Drug Screening Applications Research Articles

Large-Scale Production of Human iPSC-Derived Macrophages for Drug Screening.

Tissue-resident macrophages are key players in inflammatory processes, and their activation and functionality are crucial in health and disease. Numerous diseases are associated with alterations in homeostasis or dysregulation of the innate immune system, including allergic reactions, autoimmune diseases, and cancer. Macrophages are a prime target for drug discovery due to their major regulatory role in health and disease. Currently, the main sources of macrophages used for therapeutic compound screening are primary cells isolated from blood or tissue or immortalized or neoplastic cell lines (e.g., THP-1). Here, we describe an improved method to employ induced pluripotent stem cells (iPSCs) for the high-yield, large-scale production of cells resembling tissue-resident macrophages. For this, iPSC-derived macrophage-like cells are thoroughly characterized to confirm their cell identity and thus their suitability for drug screening purposes. These iPSC-derived macrophages show strong cellular identity with primary macrophages and recapitulate key functional characteristics, including cytokine release, phagocytosis, and chemotaxis. Furthermore, we demonstrate that genetic modifications can be readily introduced at the macrophage-like progenitor stage in order to interrogate drug target-relevant pathways. In summary, this novel method overcomes previous shortcomings with primary and leukemic cells and facilitates large-scale production of genetically modified iPSC-derived macrophages for drug screening applications.

Generation of tumor spheroids using a droplet-based microfluidic device for photothermal therapy

Despite their simplicity, monolayer cell cultures are not able to accurately predict drug behavior in vivo due to their inability to accurately mimic cell-cell and cell-matrix interactions. In contrast, cell spheroids are able to reproduce these interactions and thus would be a viable tool for testing drug behavior. However, the generation of homogenous and reproducible cell spheroids on a large scale is a labor intensive and slow process compared to monolayer cell cultures. Here, we present a droplet-based microfluidic device for the automated, large-scale generation of homogenous cell spheroids in a uniform manner. Using the microfluidic system, the size of the spheroids can be tuned to between 100 and 130 μm with generation frequencies of 70 Hz. We demonstrated the photothermal therapy (PTT) application of brain tumor spheroids generated by the microfluidic device using a reduced graphene oxide-branched polyethyleneimine-polyethylene glycol (rGO-BPEI-PEG) nanocomposite as the PTT agent. Furthermore, we generated uniformly sized neural stem cell (NSC)-derived neurospheres in the droplet-based microfluidic device. We also confirmed that the neurites were regulated by neurotoxins. Therefore, this droplet-based microfluidic device could be a powerful tool for photothermal therapy and drug screening applications.

Access to stem cell data and registration of pluripotent cell lines: The Human Pluripotent Stem Cell Registry (hPSCreg).

The value of human pluripotent stem cells (hPSC) in regenerative medicine has yet to reach its full potential. The road from basic research tool to clinically validated PSC-derived cell therapy products is a long and winding one, leading researchers, clinicians, industry and regulators alike into undiscovered territory. All stakeholders must work together to ensure the development of safe and effective cell therapies. Similarly, utilization of hPSC in meaningful and controlled disease modeling and drug screening applications requires information on the quality and suitability of the applied cell lines. Central to these common goals is the complete documentation of hPSC data, including the ethical provenance of the source material, the hPSC line derivation, culture conditions and genetic constitution of the lines. Data surrounding hPSC is scattered amongst diverse sources, including publications, supplemental data, researcher lab books, accredited lab reports, certificates of analyses and public data repositories. Not all of these data sources are publicly accessible nor associated with metadata nor stored in a standard manner, such that data can be easily found and retrieved. The Human Pluripotent Stem Cell Registry (hPSCreg; https://hpscreg.eu/) was started in 2007 to impart provenance and transparency towards hPSC research by registering and collecting standard properties of hPSC lines. In this chapter, we present a short primer on the history of stem cell-based products, summarize the ethical and regulatory issues introduced in the course of working with hPSC-derived products and their associated data, and finally present the Human Pluripotent Stem Cell Registry as a valuable resource for all stakeholders in therapies and disease modeling based on hPSC-derived cells.

High-Throughput and Integrated Chemical Proteomic Approach for Profiling Phosphotyrosine Signaling Complexes.

Phosphotyrosine (pTyr) signaling complexes are important resources of biomarkers and drug targets which often need to be profiled with enough throughput. Current profiling approaches are not feasible to meet this need due to either biased profiling by antibody-based detection or low throughput by traditional affinity purification-mass spectrometry approach (AP-MS), as exemplified by our previously developed photo-pTyr-scaffold approach. To address these limitations, we developed a 96-well microplate-based sample preparation and fast data independent proteomic analysis workflow. By assembling the photo-pTyr-scaffold probe into a 96-well microplate, we achieved steric hindrance-free photoaffinity capture of pTyr signaling complexes, selective enrichment under denaturing conditions, and efficient in-well digestion in a fully integrated manner. EGFR signaling complex proteins could be efficiently captured and identified by using 300 times less cell lysate and 100 times less photo-pTyr-scaffold probe as compared with our previous approach operated in an Eppendorf tube. Furthermore, the lifetime of the photo-pTyr-scaffold probe in a 96-well microplate was significantly extended from 1 week up to 1 month. More importantly, by combining with high-flow nano LC separation and data independent acquisition on the Q Exactive HF-X mass spectrometer, LC-MS time could be significantly reduced to only 35 min per sample without increasing sample loading amount and compromising identification and quantification performance. This new high-throughput proteomic approach allowed us to rapidly and reproducibly profile dynamic pTyr signaling complexes with EGF stimulation at five time points and EGFR inhibitor treatment at five different concentrations. We are therefore optimized for its generic application in biomarkers discovery and drug screening in a high-throughput fashion.

Biomimetic Matrix Stiffness Modulates Hepatocellular Carcinoma Malignant Phenotypes and Macrophage Polarization through Multiple Modes of Mechanical Feedbacks.

The extracellular matrix (ECM) stiffening is an important sign of local microenvironment change, which is considered as a hallmark of many diseases including hepatocellular carcinoma (HCC). The fates of both cancer cells and immune cells can be regulated by mechanical feedbacks acquired from ECM, but there is a lack of a precise study of mechanical feedback modes in different cell phenotypes following with the progressively increasing ECM stiffness. Herein, we used a biopolymeric film without further modification of adhesive molecules, as a natural local niche to mimic a gradually stiffening manner from HCC onset in liver cirrhosis to its metastasis in the spinal cord. Three distinct manners of mechanical feedbacks were found: the gradual manner in HCC cell spreading, migration and early apoptosis to oxaliplatin, the stepwise manner in HCC cell adhesion, proliferation, focal adhesion (FA) formation, drug resistance, and macrophage M1 polarization; the specific manner in the stages of the progression of epithelial-mesenchymal transition at different stiffness ranges. Further investigation of molecular mechanisms confirmed those mechanical feedback manners by signaling activation of FA kinase, phosphatidylinositol 3-kinase, and expression of pro-/antiapoptotic and pro-/anti-inflammatory genes. Our results pave a novel avenue to know about mechanical feedbacks from ECM, which could be used for future cancer studies and in vitro drug screening applications.

Metallic Nanoparticle-Enabled Sensing of a Drug-of-Abuse: An Attempt at Forensic Application.

γ-Hydroxybutyric acid (GHB) functions as a depressant on the central nerve system and serves as a pharmaceutical agent in the treatment of narcolepsy and alcohol withdraw. In recent years, GHB has been misused as a recreational drug due to its ability to induce euphoric feelings. Moreover, it has gained increasing attention as a popular drug of abuse that is frequently related to drug-facilitated sexual assaults. At the moment, detection methods based on chromatography exhibit extraordinary sensitivity for GHB sensing. However, such techniques require complicated sample treatment prior to analysis. Optical sensors provide an alternative approach for rapid and simple analysis of GHB samples. Unfortunately, currently reported probes are mostly based on hydrogen bonding to recognize GHB, and this raises concerns about, for example, the lack of specificity. In this work, we report a bioinspired strategy for selective sensing of GHB. The method is based on specific enzyme recognition to allow highly selective detection of GHB with minimum interference, even in a complex sample matrix (e. g., simulated urine). In addition, the result can be obtained by either quantitative spectroscopy analysis or colorimetric change observed by the naked-eye, thus demonstrating its potential application in drug screening and forensic analysis.

Analysis of hiPSCs differentiation toward hepatocyte-like cells upon extended exposition to oncostatin

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

A Defined and Scalable Peptide-Based Platform for the Generation of Human Pluripotent Stem Cell-Derived Astrocytes.

Astrocytes comprise the most abundant cell type in the central nervous system (CNS) and play critical roles in maintaining neural tissue homeostasis. In addition, astrocyte dysfunction and death has been implicated in numerous neurological disorders such as multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), and Parkinson’s disease (PD). As such, there is much interest in using human pluripotent stem cell (hPSC)-derived astrocytes for drug screening, disease modeling, and regenerative medicine applications. However, current protocols for generation of astrocytes from hPSCs are limited by the use of undefined xenogeneic components and two-dimensional (2D) culture surfaces, which limits their downstream applications where large-quantities of cells generated under defined conditions are required. Here, we report the use of a completely synthetic, peptide-based substrate that allows for the differentiation of highly pure populations of astrocytes from several independent hPSC lines, including those derived from patients with neurodegenerative disease. This substrate, which we demonstrate is compatible with both conventional 2D culture formats and scalable microcarrier (MC)-based technologies, leads to the generation of cells that express high levels of canonical astrocytic markers as well as display properties characteristic of functionally mature cells including production of apolipoprotein E (ApoE), responsiveness to inflammatory stimuli, ability to take up amyloid-β (Aβ), and appearance of robust calcium transients. Finally, we show that these astrocytes can be cryopreserved without any loss of functionality. In the future, we anticipate that these methods will enable the development of bioprocesses for the production of hPSC-derived astrocytes needed for biomedical research and clinical applications.

Generation of iPS clones using automated live-imaging, picking and in-process quality controls

Background & Aim Induced pluripotent stem (iPS) cells have great potential as tools for regenerative medicine, disease modeling and drug screening applications. However, current manufacturing remain highly dependent on manual tasks and subjective human decisions about which colonies to choose and progress in large-scale cell expansion process. The goal of this work is to develop automated workflow options using the Cell X™ robotic platform for: 1) Cell Source Selection based on quantitative reproducible quality attributes, 2) Precision Automation of iPS clone “picking”, “weeding”, passage and expansion, and 3) quantitative documentation of cell and clone attributes over time to define critical process parameters and critical quality attributes that can predict future performance. Methods, Results & Conclusion The Cell X™ robotic platform was used to develop and characterize imaging and analysis tools relevant to iPS populations using the DF6-9-9T.B hiPSC cell line, reprogramming of human skin fibroblasts. The Cell X platform (Fig. 1) is a multifaceted robotic device that enables: automated large field of view (LFOV) imaging using phase contrast or fluorescence; serial imaging (videomicroscopy); quantitative LFOV image analysis (Colonyze™); media changes; cell and colony “picking” (isolation and transplantation of desired cells), “weeding” (removal of undesired cells); or “biopsy (sampling of defined cells for analysis). Feasibility was demonstrated in the capture LFOV images over the reprogramming phase to track quantitative metrics of evolution and morphology of individual iPS clones (e.g. size, shape, area, perimeter roughness, texture, growth rate) (Fig. 2 & 3). Automated image processing tools were defined to segment regions of undifferentiated iPS cells from regions of differentiating and potentially differentiating cells, which could be used to guide “picking”, “weeding” or “biopsy” interventions. Picking operations were effective, and transplanted cells continued to proliferate effectively with preservation of iPS morphology. The integrated Cell X™ platform enables both fully automated iPS clone “picking” and expansion in a hands free, reproducible, and environmentally controlled setting. This system represent a potential valuable tool to automate and scale iPS fabrication with reduced expense and variation, and with rigorous documentation. Cell X™ methods can be refined for lab-specific application, core facility, and integrated in fully automated industrial GMP manufacturing facility.

Robotically handled whole-tissue culture system for the screening of oral drug formulations.

Monolayers of cancer-derived cell lines are widely used in the modelling of the gastrointestinal (GI) absorption of drugs and in oral drug development. However, they do not generally predict drug absorption in vivo. Here, we report a robotically handled system that uses large porcine GI tissue explants that are functionally maintained for an extended period in culture for the high-throughput interrogation (several thousand samples per day) of whole segments of the GI tract. The automated culture system provided higher predictability of drug absorption in the human GI tract than a Caco-2 Transwell system (Spearman's correlation coefficients of 0.906 and 0.302, respectively). By using the culture system to analyse the intestinal absorption of 2,930 formulations of the peptide drug oxytocin, we discovered an absorption enhancer that resulted in a 11.3-fold increase in the oral bioavailability of oxytocin in pigs in the absence of cellular disruption of the intestinal tissue. The robotically handled whole-tissue culture system should help advance the development of oral drug formulations and might also be useful for drug screening applications.

Functional arrays of human pluripotent stem cell-derived cardiac microtissues

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom 3D-print-molded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Cell subtype-dependent formation of breast tumor spheroids and their variable responses to chemotherapeutics within microfluidics-generated 3D microgels with tunable mechanics.

Tumor spheroids have been considered valuable miniaturized three dimensional (3D) tissue models for fundamental biological investigation as well as drug screening applications. Most tumor spheroids are generated utilizing the inherent aggregate behavior of tumor cells, and the effect of microenvironmental factors such as extracellular matrix (ECM) on tumor spheroid formation has not been extensively elucidated to date. Herein, uniform-sized spherical microgels encapsulated with different subtypes of breast tumor cells, based on tumor aggressiveness, are developed by flow-focusing microfluidics technology. Mechanical properties of microgels are controlled in a wide range via polymer concentration, and their influence on tumor physiology and spheroid formation is shown to be highly dependent on cell subtype. Specifically, the formation of polyploid/multinucleated giant cancer cells is a key early step in determining initial proliferation and eventual tumor spheroid generation within microgels with varying mechanics. In addition, chemotherapeutic screening performed on these tumor spheroids in microgels also display significantly variable cytotoxic effects based on microgel mechanics for each cell subtype, further highlighting the importance of microenvironmental factors on tumor spheroid physiology.

Apical Sodium-Dependent Bile Acid Cotransporter, A Novel Transporter of Indocyanine Green, and Its Application in Drug Screening.

Bile acid plays critical roles in the elimination of inorganic compounds such as bilirubin, heavy metals, and drug metabolites. Apical sodium-dependent bile acid cotransporter (ASBT), a solute carrier membrane transport protein, transports bile acids. Several inhibitors of ASBT have been evaluated in clinical trials. Sodium taurocholate cotransporting polypeptide (NTCP), belonging to the same family as ASBT, has fluorescein 5(6)-isothiocyanate (FITC) and indocyanine green (ICG) transportability. ICG, a Food and Drug Administration-approved fluorophore at near-infrared range, has perfect optical characteristics, so it can be applied in cell tracking and drug screening. In this study, ASBT and NTCP were transduced into the HT-1080 cell line. Nude mice were subcutaneously xenografted with control and ASBT-expressing cells. ICG transportability was observed through flow cytometry, fluorescent microscopy, multi-mode plate readers, and an in vivo imaging system. Several molecules, including taurocholate, sodium deoxycholate, cyclosporine A, nifedipine, and Primovist, were used to evaluate an in vitro drug-screening platform by using the combination of ICG and ASBT through flow cytometry. ICG and FITC were validated and shown to be transported by ASBT. NTCP had a higher ICG intensity compared with ASBT. For cell tracking, the ASBT xenograft had similar ICG signals as the control. For a drug-screening platform, the ICG intensity decreased with 186 μM taurocholate (56.8%), deoxycholate (83.8%), and increased with nifedipine (133.2%). These findings are suggestive of opportunities for the high-throughput drug screening of cholestasis and other diseases that are related to the dynamics of bile acid reabsorption.

Surface-Modified Industrial Acrylonitrile Butadiene Styrene 3D Scaffold Fabrication by Gold Nanoparticle for Drug Screening.

Biocompatibility is very important for cell growth using 3D printers, but biocompatibility materials are very expensive. In this study, we investigated the possibility of cell culture by the surface modification of relatively low-cost industrial materials and an efficient three-dimensional (3D) scaffold made with an industrial ABS filament for cell proliferation, spheroid formation, and drug screening applications. We evaluated the adequate structure among two-layer square shape 3D scaffolds printed by fused deposition modeling with variable infill densities (10–50%). Based on the effects of these scaffolds on cell proliferation and spheroid formation, we conducted experiments using the industrial ABS 3D scaffold (IA3D) with 40% of infill density, which presented an external dimension of (XYZ) 7650 µm × 7647 µm × 210 µm, 29.8% porosity, and 225 homogenous micropores (251.6 µm × 245.9 µm × 210 µm). In the IA3D, spheroids of cancer HepG2 cells and keratinocytes HaCaT cells appeared after 2 and 3 days of culture, respectively, whereas no spheroids were formed in 2D culture. A gold nanoparticle-coated industrial ABS 3D scaffold (GIA3D) exhibited enhanced biocompatible properties including increased spheroid formation by HepG2 cells compared to IA3D (1.3-fold) and 2D (38-fold) cultures. Furthermore, the cancer cells exhibited increased resistance to drug treatments in GIA3D, with cell viabilities of 122.9% in industrial GIA3D, 40.2% in IA3D, and 55.2% in 2D cultures when treated with 100 µM of mitoxantrone. Our results show that the newly engineered IA3D is an innovative 3D scaffold with upgraded properties for cell proliferation, spheroid formation, and drug-screening applications.

Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring

Directed differentiation methods allow acquisition of high-purity cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with ReWs show higher maturation including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the ReWs within the SOTRs.

Scalable Generation of Mesenchymal Stem Cells and Adipocytes from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can provide unlimited supply for mesenchymal stem cells (MSCs) and adipocytes that can be used for therapeutic applications. Here we developed a simple and highly efficient all-trans-retinoic acid (RA)-based method for generating an off-the-shelf and scalable number of human pluripotent stem cell (hPSC)-derived MSCs with enhanced adipogenic potential. We showed that short exposure of multiple hPSC lines (hESCs/hiPSCs) to 10 μM RA dramatically enhances embryoid body (EB) formation through regulation of genes activating signaling pathways associated with cell proliferation, survival and adhesion, among others. Disruption of cell adhesion induced the subsequent differentiation of the highly expanded RA-derived EB-forming cells into a pure population of multipotent MSCs (up to 1542-fold increase in comparison to RA-untreated counterparts). Interestingly, the RA-derived MSCs displayed enhanced differentiation potential into adipocytes. Thus, these findings present a novel RA-based approach for providing an unlimited source of MSCs and adipocytes that can be used for regenerative medicine, drug screening and disease modeling applications.

RapidAIM: a culture- and metaproteomics-based Rapid Assay of Individual Microbiome responses to drugs

BackgroundHuman-targeted drugs may exert off-target effects or can be repurposed to modulate the gut microbiota. However, our understanding of such effects is limited due to a lack of rapid and scalable assay to comprehensively assess microbiome responses to drugs. Drugs and other compounds can drastically change the overall abundance, taxonomic composition, and functions of a gut microbiome.ResultsHere, we developed an approach to screen compounds against individual microbiomes in vitro, using metaproteomics to both measure absolute bacterial abundances and to functionally profile the microbiome. Our approach was evaluated by testing 43 compounds (including 4 antibiotics) against 5 individual microbiomes. The method generated technically highly reproducible readouts, including changes of overall microbiome abundance, microbiome composition, and functional pathways. Results show that besides the antibiotics, the compounds berberine and ibuprofen inhibited the accumulation of biomass during in vitro growth of the microbiota. By comparing genus and species level-biomass contributions, selective antibacterial-like activities were found with 35 of the 39 non-antibiotic compounds. Seven of the compounds led to a global alteration of the metaproteome, with apparent compound-specific patterns of functional responses. The taxonomic distributions of altered proteins varied among drugs, i.e., different drugs affect functions of different members of the microbiome. We also showed that bacterial function can shift in response to drugs without a change in the abundance of the bacteria.ConclusionsCurrent drug-microbiome interaction studies largely focus on relative microbiome composition and microbial drug metabolism. In contrast, our workflow enables multiple insights into microbiome absolute abundance and functional responses to drugs. The workflow is robust, reproducible, and quantitative and is scalable for personalized high-throughput drug screening applications.

Breeze: an integrated quality control and data analysis application for high-throughput drug screening.

SummaryHigh-throughput screening (HTS) enables systematic testing of thousands of chemical compounds for potential use as investigational and therapeutic agents. HTS experiments are often conducted in multi-well plates that inherently bear technical and experimental sources of error. Thus, HTS data processing requires the use of robust quality control procedures before analysis and interpretation. Here, we have implemented an open-source analysis application, Breeze, an integrated quality control and data analysis application for HTS data. Furthermore, Breeze enables a reliable way to identify individual drug sensitivity and resistance patterns in cell lines or patient-derived samples for functional precision medicine applications. The Breeze application provides a complete solution for data quality assessment, dose–response curve fitting and quantification of the drug responses along with interactive visualization of the results.Availability and implementationThe Breeze application with video tutorial and technical documentation is accessible at https://breeze.fimm.fi; the R source code is publicly available at https://github.com/potdarswapnil/Breeze under GNU General Public License v3.0.Contact swapnil.potdar@helsinki.fi Supplementary information Supplementary data are available at Bioinformatics online.

Enzymatic outside-in cross-linking enables single-step microcapsule production for high-throughput three-dimensional cell microaggregate formation.

Cell-laden hydrogel microcapsules enable the high-throughput production of cell aggregates, which are relevant for three-dimensional tissue engineering and drug screening applications. However, current microcapsule production strategies are limited by their throughput, multistep protocols, and limited amount of compatible biomaterials. We here present a single-step process for the controlled microfluidic production of single-core microcapsules using enzymatic outside-in cross-linking of tyramine-conjugated polymers. It was hypothesized that a physically, instead of the conventionally explored biochemically, controlled enzymatic cross-linking process would improve the reproducibility, operational window, and throughput of shell formation. Droplets were flown through a silicone delay line, which allowed for highly controlled diffusion of the enzymatic cross-linking initiator. The microcapsules' cross-linking density and shell thickness is strictly depended on the droplet's retention time in the delay line, which is predictably controlled by flow rate. The here presented hydrogel cross-linking method allows for facile and cytocompatible production of cell-laden microcapsules compatible with the formation and biorthogonal isolation of long-term viable cellular spheroids for tissue engineering and drug screening applications.