It is difficult to get UV sterilization to work in closed microfluidic devices. The surface to be sterilized needs to be within a few millimeters of the lamp producing the UV. I would suggest using gamma if you have it available. Autoclaving works as long as you don't have other plastic parts in the mix. PDMS on glass works well in the autoclave but Tygon tubing will not autoclave gracefully if inserted in the ports. I pre-autoclave my Tygon and then insert it once I have it back in the biosafety cabinet. It gets a little bigger in diameter and about 10% shorter when autoclaved so make sure you cut your pieces long enough to allow for the change.

If you are plasma treating an already sealed microfluidic, you will not treat the interior of the device with most plasma systems. I would suggest using something like 1M NaOH to activate the interior of the device and then rinse thoroughly with DI before adding your fibronectin solution. You can use atmospheric plasma but you have to be careful not to let hot spots burn the fluidic.

Your flow rate and dimensions should keep sheer below about 6-8 dyne/cm2. Properly adhered HUVECs should tolerate 12-15 dyne before being damaged or detaching. Make sure that you allow enough time for your cells to adhere in stop flow before continuing with the perfusion. You should be safe with 4-6 hours of stop flow (depending on seeding density) without too much acidification or otherwise depleting the media. I have seen protocols that allow stop flow over night.

Good Luck!

Ron Reiserer thank you so much for your advice!

I have PTFE #30 AWG thin wall tubing, so will that change shape too much if I autoclave? And should it be a dry cycle autoclave? And do the channels have any liquid in them?

Is is possible/necessary to treat with NaOH and use atmospheric plasma to achieve more thorough coating?

Most PTFE tubing is pretty stable at autoclave temperatures. There are a few exceptions though and you should test your tubing to make sure. I use the dry cycle because I am putting my devices in autoclave pouches so I can transport them between the autoclave room and my biosafety cabinet. If the devices are autoclaved wet, they will get cloudy.

I think it is important to make the surface hydrophilic, I don't think the method is particularly critical. I have used corona from an Electro-Technic BD-20 to treat the inside of fluidic devices and to retreat areas that have failed to properly bond after vacuum plasma treatment. The main problem being that it is really hard to control the corona for consistent surface treatment.

I really like using NaOH for surface treatment inside already bonded devices. It gives very consistent results and is easily applied using run of the mill perfusion tools. It does take more time though. Plasma can get the work done in less than a minute while the NaOH needs 20 minutes or more.

Hello, Taylor Martinez.

I am new to culturing HUVEC in 3D collagen on a chip. I also met the same problem. 4mg/ml collagen is used to form a gel. When the collagen lumen is formed, fibronectin is injected to coat the lumen. After that, HUVEC is seeded into the lumen. After incubated at 37 degree for 1h, the EGM-2 medium is added to the inlet and outlet. After culture 1day, some cells are dead and many are still round. After culture 2 days, most of them are dead.

Do you solve it? I don't know why.

Any personal tips are welcome and helpful.

Looking forward to your reply.@Taylor Martinez