substrate holder for tissue staining and engineering
TRANSCRIPT
Biomaterials 5: Substrate Holder for Tissue Staining and Engineering
Derek Dodge, Daniel Evans, Lisa DeConti, Andrew Cohen
Junior Design: BME 4985
12/6/15
Electronic Signatures:
-Derek Dodge
-Daniel Evans
-Lisa DeConti
-Andrew Cohen
1
Authorship Page:
Contributions of each team member:
Derek: Performed calculations, wrote future work, preliminary design, and financial
aspects portions of the report and performed all photography.
Dan: Wrote list of images, and generated all SolidWorks screenshots, designs, and
drawings for the report. Also contributed to the analysis section.
Lisa: Wrote the table of contents, list of tables, abstract, executive summary, design
problem, research, references, and contributed on important constraints, analysis, ethical issues,
suggestions for future modifications, appendix and conclusion.
Drew: Wrote preliminary evaluations of each design, regulatory issues, and conclusion.
Also created objectives tree, functions-means tree, and pairwise comparison chart.
2
Table of Contents:
● Authorship Page- Page 1
● Table of Contents- Page 2
● List of Figures- Page 3
● Abstract- Page 4
● Executive Summary-Page 4
● Design Problem- Page 5
● Research- Pages 5 - 6
● Important Constraints- Pages 6 - 7
● Alternative Solutions- Pages 7-8
● Preliminary Evaluations of Each Design- Pages 9-15
● Analysis- Pages 15-17
● Final Decision Choices- Pages 17-18
● Regulatory Issues- Pages 18 - 19
● Financial Aspects- Page 19
● Ethical Issues- Page 19
● Suggestions for Future Modifications- Pages 19-22
● Conclusion- Page 22
● References- Page 23
● Appendix- Pages 23 - 26
3
List of Figures:
Tables:
● Table 1: Absorbance results from Control Device
● Table 2: Absorbance results from Modified Cage Device
● Table 3: Absorbance results from Original Cage Design
● Table 4: Calculations for relative concentrations of carry over solutions
● Table 5: Mass of transferred liquid for each design
Images:
● Image 1 & 2: Above are pictures of the dimensions for solution container (1) and
attachment point (2)
● Image 3: Cage Design from SolidWorks
● Image 4: Modified Cage Design from SolidWorks
● Image 5: Spine Design from SolidWorks
● Image 6: Initial Brainstorming Diagrams
● Image 7: Experimental setup in action
● Image 8: Manual dilutions of dye concentrations
● Image 9: Concentration of Dye vs Absorbance plot
● Image 10: Linearized Concentration of Dye vs Absorbance plot
● Images 11 & 12: show the color distribution between beakers for each prototype
(Modified cage: Left and Original Cage: Right) Even with this lighting, it is clear that
the solutions are much more green on the left.
● Image 13: The ANSI type drawing of the final design of the substrate holder
● Image 14: Different angles of the final design in SolidWorks
● Image15: Rustoleum Never Wet, a hydrophobic coating we found on the internet
● Image16: Rust-oleum High Heat, much cheaper than the Rustoleum online
4
Abstract:
The task was to design a substrate holder that attached to a Varistain 24-4 machine. The
substrate holder needed to hold 6 cell culture disks that are dipped into multiple solutions and
minimize the carryover of solutions as the layer by layer process progresses. The design team
came up with two prototypes and tested them, finding that the cage prototype best fulfilled the
user’s needs and was the final design.
Executive Summary:
The design team met with the client, Dr. Wendy Vanden Berg-Foels, to discuss the task
and solidify the objectives and constraints from the user. The team then worked together to
brainstorm possible ideas for the design of the prototype and narrowed it down to three designs.
The substrate holders were designed using the SolidWorks software to make a virtual prototypes,
which were then 3D printed. The top two prototypes were then tested to observe the amount of
carryover they allowed in a clinical setting. This was measured by dipping the prototype into a
solution of water and very concentrated green food coloring and then into three beakers of water,
mimicking the motion of the Varistain 24-4 machine that the substrate will be used in. Then the
concentration of the green food coloring found in the three beakers of water were measured to
show the carryover. The other measurement of carryover was the weight of the prototype before
the testing, when it was dry, and after the testing, including any water that stayed on the substrate
holder. The two tests revealed that the original cage design had the least carryover, both by
visual observation and from the weight of carryover liquid tests. The measured concentrations of
the final water cups from both prototypes and the control were found to be exactly equivalent.
Therefore, the final design chosen was the cage prototype.
5
Design Problem:
The user is using the Varistain 24-4 machine as an essential tool in her research, but
needs a new way to attach her cell culture slides to the machine so that they can be dipped in
various solutions more efficiently. The team was then giving the task of designing a substrate
holder that can attach to the Varistain 24-4 machine. The holder must meet the following
objectives:
● Be able to withstand sterilization
● Securely attach to the Varistain 24-4
● Be non-reactive in solutions
● Compatible with the existing geometry of the machine
● Able to hold 6 cell culture slides
● Be durable, reusable
● Minimize carryover between solutions
These objectives need to be met by the final design of the substrate holder while being
limited the following constraints:
● 100 dollar budget
● Time management
● Availability of 3D materials to print with
● Availability of testing prototypes
Research:
When designing the prototype, the team looked at how the prototype could be modified
to help expel water from the surface of the holder itself. It was found that the shape of the edges
6
of the prototype would have a large impact on the ability for the water to pool and fall off of the
prototype, instead of being carried over into the next container of solution. One main area of
focus was the shape of the bottom edges of the substrate holder. Originally, the edges were flat
and rectangular in shape. This proved to be a poor choice in design, as the solution does not as
easily roll off this shape, instead liquid pools underneath it. This is because of the solution’s
ability to adhere to the base of the substrate. Water adheres to the surface area it is touching and
the large surface area of the rectangular prototype was an ample spot for water to pool. To
minimize the carryover, the design was modified so the bottom edges came to a point. Instead of
a flat rectangle, an upside down prism formed the bottom edge. This allowed for water to run
down to the point, form drops, then drip off; therefore minimizing the liquid that stays on our
prototype. [1]
Important Constraints:
● At least 6 slots for disks, disk diameter = 22mm, thickness= 100-200µm
● Sample holders dip into specimen cups that have dimensions: diameter = 40mm, height
75 mm, solution fill height = ~40mm
● Ideally we need a non-reactive solution, but since we do not have the time and resources
to find the best material, we are 3D printing and testing the geometric designs
● Withstand sterilization
● 100 dollar budget
● Time
● Must fit in container
● Hold 22mm disks
7
Image 1 & 2: Above are pictures of the dimensions for solution container (1) and attachment point (2)
Alternative Solutions:
Originally we came up with 3 design solutions; the Cage, Modified Cage, and Spine
designs as seen below in Images 3, 4, and 5.
Image 3: Cage Design from SolidWorks
8
Image 4: Modified Cage Design from SolidWorks
Image 5: Spine Design from SolidWorks
9
Preliminary Evaluations of each Design:
Once our team understood the design problem, we were able to brainstorm some methods
of solving this problem. Some of our thoughts can be seen below in Image 6.
Image 6: Initial Brainstorming Diagrams
The Objective tree, Pairwise Comparison Chart, and the Function-Means Tree are put
together to examine possible objectives, means, and functions of possible designs. It has been
10
established that the main objectives for the substrate holder are manufacturing friendliness, user
friendliness, stability, and machine compatible. To achieve these larger goals, several sub-
objectives are defined, such as easy to build, removable, sterile, secure, small, etc. Some of the
key constraints for the holder are a budget of $100, the holder must fit in the solution container,
and it must hold 22mm disks. The pairwise comparison chart ranks the objectives from most
important to least important so that our team can focus on the most essential pieces of our design.
As can be seen above, the ability to hold the disks is the highest priority objective, while cheap
and easy to build are among the least important. Objectives such as sterility, non-reactivity, and
being secure are also among the most important. Several preliminary designs are presented in the
functions means tree. The original thought process was to think outside the box and come up
with a creative solution. This is why certain designs, such as the clips and the bag of disks, are
included in this functions means tree. After further examination, however, it soon became clear
that neither of these options were feasible, given our time and resources. The slotted box and
stack of disks, however, were further developed into some of our alternative design choices. The
cage design and modified cage design were both inspired by the original slotted box concept,
while the spine design evolved from the stack of disks framework. The functions means tree
made it easy to see which designs had potential and which designs were impractical.
Unfortunately, our spine design was unable to be tested due to our misunderstanding of
the resolution of the 3D printer and the fragile components in our design. (More on this in our
PowerPoint presentation)
To test the effectiveness of our prototypes our team ran two experiments. The first test
used the setup below in Image 7.
11
Image 7: Experimental setup in action
In this experiment, one beaker was filled up to 4 cm with a specific concentration of food
coloring. The other three beakers were filled with 4 cm of clear water. Our substrate holder
loaded with disks was placed into the dyed solution for 15 seconds. Then in a steady, constant
motion, the holder was placed in the next beaker for another 15 seconds. This was repeated for
all beakers. A light spectroscopy machine then analyzed the absorbance of each beaker. After
this experiment was complete, the wet substrate holder was then massed. The original mass of
the holder and disk were taken prior to any experimentation. Our results for the analysis of the
Cage and Modified Cage compared to the control can be seen below in Tables 1 to 3.
12
Control Device Absorbance Values
Sample Test Tube Number #
1 2 3 4
Absorbance Value #1
1.263 0.055 0.041 0.039
Absorbance Value #2
1.265 0.057 0.041 0.039
Absorbance Value #3
1.267 0.056 0.041 0.039
AVERAGE VALUE
1.265 0.056 0.041 0.039
Standard Deviation
.002 .001 0 0
Table 1: Absorbance results from Control Device
Modified Cage Absorbance Values
Sample Test Tube Number #
M1 M2 M3 M4
Absorbance Value #1
1.271 0.056 0.042 0.039
Absorbance Value #2
1.272 0.056 0.043 0.039
Absorbance Value #3
1.274 0.055 0.042 0.039
AVERAGE VALUE
1.272 0.056 0.042 0.039
Standard Deviation
.0015 .0006 .0006 0
Table 2: Absorbance results from Modified Cage Device
13
Original Cage Absorbance Values
Sample Test Tube Number #
C1 C2 C3 C4
Absorbance Value #1
1.281 0.052 0.041 0.039
Absorbance Value #2
1.281 0.052 0.041 0.039
Absorbance Value #3
1.283 0.052 0.041 0.039
AVERAGE VALUE
1.282 0.052 0.041 0.039
Standard Deviation
.0012 0 0 0
Table 3: Absorbance results from Original Cage Design
After measurements were recorded, our calculations could be completed. Brandon was
able to create equations for our team to use to make our calculations easier. This was completed
by manually diluting solutions (as seen in Image 8) to known concentrations, plotting them on a
log scale as seen in Image 9 and then linearizing the previous Figure in Image 10.
Image 8: Manual dilutions of dye concentrations
14
Image 9: Concentration of Dye vs Absorbance plot
Image 10: Linearized Concentration of Dye vs Absorbance plot
15
Plugging in our absorbance values for y and solving for x, then taking 10^x, we would be
left with the relative concentrations of each solution after the testing was complete. Table 4
shows these results.
Average concentration of solutions (1 drop food color/ X mL water)
Sample test tube # 1 2 3 4
control 3.015 (mL) 245.628 (mL) 381.41 (mL) 409.305 (mL)
Modified Cage (M) 2.991 (mL) 247.707 (mL) 364.565 (mL) 409.305 (mL)
Cage (C ) 2.96 (mL) 272.712 (mL) 381.41 (mL) 409.305 (mL)Table 4: Calculations for relative concentrations of carry over solutions
The results of the mass part of the experiment can be seen below in Table 5.
Holder Tested Wet Mass (g) Dry Mass (g) Mass of Liquid (g)
Control 14.34274 12.48438 1.85836
Modified Cage 3.69478 3.23574 .45904
Cage 7.26847 6.86771 .40076Table 5: Mass of transferred liquid for each design
Images were also taken during experimentation for visual results as well. These results
can be seen below in Images 11 and 12.
Analysis:
To decide on the final design, we analyzed the results from multiple tests. We began with
the measurements on the concentrations of dye in each beaker between both our designs and the
control. This was done using an absorbance machine by a lab technician, Brandon Mehnert. It
was found that in the final beaker from each of the prototypes and the control had the exact same
concentration of one drop of dye per 409.305 mL of water. This was concerning to us as the final
beakers for our prototypes were very clearly different colors, as shown in the images below. The
16
cage design had a much lighter final beaker than the modified cage design as shown below in
images 11 and 12. It was also concerning that they would all end up at the same concentration
considering the second and third beakers for each design had very different concentrations. The
modified cage design was far more concentrated than the cage design in the second beaker, and
both of our prototypes were less concentrated than the control group. The third beaker had the
modified cage design as the most concentrated solution and the cage design as the least, with the
control in the middle. (This data can be found in table 4) The data from these tests do not seem to
make logical sense, but it seems as though the cage prototype is the best design by this method of
measurement. Although we did use this information when making our final design choice, it was
not a large contributing factor.
Images 11 & 12: show the color distribution between beakers for each prototype (Modified cage: Left and Original
Cage: Right) Even with this lighting, it is clear that the solutions are much more green on the left.
A clear difference between designs can be seen in Table 5 with the variance in mass of
liquid between the designs. The mass of the liquid is how much fluid was left on the prototype
after its final dip in the cleaning solution. The higher the mass of the liquid, the more fluid that
17
was transferred from the final beaker. We can generalize this to how much fluid was carried over
between every beaker. Ideally that number should be as low as possible which means the
unmodified cage design did the best. It beat the modified version by ~.06 g and the control by
~1.4 g. That’s a huge difference against the control for something that weighs less than 10
grams. It’s also a very large volume of fluid. The control carried almost 1 and a half milliliters
more than the unmodified cage. However against the modified cage, it’s almost a negligible
difference. This lack of a significant difference is what inspired the final cage design to be more
like the modified cage from the test. It used less material and was easier to manufacture than the
original and was effective enough by these standards.
Final Decision Choices:
Below, our team incorporated aspects of both the Cage and Modified Cage designs into
our Final Cage design. Material was also minimized just like an aspect of our Spine design.
Image 13: The ANSI type drawing of the final design of the substrate holder
18
Image 14: Different angles of the final design in SolidWorks
Regulatory Issues:
The biggest regulatory issue in our project is to ensure that the final design does not
interfere with the research it will be used in. Dr. Liisa Kuhn is using our device to regenerate
bone tissue. Thus, it is important that our design does not react with the bone cells in any way.
While the plastic that the cage is made out of is highly unreactive, our final design does involve
the use of Krazy glue to cement the pieces together. It is not expected that the glue should ever
contaminate the solutions, however, it is possible. If this glue happens to react poorly with the
bone cells, it could affect the research outcomes. Regulating how much glue and where on the
design the glue is going to be used is a big issue that we have done our best to address. Not
enough testing has been done to determine what effects glue would have on experimentation if it
19
were to contaminate the disks. An examination of the risk of using glue in the final design is
certainly a topic for future work.
Financial Aspects:
Our cost for prototypes can be analyzed below in the following list.
● First time Printing Original Cage: 1 hour 16 min = 1.267 hours → 1.5 hours
● Second time: 21 min + 23 min + 1 hour 48 min = 2.533 hours → 2.75 hours
● $6.84 for Rust-Oleum
● $2.54 Krazy Glue
The 3D printing costs 5 dollars per hour and charges the user in 15 minute increments, so
our first and second times printing cost our team $21.25. Adding that to our Krazy glue and
Rust-oleum purchases, the team spent a total of $30.63. (This does not include cost analysis of
our full prototype being turned in. Another 2 hours is estimated for printing this prototype which
is another $10)
Ethical Issues:
In the design of the substrate holder, our team feels as though there are no ethical issues
surrounding the development of our device. The substrate holder is being used by Dr. Liisa Kuhn
at the Uconn Health Center to further develop bone regeneration. This is an ethically sound
research project that will further modern medicine.
Suggestions for Future Modifications:
For the final design, we printed the cage in three pieces. This was done so that the arms
that connect the substrate holder to the Varistain 24-4 could be properly supported while being
20
3D printed. We then super glued all of the pieces together to finalize our design. Before using the
device, it would have to be tested to ensure that the glue used will not interact with the solutions
used or impact the experiment that the design is being used for. This was not something we could
do as we were not told what exactly the solutions used in the experiment are, as well as we are
not equipped with the knowledge or materials needed to prove that the glue will not react with
them on a molecular level, impacting the ability for the experiment to proceed as the user desires.
We originally did research to look for a hydrophobic coating material to spray over our
substrate holder to reduce the amount of liquid that would stay on the holder. This would in turn
also decrease the amount of carry over solution since less liquid would be transferred from
solution to solution. This would allow for more trials to be done before the testing solution
would have to be replaced and therefore would save the lab money. Searching for a cheap
coating online, we found “Rust-oleum Never Wet” as seen in the image below. This solution
would have to be tested to make sure it is non-reactive with the solutions being used in the
experiment. Also, it is possible that the coating would have to be reapplied between trials, as the
sterilization process may remove some of the coating.
Image15: Rustoleum Never Wet, a hydrophobic coating we found on the internet
21
This would have been perfect for spraying on our substrate holder, but after shipping, the
cost of this was 25 to 30 dollars so we decided that we would go to Walmart and look for a
cheaper solution. After heading there we found a cheaper Rust-oleum in Image 16.
Image16: Rust-oleum High Heat, much cheaper than the Rustoleum online
This might not have been as efficient at repelling water as the Rust-oleum Never Wet but
it withstands high heat. This means that it would hold up to the heat of the cleaning process.
Experiments would need to be done to test the Rust-oleum’s chemical reactance in the various
solutions and cleaning chemicals. Without having the resources to test for chemical stability (or
time to run more experiments), we decided that it would be best to just put this in the future work
section as an idea.
Another modification would be to test multiple materials that the prototype could be
made out of. Our team ran out of time and had a lack of materials to choose from when making
our prototype. In the future, more materials can be tested to make sure they are unreactive and
possibly stand up to the sterilization process better than the PLA material we used to 3D print
22
with. Materials that may work are other plastics that can be 3D printed or machining the
prototype out of different metals.
Conclusion:
In conclusion, we have designed a substrate holder that meets most of the objectives. It is
unknown if the design material will be reactive with the solutions as well as the glue used to
attach the different pieces. This is the only objective not met by the design and cannot be met by
our group at this time. This issue would be addressed in the future before the design is put to use.
The final prototype was modified slightly, as to be able to attach to the Varistain 24-4 machine
and get rid of unneeded material. When compared to the control, both of our design prototypes
performed significantly better, transferring less fluid throughout the dipping process. Of the two
prototypes, the original cage design performed slightly better, transferring the least fluid during
the dipping process. Most of the design elements from the original cage design were
implemented in the final design in order to capture the effectiveness of the original design, while
at the same time enhancing its performance. The final design cut out unnecessary material from
the original cage design which is expected to allow the final design to transfer even less fluid
during the dipping process. Additionally, the original cage design had a capacity of only five
disks. The specification for the project required that the final design have a disk capacity of six.
With this in mind, the final design added an additional slot to meet this design specification.
Lastly, the final design has been engineered to fit the Varistain 24-4 machine. With compatibility
tests to follow, the final design is expected to meet all performance requirements and functions.
23
References:
[1] Goodman, Jeff. "Water Drops: Cohesion and Adhesion of Water." Appstate.edu. N.p.,
2001. Web. 06 Dec. 2015.
Appendix:
The user guide for the Varistain 24-4, this can be used to learn how to load the substrate into the
machine as well as set the machine up to have the specifications needed for each experiment.
http://www.thermoscientific.com/content/dam/tfs/SDG/APD/APD%20Documents/Product
%20Manuals%20&%20Specifications/Histology%20Equipment%20and%20Supplies/Varistain
%20Operator%20Guide%2074210099_08.pdf
Appendix 1: Receipt for Rust-Oleum
24
Appendix 2: Receipt for Krazy Glue
Appendix 3: Attempt at saving the Spine design
25
Appendix 4: Original Cage
Appendix 5: Modified Cage
26
Appendix 6: Original Cage with almost no green dye carry over
Appendix 7: Modified Cage with very visible green carry over. This adds to our confusion about the light
spectroscopy results.