Development of a Small-Diameter Arteriole Model for Drug Testing and the Study of Endothelial-Perivascular

Interactions

Rachel Straughn

Mentor: Nicole Zeinstra, Principal Investigator: Dr. Ying Zheng

June 5, 2017

Capstone Track: 402

Abstract

Vascular remodeling—the modification of preexisting vasculature—often occurs in common diseases such as

cardiovascular disease and cancer. Little is known about the factors controlling perivascular remodeling

regarding interactions with the endothelium, and current methods of studying this phenomenon in the

microvasculature have shown to be inadequate. There is a need for small-diameter in vitro microvascular

models that allow for the study of drugs, disease, and their direct effects on endothelial-perivascular

interactions under sustained flow and pressure conditions. Therefore, we propose to design a small-diameter

single channel arteriole model using a needle-based subtraction method. First, a housing device will be

designed that allows for robust and time-efficient fabrication, attachment of flow and pressure, and in situ

imaging of the vascular constructs. Then, the effects of pressure and flow will be observed on smooth muscle

migration and alignment, with those conditions being optimized for perivascular remodeling and eventually

demonstrating arteriole function. If successful, this project will have a role in increasing time and financial

efficiency of drug studies, in addition to increasing overall understanding of diseases and their effects on the

microvascular environment.
 Table of Contents

Background and Significance……………………………………………………………………………………… 3

Background………………………………………………………………………………………………………… 3

Figure 1…………………………………………………………………………………………………………... 3

Statement of Problem and Need…………………………………………………………………………………. 4

Solution and Prior Art…………………………………………………………………………………………….. 4

Figure 2…………………………………………………………………………………………………………... 6

Preliminary Data…………………………………………………………………………………………………... 6

Figure 3…………………………………………………………………………………………………………... 7

Figure 4…………………………………………………………………………………………………………... 7

Figure 5…………………………………………………………………………………………………………... 8

Consequences of Success…………………………………………………………………………………………. 8

Real-World Constraints…………………………………………………………………………………………... 8

Plan of Work…………………………………………………...…………………………………………………….. 9

Design and Hypothesis…………………………………………………………………………………………… 9

Strategy: Research and Design…………………………………………………………………………………… 10

Aim 1: Design a Housing Device for Arteriole Model……………………………………………………… 10

Aim 2: Determine the Effect of Pressure and Flow on Smooth Muscle Migration and Alignment……. 11

Figure 6………………………………………………………………………………………………………... 12

Aim 3: Optimize Conditions for Perivascular Remodeling and Demonstrate Arteriole Function…….. 13

Figure 7………………………………………………………………………………………………………... 14

Key Personnel………………………………………………………………………………………………………… 14

Equipment and Facilities……………………………………………………………………………………………. 15

References…………………………………………………………………………………………………………….. 16

Appendices……………………………………………………………………………………………………………. 17

Appendix 1: RFP with PI Approval……………………………………………………………………………….. 17

Appendix 2: Concept Sheet………………………………………………………………………………………… 18

Appendix 3: Milestones and Timeline Table………………………………………………………………………. 19

Appendix 4: Additional Figures and Tables………………………………………………………………………. 20

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 Background and Significance

Background

The human circulatory system is a complex network of vessels 25 mm to 8 µm in diameter [1].

Beginning at the aorta, vessel diameter decreases as the distance from the heart increases. As the diameter

changes, function changes as well. Arterioles, with diameters ranging from 100 µm to 300 µm in humans,

regulate blood pressure [2]. A layer of smooth muscle circumferentially surrounding the endothelium is

responsible for vasoconstriction and vasodilation (Figure 1), which affects resistance to flow and allows the

body to quickly control blood flow to locations in the body.

Figure 1. Illustration of the structure of an arteriole. The innermost layer is comprised of endothelial cells,
which are surrounded by smooth muscle cells [2].

Vascular growth and remodeling occurs in all stages of life, and includes three processes:

vasculogenesis, angiogenesis, and arteriogenesis [3]. Respectively, these describe the formation of the earliest

blood vessels, the growth of new vessels from preexisting ones due to changes in tissue oxygenation, and the

growth in size of preexisting vessels to counteract narrowing. Yet, little is known about the factors controlling

these processes regarding interactions with the perivasculature and extracellular tissues [4]. Such interactions

include signaling of growth factors for cell development, recruitment, and proliferation, and occur during both

development and a variety of pathologies [5]. Some pathologies of particular interest are cardiovascular

disease, the leading cause of death globally [6] and cancer, the second leading cause of death in the United

States [7]. Both of these are characterized by distinct changes in the 3D microenvironment and
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microvasculature [8]. A significant amount of research has been dedicated to understanding these changes and

developing effective treatments and prevention strategies.

It has been shown that 2D cultures fail to recreate the structure and behavior of vasculature observed in

vivo because they are unable to completely replicate a three-dimensional physiological environment [9]. Given

recent advancements in the field of tissue engineering, many efforts have been made to recapitulate 3D

vascular environments in vitro, as in vivo studies are limited by challenges in observation, lack of control over

physical, chemical, and biological parameters, and financial costs [4, 10]. Much of the cost of these in vivo

studies can be attributed to the equipment, facilities, and trained personnel needed to monitor and care for the

animal subjects. Yet even with progress in 3D in vitro modeling, researchers have yet to fully characterize the

perivascular interactions directly influencing microvascular remodeling and how they vary in different

environments [4]. Once this is accomplished, it will be possible to create more complex in vitro disease, organ,

and tumor models that accurately represent the microvasculature of a particular physiological region and

microenvironment.

Additionally, the development of in vitro models that recapitulate human vascular environments will

provide potential substitutes for early in vivo preclinical trials. With these models, robust (high success rate)

and time-efficient (requiring less than one day) fabrication protocols will allow for lower-cost drug screenings

and therapeutic studies. This results in the direct effect on endothelial-perivascular interactions and vascular

remodeling being easily observable and characterizable [11].

Statement of Problem and Need

There is a problem with current in vitro vascular models in that little is known about the endothelial-

perivascular interactions affecting vascular remodeling, resulting in little knowledge about how these

interactions change in various microenvironments corresponding to diseases such as cancer and

cardiovascular disease. Therefore, there is a need for small-diameter in vitro microvascular models that allow

for the study of drugs, disease, and their direct effects on endothelial-perivascular interactions under sustained

flow and pressure conditions.

Solution and Prior Art

Many methods currently exist for fabricating in vitro microvascular environments, each with their own

advantages and challenges. Current methods for housing arteriole-like models include polydimethylsiloxane

(PDMS)-based microfluidic devices that are coated with collagen, elastin, or fibrin that encourage the growth,

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migration, and realignment of vascular cells [11]. Likewise, lumen fabrication is also achieved through a

variety methods such as PDMS molding and 3D bioprinting, the 3D printing of cells and tissues [12]. Despite

progress in fabrication methods, there remain limitations regarding the geometry of these preexisting models.

Models of a single-channel geometry most easily demonstrate direct interactions between endothelium and

perivasculature. These models range in diameter from 300 µm or larger, even though arterioles can have

smaller diameters [2, 11]. It is important to study these small-diameter arterioles because their composition

and function will be unique compared to those of larger diameters. Additionally, current models that include

the attachment of flow do not have the ability to vary pressure in addition to flow rate and thus cannot

completely recapitulate different microvascular environments.

The Zheng Lab seeks to develop a small-diameter single channel arteriole model through a device that

utilizes a needle-based subtractive molding method. Collagen is a significant component of vascular and

interstitial extracellular matrix (ECM), and has been shown to have a special role in regulating endothelial cell

morphology and lumen formation [13]. Therefore, it serves as a physiologically relevant scaffold for our

proposed in vitro model. Type I collagen is cast in silicone housing around an acupuncture needle that is 180

µm in diameter (Figure 2). After removal of the acupuncture needle, Human Umbilical Vein Endothelial Cells

(HUVECs) may be seeded by hand-perfusion into the lumen, which retains its 180 µm diameter. A smooth

muscle layer can be formed by hand-perfusion of Human Coronary Artery Smooth Muscle Cells (HCASMCs)

into the lumen prior to HUVEC perfusion [11] or by suspending the HCASMCs directly into the bulk collagen

matrix. Sustained pressure and flow can be introduced to the system by varying vascular resistance to syringe

pump flow, and migration and alignment of the HUVECs and HCASMCs can be observed over time.

Additionally, the overall functionality of the arteriole can be demonstrated through 1) calcium-transient

imaging in situ to observe calcium wave propagation between smooth muscle cells, 2) permeability assays to

observe endothelial barrier function, and 3) the treatment of epinephrine to observe smooth muscle contractile

function. Upon demonstration of functionality, the model can then be used to model the microvasculature of

diseases such as cancer and cardiovascular disease, and study the effects of novel drugs and therapies on

vascular remodeling and endothelial-perivascular interactions.

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 Figure 2. SOLIDWORKS assembly detailing the exploded view of the first device iteration (left) and
fully assembled device (right). Parts include a plastic clamp surrounding a PDMS housing for
constructs.

Preliminary Data

The Zheng laboratory uses endothelialized microvascular networks for various in vitro studies [14].

These networks can exhibit complex branching geometries, and the protocol for their fabrication is widely

used. This project is a continuation of a previous undergraduate student’s senior capstone, in which he

designed the first iteration of a PDMS housing device (Figure 2) and a robust cell seeding and culture protocol,

and produced evidence that luminal patency is maintained for up to 7 days of culture under flow with

HUVECs lining the lumen (Figure 3).

My work so far includes optimizing HCASMC bulk concentration for construct compaction and cell

survival within the collagen matrix (Figure 4). I have also redesigned the housing device so that it no longer

requires PDMS, instead using acrylic or Plexiglas housing and reducing the experiment preparation and

fabrication time per round of experiments by one to two days, which is the time required to mold a fresh

PDMS piece (Figure 5).

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Figure 3. Fluorescent image proving the functionality of a HUVEC-seeded channel, with nuclei (blue), vWF
(green), phalloidin (red), and CD31 (far red) stained in this particular construct (Dominic Tran, 2016).

Nuclei
F-Actin

200 um

Figure 4. Fluorescent image captured on a confocal microscope detailing HCASMC elongation and alignment
in bulk collagen at an optimized seeding density of 5 million cells per mL of collagen.

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Figure 5. SOLIDWORKS assemblies of the current PDMS housing device (left) and the next proposed iteration
of the housing device (right) that will be manufactured solely from plastic and will require no PDMS.

Consequences of Success

Success with this project will result in a better understanding of factors controlling perivascular

remodeling and a means for observing interactions between the perivasculature and extracellular tissues.

Additionally, this project will produce a platform for a variety of studies concerning novel drugs, therapies,

and diseases.

A successful in vitro platform for studies such as the one we are proposing has the potential to increase

the time and cost efficiency of drug screenings. The ability to conduct these small-scale studies in vitro would

potentially decrease the number of unsuccessful large-scale in vivo animal studies, as researchers will have a

more physiologically accurate platform for predicting the outcome of a proposed preclinical drug study. If

they are able to stop an unsuccessful study before it begins, researchers will save on time, funding, training,

and personnel needed for preclinical trials.

On the other hand, the use of this product as a disease model will also have an impact regarding the

fundamental understanding of how diseases such as cancer and cardiovascular disease change the 3D in vivo

environment on a microvascular level. This understanding will someday lead to effective treatments or cures,

and thus our platform would have a hand in benefitting the thousands of people within the United States and

around the world affected by these diseases.

Real-World Constraints

This project will involve the use of human cell lines isolated from tissue samples. These samples should be

collected only after donor or patient consent is given [15]. The cell lines being used for this project will be
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purchased from a biotech supplier and thus tissue sample collection and cell line isolation will be completed

elsewhere. Therefore, this should not be a significant ethical concern for this project. Additionally, we should

carefully consider the source of these tissues when handling them in culture and when designing experiments

in order to avoid unnecessary waste.

This product is meant for use in academic and private research settings. Because of this, most expenses will

arise from device fabrication, tissue culture reagents and materials, and immunostaining reagents and

materials. The most notable costs that pose risks to translation to the intended users arise from device

fabrication, which is a service provided by an outside machining shop, and immunostaining reagents, such as

primary and secondary antibody solutions. Primary human cell lines are also expensive to purchase and

maintain. Funding from the National Institutes of Health (NIH) will mitigate these costs.

The FDA currently has guidelines and regulations in place regarding the use of human tissue products.

These include following HIPAA laws regarding patient confidentiality and identifying information given to

researchers using the tissue samples [16]. Since this project uses cell lines purchased from a biotech company,

this should not pose a significant concern but should be taken into consideration.

Plan of Work

Design and Hypothesis

We hypothesize that the in vivo structure and characteristic remodeling of human arterioles can be

recapitulated and studied in vitro, allowing for the creation of disease models and a platform for the testing of

novel therapeutics. Other vascular structures of different sizes and compositions have been engineered in the

past. We will design a device for these in vitro studies utilizing a needle-based subtraction method.

Aim 1: Design a Housing Device for Arteriole Model

The purpose of Aim 1 is to design a housing device for the arteriole model. The design will be done

using computer-aided design (CAD) software, and prototypes will be manufactured through 3D printing. Aim

1 will be complete once the housing device satisfies the following: it includes a single-channel geometry within

each construct, simple sterilization methods through the selection of autoclavable materials, fast assembly time

(within 30 minutes to an hour), robust cell seeding, and robust in situ imaging. Once this has been completed,
progress towards Aims 2 and 3 may begin.
Aim 2: Determine the Effect of Pressure and Flow on Smooth Muscle Migration and Alignment

The purpose of Aim 2 is to fabricate the constructs within the new device and study the effects of

pressure and flow on smooth muscle cell migration and alignment. Before studying the effects of endothelial

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interactions with the perivasculature, it is important to understand how smooth muscle cells behave in the

absence of endothelial cells in an environment with varying pressures and flow rates. This aim will be

complete once the migration and alignment of the smooth muscle has been characterized through

immunofluorescent imaging for all combinations of low and high flow rates, in addition to low and high

applied pressures.

Aim 3: Optimize Conditions for Perivascular Remodeling and Demonstrate Arteriole Function

The purpose of Aim 3 is to study the effects of pressure and flow on perivascular remodeling and

function. This will include the co-culture of endothelial and smooth muscle cell lines. At an optimized set of

experimental conditions, we hope to observe the migration and alignment of smooth muscle around the

endothelium, in addition to proving arteriole functionality through calcium transient imaging, dextran

perfusion assays, and epinephrine treatments. Aim 3 will be complete when these deliverables are collected for

all combinations of low and high flow rates in addition to low and high applied pressure.

Aim 1: Design a Housing Device for Arteriole Model

Approach. Design of a housing device will be completed in three steps: initial design using CAD software,

prototype fabrication, and proof-of-concept demonstration through feasibility experiments. The design will be

improved from a single-channel device used by a previous undergraduate student to satisfy the following

requirements: simple preparation through autoclave sterilization, an assembly time within 30 minutes to an

hour, robust cell seeding protocols, and the ability for in situ immunofluorescent staining and imaging. This

will be achieved by selecting durable heat-tolerant materials, minimizing the total number of pieces needed,

including inlets and outlets that can be used for the seeding of cells within the channel, and including space at

the bottom of the device that allows a microscope objective to approach the constructs within the device. A

concept design of this device is shown in Figure 5. Initial prototypes will be 3D printed in the Zheng lab,

functional prototypes will be fabricated on a CNC mill within the Zheng lab facility, and large-scale fabrication

of the finalized design will be ordered through the physics machine shop. Prototypes will undergo feasibility

experiments to ensure that they meet the requirements mentioned above. These experiments will involve the

fabrication of acellular constructs, in which fluorescent beads will be seeded in place of cells and imaged using

a confocal microscope. This will allow us to carry out the entire experimental process—from sterilization to in

situ fluorescent imaging—over the course of one day, in addition to seeing that the device meets all

requirements, without risking the waste of valuable cells and immunostaining reagents.

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Deliverables. At the end of Aim 1, a design for the housing device should be finalized according to the

requirements stated above and an order should be placed at the physics department machine shop for the

fabrication of several copies of the device parts. Once this is accomplished, other research within the lab from

others hoping to use the device may move forward.

Anticipated Outcomes. Aim 1 should be completed within one to two months to ensure the rest of the project

can be completed. To ensure Aim 1 is completed quickly and effectively, a 3D printer will be utilized for all

early prototypes. Machine fabrication on the mill will not be done until the design is ready for feasibility

experimentation. Other potential areas for risk in the completion of this aim come from the number of

iterations needed in order to satisfy the requirements of the device. With tissue engineered constructs and

microfluidic devices, a 100% success rate in fabrication and cell seeding is not possible because of human error.

Therefore, an acceptable average cell seeding success rate of 60-80% must be met such that Aim 1 can be

deemed complete and progress towards Aims 2 and 3 can begin.

Engineering Design Standards. There are currently many different methods for designing and fabricating

microfluidic devices. The use of extracellular matrices and other biomaterials within microfluidics is a

relatively new field, resulting in a lack of national or international design standards for devices such as these.

However, there are published guidelines for first-time users and designers of microfluidic devices [17]. Such

guidelines describe aspect ratios for channel geometry: no smaller than a 1:10 (height : width) ratio and no

larger than a 4:1 ratio. These aspect ratios help to maintain the channel geometry and overall microfluidic

device integrity. Additionally, these guidelines include suggestions for structure supports at inlets and outlets.

In the Zheng lab, device dimensions typically lie within a certain acceptable range. For example, all devices

must be able to fit within a round dish and therefore must not be more than 20 to 25mm in height. For all

devices to rest on a microscope stage, they must be within 60 millimeters in width.

Aim 2: Determine the Effect of Pressure and Flow on Smooth Muscle Migration and Alignment

Approach. Aim 2 involves the characterization of smooth muscle behavior in these constructs in the absence of

endothelial cells. The study of combined pressure and flow on smooth muscle cell migration and alignment

will be completed in the following steps: optimization of smooth muscle cell density in bulk, and observing

smooth muscle migration and alignment at various flow rates, pressures, and combined flow rates and

pressures. Human coronary artery smooth muscle cells (HCASMCs) will be suspended in bulk collagen type I

at various densities (1, 5, 10, and 15 million cells per milliliter), and cultured under static conditions. Densities

of a similar order are often found in smooth muscle constructs with type I collagen [18]. Cell survival and

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construct compaction, demonstrating the elongation and alignment of the smooth muscle cells, will be

optimized and will be quantified through bright field and immunofluorescent imaging. This optimized

concentration will be used for all future experiments. The study of the effect of pressure on smooth muscle

migration and alignment will be done under three conditions: no applied pressure, low pressure, and high

pressure, all with static flow conditions using a static pressure setup developed in the lab. Next, the study of

various static, low, and high flow rates on smooth muscle migration and alignment will be completed using a

syringe pump. A schematic of a construct with this set up is shown in Figure 6. To apply pressure at various

flow rates, a resistor or clamp will be added at the outlet. Smooth muscle migration and alignment will be

observed through bright field and immunofluorescent imaging. Barrier function will be observed through a

dextran perfusion permeability assay. In this assay, the time and distance required for fluorescently-labeled

dextran molecules to diffuse outward from the lumen and saturate the field of view of the microscope is

measured. The better the luminal barrier, the longer this diffusion time will be. Seeing as there will be no

endothelial cells within these constructs, the permeability assay will establish a baseline for comparison in later

experiments where HUVECs and HCASMCs are co-cultured.

Figure 6. Schematic of a single construct, in which flow is applied by syringe pump at the inlet, and travels
straight through the construct and out the outlet.

Deliverables. Aim 2 will be complete when each experimental condition has 𝑁 ≥ 4 constructs. Data from these

experiments will include bright field images taken at time points 0, 1, 3, and 7 days, dextran perfusion

permeability assays, immunofluorescent staining, and calcium transient imaging. This data will be processed

using ImageJ, and will result in the determination of optimal experimental conditions for smooth muscle

migration toward the lumen and alignment concentrically around the lumen, which will help in the

determination of future experimental conditions and will also help with our understanding of how these

processes occur physiologically.

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Anticipated Outcomes. To reduce the effects of batch-to-batch variation on the results within this aim, as

many replicates as possible per experimental condition ( 𝑁 ≥ 2) will be completed in one experiment. Potential

risks to the success of this aim originate in the immunofluorescent staining and imaging portion of data

collection. Even with a new device design, the constructs may be too densely seeded with cells in the bulk

collagen to allow for adequate diffusion of fluorescent molecules through the entire construct during a

reasonable amount of staining time. If this is the case, and in situ immunofluorescent imaging on a confocal

microscope is not possible, we will defer to histology sections of the constructs, in which 300 µm agarose-

embedded sections can be done on a vibratome. This is not ideal because the 3D structure of the constructs

may be compromised, but we will still be able to observe cell migration and alignment through

immunofluorescent staining.

Engineering Design Standards. This aim involves characterizing cell viability within a type I collagen

biomaterial scaffold. The American Society for Testing and Materials (ASTM) lists in ASTM 2739-16 methods

for assessing the number and distribution of viable and non-viable cells within various biomaterial scaffolds

using histology and confocal microscopy [19]. Smooth muscle cell viability will be characterized using these

standards for Aim 2. Additionally, the use of human cell lines warrants that biosafety level 2 (BSL-2)

laboratory practice rules from the University of Washington Department of Environmental Health and Safety

(UW EHS) be followed [20].

Aim 3: Optimize Conditions for Perivascular Remodeling and Demonstrate Arteriole Function

Approach. Aim 3 involves the characterization of combined endothelial and smooth muscle behavior in these

constructs as a model for perivascular remodeling, with experimental conditions varying both pressure and

flow rate as in Aim 2. For this aim, Human Umbilical Vein Endothelial Cells (HUVECs) will be seeded by

hand-perfusion into the lumen of each construct at a previously optimized concentration of 10 million cells per

milliliter, with the optimized concentration of HCASMCs from Aim 2 being suspended in the bulk collagen.

Just as in Aim 2, various flow conditions, static pressure conditions, and combined pressure and flow

conditions will be applied to these constructs. Both smooth muscle and endothelial migration and alignment

will be observed through bright field and immunofluorescent imaging. Figure 7 shows a cross-sectional

schematic of how we expect the cells to migrate and align over time. Smooth muscle contractile function will

be demonstrated through epinephrine treatments, in which the construct will undergo vasodilation and the

lumen diameter will become noticeably larger. Endothelial barrier function will be demonstrated through a

dextran perfusion permeability assay.

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 Figure 7. Cross-sectional view of expected smooth muscle cell behavior over the course of the proposed
experiments. HUVECs will form a barrier, while HCASMCs will align concentrically around the channel and
exhibit contractile behavior.

Deliverables. Aim 3 will be complete when each experimental condition has 𝑁 ≥ 4 constructs. Like Aim 2,

data from these experiments will include bright field images, dextran perfusion assays, immunofluorescent

staining, calcium transient imaging, and to epinephrine treatments. We will observe smooth muscle migration

towards the endothelium and circumferential alignment around the lumen, in addition to endothelial

alignment in the direction of flow, with their morphology showing clearly-defined junctions. We will also

demonstrate overall arteriole functionality through permeability assays and epinephrine treatment.

Anticipated Outcomes. Much like Aim 2, batch-to-batch variation between experiments will be reduced by

including many construct replicates (𝑁 ≥ 2) per experimental condition in one experiment round. In situ

immunofluorescent imaging is also a risk in this aim, and will be addressed through thick vibratome sections

done by the histology department.

Engineering Design Standards. Also like Aim 2, this aim involves the characterization of cell viability within

collagen biomaterial scaffolds. The standards described in ASTM 2739-16 for the use of histology and confocal

microscopy for the assessment of cell viability [2] will be used for studying HCASMCs and HUVECs in these

constructs. This Aim will also require that BSL-2 practices be used, as described by UW EHS, due to its

incorporation of two human cell lines [3].

Key Personnel

I will be the lead researcher on this project, with guidance from other members of the lab given as

needed. Nicole Zeinstra will provide oversight during device designing and manufacturing as well as advice

during troubleshooting. Nicole is currently a first-year graduate student with extensive research experience

and a project closely related to mine. She will be my direct mentor for the duration of this project. Christian

Mandrycky, a third-year graduate student, will provide assistance and advice for all steps involving device
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fabrication and machining. Dr. Ying Zheng will oversee the general progress of the project, and will provide

advice and feedback as needed, in addition to organizing meetings at least once a quarter for progress updates.

Equipment and Facilities

For Aim 1, design will be completed using SOLIDWORKS 2016, and manufacturing will be done either

with the lab’s own CNC mill or at the physics department machine shop. For Aims 2 and 3, materials needed

to fabricate the constructs, which include collagen, smooth muscle and endothelial cells, and hardware such as

needles and screws, will be provided by the Zheng lab. Cell culture materials, syringe pumps, and staining

reagents will also be provided by the Zheng lab. The required tools for data acquisition and analysis are a

vibratome for sectioning, bright-field and wide-field microscopes, a confocal microscope, and ImageJ software.

Small-scale device manufacturing will take place at the Zheng lab, while large-scale orders will be

completed at the physics department machine shop. Construct fabrication will take place at the Zheng lab,

with imaging taking place in the Lynn and Mike Garvey Cell Imaging Lab. Both the Zheng lab and the

Imaging lab are located at UW Medicine South Lake Union.

Experiments are funded by grants from the NIH, and all software is free or licensed by the UW College

of Engineering. Through the Institute for Stem Cell and Regenerative Medicine, equipment repair and access

to the wide-field microscope are offered at no cost and the confocal microscope is offered at an hourly rate.

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Microfluidic device design, fabrication, and testing protocols. Protoc. Exch.
18. Kim, B. S., Putnam, A. J., Kulik, T. J., & Mooney, D. J. (1998). Optimizing seeding and culture methods to
engineer smooth muscle tissue on biodegradable polymer matrices.
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Appendix 1: RFP with PI Approval
Name: Rachel Straughn
Lab/PI: Dr. Ying Zheng
Mentor: Nicole Zeinstra
Design/Research? Design and Research

Working/Tentative Project Title:

Development of a Small-Diameter Arteriole Model for Drug Testing and the Study of Endothelial-Perivascular
Interactions

This proposal responds to a request from the Zheng Research Group. The Zheng Research Group seeks to
develop a single-channel small arteriole model for the study of the effects of disease and drugs on perivascular
interactions.

The deliverables to be provided to the Zheng Research Group are:

(1) Design a device that allows for robust seeding of cells and in situ imaging,
(2) Develop a system for conducting flow and pressure experiments while maintaining luminal patency and
(2) Develop an arteriole system for sustaining pressure and flow, and
(3) Demonstrate arteriole functionality through calcium-transient imaging and drug studies.

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Appendix 2: Concept Sheet

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 Appendix 3: Milestones and Timeline Table

Device Design

Device Fabrication & Feasibility Experiments

Smooth Muscle Migration and Alignment

Observing Perivascular Remodeling

Observing Arteriole Function

Years: 2017-2018 May June Sept Oct Nov Dec Jan Feb Mar Apr May June

Figure 8. Gantt chart indicating expected completion times for each phase and subtask. The darker color
indicates the ideal completion time and the lighter color indicates the time allocated for resolving any issues
that arise within the subtask.

Table 1. Research milestones, organized in columns by their associated aim.

Aim 1
Design using CAD software
Functional Prototype Fabrication
Feasibility Experiments
Aim 2 Aim 3
Optimize smooth muscle density for Apply various flow and pressures to
compaction and cell-cell interaction system
Apply various flow and pressures to Observe smooth muscle migration
system and alignment
Observe smooth muscle migration Demonstrate arteriole functionality
and alignment

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 Appendix 4: Additional Figures and Tables

Figure 9. Flow chart of overall research and design strategy. The orange, blue, and green boxes depict steps to
be completed in Aims 1, 2, and 3, respectively. The purple box indicates steps that have already been
completed, and the red box indicates steps that may be completed once this project is complete.

Table 2. Ranked needs for all stakeholders. Ranks of 1 are given highest importance, ranks of 5 are given
lowest importance.
Need # Stakeholder Need Rank 1-5
1 Researchers Single Channel Geometry 1
2 Researchers Small-Diameter Geometry 1
3 Researchers Robust, Time-Efficient Fabrication Protocol 2
4 Researchers Cost of Fabrication 3
5 Researchers In situ construct imaging 2

Table 3. Needs-Metrics Table depicting quantification of the users’ needs. Just as in Table 2, ranks of 1 are
given highest importance and ranks of 5 are given lowest importance.
Metric # Need # Specification (metric description) Units Target Values Overall Rank
1 1, 2 Construct Radius µm <300 1
2 1, 2 Construct Length mm >10 2
3 3 Fabrication Time (Time between sterilization and hours <1.5 2
collagen gelling)
4 4 Cost per experiment (1 device) USD <200 3
5 5 Device width mm <60 3
6 5 Distance from bottom of device to center of lumen µm <150 5
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Figure 10. Functional decomposition diagram. The text enclosed in boxes represents user interactions and
inputs to the product. The magenta numbers indicate the need met with that interaction, while the green
numbers indicate the metric quantified within that interaction. The dotted lines indicate iterative processes.

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