Division of Jurkat T-Cells in a Microfluidic Device

Abstract

There are many advantages of using microfluidic devices in systems biology studies. Miniaturization allows for real-time control of the cellular microenvironment, precise instrumentation and analysis, and the use of minimal solution volumes. An area of current interest is the capability of a microfluidic device to mimic in vivo conditions. Short of this goal, but still important, is the ability of such devices to equal or surpass current in vitro techniques for lab practices such as cell culture. Although T-cell and other immunology research is ongoing in the VIIBRE laboratory, cell division of primary T cells or T cell lines inside a microfluidic device has not been accomplished. In an effort to explore the feasibility of long-term microfluidic studies of immune cells, I am studying a group of dividing Jurkat T-cells within a microfluidic device. Since the Jurkat cell constantly divides without stimulation, I propose that if the correct environment can be maintained within the device for a 24 hour period, cell division and subsequent viability can be quantified.

Significance

Studying T-cells is of great importance to science because they are the primary cells of the immune system. Greater understanding of T-cells increases the chances of finding ways to prevent and cure many types of disease. For instance, specific understanding of immune pathways opens the possibility of controlling and manipulating them to produce a desired effect. One issue in using microfluidic devices is the difficult task to mimic in vivo conditions as closely as possible. It is arguable that cells do not behave normally when in a foreign environment, and therefore in vitro studies must attempt to determine and defend the accuracy of the findings. Jurkat cells divide constantly, assuming that they are provided the correct environment. Therefore, division of Jurkat cells within a microfluidic device is important because it is an indicator of the long-term suitability of the experimental environment to foster normal behavior of the T-cells.

Milestones

Achieving division of Jurkat cells within a microfluidic device requires learning a variety of other techniques and processes in advance. While working on other projects in the VIIBRE laboratory over the last year, I learned the necessary microfabrication techniques to create the devices used in the experiment. The process includes photolithographic fabrication of an SU-8 master which serves as the mold for the device which, when plasma bonded to a glass slide, forms the microfluidic channels and traps. In addition to the fabrication steps, it is necessary to learn effective experimental techniques; in this case those developed by Kevin Seale and Shannon Faley, and then adapt them to the specific aspects of my experiment.

 

Weeks 1-3

•  Became familiar with the equipment and software in the GPFM lab

      - Zeiss Inverted Microscope with automated stage

      - Automation of the Harvard Apparatus Pico Pumps

      - Learned how to control the pumps, stage, camera, and experiment using Metamorph software. Allows for running long-term     

        experiments overnight, with programmed control of pumps, stage, and cameras specific to experimental needs

      - Exposed to ImageJ analysis of experimental results in the form of DIC, FITC, and TRITC images (programming developed by Kevin

        Seale)

•  Learned experimental setup techniques developed by Kevin Seale and Shannon Faley

      - Loading Hamilton Scientific syringes, ensuring sterility and absence of air bubbles

      - Priming the device to have no air bubbles

      - Loading cells into the PEEK tubing

      - Assembling and securing device housing for long term image acquisition and subsequent analysis

•  Developed a specific research to pursue independently

      - Refined goals to meet objectives of the T-cell group and VIIBRE

      - Adapted goals into a testable thesis

Weeks 4-7+

•  Learned cell culturing and related procedures from Shannon Faley

      - Preparing T-cells for experiment

           •  Thawing, resuspending, centrifuging

      - Upkeep of Jurkat cell line

           •  Involves cell counting, splitting, and freezing

           •  Ability to gauge and control the condition of the cells and their environment

•  Ran preliminary viability experiments independently

CD69 expression of individual T Cells activated simultaneously within a microfluidic device

Abstract

CD69, a phosphorylated disulfide-linked homodimeric cell surface protein, is one of the earliest markers of T Cell activation. Studies have shown that this activation antigen exhibits rapid expression, reaching its peak expression within about 8 hours of stimulation, followed by a rapid decay and degradation.

Figure 1: Adapted from Maino, Suni, Ruitenberg, Smith-McCollum

 

My project is to be able to show the expression of CD69 on T Cells that have been activated in a number of different ways within a microfluidic device. Current data on CD69 is based on the use of flow cytometry. Flow cytometry takes the average fluorescence over a period of time. With our microfluidic devices, we can see individual cells on a scale of thousands and observe the individual fluorescence over time.

Significance

My project is to be able to detect and record evidence showing the presence of CD69 on the surface of an activated T Cell in a microfluidic device. Using a microfluidic device allows me to analysis individual cells. All previous data on CD69 has been found by using a flow cytometry. This method takes the average fluorescence of a bunch of cells in order to come out with data that is an average of a bunch of cells. My research allows me to get on individual cells over a scale of thousands and thus detect oscillations and other movements that couldn't have been detected before.

Milestones

Visualization of Heterotrimeric G Protein Activation in Living Cells

Abstract

Chemotaxis, the method whereby a cell senses an external chemical gradient and responds to it, is a fundamental process used by many different cell types.   In the model system Dictyostelium discoideum the chemotactic signaling system is capable of recognizing two chemical chemoattractants, cyclic adenosine monophosphate (cAMP) and folic acid.   When either chemoattractant binds to its receptor on the surface of the cell the signal is transduced through an almost identical network of proteins.   Receptors that have bound chemoattractant activate heterotrimeric G proteins which dissociate and transduce the signal to other intracellular effectors.   In addition to the different receptors, the primary distinction between the cAMP and folic acid signaling apparatus is the alpha subunit of the coupled G protein.

The main goal of this project is to measure the speed at which G proteins become activated in response to chemoattractant.   This portion of the project relies on a   Fluorescence Resonance Energy Transfer (FRET) based assay [1] used in conjunction with Total Internal Reflection Fluorescence (TIRF) microscopy.   We plan to work with the G proteins coupled to both the cAMP and folic acid receptors as well as visualize G protein localization and activation during cytokinesis.   Additionally we will determine a dose-response curve for the cAMP receptor cAR1 based on its phosphorylation state.   We will determine this dose-response curve for both cAMP and other analogs.

To date we do not have any final results.   We have tested the cells we will use for the FRET-based assay and the dose curve and have begun to look at the localization of G proteins during cytokinesis.   We have very preliminary results regarding the activation speed of G proteins.   In addition we have a trial cAMP dose curve for receptors that have been phosphorylated.   Work to bring this project to completion will continue throughout the course of the year.

Significance

Heterotrimeric G proteins and the receptors to which they are coupled are integral parts of many different signaling systems and are found in many different cell types.   G proteins are activated in response to ligands ranging from neurotransmitters to chemoattractants.   Because G protein coupled receptors are the single most important class of drug targets knowledge regarding where and how quickly G proteins are activated in response to signals from a receptor is medically relevant. [2]  The biological implications of this project are widespread; the kinetics of G protein activation in D. discoideum would likely apply to the multitude of other G proteins that are involved in various signal transduction pathways.   Additionally studies on the localization of G proteins during cytokinesis could bring new functions of the G proteins to light.

Milestones

We have accomplished the following:

• We have shown that the aßgamma transformants are stable.  
• We have become familiar with TIRF microscopy and use of the fluorometer.
• We have preliminary data from aßgamma cells in an 8 well plate.
• We have begun collecting data from aßgamma cells in the perfusion chamber.
• We have preliminary data from the fluorometer for a cAMP dose curve in adapted cells.
• We have begun collecting data regarding the localization of Gß during cytokinesis.

 

Haptotactic Component of Cancer Invasion

Abstract

Cell motility is essential to cancer invasion. Cells use a variety of directed migration mechanisms in response to chemotaxis, mechanotaxis, or haptotaxis. Many cancer studies focus on chemotaxis as the main event for a cell to invade. However, little is known on the role of haptotaxis in cancer invasion. We would like to produce a quantitative understanding of the haptotactic component of cancer invasion. We prepare a flourescently labeled extra cellular matrix (ECM.) We use a hydrodynamically focused protein stream to lay down the gradient, and then cells are introduced into the microfluidic device and are allowed to adhere and spread. Haptotaxis is then followed by videomicroscopy. Fluorescent pictures are acquired to assess the slope of the gradient.   Brightfield movies are analyzed to calculate parameters such as cell speed and trajectory. Our results show that we could prepare matrix gradients using the device, which is suitable for live-cell imaging. We find that cells respond differently to matrix gradients with distinct slopes and/or concentration ranges.

Significance

Cancer invasion makes cancer a life-threatening disease by severely limiting treatment options such as surgical resection. It is unclear what triggers cancer invasion. Most human cancers arise from epithelia. A critical component of the epithelial cell microenvironment is the basement membrane (BM), which is rich in extra cellular matrix (ECM) macromolecule laminin-5. Laminin-5 (Ln-5) is exquisitely localized in BM, to which epithelial cells adhere, forming continuous sheets. Ln-5 mutations may result in blistering (in the epidermis, oral and esophageal mucosa), with severe health consequences. It will help to understand the basic mechanisms of cell adhesion to laminins by studying substrate-stimulated migration (haptotaxis.)

Quantification of intracellular junction strength using a microfabricated spring assembly

Abstract

One of the most significant challenges facing cancer biologists today is developing an understanding of the biochemical and biomechanical mechanisms the results in metastasis. In order to strengthen the understanding of such mechanisms, it is first necessary to quantify the effects of the various parameters on the strength of cellular junctions of cell lines known to be susceptible to metastasis. The propose of this project is to develop a method for quantifying the strength of intercellular junctions due to E-cadherin protein.

To this effect, two methods of measuring this force have been investigated. In the first assay, cells are brought to the tip of a micropipettes under a small negative pressure. This process is repeated for a second pipette that is attached to a pressure sensor. The cells are then brought together, and the micropipette containing the pressure sensor is removed while the cells form a doublet. The second micropipette is returns, and applies a negative pressure sufficient to separate the cell pair. By, measuring the force require to separate the cell pair. A quantitative measurement of cell adhesion strength could be obtained.

In the second approach, cell pairs may be formed at the ends of a microfabricated array of springlike structures. By using attaching on end of the cell pair to By using classical strength of materials and finite element modeling, a force displacement relationship for the springs may be obtained, and this relationship can allow for a precise measurement of the separation for of the cell pair.   Furthermore, the presence of a large of array of array of springs allows for the measurement of the cell adhesion strength in a manner that can be both rapid and efficient.

While the dual micropipette assay has proven impractical for the cell lines being used, the initial modeling and testing of the microfabricated spring array has been completed.

Milestones

The following has been accomplished so far:

• Assembly of Dual Pipette assay system
• Qualitative demonstration of separation of cell pairs with pipette system
• Fabrication of   first prototype spring array
• Development of mathematical model for spring response
• Design of second testbed structure for validation of spring model

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.

 

 

Real-Time pH Measurements in Microfluidic Devices

Abstract

A scheme has been proposed that achieves flexible control over the pH of a bioreactor.   The versatility of the system is characterized by the ability to finely adjust the pH by altering the ratio of high pH and low pH media delivered by two Cavro® XLP 6000 syringe pumps based on the pH of the effluent from the bioreactor.   The media contains relatively low concentrations of phosphate buffer, thereby permitting a detectable change in the pH of the perfusate after it has left the bioreactor.   From this measured change the metabolic activity of resident cells can be backcalculated.  Verification of the accuracy of our model for the backcalculation motivates experiments in which we measure the pH of solutions created by mixing two buffer solutions of known pH in various proportions.   In these experiments, solutions of sodium phosphate monobasic and sodium phosphate dibasic served as the low pH and high pH buffer solutions, respectively.   The pH of the mixed solutions were measured with either a typical pH meter or a microsensor depending on the prepared volumes and setup.

Milestones

• Reformulated forward-calculation of bioreactor pH based on pump flow rates, buffer solution properties, and metabolic rate of cells.
• Reformulated back-calculation of cell metabolism.
• Completely redesigned and rewrote the LabView dry lab simulation of the bioreactor.

• Demonstrated successful communication with Tecan Cavro® XLP6000 syringe pumps through serial port and control of plunger movements in half-step and microstep modes.
• Gravimetrically diagnosed accuracy of flow rates achieved by the pumps through all valve ports with connected tubing across the range of motor step rates.
• Developed protocol for priming valves and tubing and for forcing trapped air out of the syringes and valve system.

 

Abstract

  Cancer affects many pathways and processes of the cell. However, two processes have been shown to be especially important in cancer research, cytokinesis and chemotaxis. In my research, I examine these processes in the social amoeba Dictyostelium discoideum. Two key molecules in the regulation of cytokinesis and chemotaxis are the phosphoinostides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) (Devreotes and Janetopoulos, 2003; Franca-Koh and Devreotes, 2004; Janetopoulos et al., 2005). PIP2 and PIP3 levels are regulated by the enzymes PI3K and PTEN (Franca-Koh and Devreotes, Iijima et al., 2002). The presence of the phosphatase PTEN at the cell membrane prevents PIP2 from being converted into PIP3. However, the localization of PI3K to the membrane converts PIP2 to PIP3 (Franca-Koh and Devreotes, 2004). PIP3 then recruits PH domain proteins and other effector molecules that activate more proteins downstream (e.g. adenylate cyclase and Actin polymerization ).   PI3K is recruited from the cytosol to the membrane as a downstream effect of chemoattractant binding. At the same time, Pten falls off the membrane. This leads to a dramatic increase of PIP3 levels at the plasma membrane. The return of PTEN to the membrane changes PIP3 back to PIP2.

  The goal of my research is to examine the pathways through which PI3k affects the processes of cytokinesis and chemotaxis. In order to accomplish this, the triple PI3K knockout or PI3K1-2-3- cell line was transformed with PTEN-GFP. This will allow us to research how the absence of three of the PI3K's affects the production of PTEN. However, before research could be conducted on this new cell line, it was important to characterize it. The cell line PI3K1-2-3- was also characterized. Characterization of the cell lines was researched using three different assays. The first assay involved starving the cells to examine their ability to form fruiting bodies. In the second assay, cells were plated along with Ka bacteria.   Finally, cells were grown in stringent conditions. Doubling time and the ability to effectively chemotax were examined at this time.

  In the future, the role of PI3K in relation to Myosin-2 will be examined through the use of laser microscopy. It is suspected their roles are inversely proportional. Further experimentation will prove whether this hypothesis is valid.

Significance

Cytokinesis and chemotaxis are two processes vital to the function of many cells.   Cytokinesis is the final process in cell division, where daughter cells pinch off from one another.   During chemotaxis, cells detect chemical gradients in their environment and direct their movements towards or away from the source (Iijima et al., 2002; Janetopoulus et al., 2005). Defects in either of these processes can lead to development of cancer cells because of damage to chromosomal integrity, increased mobilization and metasization, and lack of proper response to normal stimuli Devreotes and Janetopoulos, 2003).   In addition, “Chemotaxis and signal transduction by chemoattractant receptors play a key role in inflammation, arthritis, asthma, lymphocyte trafficking, and also axon guidance”(http://dictybase.org).   Dictyostelium discoideum is used as a model organism to study the processes of cytokinesis and chemotaxis.

 

Milestones

The following has been accomplished at this time:

•  I have examined and characterized PI3K1-2-3- knockouts containing PTEN-GFP and PH-GFP

•  I have determined the doubling time for PI3K1-2-3-/PTEN-GFP and PI3K1-2-3- cell lines.

•  I have learned new techniques to stimulate cells with chemoattractant.

•  I have begun new experiments examining the role of RAS in relation to PI3K when stimulating developed cells with chemoattractant.

 

Cell Adhesion and pH Control Testing in Microfluidic Bioreactors

Abstract

This group project is working on resurrecting several old programs used in a senior design project and applying them to new use using bioreactors.   These programs run on Labview, and were designed with the intention to control the pH of fluid through a bioreactor in which living cells are seeded.   There are two main components of this system: the pH mixer and the bioreactor.   The programs were initially created to run as a “dry lab,” a simulation of the actual system.   After the simulator is proven to work, then a program is created, based on the simulator, to control the flow of two different solutions with different pH through the pH mixer, monitor the pH, and then flow the combined fluid into the bioreactor.   After exiting the bioreactor, it then measures the outflow pH and adjusts the input flows through a feedback controller in order to maintain a constant pH.  

Essentially, there are six specific goals.   The first is to re-work the existing simulator and bring it into fruition, so that it accurately portrays the real-life pH mixer.   Second is to create an all-encompassing user manual for the pH simulation and the pH control programs, so that future users can easily become acclimated to the project.   Third is to re-work the existing program to accurately monitor and control the real-world pH mixer.   Fourth is to determine the proper coating needed for the cells to adhere to the PDMS within the bioreactor.   Fifth is to successfully seed fibroblast cells into the bioreactor and keep them alive for an extended period of time.   Sixth is to connect the pH mixer, programs, and bioreactor and begin taking data on cell acidification rates and try to control the pH of the bioreactor.

1. Re-work existing simulator

2. Create all-encompassing user manual for simulator

3. Re-work existing program for real world monitoring and control of pH

4. Determine proper PDMS coating needed to adhere cells to bioreactor

5. Successfully seed fibroblast cells into bioreactor and keep alive for extended time period

6. Connect pH mixer, control programs and bioreactor in order to collect data on cell acidification rates

Measurement of Sarcomere Length Changes in Cardiac Myocytes

Abstract

The specific behavior of the cardiac sarcomere is thought to be indicative of many types of heart disease; however, before conclusive research can be conducted to support or deny these theories, several limitations involving the current measurement systems must be overcome. VIIBRE currently makes use of a commercial system made by the Ion Optix Corporation. While this system is designed specifically for the measurement of sarcomere length, most of the commercial and industrial market requiring such a system focuses on imaging chemical transients, requiring high resolution but not fast recording speeds (Lim 970).   With high contraction frequency and only slight length changes in a single cardiac sarcomere, it becomes necessary to develop a different system to obtain an appropriate signal-to-noise ratio at conditions that mimic physiological heart rates.

 

The immediate goals of the project addressed over the course of the summer included evaluating the best mode of imaging the cardiac sarcomere, digesting the image into a digital signal, and processing the signal to extract sarcomere length as a function of time.

Significance

The long-term goal of this project is to develop a fully packaged system capable of supporting cardiac myocyte experiments at physiological conditions in an effort to better link sarcomere behavior to heart diseases and possibly to evaluate the efficacy of proposed treatments.

 

Milestones

The following goals have been accomplished:

 

 

Development of an Automated µ-Valve Testing Controller

Abstract

On chip valves typically use solenoids or pressurized channels to collapse underlying channels. A more recent technique involves turning a screw to provide the downward force which was developed by Whitesides group and was recently reported in Anal. Chem. 2005, Vol. 77, pp 4726-4733. One problem with Whitesides' Twist valves is that the fabrication process is difficult and may result in variable or defective devices. In order to improve on this technology, VIIBRE Lab researchers have developed the TURN (Tape Underlayment Rotary Node) valve system which greatly simplifies and standardizes the process of creating multiple on-chip valves.

Thermal set glue is used to cast valve casing in which a 0-80 screw is inserted.  The bottom of the screw is separated from the channel by a thin PDMS film.  The user turns the screw clockwise, collapsing the underlying channel by depressing the film.

Advantages to the TURN valve:

One aspect of the scientific development process involves failure testing and documenting any changes in closing behavior after repeated cycling.   Such testing is impractical to perform by turning the screws by hand, so I constructed an automated testing center capable of independently turning two screws at a time and running hundreds or more cycles without the possibility of human error in turning the valves.   The system was also designed to measure the degree of channel closure and document the opening/closing characteristics of the valve system in order to establish optimum device parameters.

Milestones

• Did learn LabView
• Did write control program
• Did build testing apparatus
• Will test TURN valve
• Will find failure limit if a reasonable one exists
• Will investigate valve property changes with prolonged cycling
• Will investigate different closure arrangements and optimize valve design

 

Characterization of Slug Movement Using a Silicone Bed of Nails

Rebecca Martinie and Jeremy Walker, with the assistance of Kweku Addae-Mensah and Dr. Chris Janetopoulos

Abstract

 

One of the important phases of development in the model organism Dictyostelium discoideum is the slug phase, which occurs around fourteen to eighteen hours after the start of cell aggregation, which is mediated by cAMP signals that are sent out when the cells are starved.   Little is known about the movement of D. discoideum slugs and the mechanism behind it.   In order to learn more about this important characteristic of slugs, a silicone device with posts labeled with quantum dots, or a bed of nails, was used to characterize their movement.   The movement of quantum dots imposed by the slugs can be observed by creating time lapses on an inverted confocal fluorescent microscope using the Cy3 fluorescent setting to time lapse image the movement.   In order to obtain such data, we first had to come up with a reliable method for developing slugs from GFP-labeled wild type cells.   Then, we had to find a way to transfer the slugs onto the bed of nails without damaging the slugs (figure 1).  

Lastly, we have to successfully image the displacement of the quantum dots caused by the slugs crawling across the bed of nails towards a light source.    Of these steps, the first two have been completed.   There has been some difficulty with the imaging due to delicacy of the slugs, but we have been working to resolve this issue and are very close to obtaining the necessary data.   When the time lapses necessary have been produced, they will be given to Kweku Addae-Mensah, the maker of the bed of nails, who will interpret them using multi-particle tracking software.   Once we have obtained results about the how wild type slugs move, we will then be able to study the movement of various knockouts, and see how the loss of function of a particular protein affects slug movement.   We will use knockouts of proteins that are involved in cytokinesis and chemotaxis, which is studied by other members of the lab.   We hope to gain more information about these proteins by observing their effects on slug movement.   These proteins are analogs to the proteins involved in cytokinesis and chemotaxis in cancer cells.  In addition, D. discoideum slugs are similar to cells undergoing development;of particular interest is that it is only during this phase that D. discoideum have focal adhesions, which are similar to structures found in mammalian cells.  Therefore, through our study of slug movement we also hope to gain more understanding about focal adhesions and their purpose in both slugs and mammalian cells.

Significance

The goal of this project is to learn more about the movement of Dictyostelium discoideum slugs and the mechanism behind this.   This research is important for two reasons.   First, the reason we study Dictyostelium discoideum is because the cellular pathways are similar to cancer cells.   After we finish studying the movement of wild type slugs, we can then study knockouts and learn more about the proteins involved in the pathways by studying their effects on slug movement. Second, the slug stage is the only stage in D. discoideum where there are focal adhesions, which are similar to ones found in mammalian cells. Thus by studying slug movement we hope to learn more about these focal adhesions and their mammalian analogs.

Milestones

We have accomplished the following:

•  Designed a consistently successful method for developing D. discoideum cells into slugs.

 

Primary CD4+ T Cell CRAC Channel Dynamics

Abstract

Previous experiments have shown that ionomycin can cause an increase in intracellular calcium in human T cells (Seale & Faley).   Presumably, ionomycin when introduced to T cells permeates through the cell membrane and interacts with the Sarcoplasmic Reticulum.   This apparently causes the store operated channels on the SR to release calcium into the T cell.   With the release of this calcium from the SR, the plasma membrane bound CRAC channels of the T cells open and allow extracellular calcium to enter into the cell.   In an experimental setting, I hope to prove that these CRAC channels can not only open with the presence of calcium and ionomycin, but close over time without them.

 

The goal of this experiment is to see an intracellular calcium response oscillate over time inside the T cells.   Bathing the cells in the calcium indicator Flou-3 prior to the experiment will help me detect any intracellular calcium in the cell via a florescent image.   So by alternating between media with calcium, media with calcium and ionomycin, and media without calcium I should be able to see high and low points of intracellular calcium in the cell.   This will then give me an idea of when the CRAC channels open and close and hopefully how much ionomycin and calcium will be needed in order to accomplish that task.

 

                                     

Electron microscope images of the traps  (Soike 2006)     

Significance

The T cell CRAC channels, located on the plasma membrane, are designed to direct calcium molecules into the cytoplasm of the cell.   This response is triggered by a slight increase in intracellular calcium.   Normally in a T cell, the cell will bind to an antigen presenting molecule and thus trigger the IP3 DAG activation pathway.   IP3 binds to the IP3R channel on the ER membrane and causes the ER to pump calcium into the cytoplasm.  An increase in the intracellular calcium of the cell causes the CRAC (calcium released activated calcium) channels to open.  This in turn causes an extremely large increase in intracellular calcium and leads to the next step in gene expression.   However, in this experiment I tried to skip the IP3 DAG pathway and directly increase the intracellular calcium using ionomycin.   Ionomycin guides extracellular calcium across the plasma membrane and calcium located into the ER into the cytoplasm of the cell.   This effect also increases intracellular calcium independently of the IP3 DAG pathway.   So by proving I can open and close these CRAC channels with ionomycin I showed that I can skip the initial step of gene expression and start cell activation further down the line.   Hopefully at some point I will be able to get to the next step of gene expression and maybe someday, not using an antigen expressing cell, I can activate a T lymphocyte.

Milestones

• Learning Microfabrication so I can create my own PDMS devices.
• Learning Photolithography so I can create my own masters.
• Becoming Machine Shop trained so I could build any necessary parts for my experiments in the machine shop.
• Learning how to load the Hamilton glass tight syringes with the peet tubing and use them in the Harvard Apparatus Pico Plus pumps.
• Learning Metamorph coding in order to communicate with the microscope and be able to take DIC, TRITC, and FITC images.
• Learning Serial Data coding and in order to communicate with the Pico Plus pumps through Metamorph.
• Understanding flow and diffusion rates in spiral mixing device
• Learning how to properly coat cells in Flou-3, mix media with ionomycin, and mix media with calcium.
• Running the ionomycin titration experiment to find minimum level of ionomycin needed.
• Running an experiment to see if the CRAC channels on the T cell's membrane close.
• Running ionomycin / calcium balancing experiment to find ideal amount of ionomycin and calcium concentration to oscillate between opening and closing the CRAC channels.

Abstract

Microfluidic devices provide a closed environment to accurately and efficiently measure respiratory oscillations in yeast cells.   The devices were constructed using PDMS (polydimethylsiloxane) in conjunction with masters created via photolithography.   The PDMS bioreactors provide an appropriate medium through which to image the cells of interest via light microscopy.   Eventual measurements of interest include pH, NADH/NADPH, and Oxygen.

Significance

Short-term goals include the creation of a bioreactor to measure short-term respiratory fluctuations occurring in 1-4 hour cycles.   While short term goals do not necessarily require nanoscale bioreactors, long term goals include the possibility of measuring respiratory fluctuations in various strains of evolved yeast.   These evolved yeast could exhibit varying circardian respiratory oscillations of 18-24 hours.   As a result, several nanoscale reactors measuring different respiratory circadian oscillations simultaneously in different strains of yeast would be ideal.

Oxygen-sensitive films for microfluidics

Abstract

There exists a great need for an oxygen sensing device that is compatible with microfluidic devices. The current technologies for oxygen sensing utilize Clark electrodes, dyes imbedded in sol-gels, or some form of electronic monitor.   Unfortunately, these approaches are not stable, give a non-uniform signal, require low noise electronics, or are bulky. To circumvent these limitations, we set out a novel, non-invasive optical thin film sensor based on tetrakis(pentafluorophenyl)porphyrin (Pt-TFPP) embedded in a polystyrene (PS) support matrix. The fluorescence of the Pt-TFPP is quenched in the presence of O 2 molecules due to non-radiative energy transfer from luminophore to oxygen. This compound is ideal because of its bright luminescence, high sensitivity to O 2 , and biocompatibility. To enhance O2 permeability of PS, oxygen permeable, polydimethylsiloxane (PDMS) is added to the film to allow access to dye below the surface. A mixture of Pt-TFPP, solid PS, and PDMS is dissolved in toluene then spun onto a glass slide.   As a result, 1-2 µm thick, highly robust, smooth films with a uniform dye distribution are formed on the glass surface. Several film compositions and thicknesses are evaluated and compared to obtain optimal balance of sensitivity, O 2 permittivity, and response time.   Ultimately, these O 2 sensitive films will be used as non-invasive, label-free sensors for measurement of oxygen consumption of tissue growing in microfluidic bioreactors.

Milestones

•  Thin films of PS and PDMS were spun and analyzed

•  The best host matrix was identified

•  Pt-TFPP was integrated into the host matrix

•  The luminescent film was characterized by profilometry, spectrophotometry, and fluorimetry

•  The film was integrated into a microfluidic bioreactor

•  Response of the film to various oxygen concentrations in gas and media was documented

Evaluation of the Local Excitation Global Inhibition (LEGI) model of cell chemotaxis

Abstract

Many cell types have the ability to move in reaction to chemical gradients. This process, known as chemotaxis, is sensitive even to very shallow gradients (1). Chemotaxis plays a vital role in immunity, embryological development, and wound healing and is also involved in inflammation, allergic responses, and cancer metastasis (2, 3). In order to better understand these processes, it is necessary to understand the mechanism by which cells sense and respond to external chemical gradients. By studying chemotaxis in D. discoideum , we hope to illuminate the pathways involved in chemotaxis in humans.

D. discoideum , an amoebic slime mold found in soil and leaf litter, was chosen as the model system for studying chemotaxis because of its hardiness and because its genome can be altered with reasonable ease. Also, the pathways involved in Dictyostelium chemotaxis bear significant resemblances to the pathways involved in human neutrophil chemotaxis (4). In both cell types, it is evident that directional sensing is marked by the accumulation of phosphatidylinositol 3, , 5-trisphosphate [PI(3,4,5)P 3 ] on the plasma membrane, regulated by a heterotrimeric G-protein pathway (5). This G-protein pathway allows for significant amplification of the chemical gradient. [PI(3,4,5)P 3 ] is created by the phosphorylation of [PI(4,5)P2] by PI3 kinase (PI3K) (5). Once formed, [PI(3,4,5)P 3 ] recruits several other proteins to the membrane, ultimately leading to actin polymerization and the extension of pseudopodia. Using the GFP-labeled pleckstrin homology (PH) domains of one such protein, CRAC, we are able to visualize accumulation of [PI(3,4,5)P 3 ] on the cell membrane (4).

             

How does the cell sense the chemical gradient and convert that into a biological response? The Local Excitation, Global Inhibition (LEGI) model suggests that the chemotactic pathway is regulated by the concurrent creation and destruction of binding sites for PI3K, described above and PTEN, a phosphatase that converts   PI(3,4,5)P 3 to PI(4,5)P 2 (5). The model proposes that PI3K binding sites are created only in the area of the membrane stimulated by chemoattractant, while PTEN binding sites (PTEN binds to the membrane in the absence of chemoattractant) are localized to the back of the cell (5). The global inhibition occurs in the presence of chemoattractant. The inhibitor is thought to be diffusible and spreads evenly throughout the cell (5). So, every part of the membrane is equally inhibited at a level determined by the average receptor occupancy of the cell. The front side of the cell portion of the membrane has a receptor occupancy higher than the average occupancy of the cell, so it remains excited. Activation of these receptors can also activate adenylyl cyclase, which produces cAMP, thereby allowing a chemotaxing cell to leave a chemical trail for other cells to follow (6).

Over the course of the summer, I set out to test the LEGI model using using multiple microfluidic devices, two-needle assays, and multi-barrel pipets. Although I have made progress in many techniques and assays, many experiments remain to be performed in order to further clarify the mechanism of cell sensing and chemotaxis.

Significance

 

Milestones

Using the cell trap device, I have been able to successfully trap multiple cells in the goblets and establish a GFP gradient over the exposed surface of the cell. This indicates that I will be able to successfully use the device to further test the LEGI model. (See Figure).

 

 

 

 

Quantification of Separation Forces Between E-Cadherin Mediated Cell-Cell Pair Adhesion

Abstract

In order to create a mathematical model of cancer cell development and progression, many additional discoveries must be made concerning cellular interactions, motility and signaling pathways.   The objective of this project was to create and test a dual-micropipette system to find if it was accurate and efficient enough to quantify separation forces between E-cadherin expressing cells, namely A431 and MDCK cells.   These two cell types were chosen because they possess similar adhesion mechanisms to MCF10 cells, a human breast epithelial cell line exhibiting neoplasia, but have longer lifespans and are easier to passage, making them ideal to test the system.   The system requires using known amounts of pressure to capture a cell on the tip of each micropipette, which are then brought together to form a doublet.   Then, the suction on one pipette, which is attached to a pressure sensor, increases until the cells detach from one another.   Quantifying this change in pressure could show how strong the cellular adhesion is.   After getting the system working, our next step would be to test other variables, such as sucking one cell off a mass of cells, changing the time allowed for the cells to adhere, and determining the number of adhesion junctions formed.   In the end, it was found that the dual-micropipette system was inefficient for the scale we needed to work at.   For the small pipette tips, the amount of pressure necessary to manipulate the cells greatly exceeded the dynamic range of the pressure sensor.   This system was inefficient and inaccurate for detecting pressure differences between five micron cells while using a tip that is less than a micron in diameter.   However, the system was efficient for larger tips of about two to three microns in diameter.   Currently, a new micro-fabricated unit is being created in our lab that can bring together and pull multiple cells simultaneously with springs, while taking a more accurate pressure reading than the dual-micropipette system could have done.

Significance

Within the next ten years, the Vanderbilt Integrative Cancer Biology Center hopes to create a mathematical model describing the movement of cancer cells.   This requires the in-depth study of and the modeling of individual cell motility, the effects of other cells on individual cell motility, the effects of an adhered mass of cells on an individual cell's motility and lifespan, the effect of location within the neoplasia on cell motility and lifespan, the presence of extra-cellular nutrients in the form of oxygen or calcium, gradients in extra-cellular nutrients or signals, causes of cancer fingering, causes of cancer mutations, etc.   The clinical effects of such a model would be phenomenal – potentially, one could biopsy or analyze images of a patient's carcinoma to determine the necessary variables, plug these variables into the model, and thus determine the urgency of treatment and the best form of treatment to apply.  

Many Vanderbilt laboratories under this program have undertaken a few of these many parameters.   These laboratories are studying the variables in relation to the MCF10 cell line -- a human breast derived, E-Cadherin expressing, epithelial cell line exhibiting neoplasia.   E-Cadherin is a transmembrane protein that mediates epithelial calcium-dependent cell-cell adhesion.   According to BioCare Medical Center , this glycoprotein has been associated with metastasis and could function as a tumor suppressor protein or regulator of morphogenesis.   Some studies have shown a link between incorrect E-Cadherin expression and more advanced tumor invasiveness.   Recent research from the Mayo Clinic demonstrates extreme cellular motility and invasiveness in cells that contained p120 catenin but did not express E-Cadherin.   When these cells expressed E-cadherin, the migratory and invasive effects of the p120 were blocked.   This led to a theoretical construction of the E-cadherin cellular pathway:

Our laboratory studied the ways to quantify the separation forces between individual cells that had formed E-Cadherin adhesion junctions.   This would eventually lead to quantifying the separation forces between a mass of cells, such as the MCF10 cluster, and one of the cluster's cells.   Due to the short lifespan, difficult splitting techniques, and expense associated with MCF10 cell clumps, A431 and MDCK cell lines were used in their place as these are both E-Cadherin expressing epithelial cell lines.   Once a system has been devised to accurately quantify these forces, the lab will start measuring the forces of the MCF10 cell clusters.

Milestones

•  Learned to pull and microforge/ fire polish tips to a diameter under 1 micron

 

•  Obtained a working dual-micropipette system for tips of a few microns in diameter that was attached to a working pressure sensor

 

•  Learned the next step to a better system by exploring microfabrication