Magnetic-activated Cell Sorting Research Articles

Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells.

Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selectedsgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency forco-transfectionof CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editingstrategy could efficiently target the HBB locus and enrich for HDR-positive cells.

Aluminium phthalocyanine–mediated photodynamic therapy promotes antitumour activity in oesophageal cancer stem cells.

e15194 Background: Photodynamic therapy (PDT), a non-invasive treatment modality, has emerged as an effective treatment measure for various cancers, including oesophageal cancer. PDT has attracted much attention in the field of oncology due to its low toxicity profile and the selective accumulation of the photoactivated agent in the tumour cells. However, the therapeutic effect of Aluminium (III) Phthalocyanine Chloride Tetra sulfonic Acid (AlPcS4Cl) mediated PDT on oesophageal CSCs is limited. In this study, we evaluated the cellular localization of AlPcS4Cl and its PDT anticancer effects on oesophageal CSCs. Methods: An in vitro experimental design was employed in this study. Oesophageal cancer (HKESC-1 cells) was grown, and the CSCs were isolated using the magnetic-activated cell sorting (MACS). The isolated CSCs were characterised using a flow cytometry, Hoechst efflux side population test, and immunofluorescence. The CSCs were maintained in a serum-free culture medium and incubated at 37° C, 5% CO2 and 85% humidity. The cells were grouped into control and experimental groups. The CSCs received two-fold increasing concentrations of AlPcS4Cl and were exposed to irradiation at 673.2nm wavelength using a diode laser. After 24 hours post-PDT, the anticancer activities of AlPcS4Cl on oesophageal CSCs were examined. The MTT cell viability assay was used to determine the 50% inhibitory concentration (IC50), light microscopy for morphological changes, and adenosine triphosphate (ATP) assay for cell proliferation, lactate dehydrogenase (LDH) assay for cellular toxicity. While cellular localization of AlPcS4Cl was examined using fluorescent microscopy. All experimental and control cells were conducted in six biological replicates (n = 6). Results: Results were compiled, and statistical analysis was performed using GraphPad Prism (v5). The mean values of experimental groups were compared with the mean value of the control cells. One-way ANOVA was employed, and a p-value of less than 0.05 was considered statistically significant. Findings from cellular localization showed that the AlPcS4Cl is localized in the mitochondria and lysosomes, indicating a probable cellular death pathway. The dose-response of AlPcS4Cl on oesophageal CSCs showed an IC50 value of 4µM, suggesting the photosensitizer is efficient for the treatment of CSCs at low concentrations. The CSCs exposed to PDT showed remarkable morphological distortion, high cytotoxic activity, decreased viable cells and significant antiproliferative potential. Conclusions: This study showed that AlPcS4Cl promote PDT anticancer effects on oesophageal CSCs and could serve as an efficient therapeutic option for eradicating oesophageal CSCs.

Pretreatment of prostate cancer cells with salinomycin and Wnt inhibitor increases the efficacy of cabazitaxel by inducing apoptosis and decreasing cancer stem cells.

Cancer stem cells (CSCs) are associated with metastasis and recurrence in prostate cancer as well as other cancers. We aimed to enhance the sensitivity of cabazitaxel in prostate cancer cell therapy by targeting CSCs with a Wnt inhibitor and salinomycin pretreatment. PC3, DU-145, and LNCaP human prostate cancer cells were exposed to Wnt/β-catenin pathway inhibitor CCT036477 (iWnt) with salinomycin for 48h, followed by cabazitaxel treatment for 48h. Cell viability, mRNA, and protein expression changes were evaluated by MTT, RT-qPCR, and Western blot assays, respectively. Apoptosis was determined by image-based cytometry, and cell migration was assessed by wound healing assay. Three-dimensional culture was established to assess the malignant phenotype and stemness potential of transformed or cancer cells. CD44 + CSCs were isolated using magnetic-activated cell sorting system. Pretreatment of PC3, DU-145, and LNCaP cells with salinomycin iWnt significantly sensitized the cells to cabazitaxel therapy. Spheroid culture confirmed that the treatment modality was more effective than a single administration of chemotherapy. The pretreatment of PC3 cells increased the rate of apoptosis compared to single administration of cabazitaxel, which downregulated Bcl-2 and upregulated caspase 3, caspase 8 expressions. The pretreatment suppressed cell migration, downregulated the expression of Sox2 and Nanog, and significantly reduced CD44 + CSC numbers. Notably, the treatment modality reduced pAKT, p-P38 MAPK, and pERK1/2. The data suggest that pretreatment of prostate cancer cells with salinomycin and Wnt inhibitor may increase the efficacy of cabazitaxel therapy by inhibiting cell proliferation and migration, and eliminating cancer stem cells.

A mammalian cell display platform based on scFab transposition.

In vitro display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in a single-chain Fab (scFab) format on the surface of HEK293F cells. Immune rabbit antibody libraries encompassing ~7×107 independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells and displayed as an scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting and two rounds of fluorescence activated cell sorting, mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.

Magnetic-activated cell sorting identifies a unique lung microbiome community

BackgroundThe advent of culture-independent, next-generation DNA sequencing has led to the discovery of distinct lung bacterial communities. Studies of lung microbiome taxonomy often reveal only subtle differences between health and disease, but host recognition and response may distinguish the members of similar bacterial communities in different populations. Magnetic-activated cell sorting has been applied to the gut microbiome to identify the numbers and types of bacteria eliciting a humoral response. We adapted this technique to examine the populations of immunoglobulin-bound bacteria in the lung.MethodsSixty-four individuals underwent bronchoalveolar lavage (BAL). We separated immunoglobulin G-bound bacteria using magnetic-activated cell sorting and sequenced the 16S rRNA gene on the Illumina MiSeq platform. We compared microbial sequencing data in IgG-bound bacterial communities compared to raw BAL then examined the differences in individuals with and without HIV as a representative disease state.ResultsImmunoglobulin G-bound bacteria were identified in all individuals. The community structure differed when compared to raw BAL, and there was a greater abundance of Pseudomonas and fewer oral bacteria in IgG-bound BAL. Examination of IgG-bound communities in individuals with HIV demonstrated the differences in Ig-bound bacteria by HIV status that were not seen in a comparison of raw BAL, and greater numbers of immunoglobulin-bound bacteria were associated with higher pulmonary cytokine levels.ConclusionsWe report a novel application of magnetic-activated cell sorting to identify immunoglobulin G-bound bacteria in the lung. This technique identified distinct bacterial communities which differed in composition from raw bronchoalveolar lavage, revealing the differences not detected by traditional analyses. Cytokine response was also associated with differential immunoglobulin binding of lung bacteria, suggesting the functional importance of these communities.6btME6LTfMPeXYkHvqtMADVideo

Early stage evaluation of cancer stem cells using platinum nanoparticles/CD133+ enhanced nanobiocomposite

BackgroundCancer stem cells (CSCs) are of great diagnostic importance due to their involvement in tumorigenesis, therapeutic resistance, metastasis and relapse.MethodIn this work, a sensitive electrochemical cytosensor was successfully established to detect HT-29 colorectal cancer stem cells based on a nanocomposite composed of mesoporous silica nanoparticles (MSNs) and platinum nanoparticles (PtNPs) using a simple and fast electrodeposition technique on a glassy carbon electrode (GCE).ResultsAccording to SEM images, the PtNPs nanoparticles formed on the MSNs substrate are about 100 nm. As expected, high-rate porosity, increased surface-to-volume ratio, provides appropriate local electron transfer rate and suitable platform for the efficient formation of PtNPs. These features allow direct and stable binding of biotinylated monoclonal antibody of CD133 to streptavidin (Strep) and the subsequent availability of active sites for CSCs identification. Differential pulse voltammetry (DPV) results show that close interaction of CD133+ cells with monoclonal antibodies reduces charge transfer and electrical current, as confirmed by square wave voltammogram (SWV). Based on the recorded current versus number of CSCs, we noted that our developed system can sense CSCs from 5 to 20 cells/5 μL.ConclusionsAs a proof of concept, the designed nanobiocomposite was able to specifically detect CD133+ cells compared to whole HT-29 cells before magnetic activated cell sorting (MACS) process.

Single-Cell Proteomics with Spatial Attributes: Tools and Techniques.

Now-a-days, the single-cell proteomics (SCP) concept is attracting interest, especially in clinical research, because it can identify the proteomic signature specific to diseased cells. This information is very essential when dealing with the progression of certain diseases, such as cancer, diabetes, Alzheimer's, etc. One of the major drawbacks of conventional destructive proteomics is that it gives an average idea about the protein expression profile in the disease condition. During the extraction of the protein from a biopsy or blood sample, proteins may come from both diseased cells and adjacent normal cells or any other cells from the disease environment. Again, SCP along with spatial attributes is utilized to learn about the heterogeneous function of a single protein. Before performing SCP, it is necessary to isolate single cells. This can be done by various techniques, including fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), laser capture microdissection (LCM), microfluidics, manual cell picking/micromanipulation, etc. Among the different approaches for proteomics, mass spectrometry-based proteomics tools are widely used for their high resolution as well as sensitivity. This Review mainly focuses on the mass spectrometry-based approaches for the study of single-cell proteomics.

T-cell mitochondria exhibit a functional decline with obesity that is aggravated with weight loss

Objective: Resistance to weight loss (WL) and propensity to weight regain are major challenges to achieve long-term metabolic health for people with obesity. It is yet to be determined whether mitochondria (MITO) play a role as a counter regulatory mechanism that impedes diet-induced WL that could contribute to slowing of WL and eventually, a WL plateau. This study aimed to 1) elucidate differences in MITO sensitivity to energy demand by peripheral mononuclear cells (PBMCs) in subjects with and without obesity, and 2) compare MITO respiratory capacity in subjects with obesity (OB) who were in phases of active weight loss (OB-WL), plateau (OB-PL), or regain (OB-RG). Methods: Fresh blood samples were collected from fasted participants with BMI of 30-56 kg/m2 at either their baseline, or their 6-month, or 12-month visit following 24-weeks of behavioral WL intervention. Blood was also collected from healthy weight-control participants (HWC, n=11) (BMI of 18.5-24.9). PBMCs were isolated through density gradient centrifugation, and CD3+ (T-cell) and CD14+ (monocytes) were subsequently separated though magnetic activated cell sorting. MITO function of non-permeabilized cells was assessed by Agilent Extracellular Flux Analysis (XFe), and MITO sensitivity to energy demand was assessed using the Oroboros high-resolution respirometer (O2K) in permeabilized cells. To determine the phase of WL at each visit, body weight trajectories were tracked for each participant throughout 18 months using a remote electronic scale. Threshold regression modeling was used to determine if the participant was in OB-WL (BW loss ≥0.5lbs/wk, n=7), OB-PL (±0.25lbs/wk, n=10), or OB-RG (BW gain ≥0.5lbs/wk, n=6). Results: There was a consistent reduction in MITO capacity of T-cells in the OB group (2.0±0.6 pmol O2/106 cells) compared to HWC (2.8±0.8), in both non-permeabilized (P=0.01) and permeabilized cells (p=0.02), that was more pronounced when inhibiting complex I (P=0.02). Maximal membrane potential was also lower in the OB group (P<0.01), and membrane depolarization with ADP tended to be more sensitive in the OB group, but differences were not statistically significant (p=0.17). No significant differences were found in monocyte MITO function between the two groups.We found lower MITO respiratory capacity in T-cells from the OB-PL (1.4±0.4) and OB-RG (1.6±0.8) groups relative to OB-WL (2.5±0.4). T-cell bioenergetic health index negatively correlated with the slope of % weight loss calculated using the weight loss trajectory (r=-0.661, p=0.027). Conclusions: T-cells exhibit reduced MITO respiratory capacity in participants with obesity at baseline compared to those in the normal weight range. MITO respiratory capacity was further reduced in participants at the WL-PL. These findings suggest that reductions in MITO respiratory capacity are exacerbated rather than improved in response to diet-induced WL, potentialy indicating resistance to further WL at a plateau promoting regain. T32 HL007028 (APV), K01 HL164761-01 (APV), P30DK035816 (APV), R01DK089036 (EAS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Yi-Qi-Jian-Pi formula ameliorates immune function in acute-on-chronic liver failure by upregulating autophagy and mitochondrial biogenesis in CD8+ T lymphocytes.

A key event in the pathogenesis of acute-on-chronic liver failure (ACLF) is the imbalance in the systemic immune response; immunosuppression in patients with ACLF contributes to poor prognosis. The Yi-Qi-Jian-Pi formula (YQJPF) may improve T lymphocyte immune function in patients with ACLF. To investigate the immune mechanism of YQJPF in vivo and in vitro. An ACLF rat model was established by injection of CCl4, lipopolysaccharide, and D-galactosamine. We examined the effect of different doses of YQJPF (6.43, 12.87, 25.74g/kg) on liver injury and immune function in the ACLF rat model. Magnetic-activated cell sorting was used to sort the CD8+ T lymphocytes in the spleen for lymphocyte function detection. In primary CD8+ T lymphocytes and Jurkat cell lines, the expression of mitochondrial function and biogenesis and autophagy related markers were detected using molecular biological methods and flow cytometry analysis. YQJPF improved the peripheral blood lymphocyte count and proportion of CD8+ T lymphocytes in ACLF rats, increased pro-inflammatory factors (IL-2, IFN-λ, and TNF-α), and reduced anti-inflammatory factors (IL-10 and TGF β1). YQJPF also improved metabolism and mitochondrial homeostasis in CD8+ T lymphocytes, alleviated lymphocyte immune dysfunction by promoting autophagy, upregulated mitochondrial biogenesis by promoting PGC-1α, NRF-1, and TFAM expression, and regulated the relationship between autophagy and mitochondrial biogenesis via PGC-1α. Our results suggest that YQJPF could improve immune function in a rat model of ACLF, possibly via affecting the homeostasis of lymphatic mitochondria in CD8+ T lymphocytes. YQJPF may enhance lymphocyte mitochondrial biosynthesis and promote lymphocyte autophagy. PGC-1α is a possible upstream regulatory target of YQJPF.

How to select healthy sperm for intracytoplasmic sperm injection in samples with high sperm DNA fragmentation?

The body of evidence supports the negative impact of increased sperm DNA fragmentation (SDF) on natural fertility as well as assisted reproduction conditions. High SDF has been correlated with low pregnancy and delivery rates following intrauterine insemination. Also, high SDF is accused of reducing the rates of fertilization, implantation, pregnancy, and live birth following in-vitro fertilization (IVF). Despite no impact of high SDF on fertilization or pregnancy rates following intracytoplasmic sperm injection (ICSI), it has been correlated with poor embryo quality and a higher risk of miscarriage. Several methods have been introduced to help select sperm with the best DNA quality to be used in assisted reproductive technology procedures. These include magnetic-activated cell sorting, intracytoplasmic morphologically selected sperm injection, physiologic ICSI, and microfluidic sperm sorters, among others. This article aimed to discuss the impact of high SDF in infertile men on the reproductive outcome of couples undergoing IVF/ICSI. Additionally, this review highlights the principles, advantages, and limitations of different techniques that are currently used for the selection of sperm with intact DNA to be utilized for ICSI.

Perivascular localized cells commit erythropoiesis in PDGF-B-expressing solid tumors.

Tumors possess incessant growth features, and expansion of their masses demands sufficient oxygen supply by red blood cells (RBCs). In adult mammals, the bone marrow (BM) is the main organ regulating hematopoiesis with dedicated manners. Other than BM, extramedullary hematopoiesis is discovered in various pathophysiological settings. However, whether tumors can contribute to hematopoiesis is completely unknown. Accumulating evidence shows that, in the tumor microenvironment (TME), perivascular localized cells retain progenitor cell properties and can differentiate into other cells. Here, we sought to better understand whether and how perivascular localized pericytes in tumors manipulate hematopoiesis. To test if vascular cells can differentiate into RBCs, genome-wide expression profiling was performed using mouse-derived pericytes. Genetic tracing of perivascular localized cells employing NG2-CreERT2:R26R-tdTomato mouse strain was used to validate the findings in vivo. Fluorescence-activated cell sorting (FACS), single-cell sequencing, and colony formation assays were applied for biological studies. The production of erythroid differentiation-specific cytokine, erythropoietin (EPO), in TME was checked using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA, magnetic-activated cell sorting and immunohistochemistry. To investigate BM function in tumor erythropoiesis, BM transplantation mouse models were employed. Genome-wide expression profiling showed that in response to platelet-derived growth factor subunit B (PDGF-B), neural/glial antigen 2 (NG2)+ perivascular localized cells exhibited hematopoietic stem and progenitor-like features and underwent differentiation towards the erythroid lineage. PDGF-B simultaneously targeted cancer-associated fibroblasts to produce high levels of EPO, a crucial hormone that necessitates erythropoiesis. FACS analysis using genetic tracing of NG2+ cells in tumors defined the perivascular localized cell-derived subpopulation of hematopoietic cells. Single-cell sequencing and colony formation assays validated the fact that, upon PDGF-B stimulation, NG2+ cells isolated from tumors acted as erythroblast progenitor cells, which were distinctive from the canonical BM hematopoietic stem cells. Our data provide a new concept of hematopoiesis within tumor tissues and novel mechanistic insights into perivascular localized cell-derived erythroid cells within TME. Targeting tumor hematopoiesis is a novel therapeutic concept for treating various cancers that may have profound impacts on cancer therapy.

Association of Dipeptidylpeptidase 4 (CD26) With Chondrocyte Senescence and Radiographic Progression in Knee Osteoarthritis.

To evaluate the association of dipeptidylpeptidase 4 (DPP-4; also known as CD26) with cellular senescence of human cartilage and progression of knee osteoarthritis (OA). Articular cartilage sections and chondrocytes were acquired from 35 individuals undergoing total knee replacement for OA to evaluate the following: 1) the association between OA severity and established senescence markers (senescence-associated β-galactosidase activity and p16), which was quantified using immunohistochemistry and flow cytometry (n=19 samples); 2) the coexpression of DPP-4 with established senescence markers, which was assessed using flow cytometry; and 3) expression levels of anabolic and catabolic genes, senescence-related genes, and senescence-associated secretory phenotypes in DPP-4+ and DPP-4- cells, which were isolated using fluorescence-activated cell sorting or magnetic-activated cell sorting (n=16 samples). The concentration of soluble DPP-4 was measured in samples of synovial fluid and samples of plasma from the Prediction of Osteoarthritis Progression cohort and then evaluated for association with the severity of radiographic knee OA at baseline (n=65 samples) and the progression of structural radiographic OA (n=57 samples) over a 3-year period. DPP-4 expression was associated with higher senescence-associated β-galactosidase activity, p16 expression, senescence-related gene and catabolic gene (ADAMTS5, MMP13, IL6, and IL8) expression, higher senescence-associated secretory phenotype secretion, and lower anabolic gene (COL2A1 and ACAN) expression in primary chondrocytes. Synovial fluid DPP-4 concentration was associated with radiographic OA progression (odds ratio 105.32; P=0.015), proteases (synovial fluid matrix metalloproteinase 1 and matrix metalloproteinase 3), aggrecan degradation (synovial fluid sulfated glycosaminoglycan), indicators of activated macrophages (synovial fluid CD14 and CD163), and inflammation (synovial fluid interleukin-6). Our study identifies DPP-4 as a key surface marker in senescent chondrocytes and a predictor of radiographic OA progression.

The magnetic cell separation method reveals protective effect of melatonin on human spermatozoa from peroxide-induced apoptosis

BackgroundThe selection of non-apoptotic sperm is related to successful fertilization. This study investigated the protective effects of melatonin and the role of the MACS (magnetically activated cell sorting) method to prevent oxidative damage in vitro and improve sperm quality parameters such as viability and DNA integrity.Materials and methodsSemen analysis was performed on 20 different eligible donors participating in the study. Sperm viability and concentration were checked at time of 0 (0 h). In order to conduct more studies after 24 h (24 h), the remaining sperm suspension was divided into a control group and six experimental groups. DNA fragmentation was assessed using the TUNEL assay.ResultsThe treatment of human spermatozoa with 100 µM hydrogen peroxide for 24 h induced a significant increase in phosphatidylserine externalization and significantly increases apoptotic sperm (p ≤ 0.001). TUNEL analysis of human sperm pretreated with 100 µM hydrogen peroxide for 24 h showed that the percentage of sperm with fragmented DNA was significantly reduced after sorting by MACS (P ≤ 0.001). However, pretreated human sperm with 1 μM melatonin for 24 h could effectively maintain sperm motility and progressive motility.ConclusionsPretreated human spermatozoa with 1 µM melatonin for 24 h could be effective for maintenance of sperm motility and progressive motility. Although 100 µM hydrogen peroxide-treated sperm were used, MACS was used to retain the appropriate sperm and select high-quality sperm.

OVERLOAD-INDUCED EXTRACELLULAR MITOCHONDRIA RELEASING FROM TENDON ORGANOID LEADS TO INFLAMMATION

Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation.We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by flow cytometry and super-resolution confocal microscopy. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic-Activated Cell Sorting Technology. EM induced cytokines secretion by macrophages was analyzed by a bead-based multiplex assay panel. N-Acetyl-L-cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness.We showed mechanical load induced tendon organoid to release microvesicles including mitochondria. The size of microvesicles is mainly in the range from 220nm to 880nm. More than 75% of microvesicles could be stained by PKH26, confirming they were with lipophilic membrane. Super-resolution confocal microscopy identified two forms of mitochondria, including mitochondria encapsulated in vesicles and free mitochondria. Overload led to the degeneration of the organoid and induced microvesicles release containing most EM. Chemotaxis assay showed that EM from overloaded tendon organoid induced macrophages chemotaxis. In addition, microvesicles extracted from overloaded tendon organoid induced the production of proinflammatory cytokines including IL-6, KC (Keratinocyte-Derived Chemokine) and IL-18. NAC treatment to tendon cells could attenuate overload-induced macrophage chemotaxis.Overload induces EM releasing from tendon cells, which leads to chemotaxis of macrophages toward tendon, resulting in induction of inflammation.

Density Gradient Centrifugation Alone or the Combination of DGC with Annexin V Magnetic-Activated Cell Sorting Prior to Cryopreservation Enhances the Postthaw Quality of Sperm from Infertile Male Patients with Poor Sperm Quality

Objective. To examine whether density gradient centrifugation (DGC) alone or its combination with annexin V magnetic-activated cell sorting (DGC-MACS) can be used to process semen samples from infertile male patients with poor sperm quality prior to subjecting these to freeze/thaw process in order to optimize the outcomes of sperm freezing. Methods. This study enrolled sixteen patients with sperm concentration ≥ 20 × 10 6 / mL , sperm motility < 30 %, and/or <4% normal sperm morphology. Sperms were processed by DGC or DGC-MACS prior to the freeze/thaw process. Sperm motility, hyperosmotic swelling test (HOS), TUNEL test, and morphological analysis were performed before and after the freeze/thaw process. Results. The freeze/thaw process had a detrimental effect on sperm motility, viability, morphology, and DNA integrity in all three groups (RAW, DGC, and DGC + MACS groups). The DGC and DGC + MACS groups showed increased sperm motility, viability, and normal morphology following freeze/thaw than untreated frozen controls. The motility and viability were not significantly different between DGC-MACS-CPT (cryopreservation-thawing) and DGC-CPT groups. Moreover, almost no grade A or grade B sperm was observed in the DGC-MACS-CPT groups. The sperm selected by DGC or DGC + MACS showed decreased levels of sperm DNA fragmentation than RAW samples following freeze/thaw. Moreover, the sperm DNA fragmentation following freeze/thaw in the DGC-MACS-CPT group was significantly lower than that in the DGC-CPT group. Conclusions. Sperm preparation by DGC before cryopreservation improved the quality of sperm postthaw in infertile males with poor sperm quality. If the sperm quality following freeze/thaw is foreseen to be insufficient for artificial insemination with husband’s sperm or in vitro fertilization, or if there is high DNA fragmentation in RAW sperm, DGC + MACS should be used prior to cryopreservation to reduce sperm DNA fragmentation and improve the quality of sperm available for intracytoplasmic sperm injection.

Abstract 5639: High throughput spatially resolved laser-activated cell sorting links the genomic molecules with its spatial information

Abstract Spatial omics profiling technologies have been recognized recently for its ability to decipher the genetic molecules that are structurally relevant in pathology. Especially, in tumor biology, tumor is not the group of malignant tumor cells, but rather group of various cells such as tumor cells, immune cells, fibroblasts, etc. gathers together, constructing the tumor microenvironments. Technologies to analyze such microstructures have evolved from bulk sequencing, single cell sequencing to spatial omics profiling technologies. Spatial omics profiling technologies have highly influenced in decoding cancerous mechanisms by questioning the tumor heterogeneity, tumor microenvironment and spatial biomarkers. Most of the spatial omics technologies focus on mapping the spatial omics landscape in a large scale. They rather introduces the spatially-barcoded capture probes or fluorescence labeled target probes to spatially locate the genetic molecules. The information depth and the scalability of the techniques varies according to the purpose of the spatial assay techniques. Such technologies are capable of discovering the spatial heterogeneity and the spatial landscape of the consisting cell types due to relatively low depth of the omics information. To effectively address the target molecules for therapeutics or diagnostics, higher depth of the omics information are required. To meet the needs, region of interest (ROI)-based spatial technologies isolated the target regions and applies chemistries for higher coverage omics data. Conventional cell sorters utilizes microfluidic channels to sort cells of interest which requires cell dissociation in a solution phase. For instance, Fluorescence activated cell sorter (FACS) or Magnetic-activated cell sorting (MACS) uses fluorescence or magnetic particles, respectively, to designate the cells of interest in dissociated cell solutions. Spatially isolating techniques such as laser capture microdissection (LCM) is able to sort out the ROIs while preserving the spatial context, but it approximately takes an hour for isolating the targets. Also, it uses rather UV laser to dissect out cells or IR-activated melting of polymers to stick out cells which might cause damage to cells. Here, I developed the automated spatially resolved laser activated cell sorter that isolates the cells in target per second while preserving the spatial context of the cells. Specific region of indium tin oxide (ITO) coated slide glass evaporates when illuminated by IR laser pulse, plunging the cells into the desired reservoir. The applicability of the suggested cell sorter are demonstrated in omics profiling chemistries such as DNA sequencing, RNA sequencing, mass spectrometry, etc. Citation Format: Sumin Lee, Amos C. Lee, Sunghoon Kwon. High throughput spatially resolved laser-activated cell sorting links the genomic molecules with its spatial information. [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 5639.

Electrotaxis of alveolar epithelial cells in direct-current electric fields

PurposeThis study aims to elucidate the electrotaxis response of alveolar epithelial cells (AECs) in direct-current electric fields (EFs), explore the impact of EFs on the cell fate of AECs, and lay the foundation for future exploitation of EFs for the treatment of acute lung injury. MethodsAECs were extracted from rat lung tissues using magnetic-activated cell sorting. To elucidate the electrotaxis responses of AECs, different voltages of EFs (0, 50, 100, and 200 mV/mm) were applied to two types of AECs, respectively. Cell migrations were recorded and trajectories were pooled to better demonstrate cellular activities through graphs. Cell directionality was calculated as the cosine value of the angle formed by the EF vector and cell migration. To further demonstrate the impact of EFs on the pulmonary tissue, the human bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B cells) were obtained and experimented under the same conditions as AECs. To determine the influence on cell fate, cells underwent electric stimulation were collected to perform Western blot analysis. ResultsThe successful separation and culturing of AECs were confirmed through immunofluorescence staining. Compared with the control, AECs in EFs demonstrated a significant directionality in a voltage-dependent way. In general, type Ⅰ alveolar epithelial cells migrated faster than type Ⅱ alveolar epithelial cells, and under EFs, these two types of cells exhibited different response threshold. For type Ⅱ alveolar epithelial cells, only EFs at 200 mV/mm resulted a significant difference to the velocity, whereas for, EFs at both 100 mV/mm and 200 mV/mm gave rise to a significant difference. Western blotting suggested that EFs led to an increased expression of a AKT and myeloid leukemia 1 and a decreased expression of Bcl-2-associated X protein and Bcl-2-like protein 11. ConclusionEFs could guide and accelerate the directional migration of AECs and exert antiapoptotic effects, which indicated that EFs are important biophysical signals in the re-epithelialization of alveolar epithelium in lung injury.

Application of a Magnetic Platform in α6 Integrin-Positive iPSC-TM Purification.

The emergence of induced pluripotent stem cell (iPSC) technology has provided a new approach to regenerating decellularized trabecular meshwork (TM) in glaucoma. We have previously generated iPSC-derived TM (iPSC-TM) using a medium conditioned by TM cells and verified its function in tissue regeneration. Because of the heterogeneity of iPSCs and the isolated TM cells, iPSC-TM cells appear to be heterogeneous, which impedes our understanding of how the decellularized TM may be regenerated. Herein, we developed a protocol based on a magnetic-activated cell sorting (MACS) system or an immunopanning (IP) method for sorting integrin subunit alpha 6 (ITGA6)-positive iPSC-TM, an example of the iPSC-TM subpopulation. We first analyzed the purification efficiency of these two approaches by flow cytometry. In addition, we also determined cell viability by analyzing the morphologies of the purified cells. To conclude, the MACS-based purification could yield a higher ratio of ITGA6-positive iPSC-TM and maintain a relatively higher cell viability than the IP-based method, allowing for the preparation of any iPSC-TM subpopulation of interest and facilitating a better understanding of the regenerative mechanism of iPSC-based therapy.

Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1.

In cancer metastasis, single circulating tumor cells (CTCs) in the blood and disseminated tumor cells (DTCs) in the bone marrow mediate cancer metastasis. Because suitable biomarker proteins are lacking, CTCs and DTCs with mesenchymal attributes are difficult to isolate from the bulk of normal blood cells. To establish a procedure allowing the isolation of such cells, we analyzed the cell line BC-M1 established from DTCs in the bone marrow of a breast cancer patient by stable isotope labeling by amino acids in cell culture (SILAC) and mass spectrometry. We found high levels of the transmembrane protein CUB domain-containing protein 1 (CDCP1) in breast cancer cell lines with mesenchymal attributes. Peripheral blood mononuclear cells were virtually negative for CDCP1. Confirmation in vivo by CellSearch revealed CDCP1-positive CTCs in 8 of 30 analyzed breast cancer patients. Only EpCam-positive CTCs were enriched by CellSearch. Using the extracellular domain of CDCP1, we established a magnetic-activated cell sorting (MACS) approach enabling also the enrichment of EpCam-negative CTCs. Thus, our approach is particularly suited for the isolation of mesenchymal CTCs with downregulated epithelial cancer that occur, for example, in triple-negative breast cancer patients who are prone to therapy failure.

The Impact of TRAIL on the Immunological Milieu during the Early Stage of Abdominal Sepsis

Despite intensive scientific efforts, the therapy of peritonitis is presently limited to symptomatic measures, including infectious source control and broad-spectrum antibiotics. Promising therapeutic approaches to reduce morbidity and mortality are still missing. Within the early phase of abdominal sepsis, apoptosis of neutrophil granulocytes is inhibited, which is linked to tissue damage and septic shock. TNF-related apoptosis-inducing ligand (TRAIL) is a promising agent to stimulate neutrophil apoptosis. However, the underlying mechanisms have not been elucidated so far. The objective of the present study was to characterize the molecular mechanisms of TRAIL-stimulated apoptosis in early abdominal sepsis. Therefore, the murine sepsis model Colon ascendens stent peritonitis (CASP) was applied in wild type (WT) and TRAIL knock-out (TRAIL-/-) C57/BL6j mice. Neutrophil granulocytes were isolated from spleen, blood, bone marrow, and peritoneal lavage using magnetic-activated cell sorting. Neutrophil maturation was analyzed by light microscopy, and apoptotic neutrophils were quantified by fluorescence-activated cell sorting (FACS). Western blot and FACS were used to investigate expression changes in apoptotic proteins and TRAIL receptors. The impact of TRAIL-induced apoptosis was studied in vitro. In septic mice (CASP 6 h), the number of neutrophils in the BM was reduced but increased in the blood and peritoneal lavage. This was paralleled by an increased maturation of neutrophils from rod-shaped to segmented neutrophils (right shift). In vitro, extrinsic TRAIL stimulation did not alter the apoptosis level of naïve neutrophils but stimulated apoptosis in neutrophils derived from septic WT and TRAIL-/- mice. Neutrophils of the bone marrow and spleen showed enhanced protein expression of anti-apoptotic Flip, c-IAP1, and McL-1 and reduced expression levels of pro-apoptotic Bax in neutrophils, which might correlate with apoptosis inhibition in these cells. CASP increased the expression of intrinsic TRAIL in neutrophils derived from the bone marrow and spleen. This might be explained by an increased expression of the TRAIL receptors DR5, DcR1, and DcR2 on neutrophils in sepsis. No differences were observed between septic or naïve WT and TRAIL-/- mice. In conclusion, the present study shows that neutrophil granulocytes are sensitive to TRAIL-stimulated apoptosis in the early stage of abdominal sepsis, emphasizing the promising role of TRAIL as a therapeutic agent.