Team:EPF Lausanne/Safety

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         <li class="active"><a href="#form">Safety form</a></li>
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         <li><a href="#safetyissues">Safety issues in Microfluidics</a></li>
         <li><a href="#safetyissues">Safety issues in Microfluidics</a></li>

Revision as of 19:42, 15 October 2014

BIO SAFETY

Safety Form

The safety form can be found here.

Safety issues in microfluidics

It came to us during our project that microfluidics can be a very useful tool to improve biosafety, as it requires small amounts of cells and provides an enclosed space to culture and analyse them. This is of critical importance when handling Genetically Modified Organisms or pathogenic strains, which could potentially be harmful if released in the environment. To learn more about safety issues in microfluidics, we interviewed 2 professors working in Lausanne:

- Dr John McKinney, who is the head of the Laboratory of Microbiology and Microsystems at EPFL (Ecole Polytechnique Fédérale de Lausanne) and uses microfluidics to study pathogenic strains such as uropathogenic E. coli or M.tuberculosis

- Dr Jan van der Meer, who is the head of the Department of Fundamental Microbiology at UNIL (Université de Lausanne) and works on implementing biosensors in a microfluidic chip

(put links)

After doing these interviews, we continued brainstorming on biosafety and finally came up with a new chip design summarizing the ideas we got when talking to the professors. It includes several decontamination steps to provide total on-chip waste treatment and avoid any leakage of potentially harmful organisms in the environment. You can find more about it here.

As for the interviews, here are the main points we got out of our discussion with the professors:

Main advantages of using microfluidics regarding biosafety

The most significant advantage brought by microfluidics is size reduction, which enables one to handle only minute amounts of cells or media. Thus, when running analyses on a 96-well plate, the reaction volumes are about 100 uL, which corresponds to approximately 107 cells if we consider an overnight bacterial culture. On the contrary, microfluidics allow the manipulation of nanoliter-range volumes, corresponding to only 103-104 cells. This is of critical importance when handling pathogens as it limits the risk of contamination by drastically decreasing the exposure dose. Size reduction is also crucial when one wants to commercialize a biosensor device containing GMOs, as Jan van der Meer explained us. Indeed, regulation on such kind of devices depends not only on the type of organism used but also on the amount of cells that the device contains. Thus, designing biosensors based on microfluidics could have them accepted more easily by the regulation authorities. For more information on regulation of biosensors in Switzerland, we encourage you to visit this website.

The second advantage of using microfluidics is that it constitutes a closed environment, as cells are hermetically trapped between the glass slide and the PDMS chip. It therefore minimizes the manipulation of open liquid cultures by the operator, which reduces the risk of contamination and release in the environment. One could still argue that cells can leave the chip in case liquid is actively flowed through it (for example to continuously provide cells with fresh medium). In John McKinney’s lab, they solved this problem by connecting the outlet of the chip to a bottle containing bleach or ethanol, which allows immediate decontamination of any liquid leaving the chip. Thus, microfluidics also provide direct waste treatment during an experiment.

Remaining drawbacks

As microfluidics is a very recent tool regarding biological studies, there are still some points that need to be optimized as far as biosafety is concerned.

First of all, the chip remains a hermetically closed space as long as the bonding between the glass slide and the PDMS is perfect. It sometimes happens that the chip delaminates, i.e. that the PDMS unsticks from the glass. This is often the case when liquids are flowed into the chip with a very high pressure or when cells form clots that block the channels, leading to local increase in pressure. Delamination is a real safety problem as it leads to release of cells in the environment and thus possible contamination. Yet it can be avoided by plasma-treating both glass slide and PDMS, which makes the bonding permanent. For more information on plasma-treatment, you can visit our page on chip fabrication. Another solution that is used in McKinney’s lab is to clamp the PDMS and the glass slide together but this is generally not-suited for long-term applications or when the chip is intended to be commercialized as a biosensor.

A second issue is that the assembly and disassembly of the chip still needs to be done under a biosafety cabinet as it involves manipulation of open liquid. There are usually 2 options to load a chip with cells:

• either one can bond the glass and the PDMS and then flow cells through inlets that are punched in the PDMS, which is the option we chose in our project (see our How to use a chip? page)

• or one can spot cells on the glass slide and then bond the PDMS on top of them: this second solution requires smaller amounts of cells but is not compatible with plasma-treatment as one has to align the PDMS very precisely to the cells whereas plasma-bonding is a one-shot process

Depending on the kind of cells/media/pressures that are used, one therefore has to find a compromise between manipulating open liquids and reducing the risk of delamination.

John McKinney also pointed out 2 other shortcomings that could be problematic in some specific cases. First of all, microfluidics are not very well adapted for long-term experiments such as studies of slowly growing strains, since PDMS is known to lose its mechanical properties with time, which could increase the risk of delamination. Second, bleach or ethanol are inefficient to decontaminate spores, which could make it difficult to run microfluidic experiments with pathogenic sporulating strains such as anthrax, as spores could leave the chip without being killed.

Possible improvements

To tackle the aforementioned issues, one can think of several solutions. The professors presented some dispositions they took in their lab, then we also brainstormed on our side on how biosafety could be improved.

John McKinney first explained to us what they are doing in his lab to prevent any cell from leaving the chip, even through the outlet tubing. They actually add a dialysis membrane between the cells and the PDMS chip, so that cells are trapped between the glass slide and the membrane. The latter consists of nanometer-range pores, which prevents cells and even large proteins from leaving the chip. This is much interesting when handling sporulating species or toxin-producing strains as it would avoid any contamination by spores or harmful toxins.

To tackle this issue of keeping every cells on the chip, we thought of adding 0.2 um filters at the end of the array of chambers, so that only medium can flow out of the chip. This also seemed to be a proper solution to deal with sporulating species. But it then appears that cells could form clots just before the filters, thus blocking the liquid and finally leading to delamination. A very clever solution to this problem was engineered in van der Meer’s lab: instead of directly flowing cells through the chip, these are embedded in agarose beads which size is much larger than the size of the filters. Therefore, cells are trapped in agarose and can neither clot the filters nor leave the chip. Another advantage of this technique is that the chip can be pre-filled with beads and then stored at -20°C before running the experiment, which is of great interest if one thinks of engineering a completely portable device. The only issue when handling cells in biomaterials is to be able to control the feed the cells. Indeed, bioreporter cells need a continuous energy input to be able to produce the desired reporter proteins. When cells are trapped in a biomaterial, the reaction time can become extremely long as the diffusion of medium through the gel is hampered. Once again, one has to find a compromise between safety and efficiency.

What we thought about: - continuous lysis buffer/ethanol/bleach flow - close the device with valves but feeding issue - add a suction device that frames the chip - add a microheater to kill spores

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