Low-Cost, Controllable Hypoxia Chamber

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[[File:[[File:|thumb|300px|left]]|300px|center|alt=]]
Sponsors Dr. Nate Schiele
Team Name HYPEngineers
Duration 2020 - 2021
Faculty Advisor Dr. Dev Shrestha
Mentor Ian Glasgow
Client Dr. Nate Schiele
Team Members
  • Andrew Hartman
  • Jacob Knudson
  • Colin Marchus
  • Alexandra Morrison
  • Isabell Strawn




Problem Definition[edit]

The purpose of this project is to design and manufacture a low-cost, controllable hypoxia chamber for stem cell research.

Background[edit]

A crucial component to conducting stem cell research is maintaining a hypoxic growing environment. Stem cells naturally grow in a low-oxygen environment and simulating this environment during experiments allows researchers to more efficiently cultivate stem cells and conduct research regarding the effect of low-oxygen environments on the cells’ development. A hypoxic environment can be simulated by using cobalt chloride, but this has undesired side effects on the cells’ health. A more realistic and effective solution is to cultivate the cells in a hypoxic chamber. However, current commercial hypoxia chambers are expensive. The objective of this project is to develop a functional, simple, and low-cost hypoxia chamber for stem cell research. The results of this project will include the hypoxia chamber design and physical product as well as a published paper detailing our design and construction process. This will allow other researchers to utilize our technology and ideas.


Deliverables[edit]

The hypoxia chamber must:

  • Be designed to fit within an incubator.
  • Be capable of maintaining a hypoxic environment for a minimum period of 21 days.
  • Be made of components that can withstand being autoclaved or sterilized with a solution.
  • Have a user interface that allows monitoring and control of O2 and CO2 levels.


Specifications[edit]

Chamber specifications:

  • Must be capable of controlling oxygen levels between 1% and 21%.
  • Must be capable of maintaining carbon dioxide level at 5%.
  • Control system for chamber will have an operational voltage of 5V.
  • Chamber components must withstand temperatures of up to 50 degrees Celsius and a humidity level of 85-95%.
  • At least four 13cm x 9cm x 2.5cm well multidishes must fit inside.
  • The chamber should be easily portable.
  • When closed, it should be airtight for ease of maintaining hypoxic environment.
  • Final product should be transparent so that well multidishes are visible.
  • Total cost of chamber and manufacturing must not exceed $1000.


Design Considerations[edit]

When designing the chamber, the following must be taken into consideration:

  • Materials used must not affect culturing of stem cells.
  • Door opening must be large enough to move well multidishes in & out safely
  • Pressure must not be able to build up within chamber

Design Development[edit]

Chamber From Scratch[edit]

Initial Design Idea:
Figure Description
Hypoxia initial.png
  • This was the first chamber design that was made.
  • No idea for a door had been determined yet.
  • Planning on using acrylic for the chamber walls, so that the inside of the chamber is visible.
  • Corner support pieces will be 3D printed.
Hypoxia initial 2.png
  • Chamber dimensions for this design, measured from corner to corner (L x W x H): 9.68 in. x 6.81 in. x 5.812 in.
  • Chamber walls are 0.25 in. thick.
Updated Design Idea:
Figure Description
Hypoxia updated.png
  • Increased the overall dimensions of the chamber.
  • Added a door with an o-ring seal for airtightness.
  • Some holes will be added to the left side of the chamber for gas input/output and sensor wiring.
Hypoxia updated 2.png
  • Chamber dimensions for this design, measured from corner to corner (L x W x H): 12.25 in. x 10.02 in. x 10.74 in.
  • The door & front panel of chamber thickness was increased to 0.5 in. for manufacturing purposes.
Initial Shelf Design:
Figure Description
Hypoxia shelf initial.png
  • This shelf will have a tray of water beneath it to supply humidity to the chamber.
  • The dimensions for this shelf design (L x W X H): 4 in. x 9 in. x 3.25 in.
Shelf Design Updated:
Figure Description
Hypoxia shelf updated.png
  • The shelf length was increased to give greater surface area for tray of water, and height was reduced so that it takes up less space.
  • Supports will be fixed to the inside of the chamber, while the shelf pieces themselves will be removeable to access the tray of water.
  • The shelf pieces will have small holes/slits in them to allow for circulation of the humidity.
  • The dimensions for this shelf design (L x W x H): 10.65 in. x 8.8 in. x 1.25 in.
Shelf inside chamber.png
  • This is showing the shelf placed into the updated chamber design.
  • The shelf now runs almost the entirety of the bottom of the chamber.

Pre-built Chamber[edit]

Pre-built Chamber:
Figure Description
Pre-built chamber.PNG
  • In this design, a pre-made, airtight container will be purchased and modified to fit our needs.
  • A much cheaper option than designing and manufacturing a chamber from scratch.
Premade chamber final.png
  • This is the initial design for the premade chamber option.
  • The support stilts and the humidity shelf supports will be 3D printed.
  • The humidity shelf will be made from sheet metal.
  • Sensors & housing will be mounted in the back left upper corner.
[[File:|border|450px|center]]
  • This is the finalized version of the pre-made chamber design.
  • In this design, the chamber was turned right side up so that the lid would be easier to put on and take off.

Control System[edit]

Sensor Housing:
Figure Description
Sensor housing initial.jpg
  • This was the first model for the sensor. It is designed to be form fit around the sensors.
  • The top will have threads for a filter cap to isolate the sensors because they can not be cleaned using ethanol
  • The fitting is split into two pieces to be able to insert the sensors and wire them and then combine it together.
  • This was intended to be attached to the outside of the chamber with the filter cap inside.
Sensor housing updated.jpg
  • This was the 2nd model of the sensor housing after realizing we needed a PCB for the CO2 sensor.
  • The housing has mounting points for the board as well as a holder for the O2 sensor that allows access to the wiring.
  • The filter cap portion remained the same, except that now the whole housing is desinged to fit inside the chamber.
  • The exhaust port for the hypoxia chamber was moved into the sensor housing to force airflow over the sensors
SensorHousingV4.jpg
  • The 3rd model for the sensor housing was very similar to the 2nd model, however, it was modified to be more workable.
  • The bottom portion that the sensors attach to is now removable that way the upper portion can be attached to the Hypoxia chamber.
  • The exhaust whole was shaped to fit around the bulkhead air fitting to get a tighter seal.
  • The wires are designed to come out of the bottom of the housing using an RJ45 Adapter so when the bottom plate is removed they do not get left inside the housing.
Sensors in chamber.png
  • This shows the sensors and housing mounted into the upper corner of the hypoxia chamber.
[[File:|border|450px|center]]
  • This is the final design for the sensor housing.
Control System Circuit:
Figure Description
Breadboard view circuit 2.png
  • On the left side, solenoids are connected to diodes, BJT transistors, and resistors, and then finally to the Arduino.
  • These solenoids control the gas input/output tubes.
  • The diodes ensure that the solenoids don't send a current spike back through the circuit when they turn off.
  • The transistors ensure the Arduino can control the solenoids, and the resistors limit the current so that the transistors don't burn up.
  • On the right side, the oxygen (blue) and CO2 (green/black) sensors are connected through an adapter board to the Arduino.
  • Both sensors operate at 3.3V for their serial (Tx/Rx) communications, while the Arduino operates at 5V. The adapter board allows this communication to function by level shifting the signals.
Schematic view circuit 2.png
  • A basic level schematic of the circuit.
  • The Arduino is seen on the right, the solenoids and their components are in the middle of the schematic, and the sensors and adapter board are on the bottom right.

Prototypes[edit]

Chamber From Scratch[edit]

Initial Chamber Prototype:
Figure Description
Hypoxia initial prototype.jpg
  • This prototype is of the initial chamber design.
  • This was made for the first snapshot.
Hypoxia initial prototype 2.jpg
  • This prototype was made out of laser cut wood and wood glue.
  • This prototype is solely for displaying our initial design idea for the first snapshot.
3D printed corner piece.jpg
  • This is one of the 3D printed corner pieces.
  • The purpose of these pieces is to provide support and structural stability to the chamber.
Manufacturing Acrylic Chamber Prototype:
Figure Description
Acrlic chamber manufacturing.jpg
  • The manufacturing process for this chamber prototype began with laser cutting the acrylic pieces.
  • This image shows the acrylic inside the laser cutting machine.
Acrylic crazing.jpg
  • After the acrylic was laser cut we attempted to clean the acrylic with ethanol, the solvent which will be used for sterilization in Dr. Schiele's lab.
  • Upon applying the ethanol to the acrylic, however, we discovered that ethanol causes crazing to occur within the acrylic which can impact the structural stability.
  • Due to this unforeseen issue, we decided it was in out best interest to stop working on the custom chamber design option for the time being and focus our efforts on the pre-made chamber design.

Pre-built Chamber[edit]

Manufacturing Prebuilt Chamber Prototype:
Figure Description
Prebuilt chamber manufacturing.jpg
  • The manufacturing process for this chamber prototype began drilling holes for the through-wall fittings.
  • After drilling the holes, the through-wall fittings were inserted to ensure the holes were made properly.
  • This image shows chamber with the through-wall fittings in place, as well as the humidity tray, shelf supports, and a well plate inside.

Control System[edit]

Validation[edit]

Sensor Testing[edit]

CO2 and O2 Sensor Tests:
Figure Description
O2-CO2 candle test.JPG
  • This is a snippet of the results from the O2 & CO2 sensors test.
  • Test showed that the sensors are working properly and giving good readings.
  • A candle was burned near the sensors to reduce O2 and increase CO2 and the sensors measured the change.
Pressure Tests:
Figure Description
Pressure test setup.jpg
  • This test was to determine whether the chamber has any spots that leak air.
  • Soapy water was rubber near any areas with potential to leak and the chamber was pressurized.
  • Any bubbles formed would indicate an air leak.
  • Subsequent tests were ran to determine the airflow out of the exhaust through-wall fitting as well as the filter cap which will cover it.
Pressure test.JPG
  • This is the results of the pressure tests.
  • Tests results showed that no through-wall fittings had air leaks.
  • However, the pressure buildup in the chamber caused the lid to raise slightly and slowly release the pressure.
  • Since the chamber will be operating at a pressure equilibrium, the lid should stay airtight and not have any leaks.
  • The tests to determine airflow through the filter cap showed there is little/no impedance to the airflow.
Nitrogen & O2 Test:
Figure Description
Nitrogen O2 test.JPG
  • The purpose of this test was to attempt to reach an O2 level of 5% using purely nitrogen.
  • As the test was running, some edits were made to the control system code to change how long the solenoids were releasing gas into the chamber.
  • The results showed that the control system was able to reach the 5% O2 setpoint within about 1hr10mins.

Humidity Tray Volume Analysis[edit]

Volume ratio analysis:
Figure Description
Tray vol analysis.PNG
  • To determine whether or not the humidity tray we chose for our chamber is sufficient, the ratio of our chosen humidity tray volume to the chamber volume was compared to the ratio of the incubator humidity tray volume and the incubator volume.
  • Analysis results showed that the humidity tray chosen for our chamber will supply a sufficient amount of humidity to the chamber.

Team Members[edit]

Andrew Hartman.jpg
Andrew Hartman

Major: Electrical Engineering
Hometown:
Responsibility:
Email: hart0740@vandals.uidaho.edu




[[File: |thumb|left]] Jacob Knudson

Major: Biological Engineering
Hometown: Coeur d'Alene, Idaho
Responsibility: Team Documentation, Project Wiki Master
Email: knud1426@vandals.uidaho.edu




Colin HYPE.jpg
Colin Marchus

Major: Biological Engineering
Hometown: Coeur d'Alene, Idaho
Responsibility: Agendas, Project Paper
Email: marc8355@vandals.uidaho.edu




[[File: |thumb|left]] Alexandra Morrison

Major: Electrical Engineering
Hometown: Yakima, Washington
Responsibility: Meeting Minutes
Email: alex9269@vandals.uidaho.edu




[[File:|180px|thumb|left]] Isabell Strawn

Major: Biological Engineering
Hometown:
Responsibility:
Email: stra1551@vandals.uidaho.edu





Additional Documentation[edit]

Budget

File:Hypoxia chamber budget.pdf

Client Interview

[[File:]]

Design Validation Plan

File:Chamber Design Validation Plan.pdf

GitHub Repo

GitHub Repo

Meeting Minutes

File:Meeting Minutes Sept 2020.pdf

File:Meeting Minutes Oct 2020.pdf

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Presentations

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Project Schedule

File:Hypoxia project schedule.pdf