(up to date information on perfusion system with bioreactor focus and neurons added) |
(Updated information about trials that were ran August 3rd and 4th) |
||
Line 408: | Line 408: | ||
<li>Placed in incubator</li> | <li>Placed in incubator</li> | ||
</ul> | </ul> | ||
+ | |||
+ | 3 August 2017<br> WO RG | ||
+ | <ul> | ||
+ | <li>Ran first trial: recorded impedance, started recording voltage at 1,000Hz and injected 0.5mL of 2mM 1-hexanol olfactant, waited 7 minutes and inject 0.5mL more of olfactant, waited 7 minutes, stopped recording, pumped in 1mL of fresh media</li> | ||
+ | <li>Bioreactor and reservoirs were placed back in incubator</li> | ||
+ | </ul> | ||
+ | |||
+ | 4 August 2017<br>WO RG | ||
+ | <ul> | ||
+ | <li>Ran two trials:recorded impedance, started recording voltage at 20,000Hz and injected 0.5mL of 2mM 1-hexanol olfactant, after a total of 1 minute stopped recording, pumped out olfactant with 1mL of fresh media</li> | ||
+ | <li>ran an additional trial without recording voltage reading data, analyzed data for a total of 10 minutes</li> | ||
+ | <li>ran an additional trial without recording voltage reading data injecting 20mM of olfactant, analyzed data for a total of 10 minutes</li> | ||
+ | </ul> | ||
<a name="Programming and Electronics"></a> <br> | <a name="Programming and Electronics"></a> <br> | ||
<h3>Programming and Electronics (Bioreactor focus)</h3> | <h3>Programming and Electronics (Bioreactor focus)</h3> | ||
Line 454: | Line 467: | ||
<li>Tested program that ran impedance tests and voltage readings</li> | <li>Tested program that ran impedance tests and voltage readings</li> | ||
<li>Soldered more electrode adapters</li> | <li>Soldered more electrode adapters</li> | ||
+ | </ul> | ||
+ | |||
+ | 3 August 2017<br>WO RG | ||
+ | <ul> | ||
+ | <li>Soldered pins to electrode adapter board ensuring to have pins on the ground and reference channels on adapter board</li> | ||
+ | </ul> | ||
+ | |||
+ | 4 August 2017<br>WO RG | ||
+ | <ul> | ||
+ | <li>Set voltage reading program to read at 20,000Hz</li> | ||
</ul> | </ul> | ||
</html> | </html> |
Revision as of 19:57, 4 August 2017
USMA-West_Point
Notebook
Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.
What should this page have?
- Chronological notes of what your team is doing.
- Brief descriptions of daily important events.
- Pictures of your progress.
- Mention who participated in what task.
Inspiration
You can see what others teams have done to organize their notes:
- AM - Angela Marsh
- JG - Dr. Joel Gaston
- KP - Kanak Patel
- MM - Matthew McDonough
- PB - Payton Boylston
- RG - Rayonna Gordon
- WO - Winston Ou
Perfusion System with Bioreactor Design
Programming and Electronics (Bioreactor focus)
Neurons
9 Jun 2017WO RG AM KP MM
- Received HT-22 Immortalized Mouse Hippocampal Cell Line and related materials from West Point
WO RG AM KP MM PB
- Unfroze neurons
- Plated neurons for amplification
WO RG AM KP MM PB
- Change media
JG
- Changed media
WO RG MM PB
- Split cells - Passage 1 from receipt of neurons
WO RG MM PB
- Changed media
- Froze excess cells
JG
- Changed media
WO RG
- Made and changed media
WO RG
- Thawed and plated cells - Passage 2
WO RG
- Split cells - Passage 3
WO RG
- Changed media
WO RG
- Split cells - Passage 4
WO RG
- Changed media
JG
- Cells were killed
WO RG
- Plated Passage 2 HT22 cells that were previously frozen into a 6 well plate
WO JG
- Coated MEA with polylysine
- Seeded 20,000 HT22 cells onto coated MEA
- Incubated said cells
WO RG
- Transfected cells on MEA with 3200ng of OR2W1
- Incubated transfected cells on MEA in the bioreactor
Perfusion System with Bioreactor Design
23 May 2017WO
- Began bioreactor design based on former models to be used in a perfusion system
WO
- Used AutoDesk Inventor to design a general bioreactor
- Incorporated shape and general items such as holes for screws and cavity for neuronal culturing
- Determined location of a multielectrode array (MEA) in bioreactor design, where the neurons would be transfected and tested
WO
- Adjusted design of bioreactor design
- Began a custom design for a MEA
WO RG AM KP
- Continued MEA designing: location of electrodes, leads, contact pad spacing, materials
WO RM AM KP
- MEA
- Altered the electrode arrangement and size for a single well of a 96 well plate
- Predetermined specifications: Gold or TiN wires, embedded in glass
WO RG AM KP MM
- Determined custom MEA design specs, particularly spacing
WO RG AM KP MM
- Revised MEA spacing
WO RG AM KP MM
- Discussed the location of holes for media flow and a bubble trap
WO RG AM KP MM
- Fleshed out design for bubble trap
- Began design for clamp to keep pins on the multielectrode array (extension of bioreactor) - KP
WO RG AM KP MM
- Began discussion on removal of olfactant contaminated media while maintaining a perfusion system via syringes
- Continued clamp design-KP
- Requested a quote for custom MEA
WO RG AM KP MM
- Confirmed the use of a peristaltic pump for media flow
- Finalized MEA design
WO RG AM KP MM PB
- Contacted other MEA fabrication companies for quotes
WO RG AM KP MM PB
- Continued search of MEA vendors
- Worked on specifics regarding flow into and out of the bioreactor/media reservoirs - decided to use luer locks
WO RG AM KP MM PB
- Continued contacting MEA vendors
WO RG AM KP MM PB
- Determined best luer lock connectors for tubing
- Finished printing of clamp design
- Explored photomasking vendors
WO RG AM KP MM PB
- Determined design of media reservoirs to be conical flasks
- Explored pre-made MEA vendors
WO RG MM PB
- Determined design of media reservoirs to be conical flasks
- Explored pre-made MEA vendors
WO RG MM PB
- Ordered MEAs from Multichannel Systems
- Researched on systems to control the peristaltic pump from the computer
WO RG MM PB
- Find tubing for bioreactor and pump
WO RG MM PB
- Designed media reservoirs in Inventor, obtaining a cubic appearance, as opposed to the original conical tube appearance
WO RG MM PB
- Ordered tubing
WO RG MM PB
- Redesigned clamp system for pins using a platform and screws system
- Revised bioreactor design to have internal channels in one side of the bioreactor
WO RG MM PB
- Inserted a conical steeple for the bubble trap
- Made minor modifications to bioreactor design
WO RG
- Inserted threading into media reservoirs
- Made measurements of ordered o-rings to be fitted
WO RG
- Printed out models to help test fittings for the O-ring
WO RG
- Printed array to narrow down sizing for O-Ring
- Modified media reservoir due to space constraints
- Printed model MEAs to test pin setup and general fittings
WO RG
- Modified media reservoir for better fluid flow
WO RG
- Printed first part of bioreactor
WO RG
- Adjusted dimensions and reprinted first part of bioreactor
WO RG
- Adjusted dimensions and printed a third version of the first part of bioreactor
WO RG
- Printed array to determine necessary dimensions to fit luer locks
WO RG
- Applied dimension changes to bioreactor design and reprinted the first part of bioreactor
WO RG
- Determined screw size and lengths needed to apply appropriate pressure on pins
WO RG
- Printed out top and bottom of bioreactor and clamp
- Perforated MEA Bioreactor modified
- Verified operation of the pump
- Tested calibration of pump
WO RG
- Printed bioreactor top in Dental SG and media reservoir in clear
- Tested perfusion system with bioreactor and MEA to test for leakage and overall flow
WO RG
- Printed bioreactor bottom in Dental SG and waste reservoir in clear
- Revised bioreactor design for better hole sizing and placement
- Tested perfusion system with bioreactor, media reservoir, and MEA to test for leakage and overall flow
- Redesigned reservoir for more efficient drainage
WO RG
- Printed bioreactor top in Dental SG
- Tested perfusion system with all components to test for leakage and overall flow
WO RG
- Printed both reservoirs in Dental SG
- Tested perfusion system with Dental SG bioreactor top/bottom and MEA to test for leakage and overall flow
- Cured bioreactor pieces in UV
WO RG
- Performed dry run with cured pieces
- Autoclaved epoxy-containing piece
WO RG
- Autoclaved entire perfusion/bioreactor system
WO RG
- Enclosed MEA with transfected cells in between top and bottom piece of bioreactor
- Placed in incubator
WO RG
- Ran first trial: recorded impedance, started recording voltage at 1,000Hz and injected 0.5mL of 2mM 1-hexanol olfactant, waited 7 minutes and inject 0.5mL more of olfactant, waited 7 minutes, stopped recording, pumped in 1mL of fresh media
- Bioreactor and reservoirs were placed back in incubator
WO RG
- Ran two trials:recorded impedance, started recording voltage at 20,000Hz and injected 0.5mL of 2mM 1-hexanol olfactant, after a total of 1 minute stopped recording, pumped out olfactant with 1mL of fresh media
- ran an additional trial without recording voltage reading data, analyzed data for a total of 10 minutes
- ran an additional trial without recording voltage reading data injecting 20mM of olfactant, analyzed data for a total of 10 minutes
Programming and Electronics (Bioreactor focus)
23 May 2017WO
- Introduced to labview - use to take voltage readings from electrodes
- Determined to set peak threshold to +/- 2 SD of base activity
WO
- Built time-averaging method to determine frequency and amplitude of peaks
- Generated preliminary statistics for data
- Built code to write to a file
WO
- Inserted method to adjust thresholds before data collection
WO
- Adjusted labview program to read in data from electrodes using Intan codes (as opposed to theoretical inputs)
- Finished labview program - can select filename/type
WO
- Implemented impedance program from Intan to determine the presence of cells on electrodes
WO RG
- Soldered pins to electrode adapter boards
WO RG
- Tested program that ran impedance tests and voltage readings
- Soldered more electrode adapters
WO RG
- Soldered pins to electrode adapter board ensuring to have pins on the ground and reference channels on adapter board
WO RG
- Set voltage reading program to read at 20,000Hz