Washing Of Cells Research Articles

Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells

Membrane proteins constitute around 20–30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of “omics” techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.

RvD1 disrupts nociceptor neuron and macrophage activation and neuroimmune communication, reducing pain and inflammation in gouty arthritis in mice.

Gouty arthritis is characterized by an intense inflammatory response to monosodium urate crystals (MSU), which induces severe pain. Current therapies are often ineffective in reducing gout-related pain. Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator with anti-inflammatory and analgesic proprieties. In this study, we evaluated the effects and mechanisms of action of RvD1 in an experimental mouse model of gouty arthritis, an aim that was not pursued previously in the literature. Male mice were treated with RvD1 (intrathecally or intraperitoneally) before or after intraarticular stimulation with MSU. Mechanical hyperalgesia was assessed using an electronic von Frey aesthesiometer. Leukocyte recruitment was determined by knee joint wash cell counting and immunofluorescence. IL-1β production was measured by ELISA. Phosphorylated NF-kB and apoptosis-associated speck-like protein containing CARD (ASC) were detected by immunofluorescence, and mRNA expression was determined by RT-qPCR. CGRP release was determined by EIA and immunofluorescence. MSU crystal phagocytosis was evaluated by confocal microscopy. RvD1 inhibited MSU-induced mechanical hyperalgesia in a dose- and time-dependent manner by reducing leukocyte recruitment and IL-1β production in the knee joint. Intrathecal RvD1 reduced the activation of peptidergic neurons and macrophages as well as silenced nociceptor to macrophage communication and macrophage function. CGRP stimulated MSU phagocytosis and IL-1β production by macrophages. RvD1 downmodulated this phenomenon directly by acting on macrophages, and indirectly by inhibiting CGRP release and CGRP-dependent activation of macrophages. This study reveals a hitherto unknown neuro-immune axis in gouty arthritis that is targeted by RvD1.

Abstract 1885: Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality

Abstract Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash࣪ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows. Citation Format: Melvin Lye, Christoph Eberle, Ann Wang, Geoffrey K. Feld, Namyong Kim. Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality [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 1885.

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.

Automatic Microfluidic Cell Wash Platform for Purifying Cells in Suspension: Puriogen.

Cell wash is an essential cell sample preparation procedure to eliminate or minimize interfering substances for various subsequent cell analyses. The commonly used cell wash method is centrifugation which separates cells from other biomolecules in a solution with manual pipetting and has many drawbacks such as being labor-intensive and time-consuming with substantial cell loss and cell clumping. To overcome these issues, a centrifuge-free and automatic cell wash platform for cell purity generation, termed Puriogen, has been developed in this work. Compared with other developed products such as AcouWash, Puriogen can process samples with a high throughput of above 500 μL/min. Puriogen utilizes a uniquely designed inertial microfluidic device to complete the automatic cell wash procedure. One single-cell wash procedure with the Puriogen platform can remove more than 90% ambient proteins and nucleic acids from the cell sample. It can also remove most of the residual fluorescent dye after cell staining to significantly reduce the background signals for subsequent cell analysis. By removing the dead cell debris, it can increase the live cell percentage in the sample by 2-fold. Moreover, the percentage of single-cell population is also increased by 20% because of further disassociation of small-cell aggregates (e.g., doublets and triplets) into singlets. To freely adjust cell concentrations, the Puriogen platform can concentrate cells 5 times in a single flow-through process. The presented Puriogen cell wash solution has broad applications in cell preparation with its excellent simplicity in operation and wash efficiency, especially in single-cell sequencing.

Cell radiolabeling with acoustophoresis cell washing

Labeling immune cells with zirconium-89 (89Zr)-oxine has become a viable method to track cells in vivo by PET in various pre-clinical animal models and in clinical applications. Currently, 89Zr-oxine cell labeling is performed manually, which requires a highly trained specialist and is prone to human error. As the first phase in developing a fully automated radiolabeling system to address this problem, we assess the use of acoustophoresis cell washing to replace the centrifugal cell washing used in the current 89Zr-oxine cell radiolabeling procedure. To accomplish this, a cell radiolabeling procedure was developed in which two steps requiring a centrifuge to wash cells were replaced using acoustophoresis cell washing methods. The process was tested using murine EL4 lymphoma and T cells. The centrifuge cell labeling procedure was used as a control to compare the acoustophoresis cell washing procedure. The acoustophoresis method produced radiolabeled cells with similar properties to the centrifugal method when comparing labeling efficiency, labeled specific activity, efficacy of removing unbound 89Zr-oxine from the suspension, cell viability measured using annexin V/propidium iodide staining and activation function. This suggests that acoustophoresis cell washing can be used in the design of an automated benchtop, good manufacture practice-qualified acoustophoresis cell radiolabeling device.

Dean flow velocity of shear-thickening SiO2 nanofluids in curved microchannels

We report the effects of a curvilinear microchannel width, height, and radius of curvature, as well as the kinematic viscosity and axial velocity of shear-thickening nanofluids, on the average Dean velocity (VDe) of the secondary flow in the microchannel. Manipulation of inertial and Dean drag forces in curvilinear microchannels has enabled high-throughput and high-resolution size-based separation of microparticles and cells in various biomedical applications. VDe plays a deterministic role in the estimation of the Dean drag force and the design of these microfluidic devices. Despite the previous numerical and experimental studies on VDe of Newtonian and shear-thinning viscoelastic fluids, VDe of shear-thickening metallic nanofluids, such as SiO2 nanoparticles in water, in curved microchannels is still unknown. Such shear-thickening fluids are being used in thermal microsystem applications and are on the verge of entering the field of inertial microfluidics for particle and cell sorting. Our investigations have shown that VDe of shear-thickening SiO2–water nanofluids scales directly with the channel width and the fluid axial velocity, while being inversely proportional with the SiO2 concentration and the channel radius of curvature. Our non-dimensional analysis has led to the development of an empirical correlation that relates VDe-based Reynolds number of the nanofluid to the Dean number and the normalized kinematic viscosity of the nanofluid. It provides a significant accuracy in estimating VDe of shear-thickening fluids, compared to application of Newtonian or shear-thinning equations in the literature, which could be useful toward future design of particle and cell sorting and washing microdevices.

Applications and benefits of the CTS Rotea Counterflow Centrifugation System in cell therapy workflows

The Gibco™ CTS™ Rotea™ Counterflow Centrifugation System is a closed cell processing system designed for cell therapy workflows. It has the flexibility to perform a variety of cell processing applications across several cell types. Here, we provide some key data to highlight the Rotea system’s ability to isolate white blood cells (WBCs) starting with leukopaks, as well as perform high speed T cell wash & concentrate steps and efficient cell washing.

Quick and Easy Isolation of Immune Cells From Large-Volume Samples

Abstract Large-scale cell isolation is commonly performed in labs and core facilities for cell banking and drug discovery, and as a critical step in cell therapy manufacturing. Current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day for sample processing and cell isolation. To address this need, we have developed two methods: manual column-free isolation using the Easy 250 EasySep™ magnet, and automated cell isolation in a closed system using RoboSep™-C. The Easy 250 magnet allows users to isolate cells from a full leukopak of up to 20 billion cells in under 30 minutes by simply pipetting out their target cells. Negative selection protocols have been optimized to achieve 95.3% T cell, 97.2% CD4+ T cell, 91.9% CD8+ T cell, 99.4% B cell, 96.5% NK cell, and 91.5% monocyte purities. Positive selection protocols obtain 95.4% CD3+ cell, 94.4% CD4+ T cell, 93.9% CD8+ T cell, and 96.2% CD14+ cell purities. RoboSep™-C automates this cell isolation procedure, along with the cell washing steps for sample preparation, in as little as 50 minutes. The instrument features a scale tower, pump and clamp modules to direct fluid through defined paths, and a magnet for separation of labelled cells. The system uses a sterile single-use tubing set that incorporates a cartridge for cell washing and concentration, and a magnet chamber for cell separation. Starting with fresh leukopaks, we obtained 95.9% T cell, 95.8% CD4+ T cell, and 89.6% CD8+ T cell purities following negative selection, and 92.0% CD4+ T cell and 89.5% CD8+ T cell purities following positive selection. These approaches offer efficient and user-friendly cell isolation that allow researchers to scale up their operations, and can be easily integrated upstream of existing workflows.

Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and flow cytometry data quality

Abstract Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash™ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows.

Abstract #1153940: Specific Detection of TSAb and TBAb in Serum of Suspected AITD Patients using Two Rapid Homogenous Bioassays

Two rapid homogenous bioassays, named as Turbo TSI and Turbo TBI, have been developed to either quantitatively detect Thyroid Stimulating Antibodies (TSAb) or qualitatively detect Thyroid Blocking Antibody (TBAb) in Autoimmune Thyroid Disease (ATID) patient sera. The two bioassays do not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time to 60 minutes. This study was designed to demonstrate the high specificity of the two bioassays in comparison with the TSHR Autoantibody (TRAb) ELISA Kit (Kronus).

Tangential flow filtration facilitated washing of human red blood cells: A proof-of-concept study.

Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. The TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis. No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12. Overall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.

Assessment of the LOVO device for final harvest of novel cell therapies: a Production Assistance for Cellular Therapies multi-center study

Background aimsThe final harvest or wash of a cell therapy product is an important step in manufacturing, as viable cell recovery is critical to the overall success of a cell therapy. Most harvest/wash approaches in the clinical lab involve centrifugation, which can lead to loss of cells and decreased viability of the final product. Here the authors report on a multi-center assessment of the LOVO Cell Processing System (Fresenius Kabi, Bad Homburg, Germany), a cell processing device that uses a spinning filtration membrane instead of centrifugation. MethodsFour National Institutes of Health Production Assistance for Cellular Therapies cell processing facilities (CPFs) assessed the LOVO Cell Processing System for final harvest and/or wash of the following three different cell products: activated T cells (ATCs), tumor-infiltrating lymphocytes (TILs) and bone marrow-derived mesenchymal stromal cells (MSCs). Each site compared their current in-house, routinely used method of final cell harvest and/or wash with that of the LOVO device. ResultsFinal harvest and/or wash of ATCs, TILs and MSCs using the LOVO system resulted in satisfactory cell viability and recovery with some substantial improvement over the in-house methods of CPFs. Processing time was variable among cell types/facilities. ConclusionsThe LOVO Cell Processing System provides an alternative to centrifuge-based technologies. The system employs a spinning membrane filter, exposing cells to minimal g-forces compared with centrifugation, and is automated and closed. This small multi-center study demonstrated the ability of the LOVO device to yield satisfactory cell viability and recovery of T cells and MSCs.

Extracellular Vesicles From Paracoccidioides brasiliensis Can Induce the Expression of Fungal Virulence Traits In Vitro and Enhance Infection in Mice.

Extracellular vesicles (EVs) are cellular components involved in cargo delivery to the extracellular environment, including the fungal cell wall. Their importance in cell–cell communication, cell wall remodeling, and fungal virulence is starting to be better explored. In the human pathogenic Paracoccidioides spp., our group has pioneered the description of the EV secretome, carbohydrate cargo, surface oligosaccharide ligands, lipid, and RNA content. Presently, we studied the role of fungal EVs in the context of the virulent/attenuated model of the P. brasiliensis Pb18 isolate, which consists of variants transiently displaying higher (vPb18) or attenuated (aPb18) virulence capacity. In this model, the virulence traits can be recovered through passages of aPb18 in mice. Here, we have been able to revert the aPb18 sensitivity to growth under oxidative and nitrosative stress upon previous co-incubation with vEVs from virulent vPb18. That was probably due to the expression of antioxidant molecules, considering that we observed increased gene expression of the alternative oxidase AOX and peroxiredoxins HYR1 and PRX1, in addition to higher catalase activity. We showed that aEVs from aPb18 stimulated macrophages of the RAW 264.7 and bone marrow-derived types to express high levels of inflammatory mediators, specifically, TNF-α, IL-6, MCP-1, and NO. In our experimental conditions, subcutaneous treatment with EVs (three doses, 7-day intervals) before vPb18 challenge exacerbated murine PCM, as concluded by higher colony-forming units in the lungs after 30 days of infection and histopathology analysis. That effect was largely pronounced after treatment with aEVs, probably because the lung TNF-α, IFN-γ, IL-6, and MCP-1 concentrations were specially increased in aEV-treated when compared with vEV-treated mice. Our present studies were performed with EVs isolated from yeast cell washes of confluent cultures in Ham’s F-12 defined medium. Under these conditions, vEVs and aEVs have similar sizes but probably distinct cargo, considering that vEVs tended to aggregate upon storage at 4°C and −20°C. Additionally, aEVs have decreased amounts of carbohydrate and protein. Our work brings important contribution to the understanding of the role of fungal EVs in cell–cell communication and on the effect of EVs in fungal infection, which clearly depends on the experimental conditions because EVs are complex and dynamic structures.

Viability and developmental potential of porcine blastocysts preserved for short term in a chemically defined medium at ambient temperature.

This study developed an efficient method for liquid storage of in vitro-derived porcine blastocysts at ambient temperature for 24hr. We evaluated the effects of new chemically defined media (cell wash and preservation solution, Cellstor® -W [Cell-W] and cell suspension and preservation solution, Cellstor® -S [Cell-S]) for short-term storage. In the first experiment, in vitro-derived blastocyst were stored at 25ºC for 24hr in Cell-W solution, Cell-S solution and pig embryo culture (PBM) medium. There were no differences in the rates of survival and development of stored blastocysts between the Cell-S and Cell-W solutions, but the total cell number of embryos that survived after storage in Cell-S solution was significantly higher (p<.05) than that in the Cell-W solution. In the second experiment, Cell-S solution was used to store the in vitro-derived blastocysts at 20°C, 25°C and 30°C. Storage at 20°C resulted in a significant decrease in the rates of survival and development of stored blastocysts compared to storage at 25°C or 30°C. No differences in survival and development rates were observed between storage at 25°C and 30°C, but the damage to the embryo quality after storage and culture was significantly lower at 25°C than at 30°C. In the third experiment, Cell-S solution was supplemented with β-mercaptoethanol and curcumin, either alone or in combination, as antioxidant agents. Although the supplementation with curcumin did not improve survival, it significantly increased the development rate of stored blastocysts compared with the control blastocysts stored without antioxidants. In conclusion, when porcine blastocysts were stored at 25°C for 24hr, a Cell-S solution may be effective for maintaining the survival and development of in vitro embryos.

A Simple and Rapid Bioluminescence-Based Functional Assay of Organic Anion Transporter 1 as a D-Luciferin Transporter.

Organic anion transporter 1 (SLC22A6/OAT1) plays a key role in renal tubular excretion of endo- and exogenous anionic substances including drugs. Since the inhibition of OAT1 function by a concomitant drug may cause pharmacokinetic drug-drug interactions (DDIs) in clinical practice, an in vitro uptake study to evaluate the inhibition potency of OAT1 is useful for the prediction and avoidance of DDIs and recommended for drug candidates in drug development. In this chapter, we describe a rapid and highly sensitive functional assay of OAT1 based on bioluminescence (BL) detection using D-luciferin as a substrate in living cells. The principle of measurement simply relies on the biochemical feature of D-luciferin to be recognized as a substrate of OAT1, and the BL intensity depending on intracellular D-luciferin level and luciferase activity, thereby allowing the quantitative analysis of OAT1-mediated D-luciferin transport. The BL measurement can be completed within 1min without experimental procedures for removing extracellular uptake solution and washing cells, both of which involve in the conventional uptake studies using isotope-labeled or fluorescent compounds. The present method is applicable to high-throughput screening to identify and avoid potential OAT1 inhibitors in drug development.

A reconfigurable microfluidic building block platform for high-throughput nonhormonal contraceptive screening.

Identifying nonhormonal contraceptives will have profound impacts on avoiding side effects of hormonal birth control methods, minimizing pregnancy complications and infant mortality rates, and promoting family planning. However, phenotypic screening of contraceptives is challenging due to the diverse procedures associated with oocyte culture, biochemical assays, and molecular imaging. This study reports a multifunctional microfluidic platform comprising reconfigurable building blocks and interfaces to implement various cell-based drug screening protocols. This versatile platform has three major layers. The top layer consists of interchangeable 3D microfluidic building blocks (e.g., branching microchannels, chemical gradient generators, pumpless flow controllers, and emulsion generators) or an open interface. The middle layer incorporates a multiwell array with embedded membrane filters for live cell culture, medium exchange, enzymatic cumulus cell removal, washing, and fluorescence staining. The bottom layer is also reconfigurable for waste collection, oocyte culture, plate reader measurement, and high-resolution microscopy. We demonstrate an 8 by 16 (128 wells) system for performing the cumulus-oocyte complex (COC) expansion and oocyte maturation assays for screening nonhormonal contraceptives. The microfluidic building block platform is scalable and can be reconfigured for a variety of drug screening applications in the future.

ATP is an essential autocrine factor for pancreatic β-cell signaling and insulin secretion.

ATP has been previously identified as an autocrine signaling factor that is co‐released with insulin to modulate and propagate β‐cell activity within islets of Langerhans. Here, we show that β‐cell activity and insulin secretion essentially rely on the presence of extracellular ATP. For this, we monitored changes of the intracellular Ca2+ concentration ([Ca2+]i oscillations) as an immediate read‐out for insulin secretion in live cell experiments. Extensive washing of cells or depletion of extracellular ATP levels by recombinant apyrase reduced [Ca2+]i oscillations and insulin secretion in pancreatic cell lines and primary β‐cells. Following ATP depletion, [Ca2+]i oscillations were stimulated by the replenishment of ATP in a concentration‐dependent manner. Inhibition of endogenous ecto‐ATP nucleotidases increased extracellular ATP levels, along with [Ca2+]i oscillations and insulin secretion, indicating that there is a constant supply of ATP to the extracellular space. Our combined results demonstrate that extracellular ATP is essential for β‐cell activity. The presented work suggests extracellular ATPases as potential drug targets for the modulation of insulin release. We further found that exogenous fatty acids compensated for depleted extracellular ATP levels by the recovery of [Ca2+]i oscillations, indicating that autocrine factors mutually compensate for the loss of others. Thereby, our results contribute to a more detailed and complete understanding of the general role of autocrine signaling factors as a fundamental regulatory mechanism of β‐cell activity and insulin secretion.

A rapid homogenous bioassay for detection of thyroid-stimulating antibodies based on a luminescent cyclic AMP biosensor

Graves' disease (GD) is an autoimmune disease caused by antibodies to the thyroid stimulating hormone receptor (TSHR). The FDA-cleared Thyretain™ TSI bioassay is a highly specific method to detect thyroid stimulating antibodies (TSAb/TSI) in the blood of patients with autoimmune thyroid disease (AITD), particularly GD. To simplify the workflow of this bioassay and to support a semi-quantitative result, we have generated a stable CHO-K1 cell line expressing both a chimeric TSH receptor (TSHR-Mc4) and a luciferase-based homogeneous cAMP biosensor (GS luciferase). Here, we describe a rapid, real-time, homogenous bioassay (Turbo™ TSI Bioassay) to directly assess the functional activity of TSI and produce results in International Units of IU/L. The Turbo™ TSI bioassay works by measuring changes in the intracellular cAMP level induced by a G-protein coupled receptor (G-PCR) signaling cascade which is triggered by the binding of TSI to the TSHR. Upon binding to cAMP, the GS luciferase reporter is activated through conformational changes and generates light that can be measured in intact cells with a luminometer. The LoD and LoQ of the assay were determined to be 0.016 IU/L and 0.03 IU/L, respectively and the preliminary assay cutoff was determined to be 0.024 IU/L by ROC analysis using the Thyretain™ TSI bioassay results as reference. The analytical performance of the Turbo™ TSI bioassay is comparable to the Thyretain™ TSI bioassay as evidenced by similar EC50 values for a TSHR stimulating monoclonal antibody (M22). The specificity of the Turbo™ TSI bioassay was demonstrated by showing no response to a high concentration of a human monoclonal TSHR blocking antibody (K1–70). The precision of the assay was excellent with an overall within-laboratory precision <15% CV. When testing 198 clinical samples, the positive and negative percent agreement between the Turbo™ TSI and the Thyretain™ TSI bioassays were 98.7% and 93.5%, respectively. While both bioassays yield equivalent analytical and clinical performances, the Turbo™ TSI bioassay is much simpler to perform. It does not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time from about 21 h to 60 min. In addition, the same cell line showed its capability of detecting thyroid blocking antibodies (TBAb/TBI) in a competitive format. The Turbo™ TSI bioassay is user-friendly and is a very promising advancement to aid the diagnosis of autoimmune thyroid disease (AITD).

Optimizing Commercial Manufacturing of Tisagenlecleucel for Patients in the US: A 4-Year Experiential Journey

Background:Tisagenlecleucel (Kymriah) is an autologous CD19-directed CAR-T-cell therapy, approved in Aug-2017 for treating children and young adults with relapsed/refractory (r/r) acute lymphoblastic leukemia and in May-2018 for treating adults with r/r diffuse large B-cell lymphoma. Post-approval, a key goal has been to upscale and continuously improve manufacturing success and turnaround time in the commercial settings to meet the needs of a global patient population. Here we report accrued experience from our 4-year journey of optimizing the commercial tisagenlecleucel manufacturing process at the US site (Morris Plains, NJ), for faster and successful delivery to patients in the US.Methods:As reported previously, the tisagenlecleucel manufacturing process includes leukapheresis of the patient's peripheral blood mononuclear cells, enrichment and activation of T cells, transduction of the lentiviral vector containing the anti-CD19 CAR transgene, activation with anti-CD3/CD28 antibody-coated beads, expansion in cell culture, washing, and formulation of the viable cells into a cryoformulation medium. The final product is then cryopreserved, shipped back to the treatment center and infused to patients (Tyagarajan, 2020). Use of cryopreserved leukapheresis material as the starting point in commercial manufacturing is unique to tisagenlecleucel; this allows flexibility in terms of scheduling leukapheresis when a patient's health is optimal to provide T cells, and also helps offset logistical challenges (Tyagarajan, 2019).Results:As of Jun-2021, tisagenlecleucel has been manufactured for >5000 patients worldwide, enabled by Novartis's significantly increased global manufacturing footprint at six sites strategically located across six countries (US, France, Switzerland, Germany, Japan and Australia) and a global treatment network of >340 certified centers, including 127 centers in the US.Specifically for the US manufacturing site, between Dec-2020 and Jun-2021, 376 patients in the US had starting material available for manufacturing. Overall, the manufactured product was available for shipment for 98% of patients (shipping success rate [SSR]). The commercial manufacturing success rate (MSR) was 96%, with an out-of-specification (OOS) rate of <3% and no OOS for viability. All ten OOS batches were released for infusion as benefit:risk assessment was positive. Manufacturing was cancelled for two patients upon physician's request.Immediate manufacturing capability without waiting time was available on receipt of all apheresis starting materials. The median time from start of manufacturing to shipping was 20 days. As is evident, the COVID-19 pandemic did not appear to have significantly affected the success rate or manufacturing turnaround time.These latest success metrics, reflecting significant improvements from 2018 to 2021 in MSR (69% to 96%), SSR (93% to 98%), and overall OOS rate (26% to 2%) including viability OOS rate (from 25% to 0%), are a result of upscaling the manufacturing capabilities, enhancements with hospitals focusing on optimizing apheresis collection and cryopreservation procedures, and continuous evaluation and improvement of the manufacturing process since tisagenlecleucel was first launched (Figure). Two key process and analytical improvements that were considered to have improved robustness of manufacturing and testing processes, reduced OOS rates, and minimized variability in turnaround time were introduced towards the end of 2020. Firstly, a simplified sample preparation procedure for final product cell count and viability measurement, which is more reflective of final product at infusion. Secondly, an alternate serum source (5% plasma-derived human AB serum [PD hABs]) which further improves process robustness with a trend towards improved growth and higher peak cell counts.Conclusions:Tisagenlecleucel's current global commercial manufacturing footprint and treatment network are well-positioned to meet anticipated future increase in demand for CAR-T therapies. Recent process improvements have significantly increased the MSR (to 96%) and SSR (to 98%), and immediate product availability for patients in need of CAR-T cells. Ongoing and upcoming process improvements are anticipated to further reduce the throughput time, thus allowing more patients faster access to CAR-T therapy. [Display omitted] DisclosuresRodrigues: Novartis: Current Employment. Duran: Novartis: Current Employment. Eschgfaeller: Novartis: Current Employment. Kuzan: Novartis: Current Employment. Habucky: Novartis: Current Employment.