Methods and Protocols

Background: Developing clinically relevant experimental models of the human airway can significantly advance our understanding of the mechanisms underlying airway diseases and aid in translating potential therapies to clinical settings. The aim of this study is to establish an ex vivo human airway tissue culture model. Methods: Human donor airway tissues were obtained from clinical cases of lung transplantation. Our established method is based on the concept of scavenging metabolic activity and controlling bacterial growth and includes increased media volume, frequent media exchange, and antifungal additives to efficiently maintain the homeostatic culture environment. After a 3-day culture period, the airway was investigated, and its viability and function were compared with a standard cell culture method. Results: Control tissue exhibited significant acidosis after 3 days, suggesting high metabolic activity of airway tissue and bacterial contamination. The airway epithelial viability—after culturing in our established method for 3 days—was better than that of the controls. We only performed an acute but early investigation of the cultures as airway complications have been known to start early at the proximal bronchus after transplantation. H&E and alcian blue staining showed intact morphology of the epithelium of airway tissue and mucus layers after 3 days in our model, while controls showed remarkable damage to the epithelial layer. Newly synthesized glycoproteins were detected in the epithelial layer using metabolic labeling and the click chemistry technique, suggesting cellular protein synthesis of the airway tissue in our established ex vivo model. Conclusions: We successfully established a reproducible model of human ex vivo airway tissue culture (n = 3 independent biological samples) that may be useful for investigating airway complications and developing their therapies. Full article

In the nuclear industry, the decontamination of nuclear metallic structures is an essential process to reduce radiation exposure during maintenance or dismantling. The oxide layer, such as chromium (III) oxide (Cr₂O₃), formed on stainless steel and nickel-based alloys, contributes significantly to surface radioactivity by trapping radioactive contaminants. To address this, permanganic acid (HMnO₄) has proven to be a promising oxidizing agent for dissolving these oxide layers—particularly chromium oxide—on stainless steel and nickel-based alloys. In this study, HMnO₄ was synthesized via ion exchange using AmberLite IRN97 H resin and potassium permanganate (KMnO₄). The optimized process yielded a highly acidic solution (pH~1.6) with potassium concentrations below 0.1 ppm, indicating near-complete exchange efficiency. Dissolution kinetics were investigated at HMnO₄ concentrations ranging from 240 to 1920 ppm and temperatures from 30 °C to 80 °C. At a constant temperature, increasing HMnO₄ concentration significantly improved Cr dissolution, with up to 31% of total chromium solubilized after 33 h. Lower temperatures favored higher dissolution efficiency, likely due to improved thermal stability of HMnO₄. For durations shorter than 4 h, the influence of temperature was limited compared to the effect of acid concentration. To assess post-treatment options, HMnO₄ decomposition was studied using oxalic acid (H₂C₂O₄) at 80 °C. Results showed that a minimum H₂C₂O₄/HMnO₄ molar ratio above 2.75 was necessary to achieve effective reduction while preventing MnO₂ precipitation. However, even under strongly acidic conditions and with a large excess of reductant, Mn²⁺ yields remained below 55%, suggesting that thermal degradation of oxalic acid and possible formation of undetected manganese species limited the reduction process. Full article

Hemodynamics significantly impact the biology of endothelial cells (ECs) lining the blood vessels. ECs are exposed to various hemodynamic forces, particularly frictional shear stress from flowing blood. While physiological flows are critical for the normal functioning of ECs, abnormal flow dynamics, known as disturbed flows, may trigger endothelial dysfunction leading to atherosclerosis and other vascular conditions. Such flows can occur due to sudden geometrical variations and vascular abnormalities in the cardiovascular system. In the current study, a microfluidic system was used to investigate the impact of different flow conditions (i.e, normal vs. disturbed) on ECs in vitro. We particularly explored the relationship between specific flow patterns and cellular pathways linked to oxidative stress and inflammation related to atherosclerosis. Here, we utilized a 2D cell culture perfusion system featuring an immortalized human vascular endothelial cell line (EA.hy926) connected to a modified peristaltic pump system to generate either steady laminar flows, representing healthy conditions, or disturbed oscillatory flows, representing diseased conditions. EA.hy926 were exposed to an oscillatory flow shear stress of 0.5 dynes/cm² or a laminar flow shear stress of 2 dynes/cm² up to 24 h. Following flow exposure, cells were harvested from the perfusion chamber for quantitative PCR analysis of gene expression. Reactive oxygen species (ROS) generation under various shear stress conditions was also measured using DCFDA/H2DCFDA fluorescent assays. Under oscillatory shear stress flow conditions (0.5 dynes/cm²), EA.hy926 ECs showed a 3.5-fold increase in the transcription factor nuclear factor (NFκ-B) and a remarkable 28.6-fold increase in cyclooxygenase-2 (COX-2) mRNA expression, which are both proinflammatory markers, compared to static culture. Transforming growth factor-beta (TGFβ) mRNA expression was downregulated in oscillatory and laminar flow conditions compared to the static culture. Apoptosis marker transcription factor Jun (C-Jun) mRNA expression increased in both flow conditions. Apoptosis marker C/EBP homologous protein (CHOP) mRNA levels increased significantly in oscillatory flow, with no difference in laminar flow. Endothelial nitric oxide synthase (eNOS) mRNA expression was significantly decreased in cells exposed to oscillatory flow, whereas there was no change in laminar flow. Endothelin-1 (ET-1) mRNA expression levels dropped significantly by 0.5- and 0.8-fold in cells exposed to oscillatory and laminar flow, respectively. ECs subjected to oscillatory flow exhibited a significant increase in ROS at both 4 and 24 h compared to the control and laminar flow. Laminar flow-treated cells exhibited a ROS generation pattern similar to that of static culture, but at a significantly lower level. Overall, by exposing ECs to disturbed and normal flows with varying shear stresses, significant changes in gene expression related to inflammation, endothelial function, and oxidative stress were observed. In this study, we present a practical, optimized system as an in vitro model that can be employed to investigate flow-associated diseases, such as atherosclerosis and aortic aneurysm, thereby supporting the understanding of the underlying molecular mechanisms. Full article

This study discusses the challenges encountered in implementing a detailed protocol for upper abdominal imaging using magnetic resonance imaging (MRI), ranging from patient preparation and sequence selection to clinical applications. MRI is a valuable non-invasive imaging modality employed both in the early detection of diseases and as a complementary tool for the detailed characterization of various pathologies. Nevertheless, performing an abdominal MRI examination can be challenging; therefore, the understanding of sequences is particularly important, as changing the parameters can not only influence the quality of the images but also optimize scanning time improve patient experience during the examination. The methodology illustrates the purpose of each sequence and the critical role of appropriate patient preparation. Results highlighted the significance of these factors in the evaluation of hepatic lesions, showing that the proper choice of sequences and parameters is essential for distinguishing benign from malignant findings and for achieving an accurate diagnosis. It was also shown that MRI plays an important role as a complementary technique in investigation of upper abdominal pathologies in order to avoid overexposure to radiation. Full article

The prevalence of low-birth-weight infants has increased over the past 40 years to approximately 9–10% of Japanese live births. This study aims to identify healthy lifestyle behaviors and psychosocial factors contributing to appropriate perinatal outcomes, gestational weight gain, and postpartum weight change. The Japan Pregnancy, Eating, Activity, and Cohort study was initiated in 2020 in Tokyo, Yamagata/Miyagi, Osaka, and Fukuoka. Participants will be enrolled at approximately 12 weeks of gestation, with follow-up at 18–27 and 35–41 weeks of gestation and 1, 6, and 12 months postpartum. Approximately 3000 participants are targeted: Yamagata/Miyagi (n = 300), Tokyo (n = 1500), Osaka (n = 800), and Fukuoka (n = 400). Participants will complete questionnaires on healthy lifestyle behaviors (dietary intake, physical activity, and circadian rhythm), psychosocial factors, weight control, and behavioral intentions. Medical records will be reviewed for antenatal checkup data. The primary outcomes will include gestational weight gain, infant birth weight, perinatal complications, breastfeeding, and postpartum weight change. Demographic and psychosocial factors and lifestyle behaviors will be examined as covariates and potential confounders. Biological samples will be collected in Tokyo and Yamagata. The study’s findings will inform efforts to improve perinatal care guidelines through evidence-based recommendations. Full article

Cancer development in BRCA1 carriers is a multi-step process, which is triggered by several factors and mechanisms that are not clearly understood. Most BRCA1 carriers develop triple-negative breast cancer (TNBC)—estrogen receptor (ER)-, progesterone receptor (PR)-, and HER2 -negative cancers—which originates from ER/PR/HER2-negative breast progenitor cells. Due to a lack of ER/PR/HER2-negative cell models with BRCA mutations, the processes inducing cancer development in BRCA carriers have not been comprehensively studied. Thus, studies characterizing ER/PR/HER2-negative cells carrying a BRCA1 germline mutation are needed to gain more in-depth knowledge about the steps leading to cancer initiation in BRCA1 carriers. To study the cancer development in these patients, we established a protocol for the generation of human ER/PR/HER2-negative breast organoids carrying a BRCA1 germline mutation. We confirmed that these organoids are unresponsive to estrogen, can self-renew, and express the stem/progenitor marker CD44. In addition, we observed that these organoids contain outgrowths that resemble the mature ductal and lobular units of the mammary gland, thus making it a suitable model system to study how cancer develops in ER/PR/HER2-negative mammary cells that carry a BRCA1 germline mutation. Full article

Abnormal adenylosuccinate lyase (ADSL) activity is associated with cancer and neurodevelopmental processes. However, a cell-permeable ADSL inhibitor is not yet available. Our high-throughput screen identified NF-449 as a potential lead compound. To improve cell permeability of the lead compound, fragments of NF-449 were synthesized. This fragment, 2,2’-(1,3-phenylenebis(carbonylimino))-bisbenzenesulfonate, competitively inhibits purified human ADSL with an inhibitory constant of 0.4 micromolar. Its triethylammonium salt inhibited ADSL in HeLa cells with an IC₅₀ of 0.4 micromolar. While this compound might not be ready for in vivo applications yet, further improvement in its permeability might produce useful reagents for in vivo studies of ADSL. Full article

High variability in stem cell research is a well-known limiting phenomenon, with technical variation across experiments and laboratories often surpassing variation caused by genotypic effects of induced pluripotent stem cell (iPSC) lines. Evaluation of kidney organoid protocols and culture conditions across laboratories remains scarce in the literature. We used the original air-medium interface protocol to evaluate kidney organoid success rate and reproducibility with several human iPSC lines, including a novel patient-derived GRACILE syndrome iPSC line. Organoid morphology was assessed with light microscopy and immunofluorescence-stained maturing glomerular and tubular structures. The protocol was further adapted to four microplate-based high-throughput approaches utilizing spheroid culture steps. Quantitative high-content screening analysis of the nephrin-positive podocytes and ECAD-positive tubular cells revealed that the choice of approach and culture conditions were significantly associated with structure development. The culture approach, iPSC line, experimental replication, and initial cell number explained 35–77% of the variability in the logit-transformed proportion of nephrin and ECAD-positive area, when fitted into multiple linear models. Our study highlights the benefits of high-throughput culture and multivariate techniques to better distinguish sources of technical and biological variation in morphological analysis of organoids. Our microplate-based high-throughput approach is easily adaptable for other laboratories to combat organoid size variability. Full article

Introducing fast, reliable, and low-input technologies that utilize wholemeal wheat is essential for efficiently screening gluten quality in wheat breeding lines. Although the GlutoPeak Tester (GPT) has been widely studied for gluten assessment, its application in breeding programs remains underexplored. This study presents a comprehensive approach to optimizing a GPT protocol using a diverse set of genotypes collected over seven harvest years and multiple environments. To improve screening capabilities, a quick and simple protein fractionation (PF) technique was integrated into the workflow. Key GPT parameters—such as peak maximum time, maximum torque, and aggregation energy—along with the newly proposed PM-AM parameter, showed strong correlations with established quality traits. PF data, especially insoluble glutenin percentage and the ratio of insoluble to soluble glutenin, provided additional insights into gluten composition. This extensive dataset supports the use of GPT and PF as a dual, high-throughput screening tool. When applied within specific wheat classes and benchmarked against established checks, this method offers a robust strategy for ranking breeding lines based on gluten performance. The use of wholemeal samples further streamlines the process by eliminating the need for milling, making this protocol particularly suitable for early-stage selection in wheat breeding programs. Full article

Background: Extensive skin injuries from severe burns or chronic non-healing ulcers overwhelm the body’s natural repair mechanisms, while current therapeutic approaches relying on autologous skin grafting are limited by donor site availability. Three-dimensional epithelial spheroid cultures enhance stem cell regenerative potential, but standardized comparative methodologies are lacking. Methods: We established a comprehensive framework comparing scaffold-free and scaffold-based epithelial spheroid systems using HaCaT keratinocytes. High-throughput approaches utilized BioFloat and ELPLASIA 96-well platforms, while low-throughput 6-well ULA plates generated heterogeneous populations (holospheres, merospheres, paraspheres). Scaffold-based studies embedded spheroids in Matrigel to evaluate outgrowth capacity. ROCK1 inhibitor treatment was assessed for stemness enhancement. Results: High-throughput systems generated uniform spheroids with high reproducibility and consistent circularity. Low-throughput cultures produced heterogeneous populations with distinct size distributions (holospheres: 408.7 μm², merospheres: 99 μm², paraspheres: 14.1 μm²). In Matrigel scaffolds, merospheres and paraspheres migrated outward, forming epithelial sheets, while holospheres remained intact as BMI-1+ stem cell reservoirs. ROCK1 inhibition enhanced holosphere formation, preserved stemness markers, and reduced premature differentiation. Conclusions: This standardized toolbox demonstrates scaffold-free systems optimize scalability for screening while scaffold-based approaches enable physiologically relevant regenerative studies. Integration of both methodologies provides flexibility matching experimental design to scientific objectives, accelerating translation to clinical applications. Full article

Accurate quantification of immune cell subpopulations is essential for understanding immune responses in research and clinical settings. Flow cytometry (FC) is widely used for immune cell phenotyping, providing rapid and quantitative single-cell resolution. However, tissue-based pathological assessment offers additional spatial and morphological context that is often necessary for a comprehensive understanding of immune cell distribution. Traditionally, these methods are applied separately to different specimens, limiting direct comparative analysis. Here, we describe a simple combined approach to immune cell quantification that integrates both FC and pathology analysis within the same tissue specimen of colon biopsies. Tissue samples were divided into two portions: one processed into a single-cell suspension for FC-based characterization of CD45⁺, CD3⁺, CD4⁺, and CD8⁺ T cells and another formalin-fixed, paraffin-embedded (FFPE), and analyzed with hematoxylin and eosin (H&E) for eosinophils and immunohistochemistry (IHC) for CD4 and CD8. A pilot analysis of 10 samples shows high concordance of the results taken from the two methods, allowing for cross-validation between methodologies and improved diagnostic accuracy. This proof-of-principle study demonstrates the feasibility of an integrated workflow that combines FC and pathology for immune cell quantification, which provides assessment of immune cell populations from the limited material of intestinal biopsies with potential for improved diagnostic accuracy. Full article

Patient-derived organoids (PDOs) have emerged as powerful tools in personalized medicine applicable to both non-malignant conditions and to cancer, where they are increasingly used for personalized drug screening and precision treatment strategies in part due to their ability to replicate tumor heterogeneity. They also serve as study model systems to understand disease mechanisms, pathways, and the impact of ex vivo exposures. We present a detailed step-by-step protocol for generating organoids from normal crypts, polyps, and tumors, including methods for tissue processing, crypt isolation, culture establishment, and the transition from basolateral to apical-out polarity for co-culture and exposure-based studies. The protocol also includes immunofluorescence staining procedures for cellular characterization and quality control measures. Our standardized approach successfully generates organoids from diverse colorectal tissues with high efficiency and reproducibility. This comprehensive guide addresses common technical challenges and provides troubleshooting strategies to improve success rates across different sample types. We believe that this resource will enhance reproducibility in organoid research and expand their utility in translational applications, particularly for personalized medicine approaches in colorectal cancer. Full article

Maintaining the integrity of cryogenically preserved biological materials is critical, as even brief, undetected temperature excursions in storage can compromise sample viability. Existing monitoring systems may miss transient thaw–refreeze events, posing serious quality risks. To address this, we developed and validated frozen indicator tubes that visually signal deviations from the frozen state, serving as a cost-effective backup to electronic monitors. Our first method uses an aqueous dye solution that immobilizes the dye when frozen; any thawing causes the dye to disperse, providing a clear, external visual cue of a partial or complete thaw. For ultra-low-temperature storage (−80 °C), we introduced a second method using an ethanol-based solution calibrated to indicate thaw events. This system detects temperature rises of 10 °C or more sustained for at least fifteen minutes—conditions that may jeopardize sample stability. When paired with standard monitoring systems, these indicator tubes offer an added layer of protection by providing simple, reliable, and immediate visual confirmation of critical temperature breaches. This innovation enhances confidence in cryogenic storage protocols and supports the long-term preservation of sensitive biological materials. Full article

This study aimed to evaluate the placement accuracy and reproducibility of Surface Guided Radiotherapy (SGRT) compared with the conventional tattoo/laser method in patients undergoing radiotherapy for abdominal malignancies. A retrospective analysis was conducted on 43 patients treated with either SGRT (Group A) or the tattoo/laser technique (Group B). Patients in both groups underwent CBCT to quantify the positioning shifts in the vertical (Svrt), lateral (Slat) and longitudinal (Slng) axes, as well as the total shift. Statistical indicators including median, interquartile range (IQR), and range were calculated, and Mann–Whitney U tests were performed due to non-normal data distribution. Median values in all axes were same between groups: Svrt = 0.4 cm, Slat = 0.2 cm, Slng = 0.4 cm. Group A showed a higher total median shift equal to 0.8 cm versus 0.7 cm of Group B. However, IQRs were smaller in the Group B for all directions and total shift, indicating greater method consistency. Statistically significant differences (p < 0.05) were observed in all axes, except the vertical. These findings suggest that, while SGRT achieves comparable median alignment, its use in a highly variable anatomical region such as the abdomen may be associated with greater setup variability. Full article

The hyperinsulinemic–euglycemic clamp technique is considered the gold standard for measuring insulin sensitivity in large animals. We developed a practical method for conducting concurrent glucose clamp experiments in a pair of sedated farm swine positioned in a sling. Descriptions of customized equipment and central venous access surgical procedures for blood collection are provided. Personnel functions are described for execution of the clamp protocol. A total of 24 hyperinsulinemic–euglycemic clamp studies were performed over 6 weeks. Infusaports remained functional for 1454 blood samples. There were three CSII catheter occlusions during bolus administration, and the swine showed no signs of infection or disease. IM telazol at 1.0 mg/kg, administered 1–2 h prior (mean of 3.26 mL ± 1.59) was effective in keeping animals comfortable. SpO₂ and heart rate remained within normal ranges. Means ± SD total infused volumes for octreotide, 10% dextrose, and saline were 9.7 ± 0.93 mL, 2328.0 ± 672.8 mL, and 690.3 ± 206.8 mL. Mean blood glucose was maintained between 75.7 and 87.8 mg/dL (CV 3.17%) for the 24 experiments. The GIR infusion rate peaked between 15 and 60 min after insulin bolusing, with insulin C_max of 108.5 pmol/L and t_max at 10 min. All aspects of the protocol were effectively carried out. The animals remained in good health, and the implanted infusion ports remained patent for over 700 blood draws per animal. This method could potentially reduce the number of animals used and the costs of other similar experiments. Full article

Major depressive disorder (MDD) is a prevalent and disabling condition. Transcranial direct current stimulation (tDCS) may improve symptoms by modulating neuroplastic and inflammatory mechanisms. This randomized, double-blind, placebo-controlled trial will recruit adult outpatients with MDD showing residual symptoms despite at least four weeks of stable SSRI treatment. Participants will be randomized to active or sham add-on tDCS while continuing their antidepressant regimen. The intervention will consist of 15 sessions over 3 weeks, targeting the left dorsolateral prefrontal cortex (anode F3, cathode F4) at 2 mA for 30 min per session. The primary outcome is the reduction of depressive symptoms measured by the Hamilton Depression Rating Scale-17 (HDRS), with remission defined as HDRS-17 ≤ 7. Secondary outcomes include cognitive performance (attention, executive functioning, memory), serum biomarkers (BDNF, VEGF, NGF, NRG1, angiogenin, IGF1, IL-6, TNF-α), cortical excitability assessed by transcranial magnetic stimulation (motor threshold, silent period, intracortical inhibition/facilitation), and cerebral hemodynamics by transcranial Doppler sonography (blood flow velocity, pulsatility, resistivity). Assessments will occur at baseline, post-treatment, and 3- and 6-month follow-ups. This trial aims to evaluate the efficacy of adjunctive tDCS in MDD with residual symptoms and its biological correlates, bridging clinical improvement with electrophysiological and neurovascular mechanisms. Full article

The bioavailability of a drug is closely linked to its solubility, making its early determination essential in drug development. The saturation shake-flask (SSF) method is the gold standard protocol for this, which includes a phase separation step—either by sedimentation, filtration, or centrifugation. This step is critical, as it can directly influence the accuracy of the results. This study investigated the impact of centrifugation parameters—time and rotation speed—on solubility measurements. Additionally, we compared two sample preparation protocols: continuous stirring for 24 h versus 6 h of stirring followed by 18 h of sedimentation before centrifugation. Four model compounds were tested at three pH values using Britton–Robinson buffers. Centrifugation was conducted for 5, 10, or 20 min at either 5000 or 10,000 rpm. Results showed that pre-sedimented samples yielded solubility values closer to sedimentation-only references, while continuous stirring often led to overestimated values, particularly at higher speeds and longer durations. One such example was papaverine hydrochloride, that showed solubility values 60–70% higher than the reference after centrifugation at 10,000 rpm for 20 min without prior sedimentation. Lower standard deviations were observed with shorter, slower centrifugation, with 5 min and 5000 rpm yielding results closest to the reference values. Full article

Antimicrobial resistance is a global health issue, and the WHO has made significant efforts in the development of tools for its monitoring. However, such tools are underutilized, due to limited knowledge, technical capacity, and scarcity of economic resources. AMR surveillance can be conducted using WHOnet and R, two free-of-charge software tools widely adopted in both clinical practice and scientific research. WHOnet is designed for managing laboratory data and antimicrobial susceptibility test results, while R is a programming language dedicated to statistical computing and data visualization. The combined use of these tools enables a reproducible workflow for retrospective AMR trend analysis. This paper provides step-by-step instructions on how to perform such analysis and also provides the respective R code. The described code and software results are shown using real-world data from an Italian hospital as an example. The standardization of the analysis process and the rapid availability of data on antimicrobial resistance are critical for both clinicians and public health professionals. They would allow for empirical decisions on antimicrobial treatment based on the specific epidemiological characteristics of the hospital or community setting. Full article

Dermal tissue samples are a rich source of germplasm because they can be readily collected, frozen as part of a genebank collection, digested and cultured, and used for a variety of purposes such as genotyping or other forms of genetic research. Derived fibroblasts can also be used for somatic cell nuclear transfer, and the remaining cells can be frozen for future use. However, collection of tissues with ear notchers, scalpels, or biopsy punches can be problematic because tissue handling and the tool surfaces can contaminate the samples. Therefore, the modification of the Allflex Tissue Sampling Unit (TSU) system was explored to determine if the technology can empower rapid collection of clean samples that are easily identifiable and portable. Results indicate that the TSU system was efficient, and samples that were collected and processed for tissue culture resulted in successful derivations of fibroblasts from 7 of 11 animals. Thus, the TSU system appears to be a viable option for collecting and preserving dermal tissue for genebanking and other applications where simple, rapid collection of large quantities of samples is required. Full article