Microfluidics for Cell Biology
The Advantages of Flow Control for Cell biology and Microscopy
- Shear stress control
- Automation of multiple fluid delivery
- Long term perfusion
What are the advantages of microfluidics in biology?
The development of microfluidics has brought many advantages for biological research. Indeed, due to the miniaturization and the automation of the setups, the consumption of sample and reagent is largely decreased, the experiment times are considerably reduced and the global costs of the applications are minimized. Moreover, working with small quantities allows better carry out separations and detection with high resolution and sensitivity at reduced costs.
Finally, thanks to this new generation of techniques and equipments, new capabilities are offered for biology research since microfluidics allows to obtain more accurate results, to reduce detection limits and even to perform multiple analyses simultaneously.
To learn more about it, feel free to reed our white paper about microfluidic.
Main applications of microfluidics in cell biology
![Microfluidics for Cell Biology and DNA hibridization](https://www.fluigent.com/wp-content/uploads/2022/01/dna.jpg)
RNA and DNA Hybridization
For decades, in situ hybridization has been used with greater frequency to capture the localization, structure and expression of specific DNA and RNA sequences within tissues. The high sensitivity and specificity of this technique relies on the hybridization of labelled oligonucleotide probes to targeted DNA or RNA sequences within tissues. Chromosomal microdeletion, amplification, structure, translocation and expression can be easily detected.
Compared to PCR analysis, Northern Blotting or a DNA microarray performed on lysed cells, hybridization provides spatial information as specific RNA and DNA are detected within tissues. It has significantly improved gene mapping, cytogenetics and various diagnostic techniques (oncogenic, prenatal, viral infection, etc.).
Recently, with the use of microfluidic for cell biology, a temporal dimension was added to the technology, as it further evolved to be performed on living cells. The spatio-temporal expression, degradation and storage of RNA molecules can now be thoroughly investigated by real time imaging of the hybridization of labelled oligonucleotides in living cells.
![Microfluidics for Cell Biology and Long term perfusion](https://www.fluigent.com/wp-content/uploads/2022/01/petri-dish-1.jpg)
Drug Combination and Long-Term Perfusion
In various therapeutic areas (glaucoma, vascular, HIV, oncology), a combination of drugs is typically found to be more effective for disease treatment. Oncology, in particular, has paved the way for this approach. The emergence of next-generation sequencing technology profiling has revealed the heterogeneity in many cancers at the origin of the differential response to treatment. As a result, therapeutic strategies are evolving towards multi-targeted drug combinations that effectively inhibit the cancer cells and block the emergence of drug resistance while selectively incurring minimal side effects on healthy cells.
Drug combination therapy is not straight forward as in many cases the results do not equal the sum of the parts. Cross-reactions are observed and fall into 5 categories: low risk & synergy, low risk & no synergy, caution, unsafe, and dangerous. The contribution and dosage of each active molecule should be closely investigated in terms of dose, frequency and duration to evaluate the efficacy of the drug combination.
Therefore, microfluidics for cell biology holds great promise in cancer diagnosis and also serves as an emerging tool for understanding cancer biology. Microfluidics can be a valuable tool in cancer investigation due to its high sensitivity, high throughput, less material-consumption, low cost, and enhanced spatio-temporal control.
![Microfluidics for Cell Biology and immunostaining](https://www.fluigent.com/wp-content/uploads/2022/01/immunostaining_image-e1601471930986.jpg)
Immunostaining
Immunostaining experiments are mult-istep protocols to detect specific antigens in biological samples. The sample is successively incubated or exposed to fixation agents, washing buffers, and probes.
In conventional protocols on petri dishes or well plates, fluid deliveries are performed manually using pipettes. Solutions are directly added to the sample.
Transferring protocols from a Petri dish to a microfluidic format usually requires some minor adjustments, as cells or tissue samples are exposed to solutions differently. Therefore, the use of microfluidic for cell biology provides many advantages by replacing the use of Petri dishes with microfluidic chambers (closed systems). Consequently, solutions cannot be deposited directly on the top of the cells but are perfused over the sample at a controlled flow rate, reaching all cells homogeneously. The use of a rotary valve with a perfusion system enables one to perfuse different solutions at given time points in the chip or chamber. In addition, automating the sequential delivery of solutions saves time and avoids creating bubbles inside the chips by disconnecting and reconnecting the traditional pump to the chip.
Resources
How important is the microlfluidic flow control in cell biology?
In drug discovery, it‘s important to control the flow rate of the sample used. As multiple formulations are tested, experimenters must be able to control flow rate while keeping the same level control of fluid handling at each stage of the experiment. DNA and RNA hybridization are often performed under a microscope. Manual injection over a microscope can be risky for the following reasons:
- A touch of pipette cone can misplace the dish and misrecord the position
- Samples can be flushed away during pipetting
- Liquid can be spilled over the microscope
Flow Controllers Available on the Market
Syringe Pumps
Multiple flow control technologies are available for sub-millimeter range fluid management. Syringe pumps are commonly used for fluid control. However, in microfluidics, particularly in microfluidics for cell biology, the use of syringe pumps is often problematic because the flow is not constant. This discontinuity is due to the mechanical system that creates an oscillating flow.
Manual Injection
Current methods for multiple fluid delivery, where microscopy techniques are simultaneously required and mostly made by manual pipetting action include , Immunolabeling, and DNA and RNA hybridization.. Manual injection can lead to uncontrolled and non-homogeneous fluid velocity, which can damage samples.
Pipette | Aria | |
---|---|---|
Type of injections | Abrupt injections (up to 1mL in few seconds) Disparate injections Turbulent flow | Smooth & controled injections Identical injections Laminar flow |
Geometry at injecion tip | Conic shape: Important shear strain Unhomogeneous fluid velocity | Straight shape: No modification at the injection tip |
Fluigent Pressure-Based Flow Control
As the demand for microfluidic pumps with higher flow stability, fast response time, versatility, and automation capabilities has increased, pressure controllers have become the device of choice for many users.
The working principle of such pumps is to pressurize the sample reservoirs to control the pressure drop between the inlet and the outlet of the microfluidic system. The responsiveness of the generated flow rate depends on the responsiveness of the pressure pump.
To overcome these issues, Fluigent’s Aria automates multiple fluid deliveries. The sample is preserved as the flow rate is controlled. Reproducibility increases as inter and intra operator variability are eliminated.
![Microfluidics for Cell Biology and stability response time](https://www.fluigent.com/wp-content/uploads/2021/12/stability-response-time-1.png)
A new level of technology
Microscopy and cell biology
Flow cells or microfluidic chips combined with a fluid perfusion system and microscopy are compatible with a variety of applications, including DNA and RNA hybridization, drug combination and long-term perfusion, and immunostaining.
Cell culture and Organ-on-a-chip
This area is an ideal microenvironment to study molecular and cellular-scale activities that underlie human organ function as well as identify new therapeutic targets in vitro.
Digital PCR
Microfluidic dPCR offers a new level of insight compared to quantitative PCR (qPCR). Droplet-based microfluidics is an excellent solution for partitioning a sample.
Want to learn more about the capabilities of microfluidics in life science? See Fluigent’s CEO France Hamber discuss microfluidic disruptive discoveries
Research applications
Industrial applications
Microfluidics in biology and medicine
Microfluidics has emerged as a transformative technology, revolutionizing various areas of research, diagnostics, and therapeutics. By enabling precise manipulation and control of small volumes of fluids at the microscale level, microfluidic devices, also known as lab-on-a-chip, have opened up new possibilities in the study of cells, diagnostics, DNA sequencing, and drug discovery. In biology, microfluidics allows researchers to analyze and manipulate individual cells, paving the way for advancements in cell biology and single-cell genomics. In medicine, microfluidic platforms offer portable, rapid, and cost-effective point-of-care diagnostics, bringing healthcare to resource-limited settings, e.g. by using organ-on-chip platforms.
As an example, microfluidics in life science has accelerated RNA sequencing technologies, enabling high-throughput sequencing at reduced costs. In the application note linked below, Fluigent presents the Drop-Seq method, a high-throughput method that enables sequencing of the mRNA from a large number of cells. The Drop-Seq method pairs barcoded beads and cells in droplets, capturing cell-specific RNA on beads. Tagged cDNA is generated, amplified, sequenced, and bioinformatically analyzed. Barcode sequences identify cells, and transcript sequences are mapped to a reference genome, forming cell-specific gene-expression profiles.
Microfluidic tools for cell biological research
Cellular biology plays a crucial role in the field of medical science and drives innovation. A comprehensive understanding of how cells react during infections and pathological conditions is instrumental in the discovery of novel disease treatments. Essential to the analysis and visualization of cellular behavior are microscopy and imaging technologies. In the realm of cell biology, the integration of precise fluid flow control within microfluidic systems has revolutionized the study of cells by providing an automated platform that mimics their natural environment.
Microfluidics facilitates the delivery of fluids at controlled flow rates, minimizing errors. It finds applications in diverse areas such as RNA and DNA hybridization, enabling real-time imaging of gene expression in living cells. Microfluidics also contributes to drug combination therapies, long-term perfusion studies, and improved immunostaining protocols. By replacing traditional methods, microfluidics in life science offers several advantages including enhanced control, sensitivity, throughput, cost-effectiveness, and reduced material consumption. Its potential impact extends to cancer diagnosis, enhancing our understanding of cancer biology, and driving advancements in medical research.
Advantages of pressure-based flow controllers for life science experiments
Pressure-based flow controllers offer several advantages over peristaltic pumps and syringe pumps in microfluidics for life science applications. Firstly, it provides precise and accurate control of flow rates, allowing for better reproducibility and control of experimental conditions. Secondly, the pressure-based system is less prone to pulsations and variations in flow, resulting in more stable and consistent flow profiles. Additionally, using pressure-based controllers in life sciences allows researchers to work with much smaller volumes, which is useful when the samples to be studied are expensive or rare.
Finally, using pumps like the Flow EZ allows for use of various accessories such as reservoir mixers or block heaters, which can reproduce the physiological conditions of cells, for example, and keep the sample homogeneous, which is much more complex, if not impossible, with other types of pumps.
Organ-on-chip studies for life science
Organs-on-chips (OoC) are microdevices that mimic organ functions. They use microfluidics and 3D culture to replicate human physiology. OoC models show that dynamic culture conditions affect system maturation. They aim to recreate tissue barriers, parenchymal tissues, and inter-organ interactions. An OoC system includes a microfluidic chip with cell culture chambers, fluid channels, and optional components like membranes or gels. Biosensors and bio-actuators may be added. This technology is very useful for microfluidics in life science applications, as it offers more realistic lab modeling of organs and tissues. To that end, Fluigent has created Omi, a versatile and automated platform for organ-on-chip studies.
Omi allows for long-term cell culture with controlled shear stress conditions through continuous flow. It offers customizable and automated protocols for various functions, such as perfusion, recirculation, sampling and injection. This platform caters to the requirements of both novice cell culture researchers and experienced organ-on-chip researchers, addressing their needs for automation and reproducibility.
Microfluidic in Life Science Case Study
Hans-Knöll-Institut, New Antibiotics, Cultivation in Droplets
This project focuses on how Fluigent’s customers use microfluidics to achieve outstanding results in their technology experiments.
Our speakers are Prof. Dr. Miriam Agler-Rosenbaum (Head of Bio Pilot Plant Department), Dr. Sundar Hengoju, Dr. Dede Man from the Bio Pilot Plant Department of the Leibniz Institut for Natural Products Research and Infection Biology (Hans Knöll Institute).
- How are they trying to find new antibiotics?
- Why is this important for infection biology?
- How do they research natural products?
- What are these products exactly?
- How does microfluidics allow them to cultivate microbes in droplets?
- How does the technology increase their efficiency millions of times over?
- In which cases are pressure pumps better than syringe pumps?
- How can microfluidic droplets be stabilized?
Watch this episode to find out – and note that this is just one story told. There are many possible applications for microfluidics.
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From the life sciences to the food industry, many applications require the use of fluids driven at flow rates from nanoliters to milliliters per minute. At such low flows, the success of these applications strongly depends on the level of control and automation of the fluidic operations.
These applications require flow control systems that are adapted for ensuring their success.
About The Keyser lab – University of Cambridge
The Keyser Lab is a group of researchers at the Cavendish Laboratory, University of Cambridge, UK. Since its founding in 1874, the Cavendish Laboratory has been at the forefront of discovery in physics, with a core focus on experimental physics supported by excellence in theory. The department promotes world-leading experimental and theoretical physics in all its diversity. Scientists from the Keyser Lab study the physics of ions, macromolecules and particles, with a particular focus on particles in confined geometries at the single molecule/particle level. To exert maximum control over all parameters for their experiments, they make use of several cutting-edge techniques such as DNA self-assembly (origami), optical trapping, electrophysiology, and microfluidics and nanofluidics.
The team includes researchers with expertise in physics, engineering, physical chemistry, biochemistry/biology, and micro- and nanofabrication.
![university of cambridge logo](https://www.fluigent.com/wp-content/uploads/2022/01/univ-cambridge-logo.png)
![keyserlab logo](https://www.fluigent.com/wp-content/uploads/2022/01/keyserlab-logo.png)
What are Giant Unilamellar Vesicles (GUVs)?
Giant Unilamellar Vesicles (GUVs) are micron-sized compartments composed of lipid bilayers, serving as models for cell membranes or as encapsulating structures for biological materials within cell-like environments. These vesicles have dimensions ranging from 1 to 100 µm, mimicking the size of cells. Like natural cell membranes, GUVs are constructed from lipids, predominantly phospholipids and cholesterol. The amphiphilic properties of these lipids enable them to spontaneously arrange into spherical compartments when immersed in an aqueous solution.
They offer the advantages of having well-defined lipid compositions, being easy to image, and being controlled systems for studying transport processes. These characteristics open the door to a variety of applications in biology and biomedicine, especially membrane biophysics and synthetic biology.1,2
![Structure of Giant Unilamellar Vesicles](https://www.fluigent.com/wp-content/uploads/2022/01/guv-structure.jpg)
Working principle of GUV production
![on-chip-production-of-liposomes-using-ola](https://www.fluigent.com/wp-content/uploads/2022/01/on-chip-production-of-liposomes-using-ola.jpg)
In 2016, the Dekker Laboratory from the Delft University of Technology developed a novel microfluidics-based method called Octanol-Assisted Liposome Assembly (OLA) to create uniform, cell-sized (5–20 mm) GUVs with high encapsulation efficiency. Using Fluigent’s pressure-driven MFCS-EZ controller with the Oxygen software tool, an inner aqueous phase (IA) and an outer lipid-carrying 1-octanol phase (LO) were combined, resulting in double-emulsion droplets via hydrodynamic flow focusing. These droplets developed a side-connected 1-octanol pocket, which, due to interfacial energy minimization, separated to rapidly form fully assembled solvent-free liposomes. Microfluidic GUV production addresses the persistent issue of residual oil in vesicle bilayers.
This method allows researchers to generate GUVs with higher monodispersity and greater size control compared to traditional methods, and with a faster process than alternative microfluidic methods.3
Keyser Lab: Microfluidics for GUV generation for antimicrobial efficacy and biomimetic vesicle membrane testing
Scientists from the University of Cambridge integrated octanol-assisted liposome assembly into their microfluidic platforms (“lab on a chip” devices) for quantifying drug permeation and antimicrobial efficacy on biomimetic vesicle membranes.
In a first paper published in Lab on a Chip (2019), they reported on a microfluidic platform for testing antimicrobial peptides on artificial vesicle membranes. This platform produced vesicles with an encapsulated dye to assess the efficacy of antimicrobial peptides by measuring the time it takes for vesicles to lyse.4
This Giant Unilamellar Vesicle production technique also utilized Fluigent‘s MFCS-EZ and its accompanying software (Oxygen) for fluid control. Vesicle formation and perfusion inlets were connected, and the microfluidic chip was loaded with an inner aqueous phase base stock. The outlet was connected to Fluigent’s 2-Switch, which can switch between open or closed configurations for waste removal. Vesicles flowed through a channel to the connector chip, and 1-octanol pockets pinched off to form droplets. Density-based separation removed vesicle production waste, and the vesicles were distributed to trapping chambers. Peptide doses were controlled, replacing the inner aqueous phase buffer, while maintaining a constant input pressure. The entire process was monitored under an inverted microscope.
Validated with cecropin B on bacterial-mimicking membranes, the platform enabled the researchers to study over 1000 vesicles simultaneously. The results showed dose-dependent disruption of vesicle membranes, demonstrating the platform’s potential for controlled, quantitative assessments of the efficacy and selectivity of antimicrobial peptides. This microfluidic approach to GUV production is suggested as a new standard for pre-clinical development of membrane-active antimicrobials, offering advantages in cost efficiency and parallelization.
![membrane-active drugs testing microfluidic platform](https://www.fluigent.com/wp-content/uploads/2022/01/guv-microfluidic-platform.jpg)
![membranolytic activity of cecropin and GUV fluorescence](https://www.fluigent.com/wp-content/uploads/2022/01/microfluidic-guv-production.png)
In another paper published in Biomembranes (2020), scientists from the University of Cambridge and the University of Exeter produced GUVs with tunable binary lipid mixtures to determine lipid diffusion in OLA vesicles.5
The focus was on expanding the capabilities of this microfluidic approach to form GUVs with tunable binary lipid mixtures. An MFCS-EZ equipped with a Fluiwell-4C reservoir kit with OLA solutions was connected to the microfluidic chip. The microfluidic GUV production method allowed for a high degree of control over vesicle sizes by adjusting pressures in different channels. This was possible due to the high responsiveness, stability and repeatability of the pressure generated by the MFCS-EZ. In addition, flow speed was successfully matched to the outlet channel length to prevent residual octanol attachment to vesicles.
This study employed fluorescence recovery after photobleaching to investigate lipid lateral diffusion coefficients in GUVs produced by the microfluidic approach, finding values within the expected range. Comparisons with electroformed vesicles indicated quantitative similarity in lipid diffusion coefficients. The results served as a quantitative biophysical validation of OLA-derived GUVs, enhancing the potential application of this versatile platform in drug discovery, artificial cell production, and lipid membrane studies.
![boxplot of the lipid lateral diffusion coef](https://www.fluigent.com/wp-content/uploads/2022/01/boxplot-of-the-lipid-lateral-diffusion-coef.png)
![monodisperse-stable-and-controlled-vesicle-formation](https://www.fluigent.com/wp-content/uploads/2022/01/monodisperse-stable-and-controlled-vesicle-formation.jpg)
![Confocal images of various lipid systems](https://www.fluigent.com/wp-content/uploads/2022/01/confocal-images-of-various-lipid-systems.png)
Conclusion
To produce Giant Unilamellar Vesicles, fluids were controlled using Fluigent pressure-based flow controllers. Researchers from the Keyser Lab (University of Cambridge) typically operated the chip with input pressures of 40 mbar for the inner aqueous phase and dissolved-lipid phases and 100 mbar for the outer aqueous phase. For precise flow measurements, Fluigent Flow Units can be added on the fluidic path. Microfluidic GUV production also allowed researchers to adjust the sizes of the generated vesicles by adjusting the microfluidic pressures of the phases. This degree of control is difficult to achieve using standard methods such as electroformation or traditional syringe pumps.
![Labeled GUV](https://www.fluigent.com/wp-content/uploads/2022/01/labeled-lipids.gif)
Testimonials
![](https://www.fluigent.com/wp-content/uploads/2022/01/guillemet-150x150-1.png)
“Microfluidics presents various advantages to researchers who need small volumes and high throughput in answering their scientific questions. In our lab, we use microfluidic devices for standardization and control of experimental parameters like concentration and timing. In the complex (biological) systems we are working on, the mentioned characteristics are fundamental in collecting reliable meaningful statistics, and microfluidics in combination with light microscopy offers just that. We also heavily rely on the ability to rapidly prototype devices, as we can design bespoke solutions at minimal production cost and time.”
“We use the pressure-based pumps from Fluigent for experiments that require swift responsiveness when manipulating fluids, and fine tuning at low flow rates. We use the Fluigent systems during fabrication and running of the microfluidic chips. The ability to pump in air at high precision makes the Fluigent pressure-based systems ideally suited to selectively coat and functionalize micro-channels within a microfluidic network. After coating we then fill the devices with the experimental solutions and use the pressure controls to move fluids around, open and close valves and carefully time the introduction of small molecules in the experiments.“
Kareem Al Nahas, University of Cambridge
Expertises & Resources
References
- Naziris, N., Demetzos, C. (2021). Liposomes: Production Methods and Application in Alzheimer’s Disease. In: Vlamos, P. (eds) GeNeDis 2020. Advances in Experimental Medicine and Biology, vol 1339. Springer, Cham.
- Pereira, David & Valentão, Patrícia & Andrade, Paula. (2014). Nano- and Microdelivery Systems for Marine Bioactive Lipids. Marine Drugs. 12. 6014. 10.3390/md12126014.
- Deshpande, S.; Caspi, Y.; Meijering, A. E. C.; Dekker, C. Octanol-Assisted Liposome Assembly on Chip. Nat Commun 2016, 7 (1), 10447.
- Al Nahas, K.; Cama, J.; Schaich, M.; Hammond, K.; Deshpande, S.; Dekker, C.; Ryadnov, M. G.; Keyser, U. F. A Microfluidic Platform for the Characterisation of Membrane Active Antimicrobials. Lab Chip 2019, 19 (5), 837–844.
- Schaich, M.; Sobota, D.; Sleath, H.; Cama, J.; Keyser, U. F. Characterization of Lipid Composition and Diffusivity in OLA Generated Vesicles. Biochimica et Biophysica Acta (BBA) – Biomembranes 2020, 1862 (9), 183359.
A bit of history
Miniaturization techniques have been continuously shaping the microelectronics technology and its ability to produce devices for a broad range of applications. Downscaling the dimensions of transistors have been pursued in the industry to fulfill the demand for smaller, faster and energy-efficient systems1. In the late 1960s, a new field named microelectromechanical systems (MEMS) emerged.
This laid the foundations for the miniaturization of mechanical systems, with the development of micromachining technology based on silicon semiconductor technology. That is when engineers began realizing that silicon chips could process things such as chemicals, motion, and light.
The first implementation of inkjet printer heads demonstrated that micron-sized droplets could be generated using piezoelectric or thermal effects. In 1990, Manz et al. introduced the concept of miniaturized total chemical analysis systems (μTAS)2.
Soon after, companies were founded to utilize these systems for life science applications. Rapid prototyping and replication of polymers (such as PDMS) as an alternative to silicon processing, accelerated academic research, and new terminologies such as “microfluidics” and “lab-on-a-chip” (LOC) emerged.
Chip manufacturers
![](https://www.fluigent.com/wp-content/uploads/2022/01/transparent-1.png)
Beonchip S. L. was founded in 2016 at the University of Zaragoza by Ignacio Ochoa (specialist and PhD in biology), Luis Fernández (PhD in microtechnology) and Rosa Monge (PhD in mechanical Engineering).
Therefore, Beonchip have multidisciplinarity in its roots, and their team is proof of that. The collaboration of engineers and biologists has been key to design the most user friendly and accessible organ on chip devices that are true to the physiological environment of the body.
![](https://www.fluigent.com/wp-content/uploads/2022/01/bedoubleflow-2-600x423-1.jpg)
![](https://www.fluigent.com/wp-content/uploads/2022/01/flowjem-banner-green-transparent-2048x453-1.png)
FlowJEM technology was developed for research and development projects by microfluidic researchers and engineers like you.
Microfluidic devices fabricated by FlowJEM have a broad range of applications, including but not limited to chemistry, materials science and technology, nanoscience, pharmaceutical science, biology, bioengineering and medicine.
FlowJEM’s goal is to meet the demands for high quality, fast turnaround time and low cost. Work with us to develop your ideas into prototypes or transition your prototypes to volume production.
![](https://www.fluigent.com/wp-content/uploads/2022/01/129a4474-edit-web-600x400-1.jpg)
![](https://www.fluigent.com/wp-content/uploads/2022/01/logo-secoya-no-background-transparent-3-2048x722-1.png)
Secoya Technologies develops innovative production technologies and equipment by a smart use of intensified operational units. It results in stable and reliable processes – at any scale – producing high quality (bio-)pharmaceutical products
Using cutting-edge technologies as sub-micrometric 3D printing and micro-electro-erosion, SECOYA has developed a unique device that is able to continuously produce micro-sized droplets at a very high frequency (kHz) and with a very narrow size distribution (high monodispersity). Its use for the encapsulation of active ingredients and multiple emulsions has been demonstrated.
![](https://www.fluigent.com/wp-content/uploads/2022/01/raydrop-dessus-2-600x400-1.jpg)
![](https://www.fluigent.com/wp-content/uploads/2022/01/logo_cmyk-original.png)
microfluidic ChipShop offers a wide off-the-shelf selection of microfluidic chips from thermoplastic polymers and the complementary accessories. They furthermore support customized prototyping as well as volume production services – from one to millions of chips, from simple microfluidic devices to complex lab-on-a-chip-systems.
![](https://www.fluigent.com/wp-content/uploads/2022/01/gradientchiplogo-600x400-1.png)
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Bi/ond empowers biological innovation by engineering microchips which nourish, stimulate and monitor your cells. Bi/ond’s Organ-on-Chips are compatible with a wide variety of complex 3D tissues (organoids, patient-derived samples, cell monolayers) and they have been qualified in multiple applications such as Brain-on-Chip, Heart-on-Chip and Cancer-on-Chip. When you buy one of our OOC, you are not only purchasing a product, you are adding an engineer to your team. Bi/ond’ team will help and support you to set up your model with our extensive knowledge of microfabrication, simulations and biology.
![](https://www.fluigent.com/wp-content/uploads/2022/01/complate-600x400-1.jpg)
![](https://www.fluigent.com/wp-content/uploads/2022/01/micronit_technologies.png)
Micronit is the leading innovator and global partner in design, development and manufacturing of customer specific lab-on-a- chip solutions for life science and health applications.
With over 20 years of experience, a highly qualified team, state-of-the-art technologies and certified manufacturing facilities, we deliver innovative and competitive products to our customers for life science research towards personal health applications.
![](https://www.fluigent.com/wp-content/uploads/2022/01/large-droplet-generator-in-chipholder-600x400-1.jpg)
References
- Chiu, J. and Chie
- Temiz, Y., Lovchik, R. D., Kaigala, G. V. & Delamarche, E. Lab-on-a-chip devices: How to close and plug the lab? Microelectron. Eng. 132, 156–175 (2015).
- A. MANZ, N. G. and H. M. W. Miniaturized Total Chemical Analysis Systems: a Novel Concept for Chemical Sensing. Sensors and Actuators 17, 620–624 (1990).
I. Droplet-based microfluidics
Microfluidic generation of droplets has attracted a lot of interest, enabling high throughput experiments by generating millions of micro-reactors and chambers in a few seconds. This method produces highly monodispersed droplets of very small volumes (μL to fL) of fluids with high frequency (up to hundreds of kHz), providing better control of processes like mixing, encapsulation, sorting, and sensing. Microfluidic-based droplets have many diverse and varied applications, such as particle synthesis3 and chemical analysis4. Highly controlled droplet production also enables single-cell analysis or drug testing 5,6.
Microfluidic-based droplet generation and control allow for:
- Highly monodispersed (<2% size variation) droplet production, with potentially high generation rate (up to hundreds of kHz)
- Highly reproducible complex structures (water-in-oil-in-water emulsions, multiple encapsulations…)
- Single droplet manipulation as an individual pL scale biochemical reactor
- Miniaturization of production and bioanalytical devices
With these characteristics, droplet microfluidics has a large value, including bio(chemical) analysis, and nano and microscale generation of materials7. Several chip designs exist to generate droplets. A common design is the T-junction, where the dispersed phase is injected from a channel that is perpendicular to the channel carrying the continuous phase (figure 1).
Droplet microfluidic concepts, components, and processes are now being adopted and leveraged by end-users to enable new science and innovation. Real-world success is now evidenced through a range of mainstream commercial products that are applied to key biological and healthcare-related problems (e.g., 10X Genomics, Drop-seq, and nucleic acid quantification via Droplet DigitalTM PCR systems)8.
Today, it is possible to produce multiple emulsions with complex droplet morphologies. Production of multi-cored droplets (droplets that contain a controlled number of inner droplets at one or more hierarchical levels), Janus droplets (i.e. biphasic or triphasic droplets with two or three physically and chemically distinct surface domains) is now possible using droplet microfluidics9 (figure 2).
![](https://www.fluigent.com/wp-content/uploads/2022/01/janus-1.png)
Figure 2: Types of multiple emulsions from the simple encapsulation (a), double emulsion (b), and so on (c,d) to bi- and tri-phasic structures (e to h), multiple encapsulations (I to k), and hybrid microjet achievable using droplet-based microfluidics9
A microfluidic chip for the production of highly reproducible PLGA microparticles
In recent years, biodegradable microspheres/microparticles have gained widespread importance in the delivery of bioactive agents10. The copolymers of Poly (D, L-lactic-co-glycolic acid) (called PLGA)/poly (lactic acid) (called PLA) microparticles are one of the most successful new drug delivery systems (DDS) in labs and clinics. Because of their biocompatibility and biodegradability, they can be used in various areas, such as long-term release systems, vaccine adjuvant, and tissue engineering11.
In PLGA microparticle production for drug release and delivery, microparticle size is a key parameter as it is directly related to the microparticles degradation rate and the drug release rate12. Although PLGA microparticle synthesis appears to be a successful drug delivery system, the current processes and tools to produce PLGA microparticles have many limitations, such as wide microparticle size distribution, poor repeatability, and aggressive chemical preparation conditions11. To solve these problems, droplet-based microfluidics offers an efficient method for improvement.
Fluigent provides the Raydrop: a new breakthrough technology leading to outstanding particle size monodispersity and production flexibility. The Raydrop works as a co-flow focusing principle (figure 3). The nozzle and outlet capillary are aligned in a continuous phase chamber, the dispersed phase comes through the nozzle to create the microparticles into the continuous phase and exits by the outlet insert. Using this system, PLGA microparticles ranging from 15 to 50 µm diameters have been successfully generated.
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig3-750x200-2.png)
The PLGA microparticle production station allows excellent reproducibility and significantly improved monodispersity (CV < 2%) as compared to other methods on the market. It allows one to continuously produce PLGA microparticles (up to 10 000 droplets/s) without unwanted interruption for long-term experiments.
Single-cell mRNA-seq sequencing using droplets: Drop-seq technology
The production of highly monodispersed emulsions or more complex structures (water-in-oil-in-water emulsions, multiple encapsulations…) makes microfluidics an excellent tool for single-cell analysis or single-cell culture. The technique allows for droplet-based single-cell RNA-sequencing, such that one can characterize complex tissues with many cell types and states under diverse conditions. One of the pioneering methods is Drop-Seq technology, which entraps a single cell and a single primer-barcoded bead in each droplet (figure 4).
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig4-600x599-1.png)
Cells are thus separated into nanoliter-sized aqueous droplets, with a different barcode associated with each cell’s mRNAs. The primers on beads contain a barcode consisting of three sequences. One sequence is for PCR amplification and is common to all the beads. The second sequence consists of hundreds of individual primers that also share the same ‘‘cell barcode’’. Finally, the third part has different unique molecular identifiers (UMI), enabling mRNA transcripts to be digitally counted13 (figure 5). The droplets are sequenced altogether, allowing quick profiling of thousands of individual cells from a heterogeneous population. The power of this technology resides in the fact that during sequencing, one can distinguish where the original information came on a cell to cell basis. This allows one to make a gene expression map of the cell, or even to distinguish cell populations within a tissue.
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig5.png)
II. Microfluidic cell culture
Microfluidic cell culture has significant advantages over macroscopic culture in flasks, dishes, and well-plates14 (figure 6). The microfluidic chip fabrication process allows great flexibility in the design of microfluidic devices, permitting one to understand and control interactions between cells, substrates, and the surrounding medium, physically as well as biochemically15.
This technology offers new possibilities to accurately reproduce the cellular environment and enables the analysis of biological processes that were not accessible before. Morphology-wise, chips can be structured at the cell scale to reproduce the mechanical constraints experienced by cells. Biochemically, stable gradients can be implemented with a high spatial resolution (typically, micrometer resolution).
Finally, constant perfusion enables the continuous renewal of nutrients and oxygen to promote cell growth and maintain optimal activity during long-term cell culture. Cost reduction due to volume reduction is also a major benefit15.
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig6-1024x650-1.png)
Development and characterization of a microfluidic model of the tumor microenvironment
The physical microenvironment of tumors is characterized by heterotypic cell interactions and physiological gradients of nutrients, waste products, and oxygen. This tumor microenvironment has a major impact on the biology of cancer cells and their response to chemotherapeutic agents. Despite this, most in vitro cancer research still relies primarily on cells grown in 2D and in isolation in nutrient and oxygen-rich conditions. Ayuso et al. presented an easy to use microfluidic device that can mimic the three-dimensional architecture of multicellular spheroids, while at the same time generating a visible, live “tumor slice” that allows easy monitoring of cells in different regions of the microenvironment in real-time as well as their response to different drugs17 (figure 7).
In this study, tumor cell behavior in different regions of the microdevice was studied and analyzed in conjugation with measurements of hypoxia and glucose concentrations across the device. The differential cellular response to several well-known drugs in different parts of the microdevice emphasizes the potential of this technology for analyzing the impact of microenvironmental parameters on drug response.
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig7-350x430-1.png)
Figure 7: Microdevice operation of the tumor microenvironment. (A) 3 microdevices in a Petri dish containing a central culture chamber (detailed in C) and 6 channels. (B) One microdevice is filled with (yellowish) collagen hydrogel flowing to the microchamber from the right middle channel and blue-colored water perfused through the two lateral microchannels. (C) Culture medium perfused through the lateral microchannels provides nutrients and oxygen creating physiological gradients across the device. Cells near the ‘surrogate’ blood vessels are viable, whereas oxygen-poor cells in the center of device start to die creating a ‘necrotic core’ similar to the necrotic regions of tumors. (D) Cellular monitoring with fluorescent dye in the microdevice17.
III. Organ on a chip
Many efforts are devoted to the development of cancer metastasis models that can help in understanding the disease and the development of innovative therapeutic strategies. Current in vitro and in vivo cancer models are incapable of satisfactorily predicting the outcome of various clinical treatments on patients18. Therefore, new methods and approaches for drug discovery and health research are being developed. The concept of mimicking the organ-level function of human physiology or disease using cells inside a microfluidic chip was first published in 2004. In 2010 that the term organ-on-a-chip (OOAC) was invented by Ingber, et. al., who developed a microfluidic chip to capture organ-level functions of the human lung19. Microfluidics enables one the unique ability to control the cellular microenvironment with high spatiotemporal precision and to present cells with mechanical and biochemical signals in a more physiologically relevant context19. The manipulation of the micro-liter volumes of liquids has made these models a platform where scaling, and dynamic crosstalk between cells can be achieved. Microfluidic chips can now use geometries and structures to permit the use of physiological length scales, concentration gradients, and the mechanical forces generated by fluid flow to mimic the in vivo microenvironment experienced by cells20. These biomimetic platforms overcome many drawbacks encountered with conventional tissue culture models. OOAC engineering has attracted enormous interest and attention from the pharmaceutical industry, regulatory agencies, and even national defense agencies. This is demonstrated by the increase of OOAC research papers and by the emergence of at least 28 organ-on-a-chip companies in less than seven years21.
A microfluidic chip to reconstitue organ-level lung functions
To demonstrate that it is possible to engineer a microsystem that replicates the complex physiological functionality of living organs, Huh et al. developed a multifunctional microdevice that reproduces key structural, functional, and mechanical properties of the human alveolar-capillary interface, which is the fundamental functional unit of the living lung19. The device consisted of compartmentalized PDMS microchannels to form an alveolar-capillary barrier on a thin, porous, flexible PDMS membrane coated with the extracellular matrix and human alveolar epithelial cells and human pulmonary microvascular endothelial cells is cultured on opposite sides of the membrane (figure 8). Air is subsequently introduced into the compartment to create an air-liquid interface to mimic the lining of the alveolar air space19
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig8-1024x449-1.png)
Using this organ on a chip platform, the authors demonstrated that breathing motions, simulated by the platform, might greatly accentuate the proinflammatory activities of silica nanoparticles and contribute substantially to the development of acute lung inflammation19. This behavior could not be determined using existing in vitro models.
IV. Particle/cell sorting
Isolating and sorting cells from complex, heterogeneous mixtures represents a critical task in many areas of biology, biotechnology, and medicine. Cell sorting is often used to enrich or purify cell samples into well-defined populations to enhance efficiency in research and development applications22. Cell detection is generally performed using optical methods such as FACS (Fluorescent Activated Cell Sorting), where cells pass through a laser beam and the light scattered is characteristic to the cells and their components. Cells are subsequently sorted according to the scattered emission. It is an automated and robust solution for cell sorting and was considered a gold standard for cell sorting. The immense contributions of current commercial cell sorting platforms using FACS are tempered by several significant and persistent limitations, including limited sample throughput and processing speeds that make the generation of clinical-scale samples very difficult.
As opposed to conventional instrumentation, microfluidic chips are affordable, easy to use, and smaller. These chips make use of a wide range of techniques to sort cells, with specific speeds and efficiencies. The dimensions of the chip also allow a wide range of cell sorters to exist, from large volume cell sorters to precise single-cell sorters. Moreover, microfluidic cell sorting can be combined with additional fluidic operations within a single chip for complete lab-on-a-chip applications, diagnostic and therapeutic purposes. Microfluidic cell sorting is a promising application both in academic and industrial labs. Cell sorting is based on determining a specific cell parameter that can differ from one cell type to another, such as cell size, shape, density, surface markers. In general, a heterogeneous cell solution is injected through a microfluidic chip.
The chip is designed such that cells with different properties experience a different amount of force (that could originate from inertia, channel geometry, external sources …) while similar cells undergo equal forces. The differentiated cells are subsequently pushed into different streamlines and exit the chip from different outlets. Microfluidic cell sorting can be categorized into three distinct groups: fluorescent label-based, bead-based, and label-free cell sorting22. Label-free cell sorting in microfluidic devices is perhaps the most studied of the three approaches as it encompasses active systems (i.e., systems that rely on the use of external fields cells for sorting), and passive systems that do not rely on fluorescent labels or beads. Instead, these methods rely on the inherent differences in cellular morphology between cell groups22.
Inertial microfluidics for continuous particle separation in spiral microchannels
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig9-400x280-2.png)
Kuntaegowdanahalli et al. developed a simple inertial microfluidic device that achieves continuous multi-particle separation using the principle of Dean-coupled inertial migration in spiral microchannels.
The dominant inertial forces coupled with the Dean rotational force due to the curvilinear microchannel geometry, cause particles to occupy a single equilibrium position near the inner microchannel wall. The position at which particles equilibrate is dependent on the ratio of the inertial lift to Dean drag forces.
Using this concept, they demonstrated a spiral lab-on-a-chip for size-dependent focusing of particles at distinct equilibrium positions across the microchannel cross-section from a multi-particle mixture23.
A major benefit of this system is its high throughput (e.g., 1.5 mL/min) without sheath flow or sequential cell manipulation, which is useful for processing native biological fluids and flow cytometry.
V. Micromixers
Micromixers are important components of lab-on-chip devices for applications in drug delivery, sequencing, amplification, and biochemical reactions. Based on the actuation method, micromixers can be broadly classified as passive and active. In passive mixing, no external sources are used. Thus mixing typically relies on the microfluidic chip geometry and on fluid properties. Under laminar flow, which is the typical fluid regime in microfluidics, mixing mostly happens through diffusion. This property allows performing mixing using lamination: two or more liquids are flowing in parallel, allowing for diffusion to happen and thus permitting to precisely tune mixing between liquids. For improved and faster mixing, chaotic advection can be generated by modifying the microfluidic chip geometry, such as by altering the shape of the channel for splitting, folding, stretching, or breaking the flow of fluid.
Active mixing occurs by using an external perturbation. Many active mixing methods exist. Dielectrophoresis mixing makes use of an electric field to move particles toward or away from an electrode, creating a chaotic advection. Acoustic wave energy can also be used to mix fluids: strong acoustic waves can be produced, and interfere with each other as they propagate in the fluid, generating advection24. As the diffusion coefficient of a liquid depends on the temperature, mixing can also be enhanced by increasing the microfluidic chamber temperature25.
Submillisecond organic synthesis using a serpentine-shaped microfluidic chip
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig10.png)
In chemical synthesis, it is important to explore the synthetic pathways of an intermediate. To fully observe these pathways, control over its lifetime and mixing time is required.
The reaction mixture had to be well-mixed within the lifetime of the reactive intermediate. An efficient means of prolonging the lifetime is to lower the reaction temperature (-78°C to -100 °C), above the melting point of many organic solvents. Using microfluidic devices, mixing can be extremely fast, with mixing times unattainable by batches26. However, mixing time is increased at low temperatures, as the solvent viscosity exponentially increases.
To circumvent this issue, the authors used a three-dimensional serpentine-shaped microfluidic chip, allowing improved mixing by chaotic advection. Using this microfluidic chip, submillisecond mixing was performed.
VI. Microvalves
In the past 10 years, efforts have been devoted to the development of microfluidic platforms capable of performing several assays using programmable fluidic operations within an array of microvalves. Similar to programmable logic circuits where multiple electronic computing routines are executed on a single microdevice, programmable microfluidic platforms have been implemented27, allowing one to perform fluidic operations such as mixing, sampling, washing, and reacting automatically within a single chip by modifying the sequence/order of fluidic operations using the software. The primary advantage of microvalves over their macroscale counterparts is the significantly reduced dead volume, which is important in many applications that require precise flow control at small flow rates28. They are useful in biological and chemical applications, such as quantitative metabolic biomarker and genetic analysis29,30, protein-based biomarker detection31, or small molecule chemical and environmental analysis32. These platforms usually consist of a 2D array of microvalves that permit flow regulation, on/off switching and sealing of liquids, gases, or vacuums33. Several microvalves have been developed using pneumatic, electrokinetic and electrochemical actuators. Among these mechanisms, pneumatic actuation is often recognized as the most reliable method due to the simplicity of fabrication, ease of use, scalability, reliability, and a high degree of accuracy. Pneumatically actuated microvalves utilize the deflection of an elastomer (typically PDMS) membrane to control fluid flow34.
A fully integrated multilayer microfluidic chemical analyze for automated sample processing, labelling, and analysis
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig11b-225x520-1.png)
Capillary zone electrophoresis (CZE), is a powerful tool for chemical analysis and is widely used for environmental monitoring, astrobiology, and biosensing32. CZE assays usually require complex and manual sample processing.
Commercial platforms for automated CZE have been implemented to address this concern, but are expensive, and large, thus hardly field deployable in challenging environments. Microfluidic systems allow miniaturization, automation, and reduction in sample volume requirements for chemical and biochemical sensing.
Using membrane microvalve technology, it is possible to automate metering, transporting, routing, and mixing operations. Kim et al. introduced a microfluidic platform consisting of pneumatically actuated “lifting gate” microvalves integrated with a glass CZE microchip, providing extremely low dead volumes between components.
All the procedures, including buffer filling, labeling, and dilution, can be automated. The microchip was used to analyze diverse compound classes, such as amino acids, and oxidized biomarker compounds, like aldehydes/ketones and carboxylic acids in less than 30 min.
VII. Wearable microfluidics: an emerging application
An emerging field is the use of microfluidic concepts for wearable device applications36. Here, microfluidics presents several key value propositions. The microstructures store or handle fluids and are the core of the sensing device. Using microchannels, precise liquid amounts can be manipulated, allowing for highly accurate and reliable devices and being useful for bodily fluids that are often secreted or extracted in limited quantities. Also, wearable microfluidic devices could also stock a specific drug for precise dispensing at controlled intervals. Innovations in flexible microfluidics and electronics have led to numerous applications. Typically, a wearable microfluidic device will collect a fluid, transfer it to a localized site where detection or measurement is performed. Blood and sweat are common analytes as they provide insights into physiological states such as temperature, pH, and hydration36. Wearable microfluidics finds applications in the pharmaceutical, food, sportswear, and cosmetic fields.
A wearable microfluidic device for the capture, storage and colorimetric sensing of sweat
![](https://www.fluigent.com/wp-content/uploads/2022/01/fig12-1024x453-1.png)
As mentioned previously, sweat is an analyte of interest because of its rich content of important biomarkers. It is easy to collect compared to blood. In situ quantitative analysis of sweat is of great interest for monitoring physiologic health status (for example, hydration state) and for the diagnosis of disease37.
Existing systems for sweat collection and analysis are confined to laboratories, where standard analytical technologies can be performed. Though highly precise, the analysis is time-consuming and costly.
To address this issue, Kho et al. developed a soft wearable microfluidic system than can directly harvest sweat from pores on the surface of the skin37.
The device routes the sample to different channels and reservoirs for multiparametric sensing of markers of interest, with options for wireless interfaces to external devices for image capture and analysis.
The device can measure total sweat loss, pH, lactate, chloride, and glucose concentrations by colorimetric detection using wireless data transmission. As it is a simple, low-cost, and fast analysis point of care device, it could be used to accumulate data from individual users over time, and this could serve as an analytical approach for interpreting trends in marker concentrations, potentially providing warning signs when performing physical activity.
Conclusion
Since the introduction of microfluidics, the scope of applications has kept extending over the years. The first applications were focused on analytical chemistry, but today the field of life science and specifically point of care is in the core of microfluidics. We have presented applications where microfluidics show great advantages compared to conventional systems. The importance of these applications was illustrated by showing research papers related to these applications. Some important applications were introduced here. Microfluidics covers a wide range of applications such as microreactors, bioprinting, or fuel cells, and many more.
References
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- Paquin, F., Rivnay, J., Salleo, A., Stingelin, N. & Silva, C. Droplet Control Technologies for Microfluidic High Throughput Screening (µHTS). Muhsincan Sesen,a Tuncay Alan,a and Adrian Neild∗a 10715–10722 (2017) doi:10.1039/b000000x.
- Galas, J. C., Bartolo, D. & Studer, V. Active connectors for microfluidic drops on demand. New J. Phys. 11, (2009).
- Jullien, M.-C., Tsang Mui Ching, M.-J., Cohen, C., Menetrier, L. & Tabeling, P. Droplet break in a low capillary T-junction. in 19th Mechanical French Congress (AFM, Maison de la Mécanique, 39/41 rue Louis Blanc-92400 Courbevoie, 2009).
- Yu, L., Chen, M. C. W. & Cheung, K. C. 2010 Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing. Lab Chip 10, 2424–2432 (2010).
- N.Shembekar, C.Chaipan, R. U. & C. A. M. 2016 Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics. Lab Chip (2016) doi:10.1039/C6LC00249H.
- Shang, L., Cheng, Y. & Zhao, Y. Emerging Droplet Microfluidics. Chem. Rev. 117, 7964–8040 (2017).
- Suea-Ngam, A., Howes, P. D., Srisa-Art, M. & Demello, A. J. Droplet microfluidics: From proof-of-concept to real-world utility? Chem. Commun. 55, 9895–9903 (2019).
- Vladisavljević, G. T., Al Nuumani, R. & Nabavi, S. A. Microfluidic production of multiple emulsions. Micromachines 8, (2017).
- Soppimath, K. S. & Aminabhavi, T. M. Ethyl acetate as a dispersing solvent in the production of poly(DL-lactide-co-glycolide) microspheres: Effect of process parameters and polymer type. J. Microencapsul. 19, 281–292 (2002).
- Qi, F., Wu, J., Li, H. & Ma, G. Recent research and development of PLGA / PLA microspheres / nanoparticles : A review in scienti fi c and industrial aspects. (2018).
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- Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).
- Halldorsson, S., Lucumi, E., Gómez-Sjöberg, R. & Fleming, R. M. T. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens. Bioelectron. 63, 218–231 (2015).
- Yeo, L. Y., Chang, H. C., Chan, P. P. Y. & Friend, J. R. Microfluidic devices for bioapplications. Small 7, 12–48 (2011).
- Coluccio, M. L. et al. Microfluidic platforms for cell cultures and investigations. Microelectron. Eng. 208, 14–28 (2019).
- Ayuso, J. M. et al. Development and characterization of a microfluidic model of the tumour microenvironment. Sci. Rep. 6, 1–16 (2016).
- Caballero, D. et al. Organ-on-chip models of cancer metastasis for future personalized medicine: From chip to the patient. Biomaterials 149, 98–115 (2017).
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What is a microfluidic chip?
Microfluidic chips are the devices used in microfluidic studies in which micro-channels have been molded or patterned. The micro-channels forming the microfluidic chip are connected together in order to allow fluids to pass though different channels, going from one place to another. This network is connected to the outside environment through inlet and outlet ports. The liquids or gases are injected, managed or removed from the microfluidic chip with passive ways or external active systems (pressure controller, syringe pumps or peristaltic pumps). The channels may have different inner diameters, typically ranging from 5 to 500 µm (read our article about microfluidic tubing), and their network must be specifically designed for the application and the analysis you want to carry out (cell culture and organ-on-a-chip , DNA analysis, lab-on-a-chip, microfluidic droplets. Therefore, microfluidic chips allow the integration of several functions that generally require an entire laboratory in a single micro-sized device.
![choose a Microfluidic chip](https://www.fluigent.com/wp-content/uploads/2022/01/5-4-microfluidic-chip-1.png)
Why use microfluidics?
Microfluidic devices such as chips have many advantages as they:
- Decrease sample and reagent consumption.
- Portability and flexibility of device design
- Direct coupling to downstream analysis systems
- Increase automation capabilities: multi-step reactions requiring a low level of expertise
- Excellent data quality and substantial parameter control
- Minimize analysis time
Such devices allow applications in many areas such as medicine, biology [1], chemistry and physics. Why should you be attentive when you choose a microfluidic chip? The materials for making microfluidic chips play an important role and should also present the appropriate properties desired by the application.
Material used in microfluidic devices
Most common material used in microfluidics reviewed
Three types of materials are commonly used to create microfluidic chips: silicon, glass and polymers.
Silicon and glass were the original materials first used for microfluidic applications, however as new technological advances occurred, new materials including polymer substrates, composites or paper were used.
For some experiments, a combination of these three chip materials is needed to create the desired microfluidic chip properties. Each material has its specific chemical and physical characteristics. The choice of the material depends on:
- the needs and conditions of your applications.
- the type of solvent, samples, buffers and their polarities
- the design.
- the budget.
Typically, for research purposes, the materials used generally prioritize versatility and performance of the device whereas in commercialization, cost of production, reliability and ease of use are placed above.
Why should you choose a microfluidic chip in silicon?
The first material used in microfluidics was silicon, even though it was quickly replaced by glass and then polymers. Silicon was first selected due to:
- its resistance to organic solvents
- the ease in metal deposition
- superior thermal conductivity
- surface stability
However, a microfluidic chip in silicon is not easy to handle because of its hardness, which doesn’t make it easy to create active microfluidic components such as valves and pumps. Another drawback appears when performing optical detection as silicon shows significant optical opacity.
Combined with a higher price than other materials, silicon microfluidic chips are not widely used in microfluidic studies.
![silicon microfluidic chip](https://www.fluigent.com/wp-content/uploads/2022/01/220px-etchedwafer.jpg)
Why should you choose a Microfluidic chip in glass?
![GLASS MICROFLUIDIC CHIP](https://www.fluigent.com/wp-content/uploads/2022/01/720px-fishchip.jpg)
After the initial focus on silicon, glass was used to build microfluidic chips. Optically transparent and electrically insulating, glass is an amorphous material. This material is generally processed with standard photolithography or wet/dry etching methods. Unless special etching techniques are employed, etched glass channels have rounded sidewalls.
Glass shares with silicon the same advantages mentioned above for microfluidics experiments. But one can add:
- Well-defined surface chemistries
- Superior optical transparency
- Excellent high-pressure resistance
- Biocompatibility
- Chemically inert
- Allows for efficient coatings
Glass is compatible with most biological samples.
A microfluidic chip in glass is not permeable to gas and has relatively low non-specific adsorption. Thus, it is compatible with biological samples, but it cannot be used for long-term cell culture.
One major application of glass microfluidic chips is capillary Electrophoresis (CE). This cheaper method is more convenient than standard CE since it’seasier to perform parallel analysis, and it also offers valve-free injection by directly utilizing the electro osmotic flow, which can separate analytes within seconds.
Other typical applications include on-chip reactions droplet formation, solvent extraction and in situ fabrication.
These make it a material of choice for many applications. The main hurdle with this material remains its hardness and its rather high cost, even though prices have been significantly reduced. These limitations are the origin to the development of alternative low-cost chip materials that can be easily fabricated and are compatible for broader biological applications.
Why should you choose a Microfluidic chip in polymers?
Polymer-based microfluidic chips were introduced several years after silicon/glass chips. The vast variety of polymers grants great flexibility in choosing a suitable material with specific properties.
Polymers are a promising alternative to glass and silicon as they are easy to access, cheaper, robust and require faster fabrication processes. Many polymers can be used to build chips:
- Polystyrene (PS)
- Polycarbonate (PC)
- Polyvinyl chloride (PVC)
- Cyclic Olefin Copolymer (COC)
- Polymethylmethacrylate (PMMA)
- Polydimethylsiloxane (PDMS)
![PDMS polymer microfluidic chip](https://www.fluigent.com/wp-content/uploads/2022/01/pdms-chip-microfactory-768x433-1.jpg)
![choose a pdms microfluidic chip](https://www.fluigent.com/wp-content/uploads/2022/01/microfluidic_chip_ix-factory.jpg)
PDMS is the material of choice for fast prototyping microfluidic devices. PDMS chips are commonly used in laboratories, especially in the academic community due to their low cost and ease of fabrication. The main advantages of PDMS microfluidic chips include:
- Oxygen and gas permeability, can be advantageous for oxygen and carbon dioxide transport in cellular studies and long-term experiments
- Optical transparency
- elastomeric properties
- Robustness
- Non toxicity
- Biocompatibility
- Complex microfluidic designs can also be created by stacking multiple layers.
- Relatively low cost
One of the main drawbacks of PDMS chips is its hydrophobic nature. Consequently, introducing aqueous solutions into the microchannels is difficult and hydrophobic analytes can adsorb onto the PDMS surface, thus interfering with analysis. There are now PDMS surface modifications available to avoid issues due to hydrophobicity. Another main issue of PDMS chips is that they are not suitable for high pressure operation as it can alter channel geometry and be prone to leaking at elevated pressure. Bubble formation from passage of gas through PDMS can be problematic.
PDMS is now the most commonly used microchip material.
Key information to keep in mind when choosing a suitable microfluidic chip
- Transparent materials are favored to enable optical observation/analysis
- Materials must be biocompatible for life science applications
- Most of the chips need surface treatment to adapt their surface properties to the application and to limit non-specific adsorption
Polymeric materials for a suitable microfluidic chip
Since its introduction, microfluidics keeps advancing along with technology, and expanding its fields of application. Biological and medical applications are a major focus of current research along with other areas. In terms of materials and functions, while glass and silicon have important uses, polymeric materials have become the material of choice in this field. As described above, they each have their own advantages and disadvantages. Though PDMS is still the more commonly used microfluidic material substrate, new materials and composites presenting interesting features are created in order to make them more adapted to mass production with lower pricing and greater adaptability.
REFERENCES
[1] Beebe, D. J., Mensing, G. A., & Walker, G. M. (2002). Physics and applications of microfluidics in biology. Annual review of biomedical engineering, 4(1), 261-286.
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What is Single Cell Analysis?
Most cellular research from fundamental cell biology and microbiology to applications in biotechnology is performed using cell populations with high cell numbers. This is convenient, as a huge variety of experimental techniques exist to analyze cell behavior. However, cell population data only covers information of an imaginary average cell (i.e. the average of the microbial culture), and not the mechanistic information of one cell. (1)
The Significance of Single-Cell Analysis:
Recent research provides substantial evidence that the heterogeneity of individual cells within genetically identical populations can have a profound impact on their survival.
Methods that use average responses from a population often mask the difference from individual cells. To fully understand cell-to-cell variability, a complete analysis of an individual cell, from its live state to cell lysates, is essential (1,2).
Distinguishing Bulk vs. Single-Cell Approaches:
To understand the distinction between the bulk and the single-cell approaches, imagine you have four squares: each of them is colored with a different shade of grey (figure 1). With the single-cell approach, you will see the squares as they are, with their actual colors, whereas with the more classic approach you will see four identical grey squares, hiding the color differences between each square.
![Distinguishing Bulk vs. Single-Cell Approaches](https://www.fluigent.com/wp-content/uploads/2022/01/single-cell-analysis.png)
Unveiling the Secrets of Single Cell Properties and Analysis Technologies
Within a population, the unique properties of single cells can be quantified using various cutting-edge technologies like flow cytometry and Elispot. These techniques enable the exploration of cell-to-cell variations, unveiling the heterogeneity within the population.
However, it’s essential to recognize that this analysis captures only a snapshot, offering insights into a cell’s state at a specific moment. It doesn’t provide a historical perspective or track a cell’s temporal development.
Moreover, in cases where the analysis is single-cell, the bioassay itself is bulk, with cells interacting with one another, potentially leading to changes in individual responses to stimuli. A cell can initiate its response upon detecting an activation signal from another cell.
Advancements and the Pursuit of Spatiotemporal High-Throughput Analysis:
Beyond these conventional endpoint technologies, the field is experiencing rapid developments with the ultimate goal of achieving spatiotemporal high-throughput single cell analysis, enabling a mechanistic understanding of cellular functions. The enthusiasm in this field is evident in the exponential growth of single cell studies and conferences in recent years.
This progress is driven by technical advancements in microfluidic chip manufacturing and analytical methods, as well as the increasing demand from biologists (3).
Key Challenges and the Role of Microfluidics and Lab-on-a-Chip Technology:
The sensitive detection of multiple cellular components and high-throughput analysis of individual cells pose significant challenges in achieving this goal. In this context, microfluidics and lab-on-a-chip technology have emerged as the most promising approaches to address these challenges. These innovative technologies hold the potential to unlock new insights into single cell properties and their roles within populations.
Single‐Cell Analysis Using Droplet Microfluidics
Recent analysis of healthy and diseased tissue homogeneous at the macroscopic scale revealed striking heterogeneities at cellular levels. This variability is particularly well illustrated in polyclonal tumors which constantly undergo mutations.
In this respect, single cell analysis is necessary to fully capture the complexity of such tissue. However, working at cellular scale equally exposes many variations in gene expression: from specific biomarkers to insignificant delays in gene expression. High throughput analysis is then needed to multiply the number of profiled cells and discriminate relevant biomarkers from intrinsic population noise.
Droplet microfluidics is particularly well suited and extensively used for high throughput single cell analysis: individual cells are isolated and confined at high speed in pico-volumes to analyze biological processes at the cellular level, streamlining multiple procedures on a single chip, with scope for parallelization.
Recently developed droplet microfluidics has also emerged as a new forerunner for single-cell encapsulation and analysis with massive parallelization. High throughput screening of rare cells to a drug library has been achieved, providing additional information on cell heterogeneity response. The use of microdroplet confinement has enabled new insights into the nature of quorum sensing, suggesting it is a “cell-autonomous mechanism for diffusion or efficiency sensing”.
Why is droplet microfluidics well-suited for single-cell analysis ?
High monodispersity: unique liquid-handling capabilities of microfluidic systems
Reduced costs: volume down scaling from µL to pL compared to pipetting robots
Time saving: molecular diffusion length reduced in small volumes
Higher sensitivity: smaller molecular quantity (1.106-fold less) required to reach minimum detectable concentration
High throughput: up to 1.106 cells compartmentalized per second.
Applications
Drop-Seq:
Single-cell genome or transcriptomic sequencing aims to increase our understanding of complex microbial ecosystems and disease in multicellular organisms by isolating the contributions of distinct cellular populations.
Unlocking the Potential of Flow Cytometry:
Droplet microfluidics technology has opened the door to groundbreaking capabilities in flow cytometry and (FACS). It allows for the encapsulation of single cells within double emulsions, transforming them into miniature microbioreactors. Within these microreactors, the fluorescence signals produced by specific molecules are captured and analyzed, enabling precise assessment of protein and metabolite secretion.
Through this innovative approach, flow cytometry gains the ability to delve into the intricate world of protein and metabolite secretion. The double emulsions serve as microscale laboratories, where the fluorescence signals arising from specific molecule production can be effectively trapped and scrutinized.
Sorting with FACS for Specific Cell Types:
Beyond flow cytometry, the integration of fluorescence-activated cell sorting (FACS) technology empowers researchers to selectively collect specific cell types from complex populations. This technology facilitates the precise separation and retrieval of cells of interest, providing invaluable insights for a wide range of applications.
Microbiome culture:
Compartmentalization of single bacteria inside droplets makes it possible to start new cultures inside each droplet, preventing growth competition between the different strains and allowing even rare bacteria to grow and form their own culture
Cell sorting with microfluidics:
Single cell sorter microfluidic platforms provide numerous advantages over conventional methods by reducing the size of necessary equipment, eliminating potentially biohazardous aerosols, and simplifying the complex protocols commonly associated with cell sorting.
Personalized medicine:
Single cell analysis of a tumor’s heterogeneity associated with selection and amplification of specific corresponding T cells for personalized cancer immunotherapy.
Selected publications from our customers:
Yin, H. and Marshall, D. (2012) “Microfluidics for single cell analysis,” Current Opinion in Biotechnology, 23(1), pp. 110–119. Available at: https://doi.org/10.1016/j.copbio.2011.11.002.
Andersson, H. and van den Berg, A. (2004) “Microtechnologies and nanotechnologies for single-cell analysis,” Current Opinion in Biotechnology, 15(1), pp. 44–49. Available at: https://doi.org/10.1016/j.copbio.2004.01.004.
Yin, H. and Marshall, D. (2012) “Microfluidics for single cell analysis,” Current Opinion in Biotechnology, 23(1), pp. 110–119. Available at: https://doi.org/10.1016/j.copbio.2011.11.002.
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![](https://www.fluigent.com/wp-content/uploads/2022/01/guillemet-150x150-1.png)
“As a clinician and immunologist with an interest in imaging, I was involved in developing a new technique for multiplexed immunohistochemistry. We wanted to automate the process by adding a microfluidic solution, but I had no experience with this technology. The ARIA system fit our needs and was incredibly simple and intuitive to use. The software is well-designed and readily interfaced with our microscope. Fluigent provided support throughout setup and transformed what seemed a large undertaking, into an incredibly simple task.”
Colin Chu, University College London
Introduction
Dr. Colin Chu has recently established his own lab at University College London (UCL) Institute of Ophthalmology in partnership with Moorfields Eye Hospital. As an ophthalmologist specialized in ocular immunology, his research is focused on using in vivo and ex vivo imaging to better understand the causes of blinding diseases.
While he was a visiting fellow in the lab of Dr Ron Germain at the National Institute of Health (NIH) in Maryland, USA, Dr Chu helped establish an automated highly multiplexed immunohistochemistry approach with his colleague Dr Andrea Radtke. This work was published in Nature Protocols in February 2022.
It uses an automated immunolabeling technique called IBEX, to achieve over 30 distinct antibody markers on the same section of tissue. Previously this had only been performed manually, limiting the throughput, speed and accessibility of the technique. Working together with Fluigent to connect an ARIA microfluidics system to a Leica THUNDER microscope, the two systems were able to interface to deliver automated staining and image acquisition.
Dr. Chu is now setting up the system at UCL to apply the automated immunomarking technique, IBEX, to the retina in mice, zebrafish and human post-mortem tissue. In addition to multiplexed tissue imaging with immunofluorescence, his research topics include ocular gene therapy and in vivo immune cell imaging using adaptive optics, optical coherence tomography (OCT) and fluorescence microscopy to better understand ocular inflammation.
How to perform automated immunofluorescence
Fluigent instrument ARIA coupled to a widefield microscope to perform automated multiplexed tissue imaging
High content imaging of tissue samples was performed using an automated immunolabeling method, consisting of iterative cycles of antibody labelling, imaging, and fluorophore bleaching using lithium borohydride (LiBH4). Image processing and registration were done using the SimpleITK open-source software.
To perform an automated IBEX study, the complete setup comprised several pieces of equipment:
- a pressure source (here our FLPG)
- a single output sequential perfusion system ARIA
- a closed bath imaging chamber containing tissue sections and stage heater from Warner Instruments
- a THUNDER microscope from Leica
Using ARIA software, a method containing all the fluid injection and image acquisition steps was created. Once loaded, the sequence can be run automatically. The automated immunomarking method relies on TTL signals sent and received by ARIA, enabling a cycle of image acquisition to be launched at the end of an injection step. The software interfaced with the Leica THUNDER microscope.
Once an image was acquired, the next fluid injection step started, to bleach the previous round, commence antibody staining with the next panel, then trigger microscope acquisition again. Ultimately, the full sequence of steps gave rise to high-quality, multiplexed protein level data of over 30 markers.
Fixed human tissues including mesenteric lymph node, small intestine and skin were profiled (Figure 1, 2). These tissues are usually very challenging to image due to their delicate structure and high background autofluorescence.
As stated by the authors, the original IBEX method was limited by the requirement of manual execution (manual pipetting for antibody staining and fluorophore inactivation). Here, ARIA provides an affordable automated solution to access high-content imaging as part of the spatial biology revolution.
IBEX automated immunomarking method: Experimental procedure
a) The automated IBEX protocol uses our compact microfluidics system ARIA to deliver multiple solutions to an imaging chamber placed on an inverted microscope stage. Fluids are moved through the system with compressed air and delivered to the imaging chamber based on protocols defined by the user. The inverted microscope and ARIA device communicate via TTL signals using precise timing established by the user with the Aria control software.
b) Samples are sectioned onto coated coverslips and assembled into a closed bath imaging chamber. Following assembly, the imaging chamber is secured to a magnetic platform and mounted onto the microscope stage.
c) Automated IBEX consists of: (1) nuclear labeling with Hoechst, (2) antibody labeling for 1 h at 37 °C using a heated microscope stage, (3) imaging ROIs, (4) bleaching with LiBH4, and (5) repeating Steps 2–4 until the desired number of parameters is achieved, typically 12 h for a six-cycle, 25-plex experiment.
Partial Results
Radtke AJ. *, Colin CJ*. et al, have established a high throughput automated immunolabeling protocol recently published in Nature Protocols based on the automation power of our Fluigent ARIA instrument coupled to an image alignment open-source software package.
This powerful approach relies on iterative staining and bleaching method to perform high-resolution imaging of more than 65 parameters. This Iterative Bleaching Extends Multiplexing protocol (IBEX) represents a robust and reproducible method to perform deep phenotyping and spatial analysis of cells in complex tissues (healthy organ, infected organs, tumor microenvironment).
Examples of Images obtained using automated IBEX method in human tissues
![automated immunolabeling ibex aria human jejunum](https://www.fluigent.com/wp-content/uploads/2022/03/expertise-review-ibex-aria-jejunum-2-1.png)
![automated immunolabeling ibex aria skin](https://www.fluigent.com/wp-content/uploads/2022/03/expertise-review-ibex-aria-skin-1.png)
Conclusion
In this paper, the authors have demonstrated the automation power of our sequential perfusion system ARIA together with its ability to be synchronized with a widefield (or other) microscope to perform automated and multiplexed antibody labelling. ARIA can send and receive TTL signals to launch an image acquisition cycle and resume the perfusion protocol once the imaging cycle has been completed. Other applications such as DNA-paint, OligoSTORM, dose/response studies, automated multiplex immunofluorescence experiments or dynamic pulse-chase experiments can be automated using ARIA. .
- Chu Lab website: https://colinchulab.com/
- UCL Chu Lab website: https://iris.ucl.ac.uk/iris/browse/profile?upi=CHUXX55
- General UCL website: https://www.ucl.ac.uk/
Additional information about Aria
Aria’s technology, enabling automated sequential injection for cellular perfusion or timed injection protocol, is a new state-of-the-art technology that holds great promise for many cell biology and organ-on-chip cell culture projects. Incorporating Fluigent’s latest technologies, this instrument ensures perfusion with minimal shear stress and highly controlled flow. To find out more about this technology, we invite you to visit the product web page, or take a look at our Webinars.
Aria is the only instrument that can automate the delivery of up to 10 different solutions to a flow cell. It’s the perfect compromise between manual pipetting and an all-in-one system dedicated to one specific application. Any protocol with multiple solution delivery can be automated, saving the scientist both time and reducing variability between experiments compared to procedures that do not include perfusion systems. Users can integrate their own microscope, specific chip type and solution sets using the Aria Automated sequential injection system.
Automate Cellular Studies with Aria
Unlock your path to success with Fluigent’s cutting-edge perfusion system, Aria. Say goodbye to the complexities of manual fluid delivery, and embrace a new era of seamless experimentation.
Automate with Aria:
When your experiments demand precision in timed delivery of reagents, probes, fluorophores, and more, all while reducing the need for constant operator intervention, look no further than Aria, our next-generation instrument for automating cellular perfusion studies.
Streamlined Efficiency:
In response to the feedback from our valued customers, we recognize the time and effort that multi-solution protocols often require, coupled with substantial operator involvement. Aria simplifies your research by offering swift experimental setup, user-friendly operation, and the ability to automate even multi-day protocols. Additionally, it seamlessly interfaces with various microscopes, further enhancing reagent delivery and imaging.
Your Solution for Lab Automation:
Aria is the ultimate solution to streamline your laboratory work. Its high flexibility allows adaptation to any perfusion chamber and protocol, thanks to its intuitive software.
Elevating Live-Cell Imaging:
Beyond its versatility, Aria serves as a powerful tool for automating complex protocols, particularly in the realm of live-cell imaging. Whether it’s immunostaining, omics applications, or radiometric imaging, Aria is your partner in simplifying intricate experiments.
Automated Fluid Delivery for Cell and Tissue Imaging
If you’re looking to save time, enhance precision, and achieve reproducibility in your immunofluorescence assay or any other assay that involves injecting multiple solutions into your samples, our webinar is a must-attend event. Discover how to seamlessly integrate a flow chamber or microfluidic chip with our automated fluid delivery device, ARIA. With this setup, you can deliver up to 10 different solutions sequentially and autonomously. Additionally, ARIA can be synchronized with any microscope to initiate an image acquisition cycle and resume the perfusion protocol once the imaging cycle is complete.
This all-in-one workflow streamlines the process of injecting/incubating with reagents and acquiring images, making it particularly well-suited for complex cell and tissue imaging. During the webinar, we will showcase various applications, including multiplexed tissue imaging, DNA-PAINT, seqFISH, cell capture, and staining.
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Overview :
- General principle
- Why droplet microfluidics ?
- Process description
- Different genes, one cell ?
- Popularity
- The advantages of running pressure-controlled droplet production
- Conclusion
General principle
![Overview of the DropSeq process](https://www.fluigent.com/wp-content/uploads/2022/01/overview-of-the-dropseq-process.jpg)
The DropSeq process relies on pairing one barcoded bead and one cell in a small droplet. after droplet formation, cell lysis occurs such that the polyadenylated RNA produced exclusively by this cell can specifically be captured on the bead. Capturing RNA with a single barcoded bead will allow the reconstitution of the information coming from that cell. Next, the mRNA is reverse transcribed along with the barcode into tagged cDNA, and PCR amplified. Finally, the resulting libraries are sequenced by NGS, and analyzed using various bioinformatics tools: cell barcode sequences are identified, transcript sequences linked to the barcodes mapped to a reference genome. All transcripts linked to one barcode form the gene-expression profile of a single cell.
Why droplet microfluidics?
The conventional way to isolate cells is by using Fluorescence Activation (FACS) to sort them into barcoded well plates (96 wells, 384 or 1536). This step is quite fast with the FACS currently available, but segregating thousands of cells into plates will result in a long process of pipetting for downstream steps like RT or PCRs, even on an automated platform. Droplet microfluidics allows for fast and low volume compartmentalization, usually up to a thousand drops produced per second with a volume of one nanoliter each. This consumes a thousand times less reagents and does not need high end FACS and liquid handlers, diminishing the costs per reaction. Think of a drop as a well from the plate, but smaller.
![Plate based versus microfluidics approach](https://www.fluigent.com/wp-content/uploads/2022/01/plate-based-versus-microfluidics-approach.jpg)
While all the compartmentalized steps will happen in a smaller volume, it is also make easier to pool afterwards thanks to initial cell barcoding: while combining the wells will be done by pipetting, droplets can be merged by chemical destabilization of their interface with the carrier fluid.
Process description
The encapsulation process is done using a microfluidic flow focusing junction chip, where all the reagents are brought to a nozzle where the carrier fluid (oil) will shear the water phase into small droplets at high speeds.
![Encapsulation-chip-](https://www.fluigent.com/wp-content/uploads/2022/01/encapsulation-chip-.jpg)
Once the encapsulation process is finished, the monodispersed emulsion obtained should look like this:
![Typical DropSeq emulsion](https://www.fluigent.com/wp-content/uploads/2022/01/typical-dropseq-emulsion.jpg)
Empty drops will generate noise in the data, as they carry environmental RNA that can bind to beads once droplets get merged. For drops only containing a bead, most of the time it will not generate more noise as the unused barcodes will be destroyed later in the process. Cells that were not encapsulated will not be sequenced, as their RNA cannot be captured in drops, but will participate to the noise as the RNA released will bind to random beads. Usually, a Drop-Seq run allows one to capture around 5-10% of the input cells. This is due to the Poisson distribution, basically, this is a balance between having too many empty drops and having drops with 2 or more cells/beads, that will generate noise and unusable data during sequencing.
Special buffer components that are contained in the bead buffer will start cell lysis as soon as the drops are formed, and will carry on during droplet production. By the time the run is finished, all the cells will be lysed and RNA captured. This means the emulsion may be broken by merging all the drops and recovering the beads to start the molecular biology steps for sequencing the captured RNA. Reverse transcription (RT) will generate cDNA attached to the barcodes, and an exonuclease will be used to remove unused barcodes.
![Molecular biology workflow](https://www.fluigent.com/wp-content/uploads/2022/01/molecular-biology-workflow.jpg)
Different genes, one cell?
The power of the Drop-Seq method relies on how the microbeads are constructed: they are coated covalently with oligonucleotides that help capture RNA, barcode it in a way each bead contains unique barcode and diverse unique molecular identifiers to help quantifying captured RNA molecules.
![Barcode architecture and usage](https://www.fluigent.com/wp-content/uploads/2022/01/barcode-architecture-and-usage.jpg)
First, there is the PCR handle, which will allow for the amplification of the generated cDNA molecules at the end of the process, mainly for amplifying the signal by generating more copies.
Second comes the barcoding sequence, which will be the same on each oligo from one bead, but different from one bead to another. All the sequences that contain a given barcode will then be associated to the same cell of origin.
Third comes the UMI, which stands for Unique Molecular Identifier. All the oligos on one bead have a different UMI sequence, so that different molecules of mRNA can be distinguished at the cell level. This allows for one to know how many mRNA of a given sequence have been produced by a cell.
Finally comes the capture sequence, which is usually a poly(T) tail to match the poly(A) tail of the 3’ end of mRNA. If you are only targeting a specific gene panel, you can also use complementary sequences to only capture those.
Popularity
The Drop-Seq protocol has been made open source by their original creator, and they even encouraged people to use it by fine tuning the process, publishing tutorials, tips and tricks, and finding suppliers for the specific reagents and consumables. Their protocol has been downloaded more than sixty thousand times in four years and there is an active community that is discussing and improving the protocols as we speak https://groups.google.com/forum/#!forum/dropseq
The literature around Drop-Seq represents more than a thousand papers around the world.
The advantage of running pressure-controlled droplet production
While the original protocol was developed using syringe pumps, pressure-controlled systems have a few advantages.
First, in terms of raw performance, pressure driven systems are faster to set up, easier to control and more stable over time, which allows for an emulsion of a better quality : the droplet size will be more homogeneous and start/stop populations will be smaller. This leads to better segregation of the cells, and less reagent use and less sample loss.
![New encapsulation and capture chip](https://www.fluigent.com/wp-content/uploads/2022/01/new-encapsulation-and-capture-chip.jpg)
As the sample and beads need to be agitated throughout the experiment, it is usually done with a bulky stirring bar for syringe pumps. This leads to a dead volume that will not be injected in the chip. Pressure driven setups can be run using more conventional containers that make it possible to use an external agitation system, such asa standard lab vortexing system.
You can check out the work by M. Biocanin, et. al. from the technology development team of the LSBG lab at the Ecole Polytechnique Fédérale de Lausanne. They have adapted the chip to make it easily usable with both pressure systems and syringe pumps, re-engineered the nozzle to make it perform better, and refined the downstream process on a separate microfluidic chip that allows for robust and efficient bead processing while washing and post-processing of beads.
These optimizations lead to an increase in the number of beads recoved, diminished the contamination risks during washing steps, and allows for reliable processing of small volume samples.
Example of experimental set up
![Fluidic set up for encapsulation](https://www.fluigent.com/wp-content/uploads/2022/01/fluidic-set-up-for-encapsulation.png)
The experimental set up can be reproduced with Fluigent products, using 3 LineUp™ Flow-EZ™ or 1 MFCS-EZ™, 3 sensors for flow rate monitoring and a microscope. The chip is open source and is available from suppliers including: FlowJem, Nanoshift or uFluidix off the shelf. The beads are commercially available and can be purchased from ChemGenes.
Fluid considerations when integrating fluidics into your system
How will pulsatile flow affect system performance?
Droplet-based applications: Droplet digital PCR and cell encapsulation for single-cell analysis usually make use of droplets generated by microfluidic technology. These applications require liquid handling systems that produce flow with very low to no pulsation to obtain homogeneous droplets, which is vital for the success of these applications.
Flow cytometry and surface plasmon resonance (SPR) are based on real-time analytical measurements of samples passing by a detection area. They require a fluid handling delivery system with a stable flow rate to deliver fluids through the fluidic channels.
Live cell imaging: In live-cell imaging applications with fluid perfusion, pulsatile flow leads to variable shear stress imposed on cells, and may impact cell viability. Liquid flow should be stable and controlled.
In some applications, a steady flow rate is a prerequisite for reliable operation after fluidic system integration:
![flow pulsations for fluidic system integration](https://www.fluigent.com/wp-content/uploads/2022/04/flow-pulsations-1.png)
For these applications, pressure-based fluid delivery can deliver flow rates in the microliter- or nanoliter-per-minute range with a stable flow rate (high precision and accuracy) and no pulsation.
![syringe pump vs pressure control for fluidic system integration](https://www.fluigent.com/wp-content/uploads/2022/04/flow-rate.png)
Do you require fast settling times?
Some applications call for alternating between multiple different flow rates in a short period of time. This is common for flow cytometry, cell sorting, or fluorescent activated cell sorting (FACS). In the latter, 3 flow rate modes are usually available: slow, medium, and fast, and one can switch from one to another.
For fluidic system integration in applications like these, consider using a fluid delivery solution that allows for rapid response time (a few seconds). Pressure-based flow control has the advantage of having a quasi-immediate response. A step of several bars can be made within milliseconds, and the liquid flow rate reaction is equally fast, allowing the system to reach typical microfluidic flow rates in less than one second. This cannot be achieved by most motors used in syringe pumps, as revealed by the graph below.
![fluidic system integration performance](https://www.fluigent.com/wp-content/uploads/2022/04/flow-rate-2.png)
Is sterility required?
Many biological applications require a sterile environment, including cell culture under perfusion, immunostaining, organoid culture, organ on a chip, drug discovery, single-cell analysis and cell cytometry, among others. When using fluids such as culture media, PBS, buffers, blood or plasma, every component in the fluidic path should be disposable or sterilizable.
For these applications, pressure-based liquid delivery systems that do not contact fluids or have sterilizable components are ideal. As for liquid handling without flow rate monitoring, using pressure allows the system to use disposable tubing only between the inlet reservoirs and the application. At the fluidic system integration stage, however, you may want to plan for the ability to monitor or control the flow rate. The question of sterility arises when choosing the flow rate metering solution. Most flow sensors are too costly to be considered as disposables, yet are in contact with the fluids. A non-intrusive flow sensing technology is a particularly interesting way to tackle this issue.
Do you need to handle multiple fluid streams?
Multiple streams are useful for operations that perform several tests simultaneously, such as drug screening where different drug candidates are tested, or personalized medicine applications that make use of tumor biopsy studies. It is also sometimes convenient and cost-effective to separate a fluid originating from one liquid delivery system into two distinct paths.
It’s important to choose the right liquid delivery system when designing for multiplexing. Depending on your choice, an increased number of systems may be highly expensive, and fluidic system integration can be cumbersome. Using fluidic valves or quake valves can also be an interesting alternative, as they allow for fluid management with a reduced number of fluid delivery systems.
What are the volumes to be dispensed?
The volumes to be injected are highly application-dependent. For instance, in cell biology applications such as cellular imaging or dynamic cell culture, fluids may be injected over a period of several days. In these applications, high reservoir volumes (up to 1 L) are required. Another option could be recirculating media or reagents with a peristaltic pump, a recirculation valve or a fully integrated solution. Conversely, drug screening applications may make use of expensive liquids, and only very small amounts of fluids should be used (< 100 µL).
Depending on the liquid delivery method used in the fluidic system, reservoir size can affect precision and accuracy. For example, when using syringe pumps, smaller syringes maximize accuracy for small volumes but require frequent refilling for larger volumes. Larger syringes will increase capacity, but will lose accuracy and become pulsatile at lower flow rates. Using a peristaltic pump for fluid recirculation is relatively uncomplicated.
Do you need to control or measure flow rate?
For most liquid delivery applications, the user wants to set a desired flow rate and have a solution to reach it via fluidic system integration through use of a feedback loop algorithm. Syringe or peristaltic pumps give easy access to the real time volumetric flow rate, but pressure controllers do not.
To overcome this, the user can add a flow sensor to the configuration that can either be connected to the pressure controller for flow rate control, or used for monitoring only. There are several flow sensing technologies based on different approaches, each with their respective advantages and limitations. The best choice for a given application depends on your requirements with regard to response time, chemical compatibility, flow rate range, cost, footprint and other parameters. For microfluidic applications in particular, one must also be cautious about channel and fluid interactions, the formation of bubbles, and multi-phase flows, although these can be neglected at a larger scale.
Important considerations when integrating fluidics into your system
Time to market?
In a fast-growing market sector, it’s crucial to minimize time-to-market and launch new technologies before the competition. To accelerate development, consider pre-built components or solutions, such as liquid handling components that can be integrated quickly into your system or prototype. Fluigent’s standard and customizable liquid handling OEM components offer flexibility and ease of integration into your device. Using a readymade solution avoids the hassle of dealing with multiple fluid handling issues and leaves you more time to focus on your core expertise and application.
If you need expertise in fluidic system integration and are looking for a company that can do it for you, choose one that’s reactive, communicates with its customers, and can respect short timelines to achieve your desired launch date. At Fluigent, we design and manufacture all of our products under one roof at our headquarters in the Paris region. Our R&D and production teams work closely with each other and with our customers to ensure maximum satisfaction.
Do you have all the resources needed for in-house development?
Handling fluidic system integration by yourself is an option when developing an automated liquid handling system. The conception, design and development of the system will require engineering expertise in the mechanical, electrical, software and manufacturing domains.
When developing a system internally, you need to be sure you have the experience and engineering knowledge to translate your proof-of-concept device into a reliable and efficient automated system. The entire product development process is at risk if you are missing any of the required resources.
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