Lab on a Chip Blog

3D technology has revolutionised the entertainment industry by offering viewers the experience of being part of the action, going on in a movie rather than simply watching it. Thanks to 3D technology, we can sky walk hand in hand with George Clooney in ‘Gravity’.

Microfluidics setup for 3D bioetching

The history of 3D technology can be drawn all the way back to the invention of the stereoscope by David Brewster in 1844. Last two decades have seen 3D technology replacing 2D in all walks of life, ranging from entertainment, physics, microelectronics, tissue engineering and regenerative medicine. For e.g., microelectromechanosensors (MEMS) are 3D devices produced by using soft lithography techniques. MEMS installed in air-bags in the cars have saved thousands of lives by sensing pressure levels during accidents.

Can we use 3D technology to have a better look at the complex events happening at cellular level? One of the major challenges in tissue engineering is that the conventional approaches are mainly limited to 2D monolayers systems and do not allow manipulation of complex multilayer tissue. Cells grown on 2D substrates may respond and differentiate distinctly than those in more physiologically relevant 3D environments. The emergence of 3D technology has enabled scientists to mimic the exact cellular environments and helped to provide better insights into the cell signalling, migration and differentiation in cells.

One of the ways of mimicking the cellular architectures is bio-etching which involves subtractive manufacturing. Bioetching of monolayers of cells in response to laser cuts or scratch assays is achieved by using 2D cell culture studies. But the actual biological systems such as tissues and organs are much more complex and cannot be mimicked using simple monolayers. For long time, scientists have been working on developing better technologies to address this problem. One of the ways to achieve this is 3D bio-etching.

William C. Messner et al. from Tufts University in a recent article in Lab on a Chip explain the utility of 3D bioetching technique to create and shape 3D composite tissues using a microfluidics based approach. The ability to shape the 3D form of multicellular tissues and to control 3D stimulation will have a high impact on tissue engineering and regeneration applications in bioengineering and medicine as well as provide significant improvements of highly complex 3D integrated multicellular biosystems.

Can 3D bio-etching help us to design tissue architecture of our choice mimicking different biological events? Find out by reading the full paper for free* using the link below:

3D bio-etching of a complex composite-like embryonic tissue
Melis Hazar, Yong Tae Kim, Jiho Song, Philip R. LeDuc, Lance A. Davidson and William C. Messner
Lab Chip, 2015, Advance Article
DOI: 10.1039/C5LC00530B

*Access is free through a registered RSC account.

For more than a century, researchers have been studying how the body detects and defends itself against foreign invaders.¹ This process relies on a series of cell-to-cell signaling, starting with the detection of common pathogen components by receptors of immune cells. For example, macrophages, a surveillance immune cell, recognize bacteria using the Toll-Like receptor 4 (TLR4), which binds to lipopolysaccharide (LPS) found in the membrane of gram-negative bacteria.

Upon recognition of danger signals, immune cells engulf and degrade the invaders and activate NF-κB, an intracellular protein that, when activated, migrates into the nucleus and turns on inflammatory response genes. For immune cells, this initiates the secretion of signaling mediators such as tumour necrosis factor (TNF), which helps remove invaders by dilating local blood vessels and recruiting other immune cells.

Although our understanding of the immune system has skyrocketed in the past two decades,² researchers are still lacking the tools needed to study the signaling of inflammation in a highly controlled manner. Most approaches rely on bulk population measurements, in which important spatial and temporal patterns of signaling are obscured by biological noise from neighbouring cells. To address this challenge, Tino Frank and Prof. Savas Tay from ETH Zurich developed a microfluidic system that can propagate inflammatory signals from a single immune cell (sender cell) and to a population of fibroblast cells (receiver cells).³

The microfluidic device, formed from polydimethylsiloxane (PDMS), comprises a deflectable membrane sandwiched between a flow layer and a control layer. The flow layer houses supply channels and cell culture chambers, while the control layer regulates input, output, and segregation of these chambers using solenoid-actuated valves.

As shown in picture 1, the chambers for the sender cell (chamber A) and receiver cells (chamber B) can be segregated for mono culture or connected for co-culture, depending on the state of the reversible separation valve. In mono culture mode, the sender cell can be stimulated independent of the receiver cells, whereas in co-culture mode, the signaling mediators secreted by the sender cell can be broadcasted to the receiver cells via diffusion.

Using this system, the researchers implemented a single-cell model of inflammation arising from bacterial infection (picture 2). This model begins in mono culture mode, with a single macrophage seeded in chamber A and a population fibroblasts seeded in chamber B. Inflammation was initiated by stimulating the macrophage with short pulses of bacterial LPS, leading to the activation of NF-κB and TNF secretion. Subsequently, the device was switched to co-culture mode, allowing the secreted TNF to diffuse across the fibroblasts and activate their NF-κB signaling.

By tracking the movement of NF-κB in the nucleus of individual cells, the researchers, for the first time, mapped the spatiotemporal distribution of immune response to bacterial infection. They demonstrate that a single macrophage can activate and control over 100 fibroblasts up to 1 mm away, and that fibroblasts located farther away exhibited a time-delayed activation profile and fewer NF-κB oscillations (between the nucleus and cytoplasm). Furthermore, they observed that the NF-κB signaling in some fibroblasts can linger for up to 10 hours, demonstrating that a brief exposure to pathogenic signal can induce long-term inflammatory response in nearby cells.

In summary, Tino Frank and Prof. Savas Tay developed a microfluidic platform to study spatiotemporal dynamics of immune response in single cells. The continued investigation of these dynamics will be important for understanding of the inflammatory process and modeling of relevant signaling pathways.⁴


To download the full article for free* click the link below:
Automated co-culture system for spatiotemporal analysis of cell-to-cell communication
Tino Franka and Savaş Taya
Lab Chip, 2015, 15, 2192-2200
DOI: 10.1039/C5LC00182J


—————-
References:

1. K. M. Murphy, Janeway’s immunobiology, Garland Science, 2011.
2. L. A. J. O’Neill, D. Golenbock and A. G. Bowie, Nat Rev Immunol, 2013, 13, 453-460.
3. T. Frank and S. Tay, Lab on a Chip, 2015, 15, 2192-2200.
4. M. Junkin and S. Tay, Lab on a Chip, 2014, 14, 1246-1260.

—————-

About the webwriter

Alphonsus Ng is a postdoctoral fellow under the supervision of Dr. Aaron Wheeler in the Department of Chemistry and the Institute of Biomaterials and Biomedical Engineering at the University of Toronto. His research focuses on the development of microfluidic methods for heterogeneous immunoassays, cell-based assays, enzymatic catalysis, sample preparation for proteomics, and chemical synthesis.

—————-

*Access is free until 24/08/2015  through a registered RSC account.

These HOT articles published in June 2015 were recommended by our referees and are free* to access for 4 weeks

Microscale extraction and phase separation using a porous capillary
Thomas W. Phillips, James H. Bannock and John C. deMello  
Lab Chip, 2015,15, 2960-2967
DOI: 10.1039/C5LC00430F, Paper

Dielectrophoresis-assisted 3D nanoelectroporation for non-viral cell transfection in adoptive immunotherapy
Lingqian Chang, Daniel Gallego-Perez, Xi Zhao, Paul Bertani, Zhaogang Yang, Chi-Ling Chiang, Veysi Malkoc, Junfeng Shi, Chandan K. Sen, Lynn Odonnell, Jianhua Yu, Wu Lu and L. James Lee  
Lab Chip, 2015,15, 3147-3153
DOI: 10.1039/C5LC00553A, Paper

A microfluidic platform with digital readout and ultra-low detection limit for quantitative point-of-care diagnostics
Ying Li, Jie Xuan, Yujun Song, Ping Wang and Lidong Qin  
Lab Chip, 2015, Advance Article
DOI: 10.1039/C5LC00529A, Paper

 *Access is free until 17.08.15 through a publishing personal account. It’s quick, easy and free to register

Research into biofuels as a replacement for fossil fuels is a hugely important area of research. One particular focus is on cultivating photosynthetic organisms, such as microalgae, as these organisms have a high oil content that can be extracted and converted into biofuels. They are grown in photobioreactors in order to control the growth conditions, and there are a large amount of variable factors that need to be taken into account to find the optimal conditions.

For each new microalgal strain used the conditions need to be optimised, and irradiance screening is of particular importance. Currently, the screening process requires multiple culture flasks, each with their own light source. Recent developments have switched to using microwells, again with individual light sources.

David Sinton and co-workers at the University of Toronto have developed a microfluidic irradiance assay using liquid crystal display (LCD) technology that allows them to rapidly screen irradiance conditions and identify the conditions for optimum growth. Using this technology, they were able to control all irradiance variables (light intensity, time variance, and spectral composition) in over two hundred parallel microreactors.

The diagram below shows the design of their irradiance platform – the LCD screen is lined up so that each pixel is directly below one microreactor, with every pixel individually controlled in order to produce the correct irradiance output. The bacterial growth in each microreactor was characterised by measuring the total fluorescence, emitted by a fluorescent pigment inside the organism.

Design of the irradiance platform

Demonstration of spatial control of microalgal growth

Initially, to demonstrate that their pixel-based method worked, the authors displayed the Toronto University crest on the LCD screen by using high and low irradiance intensities, and you can see from the image that this was successful!

By studying the three major irradiance variables mentioned previously they were able to quantify several important properties, such as the saturation intensity, the threshold frequency for growth and the combined effect of spectral composition and irradiance intensity on growth.

This new method drastically reduces the time needed to screen conditions for bacterial growth and hopefully should have a significant impact on the development of microalgal biofuels.

To download the full article for free* click the link below:
Microalgae on display: a microfluidic pixel-based irradiance assay for photosynthetic growth
Percival J. Graham, Jason Riordon and David Sinton.
Lab Chip, 2015, 15, 3116-3124
DOI: 10.1039/C5LC00527B

—————-

About the webwriter

Claire Weston is a PhD student in the Fuchter Group, at the Imperial College London. Her work is focused on developing novel photoswitches and photoswitchable inhibitors.

—————-

*Access is free until 24/08/2015  through a registered RSC account.