Use of Gradients in Biological Systems to Generate Biochemical Gradients

Gradients in Biological Systems

Precise biochemical gradients are crucial in dictating a number of biological processes, including diverse cellular behaviors such as proliferation, differentiation and migration. Gradients of morphogen signaling molecules during development, for example, tightly regulate patterns of tissue development by eliciting specific cellular responses depending on local morphogen concentration ¹.

Microfluidic Gradient Generators

Since it was proposed in 1999 ² , the development of microfluidic technologies has facilitated the simulation of such gradientsin vitro, allowing the experimental manipulation of the cell microenvironment in a physiologically relevant manner. Compatible syringe pumps are incorporated into microfluidic gradient-generating systems to infuse and withdraw reagents on the microscale precisely and accurately.

A number of gradient generator systems have been developed to suit various biological applications. Flow-based gradient generators are often used to study cellular responses to chemical, pulsatile or temporal stimuli, while diffusion-based generators are preferable for investigating responses of cells sensitive to the shear stresses imposed by the former— such generators have been designed to exploit the diffusive transport of chemicals without the requirement of high-flow velocities. More recently, microfluidic platforms have been developed to generate three-dimensional concentration gradients to reflect thein vivomicroenvironment even more accurately³. Advanced microfluidic gradient-generating systems continue to be developed in line with experimental demand. Such systems have become commonplace in biomedical investigations due to their ability to generate high-resolution gradients in a statistically reproducible manner.

Gradient Generators for the Investigation of Biological Processes

Microfluidic gradient generators can be used to investigate a wide range of physiological and pathophysiological processes. One major research area that has been revolutionized by microfluidic technology is the study of cell motility in response to chemical gradients, a process known as chemotaxis. Chemotaxis plays a vital role in various biological processes, from sourcing of nutrients by bacteria to mobilization of immune cells to sites of infection.

Bacterial chemotaxis, for example, is a subject of broad scientific interest, implicated not only in disease pathogenesis whereby bacteria seek out suitable colonization sites within their host, but also on a wider scale in the processing and cycling of elements by guiding bacteria towards and away from chemicals in diverse settings. Important research regarding antibiotic resistance replicated the naturally occurring bacterial microenvironment by using Chemyx syringe pumps to generate complex nutrient and antibiotic gradients⁴. Other microfluidic studies have led to a better understanding of both the mechanisms governing bacterial gradient sensing and the large-scale consequences of chemotaxis.

Importantly, biomolecular gradients of repellant or attractant factors are known to be involved in tumor-cell invasion in metastatic cancer. Many recent studies have made use of microfluidic gradient generators to mimic cancer cell transmigration⁵. In addition, investigation of the impact of gradients in terms of treatment has yielded important insights. A recent study⁷ investigating the resistance of some tumor cells to chemotherapy used a microfluidic platform integrating a Chemyx syringe pump to demonstrate that resistance can be induced by imposing stable long-term drug gradients.

Other fields of research that have benefited immensely from gradient generators include regenerative medicine⁸ and drug cytotoxicity testing(Hosokawa et al, 2011). In such studies, microfluidics has facilitated the generation of stable gradients while allowing a hospitable environment for healthy cell growth.

Future Development and Considerations

It is clear that microfluidics is a powerful and versatile tool for the study of gradient biology, an important field for the development of drugs and therapeutic interventions. Miniaturization reduces the need for expensive reagents while yielding improved assay reliability, reproducibility and well-defined cell culture conditions.

Current designs are oftentimes limited in throughput — future systems will aim to effectively handle multiple gradients and cell populations simultaneously in view of the fact that multiple stimuli are the physiological norm in vivo. At the same time, efforts to decrease production and operation complexity will increase the adoption of gradient generators.  Undoubtedly, gradient generators will continue to evolve to meet experimental requirements as well as to address technological limitations.

References

1. Rogers, K. W., & Schier, A. F. (2011). Morphogen gradients: from generation to interpretation. Annual review of cell and developmental biology, 27, 377-407.
2. Takayama, S., McDonald, J. C., Ostuni, E., Liang, M. N., Kenis, P. J., Ismagilov, R. F., & Whitesides, G. M. (1999). Patterning cells and their environments using multiple laminar fluid flows in capillary networks. Proceedings of the National Academy of Sciences, 96(10), 5545-5548.
3. Toh, A. G., Wang, Z. P., Yang, C., & Nguyen, N. T. (2014). Engineering microfluidic concentration gradient generators for biological applications. Microfluidics and nanofluidics,16(1-2), 1-18.
4. Zhang, Q., Robin, K., Liao, D., Lambert, G., & Austin, R. H. (2011). The Goldilocks principle and antibiotic resistance in bacteria.Molecular pharmaceutics,8(6), 2063-2068.
5. Acosta, M. A., Jiang, X., Huang, P. K., Cutler, K. B., Grant, C. S., Walker, G. M., & Gamcsik, M. P. (2014). A microfluidic device to study cancer metastasis under chronic and intermittent hypoxia. Biomicrofluidics,8(5), 054117.
6. Kong, J., Luo, Y., Jin, D., An, F., Zhang, W., Liu, L., … & Lin, B. (2016). A novel microfluidic model can mimic organ-specific metastasis of circulating tumor cells. Oncotarget, 7(48), 78421.
7. Wu, A., Loutherback, K., Lambert, G., Estévez-Salmerón, L., Tlsty, T. D., Austin, R. H., & Sturm, J. C. (2013). Cell motility and drug gradients in the emergence of resistance to chemotherapy. Proceedings of the National Academy of Sciences,110(40), 16103-16108.
8. Harink, B., Le Gac, S., Barata, D., Blitterswijk, C., & Habibovic, P. (2015). Microfluidic platform with four orthogonal and overlapping gradients for soluble compound screening in regenerative medicine research. Electrophoresis, 36(3), 475-484.
9. Hosokawa, M., Hayashi, T., Mori, T., Yoshino, T., Nakasono, S., & Matsunaga, T. (2011). Microfluidic device with chemical gradient for single-cell cytotoxicity assays.Analytical chemistry, 83(10), 3648-3654.