Nonviable Cells Research Articles

Optimization design of interdigitated microelectrodes with an insulation layer on the connection tracks to enhance efficiency of assessment of the cell viability

BackgroundMicroelectrical Impedance Spectroscopy (µEIS) is a tiny device that utilizes fluid as a working medium in combination with biological cells to extract various electrical parameters. Dielectric parameters of biological cells are essential parameters that can be extracted using µEIS. µEIS has many advantages, such as portability, disposable sensors, and high-precision results.ResultsThe paper compares different configurations of interdigitated microelectrodes with and without a passivation layer on the cell contact tracks. The influence of the number of electrodes on the enhancement of the extracted impedance for different types of cells was provided and discussed. Different types of cells are experimentally tested, such as viable and non-viable MCF7, along with different buffer solutions. This study confirms the importance of µEIS for in vivo and in vitro applications. An essential application of µEIS is to differentiate between the cells’ sizes based on the measured capacitance, which is indirectly related to the cells’ size. The extracted statistical values reveal the capability and sensitivity of the system to distinguish between two clusters of cells based on viability and size.ConclusionA completely portable and easy-to-use system, including different sensor configurations, was designed, fabricated, and experimentally tested. The system was used to extract the dielectric parameters of the Microbeads and MCF7 cells immersed in different buffer solutions. The high sensitivity of the readout circuit, which enables it to extract the difference between the viable and non-viable cells, was provided and discussed. The proposed system can extract and differentiate between different types of cells based on cells’ sizes; two other polystyrene microbeads with different sizes are tested. Contamination that may happen was avoided using a Microfluidic chamber. The study shows a good match between the experiment and simulation results. The study also shows the optimum number of interdigitated electrodes that can be used to extract the variation in the dielectric parameters of the cells without leakage current or parasitic capacitance.

Inference of transcriptome signatures of Escherichia coli in long-term stationary phase

“Non-growing” is a dominant life form of microorganisms in nature, where available nutrients and resources are limited. In laboratory culture systems, Escherichia coli can survive for years under starvation, denoted as long-term stationary phase, where a small fraction of cells manages to survive by recycling resources released from nonviable cells. Although the physiology by which viable cells in long-term stationary phase adapt to prolonged starvation is of great interest, their genome-wide response has not been fully understood. In this study, we analyzed transcriptional profiles of cells exposed to the supernatant of 30-day long-term stationary phase culture and found that their transcriptome profiles displayed several similar responses to those of cells in the 16-h short-term stationary phase. Nevertheless, our results revealed that cells in long-term stationary phase supernatant exhibit higher expressions of stress-response genes such as phage shock proteins (psp), and lower expressions of growth-related genes such as ribosomal proteins than those in the short-term stationary phase. We confirmed that the mutant lacking the psp operon showed lower survival and growth rate in the long-term stationary phase culture. This study identified transcriptional responses for stress-resistant physiology in the long-term stationary phase environment.

Abstract 5332: Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development

Abstract Cell viability is one of the critical quality attributes (CQAs) for cell and gene therapy products. Characterization of cell viability, in addition to cell identity, cell count and other CQAs, are essential to ensure the quality, safety and efficacy throughout various developmental stages of cell and gene therapies. To determine cell viability, a variety of viability detection methods have been used, such as trypan blue (TB) exclusion assay, and fluorescence-based dyes including acridine orange/propidium iodide (AO/PI), acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI), all of which have been employed on flow or image-based cytometry systems. In this work, we employed the Cellaca MX high-throughput cell counter to characterize and compare AO/PI and AO/DAPI viability detection methods. First, we measured the viabilities of different ratios of viable and non-viable Jurkat cells created via two cell dying processes. Next, viabilities of the stained cells were measured multiple times within a 30 min staining time for each method in order to monitor the trend of viability changes. The viability results showed that the AO/DAPI method were generally higher than AO/PI method with differences ranging from 0% to 24%. We also observed the discrepancy of the measured viability differences between naturally-dying and heat-killed Jurkat cells, suggesting that the viability difference might be dependent on the cell dying process. In the time-dependent viability characterization, results from AO/DAPI method showed increasing dead cell counts leading to decreasing viability, revealing an on-going staining process. In contrast, results from AO/PI method showed more consistent cell count and viability measurements, indicating a more instantaneous staining process. Lastly, we conducted a cell proliferation assay as an orthogonal method to confirm replicability of the low viability cell samples. The proliferation assay results showed no significant increase in cell concentrations within the 4-day time frame. The viability results before the culture were approximately 0% for both naturally-dying and heat-killed Jurkat cells, using the AO/PI staining method, and 12% and 9% using the AO/DAPI respectively. Furthermore, spiked samples of natural-dying and heat-killed cells with healthy cells (>90% viability) were generated and measured, which showed exponential growth. The viability results of these spiked samples before the culture were 11% and 9% by AO/PI and 17% and 18% by AO/DAPI, respectively. Taken together, these results suggest that cell viability methods need to be optimized for different fluorescent stains in order to generate reliable, robust, and fit-for-purpose results for the downstream assays for cell and gene therapy products.. Citation Format: Yongyang Huang, Samir Patel, Mackenzie Pierce, Carolina Franco Nitta, Bo Lin, Leo Chan. Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development. [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 5332.

Brightfield vs Fluorescent Staining Dataset–A Test Bed Image Set for Machine Learning based Virtual Staining

Differential fluorescent staining is an effective tool widely adopted for the visualization, segmentation and quantification of cells and cellular substructures as a part of standard microscopic imaging protocols. Incompatibility of staining agents with viable cells represents major and often inevitable limitations to its applicability in live experiments, requiring extraction of samples at different stages of experiment increasing laboratory costs. Accordingly, development of computerized image analysis methodology capable of segmentation and quantification of cells and cellular substructures from plain monochromatic images obtained by light microscopy without help of any physical markup techniques is of considerable interest. The enclosed set contains human colon adenocarcinoma Caco-2 cells microscopic images obtained under various imaging conditions with different viable vs non-viable cells fractions. Each field of view is provided in a three-fold representation, including phase-contrast microscopy and two differential fluorescent microscopy images with specific markup of viable and non-viable cells, respectively, produced using two different staining schemes, representing a prominent test bed for the validation of image analysis methods.

Insights into the enumeration of mixtures of probiotic bacteria by flow cytometry

The use of flow cytometry to enumerate microorganisms is gaining traction over the traditional plate count technique on the basis of superior accuracy, precision and time-to-result. Here, we assessed the suitability of live/dead flow cytometry for the enumeration of mixed populations of probiotic bacteria (L. acidophilus, L. paracasei, L. plantarum, L. salivarius, B. lactis and B. bifidum) whilst comparing outcomes with plate counting. Using a novel gating strategy designed specifically for the enumeration of mixed populations, the application of flow cytometry resulted in the detection of higher numbers of viable bacteria with a greater level of repeatability than plate counting (RSD of 6.82 and 13.14% respectively). Across all multi-species blends tested, viable cell input was more accurately recovered by flow cytometry (101.8 ± 6.95%) than plate counts (81.37 ± 16.03%). However, when certain probiotic mixtures contained preparations with high numbers of non-viable cells in their total population, flow cytometry had the potential for overestimation of the viable population. Nevertheless, the comparative plate counts of these mixtures were low and variable, thus supporting the use of flow cytometry for the enumeration of viable bacteria in mixed populations.

In vitro detoxification of aflatoxin B1 and ochratoxin A by lactic acid bacteria isolated from Algerian fermented foods

Wheat occupies the first place in Algerian’s diet. However, because of the risk of mycotoxin contamination, its consumption can cause serious health concern to Algerian population. This study aimed to determine the in vitro detoxification properties of lactic acid bacteria (LAB) strains toward aflatoxin B1 (AFB1) and ochratoxin A (OTA), the most prevalent mycotoxins in wheat and derived products in Algeria. Eleven LAB strains isolated from Algerian fermented foods were characterized and identified using API 50 CHL and 16S rDNA sequencing methods. In order to study the ability of LAB strains to remove AFB1 and OTA, viable and heat inactivated cells of two LAB strains (Lab-L4/al and Lab-L1) selected among the eleven isolated and identified ones, as well as of the reference strain Lactobacillus plantarum (LP) R1096 were incubated individually in Citrate Phosphate Buffer (CPB 0.1 M, pH 6 or 5) containing AFB1 and/or OTA at a concentration each of 40 ng/mL for 24 h at 25 °C. Free mycotoxin concentrations were analyzed by HPLC-FLD. The isolated LAB strains were identified as Lactococcus lactis ssp lactis 1, Lactococcus lactis ssp lactis 2 and Lactobacillus paracasei ssp paracasei using API 50 CHL kit, whereas with the molecular method, the strains were identified as Enterococcus faecium and Enterococcus durans. Both viable and nonviable cells of Lab-L4/al and Lab-L1, as well as LP R1096, were able to remove AFB1 and OTA, with efficiency varying between the strains and higher for AFB1 with nonviable cells (>50 %). Removal ability of viable Lab-L4/al and LP R1096 cells increased with a decrease in pH from 6 to 5, while pH had no effect on the amounts of mycotoxins removed by viable Lab-L1 cells. In conclusion, the tested LAB strains were able to reduce the amounts of AFB1 and OTA in vitro conditions. This result suggests that these LAB strains could be used as additives or formative agents to reduce mycotoxin levels in fermented wheat foods such as sourdough bread.

Cytoskeletal orchestration of glucose uptake in Sertoli cell to support efferocytosis of apoptotic germ cells

Efferocytosis of non-viable germ cells by Sertoli cells (SCs) constitutes a sentinel for testis homeostasis, yet how SCs signal for the metabolic and cytoskeletal adaption to this energetically costly process remains unexplored. Spectrin is membrane-associated periodic skeleton assembled into an actin-spectrin-based cytoskeletal structure with an interaction with glucose transporter Glut1. The contribution of spectrin to glucose uptake and efferocytosis is unknown. In this study, we identified a cross-regulation between glucose metabolism and efferocytosis in SCs. Pharmacological or genetic inhibition of glucose uptake or glycolysis compromises efferocytosis activity. We further found that βII-spectrin is a hitherto unappreciated regulator of glucose metabolism and cytoskeletal architecture. βII-spectrin deficiency impairs glucose uptake and lactate production in SCs. Moreover, a defective assembly of cytoskeleton and a loss of blood-testis barrier integrity are also featured by SCs deficient in βII-spectrin. The disruption in glucose metabolism and cytoskeletal organization synergistically lead to a defective efferocytosis. In vivo siRNA-mediated targeting of βII-spectrin in testis causes an obvious morphological aberration in seminiferous epithelium with the presence of exfoliated germ cells and multinucleated giant cells. Importantly, a decrease in expression of αII/βII-spectrin was observed in testes of Adjudin-induced infertility model. By exploring the functional relevance of βII-spectrin to the metabolic and cytoskeletal regulation of efferocytosis, our study proposes a potential link between βII-spectrin deregulation and male infertility.

Revisiting Pyronin Y, A Classical Photostable Xanthene Dye useful for Cost Effective Multimodal Fluorescence Microscopy

In search of cost-effective alternatives for developing high-throughput imaging methods for NETosis, several classical histology dyes were revisited. It was found that the second component of the marketed premix Methyl Green-Pyronin Y (Gurr’s Certistain) has several properties compatible with this aim. Therefore, a simple purification method was developed, and the obtained Pyronin Y fraction, dried and redissolved in a biocompatible medium, was found to be photostable, selectively internalized by non-viable cells, and had spectroscopic features that make it compatible with many common fluorophores for multicolor imaging. Under certain conditions, Pyronin Y has a maximum absorption at 548nm, so it can be excited by 514nm and 561nm Laser lines, with an emission peak at 571nm. Experiments with human polymorphonuclear leukocytes (PMN) enzymatically induced to NETosis confirmed the usefulness of Pyronin Y in detecting early NETotic nuclei. By super-resolution microscopy, the compound enhances the visualization of nuclear subdomains, enabling the reduction of assay duration by detecting morphological alterations indicative of initial NETosis steps. Also, in chemically fixed resting PMN, azurophilic granules were stained by Pyronin Y, suggesting a previously unappreciated nucleic acid-independent staining mechanism. In addition, fluorescence lifetime imaging microscopy (FLIM) followed by time domain phasor analysis demonstrates the utility of the dye in differentiating between NETs released into the extracellular space and the genetic material still remaining inside the cell.

Antibiofilm Activity of 4-(Adamantyl-1)-1-(1-Aminobutyl) Benzol against Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is a widespread opportunistic pathogen, causing community-acquired and nosocomial infections with both acute and chronic recurrent courses. The process of chronicity of the disease is provided by biofilms. Features of the structure and functioning of biofilms, in particular the presence of matrix, quorum sensing systems, persistent cells, and efflux pumps, provide microbial communities with resistance to antimicrobial drugs under their action in therapeutic concentrations. The insufficient eff ectiveness of modern antimicrobial chemotherapy against biofi lm microorganisms indicates the urgency of the problem to search for compounds with antibiofilm activity that can affect various stages of the biofilm formation and the formed biofilm. The aim of the study is to establish the antibiofilm activity of 4-(adamantyl-1)-1-(1-aminobutyl) benzol against methicillin-resistant S. aureus (MRSA) and to determine the mechanism of its action. Methods. The ability of adamantane-containing compound 4-(adamantyl-1)-1-(1-aminobutyl) benzol (AM-166) to prevent biofilm formation and destroy the formed biofilm of S. aureus was investigated on polystyrene plates by the sorption of gentian violet on its structures followed with desorption of the dye into the organic solvent. The viability of S. aureus cells at the first stage of biofilm formation and in the composition of mature biofilms was evaluated using specific dyes for living (acridine orange) and non-viable (propidium iodide) cells. Detection of genes responsible for antibiotic resistance and biofi lm formation was performed by the polymerase chain reaction (PCR) with detection of PCR products in agarose gel. Evaluation of the effect of AM-166 on the expression of genes regulating the biofilm formation (ica, agrA, sarA, and sigB) was investigated by the real-time PCR and semi-quantitative PCR. Results. It was found that the compound AM-166 shows activity against S. aureus biofilm formation. The most pronounced effect was registered at a concentration of 5.0 minimum inhibitory concentration (MIC) (92.3%.) Under the action of AM-166 on the formed 2-day biofilms, their destruction was marked: the biomass decreases by 30.9% at 5.0 MIC. According to the results of fluorescence microscopy, the adamantane derivative at 5.0 MIC helps to reduce the number of viable cells at different stages of formation of the S. aureus biofilm. The results of molecular genetic studies indicate that the ica gene expression is significantly inhibited by the action of subinhibitory concentrations of the compound AM-116. No significant changes in the expression of sarA, agrA, and sigB genes were registered. Conclusions. Experiments on the effect of adamantane derivative on S. aureus biofilms showed that the most pronounced activity of AM-116 was observed at the stage of biofilm formation, as evidenced by the inhibition of transcriptional activity of the ica gene responsible for early stages of the biofilm formation, in particular the adhesion of planktonic cells to the substrate.

Spectroscopic Analysis of Candida Species, Viability, and Antifungal Drug Effects With a Microwave Flow Cytometer

New methods to rapidly detect and identify <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Candida</i> cells in patients’ blood is needed for proper candidemia treatment. In this work, a tunable microwave interferometer was used to measure single <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Candida</i> cells of <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">C. albicans</i> , <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">C. tropicalis</i> , <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">C. parapsilosis</i> , and <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">C. krusei</i> at multiple frequencies between 0.265 GHz and 7.76 GHz. The obtained permittivity values <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Δϵ'</i> and <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Δϵ”</i> of non-budding <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Candida</i> were colinear with cell volumes and had a coefficient that depends on measurement frequency, cell species and viability. Viable and non-viable cells had significant permittivity differences at lower frequency spectrum, but with substantial overlap at 7.76 GHz. For single non-budding cells, measurements at 1.32 GHz and 1.85 GHz enabled a minimum 0.875 classification accuracy with quadratic discriminate analysis (QDA). However, cell budding induced significant microwave property overlaps. Further work is needed to achieve better cell species discrimination. Additionally, it was determined that treating these yeast cells with Caspofungin (CSP) diacetate in dimethyl sulfoxide (DMSO) for 10 minutes significantly altered cell microwave properties. This change was likely due to damaged cell wall and membranes, and the level of change was <italic xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Candida</i> species dependent. Therefore, broadband microwave measurement is a promising new approach for physicians to provide personalized candidemia therapy.

Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study.

Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches.

Review on the current trends and future perspectives of postbiotics for developing healtheir foods

AbstractIt is a known fact that a number of routes explaining the health benefits of beneficial bacterial cells require vitality. However, more recent terms such as the para probiotic and postbiotic have appeared to indicate that, in addition to live cultures, nonviable microbial cells, microbial fractions, or cell lysates also have the ability to benefit the host by boosting bioactivity. According to current scientific research, postbiotics are potentially safer than their parent live cells as alternative agents. Because of their distinct clinical, technological, and economic properties, they can be used as promising tools in the food and drug industries to achieve therapeutic goals and health benefits. The postbiotic theory, proof of their health benefits, probable signaling pathways involved in their protective mechanisms, and their potential use as functional foods are all covered in this review. With the use of comparative studies on past research, it discusses the methodologies utilized to obtain and identify postbiotics. It educates about postbiotics' bioactivity and probable physiological effects. It also instructs of all possible food applications and underlines the processes involved.

Dual-mode visual detection strategies of viable pathogens for point-of-care testing

Here, we introduce a power-free foldable poly(methyl methacrylate) (PMMA) microdevice fully integrating DNA extraction, amplification, and visual detection, realized in novel dual modes – colorimetric and aggregate formation – using 4-Aminoantipyrine (4-AP) for monitoring pathogens. The microdevice contains two parts: reaction and detection zones. A sealing film was utilized to connect the two zones and make the device foldable. The FTA card was deposited in the reaction zone for DNA extraction, followed by loop-mediated isothermal amplification (LAMP) at 65 °C for 45 min. When the detection zone is folded toward the reaction zone, paper discs modified with 4-AP placed in the detection zone are delivered to the reaction zone. Specifically, in the presence of LAMP amplicons, 4-AP is oxidized into antipyrine red or generates aggregates by interacting with copper sulfate, forming copper hybrid nanostructure (Cu-hNs). In the absence of LAMP amplicons, 4-AP is not oxidized and maintains yellow color or fails to form aggregates. Furthermore, we introduced the ethidium homodimer-1 (EthD-1) to identify viable bacteria. EthD-1 penetrated the compromised membranes of nonviable cells and prevented further DNA amplification by intercalating with the DNA. In this way, only samples containing viable cells displayed color change or formed aggregates upon reaction with 4-AP. Using this method, SARS-CoV-2 RNA and Enterococcus faecium were identified by naked eye, with the limit of detection of 103 copies/μL and 102 CFU/mL, respectively, within 60 min. The introduced microdevice can be used for rapidly monitoring viable pathogens and controlling outbreaks of infectious disease in resource-limited settings.

Postbiotics: From emerging concept to application

The microbiome innovation has resulted in an umbrella term, postbiotics, which refers to non-viable microbial cells, metabolic byproducts and their microbial components released after lysis. Postbiotics, modulate immune response, gene expression, inhibit pathogen binding, maintain intestinal barriers, help in controlling carcinogenesis and pathogen infections. Postbiotics have antimicrobial, antioxidant, and immunomodulatory properties with favorable physiological, immunological, neuro-hormonal, regulatory and metabolic reactions. Consumption of postbiotics relieves symptoms of various diseases and viral infections such as SARS-CoV-2. Postbiotics can act as alternatives for pre-probiotic specially in immunosuppressed patients, children and premature neonates. Postbiotics are used to preserve and enhance nutritional properties of food, elimination of biofilms and skin conditioning in cosmetics. Postbiotics have numerous advantages over live bacteria with no risk of bacterial translocation from the gut to blood, acquisition of antibiotic resistance genes. The process of extraction, standardization, transport, and storage of postbiotic is more natural. Bioengineering techniques such as fermentation technology, high pressure etc., may be used for the synthesis of different postbiotics. Safety assessment and quality assurance of postbiotic is important as they may induce stomach discomfort, sepsis and/or toxic shock. Postbiotics are still in their infancy compared to pre- and pro- biotics but future research in this field may contribute to improved physiological functions and host health. The current review comprehensively summarizes new frontiers of research in postbiotics.

Morphophysiological alterations in fruit-eating bats after oral exposure to deltamethrin.

Deltamethrin (DTM) is a synthetic pyrethroid widely used in the cultivation and management of several crops due to its insecticidal action. Application to crops of pyrethroids such as DTM can result in the exposure of water and fruit consumed by fruit bats having a high pyrethroid content which may be harmful. Therefore theobjective of this study was to evaluate the effects of short-term oral exposure of the fruit-eating bats (Artibeus lituratus) to two concentrations of DTM (0.02and 0.04 mg/kg of papaya) on histopathology of the intestine, liver and kidney. The intestine of the animals exposed to both concentrations showed inflammatory infiltrate, degeneration, necrosis and goblet cell hyperplasia as the most frequent pathologies. Besides, the acid mucins showed an increase in the frequency of non-viable cells. The liver showed hepatocyte vacuolizatio and nuclear enlargement, as well as inflammatory infiltrate and steatosis. The kidneys of the exposed animals showed and inflammatory infiltrate, benign nephrosclerosis, vacuolization and necrosis. Also, DTM reduced nitric oxide synthesis, decreased glomerular diameter and increased glycogen percentage in the proximal tubules. Our results suggest that acute exposure to DTM at low concentrations has the potential to induce pronounced histopathological changes in vital organs, such as intestine, liver and kidney of fruit-eating bats.

Binding and removal of polycyclic aromatic hydrocarbons in cold smoked sausage and beef using probiotic strains

The aim of this study is to bio-monitor the levels of 16 polycyclic aromatic hydrocarbons (PAHs) in cold smoked beef and sausages. The ability of probiotics to remove PAHs was also investigated as function of the cell viability (viable, non-viable and acid-treated cells), bacterial counts (107, 108, and 109 CFU/mL), pH (3, 5, and 7), and incubation time (6, 12, and 24 h). The results indicated a significant difference (p < 0.05) among the analyzed sausages and beef samples for the PAHs concentration. Non-viable probiotics achieved the highest PAHs reduction rates. Limosilactobacillus fermentum EMCC 1346 presented the lowest binding activity value (i.e. 41.10–56.80 %) for all PAHs, followed by Lacticaseibacillus rhamnosus EMCC 1105 with binding percentage of 50.40–65.80 %. On the other hand, the highest removal for all PAHs was achieved by Lactobacillus bulgaricus EMCC 1102 with binding rate of 60.50–76.80 %, at 109 CFU/mL, pH 7, after incubation for 24 h. The fortified sausages results revealed that L. bulgaricus EMCC 1102 cultures exhibited the maximum and significant reduction (p < 0.05) of PAHs with values of 44.71 µg/kg for the center part, compared to control non treated sausages (82.65 µg/kg). Regarding the sensorial profile, treated samples with probiotics led to a preference from the panelists, compared to control. Consequently, the results confirm that fermented probiotic suspension is a feasible future strategy to control PAHs levels in cold smoked meat stuffs.

HMSCs in contact with DMSO for cryopreservation: Experiments and modeling of osmotic injury and cytotoxic effect.

In this study a combined analysis of osmotic injury and cytotoxic effect useful for the optimization of the cryopreservation process of a cell suspension is carried out. The case of human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) in contact with dimethyl sulfoxide (DMSO) acting as Cryo‐Protectant Agent (CPA) is investigated from the experimental as well as the theoretical perspective. The experimental runs are conducted by suspending the cells in hypertonic solutions of DMSO at varying osmolality, system temperature, and contact times; then, at room temperature, cells are pelleted by centrifugation and suspended back to isotonic conditions. Eventually, cell count and viability are measured by means of a Coulter counter and flow‐cytometer, respectively. Overall, a decrease in cell count and viability results when DMSO concentration, temperature, and contact time increase. A novel mathematical model is developed and proposed to interpret measured data by dividing the cell population between viable and nonviable cells. The decrease of cell count is ascribed exclusively to the osmotic injury caused by expansion lysis: excessive swelling causes the burst of both viable as well as nonviable cells. On the other hand, the reduction of cell viability is ascribed only to cytotoxicity which gradually transforms viable cells into nonviable ones. A chemical reaction engineering approach is adopted to describe the dynamics of both phenomena: by following the kinetics of two chemical reactions during cell osmosis inside a closed system it is shown that the simultaneous reduction of cell count and viability may be successfully interpreted. The use of the Surface Area Regulation (SAR) model recently proposed by the authors allows one to avoid the setting in advance of fixed cell Osmotic Tolerance Limits (OTLs), as traditionally done in cryopreservation literature to circumvent the mathematical simulation of osmotic injury. Comparisons between experimental data and theoretical simulations are provided: first, a nonlinear regression analysis is performed to evaluate unknown model parameters through a best‐fitting procedure carried out in a sequential fashion; then, the proposed model is validated by full predictions of system behavior measured at operating conditions different from those used during the best‐fit procedure.

Monitoring yeast regulated cell death: trespassing the point of no return to loss of plasma membrane integrity.

Acetic acid and hydrogen peroxide are the most common stimuli to induce apoptosis in yeast. The initial phase of this cell death process is characterized by the maintenance of plasma membrane integrity in cells that had already lost their viability. As loss of plasma membrane integrity is typically assessed by staining with propidium iodide (PI) after exposure of cells to a stimulus and cell viability is determined 48h after plating, the percentage of cells with compromised plasma membrane integrity and c.f.u.counts often do not correlate. Herein, wedeveloped a simple method to explore at what point after an apoptotic stimulus and plating cells do non-viable cells die as result of plasma membrane disruption,i.e., when cells surpassthe point-of-no-return and undergo asecondary necrosis.The method consisted in washing cells and re-suspending them in stimulus-free medium after acetic acid and hydrogen peroxide treatments, to mimic transfer to plating, and then assessing plasma membrane integrity through PI staining. We show that, after the stimuli are removed, cells that had lost proliferative capacity but still maintained plasma membrane integrity continue the cell death process and later lose plasma membrane integrity when progressing to secondary necrosis. After exposure to hydrogen peroxide, cells undergo secondary necrosis preceded by Nhp6Ap-GFP cytosolic localization, in contrast to acetic acid exposure, where Nhp6Ap-GFP cytosolic localization mainly occurs simultaneously with an earlier emergence of secondary necrosis. In conclusion, the developed method allows monitoring the irreversible loss of plasma membrane integrity of dying apoptotic cells after the point-of-no-return is trespassed, and better characterize the process of secondary necrosis after apoptosis.

P-015 Automated, efficient sorting of sperm using dielectrophoresis (DEP), utilizing the electrical properties of the tail as a biomarker of the viability, even for immotile cells

Abstract Study question Developing a simple and automated method and device that sorts semen cell by cell for eventual application to retrieving rare sperm from severely oligozoospermic samples. Summary answer Over 90% of the viable cells in a sample can be sorted out from non-viable sperm cells and other debris using this method. What is known already The varying electrical properties of different cell types can result in unique frequency dependent dielectrophoretic (DEP) behavior when they are exposed to alternating current (AC). They are either repelled or attracted depending on their properties and the field frequency. This has been applied to sorting rare cells such as circulating tumor cells from blood. We have previously shown that the head and tail of human sperm have different electrical properties. As a result, at certain frequencies of an AC electric field the tails of viable cells (even immotile ones) are attracted to electric field gradients while the heads are distanced. Study design, size, duration Semen samples from several patients at Rambam medical center were collected and cryopreserved. Later the thawed semen samples were then tested in our sorting device. At least one hundred live cells were evaluated in each sample. The percentage of live cells that were successfully sorted out of the original mixture of live sperm cells, dead sperm cells and other debris was assessed. Participants/materials, setting, methods After thawing the samples were stained using CFDA and PI for visual live/dead identification (in actual practice no staining is necessary). The device consists of a polydimethylsiloxane (PDMS) microchannel on a glass slide patterned with Indium Tin Oxide (ITO) transparent electrodes. In the device the sperm are transferred to a low conductivity buffer. A curved electrode then sorts them out of the stream of the cell mixture to an alternate exit. Main results and the role of chance We have successfully demonstrated a new biomarker of live sperm (even immotile ones). The tails of live sperm have a positive (attractive) DEP response while dead sperm react negatively and are repelled from field gradients. At the specific frequencies used the head is simultaneously distanced from the electric fields helping to prevent damage to the DNA during the sorting. We also showed that this effect could be used to sort sperm with over 90% efficiency from a sample with dead sperm and other debris. Since each cell passes the electrodes and is automatically sorted it could possibly be used for severely oligozoospermic and cryptozoospermic samples to find sperm and make more sperm available for more discriminatory sperm selection in such samples. Nowadays such samples are searched manually and the minute number of sperm present is often missed leading to a mistaken azoospermic diagnosis and possibly unnecessary surgery. The components are relatively low cost and the chips disposable allowing for wider implementation. Significant progress was also made in preventing sperm from sticking to the substrates using coatings and a vibrational motor. Additionally, a proof of concept parallelization of the electrodes to increase the throughput was also demonstrated. Limitations, reasons for caution The inherent difficulty with microfluidic technologies is low throughput. Further work is needed to effectively parallelize and optimize the system. Effective pre-processing to clean the sample may also help to increase the throughput. Wider implications of the findings This biomarker can potentially be useful in determining an immotile cell’s viability for ICSI. This technology can also be of potential use for testicular tissue samples where there are many other cell types and debris and it is difficult to find rare sperm. Advanced sperm selection can also be incorporated. Trial registration number not applicable

Abstract 186: Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids

Abstract Cancer cells grown in two dimensional (2D) monolayers are a convenient model to use for basic research and in generating lead candidates in the drug discovery process. However, cells grown in 2D cultures lack many of the structural, biological, and functional attributes of solid tumors, which contributes to the high clinical failure rates for biologic and small molecule drugs. Multicellular three-dimensional (3D) tumor spheroids are advanced models which more faithfully mimic the physiological tumor milieu and is more predictive of therapeutic efficacy than traditional monolayer cultures. However, the inability to generate and to analyze large numbers of intact spheroids with a simplified workflow remains a barrier in the adoption of 3D models for a wide range of applications. To overcome these barriers, we used the Velocyt® large particle flow cytometer (Bennubio) to evaluate spheroid health in a well-established small molecule spheroid killing assay. The Velocyt technology rapidly and gently analyzes particles from 3 to 300 microns and comes with a suite of novel informatics tools purpose built for large particle analysis. To exemplify the use of large particle flow cytometry for the analysis of intact spheroids we generated uniformed-sized spheroids (~180 microns) from the H1650 human lung and HCT116 human colon cancer cell lines using microwell technology. Two to three days after spheroid generation, the spheroids were treated with increasing concentrations of staurosporine and other compounds. Spheroids were harvested at one to three days post treatment (depending on drug treatment) and stained with fluorescent cell viability and apoptotic dyes. Intact spheroids were analyzed for fluorescence and size using the Velocyt flow cytometer and between one and two thousand spheroids were assessed for each treatment in approximately three minutes. In untreated control spheroids, fluorescence was detected in a subpopulation of spheroids consistent with expected low levels of background apoptosis. In treated spheroids, an increase in the mean fluorescence intensity (MFI) of each particle indicated an increase in the number of non-viable and apoptotic cells in each spheroid. MFI increased nearly 6-fold with high concentrations of staurosporine compared to control spheroids, and dead cells were detected in nearly 100% of spheroids that were administered the highest staurosporine concentration. Using the size-based measurement parameters of the Velocyt, we also showed a decrease in overall spheroid size concurrent with increased numbers of spheroid fragments and single cells with increasing drug concentration. These data highlight that the use of 3D cell models combined with the Velocyt large particle flow cytometer is a powerful tool to help advance basic and translational cancer research. Citation Format: John P. O'Rourke, Mathew Saunders, Valerie Sanchez. Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids [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 186.