Toxicity Problems
This page is devoted to discussions of toxicity when culturing cells in microfluidic devices. Several instances of toxicity have been reported, mainly coming from the PDMS and the microbore tygon tubing traditionally used at Stanford. Any material in contact with the cells, the culture media, or any other liquids that eventually contact the cells is a potential source of toxicity.
PDMS Toxicity
At the Quake Lab in Stanford, toxicity coming from the PDMS was initially reported around the start of 2008, while growing 3T3 mouse fibroblasts (Tay, S. et al. Single-cell NF-kB dynamics reveal digital activation and analogue information processing. Nature 466, 267–271 (2010).). Tay et al. solved the issue by flowing DI water through the whole flow layer of each chip for at least 24 hours, followed by autoclaving.
Before this, R. Gomez-Sjoberg and A. Leyrat had cultured a variety of cells in the automated cell culture system at Stanford without any obvious symptoms of toxicity (number in parenthesis indicates the longest time the cells were cultured in the chip): Human fibroblasts (10 days), human mesenchymal stem cells (12 days), human color cancer stem cell line in matrigel (21 days), "Kelly" human neuroblastoma cell line (4 days), mouse organ of Corti stem cells (10 days), mouse fibroblasts (7 days). For all of these experiments, the chips were sterile from the fabrication process.
PDMS Tests at UCSF
More recently, R. Gomez-Sjoberg performed tests of PDMS toxicity in collaboration with the Andino Lab at UCSF (thanks to Yinghong Xiao). The tests involved culturing 3T3 fibroblasts, HelaS3, and Vero cells on pieces of PDMS that had been autoclaved or UV-sterilized (30min of UV in an Electrocure 500 UV oven from Electro-Lite Corp, with a power of 30mW/cm^2 at 365nm). After cutting the pieces from real culture chips, they were sterilized with ethanol and then rinsed with sterile water, before coating with fibronectin.
On these tests, the cells only attached and grew well on the UV-sterilized PDMS, which is strange, given that other have routinely cultured cells in autoclaved PDMS chips. However, the PDMS always get a yellowish tint after autoclaving at UCSF, so the material is changing in some unknown way.
PDMS Tests at U. of Iceland
By Skarphéðinn Halldórsson (Fleming Lab), June 19, 2012
I ran an experiment recently where I chopped up an old chip, placed the pieces in traditional culture wells and seeded cells onto them. Some of the pieces were autoclaved, some UV treated and some both. All the pieces were coated with poly-d-lysine and collagen before cells were seeded. I tested two cell lines, one (a lung epithelial cell line) attached and spread over all the pieces while the other (SHSY5Y neuroblastoma cell line) formed large clumps on the PDMS and did not spread out. I see similar behavior in the chip, the cells do not spread out and have a tendency to clump. I did not see any difference between untreated, autoclaved or UV treated PDMS pieces. Some weeks ago I tried coating culture wells of a 48-well plate with PDMS and culturing these cells in the coated wells. The PDMS I used was SYLGARD 184 witch is supposedly more pure than the the RTV PDMS used at the foundry. Cells grew just fine. At the moment, Emre at the foundry is making a batch of chips from Sylgard 184 for me. I'll let you know if these chips turn out differently than the RTV ones.
Proliferation. I've recently been using the lung epithelial cells in the chip. These cells attach very well and can be kept alive in the chip for at least a week. I do see some cell divisions during the first 24 hours and an increase in cell numbers but after that, very few divisions and the ones that do divide often die shortly thereafter. Have any of you guys compared proliferation rates of your cells with cells grown in traditional culture? If so, how do they compare? Like most of the issues we've seen, this is probably cell specific. I have a spool of PTFE tubing and will be using that instead of tygon to carry medium to the chip for my next experiment.
Tygon Toxicity
Tygon Tests at Rockefeller U.
By Benoit Sorre (Brivanlou and Siggia Labs), June 5, 2012
In our experiments using c2c12 and hESC, I found that the regular tygon tubing used at Stanford for all the cell culture work (Saint Gobain S-54-HL) is toxic to the cells.
This problem was not easy to identify, as tygon is less toxic when washed (Price, N., et al. Toxic Effects of Latex and Tygon Tubing on Marine-Phytoplankton, Zooplankton and Bacteria. Mar. Ecol.-Prog. Ser. 34, 41–49 (1986).). I was having very inconsistent growth; sometimes it was great, sometimes really bad. I work with two clones of the same cell line and one of them was OK, but the other one was very sensitve. I usually reuse the tubing for DMEM as it is common to all experiments. One day I seed cells, feed them for 12hrs with DMEM that was in a tube connected to the chip with tygon tubing that had been used before, and the cells were fine. After 12hrs I decide to stimulate them with medium containing tgfbeta that was in new tubes. Chambers stimulated for one hour continued their growth, the one stimulated for 12hrs stopped growing for 12 hrs and started growing again when medium was switched back to the DMEM with old tubing and the cells stimulated for 40hrs just sarted dying (we of course checked that this was not due to tgfbeta itself).
Next, I took about a half a foot of tubing (regular tygon, and PTFE Microbore tubing 0.022" ID x 0.042" OD from Cole Parmer - prod# EW-06417-21) that I cut in small pieces and let infuse/float in 6cm dishes with cells. I tested 4 conditions: No tubing floating, tygon tubing, autoclaved tygon tubing, and teflon/PTFE tubing. As you can guess no tubing and teflon looked ok and tygon looked bad, especially the autoclaved one. Since then, I use only teflon and cells always grow fine.
Now, your cells might not be sensitive to that as even two clones of the same line transfected with different things had different sensitivities...
I am curious to get other people's experience on this problem.
Comments
Please use the form below to post comments.
The comments will take a while to appear in the space above, so be patient and remember to hit the refresh button on your browser.