Abstract
As the 24th year of SLAS Technology begins, we take pride in continuing our commitment to presenting impressive emerging technologies that transform how research is conducted in life sciences and biomedical research.
The work published in SLAS Technology in 2018 provides new tools that enable life sciences researchers, drug developers, and clinicians. As immunotherapy and adoptive cell transfer therapy become more clinically pervasive and researchers move beyond flat cell culture toward 3D-based life sciences research, the tools to process and analyze these cells are becoming more important. This is evidenced in particular by two special issues published in 2018—“Quantitative Imaging in Life Sciences and Biomedical Research” 1 and “Enabling Technology in Cell-Based Therapies.” 2 Furthermore, advances in Internet technology now allow the Internet to connect and coordinate many aspects of our lives, including how we conduct and manage research. It is for this reason that our special issue on “The Internet of Things in the Life Sciences Laboratory” was so timely and relevant in 2018. 3
The SLAS Technology Ten annually showcases 10 individual articles that stand out as the most innovative scientific achievements published in SLAS Technology in the past 12 months. The 2019 SLAS Technology Ten highlights articles that address a number of areas that we now recognize are important emerging technologies for the laboratory and the clinic. These include technology that automates and improves the isolation of subsets of cells with applications ranging from liquid biopsies to cell therapy manufacturing.4,6,10,11 In addition to technologies that improve the isolation and propagation of desired cells, this year’s SLAS Technology Ten also includes work that improves ways to analyze these cells through both improved hardware and software solutions.8,9,13 Staying close to the roots of SLAS Technology, additional articles highlight improved automation of a wide range of assays, with implications in everything from drug development to forensic science.5,7,12
We would like to thank the 63 authors of the 2019 SLAS Technology Ten for these groundbreaking contributions and the hundreds of other researchers who also chose to share their impressive achievements in SLAS Technology in 2018. Collectively, they demonstrate the creativity and vigor with which technology continues to evolve and advance life sciences research. We look forward to another year of presenting transformative technology that will improve our lives.
Fast and Label-Free Isolation of Circulating Tumor Cells from Blood: From a Research Microfluidic Platform to an Automated Fluidic Instrument, VTX-1 Liquid Biopsy System
By Clementine A. Lemaire, Sean Z. Liu, Charles L. Wilkerson, Vishnu C. Ramani, Nasim A. Barzanian, Kuo-Wei Huang, James Che, Michael W. Chiu, Meghah Vuppalapaty, Adam M. Dimmick, Dino Di Carlo, Michael L. Kochersperger, Steve C. Crouse, Stefanie S. Jeffrey, Robert F. Englert, Stephan Hengstler, Corinne Renier, and Elodie Sollier-Christen
Tumor tissue biopsies are invasive, costly, and collect a limited cell population not completely reflective of patient cancer cell diversity. Circulating tumor cells (CTCs) can be isolated from a simple blood draw and may be representative of the diverse biology from multiple tumor sites. The VTX-1 Liquid Biopsy System was designed to automate the isolation of clinically relevant CTC populations, making the CTCs available for easy analysis. We present here the transition from a cutting-edge microfluidic innovation in the lab to a commercial, automated system for isolating CTCs directly from whole blood. As the technology evolved into a commercial system, flexible polydimethylsiloxane microfluidic chips were replaced by rigid poly(methyl methacrylate) chips for a 2.2-fold increase in cell recovery. Automating the fluidic processing with the VTX-1 further improved cancer cell recovery by nearly 1.4-fold, with a 2.8-fold decrease in contaminating white blood cells and overall improved reproducibility. Two isolation protocols were optimized that favor either the cancer cell recovery (up to 71.6% recovery) or sample purity (⩽100 white blood cells/mL). The VTX-1’s performance was further tested with three different spiked breast or lung cancer cell lines, with 69.0%–79.5% cell recovery. Finally, several cancer research applications are presented using the commercial VTX-1 system.
Isolation of Mitochondrial DNA from Single, Short Hairs without Roots Using Pressure Cycling Technology
By Kathryn A. Harper, Kelly A. Meiklejohn, Richard T. Merritt, Jessica Walker, Constance L. Fisher, and James M. Robertson
Hairs are commonly submitted as evidence to forensic laboratories, but standard nuclear DNA analysis is not always possible. Mitochondria (mt) provide another source of genetic material; however, manual isolation is laborious. In a proof-of-concept study, we assessed pressure cycling technology (PCT; an automated approach that subjects samples to varying cycles of high and low pressure) for extracting mtDNA from single, short hairs without roots. Using three microscopically similar donors, we determined the ideal PCT conditions and compared those yields with those obtained using the traditional manual microtissue grinder method. Higher yields were recovered from grinder extracts, but yields from PCT extracts exceeded the requirements for forensic analysis, with the DNA quality confirmed through sequencing. Automated extraction of mtDNA from hairs without roots using PCT could be useful for forensic laboratories processing numerous samples.
Fully Automated Pipetting Sorting System for Different Morphological Phenotypes of Zebrafish Embryos
By Helmut Breitwieser, Thomas Dickmeis, Marcel Vogt, Marco Ferg, and Christian Pylatiuk
Systems biology methods, such as transcriptomics and metabolomics, require large numbers of small model organisms, such as zebrafish embryos. Manual separation of mutant embryos from wild-type embryos is a tedious and time-consuming task that is prone to errors, especially if there are variable phenotypes of a mutant. Here we describe a zebrafish embryo sorting system with two cameras and image processing based on template-matching algorithms. In order to evaluate the system, zebrafish rx3 mutants that lack eyes due to a patterning defect in brain development were separated from their wild-type siblings. These mutants show glucocorticoid deficiency due to pituitary defects and serve as a model for human secondary adrenal insufficiencies. We show that the variable phenotypes of the mutant embryos can be safely distinguished from phenotypic wild-type zebrafish embryos and sorted from one petri dish into another petri dish or into a 96-well microtiter plate. On average, classification of a zebrafish embryo takes approximately 1 s, with a sensitivity and specificity of 87% and 95%, respectively. Other morphological phenotypes may be classified and sorted using similar techniques.
Theranostic Nanoparticles for Tracking and Monitoring Disease State
By Cristina Zavaleta, Dean Ho, and Eun Ji Chung
The development of novel nanoparticles consisting of both diagnostic and therapeutic components has increased over the past decade. These “theranostic” nanoparticles have been tailored toward one or more types of imaging modalities and have been developed for optical imaging, magnetic resonance imaging, ultrasound, computed tomography, and nuclear imaging comprising both single-photon computed tomography and positron emission tomography. In this review, we focus on state-of-the-art theranostic nanoparticles that are capable of both delivering therapy and self-reporting/tracking disease through imaging. We discuss challenges and the opportunity to rapidly adjust treatment for individualized medicine.
Quantitative Analysis of a Multiplexed Immunofluorescence Panel in T-Cell Lymphoma
By Siok-Bian Ng, Shuangyi Fan, Shoa-Nian Choo, Michal Hoppe, Hoang Mai Phuong, Sanjay De Mel, and Anand D. Jeyasekharan
Immunohistochemistry (IHC) provides clinically useful information on protein expression in cancer cells. However, quantification of colocalizing signals using conventional IHC and visual scores is challenging. Here we describe the application of quantitative immunofluorescence (IF) in angioimmunoblastic T-cell lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity that impedes IHC interpretation and quantification. A multiplexed IF panel comprising T- and B-lymphocyte markers along with T-follicular helper (TFH) markers was validated for appropriate cellular localization in sections of benign tonsillar tissue and tested in two samples of AITL, using a Vectra microscope for spectral imaging and InForm software for analysis. We measured the percentage positivity of the TFH markers, BCL6 and PD1, in AITL CD4-positive cells to be approximately 26% and 45%, with 12% coexpressing both markers. The pattern is similar to CD4 cells within the germinal center of normal tonsils and clearly distinct from extragerminal CD4 cells. This study demonstrates the feasibility of automated and quantitative imaging of a multiplexed panel of cellular markers in formalin-fixed, paraffin-embedded tissue sections of a cellularly heterogenous lymphoma. Multiplexed IF allows the simultaneous scoring of markers in malignant and immune cell populations and could potentially increase accuracy for the establishment of diagnostic thresholds.
Identification of Angiogenesis Inhibitors Using a Co-Culture Cell Model in a High-Content and High-Throughput Screening Platform
By Shuaizhang Li, Chia-Wen Hsu, Srilatha Sakamuru, Chaozhong Zou, Ruili Huang, and Menghang Xia
Angiogenesis is an important hallmark of cancer, contributing to tumor formation and metastasis. In vitro angiogenesis models for analyzing tube formation serve as useful tools to study these processes. However, current in vitro co-culture models using primary cells have limitations in usefulness and consistency. Therefore, in the present study, an in vitro co-culture assay system was optimized in a 1536-well format for high-throughput screening using human telomerase reverse transcriptase (hTERT)-immortalized mesenchymal stem cells and aortic endothelial cells. The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC) library containing 2816 drugs was evaluated using the in vitro co-culture assay. From the screen, 35 potent inhibitors (IC50 ⩽1 µM) were identified, followed by 15 weaker inhibitors (IC50 1–50 µM). Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study, including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-kappa B (NF-κB) pathways. The co-culture model developed by using hTERT-immortalized cell lines described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.
Deterministic Lateral Displacement: The Next-Generation CAR T-Cell Processing?
By Roberto Campos-González, Alison M. Skelley, Khushroo Gandhi, David W. Inglis, James C. Sturm, Curt I. Civin, and Tony Ward
Reliable cell recovery and expansion are fundamental to the successful scale-up of chimeric antigen receptor (CAR) T cells or any therapeutic cell manufacturing process. Here, we extend our previous work in whole blood by manufacturing a highly parallel deterministic lateral displacement (DLD) device incorporating diamond microposts and moving into processing, for the first time, apheresis blood products. This study demonstrates key metrics of cell recovery (80%) and platelet depletion (87%), and it shows that DLD T-cell preparations have high conversion to the T-central memory phenotype and expand well in culture, resulting in twofold greater central memory cells compared with Ficoll-Hypaque (Ficoll) and direct magnetic approaches. In addition, all samples processed by DLD converted to a majority T-central memory phenotype and did so with less variation, in stark contrast to Ficoll and direct magnetic prepared samples, which had partial conversion among all donors (<50%). This initial comparison of T-cell function infers that cells prepared via DLD may have a desirable bias, generating significant potential benefits for downstream cell processing. DLD processing provides a path to develop a simple closed system that can be automated while simultaneously addressing multiple steps when there is potential for human error, microbial contamination, and other current technical challenges associated with the manufacture of therapeutic cells.
Continuous and Quantitative Purification of T-Cell Subsets for Cell Therapy Manufacturing Using Magnetic Ratcheting Cytometry
By Coleman Murray, Edward Pao, Andrew Jann, Da Eun Park, and Dino Di Carlo
T-cell-based immunotherapies represent a growing medical paradigm that has the potential to revolutionize contemporary cancer treatments. However, manufacturing bottlenecks related to the enrichment of therapeutically optimal T-cell subpopulations from leukopak samples impede scale-up and scale-out efforts. This is mainly attributed to the challenges that current cell purification platforms face in balancing the quantitative sorting capacity needed to isolate specific T-cell subsets with the scalability to meet manufacturing throughputs. In this work, we report a continuous-flow, quantitative cell enrichment platform based on a technique known as ratcheting cytometry that can perform complex, multicomponent purification targeting various subpopulations of magnetically labeled T cells directly from apheresis or peripheral blood mononuclear cell (PBMC) samples. The integrated ratcheting cytometry instrument and cartridge demonstrated enrichment of T cells directly from concentrated apheresis samples with a 97% purity and an 85% recovery of magnetically tagged cells. Magnetic sorting of different T-cell subpopulations was also accomplished on chip by multiplexing cell surface targets onto particles with differing magnetic strengths. We believe that ratcheting cytometry’s quantitative capacity and throughput scalability represents an excellent technology candidate to alleviate cell therapy manufacturing bottlenecks.
Achieving Reproducibility and Closed-Loop Automation in Biological Experimentation with an IoT-Enabled Lab of the Future
By Ben Miles and Peter L. Lee
A robotic cloud laboratory driven by a state-of-the-art unified laboratory operating system integrates automated hardware, humans, and sensors. This lab of the future system enables researchers to transparently and collaboratively create, optimize, and organize biological experiments to achieve more reproducible results, perform around-the-clock experimentation, and more efficiently navigate the vast parameter space of biology.
Improved Fluorescence Methods for High-Throughput Protein Formulation Screening
By Yangjie Wei, Nicholas R. Larson, Siva K. Angalakurthi, and C. Russell Middaugh
The goal of protein formulation development is to identify optimal conditions for long-term storage. Certain commercial conditions (e.g., high protein concentration or turbid adjuvanted samples) impart additional challenges to biophysical characterization. Formulation screening studies for such conditions are usually performed using a simplified format in which the target protein is studied at a low concentration in a clear solution. The failure of study conditions to model the actual formulation environment may cause a loss of ability to identify the optimal condition for target proteins in their final commercial formulations. In this study, we utilized a steady-state/lifetime fluorescence-based, high-throughput platform to develop a general workflow for direct formulation optimization under analytically challenging but commercially relevant conditions. A high-concentration monoclonal antibody (mAb) and an Alhydrogel-adjuvanted antigen were investigated. A large discrepancy in screening results was observed for both proteins under these two different conditions (simplified and commercially relevant). This study demonstrates the feasibility of using a steady-state/lifetime fluorescence plate reader for direct optimization of challenging formulation conditions and highlights the importance of performing formulation optimization under commercially relevant conditions.