High-throughput Sample Processing Research Articles

Comprehensive LC-MS/MS Analysis of Nitrogen-Related Plant Metabolites.

We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5mg of lyophilized plant material followed by fractionation of different biogenic amine forms and exploits an efficient combination of hydrophilic interaction chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry. This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labelled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to the field of plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.

Rapid high-throughput processing of tissue samples for microbiological diagnosis of periprosthetic joint infections using bead-beating homogenization.

Enrichment of periprosthetic tissue samples in blood culture bottles (BCBs) for microbiological diagnosis of periprosthetic joint infections (PJI) is more reliable than the use of an enrichment broth. Nevertheless, the extremely time-consuming homogenization of the samples for BCB processing has so far limited its use, especially in high-throughput settings. We aimed to establish a highly scalable homogenization process of tissue samples for long-term incubation in BCBs. A protocol for homogenization of tissue samples using bead beating was established and validated. In a second step, the use of the homogenate for enrichment in BCBs was compared to the use of thioglycolate broth (TB) in terms of diagnostic accuracy using clinical tissue samples from 150 patients with suspected PJI. Among 150 analyzed samples, 35 samples met the microbiological criteria for PJI. Using BCB, 32 of 35 (91.4%) PJI were detected compared to 30 of 35 (85.7%) by TB. The use of BCB had a lower secondary contamination rate (2/115; 1.7% vs 4/115; 3.5%) but the trend was not significant due to low numbers of samples (P = 0.39). The time to process a batch of 12 samples using the established homogenization method was 23 ± 5 min (n = 10 batches). We established and validated a homogenization workflow that achieves the highest sensitivity in the microbiological diagnostic of PJI. The enrichment of the tissue homogenate in BCBs showed equally good results as the use of enrichment broth and allows semi-automated high-throughput processing while demonstrating lower contamination rates in our study.

Tiny swabs: nasal swabs integrated into tube caps facilitate large-scale self-collected SARS-CoV-2 testing.

The COVID-19 pandemic spurred the development of innovative solutions for specimen collection and molecular detection for large-scale community testing. Among these developments is the RHINOstic nasal swab, a plastic anterior nares swab built into the cap of a standard matrix tube that facilitates automated processing of up to 96 specimens at a time. In a study of unsupervised self-collection utilizing these swabs, we demonstrate comparable analytic performance and shipping stability compared to traditional anterior nares swabs, as well as significant improvements in laboratory processing efficiency. The use of these swabs may allow laboratories to accommodate large numbers of sample collections during periods of high testing demand. Automation-friendly nasal swabs are an important tool for high-throughput processing of samples that may be adopted in response to future respiratory viral pandemics.

A robust pipeline for high-content, high-throughput immunophenotyping reveals age- and genetics-dependent changes in blood leukocytes

High-dimensional flow cytometry is the gold standard to study the human immune system in large cohorts. However, large sample sizes increase inter-experimental variation because of technical and experimental inaccuracies introduced by batch variability. Our high-throughput sample processing pipeline in combination with 28-color flow cytometry focuses on increased throughput (192 samples/experiment) and high reproducibility. We implemented quality control checkpoints to reduce technical and experimental variation. Finally, we integrated FlowSOM clustering to facilitate automated data analysis and demonstrate the reproducibility of our pipeline in a study with 3,357 samples. We reveal age-associated immune dynamics in 2,300 individuals, signified by decreasing T and B cell subsets with age. In addition, by combining genetic analyses, our approach revealed unique immune signatures associated with a single nucleotide polymorphism (SNP) that abrogates CD45 isoform splicing. In summary, we provide a versatile and reliable high-throughput, flow cytometry-based pipeline for immune discovery and exploration in large cohorts.

Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples

ObjectiveGrowth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults (n = 12; 34–69 years), microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods.ResultsDiversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data.

FAVIS: Fast and versatile protocol for non-destructive metabarcoding of bulk insect samples.

Insects are diverse and sustain essential ecosystem functions, yet remain understudied. Recent reports about declines in insect abundance and diversity have highlighted a pressing need for comprehensive large-scale monitoring. Metabarcoding (high-throughput bulk sequencing of marker gene amplicons) offers a cost-effective and relatively fast method for characterizing insect community samples. However, the methodology applied varies greatly among studies, thus complicating the design of large-scale and repeatable monitoring schemes. Here we describe a non-destructive metabarcoding protocol that is optimized for high-throughput processing of Malaise trap samples and other bulk insect samples. The protocol details the process from obtaining bulk samples up to submitting libraries for sequencing. It is divided into four sections: 1) Laboratory workspace preparation; 2) Sample processing-decanting ethanol, measuring the wet-weight biomass and the concentration of the preservative ethanol, performing non-destructive lysis and preserving the insect material for future work; 3) DNA extraction and purification; and 4) Library preparation and sequencing. The protocol relies on readily available reagents and materials. For steps that require expensive infrastructure, such as the DNA purification robots, we suggest alternative low-cost solutions. The use of this protocol yields a comprehensive assessment of the number of species present in a given sample, their relative read abundances and the overall insect biomass. To date, we have successfully applied the protocol to more than 7000 Malaise trap samples obtained from Sweden and Madagascar. We demonstrate the data yield from the protocol using a small subset of these samples.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

Development of a Novel, Automated High-Throughput Device for Performing the Comet Assay.

A comet assay is a trusted and widely used method for assessing DNA damage in individual eukaryotic cells. However, it is time-consuming and requires extensive monitoring and sample manipulation by the user. This limits the throughput of the assay, increases the risk of errors, and contributes to intra- and inter-laboratory variability. Here, we describe the development of a device which automates high throughput sample processing for a comet assay. This device is based upon our patented, high throughput, vertical comet assay electrophoresis tank, and incorporates our novel, patented combination of assay fluidics, temperature control, and a sliding electrophoresis tank to facilitate sample loading and removal. Additionally, we demonstrated that the automated device performs at least as well as our "manual" high throughput system, but with all the advantages of a fully "walkaway" device, such as a decreased need for human involvement and a decreased assay run time. Our automated device represents a valuable, high throughput approach for reliably assessing DNA damage with the minimal operator involvement, particularly if combined with the automated analysis of comets.

Abstract 4731: Novel epigenetics technology for high-throughput processing of limited samples to study cancer using cavitation-based pixelated ultrasound and tagmentation-indexing ChIP-Seq

Abstract Epigenetics has shown great potential in translational, clinical, and precision medicine research, but with inadequate implementation. This is due to major problems with sample availability, and processing which is laborious, costly, slow, inconsistent, and leads to sample loss. Epigenetics studies need samples with certain minimum concentrations and volumes to address all necessary questions. The samples often need to be in single cell or nuclear suspensions. For chromatin immunoprecipitation (ChIP), a consistent size of approximately 300 base pairs of DNA is essential but difficult to achieve. To address the above problems, we present novel cutting-edge epigenetics methodologies. Homogenization of various tissue types from diverse patients with different disease states is the first crucial step in sample processing. Hence, we present new protocols which are compatible with high-throughput cavitation-based pixelated ultrasound sonication. It processes limited quantities of samples in a 96-well plate where cells can be grown and antibody can be coated for ChIP. Further, 1 to 12 columns of the 96-well plate can be flexibly regulated to extract lysates. Our results show how this rapid and simple technology integrates with ChIP-sequencing (seq) and RNA-seq workflows, yielding very consistent output from limited tissue samples. Bioinformatics analysis of the ChIP-seq shows the corresponding biological significance. Certain limitations of ChIP has led to the evolution of other methods but they are mostly unsuccessful in mapping transcription factors. Hence, we next present a modified ChIP-seq combined with tagmentation, indexing and pooling, coupled to pixelated ultrasound based-cavitation. Here, individual samples are placed in each well of a 96-well plate containing unique indexing adapters fused to Tn5 transposase. Hence, each sample undergoes tagmentation - a process where DNA gets fragmented and simultaneously inserted with index adapters. Next, pixelated ultrasound helps to rapidly extract the complex of protein-tagmented DNA from 96 samples which are pooled into a single tube because they are uniquely indexed, then split up for immunoprecipitation by different antibodies. Bioinformatics analysis shows that the results are robust, consistent, and match ENCODE datasets. Hence, our methods can help to process high-throughput samples for AACR Project GENIE. Overall, we present novel technology to process limited human samples for enabling epigenetics research at the clinical interface from bench to bedside. The simple and less expensive methods yield high quality samples to enable high-throughput analysis of epigenetics including transcription factors which are important in cancer. They help us to understand targeted epigenetic regulations of genes for therapeutic developments and treating cancer. Citation Format: Rwik Sen, Ngoc Tran, Sarah Traynor, Eric Maina, Jason Poole. Novel epigenetics technology for high-throughput processing of limited samples to study cancer using cavitation-based pixelated ultrasound and tagmentation-indexing ChIP-Seq. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4731.

Evaluation of two methods for detection of viable Bacillus anthracis simulant spores in maritime environmental samples.

Analytical methods exist to detect biothreat agents in environmental samples during a response to biological contamination incidents. However, the coastal zone facilities and assets of the US Coast Guard (USCG), including response boats in diverse geographical areas and maritime environmental conditions, can pose complex and unique challenges for adapting existing analytical detection methods. The traditional culture (TC) and the rapid viability polymerase chain reaction (RV-PCR) methods were evaluated for their compatibility for maritime environmental surface and grab sample analysis to detect spores of Bacillus thuringiensis subspecies kurstaki (Btk), a surrogate for Bacillus anthracis. The representative samples collected from a USCG installation included surfaces, such as aluminum on boats, nonskid tread on decks of watercraft, computer touchscreens, and concrete piers, and grab samples of boat washdown water, soil, vegetation, and gravel from surrounding areas. Replicate samples were spiked with Btk spores at two to three tenfold increasing levels and analyzed. Out of a total of 150 samples collected and analyzed, the TC method gave 10 false-positive and 19 false-negative results, while the RV-PCR method-based analysis resulted in 0 false-positive and 26 false-negative results. An abundance of microbial background and particulates in some samples interfered with true results, while both methods gave similar results for samples with low microbial background and particulates. Improved and high-throughput sample processing methods are needed for analysis of complex environmental samples.

A comparative analysis of RNA isolation methods optimized for high-throughput detection of viral pathogens in California’s regulatory and disease management program for citrus propagative materials

Citrus germplasm programs can benefit from high-throughput polymerase chain reaction (PCR)-based methods for the detection of graft-transmissible pathogens in propagative materials. These methods increase diagnostic capacity, and thus contribute to the prevention of disease spread from nurseries to citrus orchards. High quality nucleic acids, as determined by purity, concentration, and integrity, are a prerequisite for reliable PCR detection of citrus pathogens. Citrus tissues contain high levels of polyphenols and polysaccharides, which can affect nucleic acid quality and inhibit PCR reactions. Various commercially available RNA isolation methods are used for citrus and include: phenol-chloroform (TRIzol®, Thermo Fisher Scientific); silica columns (RNeasy® Plant Mini Kit, Qiagen); and magnetic beads-based methods (MagMAX™-96 Viral RNA Isolation Kit, Thermo Fisher Scientific). To determine the quality of RNA and its impact on the detection of graft-transmissible citrus pathogens in reverse transcription (RT) PCR-based assays, we compared these three RNA isolation methods. We assessed RNA purity, concentration, and integrity from citrus inoculated with different viruses and viroids. All three RNA isolation methods produced high quality RNA, and its use in different RT-PCR assays resulted in the detection of all targeted citrus viruses and viroids with no false positive or negative results. TRIzol® yielded RNA with the highest concentration and integrity values but some samples required serial dilutions to remove PCR inhibitors and detect the targeted pathogens. The RNeasy® kit produced the second highest concentration and purity of RNA, and similar integrity to TRIzol®. MagMAX™ isolation also provided high quality RNA but most importantly produced RNA with consistent results clustered around a median value for concentration, purity, and integrity. Subsequently, MagMAX™-96 was combined with the semi-automated MagMAX™ Express-96 Deep Well Magnetic Particle Processor, for high-throughput sample processing. MagMAX™-96 enabled the diagnostic laboratory of the Citrus Clonal Protection Program-National Clean Plant Network at the University of California, Riverside to process over 16,500 samples from citrus budwood source trees between 2010 and 2019. This high-throughput approach dramatically reduced the incidence of viroids in citrus nurseries and was key to the successful implementation of the mandatory Citrus Nursery Stock Pest Cleanliness Program in California.

Proteomics, Phosphoproteomics and Mirna Analysis of Circulating Extracellular Vesicles through Automated and High-Throughput Isolation.

Extracellular vesicles (EVs) play an important role in the diagnosis and treatment of diseases because of their rich molecular contents involved in intercellular communication, regulation, and other functions. With increasing efforts to move the field of EVs to clinical applications, the lack of a practical EV isolation method from circulating biofluids with high throughput and good reproducibility has become one of the biggest barriers. Here, we introduce a magnetic bead-based EV enrichment approach (EVrich) for automated and high-throughput processing of urine samples. Parallel enrichments can be performed in 96-well plates for downstream cargo analysis, including EV characterization, miRNA, proteomics, and phosphoproteomics analysis. We applied the instrument to a cohort of clinical urine samples to achieve reproducible identification of an average of 17,000 unique EV peptides and an average of 2800 EV proteins in each 1 mL urine sample. Quantitative phosphoproteomics revealed 186 unique phosphopeptides corresponding to 48 proteins that were significantly elevated in prostate cancer patients. Among them, multiple phosphoproteins were previously reported to associate with prostate cancer. Together, EVrich represents a universal, scalable, and simple platform for EV isolation, enabling downstream EV cargo analyses for a broad range of research and clinical applications.

Abstract 1393: A Novel platform providing automated, consistent and gentle cell isolation from tissues for downstream applications

Abstract Tumor biologists have long studied tumor tissue to understand cancer progression and to ultimately treat the disease. The gold standard would be to analyze tumor and immune cells from the same sample, providing purified cells that are interacting within the tumor. Many tissue homogenization techniques have been developed to release cells for downstream analysis, however, an automated and gentle process of purifying multiple cell types from the same processed tissue has remained elusive. The industry-standard workflows, such as centrifugation, have presented long-standing performance issues in terms of low cell recovery and inefficient removal of molecular debris, which often leads to sequencing results that are difficult to interpret. To ensure the quality of sequencing data from tissues, upstream processes need to improve to get the most from the precious patient tissue. Applied Cells has developed an integrated system based on multiple physics principles to achieve cell wash, concentration, and target cell isolation, with high recovery at an unprecedented flow rate. This platform combines tunable, acoustic cell purification and in-flow, column-free immuno-magnetic separation technologies, enabling automation of the entire cell sample preparation workflow for genomics and transcriptomics analysis. By harnessing the power and precision of acoustic cell manipulation, the MARS System can process a variety of samples including dissociated solid tissue in enzymatic buffer with high cell recovery and superior molecular debris removal compared to standard methods. For instance, when purifying leukocytes from a lung cancer biopsy tissue samples, MARS can remove 99.50% debris and 91% of dead cells in one single step, which is supreme than the conventional centrifuge cell wash while having >90% leukocyte recovery at 1mL/min flow rate. The in-flow microfluidics design drastically reduces residual DNA and RNA fragments that are difficult to remove using centrifuge wash methods. Furthermore, the system enables parallel cell purification and concentration for high throughput sample processing. Tumor or TIL cells can be further enriched by means of positive magnetic selection for genomic or cell biology applications. This novel platform allows improved tumor and TIL recovery in a gentle, consistent manner while having minimal interference with downstream applications, all from a single source starting material. Citation Format: Liping Yu. A Novel platform providing automated, consistent and gentle cell isolation from tissues for downstream applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1393.

High-throughput sample processing for methylation analysis in an automated, enclosed environment.

Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated. Thus, sample processing methods still require numerous manual steps that may reduce sample throughput and precision, while increasing the risk of contamination and human error. In this work, we present an integrated, automated solution for performing the entire sample preparation process, including DNA extraction, purification, bisulfite conversion and PCR plate preparation within in an enclosed environment. The method employs silica-coated magnetic particles that eliminate the need for a centrifuge or vacuum manifold, thereby reducing the complexity and cost of the required automation platform. Toward this end, we also compare commercial DNA extraction and bisulfite conversion kits to identify a protocol suitable for automation to significantly improve genomic and bisulfite-treated DNA yields over manufacturer protocols. Overall, this research demonstrated development of an automated protocol that offers the ability to generate high-quality, bisulfite-treated DNA samples in a high-throughput and clean environment with minimal user intervention and comparable yields to manual processing.

Methods for sequencing the pandemic: benefits of rapid or high-throughput processing.

Genomic epidemiology has proven successful for real-time and retrospective monitoring of small and large-scale outbreaks. Here, we report two genomic sequencing and analysis strategies for rapid-turnaround or high-throughput processing of metagenomic samples. The rapid-turnaround method was designed to provide a quick phylogenetic snapshot of samples at the heart of active outbreaks, and has a total turnaround time of <48 hours from raw sample to analyzed data. The high-throughput method, first reported here for SARS-CoV2, was designed for semi-retrospective data analysis, and is both cost effective and highly scalable. Though these methods were developed and utilized for the SARS-CoV-2 pandemic response in Arizona, U.S, we envision their use for infectious disease epidemiology in the 21 st Century.

3D-Stacked Multistage Inertial Microfluidic Chip for High-Throughput Enrichment of Circulating Tumor Cells.

Whether for cancer diagnosis or single-cell analysis, it remains a major challenge to isolate the target sample cells from a large background cell for high-efficiency downstream detection and analysis in an integrated chip. Therefore, in this paper, we propose a 3D-stacked multistage inertial microfluidic sorting chip for high-throughput enrichment of circulating tumor cells (CTCs) and convenient downstream analysis. In this chip, the first stage is a spiral channel with a trapezoidal cross-section, which has better separation performance than a spiral channel with a rectangular cross-section. The second and third stages adopt symmetrical square serpentine channels with different rectangular cross-section widths for further separation and enrichment of sample cells reducing the outlet flow rate for easier downstream detection and analysis. The multistage channel can separate 5 μm and 15 μm particles with a separation efficiency of 92.37% and purity of 98.10% at a high inlet flow rate of 1.3 mL/min. Meanwhile, it can separate tumor cells (SW480, A549, and Caki-1) from massive red blood cells (RBCs) with a separation efficiency of >80%, separation purity of >90%, and a concentration fold of ~20. The proposed work is aimed at providing a high-throughput sample processing system that can be easily integrated with flowing sample detection methods for rapid CTC analysis.

Separation of circulating tumor cells from blood using dielectrophoretic DLD manipulation.

Circulating Tumor Cells (CTCs) play a prominent role in early cancer detection. Emerging label-free techniques can be promising to CTC detection due to advantages in preserving cell integrity and minimal sample consumption. Deterministic Lateral Displacement (DLD) is a size-based label-free technique employing laminar flow for continuous sorting of suspended cells. However, separation based solely on size is challenging as the size distributions of CTCs tend to overlap with blood cells. Moreover, the rarity of CTCs in blood requires high throughput processing of samples for clinical utility. In this work, a dielectrophoretic DLD technique is presented to segregate CTCs from blood. This technique utilizes the cell size and dielectric properties as well as particle movement caused by polarization effect to accomplish continuous separation at high flow rates. A numerical model is developed and validated to investigate the effects of various parameters related to the fluid flow, micro-post array, and electric field. It is demonstrated that the dielectrophoretic DLD with specific post arrangement can continuously separate A549 lung CTCs from WBCs by applying a field frequency close to the crossover frequency of CTCs. The analysis further indicates that such a device can perform well despite uncertainties of CTC crossover frequencies. Additionally, efficient separation with minimum clogging can be achieved by setting the electric field perpendicular to fluid flow. The presented platform offers distinct advantages and can be potentially combined with techniques such as antibody-based immune-binding methods for rapid detection of CTCs.

Development of a cost‐effective and portable aeration freezing system for germplasm cryopreservation for eastern oysters and other aquatic species

The goal was to develop a cost-effective and portable freezing system for germplasm cryopreservation of fish and shellfish. The objectives were to: (1) identify components for construction of a portable freezing system by evaluating the affecting factors of cooling rates; (2) generate a range of cooling rates through adjusting different variables; (3) compare cooling profiles generated by the aeration freezing system and a commercial programmable freezer, and (4) verify the effectiveness of the aeration freezing system by cryopreserving sperm of eastern oysters Crassostrea virginica. An aeration freezing system was constructed using a Styrofoam box as cooling chamber and an innovative aeration unit to create nitrogen vapour. A wide range of cooling rates (1.5–32.1°C/min) were achieved by combinations of sample position at 2, 5, or 10 cm above liquid nitrogen surface, the temperature of nitrogen vapour (−150 or −180°C), and aeration time of liquid nitrogen (0, 1, 2 min, or continuity). Compared to a programmable freezer, the aeration freezing system generated cooling profiles with significantly higher temperature for ice nucleation initiation. No difference in post-thaw sperm motility of eastern oysters were found between samples cooling in the aeration freezing system or a programmable freezer. Cooling rates of 10 or 15°C/min from 4°C to −80°C yielded the highest post-thaw sperm motility regardless of the cooling devices. The aeration freezing system developed in this study has the potential for high-throughput sample processing, and the design allows easy construction by multiple users.

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach.

The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

Polymer substrate with surface solvent reservoir for polymer-spray mass spectrometric analysis of hydrophilic drugs

Nonporous, hydrophobic organosiloxane (OSX) polymers with surface reservoirs were developed as sampling supports in polymer-spray mass spectrometry (polyS-MS) for the detection and quantification of hydrophilic drugs, ethyl glucuronide (logP -1.8) and vancomycin (logP -4.4), and a slightly hydrophobic drug, phenobarbital (logP 1.5). OSX polymers with surface reservoirs were prepared by solvent evaporation-induced patterning in the presence of an ionic liquid during sol-gel polymerization of a methylalkoxysilane. The surface reservoir provides a continuous supply of spray solvent, enhancing both signal stability and signal duration when conducting analysis by polyS-MS. Using OSX polymer substrates with surfaces reservoirs, calibration curves were generated with good linearity (R2>0.99) and reproducibility and low data spread over the concentration ranges used to screen each of the drugs tested as compared to paper substrates: ethyl glucuronide, 5 – 1000 ng/mL (methanol) and 50 – 10,000 ng/mL (synthetic urine); vancomycin, 2 – 100 μL/mL (plasma); phenobarbital, 50 – 50,000 ng/mL (methanol) and 50 – 5000 ng/mL (synthetic urine). Quantitative analysis by polyS-MS of phenobarbital samples in synthetic urine at concentrations of 250 ng/mL and 2,500 ng/mL showed good recoveries of 90.2% (RSD 3.51%) and 95.7% (RSD 2.57%), respectively. Compared to paper substrates, OSX polymers gave calibration curves with better linearity and reproducibility, especially at low concentrations, as well as longer signal durations, improved signal stabilities, and increased signal intensities. Little to no sample pretreatment is necessary and the required sample volume is small (1 μL to 2 μL), making polyS-MS easy to use for high throughput sample processing.