better understanding of various gene interactions. Gene chips used for expression profiling are limited by the amount of target material (mRNA)required to get meaningful results. Currently, mRNA extracted from a few thousand cells (>1000) is used per chip. This averages the gene expression at the level of single cell. Here, I present two amplification steps (Design, Methods and Results) which when used in combination will help understand gene expression in a single cell. The two steps are combined by representing each gene with a DNA barcode. The first step involves amplification of mRNA (gene) during reverse transcription by using multiple gene-specific DNA barcode primers. The second step involves the use of an emulsion method to amplify the barcodes by PCR. Emulsion PCR removes amplification bias observed when amplifying multiple templates with universal primers.

2 - Xu Li , Lutgarde Raskin

Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons to Quantify rRNA of Specific Microbial Group

4 - Rebecca Schewe , Keith Cook

In Vitro Resistance Studies in Thoracic Artificial Lung Designs

Abstract:
Current Thoracic artificial lungs (TAL) developed as a bridge to transplant, possess fluid mechanical impedance greater than natural lungs resulting in low cardiac output. A lower resistance model is needed to prevent overloading the right ventricle and inducing RV dysfunction. This study examined effects of different inlet expansion angles of a rigid TAL model at different flow conditions. In vitro testing was conducted with a circuit consisting of a pulsatile pump, artificial lung, and reservoir filled with 3.0 cP glycerol. Two artificial lungs with angles of 45° and 90° were tested at heart rates of 80 and 100 BPM and flow rates of 2, 4, and 6 LPM. Flow rate and inlet and outlet pressure were used to calculate model resistance. Results indicate that resistance increases with flow rate and was 40.8% lower in the 45° model. Preliminary CFD analysis provided a qualitative relationship between flow rate and resistance which confirmed as flow rate increases, model resistance increases.

6 - Jihyang Park , Mark A. Burns, Xiaoxia Lin

Microfabricated Systems of Synthetic Symbiotic Cells for Improving Microbial Secretion

Abstract:
Microbial secretion is important in many microbe-assisted biochemical production systems such as biofuel generation. In this project, we aim to develop high-throughput screening technologies for improving microbial secretion by combining microbial symbiosis and microfluidic devices. The basic idea is that highly secreting microbes can be rewarded in a symbiotic system with appropriate partner cells in localized mini-environments. We are designing and fabricating microfluidic devices that can compartmentalize, cultivate, and detect pairs of symbiotic cells in microdroplets, to maximize and distinguish the effect of microbial secretion on the community growth rate. Here I report my initial work with a model system of two fluorescence-labeled symbiotic E. coli auxotrophs, including: i) simulations, which facilitate the design of the microfluidic devices; and ii) experiments, which have established the feasibility of cultivation and compartmentalization on microfluidic PDMS chips.

7 - Shani Ross , Wayne Aldridge

Modulation of Wanting of a Food Reward using High Frequency Stimulation in the Ventral Pallidum

Abstract:
We are interested in studying the effects of continuous high frequency stimulation (100 Hz - 200 Hz) in the ventral pallidum on the motivational "wanting" of a food reward and the underlying neural reward circuit. In this study, we are investigating if such high frequencies can disrupt the motivational "wanting" by creating an "informational lesion" in the underlying neuronal circuit as proposed by Grill et al. (Grill et al., 2004). Computational models of high frequency stimulation shows that it masks the intrinsic firing rate of the cell resulting in a regular output firing rate that matches the stimulation frequency (Grill et al., 2004). In this study, the effects of the high frequency stimulation are compared to the effects of low frequency stimulation (10 Hz - 60 Hz) which has been shown to be rewarding (Olds and Milner, 1954) and increases food intake (Berridge and Valenstein, 1991), and to no stimulation as an additional control.

9 - Geeta Mehta , Shuichi Takayama , Jennifer Linderman

Creating In Vitro Niches for Hematopoietic Stem Cells

Abstract:
We are developing microfluidic bioreactors to study and re-create the microenvironmental hematopoeitic stem cell (HSC) niches in vitro. Using polymers such as poly(dimethyl siloxane), poly(styrene), poly(ethylene teraphthate glycol) and cyclo olefin copolymers we are fabricating microfluidic systems where a small number of primary mouse hematopoeitic stem cells can be grown in co-culture with supporting primary bone marrow stromal cells. By modulating oxygen tensions and biomaterial surface chemistry we are creating in vitro model systems for the bone marrow niche of HSCs.