Helene Andersson Svahn

Droplet Microfluidics—A Tool for Single‐Cell Analysis

Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding.

Droplet size based separation by deterministic lateral displacement—separating droplets by cell-induced shrinking

We present a novel method for passive separation of microfluidic droplets by size at high throughput using deterministic lateral displacement (DLD). We also show that droplets containing Saccharomyces cerevisiae shrink significantly during incubation while droplets containing only yeast media retain or slightly increase their size. We demonstrate the DLD device by sorting out shrunken yeast-cell containing droplets from 31% larger diameter droplets which were generated at the same time containing only media, present at a >40-fold excess. This demonstrates the resolving power of droplet separation by DLD and establishes that droplets can be separated for a biological property of the droplet contents discriminated by a change of the physical properties of the droplet. Thus suggesting that this technique may be used for e.g. clonal selection. The same device also separates 11 µm from 30 µm droplets at a rate of 12,000 droplets per second, more than twofold faster than previously demonstrated passive hydrodynamic separation devices.

High-throughput screening for industrial enzyme production hosts by droplet microfluidics

A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its α-amylase production, close to the theoretical maximum enrichment. Furthermore, a 10(5) member yeast cell library is screened yielding a clone with a more than 2-fold increase in α-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.

3D Bioprinting of Tissue/Organ Models

In vitro tissue/organ models are useful platforms that can facilitate systematic, repetitive, and quantitative investigations of drugs/chemicals. The primary objective when developing tissue/organ models is to reproduce physiologically relevant functions that typically require complex culture systems. Bioprinting offers exciting prospects for constructing 3D tissue/organ models, as it enables the reproducible, automated production of complex living tissues. Bioprinted tissues/organs may prove useful for screening novel compounds or predicting toxicity, as the spatial and chemical complexity inherent to native tissues/organs can be recreated. In this Review, we highlight the importance of developing 3D in vitro tissue/organ models by 3D bioprinting techniques, characterization of these models for evaluating their resemblance to native tissue, and their application in the prioritization of lead candidates, toxicity testing, and as disease/tumor models.

Towards high-throughput single cell/clone cultivation and analysis

In order to better understand cellular processes and behavior, a controlled way of studying high numbers of single cells and their clone formation is greatly needed. Numerous ways of ordering single cells into arrays have previously been described, but platforms in which each cell/clone can be addressed to an exact position in the microplate, cultivated for weeks and treated separately in a high‐throughput manner have until now been missing. Here, a novel microplate developed for high‐throughput single cell/clone cultivation and analysis is presented. Rapid single cell seeding into microwells, using conventional flow cytometry, allows several thousands of single cells to be cultivated, short‐term (72 h) or long‐term (10–14 days), and analyzed individually. By controlled sorting of individual cells to predefined locations in the microplate, analysis of single cell heterogeneity and clonogenic properties related to drug sensitivity can be accomplished. Additionally, the platform requires remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. By seeding single cells into the microplate it is possible to analyze the cells for over 14 generations, ending up with more than 10 000 cells in each well. Described here is a proof‐of‐concept on compartmentalization and cultivation of thousands of individual cells enabling heterogeneity analysis of various cells/clones and their response to different drugs.

Point-of-Care Vertical Flow Allergen Microarray Assay: Proof of Concept

BACKGROUND Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of <10 min before imaging and data analysis. METHOD Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of ≤10 min. Microarray images were captured by a consumer-grade flatbed scanner. RESULTS A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was <14%. The observed concordance with a clinical assay, ImmunoCAP, was R(2) = 0.89 (n = 31). CONCLUSIONS In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing.

A lateral flow paper microarray for rapid allergy point of care diagnostics

There is a growing need for multiplexed specific IgE tests that can accurately evaluate patient sensitization profiles. However, currently available commercial tests are either single/low-plexed or require sophisticated instrumentation at considerable cost per assay. Here, we present a novel convenient lateral flow microarray-based device that employs a novel dual labelled gold nanoparticle-strategy for rapid and sensitive detection of a panel of 15 specific IgE responses in 35 clinical serum samples. Each gold nanoparticle was conjugated to an optimized ratio of HRP and anti-IgE, allowing significant enzymatic amplification to improve the sensitivity of the assay as compared to commercially available detection reagents. The mean inter-assay variability of the developed LFM assay was 12% CV, and analysis of a cohort of clinical samples (n = 35) revealed good general agreement with ImmunoCAP, yet with a varying performance among allergens (AUC = [0.54-0.88], threshold 1 kU). Due to the rapid and simple procedure, inexpensive materials and read-out by means of a consumer flatbed scanner, the presented assay may provide an interesting low-cost alternative to existing multiplexed methods when thresholds >1 kU are acceptable.

Microfluidics for nano-pathophysiology.

Nanotechnology-based drug delivery systems hold promise for innovative medical treatment of cancers. While drug materials are constantly under development, there are no practical cell-based models to assess whether these materials can reach the target tissue. Recently developed microfluidic systems have revolutionized cell-based experiments. In these systems, vascular endothelial cells and interstitium are set in microchannels that mimic microvessels. Drug permeability can be assayed in these blood vessel models under fluidic conditions that mimic blood flow. In this review, we describe device fabrication, disease model development, nanoparticle permeability assays, and the potential utility of these systems in the future.

Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade

ABSTRACT Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor.

Lab on a chip technologies for bioenergy and biosustainability research

Energy production is likely to be one of the most important challenges for society in the 21st century, due to current unsustainable exploitation of global fossil fuel resources. With rapid advancements in biotechnology and metabolic engineering, it has become possible to design and develop enzyme-based factories for the production of fuels and chemicals and this fact, combined with the increasing price of oil and societal demands for the development of sustainable production processes, has resulted in an increased interest in exploiting biotechnology for the production of food, drugs, fuels and chemicals. In Fig. 1 a schematic of bioenergy conversion processes is shown. Recently, the World Wide Fund for Nature (WWF) suggested that the application of biotechnology could reduce emissions by up to 2.5 billion tons of CO2equivalent by 2030—more than the total emissions of Germany in 1990. The Organization for Economic Co-operation and Development (OECD) estimates that by 2030, biotechnology will contribute approximately 35% of manufactured chemicals and 80% of the pharmaceuticals globally. Interestingly, lab on a chip applications can play an important role in this development, including areas such as high throughput screening, protein and metabolic engineering, and minimization of reagents. In this article, we discuss some of these developments with a focus on the use of cells and enzymes.