Transcript Poster 1 - Research

Development and Characterization of Device for Galvanotactic
Response in D. discoideum
Cellular response to electric fields is a physiologically important research topic 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.
Experimental Set-ups for
Characterization of Cell Response
Voltage, pH, and Flow Characteristics
The relationship between applied and measured voltage was characterized across
several flow rates to investigate the consistency, linearity, and flow independence
properties of the response. Three trials were run for each of the flow speeds. No
data points were recorded until the voltage across the experimental area stabilized for
a given applied voltage. To assess pH and ion gradients, the designs were initially
tested for proper electrical activity at the electrodes through the use of Bromothymol
Blue (upper transition to blue at pH = 7.6, lower transition to yellow at pH = 6.0).
Visual evaluation showed the clear pH-altering effects in the immediate vicinity of the
electrodes and the potential for product diffusion. Preliminary elimination of several
flow schemes and speeds was possible through the use of this pH indicator. For cellbased assays, however, we sought to demonstrate that the two outflow pH values
remained within a narrow range over the time frame of the experiment with a Denver
Instrument pH/mV meter.
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
Galvanotaxis is the movement of
cells in a particular direction in
response to an electric field across
the cells. This movement is
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. Currently the setup for a Schematic for device we reconstructed to
study galvanotaxis (See photo, right). [1]
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 were loaded into the device and studied. Once a baseline response has been
established, simple modifications of our microfluidic platform will allow for quick and
easy specialization of experiments and a better understanding of galvanotaxis.
A successful microfluidic design for this project would have four traits:
• Create voltage fields that are required for galvanotaxis, 7-10 V/cm, using
applied potentials an order of magnitude less than current designs.
• Able to control pH in the experimental time frame using flow in place of
agar salt bridges
• Be able to fit entirely on a microscope stage without any open components
Experimental Set-Up From Literature
Before we could attempt to improve upon the difficulties associated with the current
device used to study galvanotaxis, we had to reconstruct it to establish a baseline cell
response and gain first-hand experience with the device’s problems.
In the set up above, we utilize 30 ml glass beakers filled with DB buffering solution.
The voltage is applied via two platinum electrodes connected to a Fisher Biotech
power-supply. Agar bridges are constructed from glass NMR tubes bent using a
Bunsen Burner. The field strength is measured across the cell area using a Extech
Voltmeter. No. 1 coverslip walls are cut to shape by diamond-tip pen and sealed to
glass slide using Permount adhesive. The coverslip roof provides a geometric
constraint in height.
16
14
12
10
8
6
4
2
0
-2
pH control over time (11.34 V +/- .056 V, 25 uL/min)
7
6.95
6.9
6.85
50 uL/min
100 uL/min
25 uL/min
pH positive electrode
pH negative electrode
6.8
6.75
6.7
6.65
6.6
6.55
0
6.5
5 10 15 20 25 30 35 40 45 50 55 60
0
Applied (V)
20
40
60
80
100
120
Time (min.)
Summary
Microfabricated PDMS Device Using Agar Bridges
To further constrain the dimensions of the experimental area, and raise the proportion
of the voltage across the cells, we developed a microfluidic channel with height ~50
µm, width ~250 µm, and length ~1 cm. The channel is accessible to the agar bridges
via two 3 mm biopsy punch holes, which create a small reservoir of media for the
bridges to sit in. The reservoir helps to maintain consistent fluid contact for voltage
application.
Over the course of the project we have accomplished the following:
• Reconstructed the original agar bridge model for galvanotaxis.
• Designed and microfabricated our project-specific masters and PDMS devices.
• Established resistive properties of materials used in each device
• Used COMSOL to model flow and pressure gradients in our device.
• Built our own variations on a large scale system to help establish basic cell
response.
• Built auxiliary components for a galvanotaxis system: insertable electrodes and
PDMS punches
• Applied concepts from macro experiments and VIIBRE generated designs to
develop the current, very simple iteration of microfluidic designs.
• Started and maintained a healthy cell culture
• Successfully seeded cells into devices
• Viewed random motility of healthy cells in devices
• Set up short experiments applying varying voltages to cells to view galvanotactic
response
• Limited success replicating cell motility results from literature; must improve device
parameters in order to consistently gather useful motility data
Future Direction
Application
Electrode, Pt
Measuring
Electrodes, Pt
Application
Electrode, Pt
2 mm
Punch
Microfabrication Methods
500 µm
AutoCAD was used to design the device. The design patterns were sent to
Newman Printing, Inc. to create an 8 ½ x 11 sheet of film for use in fabrication.
Photolithography techniques were used to create a silicon wafer master in SU-8
in VIIBRE class 100 clean rooms. Microfluidic devices were then made from
PDMS poured over the negative master. The devices were cut from the PDMS
mold after baking for four hours in a 60º C drying oven, and access holes were
punched through the PDMS using 16 gauge needles and 2 mm biopsy punches.
The PDMS mold and cover slip were placed in a plasma bonder for 20 seconds
and then bonded together.
Applied Voltage vs. Measured Voltage
Measured (V)
Abstract
The Janetopoulos Lab, Department of Biological Sciences
pH
Devin Henson and Arunan Skandarajah
5 mm
20 mm
75 mm
Side and Top Views of microfluidic device. Inset shows D. discoideum inside our device
The completed device is attached at the inner two access holes to a Harvard
Apparatus PHD 2000 Syringe Pump for flow input and to two waste containers at the
outer holes. Electrodes connected to a Fisher Biotech Power Supply applied a
known voltage at the two outer access holes. Another pair of electrodes attached to
an Extech multimeter measured the voltage potential across the cell area.
Plastic Becton-Dickinson syringes with a capacity of 10 mL and 23 G needles were
placed in the pump apparatus. Tygon tubing of I.D. .040” and O.D. of .080” was used
to interface with the 2 mm access holes. The I.D. 020” and O.D. 060” Tygon Tubing
was used to fit the 16 G holes.
As shown above, flow into the center access holes reliably exits from the
corresponding waste ports without causing a flow across the cells in the central area.
This mechanism allowed us to eliminate the agar bridges while still isolating the cells
from electrolytes or other factors like flow.
We hope to develop the device further through continuing work as members of a
SyBBURE summer research and VUSRP. Our main goals throughout the summer
will be to first establish the baseline cellular response and then take our microfluidic
device from the design stage into the data collection stage. With a functional device,
we will explore the mechanisms that allow cells to move in an electric field by using
genetic variants and compare these effects with the cell’s response to other
gradients, such as chemotaxis.
References
[1] Song, Gu, Pu, Reid, Zhao, Z., & Zhao M. Application of direct current electric
fields to cells and tissues in vitro and modulation of wound electric field in vivo. Nat.
Protocols 6(2) 1479-1489
Acknowledgements
Thanks to our advisers on this project: Professors Janetopoulos, Wikswo, and
King. A special thanks to Carrie Elzie for her help on biological issues and
Ron Reiserer for his technical support and assistance with training and
troubleshooting. Also, thanks to the entire VIIBRE staff for making our
research possible.