Team:MIT mmethods

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Mammalian Protocol

Microfluidics Protocol
Genetic Engineering Protocol

mammalian microfluidic protocol

The Mammalian team focused on creating a pressure-sensitive promoter and creating a standard protocol for mammalian genes.

1 HTD Preparation Protocol
    1.1 PDMS Mixture Preparation
    1.2 PDMS Pouring
    1.3 PDMS Baking
    1.4 PDMS-Device Punching and Bonding
    1.5 PDMS Device Bonding
    1.6 PDL coating
    1.7 Collagen filling
    1.8 Cell Seeding
2 Protocol for Deflection Experiments
    2.1 Tubing Setup Details
    2.2 Adding Medium to Channels
    2.3 Connecting device to pressure valve
    2.4 Microcontroller details

HTD Preparation Protocols (adapted from Yannis, Alisha)

PDMS Mixture Preparation
Materials

1. PDMS Body liquid (10 parts)
2. PDMS curing agent (1 part)
3. Negative pattern wafer
4. Microscope slides (1mm thick)

Procedure

1. Pour body liquid in a plastic cup while measuring its weight (cover the balance with tissue)
2. Reset scale, pour curing agent while measuring its weight (in 1:10 ratio to body liquid)
3. Stir the mixture
4. Remove the bubbles in the vacuum chamber

a. Turn on vacuum and close the valve for sustaining vacuum (cover the chamber with tissue)

Note: Lift container to be sure the vacuum is on.

b. De-gas for 20 minutes

Note: Open/close valve quickly to get rid of bubbles faster. When finished, close vacuum; open valve slowly.

PDMS Pouring

1. Blow the wafers with air, clear of all debris
2. Pour the PDMS onto the wafers. Make sure there is enough PDMS to completely fill the hole cut in the old PDMS and does not form a meniscus. Note that any devices that are not perfectly flat on top cannot be used with a syringe pump.
3. Let PDMS on wafer stand for 15-30mins, then blow air bubbles that have risen to the surface and put in oven. Do NOT de-gas wafer. This has lead to cracking and we only have the one.

PDMS Baking

1. Bake the poured wafers for 4-8 hours at 80C (recommended: 24hours to cross-link most of PDMS monomers)
2. Detach by cutting with razor (carefully and slowly peel it off, starting from the edges and going in the circumferential direction, in order to avoid tearing posts apart)

PDMS-Device punching and Bonding

1. Cut devices apart using a razor and the outline on the mask. Punch 4mm holes in the cell seeding and reservoir ports, a 2mm hole in the outlet port, and use the syringe to punch the gel filling ports.
2. Clean devices with transparent tape. It’s better to punch the devices off of the paper covered acrylic because the paper makes the devices very dirty.
3. Put devices in beaker with DI water, autoclave (wet / dry, 20 / 10 minutes)
4. Remove devices from beaker and place devices in empty pipette tip box, up to 6 per box. Autoclave again in box (switch setting to dry)
5. Dry in oven overnight @ 80C.

PDMS Device Bonding - Plasma Treatment

1. Clean area and all tools with ethanol
2. Place one device and one coverslip per platform, up to 3 platforms (platform looks like a small glass slide)
3. Close the door (turn black wheel until it can't turn any more) and valve, confirm seal, and turn on vacuum (“Pump” switch) for 2 minutes.
4. 1min and 40 sec plasma treatment: Turn “Power” switch on, turn radiation dial to low, medium, then high. After observing a purple glow, slowly;open the valve (black wheel on the door, spin backwards slowly, fractions of a turn!) until the glow becomes bright.
5. Wait for 1 minute 30 seconds
6. Turn off Power->RF->Pump, unscrew black wheel, and take out devices
7. Bring the devices and glass coverslip into contact with tweezers. Apply firm pressure with your fingers from one side to the other, avoiding air gaps between the coverslip and the device.
Note: When making 2-layer devices, after bonding the two layers together, bake in the oven overnight. Then punch holes for the top layer and autoclave. If desired, can fill devices with 60 ul of media after bonding, and proceed directly to the cell seeding step

PDL Coating

a. Fill the devices with PDL (~100-150µL/device)
b. Wash twice with water after 24 hours. Make sure that all regions are washed, in order to avoid heterogeneous surface coating. If you get bubbles, try to remove them by using the pipette applying suction.

Collagen Filling
Material

a) PDL-coated microfluidic devices (e.g., 6)
b) Collagen Gel
c) Complete Medium

Procedure

After PDL coating/drying, let the devices cool down for 30mins-1 hour at room temperature
1. Prepare collagen gel.
2. Draw 30µL of gel with the 200µL pipetter. Fill device from gel filling port. Fill gel most of the way from one port, but not all the way to the other gel filling port. Then, fill from the other port until gel interfaces merge. This will leave an inconsistent gel interface, but we do not have an option at this point.
3. Place each gel-filled device with the PDMS side facing up in the humidity box after it has been filled to avoid dryout.
4. Leave all devices for 1 hour in the incubator (in the humidity box) for the gel to polymerize
5. [IMPORTANT. This protocol establishes a stable pocket of air between the two medium channels at the outlet port.] Fill channels with medium from reservoir ports (~50-100µL). Gently fill medium until the channels have been filled to the cell seeding filling ports. Do NOT fill all the way to the outlet port. Instead, be sure to leave air at the channel intersection and in the outlet port. Place a droplet of medium over the outlet port to trap the air inside. Place devices in incubator. They will be ready for cell seeding in 24hours.

Cell Seeding

1. Trypsinize cells, spin down (@ 1.2 rpm, 5 min), and resuspend in medium to a final concentration of 2 million cells/ml
2. Aspirate all reservoir and cell seeding ports all the way to the glass. Ensure that the suction tip is away from the channel, so that the channel is not dried out.
3. Add 20 μL of medium to the reservoir and cell seeding ports on the top channel. (This reduces the interstitial flow rate so that not too many cells are bunched up on the gel.)
4. Add 40 ul of cell suspension to one port of each channel.
5. Let cells attach for 1.5-2 hours and then aspirate all reservoir and cell seeding ports. Place large droplets of medium on the reservoir ports. Return devices to incubator.
6. Maintain devices with complete medium for 24 hours. Make sure large droplets are always present on both ports, to prevent evaporation.

Protocol for Deflection Experiments

Tubing Setup Details

1. Connect the outlet port to a ‘dead end’ (a cap at the end of the tubing that prevents liquid flow); use a tapered connection.
2. Connect the inlet port to a syringe. Note: The tubing should be sterilized.

Adding Media to Channels

Note: before applying pressure, we're going to add medium to all control channels; don’t want to apply the pressure to air in the control channels.
1. Fill a small syringe with medium and connect to the inlet port.
2. Open the ‘dead end’ and push the syringe to fill control channel with medium.

Connecting Device to Pressure Valve

1. Find the microcontroller; connect tubing from pressure valve to appropriate inlet of microcontroller – (a small hole on the left or right side, pick a side depending on how you want to control the pressure).
2. Pick an appropriate outlet tubing from the microcontroller and attach to the inlet of your device (Note: tape device to microscope for ease of attachment).
3. Turn on the gas, turn on the valve and set the pressure to an appropriate number (anything under 10 is good)
4. Use the buttons on the microcontroller to set the pressure program.
5. Always start testing with low pressure. After 25 PSI, connectors can no longer contain the pressure.

Microcontroller details

Inlets:

Right Inlet: pressure is always applied
Left Inlet: pressure is applied according to microcontroller program.

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