Abstract
First as the Journal of Laboratory Automation, and now as SLAS Technology, we have strived to present cutting-edge developments in technology that have transformed how biomedical and life science research is performed over the last 24 years. Entering our 25th year, we have seen technology transform toward more patient-centric technology that is increasingly miniaturized and automated. This has allowed for incredible advances in how drugs are developed, evaluated, and given to patients in the face of an increasingly complex understanding of mechanisms of disease.
The work presented by SLAS Technology in 2019 demonstrated the importance of studying diseases and developing novel therapeutics within the context of more clinically relevant disease models, such as patient-derived organoids (PDOs).1,2 By pairing high-throughput drug screens with genomic sequencing in these PDOs, a better understanding of how unique genomic profiles relate to drug sensitivity is developed. The application of patient-derived materials extends beyond drug discovery and toward drug delivery. In particular, exosomes are an emerging biomaterial that may serve as both diagnostic biomarker and drug delivery vehicle. In order to translate this nanoparticle into the clinic, improved microtechnology is necessary to improve detection and isolation. 3 Advances in microfabrication, however, extend far beyond just improved diagnostic devices and can also be used to improve therapy, as is the case with microneedles for transdermal drug delivery. 4 Additionally, the increasing incorporation of microfluidics, nanowells, and other assay miniaturization technology into the drug development pipeline has led to the development of improved protocols and workflows for performing and analyzing compound screens with increasingly smaller assays.5,6 Overall, these technology platforms improve the feasibility of utilizing rare patient-derived materials for drug screens as well as lowering the overall cost of drug development through more efficient experimentation. The improvement of experimental efficiency is critical to lowering the cost and time needed to develop new drugs and discover new biological mechanisms. Automation of all aspects of biomedical and life science research has benefits to both researchers seeking to answer life’s most fundamental questions and those that seek to develop the next life-saving drug. In 2019, SLAS Technology continued to bring these advances in automation to the research community, with broad implications from the improved automated study of protein–protein interactions to the development of ultra-high-throughput mass spectrometry-based drug screens.7,8 Mass spectrometry is now an established foundation for a range of applications, including drug screens, large-scale immune phenotyping, and compound identification. In order to increase the efficiency and scale of these applications, improved reagents and automated systems are required. Improved barcoding techniques can allow for faster and more reproducible mass cytometry (CyTOF) for single-cell proteomic analysis, while automated liquid chromatography fraction collection improves the throughput rate of mass spectrometry-based compound identification.9,10 It is clear that this year’s SLAS Technology Ten encompasses a wide range of the emerging technologies that have changed how we do research.
We would like to thank the 87 authors that contributed to the work presented in this year’s SLAS Technology Ten. We would also like to thank everyone who contributed to SLAS Technology as authors, reviewers, guest editors, and editorial board members. Through their hard work and contributions, SLAS Technology continues to present emerging and cutting-edge technology in biomedical and life science research. As the gap between fundamental research and clinical applications grows smaller, SLAS Technology will be there to present how breakthroughs in technology bridge this gap and integrate real-world clinical data into drug discovery and target identification.
Human iPS Cell-Derived Patient Tissues and 3D Cell Culture Part 2: Spheroids, Organoids, and Disease Modeling
By Richard M. Eglen, Terry Reisine
Human induced pluripotent stem cells (HiPSCs) provide several advantages for drug discovery, but principally they provide a source of clinically relevant tissue. Furthermore, the use of HiPSCs cultured in three-dimensional (3D) systems, as opposed to traditional two-dimensional (2D) culture approaches, better represents the complex tissue architecture in vivo. The use of HiPSCs in 3D spheroid and organoid culture is now growing, but particularly when using myocardial, intestinal enteric nervous system, and retinal cell lines. However, organoid cell culture is perhaps making the most notable impact in research and drug discovery, in which 3D neuronal cell cultures allow direct modeling of cortical cell layering and neuronal circuit activity. Given the specific degeneration seen in discrete neuronal circuitry in Alzheimer’s disease (AD) and Parkinson’s disease (PD), HiPSC culture systems are proving to be a major advance. In the present review, the second part of a two-part review, we discuss novel methods in which 3D cell culture systems (principally organoids) are now being used to provide insights into disease mechanisms. (The use of HiPSCs in target identification was reviewed in detail in Part 1.)
Mutation Profiles in Glioblastoma 3D Oncospheres Modulate Drug Efficacy
By Kelli M. Wilson, Lesley A. Mathews-Griner, Tara Williamson, Rajarshi Guha, Lu Chen, Paul Shinn, Crystal McKnight, Sam Michael, Carleen Klumpp-Thomas, Zev A. Binder, Marc Ferrer, Gary L. Gallia, Craig J. Thomas, Gregory J. Riggins
Glioblastoma (GBM) is a lethal brain cancer with a median survival time of approximately 15 months following treatment. Common in vitro GBM models for drug screening are adherent and do not recapitulate the features of human GBM in vivo. Here we report the genomic characterization of nine patient-derived, spheroid GBM cell lines that recapitulate human GBM characteristics in orthotopic xenograft models. Genomic sequencing revealed that the spheroid lines contain alterations in GBM driver genes such as PTEN, CDKN2A, and NF1. Two spheroid cell lines, JHH-136 and JHH-520, were utilized in a high-throughput drug screen for cell viability using a 1912-member compound library. Drug mechanisms that were cytotoxic in both cell lines were Hsp90 and proteasome inhibitors. JHH-136 was uniquely sensitive to topoisomerase 1 inhibitors, while JHH-520 was uniquely sensitive to Mek inhibitors. Drug combination screening revealed that PI3 kinase inhibitors combined with Mek or proteasome inhibitors were synergistic. However, animal studies to test these drug combinations in vivo revealed that Mek inhibition alone was superior to the combination treatments. These data show that these GBM spheroid lines are amenable to high-throughput drug screening and that this dataset may deliver promising therapeutic leads for future GBM preclinical studies.
Advances in Technologies for Purification and Enrichment of Extracellular Vesicles
By Pan Zhang, Joo Chuan Yeo, Chwee Teck Lim
Extracellular vesicles (EVs) are lipid bilayer-bound vesicles secreted by cells. Subtypes of EVs such as microvesicles and exosomes are further categorized mainly by their different biogenesis mechanisms. EVs have been revealed to play an important role in disease diagnosis and intercellular communication. Despite the wide interest in EVs, the technologies for the purification and enrichment of EVs are still in their infancy. The isolation of EVs, especially exosomes, is inherently challenging due to their small size and heterogeneity. In this review, we mainly introduce the advances of techniques in isolating microvesicles and exosomes according to their approaches. Also, we discuss the limitations of currently reported technologies in terms of their specificity and efficiency, and provide our thoughts about future developments of EV purification and enrichment technology.
In Situ Generation of Zinc Oxide Nanobushes on Microneedles as Antibacterial Coating
By Sharon W. T. Chew, Yongpeng Zeng, Mingyue Cui, Hao Chang, Mengjia Zheng, Shi Wei, Wenting Zhao, Chenjie Xu
This paper introduces a facile and scalable method to generate a layer of antibacterial coating on microneedles. The antibacterial coating (i.e., zinc oxide nanobushes) is generated on the surface of gold-coated polystyrene microneedles using the hydrothermal growth method. The antimicrobial property is examined using the agar diffusion test with both gram-positive and gram-negative bacteria.
Next-Generation Compound Delivery Platforms to Support Miniaturized Biology
By Snehal Bhatt, Sue Crimmin, Jeffrey Gross, Elizabeth Nixon, Maggie Truong, Michael Weglos, Lorena Kallal
Recent advancements in science and engineering are revolutionizing our understanding of an individual’s disease, and with this knowledge we are gaining an increasingly sophisticated understanding of how discovery can be transformed to deliver personalized medicines. To reach this future state, we must reengineer our approach to enable the use of more relevant human cellular models earlier in the drug discovery process. Stem cells and primary human cells represent more disease-relevant models than immortalized cell lines; however, due to both availability and cost, their use is limited in lead generation activities. Miniaturization of cellular assays below microtiter plate volumes will enable the use of more relevant cells in screening, but this would require a change in how test molecules are introduced to the biology. With these shifting paradigms, Discovery Supply teams at GlaxoSmithKline (GSK) are modernizing our sample handling approaches. Various emerging technologies such as microarrays, nanowells, and microfluidic devices could bring fundamental changes in conventional sample handling support as we transition from microtiter plates to well-less platforms. The discussion here is exploratory in nature and reviews ongoing proof-of-concept experiments. Our ultimate goal is to industrialize the sample management platforms to support future miniaturized biological assay systems.
Quantification of In Vivo Target Engagement Using Microfluidic Activity-Based Protein Profiling
By Holly T. Reardon, Rachel A. Herbst, Cassandra L. Henry, Dylan M. Herbst, Nhi Ngo, Justin S. Cisar, Olivia D. Weber, Micah J. Niphakis, Gary P. O’Neill
Accurate measurement of drug–target interactions in vivo is critical for both preclinical development and translation to clinical studies, yet many assays rely on indirect measures such as biomarkers associated with target activity. Activity-based protein profiling (ABPP) is a direct method of quantifying enzyme activity using active site-targeted small-molecule covalent probes that selectively label active but not inhibitor-bound enzymes. Probe-labeled enzymes in complex proteomes are separated by polyacrylamide gel electrophoresis and quantified by fluorescence imaging. To accelerate workflows and avoid imaging artifacts that make conventional gels challenging to quantify, we adapted protocols for a commercial LabChip GXII microfluidic instrument to permit electrophoretic separation of probe-labeled proteins in tissue lysates and plasma, and quantification of fluorescence (probe/protein labeling ratio of 1:1). Electrophoretic separation on chips occurred in 40 s per sample, and instrument software automatically identified and quantified peaks, resulting in an overall time savings of 3–5 h per 96-well sample plate. Calculated percent inhibition was not significantly different between the two formats. Chip performance was consistent between chips and sample replicates. Conventional gel imaging was more sensitive but required five times higher sample volume than microfluidic chips. Microfluidic chips produced results comparable to those of gels but with much lower sample consumption, facilitating assay miniaturization for scarce biological samples. The time savings afforded by microfluidic electrophoresis and automatic quantification has allowed us to incorporate microfluidic ABPP early in the drug discovery workflow, enabling routine assessments of tissue distribution and engagement of targets and off-targets in vivo.
Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells
By Wenjun Guo, Sunil Kumar, Frederik Görlitz, Edwin Garcia, Yuriy Alexandrov, Ian Munro, Douglas J. Kelly, Sean Warren, Peter Thorpe, Christopher Dunsby, Paul French
We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.
Automated MALDI Target Preparation Concept: Providing Ultra-High-Throughput Mass Spectrometry-Based Screening for Drug Discovery
By Martin Winter, Robert Ries, Carola Kleiner, Daniel Bischoff, Andreas H. Luippold, Tom Bretschneider, Frank H. Büttner
Label-free, mass spectrometric (MS) deciphering of enzymatic reactions by direct analysis of substrate-to-product conversion provides the next step toward more physiologically relevant assays within drug discovery campaigns. Reduced risk of suffering from compound interference combined with diminished necessity for tailored signal mediators emphasizes the valuable role of label-free readouts. However, MS-based detection has not hitherto met high-throughput screening (HTS) requirements because of the lack of HTS-compatible sample introduction. In the present study, we report on a fully automated liquid-handling concept built in-house to concatenate biochemical assays with matrix-assisted laser desorption/ionization time-of-flight closing this technological gap. The integrated reformatting from 384- to 1536-well format enables cycle times of 0.6 s/sample for automated spotting and 0.4 s/sample for MS analysis, matching the requirements of HTS compatibility. In-depth examination of spotting quality, quantification accuracy, and instrument robustness together with the implementation of a protein tyrosine phosphatase 1B (PTP1B) inhibitor screening (4896 compounds) demonstrate the potential of the heavily inquired HTS integration of the label-free MS readout. Overall, the presented data demonstrate that the introduced automation concept makes label-free MS-based readouts accessible for HTS within drug discovery campaigns but also in other research areas requiring ultrafast MS-based detection.
A Mass-Ratiometry-Based CD45 Barcoding Method for Mass Cytometry Detection
By Hongu Meng, Antony Warden, Lulu Zhang, Ting Zhang, Yiyang Li, Ziyang Tan, Boqian Wang, Hongxia Li, Hui Jiang, Guangxia Shen, Yifan Hong, Xianting Ding
Mass cytometry (CyTOF) is a critical cell profiling tool in acquiring multiparameter proteome data at the single-cell level. A major challenge in CyTOF analysis is sample-to-sample variance arising from the pipetting process, staining variation, and instrument sensitivity. To reduce such variations, cell barcoding strategies that enable the combination of individual samples prior to antibody staining and data acquisition on CyTOF are often utilized. The most prevalent barcoding strategy is based on a binary scheme that cross-examines the existence or nonexistence of certain mass signals; however, it is limited by low barcoding efficiency and high cost, especially for large sample size. Herein, we present a novel barcoding method for CyTOF application based on mass ratiometry. Different mass tags with specific fixed ratios are used to label CD45 antibody to achieve sample barcoding. The presented method exponentially increases the number of possible barcoded samples with the same amount of mass tags compared with conventional methods. It also reduces the overall time for the labeling process to 40 min and avoids the need for expensive commercial barcoding buffer reagents. Moreover, unlike the conventional barcoding process, this strategy does not pre-permeabilize cells before the barcoding procedure, which offers additional benefits in preserving surface biomarker signals.
Compound Identification Using Liquid Chromatography and High-Resolution Noncontact Fraction Collection with a Solenoid Valve
By Willem Jonker, Koen de Vries, Niels Althuisius, Dick van Iperen, Elwin Janssen, Rob ten Broek, Corine Houtman, Nick Zwart, Timo Hamers, Marja H. Lamoree, Bert Ooms, Johannes Hidding, Govert W. Somsen, Jeroen Kool
We describe the development of a high-resolution, noncontact fraction collector for liquid chromatography (LC) separations, allowing high-resolution fractionation in high-density well plates. The device is based on a low-dead-volume solenoid valve operated at 1–30 Hz for accurate collection of fractions of equal volume. The solenoid valve was implemented in a modified autosampler resulting in the so-called FractioMate fractionator. The influence of the solenoid supply voltage on solvent release was determined and the effect of the frequency, flow rate, and mobile phase composition was studied. For this purpose, droplet release was visually assessed for a wide range of frequencies and flow rates, followed by quantitative evaluation of a selection of promising settings for highly accurate, repeatable, and stable fraction collection. The potential of the new fraction collector for LC-based bioactivity screening was demonstrated by fractionating the LC eluent of a mixture of estrogenic and androgenic compounds, and a surface water sample (blank and spiked with bioactives) combining mass spectrometric detection and two reporter gene assays for bioactivity detection of the fractions. Additionally, a mixture of two compounds was repeatedly LC separated and fractionated to assess the feasibility of the system for analyte isolation followed by nuclear magnetic resonance analysis.