Common Issues and Troubleshooting Tips

Modular Device Assembly

  1. Protective mask on component 1 and/or component 2 comes off in two layers. You will remove the blue but will see a remaining clear layer. Remove this clear layer before assembly.

    A. The blue protective liner should come off as one layer, leaving behind component 2, with the PSA exposed. B. Occasionally, the protective liner comes off as two layers. Remove both to expose component 2 PSA. If you do not remove this layer, you will see that it does not bond to component 1.

  2. The chip breaks during adhesion to component 1. The most common cause is the chip is not fully flat on fixture A1 pedestal. Sometimes it looks flat but is tilted by closer inspection. Look carefully directly from each side (see below). Tap sides with tweezers to be sure it is flat prior to adding component 1.
  3. Component 1 fits tightly in fixture A1 and does not go down straight. This causes the chip to hit the PSA early and often off center. Either carefully remove the chip with tweezers and replace on the pedestal if it is very off center, or if it hit the PSA but is centered well, push gently down on the side with tweezers then place component 1 and the chip back on the fixture. Be sure the chip is not sitting on the lips of the pedestal. Then confirm good adhesion with fixture A2. If the PSA stretches or the chip breaks during the process, toss. One way to avoid this is to test component 1 on fixture A1 before adding the chip onto the pedestal to make sure it fits.
  4. Dust particles from protective masks get in devices and cause bubbles. If you see particulates on the protective layers of the components, spray with EtOH before removing mask. If you see still particulates on the component after removing the protective layers, wet a Kimwipe with EtOH and wipe non-PSA layers to remove.
  5. Port is a tight fit for pipette on component 1. This is less common but happens on occasion. Try pipetting on the other side. If both ports are too small, do not use component.
  6. Channel is not centered on component 2. This is uncommon but happens on occasion and is more common on the strip edge components due to bowing of the strip during manufacturing. While we inspect components before shipping, it is good to do your own inspection prior to device assembly. If the channel is very off center, do not use the component.
  7. PSA on component 1 comes off either with protective layer (prior to assembly) or with component 2 removal (after assembly, for analysis). This is also uncommon but has been seen on a handful of occasions. It is unclear if it is a defect in the PSA adhesion to the acrylic of component 1 or user error.

Cell Culturing

  1. Cells do not adhere or initially adhere but do not proliferate. The most common issues with adhesion are coating concentrations and seeding densities. However other issues may cause this phenomenon.
    1. The recommended concentrations for HUVECs and hCMEC/D3 are given in the protocols: ALine Modular Device Cell Culturing: HUVEC; ALine Modular Device Cell Culturing: hCMEC/D3). However, adjustments or optimization may be necessary for different cell types or different coating molecules.
      1. Troubleshooting may include making a mix of coating coating molecules, as in hCMEC/D3 culture, or pH adjustments (see Monolayer Culture of mouse CMECs on μSIM A-Line Devices)
    2. For cell density, we recommend 40,000 cells/cm^2 for most immortalized cell lines. However, adjustments may be necessary for certain applications or other types of cells. Seeding density for primary cells or stem cells will need to be optimized in devices and may differ from tissue culture plates or Transwells.
    3. Another potential issue is too much EtOH on the Kimwipes, which are used to keep the devices from drying. We keep a box of Kimwipes in the hood and wet with sterile water to avoid the use of EtOH. However, the EtOH should evaporate before cell addition if it is added during the coating step.
    4. To troubleshoot issues with cell adhesion, we recommend having a tissue culture plate control and an immortalized cell line control. This will help determine if the issue is cells or handling. When first starting µSiM cell culturing, we recommend training on a cell line that has been used in the devices, such as HUVECs. It is common to see differences between tissue culture plates and devices, so the tissue culture plate control is to make sure it is not the cells themselves that are problematic, but rather something unique to the µSiM environment.
  2. Cells are not evenly spread. Pipet cells directly into the middle of the chip when seeding. If gaps are still seen, there may be an issue with the coating molecules.
  3. Bubble formation in channel. Always check for bubbles after pipetting into the bottom channel. With good lighting, hold the Petri dish over your head to inspect the channels of your devices. Sometimes a bubble forms in the trench that may be missed by eye. Be sure to also inspect under a microscope. A bubble in the trench will make in impossible to focus on the window.
    1. The best way to deal with bubbles is to avoid adding them to the channel.
      1. Always make sure there is no air at the end of the pipette tip before pipetting into the channel.
      2. Be sure there is a tight seal between the pipette tip and the port.
      3. Hold the device at an angle during pipetting, adding to the lower port so fluid flows upward (Bottom Channel Pipetting).
    2. However, sometimes you will find bubbles still make their way into the channel. To remove, pipet 100 µl quickly into the channel. This risks bursting the membrane but is the only way to remove bubbles. Figuring out the right speed to remove the bubble without bursting the membrane comes with practice. Sometimes it takes a couple tries to remove the bubble. This is also rather messy, so dry the bottom and sides of the device carefully with a Kimwipe afterwards. Do not dry around ports, as this can pull the media out of the channel (Bubble Removal). After the bubble is removed, pipet fresh media into the channel.