Monitoring Of Viability Research Articles

Abstract 1449: A live-cell imaging approach for assessing efficacy of immune-targeting therapies using high content imaging and analysis of 3D in vitro tumor models

Abstract Background: Immunotherapy has revolutionized cancer research, however limited clinical success to date underscores the need for novel therapeutic targets. Assessment of new targets has been challenging due to limitations of ex vivo animal platforms that only partially recapitulate tumor-intrinsic features and cell interactions. Here, we present a live-cell imaging approach for assessing efficacy of immune-targeting therapies using a 3D in vitro oncology platform in combination with high-content imaging and analysis (HCA) techniques. This study shows how effects of immune-targeting agents on tumor infiltration and killing by peripheral blood mononuclear cells (PBMCs) can be visualized and quantified with high-content endpoints. Aim: To develop and test a robust live-cell-imaging methodology for assessing efficacy of immuno-modulatory cancer therapies. Material & Methods: Lung carcinoma cell-line models were generated using GFP-A549 cells in coculture with dermal fibroblasts and PBMCs. PDX cell suspensions of lung, breast and melanoma origin were self-aggregated with cancer-associated fibroblasts (CAFs) and labeled with live-cell dye for specific monitoring of tumor cell viability. Tumor killing activity and T-cell effector function were evaluated by measuring tumor model size using automatic stage fluorescence microscopy or confocal HCA instrument. Release of inflammatory cytokines IL-6, TNF, IFNγ and GM-CSF was measured with a multiplex cytokine panel. T cell infiltration into tumor models was quantified using fluorescent staining of CD3+ cells. To generate a pro-inflammatory tumor microenvironment, PBMCs were stimulated prior to coculture either with cytokines or anti-CD3/CD28. We monitored immune cell attack on tumors in realtime (56 timepoints) and in 3 dimensions without any discernable phototoxicity. Using cell centroid tools, we mapped physical locations of individual cells belonging to each population. Finally, we calculated tumor and immune cell volumes from individual image stacks to evaluate tumor killing and proliferation/infiltration of immune cells. Results: Our realtime confocal analysis of tumor-immune cell cocultures showed activation of PBMCs with either cytokines or CD3/CD28 increased proliferation/infiltration of immune cells and enhanced tumor killing. Conclusion: Proinflammatory tumor models combined with our novel, high-content imaging analysis endpoints offer a versatile solution for preclinical translational research and a promising approach for high-content screening of immune-targeting therapeutic agents and I-O studies involving engineered T cells (e.g., CAR-T) as well as for testing immunomodulatory agents, such as bispecific antibodies and immune checkpoint inhibitors. Citation Format: Irina Agarkova, Mahomi Suzuki, Silvan Strebel, Armin Wolf, Judith Wardwell-Swanson, Francesca Chiovaro. A live-cell imaging approach for assessing efficacy of immune-targeting therapies using high content imaging and analysis of 3D in vitro tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1449.

Effect of GM maize on metabolism and performance of chicken

Effect of GM maize on metabolism and performance of chicken

Prospective inference of bioprocess cell viability through chemometric modeling of fluorescence multiway data.

In this investigation, the fermentation step of a standard mammalian cell-based industrial bioprocess for the production of a therapeutic protein was studied, with particular emphasis on the evolution of cell viability. This parameter constitutes one of the critical variables for bioprocess monitoring since it can affect downstream operations and the quality of the final product. In addition, when the cells experiment an unpredictable drop in viability, the assessment of this variable through classic off-line methods may not provide information sufficiently in advance to take corrective actions. In this context, Process Analytical Technology (PAT) framework aims to develop novel strategies for more efficient monitoring of critical variables, in order to improve the bioprocess performance. Thus, in this work, a set of chemometric tools were integrated to establish a PAT strategy to monitor cell viability, based on fluorescence multiway data obtained from fermentation samples of a particular bioprocess, in two different scales of operation. The spectral information, together with data regarding process variables, was integrated through chemometric exploratory tools to characterize the bioprocess and stablish novel criteria for the monitoring of cell viability. These findings motivated the development of a multivariate classification model, aiming to obtain predictive tools for the monitoring of future lots of the same bioprocess. The model could be satisfactorily fitted, showing the non-error rate of prediction of 100%.

Drug Screening Tools: A Spheroid‐Forming Hybrid Gold Nanostructure Platform That Electrochemically Detects Anticancer Effects of Curcumin in a Multicellular Brain Cancer Model (Small 15/2021)

In article number 2002436, Tae-Hyung Kim and co-workers demonstrate a novel multifunctional hybrid platform that enables spontaneous cancer spheroids formation on the surface and real-time non-destructive electrochemical monitoring of spheroid viability simultaneously. The platform allows for mimicking the complex three-dimensional cellular networks and multicellular characteristics of real solid tumors in vitro, which will be highly useful for high-throughput anticancer drug screening.

Direct and Label-Free Cell Status Monitoring of Spheroids and Microcarriers Using Microfluidic Impedance Cytometry.

3D cellular spheroids/microcarriers (100µm-1mm) are widely used in biomanufacturing, and non-invasive biosensors are useful to monitor cell quality in bioprocesses. In this work, a novel microfluidic approach for label-free and continuous-flow monitoring of single spheroid/microcarrier (hydrogel and Cytodex) based on electrical impedance spectroscopy using co-planar Field's metal electrodes is reported. Through numerical simulation and experimental validation, two unique impedance signatures (|ZLF | (60kHz), |ZHF | (1MHz)) which are optimal for spheroid growth and viability monitoring are identified. Using a closed-loop recirculation system, it is demonstrated that |ZLF | increases with breast cancer (MCF-7) spheroid biomass, while higher opacity (impedance ratio |ZHF |/|ZLF |) indicates cell death due to compromised cell membrane. Anti-cancer drug (paclitaxel)-treated spheroids also exhibit lower |ZLF | with increased cell dissociation. Interestingly, impedance characterization of adipose-derived mesenchymal stem cell differentiation on Cytodex microcarriers reveals that adipogenic cells (higher intracellular lipid content) exhibit higher impedance than osteogenic cells (more conductive due to calcium ions) for both microcarriers and single cell level. Taken together, the developed platform offers great versatility for multi-parametric analysis of spheroids/microcarriers at high throughput (≈1 particle/s), and can be readily integrated into bioreactors for long-term and remote monitoring of biomass and cell quality.

Seed longevity of maize conserved under germplasm bank conditions for up to 60 years.

The long-term conservation of seeds of plant genetic resources is of key importance for food security and preservation of agrobiodiversity. Nevertheless, there is scarce information available about seed longevity of many crops under germplasm bank conditions. Through germination experiments as well as the analysis of historical monitoring data, we studied the decline in viability manifested by 1000 maize (Zea mays subsp. mays) seed accessions conserved for an average of 48 years at the CIMMYT germplasm bank, the largest maize seedbank in the world, under two cold storage conditions: an active (-3 °C; intended for seed distribution) and a base conservation chamber (-15 °C; for long-term conservation). Seed lots stored in the active chamber had a significantly lower and more variable seed germination, averaging 81.4 %, as compared with the seed lots conserved in the base chamber, averaging 92.1 %. The average seed viability detected in this study was higher in comparison with that found in other seed longevity studies on maize conserved under similar conditions. A significant difference was detected in seed germination and longevity estimates (e.g. p85 and p50) among accessions. Correlating seed longevity with seed traits and passport data, grain type showed the strongest correlation, with flint varieties being longer lived than floury and dent types. The more rapid loss of seed viability detected in the active chamber suggests that the seed conservation approach, based on the storage of the same seed accessions in two chambers with different temperatures, might be counterproductive for overall long-term conservation and that base conditions should be applied in both. The significant differences detected in seed longevity among accessions underscores that different viability monitoring and regeneration intervals should be applied to groups of accessions showing different longevity profiles.

Impact of Successive Exploitation of a Saccharomyces pastorianus Starter Culture on Saccharide Uptake Dynamics from Wort.

SUMMARYResearch backgroundThe production of lager beer includes successive repitchings of a single Saccharomyces pastorianus starter culture. During the beer production process, the yeast is exposed to several stress factors which could affect the fermentation performance. An incomplete fermentation represents a waste of fermentable extract and leads to a beer with higher carbohydrate levels, which could result in a beer with an atypical flavour profile. The aim of the present study is to determine the impact of successive exploitation of a single S. pastorianus starter culture on the wort saccharide uptake dynamics.Experimental approachThe fermentation was monitored during the production of twelve batches of beer, where the starter yeast culture was reused twelve times without any further treatment. The following beer production steps were monitored: wort production, yeast starter culture propagation, primary fermentation, secondary fermentation and the final product. The work was conducted on an industrial scale employing standard process conditions.Results and conclusionsMonitoring of the starter culture viability during successive fermentations indicated no reduction in the viability and vitality of the yeast culture. Monitoring of the fermentable wort saccharide concentrations (glucose, fructose, disaccharides and trisaccharides) revealed a correlation between an improvement in saccharide utilisation and starter culture age. Saccharide uptake efficacy proportionally matched the repitching frequency. Successive exploitation of S. pastorianus starter culture has a positive impact on the dynamics of saccharide utilisation from classical hopped wort and the young beer. Furthermore, the final lager beer contains no residues of fermentable saccharides that could affect the overall quality and flavour profile.Novelty and scientific contributionResults showed the impact of twelve successive wort fermentations on the dynamics of saccharides uptake that gives brewers important information. The added value of the experiment is all the work done on the industrial scale, with control of all processes and usage of exactly the same raw materials. This study contains usable technological data on the behaviour of saccharides during brewing on the industrial scale, which is not yet found in the literature.

Nonspecific nuclear uptake of anti-MUC1 aptamers by dead cells: the role of cell viability monitoring in aptamer targeting of membrane-bound protein cancer biomarkers.

Most aptamers targeting cell-expressed antigens are intended for in vivo application, however, these sequences are commonly generated in vitro against synthetic oligopeptide epitopes or recombinant proteins. As these in vitro analogues frequently do not mimic the in vivo target within an endogenous environment, the evolved aptamers are often prone to nonspecific binding. The presence of dead cells and cellular debris further complicate aptamer targeting, due to their high nonspecific affinities to single-stranded DNA. Despite these known limitations, assessment of cell viability and/or the removal of dead cells is rarely applied as part of the methodology during in vivo testing of aptamer binding. Furthermore, the extent and route(s) by which dead cells uptake existing aptamers remains to be determined in the literature. For this purpose, the previously reported aptamer sequences 5TR1, 5TR4, 5TRG2 and S22 - enriched against the MUC1 tumour marker of the mucin glycoprotein family - were used as model sequences to evaluate the influence of cell viability and the presence of nontarget cell-expressed protein on aptamer binding to the MUC1 expressing human cancer cell lines MCF-7, Hs578T, SW480, and SW620. From fluorescence microscopy analysis, all tested aptamers demonstrated extensive nonspecific uptake within the nuclei of dead cells with compromised membrane integrities. Using fluorescent-activated cell sorting (FACS), the inclusion of excess double-stranded DNA as a blocking agent showed no effect on nonspecific aptamer uptake by dead cells. Further nonspecific binding to cell-membrane bound and intracellular protein was evident for each aptamer sequence, as assessed by southwestern blotting and FACS. These factors likely contributed to the ∼120-fold greater binding response of the 5TR1 aptamer to dead MCF-7 cells over equivalent live cell populations. The identification of dead cells and cellular debris using viability stains and the subsequent exclusion of these cells from FACS analysis was identified as an essential requirement for the evaluation of aptamer binding specificity to live cell populations of the cancer cell lines MCF-7, Hs578T and SW480. The research findings stress the importance of dead cell uptake and more comprehensive cell viability screening to validate novel aptamer sequences for diagnostic and therapeutic application.

Application of a real-time cell analysis system in the process development and quantification of Rift Valley fever virus clone 13.

Conventional cell-culture viral quantification methods, namely viral plaque and 50 % tissue culture infective dose assays, are time-consuming, subjective and are not suitable for routine testing. The viral plaque formation assay is the main method utilized for Rift Valley fever virus (RVFV) clone 13 quantification. The RVFV is a mosquito-borne RNA Phlebovirus belonging to the family Bunyaviridae. The virus comprises a single serotype and causes the zoonotic Rift Valley fever disease. The real-time cell analysis (RTCA) system has been developed for the monitoring of cell growth, cell adhesion, cell viability and mortality using electronic impedance technology. In this study, Vero cell growth kinetics and RVFV clone 13 replication kinetics were investigated in a roller bottle and RTCA systems. In roller bottles, Vero cell growth was measured by cell counts through trypan blue staining, whilst impedance expressed as the cell index (CI) was used for Vero growth measurement in the RTCA system. Similar growth patterns were observed in both roller bottle and RTCA systems. Exponential growth phase was observed between 48 and 100 h, followed by a stationary phase from 100 to 120 h, before cell death was observed. Viral plaque assay quantification of RVFV clone 13 in the roller bottle system and the time required for the CI to decrease 50 % after virus infection (CIT50) in the RTCA system were comparable. The highest RVFV clone 13 titre was obtained at 120 h in both roller bottle and RTCA systems. An increase in time for cytopathic effect (CPE) formation was observed with a decrease in the concentration of the virus used to infect the RTCA plates. A positive correlation was observed between the viral concentration and the time for a CPE and was used to calculate CIT50. A similar correlation was observed between the viral concentration and the time for a CPE in the roller bottle system. This study shows that the RTCA system can be used as an alternative method for conducting cell culture kinetics and viral quantification.

A Luminescence Assay to Quantify Cell Viability in Real Time.

Comprehensive understanding of cellular responses to changes in the cellular environment or by drug treatment requires time-dependent analysis ranging from hours to several days. Here, we describe a sensitive, nonlytic live-cell assay that allows continuous or 'real-time' monitoring of cell viability, growth, and cytotoxicity over an extended period of time. We illustrate the use of the assay for small drug molecule and antibody-dependent cytotoxicity studies using cancer cells in 384-well plates. We show that the ability to measure changes in live cells over time provides instantaneous information on the biological status of the cells, information about the mode of action of the drug, and offers an added advantage of preserving the cells for multiplexing with downstream applications.

Development and characterization of a novel human 3D model of bone metastasis from breast carcinoma in vitro cultured.

Breast cancer frequently metastasizes to the skeleton causing significant morbidity. None of the therapeutic strategies used to manage breast cancer bone metastases are really curative. Here, we set-up a novel and advanced model by using fresh tissue from human vertebral bone metastasis from breast carcinoma patients able to retain the tumor microenvironment. The tissue model is based on an ex-vivo culture for up to 40days and on a constant monitoring of tissue viability, gene expression profile (IL10, IL1b, MMP1, MMP7, PTH1R, PTH2R, TNF, ACP5, SPI1, VEGFA, CTSK, TGF-β) and histological and immunohistochemical analyses (CDH1/E-cadherin, CDH2/N-cadherin, KRT8/Cytokeratin 8, KRT18/Cytokeratin 18, Ki67, CASP3/Caspase 3, ESR1/Estrogen Receptor Alpha, CD68 and CD8). Results confirmed the development of a reliable, reproducible and cost-effective advanced model of breast cancer bone metastasis able to preserve and maintain long-term tissue viability, as well as molecular markers, tissue histomorphology, tissue micro-architecture and antigen expression. The study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumor subtypes so predicting patient specific responses.

Reagent-Free Electrochemical Monitoring of Bacterial Viability Using Novel Organic Crystals Deposited on Flexible Substrates

Monitoring bacterial viability is critical in food safety, water quality, and medical applications [1]–[8]. Specifically, having a portable, low-cost, and rapid analytical tool for bacterial viability is highly desirable in academic research, healthcare, and industrial sectors. There are various methods for probing bacterial viability [7], [9], but they usually possess drawbacks in relation to point-of-care/portable application for timely diagnosis. One of the gold standard methods is colony counting on agar plates. Despite being highly sensitive, low-cost, and simple, colony counting is quite slow, requiring at least 18-24 hours incubation [7]. Another method for monitoring viability uses polymerase chain reaction to measure the DNA of cells with normal cell-wall integrity [7]. The procedure is fast (< 1 hour [10]), but unintended biases can be introduced by intact non-viable cells and/or incomplete neutralization of exposed DNA [11]. Moreover, it requires fluorescence measurement equipment and skilled personnel, and uses expensive reagents. There are also colorimetric and fluorescent viability methods that measure change of the redox state of a reagent (such as resazurin) in response to cell metabolic activity [12]–[14]. The drawback of these methods is the need to utilize reagents that may interfere with normal bacterial physiology [14], possible interactions with dissolved compounds, as well as requiring rather bulky/expensive optical equipment for readout.In this work, we have developed low-cost, portable electrochemical sensors for in situ detection of bacterial viability without using any external redox reagent. The sensors enable monitoring bacterial metabolic activity by utilizing novel redox-active organic crystals, termed as RZx, which are deposited using a facile, environmental- and user-friendly process. We had previously demonstrated deposition of RZx on pyrolytic graphite sheets for testing the effect of the antibiotic ampicillin on growth of E. coli [15]. In this , we demonstrate that the process is scalable to deposit RZx on printed electrodes on plastic or paper substrates. Hence, flexible sensors with desirable designs can be developed for detection of various bacterial pathogens. RZx was successfully deposited on laser induced graphene (LIG) as well as screen-printed carbon electrodes on paper. LIG was fabricated directly on polyimide sheets using a 25 W desktop laser-writing system (Universal Laser system, VersaLASER VLS 2.30, 10.6 μm wavelength) by adopting the process reported elsewhere [16]. Laser power (peak power), speed, and pulse per inch (ppi) were optimized to 26%, 29%, and 1000, respectively. A schematic of LIG and RZx synthesis processes are presented in Figure 1a and 1b, respectively. Figure 1c depicts a scanning electron microscope (SEM) image of RZx on LIG; the inset shows a magnified view of an RZx crystal. The high surface coverage of the RZx crystals enhances the faradaic current used in analysis of the sensor signal. The final step in device fabrication is to spin-coat a biocompatible Nafion layer onto the RZx/LIG to minimize drift of the sensor response.Once the sensor fabrication is complete, bacterial suspension with varying cell concentration – confirmed using optical density measurement – is deposited on the sensor surface, followed by performing cyclic voltammetry. A characteristic oxidation peak appears at , as depicted Figure 1d. As cells metabolize, shifts to more positive values. Figure 1e plots the time-resolved differential signal with respect to the initial time for viable and dead E. coli K-12 cells spiked in whole blood. As illustrated, the sensor is able to monitor viability in real time. In addition, we measured the sensor response to different initial E. coli concentrations, as shown in Figure 1f for 106 and 107 CFU/mL. Finally, to demonstrate versatility of the RZx deposition on other carbon-based electrodes, we utilized screen-printed carbon electrodes on paper. The sensors consist of printed counter electrode and AgCl paste as reference electrode (inset of Figure 1g). The data measured using the on-chip paper-based sensors is shown in Figure 1g, demonstrating a similar response to LIG-based sensors in Figure 1e (which were measured using off-chip Pt counter electrode and Ag/AgCl glass reference electrode).The sensors successfully enabled reagent-free electrochemical monitoring of viability of both E. coli and Salmonella as model species. The flexibility, portability, and compactness of the as-fabricated sensors (on LIG or screen-printed carbon electrodes) lends itself to broader applications, such as use in smart-catheters that can detect bacterial infection early on, or portable probes for monitoring food or water quality in the field. Figure 1

Integrated paper-based 3D platform for long-term cell culture and in situ cell viability monitoring of Alzheimer's disease cell model

Reactive oxygen species including superoxide anion, hydrogen peroxide (H2O2) and hydroxyl radicals, as a conflicting class of biological metabolites in living organism, act crucial effect on Alzheimer's disease (AD). In this work, a facile integrated platform composed of a paper-based three-dimension (3D) cell culture system and an electrochemical sensor was developed for the construction of AD cell model in third dimensional structure and in situ cell viability monitoring by H2O2 released from PC12 cells cultured on paper scaffold were divided into three groups containing control group, amyloid beta peptide 25–35 (Aβ25-35) group and Aβ25-35+curcumin (Aβ25-35+cur) group, respectively. In addition, the paper-based 3D platform displayed excellent properties, such as sensitivity, selectivity, reproducibility and stability. The levels of H2O2 expressed in PC12 cells of the three groups were monitored through a paper-based 3D platform. The viability of cells cultured on the 96-well plate was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Results of this paper-based platform are consistent with those of MTT, both displaying improved cell viability and decreased H2O2 production in Aβ25-35+cur group compared to Aβ25-35 group, which indicates that curcumin has effective cytoprotection. The paper-based 3D platform provides a convenient, economic and universal platform for in situ cell activity monitoring by key small molecules released from living cells.

Monitoring of atrial ablation using cyclic variation of integrated backscatter

Treatment of atrial fibrillation (AF) often relies on radiofrequency ablation (RFA) of atrial tissue. However, current treatment is associated with a &amp;gt;20% recurrence rate, in part due to inadequate monitoring of tissue viability during ablation. Previous work has utilized cyclic variation of integrated backscatter (CVIB) as an early indicator of myocardial recovery from ischemia. Our aim was to demonstrate the use of CVIB to distinguish normal and ablated myocardium. An AcuNav 10F catheter was used to collect radiofrequency signals from the posterior wall of the left atrium of patients before and after RFA for AF. The normalized power spectrum was obtained and integrated backscatter (IB) was extracted across two continuous heart cycles to calculate the average CVIB. Data from 14 patients demonstrated a significant difference in the magnitude of the CVIB before and after ablation (9.0 vs 6.0 dB, p &amp;lt; 0.001). However, no significant changes were identified when evaluating ECG-gated IB values before and after ablation: ventricular end-systole (−9.8 vs −8.6, p = 0.24), atrial end-diastole (−8.2 vs −8.6, p = 0.72) or ventricular end-diastole (−7.7 vs −8.9, p = 0.18). CVIB is able to differentiate normal and ablated myocardium and may be useful in monitoring atrial ablations.

Ochratoxin A affects oocyte maturation and subsequent embryo developmental dynamics in the juvenile sheep model

The genotoxic and nephrotoxic mycotoxin Ochratoxin A (OTA) has also been reported to have adverse effects on oocyte maturation and embryo development. Previous studies on the effects of OTA on female fertility have used micromolar concentrations, but no information is available to date on effects in a more relevant nanomolar range. This study used a juvenile sheep model to evaluate the effects of oocyte exposure to low levels of OTA on maturation, fertilization, and embryo development. Further, it was investigated whether different mechanisms of action of OTA could be responsible for varying toxic effects at different levels of exposure. Cumulus-oocyte-complexes (COCs) were exposed to 10 μmol/L–0.1 nmol/L OTA during in vitro maturation and evaluated for cumulus viability, oocyte maturation, and bioenergetic/oxidative status. COCs were subjected to in vitro fertilization, embryo culture, and embryo quality assessment via morphology, viability, bioenergetic/oxidative status, and time-lapse monitoring. At micromolar concentrations, OTA induced cytotoxic effects, by reducing cumulus expansion and oocyte maturation. OTA altered temporospatial dynamics of zygote pronuclear formation and embryo morphokinetics. Blastocysts, even morphologically normal, were found to undergo collapse events, which were probably related to boosted blastocyst mitochondrial activity. At nanomolar concentrations, OTA did not affect COC morpho-functional parameters, but impaired oocyte ability to prevent polyspermy and increased blastocyst apoptosis. In conclusion, in the female germ cell, cytotoxic nonspecific effects characterize OTA-induced toxicity at high exposure levels, whereas fine tuning-mode effects, not associated with altered cell viability and integrity, characterize OTA toxic action at low levels.

Developing improved methods for cost effective viability monitoring of Aeschynomene abyssinica and A. americana

Aeschynomene americana and A. abyssinica are forage legumes that are highly valuable as livestock feed and for soil fertility improvement. Hard-seededness is a form of physical dormancy imposed by a hard seed coat and is a common phenomenon in legume seeds. We assessed dormancy breaking treatments in relation to the time and cost of performing the assessments with the objective of developing improved methods for cost effective viability monitoring in these species. Results show that scarification of seeds with a hot wire, incubating them at 25 or 30°C, and germinating them on top of paper were the most reliable and cost-effective methods for overcoming seed dormancy for viability monitoring in Aeschynomene abyssinica and A. americana. However, the species differed in their response to the dormancy breaking treatments, with no significant differences in A. americana for all the factors except accession, suggesting that efficient dormancy breaking methods in Aeschynomere is species-specific.

A novel mycobacterial growth inhibition assay employing live-cell imaging of virulent M. tuberculosis and monitoring of host cell viability

Our aim was to develop a Mycobacterium tuberculosis (Mtb) growth inhibition assay (MGIA) as a summary estimate of host immune control of virulent Mtb. Mycobacterial growth inhibition (MGI) using previously frozen human PBMCs infected with H37Rv was assessed by live-cell imaging (Incucyte©) complemented by imaging flow cytometry analysis of phagocytosis. MGI measured as relative fluorescence units (RFU) was calibrated to time to positive culture (TTP) in BACTEC 960 MGIT. At a MOI (multiplicity of infection) of 5, there was a wide range of MGI of blood donors (1.1*106–2.7*106 RFU, n = 14). Intra- and inter-assay variability were at most 17.5 and 20.7 CV%. Cell viability at day 5 was 57 and 62% monitored by the LDH and Draq7 assays respectively. There was a strong correlation between a readout for Mtb growth using CFU counts or TTP compared to RFU (r2≥0.96). Our MGIA enabling live-cell imaging and monitoring of cell viability was able to detect a wide range of Mtb growth inhibition by PBMCs and was calibrated to several readout options for bacterial growth. This MGIA may be valuable as a surrogate marker of host immunity in a personalized medicine approach.

Assessment and monitoring of liver graft viability and initial function using interstitial microdialysis

Assessing the viability and monitoring the function of liver graft in the early postoperative period are critical clinical tasks. One possible solution is to determine the changes in concentration of blood glucose, its metabolites and glycerol in the graft using interstitial microdialysis. Objective: to study the dynamics of interstitial glucose, lactate, pyruvate and glycerol in the early post-liver transplant period – depending on the initial graft function (IGF) – and to compare with the results of standard laboratory blood tests. Materials and methods. Four selected clinical observations of deceased donor liver transplantation are presented. Two of the observations showed normal IGF, one observation – early allograft dysfunction (EAD), complicated by hepatic artery thrombosis (HAT), while one observation demonstrated primary non-function (PNF). Collection of microdialysis samples began after arterial reperfusion of the liver graft and continued continuously for 7 days or until death. Standard blood biochemistry and coagulation tests were performed at least once a day. Results. With normal IGF and a smooth postoperative period, interstitial concentrations of glucose, lactate, pyruvate and glycerol remained stable throughout the observation period, ranging from 5 to 20 mmol/L, 1.1 to 7.5 mmol/L, 90 to 380 μmol/L, and 10–100 μmol/L, respectively. EAD was associated with initially higher levels of glucose, lactate, and pyruvate. With HAT development, there was a rapid (within 2–4 hours) five-fold increase in interstitial concentration of lactate with simultaneous decrease in glucose and pyruvate levels to 0.1 mmol/L and 11 μmol/L, respectively. In the case of PNF, there was an initially high concentration of interstitial lactate – 16.4 mmol/L, which increased further to 35.5 mmol/L. Glucose concentration was close to 0. Changes in interstitial glucose, its metabolites and glycerol concentrations chronologically preceded the corresponding changes in peripheral blood composition by 3–5 hours. Conclusion. Microdialysis measurement of interstitial glucose, lactate, pyruvate and glycerol concentrations facilitates real-time monitoring of liver graft viability and function. The high sensitivity of the method could help in accelerating diagnosis of vascular complications (HAT in particular), as well as graft dysfunction with other causes. Therefore, the method is feasible in clinical practice.

A High-Throughput Impedance Sensing System for 3D Cell Drug Permeability Gradient Effect Real-Time Monitoring

3D cells are encapsulated in the thick matreigel, drugs can not penetrate the matrigel evenly, resulting in the gradient effect. The gradient effect would affects the accuracy of the 3D cell-based biosensor response to drug screening. Traditional 3D cell impedance sensors is difficult to measure the gradient of 3D cell drug permeation effectively. In this study, a high-throughput 3D parallel impedance chip has been constructed for real-time drug gradient effect monitoring. Adenocarcinomic human alveolar basal epithelial cell lines (A549) are encapsulated in matrigel to construct 3D lung cancer model. The system consist of 3D parallel impedance chip with 16 vertically aligned gold electrodes, a homemade detecting instrument and a PC software based on LabVIEW. Computational finite element (FE) analysis was used to explore the interactions of adjacent electrodes. In our previous study, cell proliferation or apoptosis in the matrigel would cause variation of cell density, which shown to be inverse proportional to the impedance magnitude of the cell/matrigel entire construct. By using this system, long-term, non-invasive, real-time monitoring of cell proliferation and viability can be achieved. Moreover, after the cell proliferation reached to plateau phase, 3D cell culture system was dosed with different kind of anti-cancer drugs under diverse concentration and the viability of 3D cells at different spatial location sites can be monitored. The experiment results show that drug permeability gradient can be detected by our sensing system. This high-throughput impedance sensing system has a high potential to develop a powerful analytical platform for cancer research and drug screening. Key words: 3D cell culture, 3D parallel impedance chip, permeability gradient, cell proliferation, impedance measurement,drug screening Introduction Conventional 2D cell models have been widely introduced in various researches, but it is too simplified to truly mimic the microenvironment in vivo. Besides, 2D cell model often succumbs to treatment and drugs appear to be very effective. Recently, 3D cancer cell models have attracted increasing interests in the study of drug screening and assessment. Researchers have found that 3D cell model can form a dynamic spatial gradient of soluble factors, which affect cell migration, intercellular communication and differentiation in a more physiological manner. Compared to 2D cell model, 3D cell model are often more resistant to treatment and being better predictors of in vivo drug responses. However, there exist practical problems such as drug release, drug permeation due to the special property of 3D cell model. Traditional single-channel 3D ECIS sensing chips can real-time monitor the whole 3D hydrogel mixed with cells entirely, but have no way to distinguish the spatial distribution of cells at different spatial location sites in larger scaffolds. Confocal fluorescence microscope is used as a gold standard for analysis of 3D cells viability and differentiation. Although confocal microscope provides accurate morphological characterization for 3D cell, it is still a labor-intensive, time-consuming and endpoint analysis method ,fails to meet the high-throughput demand of drug screening. We proposes a sensor to evaluate drug permeability gradient effect in real time. Method The system was constructed for 3D cancer cell culture, assay and drug permeability evaluation. Matrigel incorporated with A549 cells seeds into cell culture plate, cell viability can be monitored by our 3D ECIS chips at the same time. Cell index (CI) is proposed as equation CI=|Zt-Z0|/Z0 to acquire a normalized results, where Z0 is the background impedance, Zt is the measured impedance at a certain moment. Our high-throughput impedance sensing chip consists of 16 pairs of electrodes, which are 0.5×0.5mm square electrodes. A549 cells suspended in cell culture medium(RPMI 1640 with 10% FBS) were mixed in matrigel and Cross-linked in a 37 °C humidified incubator with 5% CO2 for 30 minutes. Drug test conducted by cisplatin,pemetrexed and gemcitabine. Results and Conclusions When an external stimulant electrical signal was imposed to the entire structure, gap junctions of cells electrically connected, causing the increasement of conductivity. As the number of cells in the scaffold increases, the number of gap junction increases, causing a further increase in conductivity of the structure (Fig.1A).The system consists of microcontroller, direct digital synthesizer, amplification and filter module and AD sampling module. 16 vertically aligned pair of electrodes were embedded at the opposite sidewalls of the sensing chip for the on-site impedance measurement(Fig.1B,Fig.1C). The impedance-frequency plot of the 3D cell construct is showed in Fig 1D. At the frequency of 10 to 1000 kHz, the impedance magnitude presents significant difference. Based on hardware cost considerations, we choose 10KHz as the simulation source frequency. Stability test was also executed (Fig.1E). A549 cells were selected as 3D cancer cell model. Fig. 1F&Fig.1G showed the first and sixth day morphology and viability of 3D cultured A549 cells separately.Fig.1H showed the cell growth curves of 16 channels in the density of 10000 cells/well.Fig.1I shows the drug permeability gradient effect. Figure 1

Holographic Imaging of Insect Cell Cultures: Online Non-Invasive Monitoring of Adeno-Associated Virus Production and Cell Concentration

The insect cell-baculovirus vector system has become one of the favorite platforms for the expression of viral vectors for vaccination and gene therapy purposes. As it is a lytic system, it is essential to balance maximum recombinant product expression with harvest time, minimizing product exposure to detrimental proteases. With this purpose, new bioprocess monitoring solutions are needed to accurately estimate culture progression. Herein, we used online digital holographic microscopy (DHM) to monitor bioreactor cultures of Sf9 insect cells. Batches of baculovirus-infected Sf9 cells producing recombinant adeno-associated virus (AAV) and non-infected cells were used to evaluate DHM prediction capabilities for viable cell concentration, culture viability and AAV titer. Over 30 cell-related optical attributes were quantified using DHM, followed by a forward stepwise regression to select the most significant (p &lt; 0.05) parameters for each variable. We then applied multiple linear regression to obtain models which were able to predict culture variables with root mean squared errors (RMSE) of 7 × 105 cells/mL, 3% for cell viability and 2 × 103 AAV/cell for 3-fold cross-validation. Overall, this work shows that DHM can be implemented for online monitoring of Sf9 concentration and viability, also permitting to monitor product titer, namely AAV, or culture progression in lytic systems, making it a valuable tool to support the time of harvest decision and for the establishment of controlled feeding strategies.