High-throughput Screening Assay Research Articles

Orthogonal assay and QSAR modelling of Tox21 PPARγ antagonist in vitro high-throughput screening assay

Disruption of signalling mediated by the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is associated with risk of cancer, metabolic diseases, and endocrine disruption. The purpose of this study was to identify environmental chemicals acting as PPARγ antagonists. Data from the Tox21 PPARγ antagonism assay were replicated using a reporter system in HEK293 cells. Two quantitative structure-activity relationship (QSAR) models were developed, and five REACH-registered substances predicted positive were tested in vitro. Reporter assay results were consistent with Tox21 data since all conflicting results could be explained by assay interference. QSAR models showed good predictive performance, and follow-up experiments revealed two PPARγ antagonists out of three non-interfering chemicals. In conclusion, the developed QSAR models and follow-up experiments are important steps in the discovery of potential endocrine- and metabolism-disrupting chemicals.

Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro

Angiotensin AT2-receptor (AT2R) agonists have shown a wide range of protective effects in many preclinical disease models. However, the availability of AT2R-agonists is very limited due to the lack of high-throughput assays for AT2R-agonist identification. Therefore, we aimed to design and validate an assay for high-throughput screening of AT2R-agonist candidates. The assay is based on nitric oxide (NO) release measurements in primary human aortic endothelial cells (HAEC), in AT2R-transfected CHO cells (AT2R-CHO) or in non-transfected CHO cells (Flp-CHO) using the fluorescent probe DAF-FM diacetate. It is run in 96-well plates and fluorescence signals are semi-automatically quantified. The assay was tested for sensitivity (recognition of true positive results), selectivity (recognition of true negative results), and reliability (by calculating the repeatability coefficient (RC)). The high-throughput, semi-automated method was proven suitable, as the NO-releasing agents C21, CGP42112A, angiotensin-(1−7) and acetylcholine significantly increased NO release from HAEC. The assay is sensitive and selective, since the established AT2R-agonists C21, CGP42112A and angiotensin II significantly increased NO release from AT2R-CHO cells, while the non-AT2R-agonists angiotensin-(1−7) and acetylcholine had no effect. Assay reliability was shown by high-throughput screening of a library comprised of 40 potential AT2R-agonists, of which 39 met our requirements for reliability (RC ≤ 20% different from RC for C21). Our newly developed high-throughput method for detection of AT2R-agonistic activity was proven to be sensitive, selective, and reliable. This method is suitable for the screening of potential AT2R-agonists in future drug development programs.

Thiophene derivative inflicts cytotoxicity via an intrinsic apoptotic pathway on human acute lymphoblastic leukemia cells.

In an effort to identify novel anti-cancer agents, we employed a well-established High Throughput Screening (HTS) assay to assess the cytotoxic effect of compounds within the ChemBridge DIVERSet Library on a lymphoma cell line. This screen revealed a novel thiophene, F8 (methyl 5-[(dimethylamino)carbonyl]-4-methyl-2-[(3-phenyl-2-propynoyl) amino]-3-thiophenecarboxylate), that displays anti-cancer activity on lymphoma, leukemia, and other cancer cell lines. Thiophenes and thiophene derivatives have emerged as an important class of heterocyclic compounds that have displayed favorable drug characteristics. They have been previously reported to exhibit a broad spectrum of properties and varied uses in the field of medicine. In addition, they have proven to be effective drugs in various disease scenarios. They contain anti-inflammatory, anti-anxiety, anti-psychotic, anti-microbial, anti-fungal, estrogen receptor modulating, anti-mitotic, kinase inhibiting and anti-cancer activities, rendering compounds with a thiophene a subject of significant interest in the scientific community. Compound F8 consistently induced cell death at a low micromolar range on a small panel of cancer cell lines after a 48 h period. Further investigation revealed that F8 induced phosphatidylserine externalization, reactive oxygen species generation, mitochondrial depolarization, kinase inhibition, and induces apoptosis. These findings demonstrate that F8 has promising anti-cancer activity.

Putative purine nucleoside interacting residues in the malaria parasite purine uptake transporter PfENT1 are critical for transporter function.

Malaria remains a major public health threat for billions of people worldwide. Infection with obligate intracellular, unicellular parasites from the genus Plasmodium causes malaria. Plasmodium falciparum causes the deadliest form of human malaria. Plasmodium parasites are purine auxotrophic. They rely on purine import from the host red blood cell cytoplasm via equilibrative nucleoside transporters to supply substrates to the purine salvage pathway. We previously developed a high throughput screening assay to identify inhibitors of the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Screening a small molecule library identified PfENT1 inhibitors that blocked proliferation of P. falciparum parasites in in vitro culture. The goal of the current work was to validate a high-resolution model of PfENT1 predicted by the AlphaFold protein structure prediction program. We superimposed the predicted PfENT1 structure on the human homologue structure, hENT1, and developed a structure-based sequence alignment. We mutated the residues in PfENT1 aligned with and flanking the residues in hENT1 that interact with the purine analog, nitrobenzylthioinosine (NBMPR). Mutation of the PfENT1 residues Q135, D287, and R291 that are predicted to form hydrogen bonds to purine nucleosides eliminated purine and pyrimidine transport function in various yeast-based growth and radiolabeled substrate uptake assays. Mutation of two flanking residues, W53 and S290, also resulted in inactive protein. Mutation of L50 that forms hydrophobic interactions with the purine nucleobase reduced transport function. Based on our results the AlphaFold predicted structure for PfENT1 may be useful in guiding medicinal chemistry efforts to improve the potency of our PfENT1 inhibitors.

A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery.

The identification of new modulators for Cannabinoid Receptors (CBRs) has garnered significant attention in drug discovery over recent years, owing to their manifold pathophysiological implications. In the context of hit identification, the availability of robust and sensitive high-throughput screening assays is essential to enhance the likelihood of success. In this study, we present the development and validation of a Tag-lite® binding assay designed for screening hCB1/hCB2 binding, employing a dual fluorescent ligand, CELT-335. Representative ligands for CBRs, exhibiting diverse affinity and functional profiles, were utilized as reference compounds to validate the robustness and efficiency of the newly developed Tag-lite® binding assay protocol. The homogeneous format, coupled with the sensitivity and optimal performance of the fluorescent ligand CELT-335, establishes this assay as a viable and reliable method for screening in hit and lead identification campaigns.

Discovery of GPX4 inhibitors through FP-based high-throughput screening

Ferroptosis is a form of non-apoptotic cell death, regulated by phospholipid hydroperoxide glutathione peroxidase 4 (GPX4), a selenoprotein with a selenocysteine residue (sec) in the active site. GPX4 is a promising target for cancer cells in therapy-resistant conditions via ferroptosis, which can reduce the level of lipid reactive oxygen species (ROS). So far, all existing GPX4 inhibitors covalently bind to GPX4 via a reactive alkyl chloride moiety or masked nitrile-oxide electrophiles with poor selectivity and pharmacokinetic properties and most were obtained by cell phenotype-based screening. Lacking of effective high-throughput screening methods for GPX4 protein limits the discovery of GPX4 inhibitors. Here, we report a fluorescence polarization (FP)-based high throughput screening (HTS) assay for GPX4-U46C-C10A-C66A in vitro, and found Metamizole sodium from our in-house compound library inhibits GPX4-U46C-C10A-C66A enzyme activity. Structure-activity relationships (SAR) demonstrated the importance of sulfonyl group on interaction between Metamizole sodium and GPX4-U46C-C10A-C66A. Our FP assay could be an effective tool for discovery of GPX4 inhibitors and Metamizole sodium was a potential inhibitor for GPX4 in vitro.

Fluorescence-Based Peptidolytic Assay for High-Throughput Screening of MMP14 Inhibitors.

The membrane-bound matrix metalloproteinase 14 (MMP14, also known as MT1-MMP) plays important roles in the remodeling of the extracellular matrix during various cellular processes such as cancer metastasis, angiogenesis, and wound healing through its proteolytic activity. There are no known MMP14-specific inhibitors to date, and hence identification of MMP14-specific inhibitors will be beneficial for finding potential therapeutics for various diseases, including cancer and inflammation. High-throughput screening (HTS) assays have become a common way to search for new small compounds, peptides, and natural products. Enzymatic assays are highly amenable to HTS because most enzyme activities are quantifiable with the effect of many small molecules of interest on a specific target enzyme. Here, we describe a fluorescence-based enzymatic assay that can be applied as a large-scale HTS and a follow-up enzyme kinetics assay to find MMP14-specific inhibitors.

Exploring the effects of experimental parameters and data modeling approaches on in vitro transcriptomic point-of-departure estimates

Multiple new approach methods (NAMs) are being developed to rapidly screen large numbers of chemicals to aid in hazard evaluation and risk assessments. High-throughput transcriptomics (HTTr) in human cell lines has been proposed as a first-tier screening approach for determining the types of bioactivity a chemical can cause (activation of specific targets vs. generalized cell stress) and for calculating transcriptional points of departure (tPODs) based on changes in gene expression. In the present study, we examine a range of computational methods to calculate tPODs from HTTr data, using six data sets in which MCF7 cells cultured in two different media formulations were treated with a panel of 44 chemicals for 3 different exposure durations (6, 12, 24 hr). The tPOD calculation methods use data at the level of individual genes and gene set signatures, and compare data processed using the ToxCast Pipeline 2 (tcplfit2), BMDExpress and PLIER (Pathway Level Information ExtractoR). Methods were evaluated by comparing to in vitro PODs from a validated set of high-throughput screening (HTS) assays for a set of estrogenic compounds. Key findings include: (1) for a given chemical and set of experimental conditions, tPODs calculated by different methods can vary by several orders of magnitude; (2) tPODs are at least as sensitive to computational methods as to experimental conditions; (3) in comparison to an external reference set of PODs, some methods give generally higher values, principally PLIER and BMDExpress; and (4) the tPODs from HTTr in this one cell type are mostly higher than the overall PODs from a broad battery of targeted in vitro ToxCast assays, reflecting the need to test chemicals in multiple cell types and readout technologies for in vitro hazard screening.

A Robust Phenotypic High-Throughput Antiviral Assay for the Discovery of Rabies Virus Inhibitors.

Rabies virus (RABV) causes severe neurological symptoms in mammals. The disease is almost inevitably lethal as soon as clinical symptoms appear. The use of rabies immunoglobulins (RIG) and vaccination in post-exposure prophylaxis (PEP) can provide efficient protection, but many people do not receive this treatment due to its high cost and/or limited availability. Highly potent small molecule antivirals are urgently needed to treat patients once symptoms develop. In this paper, we report on the development of a high-throughput phenotypic antiviral screening assay based on the infection of BHK-21 cells with a fluorescent reporter virus and high content imaging readout. The assay was used to screen a repurposing library of 3681 drugs (all had been studied in phase 1 clinical trials). From this series, salinomycin was found to selectively inhibit viral replication by blocking infection at the entry stage. This shows that a high-throughput assay enables the screening of large compound libraries for the purposes of identifying inhibitors of RABV replication. These can then be optimized through medicinal chemistry efforts and further developed into urgently needed drugs for the treatment of symptomatic rabies.

Identification and characterization of inhibitors covalently modifying catalytic cysteine of UBE2T and blocking ubiquitin transfer

UBE2T is an E2 ubiquitin ligase critical for ubiquitination of substrate and plays important roles in many diseases. Despite the important function, UBE2T is considered as an undruggable target due to lack of a pocket for binding to small molecules with satisfied properties for clinical applications. To develop potent and specific UBE2T inhibitors, we adopted a high-throughput screening assay and two compounds-ETC-6152 and ETC-9004 containing a sulfone tetrazole scaffold were identified. Solution NMR study demonstrated the direct interactions between UBE2T and compounds in solution. Further co-crystal structures reveal the binding modes of these compounds. Both compound hydrolysation and formation of a hydrogen bond with the thiol group of the catalytic cysteine were observed. The formation of covalent complex was confirmed with mass spectrometry. As these two compounds inhibit ubiquitin transfer, our study provides a strategy to develop potent inhibitors of UBE2T.

Isolation, phylogenetic analysis and bioprospection of myxobacteria from Vietnam

Abstract. Yen NTN, Chung DD, Hong NTK, Cham NP, Nhan VT, Linh DTL, Ngoc NLB, Thai NM, Nga ND, Anh NT. 2023. Isolation, phylogenetic analysis and bioprospection of myxobacteria from Vietnam. Biodiversitas 24: 5653-5663. Myxobacteria have been considered microbial factories for producing secondary metabolites that have a variety of potential biological actions for discovering and isolating new biological molecules. Myxobacteria were isolated from soil samples collected in some provinces/cities in Vietnam. The purified isolates were identified based on morphology, biochemical test and phylogenetic analysis inferred from 16S rRNA gene. High-throughput screening assays including 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DDPH) for antioxidant properties and microdilution for antimicrobial activity were performed with myxobacterial extracts. Compounds from potential strain were predicted using liquid chromatography coupled with mass spectrometry. Forty-three myxobacterial strains were isolated and classified into seven genera of Angiococcus, Archangium, Chondromyces, Corallococcus, Cystobacter, Melittangium, and Myxococcus. The extract from CT21 strain had the highest total antioxidant activity (IC50 = 52.34 ± 1.47 and 30.28 ± 0.74 ?g/mL for the DPPH and ABTS, respectively). It is worth noting that all strains isolated myxobacterial strains show inhibitory activity against at least one of the tested microorganisms. The most potent antimicrobial strain was Myxococcus stipitatus GL41, which inhibited all tested microorganisms, and the minimal inhibitory concentration (MIC) values were 1 ?g/mL against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), Streptococcus faecalis, Candida albicans, and Aspergillus niger. Mass spectrometry analysis revealed the presence of althiomycin - the polyketide antibiotic from ethyl acetate fraction. In the present study, myxobacteria were isolated from soil sample collected from Vietnam, analyzed phylogenetically, and screened for biological activities.

Semirational engineering of Cytophaga hutchinsonii polyphosphate kinase for developing a cost-effective, robust, and efficient adenosine 5'-triphosphate regeneration system.

The adenosine 5'-triphosphate (ATP) regeneration system can significantly reduce the cost of many biocatalytic processes. Numerous studies have endeavored to utilize the ATP regeneration system based on Cytophaga hutchinsonii PPK (ChPPK). However, the wild-type ChPPK enzyme possesses limitations such as low enzymatic activity, poor stability, and limited substrate tolerance, impeding its application in catalytic reactions. To enhance the performance of ChPPK, we employed a semi-rational design approach to obtain the variant ChPPK/A79G/S106C/I108F/L285P. The enzymatic kinetic parameters and the catalytic performance in the synthesis of nicotinamide mononucleotide demonstrated that the variant ChPPK/A79G/S106C/I108F/L285P exhibited superior enzymatic properties than the wild-type enzyme. All data indicated that our engineered ATP regeneration system holds inherent potential for implementation in biocatalytic processes.

Establishment and application of high throughput screening cell model for nutrient regulation of embryonic development

Embryo development exerts far-reaching influence on pregnancy outcome, postnatal development and lifelong health. Thereafter, to select functional nutrients to improve embryo development is of great importance. Herein, a stable porcine trophectoderm cell line expressing a luciferase reporter gene driven by a 1,009 bp PCNA gene promoter was constructed through lentiviral transduction and G418 selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 225 nutrients. Seven nutrients with a minimum Z-score of 2.0 were initially identified to be capable of enhancing embryonic development. Among these nutrients, resveratrol, apigenin, and retinol palmitate were furtherly confirmed the beneficial effects for embryo development. Resveratrol significantly increased the expression of key genes involved in pTr cell proliferation and the number of S-phase cells. Resveratrol was furtherly confirmed to promote the expression of key genes in trophoblast development and increase embryo adhesion rate in vitro. Similarly, dietary 0.05% resveratrol supplementation significantly increased the number of embryo attachment and serum level of P4 and E2 in rats. Resveratrol could also improve maternal antioxidant levels and reduce intracellular ROS. Collectively, a high throughput screening cell model for nutrient regulation of embryonic development was established, which can be used to highly effectively select the potential candidates for embryo development. These findings have great implications for exploring optimal functional nutrients to improve embryo development, ultimately beneficial for pregnancy outcome, offspring postnatal development and lifelong health for human beings and mammalian animals.

Acoustic droplet ejection facilitates cell-based high-throughput screenings using natural products

Natural Products (NPs) are one of the main sources for drug discovery. Many clinical drugs are NPs or NP-inspired compounds, and recently discovered New Chemical Entities (NCEs) of NPs are emerging as promising new drugs. High-Throughput Screening (HTS) of large sample sets or libraries has grown to be vital for the drug discovery field. Industrial-scale HTS of NP libraries can be limited due to the difficulties entailed in working with tiny extract volumes and the variability in viscosity of NP extracts. For these reasons, the implementation of new technologies to miniaturize different reagent volumes grows to be fundamental. Since Acoustic Droplet Ejection (ADE) emerged as a helpful tool in HTS campaigns for the transference of compound libraries. The aim of this work was to test the effectiveness of ADE for the dispensation of NP extract libraries in cell-based HTS assays.

315 Developing innovative high-throughput screening assays based on HiBiT-tagged CFTR for identification of CFTR modulators

315 Developing innovative high-throughput screening assays based on HiBiT-tagged CFTR for identification of CFTR modulators

A fine-tuned yeast surface-display/secretion platform enables the rapid discovery of neutralizing antibodies against Clostridioides difficile toxins

BackgroundNeutralizing antibody plays a key role in protecting hosts from invasive pathogens and their virulent components. Current high-throughput assays for antibody screening are based on binding activities. However, those antibodies with high affinity may not have neutralizing activities. Subsequent functionality assays are necessary to identify neutralizing antibodies from binders with high affinity to their target antigens, which is laborious and time-consuming. Therefore, a versatile platform that can rapidly identify antibodies with both high binding affinity and neutralizing activity is desired to curb future pandemics like COVID-19.ResultsIn this proof-of-concept study, we adapted Saccharomyces cerevisiae to either display human antibodies on the yeast surface or secrete soluble antibodies into the cultivation supernatant under a controllable ‘switch’ through different carbon source induced promoters. Initially, an engineered chimeric-bispecific Fab antibody, derived from humanized nanobodies against both Clostridioides difficile toxin A and B (TcdA and TcdB), was successfully expressed either on the yeast cell surface or in the culture medium with intact bioactivity, suggesting the applicability of our system in antibody display and secretion. Next, a combinatorial Fab library was constructed from B cells isolated from a convalescent patient with a high serological neutralizing titer against TcdB. Following three rounds of magnetic bead enrichment and one round of flow cytometry sorting, antibodies against TcdB were enriched efficiently. We then sorted out single binders with high binding affinity and induced them to express soluble antibodies in culture medium. The neutralizing activity of culture supernatant was analyzed using cell-based assay immediately. This way, we rapidly identified two unique neutralizers (out of seven binders) that can neutralize the cytotoxicity of TcdB.ConclusionThe antibody screening platform described here simplifies the neutralizing antibody discovery procedure and will be an attractive alternative for screening functional antibodies against infectious diseases.

Discovery of Nedd4 auto-ubiquitination inhibitors

E3 ubiquitin ligases are critical to the protein degradation pathway by catalyzing the final step in protein ubiquitination by mediating ubiquitin transfer from E2 enzymes to target proteins. Nedd4 is a HECT domain-containing E3 ubiquitin ligase with a wide range of protein targets, the dysregulation of which has been implicated in myriad pathologies, including cancer and Parkinson's disease. Towards the discovery of compounds disrupting the auto-ubiquitination activity of Nedd4, we developed and optimized a TR-FRET assay for high-throughput screening. Through selective screening of a library of potentially covalent compounds, compounds 25 and 81 demonstrated apparent IC50 values of 52 µM and 31 µM, respectively. Tandem mass spectrometry (MS/MS) analysis confirmed that 25 and 81 were covalently bound to Nedd4 cysteine residues (Cys182 and Cys867). In addition, 81 also adducted to Cys627. Auto-ubiquitination assays of Nedd4 mutants featuring alanine substitutions for each of these cysteines suggested that the mode of inhibition of these compounds occurs through blocking the catalytic Cys867. The discovery of these inhibitors could enable the development of therapeutics for various diseases caused by Nedd4 E3 ligase dysregulation.

The ToxCast pipeline: updates to curve-fitting approaches and database structure.

Introduction: The US Environmental Protection Agency Toxicity Forecaster (ToxCast) program makes in vitro medium- and high-throughput screening assay data publicly available for prioritization and hazard characterization of thousands of chemicals. The assays employ a variety of technologies to evaluate the effects of chemical exposure on diverse biological targets, from distinct proteins to more complex cellular processes like mitochondrial toxicity, nuclear receptor signaling, immune responses, and developmental toxicity. The ToxCast data pipeline (tcpl) is an open-source R package that stores, manages, curve-fits, and visualizes ToxCast data and populates the linked MySQL Database, invitrodb. Methods: Herein we describe major updates to tcpl and invitrodb to accommodate a new curve-fitting approach. The original tcpl curve-fitting models (constant, Hill, and gain-loss models) have been expanded to include Polynomial 1 (Linear), Polynomial 2 (Quadratic), Power, Exponential 2, Exponential 3, Exponential 4, and Exponential 5 based on BMDExpress and encoded by the R package dependency, tcplfit2. Inclusion of these models impacted invitrodb (beta version v4.0) and tcpl v3 in several ways: (1) long-format storage of generic modeling parameters to permit additional curve-fitting models; (2) updated logic for winning model selection; (3) continuous hit calling logic; and (4) removal of redundant endpoints as a result of bidirectional fitting. Results and discussion: Overall, the hit call and potency estimates were largely consistent between invitrodb v3.5 and 4.0. Tcpl and invitrodb provide a standard for consistent and reproducible curve-fitting and data management for diverse, targeted in vitro assay data with readily available documentation, thus enabling sharing and use of these data in myriad toxicology applications. The software and database updates described herein promote comparability across multiple tiers of data within the US Environmental Protection Agency CompTox Blueprint.

Drug Discovery for Adult Cardiomyocyte Regeneration: Opportunities and Challenges.

Significance: Cardiovascular disease is a major contributor to human mortality and morbidity. The cardiac tissue undergoes fibrotic healing after injury because of the limited regenerative capacity of adult mammalian cardiomyocyte (CM). Extensive research has been performed to identify therapeutic targets for CM regeneration, as the success of promoting adult human CM regeneration to repair the injured heart is considered the Holy Grail in the field. Recent Advances: To date, more than 30 target genes have been shown to regulate adult mammalian CM proliferation. More than 20 targets have been validated in adult mouse myocardial infarction (MI) model in a therapeutic setting. In this review, the translational efficacy readouts from 17 selected pharmaceutical targets are summarized, among which the Hippo-yes-associated protein (Yap) pathway is the most extensively investigated and fits the criteria for a promising target for pro-CM-regeneration therapy development. Critical Issues and Future Directions: As the pro-CM-regeneration potential of current drug treatment for cardiovascular patients is limited, to help identify and fill the gap between basic research and drug discovery in this specific field, details regarding target identification, validation in mouse MI models, high-throughput screening assay development, and preclinical in vivo efficacy model optimization are discussed. Finally, suggestions and recommendations are also provided to help establish a common guideline for in vivo translational studies for drug discovery focusing on CM regeneration. Antioxid. Redox Signal. 39, 1070-1087.

Optical-based microbubble for on-demand droplet release from static droplet array (SDA) for dispensing one droplet into one tube

Static droplet array (SDA) is a pivotal tool for high-capacity screening assays, yet extraction and collection the target droplets that contain unique analytes or cells from the SDA remains one major technical bottleneck that limits its broader application. Here we present an optical-based on-demand droplet release (OODR) system by incorporating a 1064 nm laser-responsive indium tin oxide (ITO) layer into a chamber array-based droplet microfluidic chip. By focusing the 1064 nm laser onto the ITO layer, microbubbles can be created via local heating to selectively push-out the droplets from the chamber. Then the released droplet is readily exported in a one-droplet-one-tube (ODOT) manner by the inherent capillary force into pipette tip. Releasing of the droplets containing fluorescein sodium demonstrated ∼100% successful rate (9 out of 6400 droplets were successfully released) and low residual (only ∼5% of the droplet volume remains in the chamber). White or fluorescence image-based releasing of single-cell-droplets directly after cell loading or multi-cells-droplets derived from on-chip single-cell cultivation for both E. coli and yeast cells further demonstrated the wide applicability of OODR. The present system is user-friendly and has the potential to be applied in various high-throughput screening assays, including single molecule/cell analysis, drug screening, and phenotype-based cell sorting.