Lab on a Chip Blog

Lance Munn and co-workers at Massachusetts General Hospital have developed tissue isolation chambers that can be implanted into the brain or skin of mice beneath a transparent window, to allow host-tumour interactions to be observed over a timescale of weeks to months. A small tumour fragment from a donor mouse was placed within the shallow ‘tumour isolation chamber’ and implanted into another mouse, forcing any vasculature and connective tissue (stroma) to occur in an essentially 2D space. Fluorescent reporters were then used to visualise specific components of the tissue.

Different implantable tissue isolation chambers that were developed. a) 'raft' model; b) 'hole' model; c) 'pillar' model; d) transparent window models in the dorsal skin or brain

By using a shallow chamber, this new method overcomes some of the limitations of other systems used for studying tumour microenvironments and stromal remodelling processes. Other in vivo mouse models use fluorescent reporter and even transparent windows, however the penetration depth of optical microscopy is only a few hundred micrometers, preventing observations below this depth in the tissue. By using tissue chambers of around 150 µm, this issue is circumvented. Another problem can be in visualising structures that extend in the z­ direction, as they may overlap and be hidden; this is overcome in this work by allowing freedom of movement in the x-y plane, while restricting movement or growth in the z direction.

In the studies carried out by the authors, tumour angiogenesis was clearly observed and was found to show the same properties usually observed in tumours. It was also found that migrating blood vessel sprouts were closely associated with bundles of collagen fibres, providing the first evidence for matrix-guided sprouting in tumour angiogenesis. The tissue isolation chambers also allowed analysis of processes that are difficult to study through other methods, due to either the short distances involved, low frequency of occurrence, or rapid dynamics.

Image sequence showing the expansion of vessel sprouts and vascular loops in the tumour isolation chamber. D1=day 1, etc. Pillar structure is indicated by an asterix.

One potential application of this technology highlighted by the authors, is to provide vascularised tissues for transplantation, allowing good blood supply to the transplanted tissue immediately after implantation. In the experiments reported in this paper, stable and mature vasculature was formed that remained functional for more than 2 months after the tissue chambers were implanted. Although these initial findings are very positive, further studies would need to be carried out on a wide range of tissue types.

To download the full article for free* click the link below:

Implantable tissue isolation chambers for analyzing tumor dynamics in vivo
Gabriel Gruionu, Despina Bazou, Nir Maimon, Mara Onita-Lenco, Lucian G. Gruionu, Peigen Huang and Lance L. Munn
Lab Chip, 2016,16, 1840-1851
DOI: 10.1039/C6LC00237D

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Shefali Lathwal and Hadley Sikes at Massachusetts Institute of Technology have carried out an in-depth study published in Lab on a Chip comparing different colorimetric paper-based immunoassays (where a positive or negative result is shown by the appearance or absence of a certain colour). The assays used were all for the detection of an enzyme found in P. falciparum in order to diagnose malaria. The authors sought to identify the optimal readout times for the different methods in order to prevent false positives.

Time course for colour generation on negative and positive surfaces using different colorimetric methods

One of the main purposes of this study was to compare a new paper-based assay that had recently been developed by Sikes in collaboration with George Whitesides at Harvard (Lab on a Chip, 2015) with other state-of-the-art methods.

The new method in question is a colorimetric assay that utilises a photo-initiated polymerisation reaction to amplify the signal when the P. falciparum enzyme is present. The reaction only occurs when the sample is being irradiated, so by using an automated timing switch the reaction time can be accurately controlled and no further signal amplification will take place once the light has been turned off. In contrast, other methods require accurate manual time keeping as they are thermally rather than photochemically controlled. This means that if the sample is left too long, it may lead to false positives due to colour forming in a negative sample.

This can be seen in the figure on the right, where positive and negative controls detected using the different colorimetric methods were photographed at various time points. All the methods tested had the same binding events in order to allow a fair comparison (i.e., all assay steps were the same apart from the detection method). A diagram included in the manuscript (Scheme 2) shows the key steps to all the assays, and how the colour is formed.

Three of the methods were enzymatic amplifications; for these reactions t=0 was taken as when the substrate solution was added to the surface of the paper. Another method was silver deposition, and t=0 was taken as when the silver enhancement solution was added to the surface. For the polymerisation-based amplification (PBA) method, the aqueous monomer was added to the surface and after illumination a basic solution was added, which led to formation of colour in the positive samples; t=0 was taken as when the basic solution was added.

In all cases other than the photo-controlled reaction, colour developed in the negative controls over time, leading to very similar results as in the positive controls. These assays are usually used at the point of care in resource limited settings, therefore the readouts are carried out by eye and there is often not a negative control to compare to. Instead, the result is compared to a colour chart, making it even easier to obtain a false positive if the readout time is not correct.

For the enzymatic amplification and silver deposition the optimal readout times varied considerably and in some cases the time window was very narrow, in order to prevent false positives. In the PBA reaction however, no colour developed in the negative control over 40 minutes, and at all time intervals there was a clear difference between the positive and negative controls. In addition to this, the visual limit of detection for the PBA reaction was much higher than that of the enzymatic amplifications and silver deposition.

This study is the first to compare multiple colorimetric methods for paper-based immunoassays with carefully controlled variables. Previously, different binding reagents, imaging techniques and methods of quantification have meant that meaningful comparisons could not be obtained. The results clearly highlight the benefits of using a photo-controlled reaction, where the reaction time can be carefully controlled with an automated timer without the requirement of accurate manual time keeping.

To download the full article for free* click the link below:

Assessment of colorimetric amplification methods in a paper-based immunoassay for diagnosis of malaria
Shefali Lathwal and Hadley D. Sikes
Lab Chip, 2016, Advance Article
DOI: 10.1039/C6LC00058D
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William Sewell at Massachusetts Eye and Ear Infirmary and Harvard Medical School and Jeffrey Borenstein at the Charles Stark Draper Laboratory in Massachusetts have developed an automated micropump device for direct delivery of drugs into the perilymph fluid within the cochlea. This has potential for use in the treatment of sensorineural hearing loss and would remove the toxicity issues that are common when drugs are administered systemically. This is one of the most common forms of hearing loss and is caused by damage to the sensory hair cells or to the auditory nerve.

Components of the device and process flow for one drug delivery cycle

Due to the small volumes of perilymph fluid within the cochlea (~ 0.2 mL) and the sensitivity of the ear, the authors have developed a reciprocating delivery system, where an accurate volume of the concentrated drug can be infused and, once given time to distribute, the same volume of fluid can be withdrawn, resulting in zero overall net increase in cochlear fluid. The specific design also minimised the dead volume present in the device in order to reduce the amount of pumping needed, and by incorporating capacitors, prevented high flow rates during pumping, which can lead to cochlear damage.

The authors emphasise the need for a device that is small and lightweight enough to be implanted near to the cochlea and that is also able to administer precise sub-microliter volumes of fluid over several days or months. The microfluidic device presented in Lab on a Chip has been fabricated onto a ~4 x 3 cm chip and is capable of delivering accurate and repeatable volumes of fluid over more than 1000 pump strokes. The authors highlight that by incorporating the device onto a head mount, this particular design could be used in animal models for preclinical drug characterisation, where extensive studies are required.

All the fluidic components of this system have been incorporated into the chip, so that, if battery operated, it could be used as a stand-alone device. In this design, a separate controller was used; however, it is stated that the control circuitry could also be miniaturised and incorporated into the chip, for use with a battery. Efforts were also made to minimise the power consumption of the pump for this purpose. The main components of the device are a drug reservoir, a fluid storage capacitor which contains artificial perilymph for flushing the system, an infuse-withdraw line, and multiple valves to control the different steps of the drug delivery process, as shown in the diagram.

Dose control was successfully demonstrated by loading the pump with fluorescein as the test drug and monitoring the fluorescence of the aliquots collected following different dosage schemes. Several studies were also carried out on guinea pigs using a glutamate receptor antagonist as the test drug. This compound reversibly suppresses compound action potentials (CAPs) in the cochlea – monitoring changes in CAP amplitude and threshold can be used to test for hearing loss.

The results showed that fully reversible hearing loss was induced and this was used to estimate the optimum wait time between infusion and withdrawal for the reciprocating delivery. The distribution of the drug in the ear was also monitored by measuring changes to CAPs at different frequencies and comparing these to the known tonotopic organisation of the cochlea. To test for cochlear damage, the authors monitored another hearing response (distortion product otoacoustic emission) that was not expected to change, and determined that there was no acute mechanical damage.

This drug delivery system has excellent potential for use in clinical and preclinical trials and also for long term treatment of hearing loss using existing drugs. The potential for battery operation is particularly important, and is an aspect that the authors are now focusing on for future work.

To download the full article for free* click the link below:

Microfabricated reciprocating micropump for intracochlear drug delivery with integrated drug/fluid storage and electronically controlled dosing
Vishal Tandon, Woo Seok Kang, Tremaan A. Robbins, Abigail J. Spencer, Ernest S. Kim, Michael J. McKenna, Sharon G. Kujawa, Jason Fiering,  Erin E. L. Pararas, Mark J. Mescher, William F. Sewell, Jeffrey T. Borenstein
Lab Chip, 2016, 16, 829-846
DOI: 10.1039/C5LC01396H

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If the Lab on a Chip HOT articles are anything to go by, using microalgae as a feedstock for biofuel is definitely a flourishing research area.  Microalgae is a particularly attractive feedstock as it grows rapidly, has a large oil content, and can be grown pretty much anywhere.

David Sinton and co-workers at the University of Toronto have previously published in Lab on a Chip on this topic and have now reported their work on optimising the conditions for converting microalgae ‘biomass’ into crude biofuel (‘biocrude’). The process by which this is achieved is known as hydrothermal liquefaction. High temperatures and pressures are employed to break down the organic compounds from the biomass into the oils that make up biocrude.

a) Fluorescence images at increasing reaction time; b) Fluorescence and dark-field imaging of fluids at inlet and outlet.

The Sinton lab have developed a microfluidic chip in order to accurately control the reaction conditions of this process and also to study the effect of changing conditions on the biofuel that is formed. The continuous flow and small volume of the chip allow very fast heating of the algal slurry so reaction times can be accurately studied – in fact the heating rate achieved is the fastest reported to date. The slurry was analysed in situ by fluorescence imaging and changes to the fluorescence signature were monitored. Over the course of the reaction, the fluorescence signal due to chlorophyll disappeared and a new peak developed, indicating the formation of the aromatic compounds that are a characteristic component of crude oil and plant based oils.

Further analysis of the samples collected from the chip outlet found that the energy content (measured by the elemental composition) of the biocrude reached saturation after short reaction times – much before the fluorescence signal stopped changing. In addition to this, non-fluorescent droplets could be seen inside the reaction chamber, as shown in the diagram on the left, which were presumed to comprise of aliphatic oils. These findings indicate that analysis of the elemental composition alone is insufficient to measure chemical conversion to biocrude and methods such as fluorescence imaging should also be employed.

This work is the first example of using a microfluidic platform in hydrothermal liquefaction research and just goes to highlight the versatility of lab-on-a-chip systems.

To download the full article for free* click the link below:

Biomass-to-biocrude on a chip via hydrothermal liquefaction of algae
Xiang Cheng, Matthew D. Ooms and David Sinton
Lab Chip, 2016, 16, 256-260
DOI: 10.1039/C5LC01369K

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About the webwriter

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

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The Biomicrofluidics System Group at the Dalian Institute of Chemical Physics in China have published an exciting paper in Lab on a Chip where they have used paper as a material to grow and differentiate human pluripotent stem cells.

Recently, there has been much research into generating biocompatible materials for creating microenvironments for the growth of stem cells, with the aim of improving their regenerative potential. Using paper as the material has several advantages over the conventional polymers – it is cheap and readily available, it is biocompatible, and the bundles of cellulose microfibers that make up paper provide a porous 3D structure.

Identification of cardiomyocytes derived from pluripotent stem cells on paper

The authors used three different types of paper to identify which were best for stem cell growth – printing paper, filter paper, and nitrocellulose membrane. The paper was pre-coated with the required gels and the stem cells were seeded onto the surface. Initially, the stem cells were differentiated into cardiomyocytes prior to being added to the paper to test if the differentiated cells were able to grow on the different types of paper. The cells aggregated on both printing and filter paper and demonstrated spontaneous beating function, but not on the nitrocellulose membrane. These tissues also maintained their beating function for up to three months. The stem cells were then added to the paper prior to differentiation and the required cardiac differentiation procedures were carried out. The cells differentiated to the cardiomyocytes on all three paper types, however the cardiac-specific marker was only expressed weakly on the nitrocellulose membrane. Within two weeks a strong beating function was observed for the printing paper, but not the other paper types. The authors suggest the printing paper had a better pore size to support the cells than the filter paper, while the nitrocellulose membrane didn’t have a favourable microstructure to support growth of cardiac tissue.

Along with this article, there are some impressive videos showing the cardiac tissue beating that are well worth a watch!

To download the full article for free* click the link below:

Human induced pluripotent stem cell-derived beating cardiac tissues on paper
Li Wang, Cong Xu, Yujuan Zhu, Yue Yu, Ning Sun, Xiaoqing Zhang, Ke Feng and Jianhua Qin
Lab Chip, 2015, 15 , 4283-4290
DOI: 10.1039/C5LC00919G

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

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The device

The study and optimisation of microalgal growth is a hot topic at the moment due to the use of microalgae in many industrial processes, as well as its potential use as biofuel. Previously, I have written about a Lab on a Chip article from the Sinton lab on optimising microalgal growth by varying irradiance conditions.

Now Mingming Wu’s group, from Cornell University, have published an article focused on the effect of nitrogen concentration on cell growth rates. Wu has developed a platform based on agarose gel, as shown in the diagram. The nutrient media can flow through this gel while the cells can’t, maintaining separate microhabitats.

The authors decided to study the effect of nitrogen concentration gradients on the microalgae (C. reinhardtii), using ammonium as the nitrogen source. Nitrogen is essential for microalgae, as it is required for protein and nucleic acid synthesis, and ammonium is the preferred source for this particular strain.

An ammonium gradient was obtained by flowing ammonium-containing media through the source channel, and ammonium-free media through the sink channel (diagram C). As expected, increasing the concentration (within the micromolar range) increased the microalgal growth rates. Fluorescence imaging allowed the authors to quantify the growth kinetics using the Monod equation (similar to the Michaelis-Menten equation for enzyme kinetics). This is the first time this has been achieved for this particular microalgal strain with nitrogen concentration as the variable.

Another interesting find was that when the microalgae were subjected to millimolar ammonium concentrations, growth inhibition was seen. The standard medium for microalgae contains 7.5 mM ammonium, so these results suggest that these concentrations need to be reduced by several orders of magnitude in order to maximise growth rates!

Wu and co-workers have nicely demonstrated the capablilty of their agarose-based platform in quantifying growth kinetics and they highlight that it is 50-fold faster, and more cost effective, than the standard chemostat system. They also observed cell heterogeneity during their experiments and plan to use their system to study this further, along with other aspects of cellular behaviour such as quorum sensing.

To download the full article for free* click the link below:

An array microhabitat system for high throughput studies of microalgal growth under controlled nutrient gradients
Beum Jun Kim, Lubna V. Richter, Nicholas Hatter, Chih-kuan Tung, Beth A. Ahner and Mingming Wu
Lab Chip, 2015,15, 3687-3694
DOI: 10.1039/ C5LC00727E

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

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For effective treatment of many illnesses, in particular cancer, long-term monitoring of certain biomarkers is required. A needle probe with a chemically sensitive tip can be used, however this is invasive as it has to be inserted every time a new measurement is taken. There are also issues with tissue heterogenicity, as it is possible that changes in the measurements are solely due to a different local environment within the tissue.

Size of sensor

Previously the Cima lab at MIT reported an improved alternative for long-term in vivo monitoring. A capsule containing an NMR contrast agent was inserted in vivo and measurements were recorded using an MRI scanner. They have now done one better and eliminated the need of very costly MRI equipment by developing a small NMR sensor that simply requires a small external reader coil.

Both the sensor and reader contain a circuit with a coil and when both are in range magnetic inductance occurs, causing field amplification inside the chamber of the sensor.  This effectively means that a reading is taken only from tissue within the sensor, rather than surrounding tissue, and this is responsible for the high selectivity seen.

Different components of the system

In order to test their wireless sensor, Cima and coworkers separately measured pH and oxygen tension, both in vivo and in vitro. For the pH experiments, a polymer gel was used as the NMR contrast agent that had an exchangeable H atom with an appropriate pKa value. Using a tumor mouse model, pH readings were found to be lower when the sensor was nearer the tumor, as expected from the acidic nature of tumors. For the oxygen experiments, silicone was used as the contrast agent. The paramagnetic nature of molecular oxygen alters the relaxation time and this can therefore be used to determine the concentration of oxygen in the sensor.

From the success of their experiments, the authors conclude that they have demonstrated the flexibility of the sensor with these two measurements, and indeed there is huge potential for this NMR probe to greatly simplify in vivo monitoring.

To download the full article for free* click the link below:

Miniaturized, biopsy-implantable chemical sensor with wireless, magnetic resonance readout
C. C. Vassiliou, V. H. Liu and M. J. Cima
Lab Chip, 2015, 15, 3465-3472
DOI: 10.1039/C5LC00546A

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Researchers from the Kaneko Higashimori Lab at Osaka University and the Arai Lab at Nagoya University have observed an interesting phenomenon when studying red blood cells in microfluidic channels. Instead of flowing along the channel in a smooth motion as expected, some cells bounce back and forth between the channel walls in a pinball-like motion at much slower speed. In addition to these ‘cell pinballs’, there are also cells that move at a similar reduced speed, but don’t hit the channel walls.

This altered behaviour could have detrimental effects on microfluidic devices, caused by non-uniform movement of the cells in the channels. In order to prevent these potential problems, the authors have investigated the cause of this behaviour. They noted that cell pinballs only occur when the saline medium is hypotonic, as this causes the cells to inflate due to intake of water. By attaching microbeads to the cells and using a high speed camera, the motion of the cells were studied in more detail. The pinball cells rotated clockwise as they moved to the left of the channel and anticlockwise as they moved to the right of the channel (relative to the direction of the flow).

This observation, combined with the knowledge that the cells were inflated in the hypotonic solution, led the authors to believe that the pinball-motion was occurring due to both the shape of the red blood cell and contact with the channel walls. 3D images obtained using confocal microscopy showed that the upper and lower surfaces of the cells were flattened, confirming that the cells were in contact with the walls.

By studying the different possible deformations of the inflated red blood cells when subjected to flow, the authors found that the contact line (between the cell and wall) and the centre line of the cell were not the same. This explains both types of unexpected cell motion – if the contact line is downstream of the centre line, the cell is unstable to rotational motion and this causes it to move at an angle to the flow, leading to the pinball cells, whereas if the contact line is upstream of the centre line the cell is stable to rotational motion and no displacement occurs, leading to the slow moving non-pinball cells.

From these studies, the authors were able to propose mechanisms that successfully explained the two types of altered red blood cell behaviour in hypotonic solutions, and hopefully in the future this should allow microfluidic systems to be used which will avoid this pinball-motion occurring.

To download the full article for free* click the link below:
Cell pinball: phenomenon and mechanism of inertia-like cell motion in a microfluidic channel
Ryo Murakami, Chia-Hung Dylan Tsai, Makoto Kaneko, Shinya Sakuma and Fumihito Arai
Lab Chip, 2015, 15, 3307-3313
DOI: 10.1039/ c5lc00535c

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About the webwriter

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

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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

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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.

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