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Making Micro-Volume Biology Work: Tools, Techniques and Secrets

The field of microfluidics has been evolving rapidly in the past few years. From the perspective of someone looking for technologies to improve high-throughput screening and compound profiling, the state of microfluidic applications is more interesting and attractive than ever. So says SLAS2017 Session Chair Daniel Sipes, M.S., director of automation technologies at the Genomics Institute of the Novartis Research Foundation (GNF), San Diego, CA. 

Sipes has been exploring microfluidic technologies in search of ways to impact GNF’s research and development programs. As reported in a previous SLAS ELN article, early in his career Sipes developed an “aptitude for the mechanics of science” that altered his trajectory from pure science toward the application of automation technologies to improve work processes, and that path continues to be rewarding.

While exploring many topics for the SLAS2017 session, Sipes ran across some eye-opening work that has further broadened his perspective. Ultimately, he chose a few outstanding topics that were either “practical, meaningful and broadly applicable to a number of applications, or those that were more narrow but potentially very helpful and interesting to people looking to answer specific scientific questions.” All of the technologies presented in this session involve working at the single-cell level in automated and high-throughput formats.

Interactive Cellular Biology Using Visible Light

Kevin Chapman, Ph.D., chief scientific officer at Berkeley Lights, Inc. (BLI), Emeryville, CA discusses applications of the company’s optically actuated microfluidic systems that may be applied to biopharmaceutical, genomic and cell therapy workflows. The microfluidic environment in chip format that they have created can be used for cloning, culturing, assaying and exporting mammalian cells. The systems allow scientists to manipulate individual cells using optical actuation and computer controls in real time.

BLI’s technology uses low-intensity visible light to manipulate individual cells, beads and reagents. The technology section of the website explains that there are two different methods of optical actuation. Using OptoElectroPositioning (OEP) cells in culture medium can be manually or automatically selected based on cell surface markers, morphology and real-time protein secretion levels. OptoElectroWetting (OEW) enables the manipulation of aqueous droplets or cells in droplets of oil allowing researchers to “aliquot, measure, titrate, combine, split and dilute at nanoliter volumes.”

BLI’s primary areas of interest are in the areas of antibody discovery, single cell annotation and genomics and production cell line generation. According to their website, they have developed workflows to optimize production cell line generation and run secretion-based assays on clones in real-time using their opt-fluidic chip.

Sipes thinks “this is really interesting because it can be used so many different ways. It is already available, and what they essentially do is sort cells into bins, grow them as clones, and then screen those clones with functional readouts very early, when they are just a few cells. That can be quite enabling.” He thinks there are many different areas where this technology potentially can be applied and anticipates learning more during Chapman’s talk.

Relative ranking of 5 cell lines with different expression levels and a negative control cell line.

Profiling Anti-Tumor Immune Responses with Microfluidic Cell Pairing

Tania Konry, Ph.D., assistant professor, Northeastern University, Boston, MA is using droplet array-based microfluidic platforms to study live cell-cell interactions at the single cell level. Konry, a finalist for the 2017 SLAS Innovation Award, states in the session abstract that studies of this type are necessary “to uncover fundamental biological principles and ultimately improve the detection and treatment of disease.” The technology Konry is using relies on a new lab-on-a-chip (LOC) microfluidic technology and Sipes notes “that while the approach is entirely different [from Berkeley Lights], you can still co-localize cells, look at how they interact and do phenotypic screening.”

With the LOC technology, Konry’s group is able “to acquire live functional phenotyping of single cells of the human immune system to monitor and regulate their interactions with tumor cells in hematologic cancer-relevant system.” Konry explains that the technology “allows: 1) conducting dynamic and simultaneous multi-parameter analysis of both cell surface and secretions; 2) controlled delivery of regulatory agents and therapeutics to study their effect on functional phenotype of the cell; and 3) monitoring of cell-cell interactions on a single cell based level.”

Konry feels “this approach should have a broad impact on diverse biological systems for the characterization of cell surface and secretion proteins as potential biomarkers and targets for diagnostics and therapeutics as well as cell-cell interactions in immune response to malignancy, autoimmune diseases, immunotherapy and biomarker discovery.” Her lab is interested in developing diagnostic tools and detection tools as well as the development, evaluation or delivery of new therapeutic strategies. Primary areas of interest involve the immune response in cancer patients. 

Studying interactions of immune cells with cancer cells.

The technologies presented by Chapman and Konry represent different approaches that promise to have broad applicability, according to Sipes. In contrast, he feels that the next two technologies, are extremely interesting for researchers in more specific niches.  

Monoclonal Cell Line Generation with Automated Single-Cell Electroporation

Vincent Lemaitre, Ph.D., research bioengineer at infinitesimal LLC, Skokie, IL, is helping to make the process of generating stably-transfected monoclonal cell lines more efficient using the Nano Fountain Probe (NFP) single-cell electroporation systems in conjunction with pre-patterned array of cell colonies, as described in his session’s summary. Sipes is fascinated by this technology and feels “the ability to very precisely localize and transfect the cell that you are interested in without having to go through limiting dilutions can be quite enabling, especially for cells that don’t do well when isolated.” Infinitesimal is one of the entrepreneurial companies earning a spot on SLAS2017 Innovation AveNEW.

infinitesimal's Nano Fountain Probe (NFP) 

In addition to the cell line generation, the technology, previously reported in JALA (now SLAS Technology), can manipulate and precisely deliver molecules to individual cells in an aqueous environment conferring an advantage over oil-based systems. The website provides examples of how automated precise intracellular delivery can be applied to studies of single cell gene expression or cell-specific gene editing (using a CRISPR/Cas9 system). This is also an advantage in delivering molecules to sensitive or differentiating cells or cells lines that are known to be hard to transfect.

Lemaitre indicates in the session abstract that a variety of cells have been transfected with high efficiency and cell survival and the technique is amenable to full automation. The technology enables new capabilities for fundamental research and cellular engineering and there is potential for applications in academia, biotechnology, pharma and healthcare.

High-Throughput Drug Screening of Cellular Force Generation

Ivan Pushkarsky, a graduate student researcher in the Department of Bioengineering at University of California, Los Angeles is studying cell-generated forces. Pushkarsky was one of nine finalists for the 2016 SLAS Innovation Award and received an SLAS Tony B. Academic Travel Award in 2016 and 2017. As part of the Di Carlo laboratory, Pushkarsky has developed a high-throughput platform to assess the ability of single cells to exert forces on their environment.

A proof of concept evaluation of phagocytic forces by human macrophages, presented at the 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences in 2015, demonstrates simultaneous profiling of a large number of cells using cell contractility measurements.  In the meeting’s proceedings, Pushkarsky explains that prior technology has been limited to single cell evaluations in a serial fashion with corresponding low-throughput potential. This technology has been implemented in 96- and 384-well plates. Pushkarsky’s lab has developed a fabrication process to embed any extracellular matrix protein onto a glass-backed elastomeric surface with uniformly shaped patterns, enabling study of a variety of physiological environments. Image analysis measures displacements (amount of cell force/contraction) and compares with internal controls (see figure).

What makes this interesting, according to Pushkarsky’s session abstract, is that malfunctioning cellular force generation has been implicated as a cause of some chronic conditions such as asthma, hypertension and bowel disease for which there is a paucity of effective drugs. In a recently published paper, this testing platform is used to assess cellular forces generated by human airway smooth muscle cells in response to a panel of bronchoconstrictive agents. Additionally, the platform’s drug discovery potential is validated by testing comparing results of a known drug (formoterol) with bronchodilation effects to a phosphoinositide 3-kinase inhibitor.

Sipes noted that, as understood in the industry and accurately stated in the abstract, “cell generated forces are being recognized as playing key roles in many physiological systems. Having a means to screen for these cell-generated forces is key. Having a scientific model for these chronic diseases is enabling, and it can scale up to a plate format.” Pushkarsky feels the platform has the potential to “strengthen all drug development efforts focusing on the restoration of normal cellular force generation at the high-throughput screening, lead generation and toxicity screening stages.” 

Figure 3 from Koziol-White, C. J., Yoo, E. J., Cao, G., Zhang, J., Papanikolaou, E., Pushkarsky, I., Andrews, A., Himes, B. E., Damoiseaux, R. D., Liggett, S. B., Di Carlo, D., Kurten, R. C., and Panettieri, R. A. Jr. (2016) Inhibition of PI3K promotes dilation of human small airways in a rho kinase-dependent manner. British Journal of Pharmacology, 173: 2726–2738. doi: 10.1111/bph.13542.

Ongoing Challenges

Sipes identifies a number of challenges in making micro-volume biology work. He talks about some of the technical problems that arise when working at such a minute scale where small changes in the geometry of a platform may result in clogging or sticking. Sipes also notes that in making the shift from aqueous-based macro assays to droplets, “you may find that the oil or surfactant interferes with the assay’s function.” Regardless of the hurdles, Sipes says there are benefits beyond obvious things like throughput and decreased reagent usage. One of these is physics-based and appreciated with reduced incubation times and the elimination of mixing steps because diffusion occurs so much faster at the nanoscale.

One of the larger hurdles Sipes mentions for research groups implementing new technologies is the issue of reproducibility. He explains that microfluidics systems are complex and hard to implement efficiently from the published literature, requiring trial and error and multiple runs before attaining reliable results. The hurdles involved in creating reproducible results from published information may be a barrier to adoption for some companies and research groups. He feels that companies that are reducing the science to a platform with established workflows, such as Berkeley Lights, help to increase reproducibility for their applications and Sipes sees value in leveraging technology to broader applications.

An even broader conversation about reproducibility in research is ongoing and the focus of a SLAS2017 session entitled “Whose Responsibility is Research Reproducibility?”. Current discussion of the topic is happening in the SLAS ELN article entitled “Remedy for Reproducibility: Opening a Dialog to Explore the Complexities.” Sipes feels that solutions that increase reproducibility are of interest to the drug development community, whether in academia or industry.

Learn More

Sipes’ session is just one of several sessions in the SLAS2017 Micro- and Nanotechnologies Track.

Related to this topic, there is a SLAS2017 Short Course, Lab-on-a-Chip: From Technology to Bioanalysis on Chip. Course topics include information on new materials and fabrication techniques, miniaturized instrumentation and comparison of microdroplet and continuous-flow technologies. Finally, access the SLAS On-Demand Webinar, “Droplet Microfluidics: Amphiphilic Nanoparticles as Droplet Stabilizers for High-Fidelity and Ultrahigh-Throughput Droplet Assays.”


About the Banner Images

Many thanks to Chapman, Konry, Lemaitre and Pushkarsky for sharing their work with us for this story and the banner. Image credits:

A = Kevin Chapman, Berkeley Lights, Inc.

B = infinitesimal LLC (

C = Tania Konry, Northeastern University

D = Koziol-White, C. J., Yoo, E. J., Cao, G., Zhang, J., Papanikolaou, E., Pushkarsky, I., Andrews, A., Himes, B. E., Damoiseaux, R. D., Liggett, S. B., Di Carlo, D., Kurten, R. C., and Panettieri, R. A. Jr. (2016) Inhibition of PI3K promotes dilation of human small airways in a rho kinase-dependent manner. British Journal of Pharmacology, 173: 2726–2738. doi: 10.1111/bph.13542.

January 24, 2017