I am trying to culture cells inside a PDMS microchannel, but it doesn't seem to be working as it normally does on a culture plate. I introduced around 40,000 cells into a channel with a surface area of 2.5 millimeter square. I am unsure if the death is because of overcrowding of cells or if there is not enough nutrients reaching the cells because there are no signs of any contamination inside the channel. The cells that I use are porcine meniscus cells and the medium is DMEM with 10% FBS. I intend to culture the cells for less than a week, and would like it to attain confluence. Could anybody help me understand the reason behind cell death?
Are you perfusing your system? Simple diffusion may not provide adequate transport of nutrients to keep your cells alive. Have you been able to successfully culture your cells on a 2D PDMS substrate functionalized by a similar method? Please provide some more information about what, if any ECM, you include, and how you attach it to your PDMS.
can cells attach and spread on the surface of the channel? You should verify this point, otherwise cells could die by anoikis
I agree to Claudio cell attachment is first thing you should check. As for efficient attachment you may consider modifying your surface with collagen, lysine or fibronectin etc. Plus you should consider growing in 7.5% CO2, Also creates some micropores in the PDMS roof of the chamber. Usually cells follow similar pattern of growth in PDMS micro-channel bonded to glass surface coated with cell-attachment matrix as they would show for standard cell-culture petridishes.
Are you perfusing your system? Simple diffusion may not provide adequate transport of nutrients to keep your cells alive. Have you been able to successfully culture your cells on a 2D PDMS substrate functionalized by a similar method? Please provide some more information about what, if any ECM, you include, and how you attach it to your PDMS.
I would suggest pre-wetting the channels with media for several hours before plating the cells. This would allow proteins to be adsorbed onto the PDMS surface and assist in cell attachment. It is a commonly used technique for plating cells on hydrophobic tissue engineering scaffolds.
@Dr. Daniel: I am not using any perfusion system at the moment. I am currently relying on diffusion. I have tried using the hydrogel of hyluaronic acid, but cell proliferation does not seem to be happening much.
Thank you everyone for your inputs. I really appreciate this.
Hi Nishanth. HA gels generally don't support cell adhesion unless you functionalize them with fibronectin or another ECM. Also, cell proliferation on HA gels is quite dependent on the cell type and the stiffness of your hydrogel. You should make sure you can get good cell attachment and proliferation in a flat, non-channeled environment before testing out your final system. Otherwise, it is unclear whether the lack of the desired cell response is due to your hydrogel, a problem with insufficient media perfusion, or both.
Thanks a lot for the help Dr. Daniel. I will certainly figure out what the problem is. Thank you!
This could be a number of things. Have you tried culturing cells at the same concentration in a sterile petri dish with the same media (ie 10,000 cells/mL)?
If they proliferate in a petri dish with the same media then the problem is going to be with the micro channels. Have you modified or functionalized the surface so that the cells may adhere? If not, you could try modify the surface with oxygen plasma to promote adhesion.
How often are you changing your medium? In long terms cultures in PDMS devices (i.e. over a month) I may change medium everyday. I check the color and if it's yellow I change it.
@ Mr. Kerman I do change the medium everyday and normally the color of the medium does not turn yellow before I change it.
What's your device desing? You can try to add different levels of medium to the reservoirs. That way there will be a flow of medium through the channel. If you don't notice medium flow one reservoir to the other one then somehow you may be blocking the channel. When cells grow at the entrance of channel they block the channel.
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