Exploring operational boundaries for acoustic concentration of cell suspensions.

The development of a standardized, generic method for concentrating suspensions in continuous flow is challenging. In this study, we developed and tested a device capable of concentrating suspensions with an already high cell concentration to meet diverse industrial requirements. To address typical multitasking needs, we concentrated suspensions with high solid content under a variety of conditions. Cells from Saccharomyces cerevisiae, Escherichia coli, and Chinese hamster ovary cells were effectively focused in the center of the main channel of a microfluidic device using acoustophoresis. The main channel bifurcates into three outlets, allowing cells to exit through the central outlet, while the liquid evenly exits through all outlets. Consequently, the treatment separates cells from two-thirds of the surrounding liquid. We investigated the complex interactions between parameters. Increasing the channel depth results in a decrease in process efficiency, attributed to a decline in acoustic energy density. The study also revealed that different cell strains exhibit distinct acoustic contrast factors, originating from differences in dimensions, compressibility, and density values. Finally, a combination of high solid content and flow rate leads to an increase in diffusion through a phenomenon known as shear-induced diffusion. KEY POINTS: • Acoustic focusing in a microchannel was used to concentrate cell suspensions • The parameters influencing focusing at high concentrations were studied • Three different cell strains were successfully concentrated.

Optimum quorum quenching bacteria concentration in the better-quality cell entrapping beads to control biofouling in membrane bioreactor.

Quorum quenching (QQ) by cell entrapping beads (CEBs) is known to inhibit biofouling by its biological and physical cleaning effect. Although there are better QQ media reported, due to the ease of fabrication of QQ-CEBs, this study focused on improving the quality of CEBs by comparing two distinct bead-making methods - polyvinyl alcohol-alginate (PVA-alginate) and phase inversion - and on finding the optimum concentration of QQ bacteria in the CEBs. The evaluation of PVA-alginate bead showed better uniformity, and higher mechanical and chemical strength in comparison with the phase inversion bead. Through the operations of two control membrane bioreactors (MBRs) (no bead, vacant bead) and four QQ-MBRs with different Rhodococcus sp. BH4 concentrations (2.5-15 mg cell ml-1) in PVA-alginate CEBs, the maximum QQ effect was observed by 5 mg ml-1 BH4 concentration beads. This implies that an optimum cell concentration of QQ-CEBs is crucial to economically improve MBR performance using QQ.

Projection Micro-Stereolithography to Manufacture a Biocompatible Micro-Optofluidic Device for Cell Concentration Monitoring.

In this work, a 3D printed biocompatible micro-optofluidic (MoF) device for two-phase flow monitoring is presented. Both an air-water bi-phase flow and a two-phase mixture composed of micrometric cells suspended on a liquid solution were successfully controlled and monitored through its use. To manufacture the MoF device, a highly innovative microprecision 3D printing technique was used named Projection Microstereolithography (PµSL) in combination with the use of a novel 3D printable photocurable resin suitable for biological and biomedical applications. The concentration monitoring of biological fluids relies on the absorption phenomenon. More precisely, the nature of the transmission of the light strictly depends on the cell concentration: the higher the cell concentration, the lower the optical acquired signal. To achieve this, the microfluidic T-junction device was designed with two micrometric slots for the optical fibers' insertion, needed to acquire the light signal. In fact, both the micro-optical and the microfluidic components were integrated within the developed device. To assess the suitability of the selected biocompatible transparent resin for optical detection relying on the selected working principle (absorption phenomenon), a comparison between a two-phase flow process detected inside a previously fully characterized micro-optofluidic device made of a nonbiocompatible high-performance resin (HTL resin) and the same made of the biocompatible one (BIO resin) was carried out. In this way, it was possible to highlight the main differences between the two different resin grades, which were further justified with proper chemical analysis of the used resins and their hydrophilic/hydrophobic nature via static water contact angle measurements. A wide experimental campaign was performed for the biocompatible device manufactured through the PµSL technique in different operative conditions, i.e., different concentrations of eukaryotic yeast cells of Saccharomyces cerevisiae (with a diameter of 5 µm) suspended on a PBS (phosphate-buffered saline) solution. The performed analyses revealed that the selected photocurable transparent biocompatible resin for the manufactured device can be used for cell concentration monitoring by using ad hoc 3D printed micro-optofluidic devices. In fact, by means of an optical detection system and using the optimized operating conditions, i.e., the optimal values of the flow rate FR=0.1 mL/min and laser input power P∈{1,3} mW, we were able to discriminate between biological fluids with different concentrations of suspended cells with a robust working ability R2=0.9874 and Radj2=0.9811.

Non-invasive detection of the content of white blood cells in the blood of humans based on dynamic spectrum.

Objective. Changes in white blood cell content have been shown to be useful in determining whether the body is in a healthy state. We propose an improved data processing and modeling approach, which helps to accommodate blood component content detection and improve prediction accuracy.Approach. In this experiment, the finger-end transmission method was used for spectral measurement, and we collected a total of 440 sample data. In this paper, we first use the method of CEEMDAN combined with wavelet threshold to denoise the PPG signal, and then use the integral method to extract the spectral features, which makes up for the defects of the single-edge method using incomplete data and the deviation of the slope of the rising segment from the actual signal. We further improve the screening of samples and wavelengths, and used PLS regression modeling combine the double nonlinear correction method to build the most stable and universal model.Main results. The model has been applied to 332 subjects' finger transmission spectral data to predict the concentration of leukocytes. The correlation coefficient of the final training set result was 0.927, and the root mean square error (RMSE) is 0.569×109l-1, the correlation coefficient of the prediction set result is 0.817, and the RMSE is 0.826×109l-1, which proves the practicability of the proposed method.Significance. We propose a non-invasive method for detecting leukocyte concentration in blood that can also be generalized to detect other blood components.

SpinTip: A Simple, Robust, and Versatile Preanalytical Method for Microscale Suspension Cell Proteomics.

Sample loss and contamination are critical preanalytical pitfalls in microscale proteomic applications of nonadhering cells. Common assays and workflows are not easily adoptable to microscale sample sizes of suspension cells due to inadvertent sample loss. This impedes preanalytical experimental manipulation of limited suspension cell samples for microscale proteomics applications, such as encountered for primary human materials. Here, we describe and test a simple manual batch technique for single-step 100-fold concentration of scarce numbers of diluted suspension cells (down to 5000 cells) by volume reduction, facilitating microscale experiments with suspension cells. Pipette tips with heat-sealed orifices (SpinTips) are manufactured within 1 min and serve as versatile microcentrifugation vessels from which supernatant can be aspirated with minimal cell loss. A residual volume of approximately 3 µL can be achieved without visualization of the cell pellet. The results show that SpinTips enable the concentration, medium exchange, washing, and culture of highly limited amounts of suspension cells for functional manipulation and microscale proteomics and are readily incorporated into standard workflows. The application is illustrated by profiling ex vivo responses of primary acute myeloid leukemia (AML) cells from one AML patient to daunorubicin (DNR) to a depth of 3462 quantified proteins with excellent repeatability.

Non-invasive real-time monitoring of cell concentration and viability using Doppler ultrasound.

Bioprocess optimization towards higher productivity and better quality control relies on real-time process monitoring tools to measure process and culture parameters. Cell concentration and viability are among the most important parameters to be monitored during bioreactor operations that are typically determined using optical methods on an extracted sample. In this paper, we have developed an online non-invasive sensor to measure cell concentration and viability based on Doppler ultrasound. An ultrasound transducer is mounted outside the bioreactor vessel and emits a high frequency tone burst (15 MHz) through the vessel wall. Acoustic backscatter from cells in the bioreactor depends on cell concentration and viability. The backscattered signal is collected through the same transducer and analyzed using multivariate data analysis (MVDA) to characterize and predict the cell culture properties. We have developed accurate MVDA models to predict the Chinese hamster ovary (CHO) cell concentration in a broad range from 0.1 × 106 cells/mL to 100 × 106 cells/mL, and cell viability from 3% to 99%. The non-invasive monitoring is ideal for single use bioreactor and the in-situ measurements removes the burden for offline sampling and dilution steps. This method can be similarly applied to other suspension cell culture modalities.

Cell-laden bioink circulation-assisted inkjet-based bioprinting to mitigate cell sedimentation and aggregation.

Three-dimensional (3D) bioprinting precisely deposits picolitre bioink to fabricate functional tissues and organs in a layer-by-layer manner. The bioink used for 3D bioprinting incorporates living cells. During printing, cells suspended in the bioink sediment to form cell aggregates through cell-cell interaction. The formation of cell aggregates due to cell sedimentation have been widely recognized as a significant challenge to affect the printing reliability and quality. This study has incorporated the active circulation into the bioink reservoir to mitigate cell sedimentation and aggregation. Force and velocity analysis were performed, and a circulation model has been proposed based on iteration algorithm with the time step for each divided region. It has been found that (a) the comparison of the cell sedimentation and aggregation with and without the active bioink circulation has demonstrated high effectiveness of active circulation to mitigate cell sedimentation and aggregation for the bioink with both a low cell concentration of 1 × 106cells ml-1and a high cell concentration of 5 × 106cells ml-1; and (b) the effect of circulation flow rate on cell sedimentation and aggregation has been investigated, showing that large flow rate results in slow increments in effectiveness. Besides, the predicted mitigation effectiveness percentages on cell sedimentation by the circulation model generally agrees well with the experimental results. In addition, the cell viability assessment at the recommended maximum flow rate of 0.5 ml min-1has demonstrated negligible cell damage due to the circulation. The proposed active circulation approach is an effective and efficient approach with superior performance in mitigating cell sedimentation and aggregation, and the resulting knowledge is easily applicable to other 3D bioprinting techniques significantly improving printing reliability and quality in 3D bioprinting.

Regrowth potential of chlorine-resistant bacteria in drinking water under chloramination.

The regrowth of chlorine-resistant bacteria in drinking water can deteriorate water quality. The study evaluated the relationship between organic carbon and the regrowth potential of chlorine-resistant bacteria remaining in chloraminated water samples. The results showed that the community structure of bacteria changed with the increase of chloramine dosage. The order in which organic carbon utilized by bacteria was affected by the composition of bacterial community. The biodegradable dissolved organic carbon (BDOC), assimilable organic carbon (AOC), bacterial regrowth potential (BRP) and total cell concentration (TCC) in cultivated water sample after disinfection with 1.8 mg/L chloramine increased form 0.22 mg/L, 33.68 µg/L, 2.70 × 105 cells/mL and 3.48 × 104 cells/mL before cultivation to 1.20 mg/L, 193.90 µg/L, 4.74 × 105 cells/mL and 1.46 × 105 cells/mL, respectively. The increase of TCC did not result in the decrease of BDOC, AOC and BRP in the cultivated water samples. The results showed that other biodegradable organic carbon in chloraminated water samples assimilated by residual chlorine-resistant bacteria besides AOC, BDOC, and organic carbon assimilated by indigenous bacteria. AOC, BDOC, and BRP indicators used to characterize the biostability of drinking water were not enough to accurately assess the regrowth potential of chlorine-resistant bacteria remaining in drinking water. It is suggested to supplement the index of TCC in cultivated water samples, which might be able to more accurately evaluate the regrowth potential of chlorine-resistant bacteria remaining in drinking water.

Distinctive correlations between cell concentration and cell size to microalgae biomass under increasing carbon dioxide.

Carbon capture and storage (CCS) via microalgae cultivations is getting renewed interest as climate change mitigation effort, owing to its excellent photosynthetic and CO2 fixation capability. Microalgae growth is monitored based on their biomass, cell concentrations and cell sizes. The key parametric relationships on microalgae growth under CO2 are absent in previous studies and this inadequacy hampers the design and scale-up of microalgae-based CCS. In this study, three representative microalgae species, Chlorella, Nostoc and Chlamydomonas, were investigated for establishing key correlations of cell concentrations and sizes towards their biomass fluctuations under CO2 influences of 0% to 20% volume ratios (v/v). This revealed that Chlorella and Chlamydomonas cell concentrations significantly contributed towards increasing biomass concentration under CO2 elevations. Chlorella and Nostoc cell sizes were enhanced at 20% (v/v). These findings provided new perspectives on growth responses under increasing CO2 treatment, opening new avenues on CCS schemes engineering designs and biochemical production.

A Micro-Scale Non-Linear Finite Element Model to Optimize the Mechanical Behavior of Bioprinted Constructs.

Extrusion-based bioprinting is an enabling biofabrication technique that is used to create heterogeneous tissue constructs according to patient-specific geometries and compositions. The optimization of bioinks as per requirements for specific tissue applications is an essential exercise in ensuring clinical translation of the bioprinting technologies. Most notably, optimum hydrogel polymer concentrations are required to ensure adequate mechanical properties of bioprinted constructs without causing significant shear stresses on cells. However, experimental iterations are often tedious for optimizing the bioink properties. In this work, a nonlinear finite element modeling approach has been undertaken to determine the effect of different bioink parameters such as composition, concentration on the range of stresses being experienced by the cells in the bioprinted construct. The stress distribution of the cells at different parts of the constructs has also been modeled. It is found that both bioink chemical compositions and concentrations can substantially alter the stress effects experienced by the cells. Concentrated regions of softer cells near pore regions were found to increase stress concentrations by almost three times compared with stress generated in cells away from the pores. The study provides a method for rapid optimization of bioinks, design of bioprinted constructs, as well as toolpath plans for fabricating constructs with homogenous properties.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples.

BACKGROUND: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. METHODS: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. RESULTS: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. CONCLUSION: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Circulating tumor cells can predict the prognosis of patients with non-small cell lung cancer after resection: a retrospective study.

BACKGROUND: The development of metastasis is the primary cause of death in patients with non-small cell lung cancer (NSCLC). However, identifying those NSCLC patients who will have loco-regional or distant disease recurrence after surgery is still challenging. Circulating tumor cells (CTCs) can accurately reflect the impact of micro-metastasis of tumor cells in circulating blood on patients' treatment and prognosis. The aim of the present study was to explore the value of preoperative CTC concentration in predicting postoperative metastasis and recurrence risk in patients with NSCLC. METHODS: This study enrolled 347 patients with stage I-IIIA NSCLC. The CTCs were isolated using folate receptor (FR) positivity from peripheral blood samples before surgery, and then enriched and analyzed. Patients were divided into two groups for retrospective survival analysis based on the geometric mean of CTC concentration. The primary study endpoint was recurrence-free survival. Spearman's correlation was used to evaluate the relationship between CTC concentration and clinical characteristics of NSCLC patients. A nomogram based on the multivariate Cox regression model was developed to predict recurrence and metastasis in the NSCLC patients. The performance of the nomogram was evaluated using the concordance index, calibration curve, and Hosmer-Lemeshow test. RESULTS: The median follow-up time was 38 months. Preoperative CTC concentration was not significantly related to tumor-node-metastasis staging (P>0.05) and was an independent prognostic factor for NSCLC patients [hazard ratio (HR), 5.489; 95% confidence interval (CI): 2.660-11.326, P<0.001]. The nomogram based on preoperative CTC concentration had a concordance index value of 0.82. Validation revealed that the nomogram possessed excellent predictive ability and calibration. CONCLUSIONS: Preoperative CTC concentration is an independent and sensitive biomarker of prognosis in patients with NSCLC. Our nomogram based on preoperative CTC concentration is an effective and non-invasive tool for predicting the recurrence and metastasis of NSCLC.

Miniature auto-perfusion bioreactor system with spiral microfluidic cell retention device.

Medium perfusion is critical in maintaining high cell concentration in cultures. The conventional membrane filtration method for medium exchange has been challenged by the fouling and clogging of the membrane filters in long-term cultures. In this study, we present a miniature auto-perfusion system that can be operated inside a common-size laboratory incubator. The system is equipped with a spiral microfluidic chip for cell retention to replace conventional membrane filters, which fundamentally overcomes the clogging and fouling problem. We showed that the system supported continuous perfusion culture of Chinese hamster ovary (CHO) cells in suspension up to 14 days without cell retention chip replacement. Compared to daily manual medium change, 25% higher CHO cell concentration can be maintained at an average auto-perfusion rate of 196 ml/day in spinner flask at 70 ml working volume (2.8 VVD). The auto-perfusion system also resulted in better cell quality at high concentrations, in terms of higher viability, more uniform and regular morphology, and fewer aggregates. We also demonstrated the potential application of the system for culturing mesenchymal stem cells on microcarriers. This miniature auto-perfusion system provides an excellent solution to maintain cell-favorable conditions and high cell concentration in small-scale cultures for research and clinical uses.

Hand-Powered Inertial Microfluidic Syringe-Tip Centrifuge.

Conventional sample preparation techniques require bulky and expensive instruments and are not compatible with next-generation point-of-care diagnostic testing. Here, we report a manually operated syringe-tip inertial microfluidic centrifuge (named i-centrifuge) for high-flow-rate (up to 16 mL/min) cell concentration and experimentally demonstrate its working mechanism and performance. Low-cost polymer films and double-sided tape were used through a rapid nonclean-room process of laser cutting and lamination bonding to construct the key components of the i-centrifuge, which consists of a syringe-tip flow stabilizer and a four-channel paralleled inertial microfluidic concentrator. The unstable liquid flow generated by the manual syringe was regulated and stabilized with the flow stabilizer to power inertial focusing in a four-channel paralleled concentrator. Finally, we successfully used our i-centrifuge for manually operated cell concentration. This i-centrifuge offers the advantages of low device cost, simple hand-powered operation, high-flow-rate processing, and portable device volume. Therefore, it holds potential as a low-cost, portable sample preparation tool for point-of-care diagnostic testing.

No Effect of Microgravity and Simulated Mars Gravity on Final Bacterial Cell Concentrations on the International Space Station: Applications to Space Bioproduction.

Microorganisms perform countless tasks on Earth and they are expected to be essential for human space exploration. Despite the interest in the responses of bacteria to space conditions, the findings on the effects of microgravity have been contradictory, while the effects of Martian gravity are nearly unknown. We performed the ESA BioRock experiment on the International Space Station to study microbe-mineral interactions in microgravity, simulated Mars gravity and simulated Earth gravity, as well as in ground gravity controls, with three bacterial species: Sphingomonas desiccabilis, Bacillus subtilis, and Cupriavidus metallidurans. To our knowledge, this was the first experiment to study simulated Martian gravity on bacteria using a space platform. Here, we tested the hypothesis that different gravity regimens can influence the final cell concentrations achieved after a multi-week period in space. Despite the different sedimentation rates predicted, we found no significant differences in final cell counts and optical densities between the three gravity regimens on the ISS. This suggests that possible gravity-related effects on bacterial growth were overcome by the end of the experiment. The results indicate that microbial-supported bioproduction and life support systems can be effectively performed in space (e.g., Mars), as on Earth.

Development of Digital Image Processing as an Innovative Method for Activated Sludge Biomass Quantification.

Activated sludge process is the most common method for biological treatment of industrial and municipal wastewater. One of the most important parameters in performance of activated sludge systems is quantitative monitoring of biomass to keep the cell concentration in an optimum range. In this study, a novel method for activated sludge quantification based on image processing and RGB analysis is proposed. According to the results, the intensity of blue color in the macroscopic image of activated sludge culture can be a very accurate index for cell concentration measurement and R2 coefficient, Root Mean Square Error (RMSE), Mean Absolute Error (MAE), and Mean Absolute Percentage Error (MAPE) which are 0.990, 2.000, 0.323, and 13.848, respectively, prove this claim. Besides, in order to avoid the difficulties of working in the three-parameter space of RGB, converting to grayscale space has been applied which can estimate cell concentration with R 2 = 0.99. Ultimately, an exponential correlation between RGB values and cell concentrations in lower amounts of biomass has been proposed based on Beer-Lambert law which can estimate activated sludge biomass concentration with R 2 = 0.97 based on B index.

Assessment of oxygenation with polarized light spectroscopy enables new means for detecting vascular events in the skin.

INTRODUCTION: Impaired oxygenation in the skin may occur in disease states and after reconstructive surgery. We used tissue viability imaging (TiVi) to measure changes in oxygenation and deoxygenation of haemoglobin in an in vitro model and in the dermal microcirculation of healthy individuals. MATERIALS AND METHODS: Oxygenation was measured in human whole blood with different levels of oxygenation. In healthy subjects, changes in red blood cell concentration (CRBC,TiVi), oxygenation (ΔCOH,TiVi) and deoxygenation (ΔCDOH,TiVi) of haemoglobin were measured during and after arterial and venous occlusion using TiVi and were compared with measurements from the enhanced perfusion and oxygen saturation system (EPOS). RESULTS: During arterial occlusion, CRBC,TiVi remained unchanged while ΔCOH,TiVi decreased to -44.2 (10.4) AU (p = 0.04), as compared to baseline. After release, CRBC,TiVi increased to 39.2 (18.8) AU (p < 0.001), ΔCOH,TiVi increased to 38.5. During venous occlusion, CRBC,TiVi increased to 28.9 (11.2) AU (p < 0.001), ΔCOH,TiVi decreased to -52.2 (46.1) AU (p < 0.001) compared to baseline after 5 min of venous occlusion. There was a significant correlation between the TiVi Oxygen Mapper and EPOS, for arterial (r = 0.92, p < 0.001) and venous occlusion (r = 0.87, p < 0.001), respectively. CONCLUSION: This study shows that TiVi can measure trends in oxygenation and deoxygenation of haemoglobin during arterial and venous stasis in healthy individuals.

Daily Preventive Zinc Supplementation Decreases Lymphocyte and Eosinophil Concentrations in Rural Laotian Children from Communities with a High Prevalence of Zinc Deficiency: Results of a Randomized Controlled Trial.

BACKGROUND: Zinc deficiency impairs immune function and is common among children in South-East Asia. OBJECTIVES: The effect of zinc supplementation on immune function in young Laotian children was investigated. METHODS: Children (n = 512) aged 6-23 mo received daily preventive zinc tablets (PZ; 7 mg Zn/d), daily multiple micronutrient powder (MNP; 10 mg Zn/d, 6 mg Fe/d, plus 13 other micronutrients), therapeutic dispersible zinc tablets only in association with diarrhea episodes (TZ; 20 mg Zn/d for 10 d after an episode), or daily placebo powder (control). These interventions continued for 9 mo. Cytokine production from whole blood cultures, the concentrations of T-cell populations, and a complete blood count with differential leukocyte count were measured at baseline and endline. Endline means were compared via ANCOVA, controlling for the baseline value of the outcome, child age and sex, district, month of enrollment, and baseline zinc status (below, or above or equal to, the median plasma zinc concentration). RESULTS: T-cell cytokines (IL-2, IFN-γ, IL-13, IL-17), LPS-stimulated cytokines (IL-1ß, IL-6, TNF-α, and IL-10), and T-cell concentrations at endline did not differ between intervention groups, nor was there an interaction with baseline zinc status. However, mean ± SE endline lymphocyte concentrations were significantly lower in the PZ than in the control group (5018 ± 158 compared with 5640 ± 160 cells/µL, P = 0.032). Interactions with baseline zinc status were seen for eosinophils (Pixn = 0.0036), basophils (Pixn = 0.023), and monocytes (P = 0.086) but a significant subgroup difference was seen only for eosinophils, where concentrations were significantly lower in the PZ than in the control group among children with baseline plasma zinc concentrations below the overall median (524 ± 44 compared with 600 ± 41 cells/µL, P = 0.012). CONCLUSIONS: Zinc supplementation of rural Laotian children had no effect on cytokines or T-cell concentrations, although zinc supplementation affected lymphocyte and eosinophil concentrations. These cell subsets may be useful as indicators of response to zinc supplementation.This trial was registered at clinicaltrials.gov as NCT02428647.

Magnetic ionic liquids: interactions with bacterial cells, behavior in aqueous suspension, and broader applications.

Previously, we demonstrated capture and concentration of Salmonella enterica subspecies enterica ser. Typhimurium using magnetic ionic liquids (MILs), followed by rapid isothermal detection of captured cells via recombinase polymerase amplification (RPA). Here, we report work intended to explore the broader potential of MILs as novel pre-analytical capture reagents in food safety and related applications. Specifically, we evaluated the capacity of the ([P66614+][Ni(hfacac)3-]) ("Ni(II)") MIL to bind a wider range of human pathogens using a panel of Salmonella and Escherichia coli O157:H7 isolates, including a "deep rough" strain of S. Minnesota. We extended this exploration further to include other members of the family Enterobacteriaceae of food safety and clinical or agricultural significance. Both the Ni(II) MIL and the ([P66614+][Dy(hfacac)4-]) ("Dy(III)") MIL were evaluated for their effects on cell viability and structure-function relationships behind observed antimicrobial activities of the Dy(III) MIL were determined. Next, we used flow imaging microscopy (FIM) of Ni(II) MIL dispersions made in model liquid media to examine the impact of increasing ionic complexity on MIL droplet properties as a first step towards understanding the impact of suspension medium properties on MIL dispersion behavior. Finally, we used FIM to examine interactions between the Ni(II) MIL and Serratia marcescens, providing insights into how the MIL may act to capture and concentrate Gram-negative bacteria in aqueous samples, including food suspensions. Together, our results provide further characterization of bacteria-MIL interactions and support the broader utility of the Ni(II) MIL as a cell-friendly capture reagent for sample preparation prior to cultural or molecular analyses. Graphical abstract.

Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.

BACKGROUND AIMS: In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards. METHODS: Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures. RESULTS: Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells. CONCLUSIONS: The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.