Adit Dhummakupt

The Effect of Selective Pressure to selectively breed protozoa with certain phenotypic traits

Introduction

The use of microfluidic devices to observe protozoa has allowed researchers to view them in a close simulation of their native micro-pore environment. These protozoa normally live in the network of pores in the muddy sediments of estuaries. The advantages of using microfluidic devices are that they are optically clear and permeable to gases, which makes it an ideal living condition for the protozoa while making them observable. The device can be fabricated very precisely to ensure reproducibility and ease of data recording. Previous experiments within microfluidics devices have elucidated the behavior of protozoa when encountering channels of different heights, widths and angles.

The purpose of this experiment is to take the previous experiments one step further and determine whether the protozoa will adapt and evolve in a situation where a selective pressure to explore and traverse channels rapidly is applied.

The protozoa that we are using is cyclidium because they are small, have short doubling time and are easily maintained.

Objectives

Experimental Objectives

• To selectively breed protozoa so that they will be more prone to explore the channels and traverse the channels quicker than cyclidium grown in flasks. This is done by allowing cyclidium to run through a series of circuits, allowing the faster cyclidium to live and killing off the rest.
• To maintain protozoa in the microfluidics device over an extended period of time so that subsequent generations will reveal a phenotypic change.

Device Fabrication Objectives:

• To create a temporary holding well where the cyclidium can be injected and multiply.
• To create a gate or valve that prevents the cyclidium from running the circuit until they’re ready and to keep them in the holding wells.
• To create a circuit that can easily be accessed by the cyclidium when the valve is opened and is long enough to allow the researcher to positively identify the fastest protozoa.
• To create another valve that will prevent the cyclidium from backtracking after it has reached to target well.
• To create an array of these devices so that several runs can be made on the same device, and to fit this device on a 3” by 2” slide.

Significance

Understanding the behavior of protozoa in their natural habitat is important to control a healthy population of bacteria. The applications of such populations include optimizing bioremediation and bacterial control.

Method

To create the device, we designed the microfluidics array in AutoCAD and printed it off onto a piece of opaque film to create a film mask. We then created a master using photolithography. By pouring a thin layer of PDMS on top of this master, we were able to insert a screw valve into the device before pouring the rest of the device.

As to the design of our device, we decided to create an array of wells connected by a long, winding circuit. These circuits are cut off from the wells by screw valves. There are several advantages to this design. Since all of the wells must be an equal distance away from the edge for equal oxygen diffusion, the space in the middle of slide can be used for the circuit. This saves a lot of space and allows for more runs to be made per device. The screw valve entry from two different wells at once, saving space while efficiently blocking both channels. Finally, the winding design in the circuit allows enough time for the fastest protozoa to separate themselves from the others. This allows for more precise selections.

The procedure of the experiment is as follows:

• Fill the device with artificial seawater for protozoa (ASWP).
• Close every valve.
• Remove the ASWP from the first and second well. Add bacteria to the second well.
• Insert the appropriate amount of cyclidium.
• Open the first valve and allow the cyclidium to run its course.
• Close the first valve when the leader cyclidium has made it to the second well.
• Allow that cyclidium to multiply.
• Repeat the procedure until the cyclidium reach the last well. Remove the cyclidium and place it in a flask to allow it to recover, then repeat with another device.

Results

My focus for the summer was to test the efficiency of the valve to stop cyclidium and the cyclidium’s willingness to enter a channel. This was done by creating a basic design involving 2 wells connected by 2 40 micron wide channels. A screw valve was placed on top of the channels. I closed the valve, injected about 500 cyclidium into the source well and allowed the cyclidium to swim around for 20 minutes. The valve did indeed work properly, as no cyclidium entered the channels. I then opened the valve and determined the rate at which cyclidium crossed the channels. What was unexpected in the result is that the opening of the valve created a sudden change in pressure which sucked a fair number of protozoa into the channels without them having to swim.

Milestones

I have accomplished the following:

• I learned how to make AutoCAD designs
• I learned how to do photolithography and microfabrication with screw valves
• I learned how to transfer and create new protozoa cultures in flasks
• I learned how to filter large flocks and bacteria out of protozoa cultures to use in experiments.
• I determined the efficiency of screw valves in preventing protozoa from entering channels
• I learned how to use the inverted microscope and how to take a multidimentional aquisition on the computer.

Name

Title of my work

Abstract

Insert Abstract Text Here.

Significance

Insert Significance of Work Here.

Milestones

I have accomplished the following:

• A
• B
• C

Andy Hogan

Trapping cells for imaging by non-adhesive spatial confinement

Abstract

A regulatory circuit involving PI(3,4,5)P₃ controls the polarity of Dictyostelium discoideum during cytokinesis. Two enzymes, PI3K and PTEN, that exert great influence over PI(3,4,5)P₃ are carefully regulated during the process of cell division. By eliminating both PTEN and two types of PI3K (PI3K1 and PI3K2) in a cell line, the process of cytokinesis can be disrupted and cells will fail to divide in shaking suspension (which prevents their ability to undergo traction-mediated cytokinesis).¹ As a result, the cells swell and multinucleate. However, shaking suspension is not ideal for imaging cells. The goal of my project was to trap samples of these triple mutant D. discoideum in non-adhesive wells with fluidic interface for the introduction of media to simulate the conditions of shaking suspension in an environment more conducive to imaging.

Objective

The idea for this project stemmed from the frequent use of a device known as a "rotocompressor" in the Janteopoulos lab. This device essentially consists of a pair of circular brass fittings that interface in a screw-like fashion. The bottom piece is attached semi-permanently to a glass slide. A drop of fluid containing cells to be imaged is placed upon the glass side in the center of the bottom brass fitting. The top brass fitting interfaces with a brass disk which holds a circular glass coverslip in place via Newton rings. By tightening the Newton rings, the coverslip within the top brass fitting bows slightly to form a convex shape. By rotating the entire top brass fitting, the two fittings screw together and lower the convex coverslip. By carefully controlling the whole process, individual cells within the drop of fluid can be trapped and slightly compressed between the coverslip and the glass slide. They can then be imaged on a microscope.

Initially, the project's goal involved adapting the rotocompressor to include a fluidic interface. Eventually, it was posited that the coverslip and glass slide could be coated with a pluronic substance ² to prevent cell adhesion and potentially image a cell line of triple mutant Dictyostelium discoideum. After some investigation, the production of more rotocompressors was deemed too expensive due to the fact that Vanderbilt's machine shops do not have computer guided lathes. This situation would have hampered experimentation with the device, so other alternatives were pursued.

After coming across an existing VIIBRE pneumatic cell trap known as Bambi, I decided to modify the device for use with D. discoideum, particularly the triple mutants that multinucleate in shaking suspension. According to the initial plan, the interior of the microfluidic device would be treated with some sort of pluronic coating to prevent cell adhesion. Later, it was decided to try several simpler and more cost effective approaches before the pluronics including 1% agarose solution and polyethyleneoxide.

Methods

Using Autodesk's AutoCAD computer software and referencing the original file for the Bambi device, I drew up plans for a version to accomodate the attributes of D.Dictyostelium, an amoeboid cell quite different from the T-cells originally intended for use in Bambi. I dubbed the device "BAMBO" (after RAMBO) since this version was beefier and more rugged than its gentler predecessor. The device was to consist of an open chamber into which cells in media would be introduced. A pattern of channels and wells was embedded into the ceiling of this chamber.

A pneumatic chamber (170 microns thick) was located above the fluidic chamber. As air entered the pneumatic chamber, the ceiling would lower, trapping individual cells within the wells in the ceiling. The channels would still allow perfusion to the trapped cells. The wells were evenly distributed between sizes of 35, 75, and 100 microns to accomodate different sizes of D.discoideum ranging from the standard AX2 wild-type (roughly the size of the smallest wells) to the swelling triple mutants (the largest wells).

The blueprints for each layer BAMBO were printed onto several strips of 35mm opaque film for use as photolithographic masks. The designs were then etched onto chrome masks to create a more permanent mask for future use and to achieve higher resolution of features in the finished device. A master was made by spinning several layers of SU-8 polymer onto a silicon wafer and baking and exposing the layers to UV light in a highly specific order due to the device's multi-layer nature.

The master for the pneumatic chamber was recycled from Bambi for the first batch of devices. This master was contained on a separate silicon wafer. Due to the chamber's insufficient high, several replicas of the final device collapsed, so a new master was made in the machine shop.

The actual devices were fabricated from the polymer polydimethylsiloxane (PDMS) using techniques of soft lithography. Liquid PDMS was poured onto the SU-8 master, air was removed from the device by placing it in a vacuum, and the device was cured overnight in an oven. After curing, the device was bonded to a glass slide by exposing it to a plasma arc.

Three initial test devices were created and were not treated to prevent cell adhesion. To test the pneumatic action of the device, holes were punched through the two fluid access ports of the device and through the access port of the pneumatic chamber. Syringes were used to introduce green food coloring to the fluidic chamber and to apply air pressure to the pneumatic chamber.

Several batches of 1%w/v agarose solution were prepared by dissolving agarose powder in DI water. The agarose did not dissolve readily at room temperature, so the test tube was heated in a beaker of water on a hot plate. The beaker never reached a boiling temperature despite reaching the plate's maximum observed temperature of 152 Celsius. Thus, the agarose crystals were not sufficiently dissolved for the purposes of the experiments (as was later observed).

For the next two devices, the agarose solution was applied after exposing the PDMS device to a plasma arc to make its surface hydrophilic. The beads of agarose solution evenly spread across the fluidic chamber surfaces and then the devices were bonded to glass slides. Introducing actual D. discoideum cells into the device was the next step in the testing process. An unaltered control sample was obtained from a long-standing AX2 culture and a high density solution of cells was introduced via syringe into the entrance port of the device. Pictures were obtained at several magnifications up to 100x. The pneumatic action of the device was also tested on the cells.

Conclusions, Difficulties, and Future Directions

While the original goal focused on utilizing the capillary action caused by compression to force cells into the wells, the desired effect was not achieved on standard AX2 cells and experimentation never progressed to using the triple-mutant D.discoideum cells. Additionally, the agarose did not sufficiently dissolve in solution to be useful in coating the wells to form non-adhesive surfaces. More costly options yet to be tested include coatings of polyethyleneoxide (PEO) or pluronics.

Though the triple mutants were not tested within the device, it was decided to move the focus from trapping the multinucleate mutant cells for imaging to experimenting with microcontact printing on surfaces to test the same intracellular processes on regular Dictyostelium.

SyBBURE Milestones

I have accomplished the following:

• I learned how to design microfluidic devices using AutoCAD software.
• I created masters for my device using photolithography.
• I practiced photolithographic techniques for fabricating complicated multilayer devices
• I fabricated replicas of my device using soft lithography.
• I practiced advanced techniques of microfabrication including partial-cure bonding of multiple PDMS layers.

• I learned to culture D. Discoideum cells.

• I learned how to use the inverted microscope in the Janetopoulos lab (and Slidebook)

References

Arunan Skandarajah

SyBBURE 2007

• A Microfluidic Device for Galvanotactic Response in Dictyostelium discoideum

Abstract

Cellular response to electric fields is a physiologically important field that has been under-explored because of deficiencies in the current devices available. In the course of this project we seek to develop a device on the microfluidic scale that will address the current problems and promise a platform for rapid production of experiment-specific devices.

We have followed the development process through several iterations – constructing prototypes based on designs provided by senior SyBBURE members, modified designs used for other purposes, and designs built explicitly on the results of our experimentation and experience. Our work has cycled through varying levels of complexities, but the most promising solution yet has been the simplest.

Introduction and Significance

Unicellular organisms and migratory cells in multi-cellular creatures have evolved triggers to motility by several environmental indicators. Signals can take several forms including variations in light intensity, reagent concentration, or electrical potential. While there has been extensive investigation in the detection, amplification, and response to of chemical gradients in chemotaxis, similar work in the field of voltage potential for galvanotaxis is more limited. Voltage potentials are significant in the physiological realm because fields have been associated with cell activity in wound healing, neural cone growth, and embryonic reorganization. The role of voltage potentials in tumor metastasis also presents a target for future treatment. Work in this field, however, is currently constrained by technological limitations and operational safety issues. The basic design has not been supplanted by a new universal standard despite several long-standing issues: the design is too bulky to fit on unmodified inverted microscope stages; the applied voltages necessary for galvanotaxis are dangerously high and exposed to the experimenter; and the timeframe of the experiment could be limited by diffusion of toxic electrode products across agar bridges.

To address these problems, we use the techniques of photolithography and microfabrication to bring microfluidic devices to the testing stage. Once a basic functional design is well-tested, the next step is to investigate evidence of similarities across several types of taxis in the signal transduction pathways, the cell’s machinery utilized in interpreting extra-cellular information. The process of modifying the device would make customizable, reproducible devices capable of testing the interaction and competition of different signals a reality.

Completed Program Goals

I have accomplished the following general objectives that are part of participation in SyBBURE:

• Completed photolithography and soft lithography training.
• Developed relevant designs for galvanotaxis studies in AutoCAD.
• Attended weekly journal club for discussions in various associated topics.
• Attended workshops on skills to be integrated into our research processes.
• Attended biological laboratories providing experience and exposure to fundamental skills.

Project Milestones

We have reached the following project-specific landmarks:

• Reconstructed the original agar bridge model for galvanotaxis.
• Microfabricated our project-specific masters and PDMS devices.
• Did basic modeling of our device in COMSOL.
• Built our own variations on a large scale system.
• Built auxiliary components for a galvanotaxis system: y-connector electrodes, device clamp, PDMS punches
• Applied concepts from macro experiments and VIIBRE generated designs to develop the current iteration of microfluidic designs.
• Established a relationship between applied voltage and the field strength across the cell area.
• Preliminary success in goal of developing a field strength of 7-10 V/cm

Future Work

We hope to develop the device further through continuing work as members of SyBBURE sponsored Senior Design team. Our first step will be running multiple trials of the preliminary work done in the summer to determine the effects of varying flow rates on the pH control capabilities of our device. We also hope to streamline our device and eliminate the obstructive electrodes that currently rest above our device with sputtered electrodes placed directly on the plane of the device.

After making these qualitative improvements on the current prototype, one major avenue of our work will involve observing the activity of live cells in our device. With success on this front, we hope to return to the development process to adapt our basic galvanotaxis platform to build a device that will allow biologists to observe the integrated effects of multiple, simulataneous cell signals.

References

ArunanSkandarajah

Charlie Wright

The use of micromirrors to obtain three-dimensional images of cells

Introduction

Techniques such as confocal scanning laser microscopy and multiphoton microscopy are currently used to recover three-dimensional data from biological specimens, but with limited precision along the z-axis. This uncertainty places restrictions upon the conclusions which can be drawn from quantitative analysis of measurements defined by three-dimensional parameters, such as volume. Microscale mirrors can be used to obtain three-dimensional images of living cells with classical widefield microscopy, and optimization of this system may result in the ability to obtain more precise three-dimensional data.

Objectives

The ultimate goal of this project involves making precise measurements of the volume of an individual budding yeast cell as it progress through the cell cycle. The first step towards meeting this objective requires the optimization of the image acquisition process. Once data points from the reflections of the cell are extracted from the images, a back-projection algorithm can be used to reflect them onto a set of points in three-dimensions. Due to the roughly prolate spheroidal shape of the yeast cell, data from this organism is suitable for fitting to a simple three-dimensional surface, and spherical harmonics should provide a reasonable method for creating such a model. Finally, integration of this surface will provide the volume of the cell over time.

Results

The introverted mirrored wells, etched from silicon and coated with aluminum, contain five flat mirrors per well, each of which creates a reflection visible under the microscope. The wafer containing the wells was submerged in a small volume of water to which Saccharomyces cerevisiae cells were then added. In the first setup they settled into wells randomly and only those wells with one cell were examined, and in the second setup a cell was adjoined to the end of a pulled glass tip and positioned inside a well using a micromanipulator. I obtained images of individual and multiple yeast cells within the mirrored wells, as well as images of individual cells adjoined to a pulled glass tip and positioned within a mirrored well. I also took image stacks of 1 μm fluorescent beads in mirrored wells using a confocal microscope, which will be used to check the reflection matrices governing the construction of a single three-dimensional image from the data. In addition, I helped further develop a method for forming a hexagonal mirrored well on the end of an aluminum rod. Such an extroverted mirror which could be placed in the optimal position above a cell, thus obviating the need for the cell to sit within a well. Tungsten carbide rods were polished to resemble a lead pencil with a flat on the end, but their smoothness will be checked with an SEM before imprinting the aluminum.

Future Directions

Although images have been captured on the time scale of minutes, no yeast cell has yet been observed undergoing budding within one of the wells, which will be addressed by substituting growth media for in the experimental setup. Additional manipulation will still be necessary to obtain suitable data for fitting to a surface. In order to ensure the accuracy of the results, fluorescent images will also be used for a separate analysis, and options are being investigated as the yeast cells have exhibited minimal autofluorescence. Another method for obtaining more precise results involves the ability to place a mirrored well in the exact center of a rotary stage. Images of a yeast cell suspended above the center of such a well by a pulled glass tip could be gathered in 1° increments as provided by the rotation of the stage instead of 90° increments as currently provided by the geometry of the mirrored wells.

Milestones

I also learned how to use the following:

• Zeiss Axiotech vario microscope, Zeiss Upright LSM510 META confocal microscope, Hitachi S4200 Field Emission scanning electron microscope
• Polishing wheel, puller, microforge, micromanipulator
• ImageJ, MATLAB

Acknowledgments

• Dr. Kevin Seale
• Ron Reiserer
• Dr. John Wikswo

David Hall

Cardiac Stuff

Abstract

Played with hearts.

Significance

Hearts are cool.

Milestones

I have accomplished the following:

• I went to 8 journal clubs.
• I went to workshops on things having nothing to do with my project.
• I set up the wiki site.

References

1. asdoifj
2. asdfoj

Devin Henson

Design for a low-voltage microscope-ready microfluidic device for studying galvanotaxis in ''Dictyostelium discoideum

Abstract

Galvanotaxis is the movement of cells in a particular direction in response to an electric field across the cells. Currently the setup for a galvanotaxis experiment requires large beakers of media, long agar salt bridges, dangerously high voltages to generate the necessary field across the cells, and generous cross flow to control pH and ion gradients. Such a cumbersome design makes cell study under a microscope very difficult. A microfluidic device for galvanotaxis will reduce the amount of media used, lower the flow rate necessary to control pH and ion flow, and decrease the required voltage by an order of magnitude to a value deemed safe. Additionally the device can sit entirely on the stage of a microscope with flow controlled by a nearby syringe pump. Following the development of a successful device, Dictyostelium discoideum cells will be loaded into the device and studied. Easy device modifications will allow for quick and easy specialization of experiments and a better understanding of galvanotaxis.

Methods

AutoCAD is used to create various device designs, each modified over time to account for several unexpected problems. Photolithography techniques are used to create silicone wafer masters, and soft lithography using PDMS creates the microfluidic devices. Each device is punched appropriately to create tubing inserts and then plasma bonded to a glass cover slip to guarantee perfect adhesion. Tygon tubing connected to a syringe pump fits into the punches to set up the profusion system. Currently Y-connectors allow platinum electrodes to enter the tubing without leaking, but thin metal deposition techniques are planned when a more complete and successful device is ready. To check the control of pH and ion gradients, devices are tested with pH indicators such as phenol red and bromothymol blue.

Accomplishments and Conclusions

Through this project, I have obtained a solid understanding of photolithography and soft lithography techniques. We have shown in our devices that the applied voltage can be reduced by an order of magnitude compared with the current macroscale device and still provide the proper field strength across the cells (approximately 7-10 V/cm). Experiments with bromothymol blue show pH changes around the platinum electrodes at low flow rates, but the low flow rates are still able to keep the cell area from changing color. At higher flow rates color changes are not seen, indicating that pH is controlled even around the electrodes.

Further Research

Further experiments with bromothymol blue and phenol red must be done to gain a complete understanding of the pH and ion gradients in the device. Optimal flow rates for controlling pH while using only a small volume of media are still undetermined. Simultaneously, it will be important to measure the voltage across the cell area to verify that the desired 7-10 V/cm is possible. Overall, a full experiment that addresses all of our initial goals must be setup and repeated before we will be ready to test our device with cells.

Elly Sinkala

SyBBURE 2007

• Modifying Tracking in the Magnetic Tweezers System

Abstract

Viscoelasticity is an important contributor to cell behavior. It is determined by lipid-protein bilayer, microtubles, microfilaments, and other cellular structural components, and evaluating the viscoelasticity can provide insight into the cell’s internal structure. Furthermore, this information can be beneficial for understanding cell behavior under the influence of cancer and drugs.

In epithelial cells, the molecule E-cadherin plays an important role in cell-cell adhesion. Previous research shows that E-cadherin disregulation is an significant component in many invasive cancers. To understand the effect cancer has on the cell structure, Fc-E-cadherin coated beads and the magnetic tweezers system are used to measure the force between Madin-Darby Canine Kidneys cells (MDCKs) and the beads. A series of creep response curves are created from the experiments involving the deflection of the bead attached to cell. In order to create the response curves, a tracking system must be employed to record the displacement of the bead by the magnetic tweezers system. Velocity and position are the primary measurements needed to verify the tracking system and measure the force exhibited on the bead. It is important to have or create a tracking system that can accurately measure the velocity and force so that the resulting response curves better represent the cell-cell adhesion.

In an attempt to improve the tracking system, the implementation of the program Slidebook and fluorescent paramagnetic beads are used in conjunction to provide better insight into the viscoelasticity and cell-cell adhesion of cancer cells.

Project Objectives

The first objective is to learn how to track objects in the program Slidebook. It will run through a calibration test to confirm that the tracking system is working and is accurate. Accurate measurements will provide better response curves that can better predict the performance of the cells. Also, the replacement of fluorescent beads will enhance the tracking system since the bead is clearly distinguished from the cell and background. This will help to make bead tracking easier since the only thing in the image is the bead. These two modifications should provide better force calibrations curves such that the future experiments will be accurate.

Methods

General Procedure

• Trypsinizie cells with 0.25% Trypsin EDTA
• Two hour incubation for cell adherence to well
• Add fluorescent paramagnetic beads
• Incubation for 30 min
• Place on micromanipulator
• Turn on magnet and response cell response

Project Milestones

We have reached the following project-specific landmarks:

• Incorporated the use of fluorescent beads into the experiment
• Coated the fluorescent beads with protein-A
• Successfully learned and operated Slidebook tracking system
• Began doing force calibrations

Problems Encoutered

Variation in bead diameter (4.0-4.8um) may have resulted in the bead vs. velocity graph not to consistently decrease .

The carboxyl groups on the bead caused it to stick to the well.

Future Work

Using the same bead in the force calibration can help to alleviate velocity variations and better show if the Slidebook tracking system is accurate.

Force Calibrations can be used to begin cell pulling experiments to measure cell-cell adhesion.

Name

Title of my work

Abstract

Insert Abstract Text Here.

Significance

Insert Significance of Work Here.

Milestones

I have accomplished the following:

• A
• B
• C

Eric DeLong

Construction of a Microfluidic Device for Galvanotaxis of '''''Dictyostelium discoideum'''''

Abstract

When charged cells are exposed to an electric field, they migrate from their current position to either the cathode or the anode, depending on the type of cell. The study of this migratory event, known as galvanotaxis, has many medical applications. Cell migration has been shown to be influential in cell metastasis, wound healing, embryogenesis, morphogenesis, and neuronal guidance.

While it is indeed possible to obtain experimental data from galvanotactic processes, the current process is not entirely safe or simple to reproduce. These experimental rigs sometimes require a voltage input of several hundred volts, are too large or unwieldy to place on a microscope stage, and can require very large portions of buffer medium. This project is seeking to construct a microfluidic device which will provide biologists with the opportunity to easily study galvanotaxis. The project currently focuses on constructing a device designed specifically for use with Dictyostelium discoideum, a fairly cooperative cell used often to study galvanotaxis.

Project Objectives

-The primary concern of the project is to significantly lower the required applied voltage in order to attain a potential across the cell area in the range of 7-10 V. This value has been shown to be a sufficient condition for galvanotactic activity. An applied voltage somewhere under 50 V with a current somewhere below 6 mAmps should eliminate any risks to serious shocks.

-Additionally, it is desirable that a device be fabricated that will easily fit on a microscope stage.

-In order to remove undesirable byproducts of activity at the electrodes, a certain rate of perfusion must be utilized within the device. Preferably, a flow rate of around 8.3 x 10-7 ml/s. This activity at the electrodes will result in a pH gradient if left unchecked, possibly killing the dictyostelium. The presence of ion and pH gradients will also introduce an undesired additional variable which can affect cell migration.

Methods

Devices have been constructed using photolithography and microfabrication techniques. A positive relief pattern of a fluidic was placed upon a chrome wafer by exposing SU-8 photoresist through a photomask. This master wafer was then covered in polydimethylsiloxane (PDMS) and cured at 60o C for four hours. The PDMS was cut off the wafer, resulting in a pliable piece of PDMS with a negative relief of the microfluidic channel. Holes were punched in the PDMS for tubing ports and the device was cleaned and bonded to No. 1 cover glass after being activated in a plasma cleaner. At this point, Tygon tubing could be top-loaded through the ports, finishing the fabrication process.

Problems Encountered

-A discrepancy related to the voltage has been observed when platinum wire electrodes are placed directly in the tubing ports and when they are inserted inside the tubing. The voltage output with the electrodes placed directly in the ports is around 50% of the input voltage. With the wire electrodes inserted into the tubing and then placed in the ports, the output is around 10% of the input.

-When perfusing the buffer medium, it is difficult to control the flow rate equally in both tubing pathways (two separate tubing pathways are being used to guarantee an elimination of pH and ion gradients). This problem is currently being approached by standardizing the tubing hookups along with the tubing ports. Hopefully this will equilibrate the pressure throughout the device.

-Initially, agar plugs were used to provide a barrier to ion byproducts. These agar plugs could not withstand the pressure provided by the necessary flow rate and would pop out of the ports. This was never solved so the agar plugs were discarded from the design.

Milestones

-A macro-device was constructed consisting of a chamber constrained by glass slides and including agar plugs. This device functioned properly and a high voltage output efficiency was obtained. Unfortunately, it has been difficult to shrink this device down to a microscale.

-Problems with bonding the device to the cover glass were encountered and overcome. The reason is not clearly evident; however, it may be due to a lack of pressure in forcing the bond.

-All galvanotaxis group members were inexperienced as to photolithography and microfabrication prior to the project. Throughout the duration of the project, a considerable amount of knowledge and expertise have been gained in this area thanks to trial and error, as well as the microfabrication expert for Viibre: Ron Reiserer.

-The group members have been able to sucessfully control the pH inside the device in order to eliminate another variable that may affect data collection processes. This is a wonderful achievement, as pH changes may be considered a very large problem inside a microfluidic device due to its small volume. This problem was soved by placing the applied electrodes in the output interface of the tubing system.

Future Prospects

Eventually, the group will have a microfluidic device which will have an appropriate voltage conducive to the study of galvanotaxis which will be both safe and manageable in size. The physical parameters of this device will be easily manipulated by microfabrication in order to account for the needs of spinoff experiments, such as those which would require a different voltage output, pH range, cell area, or flow rate. Shear forces have not been accounted for at present; this could be a future problem, and the group hopes to account for this and acquire a quantified data set that would account for this in order to relay this information to experimental biologists. The final design of the device will desirably include a set of both applied and measuring electrodes plated out on a cover slip so that the voltage will be easily measured across the cell area, making it simple to manipulate the voltage for experimental purposes.

Name

Title of my work

Abstract

Insert Abstract Text Here.

Significance

Insert Significance of Work Here.

Milestones

I have accomplished the following:

• A
• B
• C

Jason McGill

A Microfluidic Interface to Screen Printed Platinum Electrodes

Abstract

As the scale which biology is studied on shrinks, it is becoming increasingly necessary to develop methods of cellular measurement that are also small. It is important to understand the function of a small number of cells, such as in a bioreactor, rather than a Petri dish containing millions. Glucose is one compound that is important to measure since it plays a key role in cellular metabolism. Instead of developing a completely new glucose electrode system, a microfluidic PDMS device was interfaced with a commercially manufactured screen printed electrode. Eventually, it would be hooked up to a bioreactor to measure continuous glucose levels when subjecting cells to a variety of insults.

Materials and Methods

An initial design was created to interface Polydimethylsiloxane (PDMS)with an electrode produced by Pine Research Instruments. The electrode had platinum working and counter electrodes and a Ag/AgCl reference electrode. Using acrylic plates, a clamp was machined which allowed for four screws to tighten a piece of PDMS interfaced with the Pine electrode (shown on right). There were two ports drilled for input and output tubing. Using scotch tape, a simple design was cut out and positioned on a Pine electrode. The electrode was submersed in ~20g of PDMS in a Petri dish for 4 hours. The cured PDMS was then cut to the size of the clamp.

To measure glucose, a layer of a glucose oxidase matrix was placed on the working electrode surface. Before any deposition occurred, the Pine electrode was cleaned by cyclic voltometry in 0.5mM sulfuric acid solution between -0.6V and 1.0V. To make the glucose oxidase solution, 50mg of bovine serum albumin (BSA) was added to 800ul of 50mM phosphate buffer solution (PBS) pH 7. Next, 1mg of glucose oxidase was added to 700ul of the BSA/PBS solution. Finally, 7ul of gluteraldehyde was added to the solution. After each step, the solution was thoroughly mixed with a vortexer. The final solution was applied to the working electrode surface by pressing a finger on a pipette tip to suck in and push out the solution. The deposited layer was given at least 20 minutes to dry in the dark.

A test without a microfluidic device was done first to determine that the electrode worked correctly. This was done by adding 20ul of 500mM glucose/50mM PBS/100mM NaCl solution to a jar filled with 10ml 50mM PBS/100mM NaCl. Each addition made the total solution increase glucose concentration by 1mM. Concentrations were run from 1mM to 10mM. All experiments using the Pine electrode were run at a potential of 0.7V.

The experiment was run slightly different when interfacing the Pine electrode with the PDMS clamp. Concentrations from 1mM to 9mM of glucose were premixed. Taigon tubing was inserted into the PDMS channels and out holes in the acrylic plate. Glucose mixtures were injected one at a time, using individual syringes, to measure glucose concentration. Prior to injection, glucose mixtures were vortexed to ensure uniform concentration. A control was run first, with no glucose oxidase coating. Later, experiments were run with the glucose oxidase coating on the working electrode.

Conclusions

So far, my research shows that it is possible to interface a Pine electrode with a channel in PDMS and clamp it without leaks. Pumping with a syringe pump works best to prevent leaks, but pumping by hand is also possible. In one experiment, the clamp showed flow characteristics up to 300ul/min. No statistical analysis has been performed on the data collected from the preliminary studies yet. When the Pine electrode was used without the PDMS clamp, the data appeared to be linear. Jennifer Merritt, who works in the Cliffel lab, has already done extensive testing on this subject. Results from experiments run with the PDMS clamp (shown on right) appear not to be as linear and are not as easily reproduced. Several tests yielded very poor results for reasons relating to the design of the PDMS clamp. There was some difficulty in getting the flow through the channel to be uniform due to a collapse under pressure from the clamp. Scotch tape is likely not the best way to create a reliable channel to interface with the Pine electrode. Alterations will have to be made to the design of the microfluidic channel to yield better results.

Future Work

• Perform enough experiments to be able to statistically analyze the data collected.
• Investigation of flow patterns through microfluidic channels on the Pine electrode.
• Design in AutoCAD a channel to fit a Pine electrode.
• Fabrication of a duplicate electrode which would bind SU-8 to create a master for the AutoCAD drawing mentioned above.
• Creation of a system using a Harvard Apparatus machine to flow glucose through the fabricated channel at desired concentrations and a uniform rate.

Milestones

I have accomplished the following:

• Training in machining as well as access to the machine shop.
• Fabrication of a PDMS clamp.
• Deign and testing of a microfluidic channel to interface with a screen printed electrode.
• Presentation on cell culture to the SyBBURE group.
• Training in experimentation and maintenance of Pine Institute electrodes.
• Training in soft lithography and microfabrication of microfluidic devices.

Acknowledgments

Dr. Kevin Seale, Jennifer Merritt, Ron Reiserer, Dr. John Wikswo, Dr. Franz Baudenbacher

Jenny Lu

SyBBURE summer 2007

Maintaining Long-term Cell Viability in a Perfused Bioreactor System

Abstract

Fabrication of a small scale bioreactor system with improved delivery of drugs or assay reagents can be highly beneficial to the development of an anti-tumor therapy, and key cellular functional signatures will be identified to predict the response to anti-tumor therapy. MCF10A human mammary cells were chosen as the cell line due to their distinct formation of hollow "mammospheres", creating an excellent model to validate bioreactor culture capabilities. Experiments show that 2000 cells/chamber of MCF10A cells in chamber slide is the optimal concentration for the formation of 3D mammospheres during 20 days, and that is used to correlate the optimal concentration of cells in the parallel wells of the bioreactor. Four cell lines are used for the bioreactor, and after comparing the gene expression profiles from Microarray Data Set, there seem to be only a few protease family classes are differentially expressed by cell system. Protease Inhibitor cocktail that inhibit multiple classes of matrix proteases inhibit invasion but may be toxic for long term treaments. The effects of individual protease inhibitors on the cell system are compared. 1μM of Calcein AM and 2μM of Ethidium homodimer-1 are the optimal concentrations for the live-dead assay, which can be used to compare viability of mammosphere at different stages. Tumor xenografts are grown in mice for tumor biopsies, which will later replace the MCF10A cell lines in order to identify key functional signatures of the tumor cells.

Methods

Soft lithography

• Mix curing agent and elastomer base
• Pour PDMS over master
• Cure PDMS in oven or slow cure on countertop
• Cut and peel PDMS
• Attach PDMS to glass using plasma bonder

Matrigel coating of cells in chamberslide

• Trypsinize, centrifuge, and suspend MCF10A cells
• Coat each well of the chamberslides with 30 μL of Matrigel
• Let Matrigel polymerize for 15-20 minutes
• Count cells
• Add 2000 cells with 70μL of Matrigel for each well
• Let Matrigel polymerize for 25-30 minutes
• Add in 500 μL of MCF10A growth media and appropriate amount of protease inhibitors.

Problem and Solution

Problem: The old, bulky design is not suitable for optical imaging, and it only accommodates one cell line during one experimental run.

Solution: The new bioreactor design allows optical imaging capability due to its smaller clamps and 1mm thick glass slide, and the parallel channels and wells allow for higher thru-put.

Significance

The microfluidic bioreactors are designed to offer significant advantages over traditional cell culture techniques by reducing size and thereby decreasing amount of reagents used while generating physiologically relevant environments for correlating cellular concentrations. Fabrication of a small scale bioreactor system with improved delivery of drugs or assay reagents can be highly beneficial to the development of an anti-tumor therapy, and key cellular functional signatures will be identified to predict the response to anti-tumor therapy. Thus my research can make great impacts in the medical research and biomedical engineering.

Milestones

I have accomplished the following:

• Perfected my cell culturing techniques
• Compiled and written my cell culturing techniques into a booklet
• Grew mammospheres in a bioreactor
• Finished photolithograhpy and soft lithography training
• Designed and tested new parallel bioreactor
• Received confocal microscopy training
• Helped bridge cancer biology to biomedical engineering

Future Works

• Grow mammospheres using MCF10A cell lines in the tissue bioreactor with perfused channel system.
• Collect biopsy samples of MCF-10CA xenografs and grow tumor biopsies in the improved tissue bioreactor clamp system.
• Try additional and combined protease inhibitors or anti-tumor reagents on the cells in the bioreactor system.

Mary Marschner

Pinocytic Loading of Primary T-cells

Abstract

Experiments often hinge on the introduction of foreign substances into a cell. Pinocytic loading is one successful method to get desired substances into the cytosol of a cell. The objective of this project was to continue the study of pinocytic loading in microfluidic devices and to extend the study from jurkat cells to primary T-cells. Using a system of pumps to alternate the environment around the cells from regular media to a hypertonic fluorescent media the experiment was able to show an uptake of fluorescent dye into the cytosol of a primary T-cell.

Fluorescing primary cell

Significance

Understanding the immune response as it starts with a single cell is needed to understand the immune system as a whole. Microfluidic devices enable a single cell to be studied while their environment changes. Still, how a cell functions is often much better understood when a comparison can be made between a properly working cell and a cell that has a specific gene missing. The eventual goal of this project is to be able to easily and quickly insert siRNA into a cell within a microfluidic device. The siRNA will interfere with the expression of a particular gene and this would enable observations to be made between normal cells and cells with particular genes turned off.

Milestones

I have accomplished the following:

• I made a silicon wafer master of a microfluidic device
• I used micro-fabrication to create a device out of PDMS and a cover slip
• I used the microscope and learned how to program Metamorph to observe experiments
• I successfully loaded cells into a device
• I assembled and used air-tight syringes
• I learned how to use a system of pumps to change the environments of the trapped cells
• I learned how to keep a Jurkat cell line going
• I learned how to thaw primary T-cells to use in experiments

Matt Houston

The effect of media flow rate on CD4+ T cell viability in microfluidic devices

Abstract

CD4+ T cells, like all living organisms, require nutrients. The nutrients for cells in microfluidic devices are provided by the media in which the cells live. When cells remain in stagnant media, the organisms will eventually metabolize all of the nutrients from that media, and then continue on to starve and die. When cells are in a microfluidic device, it is beneficial to any experimenter to know the ideal situation for maximum cell viability. Solving part of this enigma is the purpose of my experiments. I wish to determine the effects that different flow rates of normal media will have on trapped CD4+ T cell viability, with a long term goal of establishing an ideal flow rate for maximum cell viability.

In the course of this project, many interesting results have been obtained. Cells were observed dieing in microfluidic devices, and were observed to survive surprisingly well in PEEK polymer tubing. The PEEK tubing is used to load cells into the device, as well as pump media, YOPRO, or other substances into the device. An experiment was done to test the death rate of these cells inside PEEK tubing, to try to measure the extent of this variable. The results proved that very few cells die inside the PEEK tubing, even when the cells are held for long periods of time (up to 17 hours). Once in the device, experiments were run with media flowing at different rates, and cells were successfully observed dieing. To date, a significant trend cannot be established for death rate at variant flow rates, but this feat should be overcome in the near future.

Significance

T Cells are an obligatory part of the human immune system. Still, there is much to be learned about and from T Cells. An important method of T cell research is that which uses microfluidic devices, because it allows the experimenter to observe each individual cell. My experiment will inform other experimenters of how to achieve ideal cell viability within a device. The data should tell the best flow rate for T cells to maximize their lifetime, allowing the researcher to experiment on the cells under an ideal environment, and for a longer period of time. Thus, my experiment is significant to medical research because of its relevance to other experimenters using T cells in microfluidic devices.

Milestones

I have accomplished the following:

• I designed and fabricated microfluidic devices using soft lithography.
• I made silicone master wafers using photolithography techniques.
• I simultaneously controlled multiple Harvard Apparatus pumps using serial communication.
• I learned to properly load gas-tight syringes.
• I learned proper cell culture techniques.
• I received training in machining.
• I worked with T cells, Dendritic cells, and Jerkit cells.
• I used an electron microscope to capture SEM images of the microfluidic devices.

Priya Sivasubramaniam

Toxicology Studies Using Microfluidic Devices and T Cells

Abstract

Microfluidic devices were used in order to trap primarily Jurkat cells in order to then inject a given toxin and observe its effects upon individual cells that have been trapped in the traps of these devices. This is key as although several effects are known on a macroscopic scale in regards to these toxins, little is known on a one-cell, microscopic scale in these directions.

Methods

Various microfluidic trap devices were created using the process of microfabrication with chrome masks formed via photolithography techniques. In doing so, PDMS was poured on top of these chrome masks and this was then placed in a degasser. Nitrogen gas was used to rid of all remaining air bubbles upon being taken out of the degasser. This was then placed in an oven so as to solidify. This substance was then removed from the oven, cut out of the petri dish that it had been poured and baked in, and holes were punched in its input and output holes. One-hole devices were not used in these experiments; three-hole devices seem to be the most useful in this particular field of study. These devices were then plasma bonded to cleaned cover slips and saved to be used for later experiments.

An ionomyocin experiment was performed in order to observe calcium intake in Jurkat cells. Calcium chloride was mixed into the cell media that was used, with 18 microliters of calcium chloride per 1 mL of media. Two different syringes were used, which called for two Harvard Pico Plus pumps. Jurkat cell media was drawn into the first syringe syringe.The ionomyocin that was used was diluted two times. The first dilution involved taking 5 microliters of ionomyocin and mixing that with 1,000 microliters of media. The second dilution involved taking 5 microliters of this newly made diluted solution and mixing that with 1,250 microliters of media. This solution was then put into a second syringe. 50 micrograms of Fluo3AM Dye was mixed with 50 microliters of DMSO. This solution was pipetted up and down. 5 microliters of this stock solution was then put into 1 microliter of Jurkat cells. These cells were suspended for 20 minutes. After priming a two-hole trap device with the calcium chloride media that was previously created, these Jurkat cells were spun down using a centrifuge and the pellet of Jurkat cells that was thus formed was aspirated into the Pico tubing of the first syringe filled with media. As the three syringes were securely attached to the Harvard Pico Plus pumps, and fastened to the device, the first syringe was run forward at 0.250 microliters so as to load the aspirated Jurkat cells into the designated traps. This was done for approximately 20 minutes, ensuring numerous trapped cells for observation. Ionomyocin from the second syringe was then pumped into the device, also at 0.250 microliters.

Initially, two-hole microfluidic devices were used. This called for two seperate syringes, and two Harvard Pico Plus pumps. Jurkat cell media was drawn into the first prepared syringe, and 2% formaldehyde was drawn into the second syringe. This formaldehyde had been diluted from 38% to the given concentration. A 2-hole device would be primed with solely the media of the first syringe. Upon doing so, the primed device was temporarily stored in a petri dish containing a humid atmosphere, manifested by slightly watered Kimwipes. 1 mL of Jurkat cell solution, typically on Passage 6, were spun down using a centrifuge so as to form a pellet of Jurkat cells. These cells were then aspirated into the Pico tubing of the first syringe filled with media. To do so, the media-filled syringe was attached to a Harvard Pico Plus pump with a clamp atop it. The pump was run anywhere from 0.200 microliters to 1.000 microliters forward in order to ensure forward flow and a lack of air bubbles at the beveled end of the tubing. After successfully running this pump as such, the end of the tubing was placed into the tube containing the centrifuged Jurkat cells, while the pump was still being run in the forward direction. Once the beveled tip of this tubing was safely in this solution, the direction of the pump was reversed so as to aspirate these cells. This was done for approximately 4.5 minutes during each experiment, given that the tubing measured approximately 24 inches. After sufficient aspiration of Jurkat cells, usually at 0.250 microliters per minute, the direction of the pump was switched to forward, so as to prevent air bubble intake into the Pico tubing, and the tubing was drawn from the cells and placed into the stored device. This device was then also connected to the second syringe containing formaldehyde. Cells were then loaded into the device at approximately 0.200 to 0.250 microliters per minute, until a sufficient amount of cells had been trapped in the traps of the given microfluidic device. These cells were run through the device for approximately 20 minutes. Formaldehyde was then run through the device so as to fix the cells.

=== Results === With the ionomyocin experiment, as this took place, the crac channels in the cells opened up, causing an influx of calcium into the cells. The ionomyocin locked the crac channels in a semi-open position, meaning that the sarcoplasmic reticulum, which generally allows calcium to be put back out of the cell, was thereby ineffective. This was observed by the calcium indicator seen within the Fluo3 fluorescent dye, as the cells with a heavy influx of calcium lit up.

Observing data gained from the formaldehyde experiment, one can assert that formaldehyde fixes Jurkat cells but perhaps is useless in regards to Primary T cells as their movement is already limited in comparison to Jurkat T cells. Jurkat cells move slightly and their stop in movement is noticeable when formaldehyde is added. However, one must question the movement of some Jurkat cells during observation with the consideration that this movement may be due to media flow from the syringe.

Problems

Primary cell aspiration had to be done a bit differently from Jurkat cell aspiration. This problem was dealt with as constant experimentation indicated that while Jurkat cells can be aspirated slowly at a steady rate, primary cells must be aspirated relatively quickly, somewhat all at once, so as to allow a sandstorm of these cells into the device upon loading, in order to allow enough cells to be trapped, as these cells are smaller and harder to trap than Jurkats. Primary cells must be aspirated for approximately 4.5 minutes when using a 24 inch segment of Pico tubing.

Air bubbles were an issue that has somewhat sufficiently been dealt with. Most of the air bubbles that would enter the device, thereby killing the loaded cells, would be drawn in through the ends of the green sleeves used when putting together a syringe. This problem was finally observed and approximately 1 mm of each end of the green sleeve is cut off prior to attaching it to a syringe, preventing most air bubbles.

2 microliters of DAPI were mixed with 2,000 microliters of media. Granted that the proper Zeiss filter was unavailable during these experiments, and a halogen lamp was used instead, problems seeing the predicted blue stain upon the nuclei of these fixed cells could be due to either not enough DAPI used, or the current lack of proper viewing equipment.

Some of the microfluidic trap devices that were used seemed to be quite inefficient. One specific trap device was not used during these experiments; instead, several different devices were used. Some of these had poor trapping ability and efficiency, while others trapped too many cells, causing less flow of formaldehyde through the device, and too many cells stuck in all parts of the given device.

Future Directions

With a provided list of toxins, a vast number of toxins with unknown microscopic effects can be observed through similar procedures as the aforementioned ones, using microfluidic trap devices and Jurkat cells.

Formaldehyde will be injected in segments, instead of the 100% switch from media to formaldehyde that was used in these performed experiments. This will allow experimenters to see the effect of formaldehyde upon Jurkat cells when injected at 10% increments.

References

1. Kevin Seale
2. Eric Kim

Sam Nackman

The use of microfluidic devices for the selective breeding of protozoa

Introduction

Protozoa are a crucial part of our ecosystem. Understanding protozoan behavior, including growth rates, mobility, and adaptability, will contribute to the fields of ecology and microbiology, while also benefiting practical applications, such as bioremediation. Understanding protozoa a

Significance

T Cells are an obligatory part of the human immune system. Still, there is much to be learned about and from T Cells. An important method of T cell research is that which uses microfluidic devices, because it allows the experimenter to observe each individual cell. My experiment will inform other experimenters of how to achieve ideal cell viability within a device. The data should tell the best flow rate for T cells to maximize their lifetime, allowing the researcher to experiment on the cells under an ideal environment, and for a longer period of time. Thus, my experiment is significant to medical research because of its relevance to other experimenters using T cells in microfluidic devices.

Milestones

I have accomplished the following:

• I designed and fabricated microfluidic devices using soft lithography.
• I made silicone master wafers using photolithography techniques.
• I simultaneously controlled multiple Harvard Apparatus pumps using serial communication.
• I learned to properly load gas-tight syringes.
• I learned proper cell culture techniques.
• I received training in machining.
• I worked with T cells, Dendritic cells, and Jerkit cells.
• I used an electron microscope to capture SEM images of the microfluidic devices.

Silviu Diaconu

Dynamic Changes of Cardiac Conduction Velocity During Rapid Pacing in EMD and TNT Knockout Mice

Abstract

The conduction velocity of different mouse hearts was calculated from previously recorded optical data. Using Matlab, a CV-restitution curve was constructed for each mouse heart. Three different types of hearts were analyzed: Control, EMD and TNT-mutants. This analysis showed that EMD mice conducted the action potential slower and had a faster fall off in conduction velocity than the control mice.

Significance

One of the leading causes of death occurs due to lethal cardiac arrhythmias such as ventricular fibrillation. It is well known that a premature ectopic beat can lead to lethal re-entry and cause fibrillation. However patients susceptible to fibrillation can have up to 1 million ectopic beats per year but only one of those might induce sudden cardiac death. Recent research has focused on determining on what makes that one beat so different from the rest. It is believed that it’s not the beat that special but rather the dynamic state in which the heart is; characterized by slow conduction and dispersion of refractoriness. It has been shown that when the heart is in a state where it’s in the steep portion of the CV-restitution curve initiation of lethal arrhythmias is much more common. Therefore understanding the effects of EMD on the CV-restitution curve can help identify the characteristics required for lethal arrhythmias.

Methods

The mouse heart was paced at a specific frequency. As the action potential traverses the heart the region of the heart that is activated turns darker than the surroundings due to a calcium dependent die. This allows the action potential propagation to be recorded across the heart. The first step in analysis is to determine when each point of the heart is “activated” by the action potential. Our definition of this event is when dI/dt is at its maximum where I is the intensity of light emitted from the heart. From this data we can construct an isochoric graph that shows regions of the heart activated at the same time. The conduction velocity is calculated for different vectors across the heart. This is done by plotting the distance of the points on the vector from the stimulation point versus the time of activation for each of the point. The slope of this graph is then the conduction velocity of the heart along that vector. For each heart, the conduction velocity is calculated for 60 to 70 vectors and then averaged to get the overall conduction velocity for that specific pacing frequency. This method is repeated for different pacing frequencies such that we can plot conduction velocity versus pacing frequency also known as a CV-restitution curve.

Milestones

I have accomplished the following:

• I was successful in developing a method for quantifying the conduction velocities from optical data for different pacing frequencies.
• I successfully determined the CV-restiution curves for control and EMD mice
• I showed the dependence of conduction velocity on heart fiber orientation.
• I demonstrated that conduction velocities are slower in EMD mice when compared to control mice.

Future Goals

• Determine the CV-restitution for TNT-mice
• Compare the conduction velocities between EMD and TNT mice
• Possibly apply my method to rabbit heart recordings.

Stephen Arndt

The rate of flourescent dye loading in T cells

Abstract

Flourescent dyes are one of the most valuable tools for the investagation of cellular processes, as they allow for the cell to be studied in unique ways. However the use of these dyes in an environment like the nanophysiometer is relatively new and many parameters still need further study. For many experiments flourescent dyes are loaded into the cells macroscopically, with little being known about how quickly the cells load the dyes individually, or if the rate of flow in the device effects loading of the dye, as well as maximum flourescence. This experiment seeks to ascertain the rates of flourescent dyes loading into individual cells in a microfluidic device, as currently we are unsure if there is much variance in the population, and if placement in the device can be corelated to flourescence.

In the course of this project, many interesting results have been obtained. Cells were thought to have died in the microfluidic devices, however YOPRO staining has not yet been used to ascertain the rate or times. Cells were observed to have ocillating flourescence, but the period of the oscillation has not yet been determined.

Significance

T Cells are an obligatory part of the human immune system. Still, there is much to be learned about and from T Cells. An important method of T cell research is that which uses microfluidic devices, because it allows the experimenter to observe each individual cell. My experiment will inform other experimenters of how to achieve ideal cell viability within a device. The data should tell the best flow rate for T cells to maximize their lifetime, allowing the researcher to experiment on the cells under an ideal environment, and for a longer period of time. Thus, my experiment is significant to medical research because of its relevance to other experimenters using T cells in microfluidic devices.

Milestones

I have accomplished the following:

• I designed and fabricated microfluidic devices using soft lithography.
• I learned how to create metamorph macros to fully automate my experiments.
• I simultaneously controlled multiple Harvard Apparatus pumps using serial communication.
• I learned to properly load gas-tight syringes.
• I learned proper cell culture techniques, including thawing and freezing jurkats and reviving primary T-cells
• I worked with T cells,both primary and Jurkat.
• I used an electron microscope to capture SEM images
• I learned how to use ImageJ to process the captured images and get data from the experiment