Difference between revisions of "Microfluidics"

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(Controlling O2 and CO2 concentration during experiment)
 
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= Assembling the chip =
 
= Assembling the chip =
* Open [[https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 Wiki lab notebook] on computer to make notes of observations and deviations from this protocol
+
* Open [https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 Wiki lab notebook] on computer to make notes of observations and deviations from this protocol
 
* Clean glass slide with IP and N2
 
* Clean glass slide with IP and N2
 
* Punch holes for fluid inlets
 
* Punch holes for fluid inlets
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= Filling the chip =
 
= Filling the chip =
* Open [[https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 Wiki lab notebook] on computer to make notes of observations and deviations from this protocol
+
* Open [https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 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  
 
For sterilizing and filling the chip with water and fibronectin you need  
 
* 1ml syringe with  
 
* 1ml syringe with  
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=== In cell lab 431 ===
 
=== In cell lab 431 ===
* Open [[https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 Wiki lab notebook] on computer to make notes of observations and deviations from this protocol
+
* Open [https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 Wiki lab notebook] on computer to make notes of observations and deviations from this protocol
 
* Follow protocol for [[https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Cell_lab_431#Cell_culturing cell passaging]]
 
* Follow protocol for [[https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Cell_lab_431#Cell_culturing cell passaging]]
 
* Transfer the some of cell suspension to a sterile centrifuge tube of appropriate size (Eppendorf or other depending on volume)
 
* Transfer the some of cell suspension to a sterile centrifuge tube of appropriate size (Eppendorf or other depending on volume)
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=== In Giaver lab 420 ===
 
=== In Giaver lab 420 ===
 
You now need to have  
 
You now need to have  
* [[https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 Wiki lab notebook] open to make notes of observations and deviations from this protocol
+
* [https://wiki.uio.no/mn/fysikk/laglivlab/index.php/Lab_and_Planning_Notes#Spring_2022 Wiki lab notebook] open to make notes of observations and deviations from this protocol
 
* Computer with microscope and camera ready
 
* Computer with microscope and camera ready
 
** turn on computer, microscope and start flycap
 
** turn on computer, microscope and start flycap
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= Controlling O<sub>2</sub> and CO<sub>2</sub> concentration during experiment =
 
= Controlling O<sub>2</sub> and CO<sub>2</sub> 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]].
+
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 CO<sub>2</sub> in the cell media =
 
= Measuring CO<sub>2</sub> in the cell media =
 
Here we have not started the planning notes. Testing the electronics is the next step.
 
Here we have not started the planning notes. Testing the electronics is the next step.

Latest revision as of 14:33, 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.