DEPARTMENT OF BIOMEDICAL ENGINEERING TULANE UNIVERSITY Biomedical Engineering Undergraduate Research and Design Conference

Conference Proceedings January 30, 2010

Sponsored by Tulane University’s Department of Biomedical Engineering Copyright© 2010 Department of Biomedical Engineering at Tulane University

Department of Biomedical Engineering Tulane University

Biomedical Engineering Undergraduate Research and Design Conference

Conference Proceedings January 30, 2010

Welcome to the 8th Annual Biomedical Engineering Undergraduate Research and Design Conference! Tulane University has one of the nation’s elite and most mature undergraduate departments of Biomedical Engineering. The department evolved from joint research efforts among faculty in the School of Engineering and the Schools of Medicine at Tulane and the Louisiana State University Medical Center in New Orleans. Research interests led to educational programs, and undergraduate BMEN degrees were first awarded at Tulane in 1972. In 1977 a separate Department of Biomedical Engineering was formed in the School of Engineering to offer the B.S., M.S., and Ph.D. degrees. The undergraduate program, ABET accredited since 1981, is focused in five ‘domains’ of biomedical engineering: biomechanics, bioelectronics,, biomaterials, cell and tissue engineering and design. As one of the first and most highly acclaimed departments in the country, our faculty and students have pioneered many of the curricular innovations that set the standard for educational excellence in this exciting and rapidly growing field. As is common for all accredited engineering disciplines, our students have participated in a required team design project since the department’s founding in 1977. Since 1987, these projects have all been related to assistive technology for local individuals with specialized needs. Biomedical Engineering students work on these designs during their senior year, culminating in a public design show in the spring. Starting in 2006, the outstanding design team has been presented with the Kenneth H. Kuhn Memorial Award, given in memory of our former Lab Coordinator/Instructor who served the department and assisted students with design projects from 1993-2005. However, unique among the nation’s departments of Biomedical Engineering, all Biomedical Engineering undergraduates have also participated in a required year-long independent research or design project since the department’s founding in 1977. Students work with departmental faculty mentors or our counterparts throughout Tulane and at affiliated institutions in the New Orleans area and across the nation. The projects are not merely academic exercises – they are genuine and significant contributions to the field of biomedical engineering complete with scholarly undergraduate theses and, in many instances, peer-reviewed publications. The presentations to follow today represent in a few minutes what has taken each student countless hours in the research and/or design laboratory to generate. The conference presentation is thus the punctuating culmination of a sustained effort of scholarship worthy of admiration at the highest levels. We are hopeful that this conference will highlight the efforts and results of our undergraduates, and will serve to communicate the excitement and potential of the field to the larger university community. As you look at the conference proceedings and attend the talks, I’m certain that you will be astounded by the breadth and depth of the educational experience each of our

students has achieved, and I hope you will be excited about their potential as they prepare to graduate this Spring and begin to make their marks as leaders in the field of biomedical engineering. We think you will agree that congratulations are due the members of the Biomedical Engineering Class of 2009 for their achievements, and determination to leave their mark on biomedical engineering research and practice. Thank you for attending!

Donald P. Gaver, Ph.D. Alden J. ‘Doc’ Laborde Professor and Department Chair of Biomedical Engineering

Department’s Mission Statement Our mission is to inspire and work with students as we develop and apply engineering methods to confront health science challenges. Departmental Vision The Department of Biomedical Engineering is committed to being a global leader in biomedical engineering scholarship. Our faculty, staff, and students are all important parts of the team that provide distinctive opportunities for creative solutions to biomedical engineering research and design problems. We aim for: excellence in undergraduate and graduate education; meaningful and innovative research; and service dedicated to advancing the field of Biomedical Engineering. Undergraduate Program Objectives Our undergraduate program provides students with the breadth required for participation in the interdisciplinary field of biomedical engineering and the depth required by engineers to advance the practice in our discipline. Our objective is to prepare graduates who are able to successfully pursue: • advanced studies leading to research or professional practice in biomedical engineering • advanced studies leading to research or professional practice in the health and medical sciences • practice in biomedical engineering industries or related technical and professional fields

OPENING REMARKS Donald P. Gaver, III Professor & Laborde Chair In Biomedical Engineering 10:15 – 10:30 am, Boggs Room 104 MORNING SESSION: Boggs Room 104 MODERATORS: Michael J. Moore, Christopher B. Rodell 10:45 – 11:00

DESIGN OF A CONE AND PLATE BIOREACTOR SYSTEM Austin S. Dobbins and Tabassum Ahsan

11:00 – 11:15

DEVELOPMENT OF AN ANTIBODY INDEPENDENT METHOD FOR THE SELECTION OF EMBRYONIC STEM CELLS Todd D. Johnson, Tabassum Ahsan, Ronald C. Anderson, and W.T. Godbey

11:15 – 11:30

THE EFFECTS OF SHEAR STRESS ON MOUSE EMBRYONIC STEM CELLS: POINT OF APPLICATION Jardin A. Leleux, Tabassum Ahsan, Walter L. Murfee, and Robert Dotson

11:30 – 11:45

ANALYSIS AND AUTOMATION OF THE QUANTIFICATION OF ASYMMETRIC SELF RENEWAL KINETICS OF HUMAN LIVER STEM CELLS Benjamin D. Cappiello, Krishnanchali Panchalingam, and James L. Sherley

11:45 – 12:00

INVESTIGATION OF LYMPHATIC/BLOOD ENDOTHELIAL CELL CONNECTIONS DURING MICROVASCULAR REMODELING STIMULATED BY CHRONIC HYPOXIA Garrett M. Gros, Jennifer L. Robichaux, and Walter L. Murfee

12:00 – 1:00

LUNCH BREAK – BOGGS ATRIUM

AFTERNOON SESSION A: Boggs Room 104 MODERATORS: Damir Khismatullin, Elaine Horn-Ranney 1:00 – 1:15

DIGITAL LIGHT MICROSCOPY FOR HIGHLY-RESOLVED SPATIOTEMPORAL CONTROL OF NEURAL ACTIVATION Joseph A. Majdi, Benjamin J. Hall, and Michael J. Moore,

1:15 – 1:30

USING PHOTOPROTECTED CYSTEINE FOR THE DESIGN OF PHOTOLABILE HYDROGELS Donald R. Campbell, Michael J. Moore, Ronald C. Anderson, and Walter L. Murfee

1:30 – 1:45

THE SYNTHESIS AND CONTROLLED RELEASE STUDIES OF A NOVEL BIODEGRADABLE DISULFIDE-BASED CROSS-LINKED HYDROGEL Lin Bai, Muhammad Ejaz, and Scott M. Grayson

1:45 – 2:00

QUANTITATIVE ASSESSMENT OF HEMOGLOBIN USING DIGITAL IMAGE ANALYSIS DURING ROBOTIC KIDNEY CANCER SURGERY: ASSESSMENT OF OXYGEN PERFUSION TO THE KIDNEY James W. Gallagher, Sergey Shevkoplyas and Benjamin R. Lee

2:00 – 2:15

DSP ANALYSIS OF A PROPOGATING AIR-LIQUID INTERFACE IN A MODEL OF PULMONARY AIRWAY REOPENING Joshua W. Thieman, Bradford J. Smith, Eiichiro Yamaguchi and Donald P. Gaver

2:15 – 2:30

BREAK

AFTERNOON SESSION B: Boggs Room 104 MODERATORS: Tabassum Ahsan, Kristen Lynch 2:30 – 2:45

THERMOTHERAPY DEVICE FOR TREATMENT OF CUTANEOUS LEISHMANIASIS Alison M. Douglas, David A. Rice, Richard Witzig, and Walter L. Murfee

2:45 – 3:00

AN OBSTETRICAL DEVICE CAPABLE OF CLAMPING, CUTTING AND SEALING UMBILICAL CORDS TO REDUCE INCIDENCES OF INFECTIONS IN RESOURCE POOR AREAS Michael Liu and David A. Rice

3:00 – 3:15

DESIGNING A CURRICULUM TO TEACH THE PRINCIPLES OF ELECTRONICS TO STUDENTS INTERESTED IN FIRST ROBOTICS Shanna K. Connolly and Cedric F. Walker

3:15 – 3:30

QUANTIFYING AQUEOUS HUMOR OUTFLOW TO DETERMINE THE VALIDITY OF A TONOMETER THROUGH TONOGRAPHY Christian T. Elrod and Ronald C. Anderson

3:30 – 3:45

ENHANCING FEATURES OF A REMOTE-CONTROLLED ROBOT TO PLAY MINIATURE GOLF John B. Huck and Cedric F. Walker

Department of Biomedical Engineering Tulane University

Morning Session Lindy Boggs Center Room 104

Biomedical Engineering Undergraduate Research and Design Conference Abstracts

DESIGN OF A CONE AND PLATE BIOREACTOR SYSTEM Austin Dobbins, Taby Ahsan1 Tulane University Department of Biomedical Engineering

1

Results Introduction Multi potent cells, proliferative unspecialized cells, have shown great promise in fields such as bioengineering and regenerative medicine. Research into the function and development of these cells will one day provide new and unique medical therapies; however, before this can occur, cell response to a variety of stimuli must be understood. These stimuli guide and encourage the development or lack of development in these cells, understanding and controlling them will allow for the guided and controlled growth and specialization of multi potent cells. Methods & Materials Shear stresses can be applied using several different methods; however, one of the most common methods uses fluid flow to create shear stress. The advantage to using fluid flow to apply shear stress could be the ease with which other growth factors can be added in conjunction with the physical stimuli. Also, fluid flow does not add extra forces that must be accounted for in results. Fluid flow requires very little force to apply a shear stress to a sample. Fluid flow can also be precisely controlled, depending on device design. Starting with a simple equation for fluid flow we derive the equation describing fluid flow in the bioreactor:

u y y y  u0 z

 

Equation 1

Equation 2 Our boundary conditions are defined as u (0) = 0 and u (y) =Vcone where u is the velocity of the fluid and y is the distance in the vertical direction from the bottom of the plate. Z is the overall distance from the bottom of the plate to the cone. At R=50 mm we see that z = h + R*sin (α), but for small α it is reasonable to assume that sin (α) = α. We then take the derivative of Equation 2 with respect to y.

Figure 1 3D view of the Device Assembly

The device was designed using Solid Works 2007 Edition. The device uses a single motor to rotate four separate cones suspended above tissue culture. This is accomplished by adding a gear to each drive shaft; this effectively transmits power from one shaft to two others. The rotating cones create fluid flow in the tissue culture dish; this in turn causes fluid shear stress at the bottom of the plate. A vacuum system keeps each tissue culture plate in place; this system runs through the bottom of each well in the base of the assembly and a rubber oring lies in a groove below each plate. A simulation was done using Flow works 2007. All pieces were created using Solid Works 2007. The material used for the cone and plate was a high density plastic with default materiel surface roughness. The fluid used for this simulation was water, and the cone was spun at ~400 RPM. According to the equation this should cause a shear stress of ~10.5 dynes/cm^2. A velocity surface plot was made using the bottom of the plate as reference.

u u 0  y Z

Equation 3 By substituting Equation 3 back into Equation 1, we find:



 * u0

Z Equation 4 Now we determine our initial speed in terms of the variables R, α and ω with ω being the angular velocity of the cone.

u0   * R

Equation 5 We then substitute Equation 5 back into Equation 4, and then substitute Z = h + α R to find an equation for shear stress. R Equation 6   h  R R, α, μ, ω and h will all be set at the beginning of each experiment, making them constants. This means that Equation 6 uses only known quantities to determine shear; from these it is possible to calculate shear stress at any point along the radius, R.

Figure 2 Velocity Plot of Tissue Culture Dish Surface Discussion While the device should operate as planned, this does not mean there is no room for improvement. The device is limited, because it cannot be used without a separate incubator. It could be possible to integrate the incubator with the device. There is also room for improvement in size and weight. Construction and analysis of a prototype could lead to smaller parts being designed. With analysis, it may prove possible to use lighter materials for some parts as well. Acknowledgments Special thanks to the Tulane Department of Biomedical Engineering for their support on this project.

DEVELOPMENT OF AN ANTIBODY INDEPENDENT METHOD FOR THE SELECTION OF EMBRYONIC STEM CELLS Todd D. Johnson1, Tabassum Ahsan1 Tulane University, Department of Biomedical Engineering

1

Introduction A major challenge in the field of tissue engineering and regenerative medicine is the need for an ideal cell source for research and medical applications. One of the common ideas for this is to use embryonic stem cells (ESCs). ESCs can differentiate into specific functional cells, such as osteoblasts, cardiomyocytes, or fibroblasts. The challenge that needs to be overcome is related to the process of isolating and expanding ESCs in vitro. Currently, the preferred method of expanding murine ESCs is to grow them in co-culture on a feeder layer of murine embryonic fibroblasts (MEFs). Thus, when a stem cell population is needed for a medical application or research, the presence of the feeder layer may be undesirable. One approach would be to simply devise a method of separation, to remove the un-wanted cells of the feeder layer and target the desired ESCs. Current methods of separation based on physical properties such as density have been used in hematology and histology. Centrifugation is used to separate out cells based on subtle differences in densities. The goal is to apply this concept to develop a method of selecting mESCs from a co-culture.

Results

Materials and Methods A method of separation was devised using a discrete density gradient made with Percoll™, a product of GE Healthcare, which is commonly used for blood cell separation. Made of colloidal silica and coated with polyvinylpyrrolidone (PVP), Percoll™ is stable and nontoxic1. The mESCs were incubated at 37° in 5% CO2, and grown in medium containing DMEM, 15% ESQ-FBS, 2mM Lglutamine, 100ug/ml Penn-Strep (PS), 0.1 mM non-essential amino acids (NEAA), 0.1mM beta-mercaptoethanol, and 1uL of 1000ug/ml leukemia inhibitory factor (LIF) per 1ml of medium. The medium was changed daily and the cells were harvested by washing with PBS, trypsinized for 3 minutes in the incubator, and centrifuged for 4 minutes at 200 x G to form a cell pellet. The MEFs were grown and harvested similar to the mESCs, but were fed every other day with medium containing ATCC DMEM, 15% FBS, and 100ug/ml PS. The MEFs were stained with a solution of Calcein AM with PBS w/ w/ 2mM dextrose and incubated for 20 minutes, which fluoresces the cytoplasm of the stained cells. The mESCs and the fluorescent MEFs were combined to create a controlled heterogeneous population.

Figure 2: Typical flow cytometry histograms of mixed cell populations before and after the discrete PercollTM gradient. Each histogram is a plot of 10,000 events.

Centrifuge at 400 x G for 20 minutes a 50:50 solution of non-fluorescent mESCs and fluorescent MEFs through a discrete PercollTM gradient of (1.030-1.055-1.07 g/ml).

The top flow cytometry histogram is a typical control 50:50 solution of non-fluorescent mESCs (left peak) and fluorescent MEFs (right peak). After using the developed density separation, the harvested cell population from the interface between 1.055g/ml and 1.070g/ml produced the bottom histogram. The targeted unstained mESCs (left peak) were 90% of the harvested subpopulation. Discussion This method has benefits of being cheaper, faster, and easier than other methods such as fluorescent activated cell sorting (FACS). For this method centrifugation is applied, which uses a common piece of lab equipment, unlike with FACS, which requires a significantly more complex and costly machine along with expensive antibodies. Also, FACS sorts cells one at a time, where the developed method sorts the cells in larger batches leading to a dramatic decrease in time of the process. Thus, this method using PercollTM is initially showing promise to improve the selection of embryonic stem cells for applications towards various tissue engineering and regenerative medicine research. The method could be expanded further and applied to adult stem cells, bone marrow, or partially differentiated cell populations. References 1. GE Healthcare Bio-Sciences AB. Cell Separation Media: Methodology and applications. Handbook 18-115-69 AD. May 2007. General Electric Company. 13 Mar. 2009 . 2.

Figure 1: Summary of the Approach of Separaion2.

Acknowledgements Research was funded by Tulane University through the STEM Cell Laboratory of Tabassum Ahsan.

THE EFFECT OF SHEAR STRESS ON MOUSE EMBRYONIC STEM CELLS: POINT OF APPLICATION Jardin A. Leleux1, Taby Ahsan1 Tulane University, Department of Biomedical Engineering

1

Introduction Embryonic stem cells (ESCs) have been the topic of research for regenerative medicine applications due to their potential to differentiate into all kinds of cells and their long-term proliferative capabilities; one of these applications is treatment of vascular disease using endothelial cells (ECs). However, there is not currently an adequate endothelial cell source that can be used for therapeutic purposes. It has been demonstrated that applying shear stress to mouse embryonic stem cells (mESCs) can direct differentiation of the ESCs to an endothelial cell lineage. The objective of this study is to test how fluid shear stress applied to mouse embryonic stem cells using a parallel plate system affects the differentiation of the cells at different stages in their development; the motivation is to eventually develop a procedure for producing endothelial tissue in vitro that can be used in vivo to aid in repair or replace damaged or diseased tissue. Materials and Methods mESCs are seeded on a gelatin coated T-175 flask and grown for approximately 4 days in ESC medium. The cells are then removed from the flasks and are seeded at 10,000 cells/cm2 onto collagen IV-coated glass slides. These cells are then cultured in an incubator at 37 in 25ml of medium for 1, 2 or 3 days before being sheared. Fluid shear stress is applied using a parallel plate system.

reaction (RT-PCR) method, which measures gene expression levels of a given gene in a sample. Results Specific genes were analyzed to determine whether the expression levels in the experimental samples varied from control samples, and also whether the experimental samples that were stressed at each time point exhibited different levels of gene expression. Genes analyzed include OCT4, NESTIN, BRACHYURY, AFP, CD41, SCL, RUNX1, FLK1. OCT4 is a gene marker for pluripotency, high level of which would indicate that the cells were highly undifferentiated. NESTIN, BRACHYURY and AFP are markers for the three germ lineages; ectoderm, mesoderm and endoderm, respectively. , CD41, SCL and RUNX1 are genes that are expressed at points throughout differentiation to the hematopoetic cell phenotype, which is closely related to the endothelial cell lineage. FLK1 is expressed in cells that have begun to differentiate towards an endothelial phenotype. Gene expression of BRACHYURY, RUNX1 and FLK1 significantly changed (p