SortoStat/Operation

Materials

• Microfluidic chemostat chip
• Lysis Buffer
  • prepare lysis buffer by adding 10mg/ml Lysozyme to Pierce B-per Reagent.
• Media (+ antibiotic if necessary) (+BSA 2mg/ml)

Both lysis buffer and media should be filter sterilized to remove any debris which might clog the channels of the chip.

Setup the device

If the tubing and pins have not yet been attached to the chip then start here first.

Prime the control channels

1. Ensure that the valve leading to the 3-way split is in the off position so that no pressure is getting to microfluidic tubes.
2. Disconnect the large tube leading from the top pressure nozzle to the media/lysis buffer jars, cover with parafilm, and turn the valve counter-clockwise until it stops. (OFF position)
3. Load the tubes leading to the control channels with sterile h20
   • Seem to always get some growth in the control channel anyway, don't suspect that it seriously affects valve performance.
   • If you have recently run the chip many of the control tubes will already be full (not sure how they leak, but they do), so to save time you can pressurize the control tubes and then visually inspect the chip to see which control valves are not filled with water. Then can selectively fill those rather than doing all 32.
   • May also consider just leaving pressure on the control lines if you have <2-3 days between experiments, the loss of gas will be worth your time.
4. Place the chip on the stage and turn the valve before the 3-way split to the ON position.
   • BE SURE that the regulator leading to the media/lysis jars is off and disconnected, otherwise you are going to start spraying media around.
5. Close all the valves and leave the chip for ~15 minutes to ensure that all the control channels have filled with water.
6. Visually confirm that the control channels are filled with water and open and close each channel individually to confirm function.
   • (may want to make this a feature in labview, so you can just watch while it opens and closes everything)

Prime the media and lysis channels

1. Insert tubes into the lysis buffer and media bottles and then insert the pin into the appropriate inlet channels
   • Lysis buffer is typically placed in the left input channel
     • be sure to update the 'experimental parameters' array if the lysis channel is changed.
   • Be sure to define the media inlet in Labview
2. Insert the 2 waste tubes (center and lower) and place the ends in the microfluidic waste jar.
3. Hook up the large tubes to the media and lysis jars and then turn on the pressure. Open appropriate valves to create a clear path between the inlet and the outlet in order to prime the media and lysis tubes. (should take about 1 min to prime the tube).
4. Clean out the chemostat with lysis buffer and then media. (particularly if re-using the chip)

Load the cells into the chemostat

1. Spin down cells at 2000rpm for 2 minutes and then suck up some of the suppernatant by attaching a small tube with a pin to a syrinnge.
   • This helps reduce the likelihood that a large piece of debris clogs one of the channels.
   • Use a long tube since it helps to be able to sit at the computer and hold the syringe.
2. Close all valves - once you insert the cells tube be very careful of contaminatoin of the fluid inlets.
3. Remove the center waste tube and insert the cells tube there
4. Open channels leading from the center waste channel to the lower waste channel and going all the way around the reactor ring.
   • Need to write a button into labview to open the appropriate channels automatically
5. Depress the syringe and you should see a blast of air clear out the reaction chamber followed by the cells (which may be moving to quickly to be visible).
   • NOTE: This is the most likely place to get contamination in the media feed line (which renders that port useless as a future media inlet - although it can be used for lysis buffer). To avoid contamination, DO NOT press heavily on the syringe - you should need very little pressure to insert the cells.
6. Seal the exit channel then the entrance channel to the reactor ring to pressurize the ring. You should be able to see the cells clearly now.
7. When removing the cells tube detach the syringe first so that there wont be pressure behind the tube, this keeps the cells from spraying out when you pull out the pin.
8. Double check the media inlet for contamination.
9. Clear the outer ring with lysis buffer.
10. Flow media through outer ring for ~5min in case of accidental, undetected contamination.

Test the device operation

1. Pump
   • Test if the cells move well in response to the pump
2. Clean chamber event (on the chamber with the pumps - chamber 8)
   • Anecdotally, if the pumps are not working well this seems to help, why?
     • should try just cleaning a random chamber to see if it has same effect, might be a pressure issue, since the channel is higly pressurized following the inputting of cells.
3. Screening event
   • confirm that the images in the 2 debug windows show up (both FL and BF). If they don't try restarting the camera. Also in the device section of 'my computer' in windows you can check if the camera is working properly.
   • If you are getting a "another program is using this device error" when you click on camera in My Computer, you may wnt to stop the LabView VI (don't close entire program!) you are currently running and try again.
4. Sorting event
   • There is a known issue with the reliability of he sorting event. When the CW and CCW valves are closed the increased pressure in the reactor ring can burst one or several of the Exit channel valves.
     • First, close the CW and CCW valves and then check the chip to see if any of the exit valves burst. Then test the automated procedure as decribed below.
   • be sure the correct chemostat has been selected so the appropriate 'specal valve' will be opened
   • check that no cells escape when all the valves around the sorting chamber are closed. Sometimes if the push-up pressure is too great (or push-down is too low) the special valve will be forced open and some cells will exit to the feeder channel.

Operate the device

1. Prepare lysis buffer by adding 10mg/ml Lysozyme to Pierce B-per Reagent.
2. Spin down an overnight culture of cells at 2000rpm for 2 minutes to bring down large clumps of debris. The cells in the supernatant (which should still be cloudy) will serve as the cells to innoculate the device.
3. Test the sort and screen, etc.
4. What other tests should be run?

• Turn MatLab on first

Operating Parameters

These will vary somewhat by chip, however have had success with

• fluid: 4.5psi
• push-down: 15psi
• push-up: 16psi

Feeder Flow Settings

Feeder Fill Period: 10s Wash Period: 10s Clean Period: 15s Pressurize Period: 2s

Labview Settings

• Be sure to set the chemostat position.

Shutdown

Chemostat chambers can be re-used if cleaned properly. (Would like this to be automated in labview as well at somepoint) Microfluidic chemostat labview program

1. Flow lysis buffer through the entire chemostat
   • Make sure to clear out any cell debris, flow for ~5min through the ring of the chemostat, as well as each individual entrance and exit to the ring. (really needs to be automated)
2. Flow lysis buffer into the media tube by removing the media tube from its back pressure source first.
3. Remove the lysis buffer inlet tube and the media tube and replace one with an empty tube with a pressure source.
4. Drive air through the system and clean out the entire chip.

Notes

Jen & Caitlin have been running their chemostat at a dilution rate of .75 and reaching ODs of 1-1.6. This would be roughly 10K cells in the chip, i think. Haven't actually gotten a steady-state counted yet to measure what we are actually seeing.

Contact: Jason Kelly