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.
The recommended concentrations for HUVECs and hCMEC/D3 are given in the protocols: µSiM Cell Culturing: HUVEC; µSiM Cell Culturing: hCMEC/D3). However, adjustments or optimization may be necessary for different cell types or different coating molecules.
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.
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. UPDATE: We no longer use EtOH with Kimwipes but a small Petri dish or 50 ml conical cap and fill with sterile water.
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.
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.
Excess evaporation of media. This can be seen in different laboratories, that excess media is lost from the well and the bottom channel even after less than 24 hours. This can lead to cell death or cell loss, and makes it easier to add bubbles into the channel if there is an air gap between the media height and top of the port. First, check the temperature and humidity of the incubator. Devices are more sensitive than tissue culture plates. In the interim, you can add excess media into the well and over the ports for overnight incubations and switch media daily or twice daily. If you do see significant evaporation from the channel, to avoid pipetting in a bubble, first pipet media into one port until you start to see media coming out the opposite port, then pipet from that new port to switch media in the channel.
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 (see image below, if all edges are rounded, you have one bubble filling the whole trench). If there is a bubble in the channel under the membrane window, you will need to remove the bubble via pipetting.
The best way to deal with bubbles is to avoid adding them to the channel.
Always make sure there is no air at the end of the pipette tip before pipetting into the channel and that the channel media line is at the top of the port.
Be sure there is a tight seal between the pipette tip and the port.
Hold the device at an angle during pipetting, adding to the lower port so fluid flows upward. As media comes out the top port, flatten the device (Bottom Channel Pipetting Technique Video).
However, sometimes you will find bubbles still make their way into the channel (see image below). One method to remove is to clear most of the media from the top well and pipet 100 µl quickly into the channel, allowing the media to spill into the well. Then replace the well with fresh media. This method is very effective, but it can be harsh on cells and may risk bursting the membrane. Figuring out the right speed to remove the bubble without bursting the membrane comes with practice, and sometimes it still takes a couple tries to remove the bubble. (Video Showing Bubble Removal). Another drawback to this method is for co-culture or directional treatment setups, where you do not want to mix media between wells.
An alternative method for bubble removal is to pipet the media out of the channel first by pulling from one port, and then pipet ~50 µl back into the channel via the normal bottom channel pipetting technique. This method is gentler on the cells but can be less effective. I would recommend trying both methods and using what works best in your hands with your materials.
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