Density Gradient Centrifugation Research Articles

Dual targeting negative enrichment strategy for highly sensitive and purity detection of CTCs

Circulating tumor cells (CTCs) have significant clinical value in early tumor detection, dynamic monitoring and immunotherapy. CTC detection stands out as a leading non-invasive approach for tumor diagnostics and therapeutics. However, the high heterogeneity of CTCs and the occurrence of epithelial-mesenchymal transition (EMT) during metastasis pose challenges to methods relying on EpCAM-positive enrichment. To address these limitations, a method based on negative enrichment of CTCs using specific leukocyte targets has been developed. In this study, aiming to overcome the low purity associated with immunomagnetic beads targeting solely the leukocyte common antigen CD45, we introduced CD66b-modified immunomagnetic beads. CD66b, a specific target for neutrophils with abundant residues, was chosen as a complementary approach. The process involved initial collection of nucleated cells from whole blood samples using density gradient centrifugation. Subsequently, magnetically labeled leukocytes were removed by magnetic field, enabling the capture of CTCs with higher sensitivity and purity while retaining their activity. Finally, we selected 20 clinical blood samples from patients with various cancers to validate the effectiveness of this strategy, providing a new generalized tool for the clinical detection of CTCs.

In vitro investigation of the mechanics of fixed red blood cells based on optical trap micromanipulation and image analysis

Erythrocyte deformability correlates with various diseases. Single-cell measurements via optical tweezers (OTs) enable quantitative exploration but may encounter inaccuracies due to erythrocyte life cycle mixing. We present a three-step methodology to address these challenges. Firstly, density gradient centrifugation minimizes erythrocyte variations. Secondly, OTs measure membrane shear force across layers. Thirdly, MATLAB analyzes dynamic cell areas. Results combined with membrane shear force data reveal erythrocyte deformational capacity. To further characterize the deformability of diseased erythrocytes, the experiments used glutaraldehyde-fixed erythrocytes to simulate diseased cells. OTs detect increased shear modulus, while image recognition indicates decreased deformation. The integration of OTs and image recognition presents a comprehensive approach to deformation analysis, introducing novel ideas and methodologies for investigating erythrocytic lesions.

The effect of temperature shock on enhancing sperm motility: A new insight into molecular and cellular mechanisms through current procedures

Asthenozoospermia, characterized by reduced progressive motility (below 32%), is a leading cause of male infertility. Intrauterine Insemination (IUI) serves as the first-line treatment in sperm-related processes, aiming to increase sperm motility. Although Density Gradient Centrifugation (DGC) and swim-up procedures have become primary treatment options, considered the gold standard for IUI, there is a need to optimize these methods with additional factors to enhance motility, particularly for Asthenozoospermia patients.We hypothesize that a brief cold shock, lasting approximately 15–20 min at 17 °C, followed by incubation and sperm processing, can substantially enhance motility. For the first time, the hypothesis and evaluation of post-exposure incubation following a short-term cold shock, within the temperature range of 17–20 °C, were explored and discussed.This hypothesis appears valid and holds importance for several reasons. Firstly, there is a significant relationship between cold shock and its impact on intracellular components and the molecular organization of the cell membrane. Additionally, this process regulates various physiological cellular processes, including transbilayer distribution, acrosome reaction, and sperm motility. Moreover, lowering the temperature induces a molecular flux, facilitating calcium uptake by sperm. This process significantly impacts the quantity of calcium accessible to the sperm, particularly within the flagellum. Choosing the temperature below 20 °C temperature can lead to the activation of calcium-absorbing protein channels in the shortest possible time. Furthermore, a crucial reaction in IUI is sperm capacitation, which occurs when the protein tyrosine phosphorylation pathway, a key factor, is activated. Alternatively, the cold incubation of sperm reveals an increased level of tyrosine phosphorylation and a higher percentage of induced Acrosome Reaction (AR) compared to sperm incubated at normal temperatures. Hence, when sperm is cooled, it initiates a process called tyrosine phosphorylation, ultimately leading to sperm capacitation. Besides these logical reasons, there is limited empirical and incidental support in two cases that confirmed the hypothesis well

A Comparative Study of B Cell Blast Isolation Methods from Bone Marrow Aspirates of Pediatric Leukemia Patients.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

Extracellular vesicles isolated from Arabidopsis thaliana leaves reveal characteristics of mammalian exosomes.

Plant-derived extracellular vesicles (EVs), containing a myriad of bioactive proteins, microRNAs, lipids, and secondary metabolites, have recently become the focus of rising interest due to their important roles in various applications. The widely accepted method for isolating plant EVs is differential ultracentrifugation plus density gradient centrifugation. However, the combination of differential ultracentrifugation and density gradient centrifugation for the isolation of plant EVs is time-consuming and labor-intensive. Hence, there is a need for more efficient methods to perform the separation of plant EVs. In this study, EVs were separated from Arabidopsis thaliana leaves by a cost-effective polyethylene glycol (PEG)-based precipitation approach. The mean size of purified Arabidopsis thaliana EVs determined by dynamic light scattering was 266nm, which is consistent with nanoparticle tracking analysis. The size was also confirmed via transmission electron microscopy with morphology of a cup-shaped appearance which is the typical mammalian exosome's morphology. Additionally, Western blotting of the purified Arabidopsis thaliana EVs, using commercially available mammalian exosomal kits, displayed surface marker tetraspanin proteins (CD9, CD63, and CD81), and endosomal sorting complexes required for transport (ESCRT)-associated proteins (TSG101 and ALIX). This demonstrates that the purified Arabidopsis thaliana EVs reveal the typical proteins reported in mammalian exosomes.

Interleukin (IL)-21 and IL-21 receptor expression in peripheral T and B cells of patients with breast cancer.

IL-21 is a cytokine with versatile antitumor and pro-tumorigenic activities. It is mainly produced by CD4+ T cells and B cells are one of its pivotal targets. In this study, we assessed and compared the expression of IL-21 by CD4+ T cells and the IL-21 receptor (IL-21R) on B cells in the peripheral blood of women with breast cancer and healthy individuals. Blood samples were taken from both patients and controls. Mononuclear cells were seperated using Ficoll-Hypaque density gradient centrifugation. These isolated cells were then stained with either anti-CD19/anti-IL-21R or anti-CD4/anti-IL-21 antibodies and analyzed using flow cytometry. The results showed that there was no significant difference in the percentage of IL-21R+ B cells and IL-21+CD4+ T cells between patients and controls. However, the percentage of CD4+ T cells decreased significantly in patients with breast cancer (P=0.003). This decline was observed from the early stage and before lymph node (LN) involvement. In comparison to the control group, IL-21R+ B cells were relatively lower in patients with stages I+II and those with fewer than 4 involved LNs. The intensity of IL-21 expression in T cells was associated with HER2 expression (P=0.029). Furthermore, we found that the majority of IL-21R+ B cells exhibited a naïve phenotype and most of IL-21+CD4+ T cells did not produce IFN-γ or IL-17. In conclusion, breast cancer from the early stages leads to a significant reduction in the proportion of peripheral CD4+ T cells. However, we did not find a significant change in IL-21 and its receptor expression during disease progression.

Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing

Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.

SARS-Cov-2 spike induces intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9.

Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), as a typical tumor marker, has been found to exert immunomodulatory effects in many diseases. We previously reported the clinical and molecular evidences supporting that SARS-Cov-2 infected the gastrointestinal (GI) tract and found a reduction of CEACAM5 in COVID-19 patients' feces which associated with gut dysbiosis. Yet the role of CEACAM5 in GI infection is ill-defined. Mice models were established through intraperitoneally injecting with recombinant viral spike-Fc to mimic the intestinal inflammation. We collected duodenum, jejunum, ileum and colon samples after 6h, 2 days, 4 days and 7 days of spike-Fc or control-Fc injection to perform proteomic analysis. Blood was collected from healthy donors and peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, then CD4+ T cells were isolated with magnetic beads and co-cultured with Caco-2 cells. In addition to intestinal CEACAM5, the expression of tight junction and the percent of CD4+ T lymphocytes were significantly decreased in spike-Fc group compared to control (p < 0.05), accompanied with increased level of inflammatory factors. The KEGG analysis revealed differentially expressed proteins were mainly enriched in the coronavirus disease (COVID-19), tight junction, focal adhesion, adherens junction and PI3K-Akt signaling pathway. Protein-protein interaction (PPI) network analysis identified the interaction between CEACAM5 and Galectin-9 that was also verified by molecular docking and co-IP assay. We further confirmed a reduction of CEACAM5 in SARS-CoV-2 spike stimulated enterocytes could promote the expression of Galectin-9 protein in CD4+T cells. Then it gave rise to the increasing release of inflammatory factors and increased apoptosis of CD4+T cells by inhibition of PI3K/AKT/mTOR pathway. Ultimately intestinal barrier dysfunction happened. Our results indicated that CEACAM5 overexpression and Galectin-9 knockdown played a protective role in intestinal barrier injury upon spike-Fc stimulation. Collectively, our findings identified firstly that SARS-CoV-2 spike induced intestinal barrier dysfunction through the interaction between CEACAM5 and Galectin-9. The result provides potential therapeutic targets in intestinal barrier dysfunction for treating severe COVID patients.

A promising area of research in medicine: recent advances in properties and applications of Lactobacillus-derived exosomes.

Lactobacillus-derived exosomes, small extracellular vesicles released by bacteria, have emerged as a promising area of research in recent years. These exosomes possess a unique structural and functional diversity that allows them to regulate the immune response and promote gut health. The isolation and purification of these exosomes are crucial for their effective use as a therapeutic agent. Several isolation and purification methods have been developed, including differential ultracentrifugation, density gradient centrifugation, and size-exclusion chromatography. Lactobacillus-derived exosomes have been demonstrated to have therapeutic potential in various diseases, such as inflammatory bowel disease, liver disease, and neurological disorders. Moreover, they have been shown to serve as effective carriers for drug delivery. Genetic engineering of these exosomes has also shown promise in enhancing their therapeutic potential. Overall, Lactobacillus-derived exosomes represent a promising area of research for the development of novel therapeutics for immunomodulation, gut health, and drug delivery.

Fractionating microplastics by density gradient centrifugation: a novel approach using LuerLock syringes in a low-cost density gradient maker

Abstract Microplastics are now ubiquitous in the environment and are even considered “technofossils” of the Anthropocene. Given their omnipresence and potential impact, identifying and analyzing these particles becomes increasingly crucial. Novel approaches suggest density gradient centrifugation for simultaneous extraction and fractionation of microplastic particles based on their plastic-specific densities. In this article we describe a cheap and harmless experimental setting to fractionate microplastic particles by density gradient centrifugation. An innovative low-cost Do-It-Yourself (DIY) gradient maker using Luer-Lock syringes is presented. With this gradient maker it is possible to produce density gradients with water and sucrose solutions, covering a density range of 1.00–1.32 g/cm3, as well as with water and saturated potassium carbonate solutions, covering a density range of 1.06–1.53 g/cm3. The separation performance was tested with the most broadly used plastics polyamide, polyurethane, polycarbonate, polyethylene terephthalate and polyvinylchloride. Both density gradients show centrifugation stability and clear banding patterns after centrifugation. Due to its cheap and easy-to-build-easy-to-use nature, this experimental setting for microplastic fractionation by density gradient centrifugation offers an approach for schools not only to address the microplastic problems, but also to integrate new methods of microplastic analysis in upper secondary school laboratories.

Monodispersed Renewable Particles by Cascade and Density Gradient Size Fractionation to Advance Lignin Nanotechnologies.

Control over particle size and shape heterogeneity is highly relevant to the design of photonic coatings and supracolloidal assemblies. Most developments in the area have relied on mineral and petroleum-derived polymers that achieve well-defined chemical and dimensional characteristics. Unfortunately, it is challenging to attain such control when considering renewable nanoparticles. Herein, a pathway toward selectable biobased particle size and physicochemical profiles is proposed. Specifically, lignin is fractionated, a widely available heterogeneous polymer that can be dissolved in aqueous solution, to obtain a variety of monodispersed particle fractions. A two-stage cascade and density gradient centrifugation that relieves the need for solvent pre-extraction or other pretreatments but achieves particle bins of uniform size (~60 to 860nm and polydispersity, PDI<0.06, dynamic light scattering) along with characteristic surface chemical features is introduced. It is found that the properties and associated colloidal behavior of the particles are suitably classified in distinctive size populations, namely, i) nanoscale (50-100nm), ii) photonic (100-300nm) and iii) near-micron (300-1000nm). The strong correlation that exists between size and physicochemical characteristics (molar mass, surface charge, bonding and functional groups, among others) is introduced as a powerful pathway to identify nanotechnological uses that benefit from the functionality and cost-effectiveness of biogenic particles.

The M2 Macrophages Derived Migrasomes From the Surface of Titania Nanotubes Array as a New Concept for Enhancing Osteogenesis.

As newly discovered substrate anchored extracellular vesicles, migrasomes (Migs) may bring a new opportunity for manipulating target cells bioactivities. In this study, the M2 macrophages derived Migs are obtained by titania nanotubes surface (NTs). Due to the benefits of nanostructuring, the NTs surface is not only able to induce RAW264.7 for M2 polarization but also to generate more Migs formation, which can be internalized by following seeded mesenchymal stem cells (MSCs). Then, the NTs surface induced Migs are collected by density-gradient centrifugation for MSCs treatment. As indicated by immunofluorescence staining, alkaline phosphatase activity, and alizarin red staining, the osteogenic differentiation capacity of MSCs is significantly enhanced by Migs treatment, in line with the dosage. By RNA-sequence analysis, the enhancement of osteogenic differentiation is correlated with PI3K-AKT pathway activation that may originate from the M2 polarization state of donor cells. Finally, the Migs are coated onto Ti surface for therapeutic application. Both the in vitro and in vivo analysis reveal that the Migs coated Ti implant shows significant enhancement of osteogenesis. In conclusion, this study suggests that the nanosurface may be a favorable platform for Migs production, which may bring a new concept for tissue regeneration.

Precise analysis of single small extracellular vesicles using flow cytometry

Methods that enable specific and sensitive quantification of small extracellular vesicles (sEVs) using flow cytometry are still under development. Aggregation or adsorption of antibodies causes sub-nano sized particles or non-specific binding and largely affects the results of flow cytometric analysis of single sEVs. Comparison of control IgG and target-specific IgG is inappropriate because they have different characters. Here, we evaluate four preparation methods for flow cytometry, including ultracentrifugation, density gradient centrifugation, size exclusion chromatography (SEC), and the TIM4-affinity method by using tetraspanin-deficient sEVs. The ultracentrifugation or density gradient centrifugation preparation method has large false-positive rates for tetraspanin staining. Conversely, preparation methods using SEC or the TIM4-affinity method show specific detection of single sEVs, which elucidate the roles of sEV biogenesis regulators in the generation of sEV subpopulations. The methods are also useful for the detection of rare disease-related markers, such as PD-L1. Flow cytometric analysis using SEC or the TIM4-affinity method could accelerate research into sEV biogenesis and the development of sEV-based diagnostics and therapies.

CGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

Abstract 2743: Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of non-small cell lung cancer patients

Abstract Introduction: Disseminated cancer cells (DCC) can be detected years before metastatic manifestation in bone marrow of lung cancer patients. Although a consensus protocol has been established, the impact of markers and methods for DCC detection are still not settled. We recently found that EpCAM+ DCC detected by immunofluorescence (IF) outperform the classical immunocytologic (IC) detection by cytokeratin (CK) antibodies regarding prognostic impact. However, it remained unclear whether this difference resulted from biological traits associated with EpCAM+ vs CK+ DCC or from methodological reasons. We therefore performed a side-by-side comparison of IC vs IF for CK and EpCAM. Methods: We prospectively collected bone marrow (BM) aspirates from 56 non-metastasized NSCLC patients that underwent tumor resection with curative intention. After density gradient centrifugation, we analyzed the mononucleated cells by (i) IC using the anti-CK antibody A45-B/B3 and (ii) by IF using the anti-CK antibody A45-B/B3 and the anti-EpCAM antibody HEA-125. IF signals were captured and objectively quantified using ImageJ, whereas IC was evaluated by two independent observers. Marker-positive cells were isolated and subjected to Whole Genome Amplification (WGA) and whole genome sequencing for assessment of copy number alterations (CNA). Survival analysis was performed for progression-free survival (PFS), tumor-specific survival (TSS) and overall-survival (OS) after a median follow-up time of 72.5, 78.7, 78.7 months, respectively. Results: IC and IF revealed the presence of CK+ DCCs in 37.5% and 41.1% of cases, respectively (P=0.34). IF identified EpCAM+ DCCs in 55.4% of patients, resulting in a 63.8% DCC-positivity rate found by IF, when combined with CK. The average number of DCCs per million MNC, the DCC density (DCCD), was 0.92, 0.93 and 1.44 for CKIC and CKIF and for EpCAMIF, respectively. No association was found between CKIC + DCCs and PFS, TSS or OS. In contrast, detection of EpCAMIF + and/or CKIF + DCCs showed a trend towards reduced PFS (P=0.055), TSS (P=0.225) and OS (P=0.155). Subgroup analysis revealed that both EpCAMIF + and CKIF + cells were correlated with worse PFS (P=0.016 and P=0.040, respectively) and TSS (P=0.033 and P=0.008, respectively). CKIF-positivity was also associated with worse OS (P=0.014), whereas EpCAM was not. CKIF + DCCs remained an independent prognostic variable for TSS and OS upon multivariate testing (hazard ratio: 6.45 and 3.31, respectively), while EpCAMIF + was not. The IF-marker-defined cell phenotypes exhibited comparable frequencies and distributions of CNAs across the entire genome (~50% of cells). Conclusions: Detection of CK+ DCC by quantified IF analysis apparently outperforms CK detection by IC in survival analysis. The impact of CK and EpCAM expression levels on outcome are under investigation. Citation Format: Daniel Spitzl, Tobias Mederer, Julius Zank, Severin Gütter, Michael Ried, Hans-Stefan Hofmann, Christoph A. Klein. Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2743.

Abstract 6561: Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer

Abstract Background: Ovarian Cancer (OC) is one of the leading causes of death among gynecological tumors with an insufficient understanding of OC disease evolution. Since tumor tissue is not always available, blood allows the investigation of single circulating tumor cells (sCTC) over time. We combined immunomagnetic enrichment and image-based sCTC sorting for phenotypic and genotypic analysis of sCTCs in the follow-up of OC to provide insights into sCTC heterogeneity and their malignant character. Patients and Methods: 10 ml blood of high-grade serous OC patients (pts) at the time of primary diagnosis (n=42), after chemotherapy (n=23) and after Avastin (n=11) was analyzed for sCTCs using density-gradient centrifugation to further apply MACS® using CD45 and CD235a antibodies coupled to MACS® MicroBeads. For image-based sCTC sorting (DEPArray®), an immunofluorescence protocol staining for Hoechst, cytokeratin (CK), the folate receptor alpha (FRa), Vimentin (Vim) and CD45 was applied. Whole genome amplification (WGA) was performed in recovered single cells, comprising cell lysis, DNA digestion, adaptor ligation and an amplification PCR for subsequent copy number alteration (CNA) analysis. Ampli1TM low-pass analysis was used to analyze single cell WGA products. 21 primary tumors (PT) were assessed for whole-genome CNA and the Ampli 1TM OncoSeek panel was used for deep sequencing of sCTCs. Results: Phenotypic assessment revealed FRa-positive cells in 28/42 (67%) pts, CK-positive cells in 32/42 (76%) pts and CK-FRa-double-positive cells in 15/42 (36%) pts, respectively, with a significant (p= 0.0205) reduction of cells after chemotherapy only documented for FRa-positive cells. Interestingly, cells with a high nuclear staining but no expression of target antigens were found in 37/42 pts (88%) and significantly (p=0.002) expanded during treatment. Genotyping revealed that only 7% (19/289) of single cells deriving from 13 different pts were aberrant. Whole genome CNA analysis in all pre-treatment sCTCs revealed enriched CNAs in chromosomes 2, 7 and 12. In post-treatment sCTCs, CNA dynamics highlighted the adaptive potential of the homologous recombination pathway and suggested further investigation of MAPK signaling in resistant OC. Low-pass CNA comparison between sCTCs and the PT was feasible in nine pts, 2/9 with persistent PT characteristics in sCTCs at different sampling times. TP53 variants were the most frequently detected ones, persistent CNA in the CDK4 oncogene and emerging CNA in the ALK oncogene were found in sCTCs from two pts. Conclusion: We identified sCTCs in the follow-up of OC and demonstrated interpatient as well as intrapatient heterogeneity of sCTCs. Matched sCTCs and PT data showed similar CNAs to a certain amount. Our low percentage of aberrant cells suggests that higher patient numbers are needed to confirm our findings to understand disease progression. Citation Format: Sabine Kasimir-Bauer, Nikolas H. Stoecklein, Rui P. Neves, Sven T. Liffers, Jan D. Kuhlmann, Pauline Wimberger, Paul Buderath, Rainer Kimmig, Carolin Salmon. Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6561.

Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. The study is funded by the Iran University of Medical Sciences and no competing interest exists. N/A.

Methods to Isolate Muscle Stem Cells for Cell-Based Cultured Meat Production: A Review.

Cultured meat production relies on various cell types, including muscle stem cells (MuSCs), embryonic stem cell lines, induced pluripotent cell lines, and naturally immortalized cell lines. MuSCs possess superior muscle differentiation capabilities compared to the other three cell lines, making them key for cultured meat development. Therefore, to produce cultured meat using MuSCs, they must first be effectively separated from muscles. At present, the methods used to isolate MuSCs from muscles include (1) the pre-plating method, using the ability of cells to adhere differently, which is a biological characteristic of MuSCs; (2) the density gradient centrifugation method, using the intrinsic density difference of cells, which is a physical characteristic of MuSCs; and (3) fluorescence- and magnetic-activated cell sorting methods, using the surface marker protein on the cell surface of MuSCs, which is a molecular characteristic of MuSCs. Further efficient and valuable methods for separating MuSCs are expected to be required as the cell-based cultured meat industry develops. Thus, we take a closer look at the four methods currently in use and discuss future development directions in this review.

Isolation and usage of exosomes in central nervous system diseases.

Exosomes are vesicles secreted by all types of mammalian cells. They are characterized by a double-layered lipid membrane structure. They serve as carriers for a plethora of signal molecules, including DNA, RNA, proteins, and lipids. Their unique capability of effortlessly crossing the blood-brain barrier underscores their critical role in the progression of various neurological disorders. This includes, but is not limited to, diseases such as Alzheimer's, Parkinson's, and ischemic stroke. Establishing stable and mature methods for isolating exosomes is a prerequisite for the study of exosomes and their biomedical significance. The extraction technologies of exosomes include differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polymer coprecipitation, immunoaffinity capture, microfluidic, and so forth. Each extraction technology has its own advantages and disadvantages, and the extraction standards of exosomes have not been unified internationally. This review aimed to showcase the recent advancements in exosome isolation techniques and thoroughly compare the advantages and disadvantages of different methods. Furthermore, the significant research progress made in using exosomes for diagnosing and treating central nervous system (CNS) diseases has been emphasized. The varying isolation methods result in differences in the concentration, purity, and size of exosomes. The efficient separation of exosomes facilitates their widespread application, particularly in the diagnosis and treatment of CNS diseases.

Expression of neutrophil extracellular traps and phagocytic functions among patients with hepatic alveolar echinococcosis

To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis (HAE), and to examine their correlations with clinical inflamma tory indicators and liver functions. A total of 50 patients with HAE admitted to Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qinghai University from August 2022 to June 2023 were enrolled, while 50 age- and gender-matched healthy individuals from the Centre for Healthy Examinations of the hospital during the same period served as controls. The levels of NETs markers neutrophil myeloperoxidase (MPO) and neutrophil elastase (NE) were measured using enzyme-linked immunosorbent assay (ELISA). Peripheral blood neutrophils were isolated using density gradient centrifugation, stimulated in vitro using phorbol 12-myristate 13 acetate (PMA), and the levels of MPO and citrullination histone H3 (CitH3) released by neutrophils were quantified using flow cytometry. The phagocytic functions of neutrophils were examined using flow cytometry. In addition, the correlations of MPO and NE levels with clinical inflammatory indicators and liver biochemical indicators were examined using Spearman correlation analysis among HAE patients. The peripheral blood plasma MPO［(417.15 ± 76.08) ng/mL vs. (255.70 ± 80.84) ng/mL; t = 10.28, P < 0.05］, NE［(23.16 ± 6.75) ng/mL vs. (11.92 ± 3.17) ng/mL; t = 10.65, P < 0.05］and CitH3 levels［(33.93 ± 18.93) ng/mL vs. (19.52 ± 13.89) ng/mL; t = 4.34, P < 0.05］were all significantly higher among HAE patients than among healthy controls, and a lower phagocytosis rate of neutrophils was detected among HAE patients than among healthy controls［(70.85 ± 7.32)% vs. (94.04 ± 3.90)%; t = 20.18, P < 0.05］, and the ability to produce NETs by neutrophils was higher among HAE patients than among healthy controls following in vitro PMA stimulation. Pearson correlation analysis showed that the phagocytosis rate of neutrophils correlated negatively with platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), interleukin-6 (IL-6) level and C-reactive protein (CRP) level (rs = -0.515 to -0.392, all P values < 0.05), and the MPO and NE levels positively correlated with inflammatory markers NLR, PLR, CRP and IL-6 (rs = 0.333 to 0.445, all P values < 0.05) and clinical liver biochemical indicators aspartic transaminase, alanine aminotransferase, direct bilirubin and total bilirubin among HAE patients (rs = 0.290 to 0.628, all P values < 0.001). Excessive formation of NETs is found among HAE patients, which affects the phagocytic ability of neutrophils and results in elevated levels of inflammatory indicators. NETs markers may be promising novel biomarkers for early diagnosis, monitoring, and severity assessment of liver disease.