Design of an integrated microfluidic system for culturing patterned tissue

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Design of an integrated microfluidic system for
culturing patterned tissue Allyson Fry1, Bryan
Gorman1, Jonathan Lin2, William Wong1 Advisors
John Wikswo1, Dmitry Markov1, Lisa McCawley3,
Phil Samson1 Departments of 1Biomedical
Engineering, 2Electrical Engineering, and 3Cancer
Biology at Vanderbilt University.
Introduction
Microfluidic pH Control
Characterization of Tissue Culture Model
The primary limitation of growing thick tissues
in vitro is lack of proper vascular supply for
nutrient and metabolite transport. Here, we
report progress in developing micro-bioreactors
with the ultimate goal of guiding the formation
of externally perfused capillary beds in vitro.
This project involves engineering an environment
in which microvascular endothelial cells can
differentiate and form tubular, capillary-like
structures.  Currently available micro-bioreactor
designs 1-3 do not provide physiologic
perfusion over large areas, while allowing
manipulation of the local microenvironment.
While in situ bioreactors represent the future of
tissue engineering and rehabilitation, our
approach is directed towards massively parallel
microenvironments for basic research in cellular
and systems biology. We continue work begun last
year to develop components of the bioreactor with
the goal of bringing perfused microenvironments
to a new level of sophistication and practical
laboratory utility.
The metabolic processes of living cells cause
significant changes in extracellular pH levels.
Left alone, such acidification renders the
microenvironment inside the bioreactor
inhospitable, adversely affecting cell growth. A
system to monitor and compensate for significant
deviations in pH was thus developed. It consists
of a pH sensor placed at the bioreactor exit and
two syringe pumps with low- and high-pH RPMI
media. The control program constantly monitors
pH levels and adjusts the mixing ratios of the
media from both pumps to maintain optimal cell
growth conditions. In contrast to using a
buffered perfusate, this process allows us to
monitor the acidification rate of the cells and
thus assess the metabolic status of the tissue
culture.
The organotypic culture used in this project is
adapted from a model developed by Manuela
Martins-Green 4. Our cultures contain two
layers of human dermal fibroblasts embedded in a
collagen matrix and separated by a layer of human
microvascular endothelial cells. This culture is
constructed in a transwell chamber in a normal
12-well culture plate. The culture is fed from
both top and bottom as shown in the diagram
below. In this tissue culture model the,
molecular cross-talk between two types of cells
results in cell rearrangement and formation of
microvessels, which can be visualized using
standard staining techniques.
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Project Goals
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• Our team addressed the following four problems
• Development and fabrication of several classes of
  microfabricated capillary scaffolds
• Development of a microfluidic pH control system
• Histological characterization of an angiogenic
  tissue culture model
• 4. Integration of scaffolds into tissue culture

• The long-term goals of the VIIBRE bioreactor
  project
• Develop an in vitro microvascular network that
  will
• Create a realistic extracellular matrix
• Allow evaluation of angiogenesis mechanisms
• Provide environment for endothelial cell
  differentiation
• Facilitate formation of stable tubular structures
• Support a flow of perfusate
• Allow study of transendothelial migration

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Diagram of a transwell culture plate used to promote formation of microvessels in the intermediate layer, thus making it an ideal platform for scaffold-guided vessel development and study of angiogenesis.
Social Impact
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In order to study the functions and mechanisms of
a system, scientists commonly use animal models.
However this practice has several shortcomings.
First, use of animals in research is fraught with
ethical implications concerning the value of
animal life. Second, results can never fully
predict the response of human tissue to
treatment. Finally, an animal system is
relatively difficult to manipulate. Our project
overcomes these shortcomings by creating a tissue
microenvironment completely based upon human
cells, which can also be instrumented to a
greater degree possible than in animal models.
Diagram of the perfusion system providing nutrients to the cells and maintaining pH and oxygen (future) levels
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Physical constraints for pH control
Cells Media needed (ul/cell/hr) Flow rates (ul/hr) Volume of bioreactor (ul) Desired pH range
45,000 4e-4 5-20 1-10 7.2-7.6
Bioreactor Design
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Vimentin
The function of our bioreactor is to add another
spatial dimension to transwell plate cocultures.
The design consists of parallel nutrient supply
networks perfusing layers of collagen seeded with
fibroblasts. Nano-pore filters support the
collagen and allow nutrient/waste exchange. A
scaffold seeded with endothelial cells is placed
between the collagen layers. The inspiration for
the scaffold design is the fractal pattern of
vasculature occurring in nature. Cells grown in
fractal flows are exposed to similar nutrient,
waste, and shear stress levels. Once the
endothelial cells in the capillary perfusion
system have grown to confluence, the system can
support internal flow.
Proportional Differential Integral (PID) control
scheme
Developed tissue cultures stained with HE and three antibodies. Arrows indicate microvessels. For reference, the filter is 10 mm thick.
Modeling and experimental results
The design and dimensions of the capillary scaffold
The bioreactor design concept
For experimental tests of the pH control system,
a third pump was used to inject a low pH solution
into the bioreactor in order to perturb the pH
and imitate the presence of living cells.
Additionally, a numerical simulation was
constructed in order to verify the experimental
results.
Scaffold Fabrication and Tissue Culture
Integration
H E stained sections of transwell cultures showing healthy fibroblasts as well as microvessel structures in two adjacent sections separated by 15 mm.
A microvessel sectioned semi-longitudinally. For reference, the filter is 10 mm thick. HE Stain.

• We were able to fabricate the following three
  different types of tissue culture scaffold
• The PDMS Egg-crate scaffold
• Advantage supports arterial and venous branches
  in
• rigid network
• Disadvantages difficult to construct, not
  biodegradable
• The patterned Matrigel scaffold
• Advantage biodegradable
• Disadvtantage poor mechanical features
• The SU-8 scaffold
• Advantages good mechanical properties, easy
  fabrication
• Disadvantage not biodegradable
• We then integrated each type of scaffold into an
  organotypic tissue culture model and analyzed the
  results.

Control signal and resulting changes in pump
rates during the same experiment
Real time pH control following onset of low pH
perturbation
Simulated pH response
Accomplishments We have grown and characterized
an angiogenic tissue culture model by employing
several histological labeling methods.
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Real - time experimental pH response
300
Pump Rate of High pH Buffer
Pump rate uL/hr
Conclusion
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• In conclusion, we have accomplished the
  following
• We microfabricated tissue culture scaffolds
  using three different biomaterials.
• We integrated each type of scaffold into a known
  tissue culture model.
• We developed LabVIEW software which integrates a
  sensor and syringe pumps for real-time pH
  control.
• We experimentally verified the performance of a
  real-time pH control program in a microfluidic
  environment,
• We compared our pH control experiments with
  numerical simulations,
• We implemented a multi-cell angiogenic tissue
  culture model, and
• We have applied various histological staining
  methods to characterize the developed tissues
• We have met all four specific project goals
  outlined in the introduction.

Desired pH range 7.2 7.6
Pump Rate of Low pH Buffer
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The photolithographic mask used to define the structures of the scaffold and supporting fluidics
Control Signal (PID)
Accomplishments We have demonstrated computer
control of pH in a microfluidic environment.
Experimental results are in excellent agreement
with numerical simulations.
Acknowledgements
Cost and Market Analysis
We would like to thank Jason Greene, who
contributed suggestions for device design and
testing. This project is a continuation of a
senior design project started in 2004-2005 by
Barnett, Garrett, Harvill, Mayer, and McClintock.
This project was made possible by use of
resources provided by the Vanderbilt Institute
for Integrative Biosystems Research and Education
(VIIBRE) directed by Dr. John Wikswo. Dr. Paul
King is the course instructor for BME 272.
The predicted market for this product is cellular
and systems biology researchers and drug
companies desiring to perform massively parallel
assays on realistic in vitro human models.
Although the pumps, sensors, and computer
hardware cost over 8,000, the plastic
microfluidic chips are disposable and can be
batch-produced off a single mold for less than 1
each. The minimal cost of the microfluidics and
supporting framework is estimated at 500. Thus,
the system represents an excellent ratio of cost
to high-throughput research benefit.
Raw Material Expenses of Project
Microfabrication 1,325.26
Tissue Culture 3,880.00
Control Systems 8,880.00
Total 14,047.39
References
A free-standing PDMS scaffold on glass
The PDMS scaffold in cross-section
SU-8 mold of capillary scaffold
Accomplishments We have fabricated three
different types of tissue culture scaffold and
integrated them into a tissue culture model.
3. Shin M et al. Biomedical Microdevices 6,
269-278, 2004.5 4. Martins-Green M, Li QJ, and
Yao M. FASEB 2005 19222-224.

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