Methods and Protocols最新文献

{"title":"Agarose-Based 3D Invasion Assay for Simultaneous Quantification of Tumor Cell Invasion and Extracellular Matrix Degradation.","authors":"Andreas R Thomsen, Pascaline Kouam-Daniel, Bettina Priesch-Grzeszkowiak, Anja Grillenberger, Sandra Kumbruch, Ali H Acikelli, Helmut Bühler, Christian Baues","doi":"10.3390/mps9040112","DOIUrl":"10.3390/mps9040112","url":null,"abstract":"<p><p>Tumor cell invasion is a critical step in local tumor progression, recurrence, and metastasis. Conventional two-dimensional migration assays and many existing three-dimensional invasion models often assess cell migration, invasion into the extracellular matrix and matrix degradation as separate endpoints, although these processes are tightly coupled in vivo. Therefore, robust and reproducible in vitro models are needed to investigate tumor cell invasion under defined extracellular matrix conditions. We developed an agarose-based three-dimensional invasion assay, termed the Freiburg 3D invasion assay, for the simultaneous analysis of tumor cell migration, invasion, and extracellular matrix degradation. The system consists of a 2.8% agarose matrix containing defined microcavities connected by a common loading channel. Tumor cells are seeded into these microcavities, where they form compact cell aggregates. The cavities are subsequently filled with collagen type I or extracellular matrix gel. After polymerization, the matrix-containing agarose strips are transferred into parking pockets, cultured for several days, and monitored by microscopy. Invasion distance, single-cell migration, and ECM-cleared area are quantified from serial microscopic images using image analysis software. The system distinguished weakly invasive MCF7 breast cancer cells from highly invasive MDA-MB-231 cells. In addition, treatment with a protease inhibitor and irradiation reduced tumor cell invasion and extracellular matrix remodeling, demonstrating the suitability of the assay for pharmacological and radiation-response studies. The Freiburg 3D invasion assay provides a practical and reproducible three-dimensional in vitro model for analyzing tumor cell invasion and protease-associated extracellular matrix degradation.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515452/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single Radial Immunodiffusion (SRID) Assay for Quantitative Determination of Recombinant SOD Protein in a Brucellosis Vaccine Candidate: Method Development and Validation.","authors":"Gulnur Nakhanova, Olga Chervyakova, Kamshat Shorayeva, Aigerim Zhakypbek, Sabina Moldagulova, Aknur Ulankyzy, Alisher Omurtay, Yeraly Shayakhmetov, Temirlan Baiseit, Zharkinay Absatova, Aisha Issabek, Gaukhar Shynybekova, Sandugash Sadikaliyeva, Aziz Nakhanov, Kuanysh Jekebekov, Ainar Kossylganova, Karlygash Zhaparkulova, Assem Kalykova, Tolkyn Bekezhanova, Albina Atakanova, Zakir Yershebulov, Kuandyk Zhugunissov","doi":"10.3390/mps9040113","DOIUrl":"10.3390/mps9040113","url":null,"abstract":"<p><p>Recombinant superoxide dismutase (SOD) protein is a promising antigen candidate for brucellosis vaccines, and its quantitative determination is essential for vaccine quality control, standardization, and regulatory compliance. Although enzyme-linked immunosorbent assay (ELISA) is widely used for antigen quantification, its application to vaccine formulations may be limited due to chemical modifications of antigenic determinants occurring during detoxification and adjuvantation processes. Therefore, alternative analytical methods, such as single radial immunodiffusion (SRID), which enable direct antigen quantification independent of antigenic determinant modifications, represent valuable tools for vaccine analysis. The aim of this study was to develop and validate a single radial immunodiffusion (SRID) assay for the quantitative determination of recombinant SOD protein and to obtain hyperimmune sera in sheep. Sheep were immunized with recombinant SOD protein formulated with aluminum hydroxide or AddaS03 adjuvants. Antibody responses were evaluated using agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). Immunization with aluminum hydroxide induced higher antibody titers than AddaS03. By day 42, ELISA titers reached 1:25,600 in the aluminum hydroxide group compared with 1:3200 in the AddaS03 group. The obtained hyperimmune sera were used for SRID assay development. The optimized SRID assay demonstrated high reproducibility and specificity, with no cross-reactivity against unrelated proteins. A linear relationship was observed between the square of the precipitation ring diameter (D²) and antigen concentration (R² = 0.956-0.989). The assay showed a limit of detection of 0.312 μg/mL, a linear range of 0.625-10 μg/mL, high precision (CV < 2%), and robustness under varying analytical conditions. The developed SRID assay represents a specific, reproducible, and robust tool for quantitative control of recombinant SOD protein in vaccine formulations.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515976/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830543","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A One-Step RT-PCR-Coupled Cysteamine-Functionalized Gold Nanoparticle Assay for Colorimetric Detection of Tobacco Mosaic Virus.","authors":"Thuy-Duong Thi Tran, Quy Thi Vu, Hoa Thi Hoang, Phan Thi Ngoc Hoa, Nguyen Pham Thi Thao, Truong T N Lien","doi":"10.3390/mps9040111","DOIUrl":"10.3390/mps9040111","url":null,"abstract":"<p><p>Viral diseases are one of the most destructive threats to global agriculture. Among plant viruses, tobacco mosaic virus (TMV) is a highly contagious pathogen infecting numerous economically vital crops. With no curable treatments, the only strategy to mitigate virus spread is early detection and plant removal. The gold standard for TMV detection is reverse transcriptase-polymerase chain reaction (RT-PCR), followed by agarose gel electrophoresis. To reduce TMV diagnosis time and eliminate the requirement for expensive equipment, this study developed and optimized a one-step RT-PCR-coupled cysteamine-functionalized gold nanoparticle assay for the colorimetric determination of the virus. By integrating cDNA synthesis and PCR amplification into a single tube and analyzing the results using cysteamine- functionalized gold nanoparticles (Au@Cys), the diagnosis turnaround time was significantly reduced. Furthermore, this AuNPs-based colorimetric assay enabled straightforward visual inspection with the naked eye, eliminating the need for costly optical devices. Additionally, our regression equation linking RT-PCR product color values, extracted as mean A value based on the CIELAB color space (green-red axis), and viral load allows for the accurate quantification of TMV infection levels in field samples. Our research lays the groundwork for the further development of more cost-effective, quantitative and rapid plant virus diagnosis methods.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515312/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gas Chromatography Method for Quantitation of Residual Solvent Impurities in Nanoformulations.","authors":"Krishna Kattel, Rachael M Crist, Jeffrey D Clogston","doi":"10.3390/mps9040110","DOIUrl":"10.3390/mps9040110","url":null,"abstract":"<p><p>The development and validation of a sensitive, rapid, and specific gas chromatography method for the evaluation of 19 common Class 2 and Class 3 solvents frequently used in nanomedicine formulation is described. Method validation was performed using PerkinElmer's headspace gas chromatograph system with flame ionization detection and an Elite 624 Crossbond 6% cyanopropylphenyl-94% dimethylpolysiloxane or DB-Fatwax-Ultra Inert column with helium as the carrier gas. Validation characteristics such as linearity, spike recovery, method precision, specificity, sensitivity, limit of detection/quantitation, and analyte stability were evaluated. The validated methods showed excellent linearity, with a correlation coefficient > 0.99, and good precision, with intra-day precision < 7.4% for all tested analytes. The percent recoveries ranged 83-104% within the method's quantitation range. In comparison to previously reported methods, the current method has a much shorter equilibration time, higher sensitivity, better separation for many solvents, and a wide concentration detection range. The current method is also perfectly suitable to analyze short chain fatty acids such as formic acid, acetic acid, butyric acid, and valeric acid without requiring additional extraction or derivatization steps. Notably, the method was found to be suitable for analysis of formic acid-a common solvent in certain nanoformulations and one in which there is no prior gas chromatography method available which does not require this additional sample manipulation-down to approximately 75 ppm. Herein, the method is demonstrated using various nanoformulations, including the commercial Doxil formulation as well as several research nanoformulations, including polymeric, cross-linked polymeric, and dendrimer platforms.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13515910/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148830259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Simple Method to Evaluate the Length of Poly(A) Tails in mRNA.","authors":"Jonas Mumenthaler, Maximilian Feldmann, Shahab Mamaghani, Rocco Roberto Penna, Julia Frei, Natalia Jarzebska, Mark Mellett, Emmanuella Guenova, Thomas Kündig, Severin Lauchli, Steve Pascolo","doi":"10.3390/mps9040109","DOIUrl":"10.3390/mps9040109","url":null,"abstract":"<p><p>Synthetic mRNA produced by in vitro transcription is the active pharmaceutical ingredient of approved vaccines and of many drugs under development. It typically contains a 3' poly(A) tail required for optimal stabilization and translation of the mRNA. The average length of the poly(A) tail in the produced mRNA cannot be precisely predicted and consequently must be measured using complicated technologies such as reverse transcription and sequencing or cleavage of the poly(A) tail and analysis by capillary electrophoresis or mass spectrometry. We report an accelerated method to evaluate the average poly(A) tail length in synthetic mRNA, which benefits from the fact that thiazole orange is fluorescent only when in close proximity of nucleic acid. An oligo(dT)₁₂ oligonucleotide having thiazole orange at its extremities emits a fluorescence signal proportional to the amount of poly(A) sequence available. Using a titration curve made with known amounts of poly(A) RNA oligonucleotide, the thiazole orange oligo(dT)₁₂ oligonucleotide can instantly indicate the average length of the poly(A) tail in an mRNA sample. This fast and easy method can be used in any laboratory to determine the size of the poly(A) tail in in vitro-transcribed mRNA produced for research, pre-clinical studies and clinical trials.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398250/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579371","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preliminary Evaluation of the Virtual Reality-Based Gait Sensory Interaction Test (GaitSIT) for Quantifying Sensory Reweighting During Walking Balance.","authors":"Priyo Ranjan Kundu Prosun, Shafique Chaudhry, Masudul H Imtiaz, Poorna Raavi, David DiSalvo, Kwadwo O Appiah-Kubi","doi":"10.3390/mps9040108","DOIUrl":"10.3390/mps9040108","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;b&gt;Background:&lt;/b&gt; Walking is a dynamic activity that relies on inputs from the multisensory system, i.e., somatosensory, vision, and vestibular. These inputs are processed and integrated in the central nervous system to produce motor impulses for efficient walking balance. The Sensory Organization Test (SOT) is established as the gold standard for assessing sensory contributions to standing balance. However, no comparable assessments have been developed for the clinical evaluation of balance during gait. This study evaluated the Gait Sensory Interaction Test (GaitSIT), a novel virtual reality (VR)-based assessment for characterizing sensory-condition-specific changes in walking balance. &lt;b&gt;Methods:&lt;/b&gt; The GaitSIT comprises a VR environment with a physical compliant foam walking surface that evaluates gait-balance by systematically manipulating and evaluating the sensory systems. Twenty-nine healthy young adults (mean age 24.9 ± 6.4 years) were instructed to complete 6 m walking trials under six standardized conditions (C): eyes open, eyes closed/dark scene, and rotating visual scenes on a firm surface, then repeated on a foam surface. Wearing an Oculus VR headset, participants were instructed to walk in a straight line at their preferred speed, as naturally as possible, in two test sessions on the same day, followed by a third test session 24 h later. Headset-derived sway measures, including position, velocity, and acceleration data, were recorded, and the continuous trajectory deviation angle (i.e., directional control) and sensory ratios were calculated. Linear mixed-effects models included trial-level walking speed as a covariate. Additionally, participants completed the modified Clinical Test of Sensory Interaction on Balance (mCTSIB) as a clinical standing-balance reference measure; its concurrent-validity findings will be reported separately. &lt;b&gt;Results:&lt;/b&gt; Significant condition effects were observed for position, velocity, acceleration, and CTDA after adjustment for trial-level walking speed (all p&lt;0.001), indicating that the six sensory conditions elicited distinct gait-balance responses. Significant differences relative to the baseline condition (C1) were observed across conditions C2-C6 for position, C3-C6 for velocity, and C2 and C5 for acceleration. Session effects were not significant for any primary kinematic outcome after speed adjustment. A significant condition × session interaction was observed for position (p&lt;0.001), whereas velocity, acceleration, and CTDA demonstrated no significant interactions. Walking speed was significantly associated with position, acceleration, and CTDA, but not velocity. Sensory-ratio analyses revealed larger visual and vestibular ratios relative to somatosensory ratios, with the visual and vestibular ratios generally decreasing across sessions. &lt;b&gt;Conclusions:&lt;/b&gt; GaitSIT successfully manipulated sensory conditions during overground walking and produced significant changes in gait-related swa","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398260/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579374","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Selection and Characterization of Cell Line-Virus Pairs for Sensitive Viral Detection Assays in Biopharmaceutical Testing.","authors":"Agnieszka Staniszewska, Agnieszka Piastowska-Ciesielska","doi":"10.3390/mps9040107","DOIUrl":"10.3390/mps9040107","url":null,"abstract":"<p><p>Ensuring viral safety is a critical aspect of biopharmaceutical production, requiring sensitive and reliable methods for detecting adventitious agents. In this study, we systematically evaluated the performance of selected cell line-virus combinations to identify optimal models for in vitro viral detection assays. Three cell lines (Vero, MRC-5 and BHK-21 [C-13]) and representative model viruses (Reovirus type 3, Adenovirus type 5, Human parainfluenza virus type 3, and Herpes simplex virus) were analyzed in terms of cytopathic effect (CPE) kinetics, morphology, and detection sensitivity. All tested systems demonstrated high analytical sensitivity, with limits of quantification (LOQ) reaching 0.01 TCID₅₀/mL for selected viruses. However, substantial differences were observed in infection dynamics and CPE morphology depending on the cell line-virus combination. BHK-21 [C-13] cells exhibited the most rapid and pronounced CPE for Reovirus type 3, enabling early and unambiguous detection. Vero cells provided robust and reproducible detection of Adenovirus type 5, characterized by well-defined cytopathic progression. MRC-5 cells showed controlled and consistent infection kinetics for both Human parainfluenza virus type 3 and Herpes simplex virus, allowing improved temporal resolution and interpretability. These findings demonstrate that assay performance depends not only on sensitivity but also on the kinetics and morphology of infection. Based on combined evaluation criteria, the following optimal cell line-virus pairs were identified: BHK-21 [C-13]/Reovirus type 3, Vero/Adenovirus type 5, and MRC-5/Human parainfluenza virus type 3 and Herpes simplex virus. The proposed approach supports rational selection of detection models and provides a preliminary descriptive framework for the development of routine visual screening assays in biopharmaceutical quality control.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398311/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579343","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simplified and Rapid Preparation Protocol for Producing <i>Aloe Vera</i>-Based Natural Coagulant for Water Treatment.","authors":"Danieli Soares de Oliveira, Clainer Bravin Donadel","doi":"10.3390/mps9040106","DOIUrl":"10.3390/mps9040106","url":null,"abstract":"<p><p>Natural coagulants have emerged as potential alternatives to synthetic chemicals in water treatment, especially for decentralized and low-resource applications. However, many previously reported <i>Aloe vera</i>-based coagulant preparation methods rely on drying, powder production, distilled water extraction, refrigeration, or other laboratory-dependent procedures that increase operational complexity and limit practical implementation. This study presents a simplified and rapid protocol for producing an <i>Aloe vera</i>-based natural coagulant using accessible materials and simplified preparation steps. The proposed methodology consists of extracting <i>Aloe vera</i> g13el, homogenizing 2 g of fresh gel with 50 mL of tap water using a household blender, and applying simple paper filtration to obtain the liquid coagulant. The protocol can be completed in less than 10 min without specialized laboratory infrastructure, energy-intensive processing, or laboratory-grade reagents. Coagulation performance was evaluated using synthetic turbid water with initial turbidity levels of 100, 200, and 300 NTU. Significant turbidity reduction was observed under all tested conditions, with several samples reaching residual turbidity values close to or equal to 0 NTU after 50-60 min of sedimentation. The results demonstrate the potential of the proposed protocol as a rapid, reproducible, and accessible approach for future investigation in point-of-use and decentralized water treatment applications.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13397813/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Improved Method for the Isolation of Extrachromosomal DNA from the Pathogenic Free-Living Amoeba <i>Naegleria fowleri</i>.","authors":"Colm P Roster, James C Morris","doi":"10.3390/mps9040105","DOIUrl":"10.3390/mps9040105","url":null,"abstract":"<p><p>The pathogenic free-living amoeba <i>Naegleria fowleri</i> is the cause of primary amebic meningoencephalitis (PAM), a central nervous system infection that is almost always lethal. One of the unusual features of the amoebae is the presence of ~4000 copies of a nucleolar-localized closed circular extrachromosomal ribosomal DNA element (CERE) that encodes the cell's ribosomal RNA repertoire. It has historically been challenging to purify large quantities of CERE, limiting our understanding of the nucleic acid. Here, we describe a methodology for CERE purification that improves yield, reduces processing times, and maintains the integrity of the plasmid. This approach will enable the study of this unique DNA architecture, advancing our understanding of the pathobiology of the organism.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398248/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579416","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Non-Healing Wound Model in Diabetic C57BL/6 Mice.","authors":"Lyubov A Rzhanova, Ekaterina V Kuzmenko, Alena A Permyakova, Andrei A Riabinin, Evgenii S Ruchko, Maria B Chernysheva, Ekaterina A Vorotelyak, Elena I Morgun","doi":"10.3390/mps9040104","DOIUrl":"10.3390/mps9040104","url":null,"abstract":"<p><p><b>Background</b>: This study focuses on developing a model of a non-healing wound that recapitulates the pathogenesis of the corresponding human pathology. <b>Methods</b>: A non-healing wound was modeled in mice with streptozotocin-induced diabetes. The following parameters were assessed: re-epithelialization, epidermal hypertrophy, wound contraction, relief index, angiogenesis, and granulation tissue maturation. These parameters were compared between diabetic mice and healthy controls. <b>Results</b>: The proposed model demonstrated a significant delay in regenerative processes compared to healthy animals. <b>Conclusions</b>: These findings <b>support the relevance</b> of this model to human pathology and <b>indicate that it may be applicable</b> for preclinical studies of drugs aimed at promoting wound regeneration.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"9 4","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13398050/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148579385","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}