PDMS in biology research: A critical review on PDMS lithography for biological studies

Introduction on PDMS for microfluidics

PDMS
Crosslinked PDMS block

From a technological point of view, the ability to make microfluidic devices in a few hours, without needing clean room equipments, remains very attractive for research teams beginning in microfluidics.

Additionally PDMS shows numerous advantages coming from its intrinsic properties:

  • the PDMS is biocompatible [1].
  • it is cheap.
  • it is transparent (240nm-1100nm).
  • it has low autofluorescence [2].
  • it can be molded with a resolution down to a few nanometers using modified PDMS [3].
  • PDMS replica can be covalently stuck to a glass substrate using simple plasma treatment to form a sealed microfluidic device.
  • PDMS deformability enables easy connections of leak-proof fluidic connections, integration of fluidic valves through PDMS microchannels and it is used to detect very low forces like biomechanics interactions from cells [4].
  • This elastomer is also permeable enough to gas [5] to allow gas supplying for on-chip cells culture [6].
  • PDMS, during cross-linking, can be coated with a controlled thickness on a substrate using a simple spincoat. This allows the fabrication of multilayer devices and the integration of micro valves.

PDMS drawbacks for biology

Microfluidic chip made of PDMS/glass with electrodes

PDMS also shows some drawbacks like:

  •  It is difficult to integrate electrodes or to carry out deposition directly on its surface.
  • One the main drawbacks for cell biology is that PDMS can absorb small hydrophobic molecules like biomolecules and drugs from the solution. Also, many researchers noticed adsorption of proteins on the PDMS surface which has been identified as a major problem for molecular biology. For experiments on cell signaling and determination of drug dose response, the use of PDMS can strongly bias the final result. To overcome this problem, numbers of PDMS surface treatments have been developed depending on the application.
  • Reciprocally, incomplete reticulated PDMS is suspected to migrate within the channel [7]. For that matter, this polymer has been used as an extraction matrix to remove traces of organic compounds from solutions.
  • Another problem met during cell biology experiment in PDMS devices is the permeability of PDMS to water vapor leading to the evaporation of the water contained in the channel as time goes by. This effect can lead to complete device drying or changes in medium osmolarity. It is generally possible to overcome this problem using hydratation channel networks, medium renewal systems or hygrometry controlled environments. Ideally, PDMS devices should be conditioned several hours before use to stabilize the device hygrometry.
  • PDMS is sensitive to exposure at some chemicals (see below).
  • PDMS is a material that ages, therefore after a few years the mechanical properties of this material may change.

Despite those limitations, PDMS microfluidic devices are widely used for cell studies and will probably be used more and more for researches in cell biology.

It is then necessary to understand the effects of microscale environment to integrate the results obtained in microfluidic devices with biological experiments obtained using traditional methods.

Biological results obtained in microfluidic devices have already been of great interest for cell biology but the careful understanding of condition changes generated by miniaturization and properties of PDMS will lead to a better understanding of those results.

There are significant differences in proliferation, glucose consumption, gene expression patterns and mitosis defects between traditional well plates and PDMS micro cell culture. Paguirigan & al used In-Cell western (ICW), which allows to quantify protein expression changes, to show differences signaling pathway activation and protein expression levels between experiments in PDMS microsystems and traditional in-vitro experiments [7]. These studies also show significant inhibition of mouse fibroblast proliferation with 3 times higher glucose consumption. Observations in PDMS microchannel show smaller amounts of cells performing division and show several different cell cycle progression problems with a lot of arrest in S/G2 phase.

Possible causes of cells behaviours

Microfluidic cell culture perfusion

One explanation of this difference in behavior could be that microscale culture generally increases the cell volume density (cell by unit of medium) compared to multiwall plates leading to more rapid waste products accumulation and medium consumption. But using higher concentration media has little impact on proliferation; suggesting that the volume density is not the predominating factor [7].

Main reasons for these differences between macro and micro scaled culture systems could be:

  • Uncrosslinked low molecular weight polymer may also leach from the polymer to the medium and monomer could interact with hydrophobic parts of the cell membrane.
  • Absorption of media components into PDMS, particularly hydrophobic molecules. Hydrophobic growth factors or lipids from the cell culture medium can migrate into the PDMS bulk. This loss of lipids, which is a source of energy for cells, in PDMS may explain the increase in glucose consumption.

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One solution to overcome those problems is to perform cell culture with a proper renewal of cell medium which allows evacuation of wastes and renewal of nutriments.

Indeed, Leclerc & al showed that, in the case no media change, glucose and albumin concentrations within the device drop after three days, leading to cells death; but a renewal of media leads to constant albumin and glucose concentrations and long term cells viability [6].

Most of the microfluidic cell culture systems presented before included convective or diffusive medium renewal, depending on the application. Nevertheless, this method may thus not be sufficient for studies like cell signaling studies and determination of drug dose-response where precise molecules amount absorbed or produced by the cell are crucial. In those cases, medium non-renewal and PDMS absorption could strongly bias the results.

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Concerning medium gas composition, since PDMS is permeable to gas, this material allows enough O2 renewal by diffusion through a PDMS wall for cell culture.

Leclerc & al showed that gas diffusion through PDMS walls of 200 µm is sufficient for long term culture of hepatocarcinoma [6]. However, many publications cited before in this review described cell proliferation and growth for weeks. All of them used a medium renewal system to allow proper cells growth for long term experiments.

Conclusions

It is possible to perform relevant biological studies in PDMS microsystems when taking into account limitations of this technology. Additional studies will be required to identify which kind of results obtained in such kind of microsystems will be exploitable and in which conditions. Instead of trying to correct intrinsic characteristics of PDMS like its surface chemistry, some laboratories develop and test new polymers which could allow same technological potentialities than PDMS with better chemical properties. For more detailed critical information on PDMS as a material for microfluidic devices, one can read [8].

For more tutorial about microfluidics, please visit our other tutorials here: «Microfluidics tutorials». The photos in this article come from the Elveflow® data bank, Wikipedia or elsewhere if precised. Article written by Guilhem Velvé Casquillas and Timothée Houssin and revised by Lauren Durieux.

References

[1] Belanger, M. C.; Marois, Y. J Biomed Mater Res (2001), 58, 467-77.

[2] Piruska, A.; Nikcevic, I.; Lee, S. H.; Ahn, C.; Heineman, W. R.; Limbach, P. A.; Seliskar, C. J. Lab Chip (2005), 5, 1348-54.

[3] Hua,F.; Sun,Y.; Gaur,A.;Meitl,M. A.; Bilhaut, L.; Rotkina, L.; Wang, J.; Geil, P.; Shim, M.; Rogers, J.A. Nano letters (2004), 4, 2467-2472.

[4] du Roure, O.; Saez, A.; Buguin, A.; Austin, R. H.; Chavrier, P.; Silberzan, P.; Ladoux, B. Proc Natl Acad Sci USA (2005), 102, 2390-5.

[5] Charati, S. G.; Stern, S. A. Macromolecules (1998), 31, 5529-5535.

[6] Leclerc, E.; Sakai, Y.; Fujii, T. Biomedical Microdevices (2003), 5, 109-114.

[7].Paguirigan, A. L.; Beebe, D. J. Integrative Biology (2009), 1, 182-195.

[8] Mukhopadhyay, R. Analytical Chemistry (2007), 79, 3248-3253.