Microfluidic platform for the simultaneous generation of four independent gradients: towards the high throughput screening of trace elements for bone tissue engineering

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MICROFLUIDIC PLATFORM FOR THE SIMULTANEOUS

GENERATION OF FOUR INDEPENDENT GRADIENTS:

TOWARDS THE HIGH THROUGHPUT SCREENING OF TRACE

ELEMENTS FOR BONE TISSUE ENGINEERING

Björn Harink

1

*, Séverine Le Gac

2

, Clemens van Blitterswijk

1

and Pamela Habibovic

1 1

Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine

2

BIOS – The Lab-on-a-Chip Group, MESA+ Institute for Nanotechnology

1,2

University of Twente, Enschede, The Netherlands

ABSTRACT

We propose a microfluidics-based cell-culture platform for the screening of trace elements in bone tissue engineering. With this platform, it is possible to create four stable, independent and perpendicular diffusion-based concentration gradients in a single square chamber, in which cells are cultured. This allows examining the combined effect of four compounds in a single assay.

KEYWORDS: Gradient, Trace Element, Bone, Tissue Engineering INTRODUCTION

Current strategies in bone tissue engineering are based on combinations of a biomaterial carrier with bone forming cells and/or growth factors. Currently, growth factors present serious drawbacks, such as limited availability, biological instability and high costs. Therefore, novel strategies that reduce the use of expensive inductive biomolecules are desirable. One class of simple compounds appears as an attractive alternative: trace elements (such as Strontium [1], Lithium [2]). These are naturally present in humans and have shown to be active in various biological processes. Although, trace elements play an important role, their application in bone tissue engineering has not yet been systematically studied.

We hypothesize that mixtures of trace elements exist that, at low concentrations, stimulate bone formation. Since there are over seventy known trace elements, the systemic investigation of thousands of different combinations needs an approach different from classical cell culture. Therefore, we propose a new microfluidic cell-culture platform that allows for high-throughput live imaging and analysis, to use in various setups.

The platform consists of a microfluidic chip (Figure 1) inserted into a chip-manifold (Figure 2), which also functions as an incubator. The microfluidic chip includes a square chamber, in which cells are cultured, with four surrounding independent concentration gradient generators and two cell-loading channels. Supply channels, on the sides of the chamber, are connected to the chamber through arrays of smaller channels that have a low µm2cross-section. This allows for the diffusion-based delivery of chemicals introduced into the supply channels [3, 4]. As a result, four perpendicularly overlapping concentration gradients form across the chamber.

Figure 1. Illustration of the microfluidic platform, which includes a 500x500 µm2chamber, two cell-loading channels and four independent concentration gradient generators.

Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands

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Figure 2. Photo of the chip-manifold inside an automated confocal plate microscope (BD Pathway 435). The manifold is temperature-controlled and culture gas is flushed through the gas-tight chamber, where the chip is connected through

gas permeable capillaries. Insets: close-up of capillary connections (bottom) and chip (top).

THEORY

While designing the microfluidic chip, its dimensions were optimized through running iterative COMSOL-MEMS simulations, to ensure that diffusion is predominant over convection (Figure 3). The generated concentration gradients show a logarithmic decay from source to sink, and they can be compressed or spread by increasing or decreasing the flow rate in the side channels, respectively.

Figure 3. Modeled concentration gradient profile from one concentration gradient generator propagating from right (source) to left (sink). Flow rate in the supply channels is 0.5 µL min-1(Rhodamine 6G 100 µM).

EXPERIMENTAL

All microfluidic features and access holes were etched in a single silicon wafer using dry-etching (Figure 4), and bonded to a borosilicate substrate. The chip is placed within an in-house built manifold that provides temperature control and gas exchange. Since the chip is not gas permeable, gas exchange is facilitated using gas permeable capillary tubing. The whole platform is the size of a micro-titer plate, making it flexible in use.

Figure 4. SEM image of a corner of the cell-culture chamber with the supply channels connected to the chamber through diffusion channels. Inset: magnification of the diffusion channels.

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RESULTS AND DISCUSSION

We correlated our simulation results with experimental data and used two fluorescent dyes (Rhodamine 6G and Fluorescein) to visualize the generation of overlapping gradients (Figure 5). Stable gradients were formed after roughly 10 minutes. To investigate cellular viability, the platform was used for time-lapse imaging of the proliferation of C2C12 cells over three days (Figure 6). During the experiment, the cells were continuously supplied with nutrients by diffusion of medium through the supply channels.

Figure 5. Middle: fluorescence microscopy overlap image of overlapping fluorescent concentration gradients starting from right and bottom (source) to the left and top (sink). Right: Fluorescein channel (green). Left: Rhodamine

6G channel (red). The solutions are pumped at a flow rate of 1 nL min-1and both concentrations are 100 µg mL-1.

Figure 6. Time-lapse microscope images showing Murine Myoblast (C2C12) proliferation inside the culture chamber, starting with 11 cells (t=0) and finally achieving a confluent layer (t=60 hours).

CONCLUSION

We are currently testing relevant osteogenic models on cell monolayers in our integrated platform. To the best of our knowledge, this is the first microfluidics platform, fabricated in silicon that allows the simultaneous generation of four diffusion-based independent gradients on a cell monolayer.

REFERENCES

[1] Liang Yang et al. "The effects of inorganic additives to calcium phosphate on in vitro behavior of osteoblasts and osteoclasts" Biomaterials 31(11): 2976-2989 (2010)

[2] Jan de Boer et al. "Wnt signaling inhibits osteogenic differentiation of human mesenchymal stem cells" Bone, 5, 34 (2004)

[3] Paul J. Hung et al. "Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays" Biotechnology and Bioengineering, 1, 89 (2005)

[4] Atencia, J., J. Morrow, et al. "The microfluidic palette: A diffusive gradient generator with spatio-temporal control" Lab on a Chip 9(18): 2707-2714 (2009)

CONTACT

*Björn Harink, tel: +31-53-489-3116; m.b.m.harink@utwente.nl