Difference between revisions of "Microfluidics"

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(In Giæver lab 420)
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* Stop all flow until cells have adhered to surface
 
* Stop all flow until cells have adhered to surface
 
* Start slow flow of media through chip
 
* Start slow flow of media through chip
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 +
= Controlling O2 and CO2 concentration during experiment =
 +
This is still on the planning stage. Check out [https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Controlling_O2_and_CO2_level planning notes here]].
 +
 +
= Measuring CO2 in the cell media =
 +
Here we have not started the planning notes. Testing the electronics is the next step.

Revision as of 14:25, 6 April 2022

Designing Microfluidic masks

  • Stanford microfluidics foundry has a good guide for designing your own device. That includes a guide to use AutoCAD.
  • AutoCAD (a program from Autodesk) is available free.
Some microfluidic circuit designs for a 3" wafer
  • You can use Klayout but we have more support for using Autocad.
  • Mask template for placing your designs
  • This guide for designing masks has some important tips
    • All fluid pathways have to be inside one or more closed contour(s)
    • The outer contour should be drawn in one layer (Give it a name like "Flow")
    • Any obstacles inside this outer contour must be drawn in another layer. Give it a name like "Flow inner polygons")
  • Displaying your design as PDF or otherwise is not straight forward because the resolution needed. You get a fair impression using CloudConvert which is much better than AutoCads own pdf export.
  • Example of a design where white lines (in screen shot below, black lines in PDF) are contours containing flow and green lines are inner polygons. Here is a PDF version of the file and the DWG file.
  • Rounding corners on a region
    • Command EXPLODE makes region into lines
    • Command JOIN joins lines into polylines
    • Command FILLET, downarrow to get options RADIUS and POLYLINE

Photolithography

Photolithography procedures

PDMS casting

  • PDMS preparation
  • Bring cup with PDMS close to wafer in Petri dish
  • Pour slowly and avoid introducing air bubbles
  • Remove air bubbles by puncturing them
  • Fill petri dish to 5-10 mm above wafer surface

Assembling the chip

  • Open Wiki lab notebook on computer to make notes of observations and deviations from this protocol
  • Clean glass slide with IP and N2
  • Punch holes for fluid inlets
    • For 1/16" tubes use OD?? punch
    • For thin teflon tube use OD?? punch
  • Place both PDMS and glass slide with clean sides up on glass plate in plasma cleaner
  • Follow instructions for Plasma treatment
  • Place the PDMS on top of the glass slide and watch it attach
  • If necessary, apply a gentle pressure to the PDMS

Filling the chip

  • Open Wiki lab notebook on computer to make notes of observations and deviations from this protocol

For sterilizing and filling the chip with water and fibronectin you need

  • 1ml syringe with
  • 20G needle and
  • 2cm long 1/16" teflon tube on the needle.
  • small beaker
  • ethanol
  • distilled water
  • Eppendorf tube of fibronectin
  • Cells in fresh media
  • Inlet and outlet tubes
    • Measure lengths of inlet and outlet tubes and cut
    • Sterilize the tubes

Sterilizing

  • If you want a sterile chip move each object into LAF bench once they have been sterilized
  • Spray chip with ethanol and rub off glass side
  • Assemble syringe, needle and teflon tube
  • Spray ethanol and rub outside
  • Rinse beaker in ethanol and pour in a small amount of ethanol
  • Retract syringe half way in air, and fill teflon tube with ethanol
  • Empty syringe of ethanol and air, make sure it is dry
  • Empty beaker of ethanol

Water filling

  • Fill a small amount of distilled water in beaker
  • Retract <1ml distilled water into tube/needle/syringe
  • Fit in inlet of chip and fill slowly
  • Empty syringe and needle to be ready to use for fibronectin

Fibronectin filling

  • Take one Eppendorf with 0.5 ml fibronectin form freezer
  • Thaw the fibronectin
  • Retract fibronectin into tube/needle/syringe
  • fit in inlet of chip and fill slowly
  • Attach inlet and outlet tubes
  • Incubate for 30 min in incubator
  • Rinse with PBS
    • Pour a few ml PBS in sterile beaker
    • Fill sterile 1 ml syringe+needle with PBS
    • Fit correct length of (sterile) inlet tube and outlet tube to chip
    • Slowly flow PBS through chip

Cell filling

In cell lab 431

  • Open Wiki lab notebook on computer to make notes of observations and deviations from this protocol
  • Follow protocol for [cell passaging]
  • Transfer the some of cell suspension to a sterile centrifuge tube of appropriate size (Eppendorf or other depending on volume)
  • Centrifuge for 10 minutes at 800 × g.
  • Note: Certain cell lines are very sensitive to centrifugal force.
  • Carefully remove the supernatant without disturbing the cell pellet.
  • Add the desired volume of fresh medium gently to the side of the tube and slowly pipette up and down 2 to 3 times to resuspend the cell pellet.
  • Transfer the cells to a sterile syringe or sample holder
  • Fill another syringe or sample holder with fresh media

In Giaver lab 420

You now need to have

  • Wiki lab notebook open to make notes of observations and deviations from this protocol
  • Computer with microscope and camera ready
    • turn on computer, microscope and start flycap
    • test imaging on an old chip (start with 4x, turning the objective turret CCW: 4X - 10X - 20X - 40X)
  • 2 syringe pumps or pressure controller ready
    • Syringe pumps: adjust syringe size and flow rates to 1-60 ul/min
    • Pressure controller: set initial pressure to zero on both channels
  • The sterile chip
    • with fibronectin on the glass
    • filled with PBS
    • inlet and outlet tubes connected
  • A syringe or sample holder with cells suspended in fresh media
    • If possible: bubble correct air/CO2 mixture through media before starting

Now it's time to fit everything together on top of the microscope

  • Place the chip on the microscope, fasten with gum and focus on area by inlet
  • Connect syringe pumps or pressure regulator:
    • Syringe pumps: Place syringes in syringe pumps and test that they engage
    • Pressure regulator: Connect air tubes from regulator to sample holders
  • Connect sample holders/syringes to the chip via the T-connector (see figure)
  • Connect output tube to a waste container
  • Press media through T-connector, chip and to cell syringe/sample holder until all air bubbles have been removed
  • Stop flow from media container
Injection of cells and media using pressure control or syringes

Running experiment

  • Start slow flow from cell container to chip
  • Follow images. Stop flow now and then to check if cells have reached chip
  • Start recording image sequence to D:\Microfluidic\2022...\
  • Stop all flow until cells have adhered to surface
  • Start slow flow of media through chip

Controlling O2 and CO2 concentration during experiment

This is still on the planning stage. Check out planning notes here].

Measuring CO2 in the cell media

Here we have not started the planning notes. Testing the electronics is the next step.