Ex-vivo Model of Breast
Cancer Bone Metastasis
Keagan Collins, Laura Graefe, Chris Hubley, Jaymin Modi,
Bethany Porter and Ian Roberts
Sponsor: Dr. Anja Nohe
Advisor: Dr. Liyun Wang
1
TABLE OF CONTENTS
Title Page………………………………………………………………………………………………………………………………..…1
Abstract……………………………………………………………………………………………………………………………………..3
Introduction……………………………………………………………………………………………………………………………….4
Background and Significance………………………………………………………………………………………….4
Project Scope…………………………………………………………………………………………………………………4
Wants and Constraints…………………………………………………………………………………………….…….4
Design Metrics…………………………………………………………………………………………………………..…..5
Concept Generation and Selection…………………………………………………………………………………………….5
Benchmarking………………………………………………………………………………………………………………..6
Preliminary Concepts……………………………………………………………………………………………………..7
Concept Selection…………………………………………………………………………………………………………..9
Final Design……………………………………………………………………………………………………………………………..11
Overview………………………………………………………………………………………………………………………11
Design Details……………………………………………………………………………………………………………….15
User Instructions………………………………………………………………………………………………………….16
Cost Analysis……………………………………………………………………………………………………………..…16
Design Validation……………………………………………………………………………………………………………………..17
Failure Analysis…………………………………………………………………………………………………………….17
Testing………………………………………………………………………………………………………………………….17
Validation Results…………………………………………………………………………………………………………19
Conclusions………………………………………………………………………………………………………………………….....19
Design Evaluation……………………………………………………………………………………………………......19
Deliverables………………………………………………………………………………………………………………….20
Path Forward………………………………………………………………………………………………………..……..20
Appendices………………………………………………………………………………………………………………………………21
Appendix A…………………………………………………………………………………………………………………..21
Appendix B…………………………………………………………………………………………………………………..24
Appendix C……………………………………………………………………………………………………………………26
References……………………………………………………………………………………………………………………………….27
Team Resume………………………………………..………………………………………………………………………………..29
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ABSTRACT
The purpose of this project is to develop a bioreactor system to aid in development of a more
in-depth model of metastatic breast cancer cell activity and its effects on bone. If successful,
this project could allow for a decrease in human trials, as the bone in the bioreactor will closely
simulate an in vivo model. This will be achieved through the design and construction of a
functioning perfusion bioreactor capable of maintain cell viability within a femoral head, such
that metastatic breast cancer cells can be introduced and studied. The final design consists of
four components: a vibrating table responsible for applying mechanical stress, a perfusion
pump to allow for proper vasculature perfusion, a container to hold the bone and media, and
all components necessary to lock the bone in place. After prototype assembly, an initial trial
was conducted using a juvenile bovine femoral head; the trial lasted for ten days. Overall, the
prototype was able to maintain a majority of the initial cell viability within the bone. Of issues
encountered, the most notable was formation of fungus at the top of the media. The fungus did
not appear to contaminate the bone sample, and the issue was resolved by the addition of an
anti-fungal to the media. Following this trial, the bioreactor was cleaned and sterilized in
preparation for a second trial using a human femoral head. Though adjustments were made for
the different tissue type, some compatibility issues were apparent and the trial had to be
concluded earlier than anticipated. In the future, more trials will be conducted using human
tissue and ultimately, the human femoral head will be introduced to metastatic breast cancer
cells and the effects will be studied.
3
INTRODUCTION
Background and Significance
The sponsor for this project is Dr. Anja Nohe, an Assistant Professor in the Department of
Biological Sciences at the University of Delaware. One of the current objectives of the Nohe
Laboratory is to develop a bioreactor that can be used to study the development of metastatic
breast cancer in human bone. More specifically, Dr. Nohe is interested in creating a bioreactor
that can function to maintain cell viability within a femoral head for a certain period of time,
enough to allow for perfusion of cancer cells into the bone. By perfusing cancer cells into an
otherwise healthy bone, it will be possible to follow the progression of metastasis from the
beginning, as opposed to the commonly used models where metastasis has already been
developed. This perfusion bioreactor system will allow the mechanisms of cancer growth
within bones to be studied and new treatments to be developed.
Project Scope
The team will work to develop a perfusion bioreactor that can maintain cell viability within a
femoral head that closely mimics the environment in vivo. Once a viable state has been
achieved, cancer cells will be introduced and bone metastasis can be properly studied.
Wants and Constraints
Wants and constraints were generated with regard to existing bioreactors and the project
goals. A successful bioreactor will be defined by its capability to fulfill the defined constraints.
Wants and constraints are prioritized below, beginning with the components given the most
consideration.
Constraints:
1. Bone remains viable for at least ten days – Ten days is the minimum amount of time the
femoral head must be kept alive (viable) to observe cancer cells in the bone. Bone
viability will be maintained through proper perfusion and environmental controls.
2. Project deadline: Project must be completed by the deadline set forth by the advisor,
December 11, 2013.
3. Sterility of bioreactor - Bioreactor must be sterile to avoid the addition of extra variables
that could alter the results.
4. Ability to test for cell viability - The purpose of the project is to keep the bone alive for a
minimum of 10 days, so a simple way to test for life is absolutely necessary.
5. Integrity of vasculature - Necessary blood vessels for perfusion of blood and nutrients
must be kept intact when obtaining the femoral head and culturing.
Wants:
1. Ease of use – Bioreactor should allow for easy placement of bone, as well as the ability
to test cell viability. Bioreactor must allow for an easy method for changing the media.
Setup of bioreactor should be straight forward, if applicable.
2. Efficient use of resources – Bioreactor should be able to perfuse efficiently, not using
more than is necessary to keep cells alive. Efficient use of media is especially important.
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3. Ideal size – Size should allow for the size of the bone but should not have a lot of excess
room, which would allow for excess contamination.
4. Cost effective – Bioreactor should not cost more than similar models currently on the
market.
Metrics
The design of the bioreactor must take into consideration two different kinds of metrics. These
metrics are the mechanical design on the bioreactor itself, and the biological conditions that it
must be able to mimic. The “life-support” function of the bioreactor is prioritized over the
mechanical design. An important subset of the biological metric system of the design is the
ability to test to ensure if the bone cells are still alive, and therefore still valid for breast cancer
metastasis testing.
Biological Metrics
Metric [Applicable
Constraint/Want]
Time Bone Kept Alive
[C1]
Temperature [C1]
Ph [C1]
Nutrient
Levels/Concentration
[C1]
Holding Solution
Concentration [C1]
Mechanical Stress [C1]
Ability to test for cell
viability [C3]
Perfusion Locations [C4]
Media flow [C4]
Media volume [W2]
Other Metrics
Metric [Applicable
Constraint/Want]
Sterility [C2]
Description
Bone must be kept
"alive" for at least 10
days
Normal body
temperature
Normal blood pH
Target Value
37° C
7.4
Specified by
Sponsor
common
knowledge
[2]
Perfusion liquid
Neutral solution for the
bone to rest in during
the trial
Vibration to simulate
mechanical stress
Need to be able to test
for cell being alive/dead
Analogous to blood
Preference
Neutral saline solution
60 Hz, 1 G vertical
acceleration
Able to determine cell
viability
Close to natural vessels
as possible
Preference
>10 days
Normal vessel locations
Flow of media through
vessels in femoral head
3.5 +/- 1.2 ml/min/100g
Volume of media flowing
through vessels
3.0 +/- 1.3 ml/100g
Description
Able to completely
sterilize all components
Target Value
<1 hr to sterilize
Reference
[3]
Specified by
Sponsor
common
knowledge
[4]
[4]
Reference
Specified by
Sponsor
5
Size of chamber [W3]
Size of entire apparatus
[W3]
Large enough to hold
normal human femoral
head
Size of chamber and
support vessels
500 mL
2' x 3'
Cost [W4]
Cost of bioreactor
<$5000
*Metrics in bold specified as most important by sponsor
Specified by
Sponsor
reasonable
range
Specified by
Sponsor
CONCEPT GENERATION AND SELECTION
Benchmarking
From academic research labs to pharmaceutical companies testing vaccines, drugs, and various
other treatments, the use of bioreactors is widespread [5]. Many of these bioreactors are
single use, which simplifies the design considerably. In the next ten years, it is expected that
the market for single use bioreactors will increase significantly [6]. There are many different
types of perfusion bioreactors available, split into different categories based on needs such as
tissue types [7]. An example of a perfusion bioreactor is shown in Figure 1.
The bioreactor for this project will maintain a femoral head that will eventually be perfused
with metastatic breast cancer. Although the field of breast cancer research is very populated,
much of the current literature focuses on the cancer specifically, and not its effects on bone.
Research focused on the effect of metastatic breast cancer on the bone is still in its early stages,
primarily because bone injury is usually only found after cancer has been eradicated from the
breast. There is some treatment available for metastatic breast cancer, however the success
rate is not promising and not making huge strides [8].
Figure 1: Simplified Illustration of Perfusion Bioreactor
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Preliminary Concepts
Once research into the different aspects of bioreactors was completed, initial concepts could be
discussed. Three concepts were formed based on the available research, as well as the metrics
and wants and constraints specified by Dr. Nohe. The concepts may have differences, but the
fundamental design is preserved in each. Perfusion into the bone occurs at a similar location in
all three concepts. Currently, the exact vasculature within the femoral head is not documented
in both bovines and humans. However, there are several main arteries that are known, such as
the deep femoral artery and the caudal gluteal artery. Further testing will confirm where this
bioreactor will perfuse a femoral head. This simplified drawing depicts perfusion tubes
traveling through the bone, but in actuality the tubes will approach the bone and cannulation
will occur. The saline solution is maintained and will surround the bone in the bioreactor. In
keeping the main idea the same, small details could be focused on, such as the method of
mechanical stress or the placement of the bone. For all three design concepts, perfusion will be
through the vasculature or through fabricated entry points. Testing will decide which of these
options is most feasible.
In the first concept generated, mechanical stress is applied via direct force. Perfusion out
occurs from a pump present within the structure applying force. A detailed drawing of the first
concept is shown in Figure 2.
Figure 2: Initial Design Concept with Direct Mechanical Stress
After speaking with Dr. Nohe, a second concept was generated on the basis of a different
mechanical force. Here the bone is being stimulated via a vibrating table, attached beneath the
main structure. The rest of the design remains much the same. A detailed drawing of this
concept is shown in Figure 3.
7
Figure 3: Initial Design Concept with Vibration as Mechanical Stress
A final concept was designed that again includes vibration as the mechanical stress but varies
slightly in the shape of the foam brace. Here the bone is elevated via foam support beams,
allowing for a greater submersion in the saline solution. Furthermore, the bone is flipped such
that the exposed trabecular bone is facing the top of the structure. A detailed drawing of this
concept can be found in Figure 4.
Figure 4: Initial Design Concept with Elevated Bone
8
Concept Selection
Via comparison and further research, a final design was chosen from the three initial concepts.
After a lengthy discussion with the sponsor, direct force as the main form of mechanical stress
was rejected. In choosing vibration as the principle mechanical stress, the first concept was
eliminated. The main difference between the second and third concepts is the location of the
bone. In the third concept, the bone is raised further up, away from the vibration platform.
After some discussion, it was decided that this could dampen the effects of the vibration and
thus, the third concept was eliminated. With two concepts eliminated, the second design was
chosen. A more detailed drawing of the final design is shown in Figure 5.
Furthermore, research has indicated that there are two possible methods of cannulation and
thus, perfusion. The first possible method would involve direct cannulation and is only possible
with exposed arterial entrances. If the arteries are known, perfusion can occur directly,
allowing for the most realistic and accurate perfusion path within the bone. Figure 6 better
illustrates this method. However, this method will not always be possible, as it is difficult to
obtain a bone with exposed arteries and so, the second possible method of perfusion is
indirect. Using this method, small holes would be drilled into the bone and a catheter would be
inserted in these holes. There would most likely be four holes drilled with this method, allowing
for a higher chance of locating some of the initial vasculature. This method is shown in Figure
7. The chosen perfusion method will be dependent on each particular bone sample.
*All dimensions are in inches
Figure 5: Final Design
9
Figure 6: Perfusion through Vasculature
Figure 7: Perfusion through Drilled Entry Points
The final design for this project is consistent with most of the metrics set by the team. In order
to satisfy the main biological constraint, keeping the bone alive for at least ten days, many of
10
the other metrics are also satisfied, such as maintaining temperature at 37°c, pH of 7.4,
mechanical stress at low levels via a vibration table, and physiological nutrient levels, similar to
those found in bovine blood. Temperature will be maintained by the incubator containing the
bioreactor. pH will be maintained by the media perfusing through the bone, as will
physiological nutrient levels. Further research and discussion with Dr. Nohe has led to
reconsideration of using a neutral saline solution as the holding solution, as the saline solution
may cause the cells to swell and lyse. Thus, an alternative solution was proposed. The holding
solution will be identical to the perfusion media, as per sponsor recommendation. The
perfusion media will be consistent with current products on the market with a goal of
maintaining cell viability. Perfusion location will be dependent on individual bones and will
occur within the vasculature of the bone when possible. If vasculature was not maintained,
perfusion will occur via holes drilled into the cut surface of the bone. Flow rate has been
determined on the basis of human flow rate and preference from the project’s sponsor. From
basic engineering calculations, the flow rate for perfusion of the femoral head is 10 ml/hr. The
media volume was also calculated, with a final value of 900 ml. The used media was exchanged
for 900 ml of fresh media every 36 hours, due to a limitation in resources.
All mechanical metrics have been met by this design. To ensure a successful bioreactor, sterility
will need to be maintained, as is consistent with the metrics. All precautions will be taken, such
as sterilizing all components in either an autoclave or ethanol solution, in addition to sterilizing
all areas in use. The size of both the chamber and apparatus are also expected to fall within the
target values. As many of the parts used to construct the bioreactor will be recycled from the
Nohe lab, the cost is expected to come in well below the target value, with the largest costs
coming from media use and bones used for initial testing.
FINAL DESIGN
Overview
The final design of the perfusion bioreactor includes one container (the main component of the
bioreactor) containing the bone, the media and placement holding foam; a pump, used to
pump media into the bone; and a vibrating table, used to simulate mechanical stress. The
vibrating table was assembled by the team using a motor found in the Nohe lab and is shown in
Figure 8. It vibrates with a frequency of 60 Hz and has a vertical acceleration averaging 0.4 G. A
3D rendering of the container is shown in Figure 9. The pump, which was supplied by the
sponsor, is not shown but resembles many other media pumps found on the market.
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Figure 8: Vibrating Table (60 Hz, .4 G vertical acceleration)
Figure 9: 3D Rendering of Bioreactor
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Design Details
The final design for the perfusion bioreactor is shown in detail in Figures 10 and 11. The design
consists of a vibrating table, main container, and perfusion pump. The vibrating table consists
of a motor epoxied to a biologically inert platform (as shown in Figure 8). This table will be kept
in a bio-hood and will be sterilized with ethyl alcohol before each use. To simplify the diagram,
the table is shown as if in use underneath the bioreactor in the 2D rendering (Figure 10).
The main component of the bioreactor is the 4L polystyrene container, which will be kept in an
incubator while in use. This container acts as both the holding cell for the bone and the
reservoir for media perfusion. The container consists of a bottom layer of biologically inert
plastic. Through this plastic, which is epoxied to the bottom of the container, holes have been
drilled such that long bolts can be attached. These bolts serve to hold the polyurethane foam in
place during use. The polyurethane foam is in the bioreactor to ensure secure positioning of
the bone. The foam is cut into four sections, two placed below the bone and two above, with
semicircles cut out to help with securely holding the bone. If the bone is too large, the foam
above the bone can be removed with no large repercussions, though the bone will not be held
as securely during the vibration period. As per the resources available, the foam on the bottom
has a density of 20 pcf and the foam on the top has a density of 12 pcf. Having the more dense
foam on the bottom should help resist dampening of the vibrations when using the vibrating
table. Furthermore, the foam is biologically inert and autoclavable, as are all materials that will
enter the incubator.
In the center of the container rests the bone itself. The bones being used for this project are
juvenile bovine femoral heads. Initially, Dr. Nohe had requested adult bovine femoral heads for
use in this project. However, it has proven near impossible to obtain adult bovine femoral
heads in which the cells are viable due to FDA regulations of bovine meat. However, juvenile
bovine meats are not subject to the same restrictions and, thus the cells within the femoral
head should remain viable upon delivery. The bones are prepared before being given to the
team. The bone comes with all meat removed and is cut along the femoral neck, such that no
further cuts need to be made by the team. The bone has been left intact in all other ways.
Perfusion media is also present in the container of the bioreactor. This media serves both as a
reservoir and also as a possible source for perfusion of the bone directly. This media will be
replaced regularly, so as to ensure accurate levels of necessary nutrients and proper removal of
waste. Furthermore, this media will be pumped from the main container using the perfusion
pump into the bone directly via the cannulated entry points. This pump will run at a specified
setting to ensure proper flow rate to maximize cell viability, back calculated using the pump
itself and engineering principles.
The final components of the bioreactor are perfusion tubes. The tube that is not attached to
the bone is responsible for perfusion out of the bioreactor. This tube pulls media from the
general container (i.e. not directly from the bone). This is because perfusion out of the bone is
not entirely understood and occurs in many places. The remaining tubing is used to perfuse
media into the bone. Perfusion into the bone will occur directly through vasculature, when
possible. The perfusion tubing will be attached using a catheter. If necessary, perfusion can
occur indirectly, via small holes drilled into the exposed surface of the femoral neck. These
13
holes will be perfused using needles attached to the tubing, allowing media to enter the bone.
A full description of the method used to arrive at the decision for perfusion method is shown in
Appendix A.
All components of the bioreactor can be sterilized, either through autoclaving or soaking in an
ethanol solution. Most components will be kept in an incubator throughout the trial, with the
only exception being the vibrating table, which will be held in a bio-hood. This is possible
because mechanical stress does not need to be constant. The table will be sterilized prior to
each use and should not cause contamination in the bioreactor. Because the perfusion pump
does not directly touch anything entering the bioreactor, it is not a concern with regards to
sterilization.
This project does not have an explicit bill of materials, as the document would be equivalent to
the cost analysis, shown below. However, final material costs are expected to remain below
$200.00, as most of the materials were provided by the sponsor, Dr. Nohe and her lab.
Figure 10: 2D Rendering of Final Design
14
Figure 11: Photo of Working Prototype on Day 1, Trial 1 (vibration table not shown)
User Instructions
Femoral Head Preparation:
After procuring from supplier, place bone in ice until ready to insert into the bioreactor.
Observe the bone to determine the method of perfusion necessary. If perfusion through the
vasculature is possible, the catheter and tubing should be rinsed in media and the final entry
point should be chosen. Using the trial and error method, the catheter should be inserted into
the vessel until no backflow is observed. At this point, the bone is ready to be perfused.
If perfusion through vasculature is not possible, perfusion through drilled entry points will be
necessary. After rinsing in media, use a sterilized surgical drill to drill four 5 mm hole 40 mm
deep into the bone, far enough in that the holes have passed any possible growth plates. The
holes should be drilled in a zigzag pattern, starting in one corner of the trabecular bone. A
catheter will be inserted into the drill sites and perfusion tubing will be connected to the
inserted catheter. As there will still only be one tube with media pumped through, this tube
will need to be split twice, resulting in four tubes that can be attached to the catheters for
perfusion. One or two holes can also be drilled on the non-cut side of the bone to aid in
draining of the media after having traveled through the bone.
Reactor Preparation:
All components that will be put inside of the incubator (reactor chamber and lid, polyurethane
foam, and tubing) must be sterilized using standard autoclave procedures. A clean biosafety
cabinet must be prepared, and the vibrating table should be sterilized using ethyl alcohol and
15
placed into the hood. Fill reactor chamber with 1.25L of prepared media solution. Place the
catheterized bone into the reactor chamber, and surround with sterilized polyurethane foam to
secure bone inside chamber. Then place the reactor chamber into the incubator ensuring that
the lid is properly sealed and the tube inserted during cannulation is fed out through an
opening in the incubator. Next, connect the perfusion tubing to the pump tubing, which should
be located outside of the incubator. Run the pump at a setting of “25” until the media is about
to start perfusing the femoral head, then reduce to a setting of “6”, which corresponds to a
flow rate of 10 mL/hr. Continue pumping through, changing media every 36 hours due to
resource constraints. If possible, media should be changed every 24 hours. Following media
change, disconnect reactor from pump, and vibrate on the vibrating table at 60 Hz and .4 G
vertical acceleration for 30 min, then place back into the incubator, reattach perfusion tubing to
pump, place the reactor chamber into the incubator and resume perfusion.
Cost Analysis
Due to the nature of this project, the cost analysis will be the same as the bill of materials. This
is due to the fact that this prototype will not be replicated with the goal of mass manufacture.
Instead, this bioreactor is being built with the purpose of use by the Nohe Lab at the University
of Delaware only. As such, items being supplied by the lab do not factor in to the final cost. A
full cost analysis is shown below.
Materials
Specific
Comment
Cost
Motor (provided by Nohe Lab)
epoxied to platform (purchased $10.00
by Nohe Lab)
Vibration Table
Constructed
Polyurethane Foam
(12.5 pcf)
12'' x 12'' x 4''
Provided by UD biomedical
engineering department
$39.75
Polyurethane Foam
(20 pcf)
12'' x 12'' x 4''
Provided by UD biomedical
engineering department
$55.50
Perfusion Media
DMEM
Provided by Nohe Lab
$0.00
Perfusion Tubing
Tygon E-3603 Laboratory
Tubing
Provided by Nohe Lab
$0.00
Perfusion Pump
Delta T Micro-Perfusion Pump
Provided by Nohe Lab
$0.00
Cannulation Needles
BD 30 gauge cannulation
needles
Provided by Nohe Lab
$0.00
Bioreactor Chamber
Fischer 4L polystyrene
container
Purchased by UD biomedical
engineering department
$50.00
16
Bone Samples
veal femoral head
Purchased by Nohe Lab
$35.00
Actual Cost
$190.25
DESIGN VALIDATION
Failure Analysis
One of the largest threats to the function of the bioreactor is that of contamination,
from the bioreactor itself, the sample, or the surrounding environment. The bioreactor was
sterilized using standard techniques, and was assembled in a clean hood. The other factors are
less easily controlled, but sterilization and cleaning techniques were used to ensure the best
possible trial. Another possible issue is that of initial cell viability of the femoral head. This is
dependent entirely on the provider of the bone, but as long as the sample is placed into the
reactor less than 12 hours after it is removed from the body, high enough viability is maintained
for a successful trial.
Another possible issue is the placement of the catheter in an incorrect location or in an
incorrect fashion. The group used techniques learned from Dr. Robert Sikes, who has
experience in this field, to ensure that neither of these issues would occur. The final issue that
was tested for was the sufficiency of the media. The media had to be completely sterile and had
to have proper nutritional components. With the help of graduate students in Dr. Nohe’s
laboratory we were able to ensure that both of these requirements were met initially. The old
media glucose and pH levels were tested when the media was changed to ensure that it met
our specifications as well.
Finally, the media was exchanged in the initial trial every 36 hours due to resource
limitations. However, it was clear from the testing conducted that this was not often enough to
ensure proper nutrients for the cells in the bone. Going forward, the time will be optimized to
24 hours or less between media exchange. This should allow for higher cell viability at the
conclusion of the next trial.
Testing
Testing for this project looked at determining cell viability and some design aspects of the
reactor itself. For cell viability, originally a live/dead cell viability assay was to be conducted.
However, due to logistical issues it had to be scrapped, and a secondary testing plan of media
level testing was conducted. The pH and glucose level of the media was taken when the media
was changed, every 36 hours. Also, the correct perfusion location was tested for placement
and how to hold the needle itself into the bone. The placement of bone within the reactor was
also tested.
 Glucose Level Testing – Glucose level testing was conducted every 36 hours using a
standard off-the-shelf diabetic glucose meter. The end value was compared to the
known starting glucose level of the media (4500 mg/L). The glucose was almost entirely
depleted after each changing, and showed no decrease in usage throughout the ten
17




Glucose Usage (mg/h) at a function of
Time
140
Glucose Usage Rate (mg/h)

days. It can indirectly measure cell viability through the media consumption. Figure 12
shows the data collected from these tests.
pH Level Testing – pH level was taken every 36 hours following the exchange of media.
pH consistently decreased by 1 to 1.5. This is within an acceptable range for tissue
culture and shows the nutrients are being used by the cells. The data collected is shown
in a chart in Figure 13.
Contamination Testing – Using a standard cell nucleus dye (trypan blue), the media was
checked for contamination after 5 days and at the end of the experiment. With the
expertise of Dr. Nohe, it was determined that there was no media contamination. There
was a small fungal contamination, but it was separate from the media itself should not
affect the results of the trial. Following this diagnosis, an anti-fungal was added to the
media.
Cannulation/Perfusion Testing – With the help of Dr. Robert Sikes, the correct
perfusion site and technique was chosen through trial and error. The site chosen
worked well with the bone orientation that was chosen. The needle was firm enough
inside the tissue to stay in place without any additional securing process.
Bone Orientation Testing – Prior to the trial, bone samples were procured from the
supplier, and examined to look at how it should be oriented inside the reactor. An
upright orientation was chosen, as the designated vessel for cannulation was the branch
of the obturator artery, leading into the top of the femoral head.
Vibration Table Testing– The vibration table was used for 30 minutes every time the
media was exchanged (36 hours). The frequency was determined via manufacturer
survey as 60 Hz. The vertical acceleration was measured via iPhone application, and was
found to be an average of 0.4 g with spikes of up to 0.5 g. This is not as high as specified
in the metrics, but should be sufficient for our purposes.
120
100
80
60
40
20
0
0
50
100
150
200
250
Time (hours)
Figure 12: Chart of Glucose Levels throughout Ten Day Trial
18
Bioreactor Media pH as a Function of
Time
9
8.5
8
pH
7.5
7
6.5
6
5.5
5
0
50
100
150
200
250
Time (hours)
Figure 13: Graph of pH Levels throughout Ten Day Trial
(Note: There is no standard deviation for either chart because only 1 trial was conducted)
Validation Results
Overall, the design functioned very well considering the time and cost constraints. Using mostly
available components in our sponsor’s laboratory, we were able to culture a juvenile bovine
femoral head for 10 days, from November 15th through November 26th. Our sponsor was
satisfied with the level of cell viability we are able to suggest occurred in the preliminary ten
day trial. Additionally, due to the glucose and cell viability tests conducted and described above,
we are able to suggest that there was not a significant decrease in viability. We cannot directly
comment on overall cell viability due to the indirect nature of media glucose testing. We were
not able to compare the effects of the vibration table on these results, as that would require
further testing and the time constraint will not allow for it. Some of our metrics have changed
since the initial design was created, such as holding the femoral head sample in media rather
than a neutral saline solution. However, none of the crucial metrics were sacrificed with this
design and the most important metrics, such as low cost and maintaining viability for at least
ten days, were upheld.
CONCLUSION
Design Evaluation
In evaluating the final design, the ten day trial of the bioreactor prototype met all major metrics
established in the initial phases. The bioreactor was able to culture the femoral head for 10
days. Using the vibration table, mechanical loading conditions were mimicked in order to inhibit
osteocyte apoptosis and similar issues. The bioreactor was placed on the vibration table every
36 hours for 30 minutes at a time, following media exchange. Fungal contamination was
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observed in the bioreactor, but the spores remained airborne and never contaminated the
bone itself; thus they did not pose a serious problem to the function of the bioreactor.
Following this observation, an anti-fungal was added to the media. No signs of bacterial or viral
infection were detected within the bone, leading to the assumption that the sterility
requirements were achieved. Media was changed consistently every 36 hours and glucose and
pH levels were measured with each changing. Overall, the prototype achieved the necessary
goals set out, thus the trial was completed without failure and opened the door for extended
testing with human tissue with minor alterations to media contents and flow rate. One change
that should be made not to the design but to our protocol will be to change media more often,
such as every 24 hours rather than every 36 hours. This will allow us to have a measurable
glucose concentration, resulting in a non-static glucose consumption rate over time.
Deliverables
At the conclusion of this project, multiple items will be handed over to Dr. Nohe and the Nohe
Research Lab. The items given include all components necessary to build the bioreactor and
the report, which contains the user manual for assembly and conducting a trial and the data
collected during the initial trial. For a successful trial to be conducted the chosen bone will
need to be obtained on the first day of the trial and media will need to be prepared within a
week of the trial’s initiation. A list of the physical components being given is shown below.
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4L polystyrene container with lid
o Polycarbonate sheet epoxied to bottom on the inside with bolts attached to hold
foam
o Bolts, washers, and wing nuts to hold foam in place
4 pieces of polyurethane foam, with semicircles cut out where the bone will be held
Perfusion tubing
Perfusion Pump
60 Hz, .4G vertical acceleration vibration table with attached straps
Report containing user’s manual and data from initial trial
Path Forward
At the conclusion of this project, cell viability of a juvenile bovine femoral head was maintained
within the bioreactor over the ten day trial. Furthermore, a four day trial was attempted using
a human femoral head. Unfortunately, backpressure from a clogged filter forced the trial to
conclude after 12 hours, as perfusion had been compromised. The results from this trial are
detailed in Appendix B. Going forward, the Nohe Lab will continue conducting trials, beginning
with a ten day trial to sustain cell viability in a human femoral head. Following a successful trial,
a third trial may be conducted with the purpose of determining how long sufficient cell viability
can be maintained in a human femoral head. With cell viability properly tested, the focus of the
project will move to metastasizing breast cancer. Having this goal in mind, trials will be
conducted where cell viability is maintained and breast cancer cells are perfused through the
femoral head. Following the conclusion of these trials, the bones will be thoroughly examined
for evidence of metastasis and the effects on the bone. Depending on the results of these
trials, a functional bioreactor may be purchased. This could help to minimize contamination
20
and may contribute to maintaining higher cell viability, allowing for longer, more in-depth trials
to be conducted.
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Appendix A: Determination of Perfusion Method
Trial 1 – October 25, 2013
The group obtained two juvenile bovine femoral heads, sliced through the femoral neck. The
cow had been sacrificed earlier that day and the bone appeared intact. Before any testing was
conducted, the bones were observed with an emphasis on finding any possible entry points
that followed the known vasculature. The bones were examined by four team members, as
well as one of Dr. Nohe’s graduate students. After the initial observation, it was determined
that perfusion through an entry point related to the known vasculature would not be possible,
at least for these two bone samples.
A mannitol solution was made up and the smallest possible needle was chosen, as any possible
entry points were very small. Because there were no known entry points, two members of the
team attempted to find an entry point using the needle and solution. Though the needle was
able to enter the bone at a few different locations, none of the entry points allowed for
sufficient perfusion. One hole showed evidence of the solution spreading throughout
approximately half the trabecular bone, but this does not appear to be consistent for every
bone, as it has not been able to be replicated since.
After attempting to perfuse the bones, the bones were cut in half and flushed out using water.
The goal of this experiment was to determine if vasculature could be observed within the
femoral head. However, testing indicated this was not be possible. This test did indicate that
both bones contained a growth plate, which could impact perfusion method of the final bone.
Trial 2 – November 1, 2013
In an effort to ensure the first two bones were an accurate representation of the bones to be
inserted in the bioreactor, the team collected two more juvenile bovine femoral heads one
week after the first trial. These bones were sliced further from the femoral head and required
the team to cut them before testing. The cut was made through the femoral neck, as similar to
the other bones received as possible. It should be noted that this cut contributed some
damage to the bone and may have affected the results of the trial.
After cutting the bone, four holes were drilled into the exposed side of the femoral head. Holes
were drilled in a zigzag pattern, similar to the pattern to be used for indirect perfusion. Needles
were then inserted through these holes and perfusion was attempted. Though most of the
solution perfused exited the bone through the drilled hole and did not spread through the
bone, it is believed that some spread throughout the vasculature. This method of perfusion will
most likely not provide sufficient media to the bones, but may help spread some of the media.
The conclusion from this trial is that perfusion through drilled entry points is to be the backup
plan when perfusion through vasculature is not possible.
Trial 3 – November 8, 2013
Through discussion with Dr. Nohe and Dr. Robert Sikes of the Biology Department, it was
determined that Dr. Sikes may be able to identify vasculature in the femoral head. Five bone
samples were obtained on the day of slaughter and Dr. Sikes sat down with two team
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members. After some initial observations, Dr. Sikes started removing some of the excess fat
and meat from the bone. After carefully observing the bone, he was able to identify three veins
that could be used for perfusion. Two team members then practiced finding these veins on the
other bone samples and were able to find the vessels in similar areas.
Conclusion
With the data collected from the three trials conducted, it was determined that perfusion
through known vasculature is preferable. Though the perfusion method will have to be
determined on the day of the trial, as it is dependent on each particular sample, the team is
confident that one of the bones collected from the supplier will have enough vasculature intact
for proper perfusion. As a backup plan, the team will continue to practice drilling holes into the
cut side of the femoral head. If necessary, perfusion will then occur in each of the four drilled
holes with the hope that enough of the media perfused will reach the internal vasculature.
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Appendix B: Four Day Human Femoral Head Trial
Graduate students from Dr. Nohe’s lab attempted to conduct a four day trial using all
components of the bioreactor and a human femoral head sample. Due to differences in
vasculature, the current design was slightly modified such that four entry points could be
cannulated and perfused. Due to the increase in tubing and entry points, the flow rate was
increased proportionately. The graduate students were instructed on the methods for
preparing the bioreactor and the methods of cannulation used in the initial trial. A sample was
removed from a femoral head such that a live/dead assay could be conducted to determine
initial cell viability. The protocol for staining this bone can be found in Appendix C. The images
were found using a Zeiss Axioscope. The resulting images are shown in Figure 14.
At hour six of the trial, the pressure from the pump caused the catheters to be removed from
the entry points. The catheters were placed once more and the trial continued. At hour
twelve, the catheters had again been forcibly removed and the filter appeared clogged. It is
hypothesized that the clogged filter resulted in an excess of pressure. At this point it was
decided that the trial should conclude and adjustments should be made to the design to more
properly accommodate human tissue. When the bone was removed at hour twelve of the trial,
a sample was removed and stained following the same procedure used to test initial cell
viability. The resulting images are shown in Figure 15.
From the data obtained, it appears that cell viability was largely maintained throughout the
trial. However, it must be acknowledged that the trial was much shorter than anticipation and
thus, the results are not particularly significant.
Figure 14: calcein stained live cells at time zero hours for human femoral head trial (left);
propidium iodide (PI) stained dead cells at time zero hours for human femoral head trial (right)
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Figure 15: calcein stained live cells at time twelve hours for human femoral head trial (left); PI
stained dead cells at time twelve hours for human femoral head trial (right)
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Appendix C: Procedure for Live/Dead Staining
Solution A: Calcein-AM solution, 1 mM in DMSO
Solution B: PI solution, 1.5 mM in H2O
1. Add 10 μl Solution A and 5 μl Solution B to 5 ml PBS to prepare the assay solution.
2. Prepare a cell suspension with a trypsin-EDTA treatment if cells are adhered to a culture
plate.
3. Centrifuge the cell suspension at 1,000 rpm for 3 minutes.
4. Wash the cell pellet with PBS several times to remove residual esterase activity.
5. Prepare a cell suspension with PBS in which the cell density is 1 x 105 to 1 x 106 cells/ml.
6. Mix 200 μl of cell suspension and 100 μl of assay solution and incubate the mixture at 37 ºC
for 15 minutes.
7. Detect fluorescence using a fluorescence microscope with 490 nm excitation for
simultaneous monitoring of viable and dead cells. With 545 nm excitation, only dead cells can
be observed.
a) The concentration of each reagent should be optimized. Following steps may be necessary to
determine the suitable concentration of each reagent:
1. Prepare dead cells by 10 minutes incubation in 0.1% saponin or 0.1 - 0.5% digitonin or by 30
minutes incubation in 70% ethanol.
2. Stain dead cells with 0.1 - 10 μM PI solution to find a PI concentration that stains the nucleus
only and does not stain the cytosol.
3. Stain dead cells with 0.1 - 10 μM Calcein-AM solution to find a Calcein-AM concentration that
does not stain the cytosol. Then stain viable cells with that Calcein-AM solution to check
whether these viable cell can be stained.
b) Or you may remove culture medium and wash cells with PBS several times. Add assay
solution and incubate at 37 ºC for 15 minutes.
26
References
[1] Carreau, A., B. El Hafny-Rahbi, A. Matejuk, C. Grillion, and C. Kieda. "Why Is the Partial
Oxygen Pressure of Human Tissues a Crucial Parameter? Small Molecules and Hypoxia." J Cell
Molecular Medicine (2011): 1239-253. Pubmed. Web.
[2] Kellum, John A. "Determinants of Blood PH in Health and Disease." Determinants of Blood
PH in Health and Disease (2000): 6-14. Pubmed. Web. 3 Sept. 2013.
[3] De Oliveira, Mônica Longo, Cássia T. Bergamaschi, Orivaldo Lopes Silva, Keiko Okino Nonaka,
Charles Chenwei Wang, Aluízio Barbosa Carvalho, Vanda Jorgetti, Ruy R. Campos, and Marise
Lazaretti-Castro. "Mechanical Vibration Preserves Bone Structure in Rats Treated with
Glucocorticoids." Bone 46.6 (2010): 1516-521. Print.
[4] Kubo, T., K. Kimori, F. Nakamura, and S. Inoue. "Blood Flow and Blood Volume in the
Femoral Heads of Healthy Adults According to Age: Measurement with Positron Emission
Tomography (PET)." Ann Nucleic Medicine (2001): 231-35. Pubmed. Web. 3 Sept. 2013.
[5] Eibl, R., Eibl, D., Kaiser, S., & Lombriser, R. (2010). “Disposable bioreactors: the current stateof-the-art and recommended applications in biotechnology.”. Applied Microbiology and
Biotechnology, 86(1). Retrieved September 5, 2013, from
http://www.ncbi.nlm.nih.gov/pubmed/2009
[6] Single-use Bioreactors Market Will Expand Rapidly Over the Next Decade, Visiongain
Predicts. (n.d.).Vision Gain. Retrieved September 5, 2013, from
www.visiongain.com/Press_Release/421/Single-use-bioreactors-market-will-expand-rapidlyover-the-next-decade-visiongain-predicts
[7] Fisher, J.P., & Yeatts, A.B. (2011). “Bone tissue engineering bioreactors: Dynamic culture
and the influence of shear stress.” Bone, 48 (2), 171-181
[8]Metastatic Breast Cancer. (n.d.). Patient Resource. Retrieved September 6, 2013, from
http://www.patientresource.com/Metastatic_Breast.aspx
[9] Davies, CM; Jones, DB; Stoddart, MJ; Koller, K; Smith, E; Archer, CW; Richards, RG,
“Mechanically Loaded Ex Vivo Bone Culture System ‘Zetos’ Systems and Culture Preparation,”
European Cells and Materials, Vol. 11, 2006, pages 57-75
[10] Vivanco, J; Garcia, S; Ploeg, HL; Alvarez, G; Cullen, D; Smith, EL, “Apparent elastic modulus
of ex vivo trabecular bovine bone increases with dynamic loading,” Proceedings of the
Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, 14 May 2013
[11] Arora, Meenakshi, “Cell Culture Media: A Review,” Mater Methods 2013;3:175, 30 Oct
2013
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[12] Howlett, C.R.; Bryden, M.M., “Anatomy of the arterial supply to the hip joint of the ox,”
Departments of Veterinary Pathology and Veterinary Anatomy, University of Sydney, New
South Wales, 2006, Australia, 14 September 1971
[13] Davidson, EH; Reformat, DD; Allori, A; Canizares, O; Wagner, Janelle I.; Saadeh, PB; Warren,
SM, “Flow perfusion maintains ex vivo bone viability: a novel model for bone biology research,”
J Tissue Engineering Regenerative Medicine 2012, Nov;6(10):769-76, 3 Nov 2011
[14] Alexander, Sailon M, et al. "A Novel Flow-Perfusion Bioreactor Supports 3D Dynamic Cell
Culture." A Novel Flow-Perfusion Bioreactor Supports 3D Dynamic Cell Culture. N.p., 2009. Web.
10 Oct. 2013. <http://www.hindawi.com/journals/bmri/2009/873816/>.
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Nohe Lab Senior Design Team
Keagan Collins
Laura Graefe
Chris Hubley
Jaymin Modi
Bethany Porter
Ian Roberts
(484) 225-3605
(610) 906-6681
(302) 853-5697
(302) 824-2529
(253) 225-8820
(856) 417-5232
keaganc@udel.edu lgraefe@udel.edu chubley@udel.edu jaymodi@udel.edu bsporter@udel.edu iroberts@udel.edu
Education
Fall 2010 – Spring 2014
University of Delaware – Bachelor of Science in Biomedical Engineering
Minors: Chemistry, Biology, Bioelectrical, Electrical and computer Engineering
Mission Statement: To develop a working bioreactor for the study of metastasis in the femoral head.
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Capabilities
Microsoft Office
Matlab
AutoCad
Finite Element Analysis (FEA)
Cell Culture
Statistics
Minitab
Laboratory Management
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Work Experience
Ian Roberts
University of Delaware Medical Technology Department
Received Summer Fellowship funding for research
Cell culture, nanotechnology development, microscopy
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Jaymin Modi
University of Delaware Dept. of Material Science Engineering – Pochan Lab
Worked on Hydrogel drug delivery technology
Fluorescent Microscopy analysis
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Keagan Collins
Regeneron Pharmaceuticals
Statistical process control analysis of soy media for optimization of CHO cell bioreactors
Applied LEAN Six Sigma and 5S Techniques within Commercial Manufacturing
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Laura Graefe
Eastern Technologies
Maintained the lab and actively trained the future lab manager
Tested customer samples to maintain accuracy of product levels
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Chris Hubley
University of Delaware Dept. of Material Science Engineering – Martin Lab
Chemical Polymerization and Electrochemical Deposition of polymers
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Bethany Porter
Mathematics Tutoring and Music Instruction
Instructing elementary students in piano
Tutored elementary through high-school level mathematics
(Research Laboratory Manager 2012 – 2013)
(Research Assistant 2012-2013)
(Summer Intern 2013)
(Summer Intern 2012 -2013)
(Research Assistant 2013- Present)
(2007 - present)
Interests
Orthopedics, tissue engineering, prosthetics, 3D printing, physiology, biomechanics, bioelectrics, nanotechnology, anatomy, material
science, medical imaging, medical instrumentation, pharmaceuticals, drug delivery
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