Jove-Journal of Visualized Experiments最新文献

{"title":"Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates.","authors":"Matthew J Johnson, Anthony P DeFeo, Nicholas J Slipek, Timothy D Folsom, Tom Henley, Modassir S Choudhry, Beau R Webber, Branden S Moriarity","doi":"10.3791/68150","DOIUrl":"https://doi.org/10.3791/68150","url":null,"abstract":"<p><p>Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mouse Model of Coronary Collateral Growth Through Repetitive Ischemia.","authors":"Molly Enrick, James Gadd, Katie Yanez, Liya Yin","doi":"10.3791/66127","DOIUrl":"https://doi.org/10.3791/66127","url":null,"abstract":"<p><p>Coronary collaterals are a natural bypass in ischemic heart diseases (IHD), and so for many years, coronary collateral growth (CCG) has been a promising therapeutic target for IHD, particularly in patients with type 2 diabetes or metabolic syndrome in which CCG is impaired. However, this process is understudied, partly due to the lack of mouse models of CCG, even though other animal models, such as pigs, dogs, and rats, have been established. A mouse model can take advantage of the many genetic modifications available for the species, including lineage tracing and gene regulation (overexpression or knockout), to elucidate the process and mechanism of CCG, including the pathways and cell types involved.  We, therefore, set out to develop a mouse model of CCG induced by repetitive ischemia (RI) via transient, repetitive occlusion of the left anterior descending artery (LAD). This manuscript provides details of this mouse CCG model, including the RI surgery to implant a pneumatic occluder on the LAD, the automated pressure-based inflation system used for controlling the pressure and timing of inflation, and the sequence of the RI protocol. This method has already generated one publication to elucidate the process of CCG induced by RI, showing that sprouting angiogenesis gives rise to mature coronary arteries in CCG in adult mouse hearts.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152475","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of a Novel Hyaluronan Hydrogel for Three-Dimensional Follicle Culture and Methodology for Mouse Ovarian Follicle Cryopreservation.","authors":"Nina Desai, Megan Attard, Maribeth Spangler, Arsela Gishto, Alyssa Brown, Megan Wirtjes","doi":"10.3791/67786","DOIUrl":"https://doi.org/10.3791/67786","url":null,"abstract":"<p><p>The 3-D architecture of the ovarian follicle and the complex interactions between somatic cell components and the oocyte that are necessary for cytoplasmic and nuclear maturation are difficult to maintain in conventional two-dimensional (2-D) culture systems. We describe a novel 3-D culture model using a tyramine-linked hyaluronan hydrogel for encapsulation and culture of mouse ovarian follicles. The hyaluronan encapsulation technique allows 3-D growth of follicles and retention of trophic factors in close proximity to the developing follicles. This hydrogel is highly versatile and can be applied to isolated follicles as well as ovarian tissue fragments. The viscoelastic properties of the HA gel enable adjustment of rigidity as well as moldability based on gel concentration. Preantral follicles developing in this culture model are able to complete meiotic maturation within 10-12 days of culture and ovulate a metaphase II oocyte upon triggering with hCG. This paper also details two approaches to ovarian follicle cryopreservation by vitrification.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152463","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors.","authors":"Vitalis Mbayo, Penny P Govender, Ephraim F Marondedze, Krishna K Govender","doi":"10.3791/67457","DOIUrl":"https://doi.org/10.3791/67457","url":null,"abstract":"<p><p>The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in Southern Africa. The development of resistance to non-nucleotide reverse transcriptase inhibitors (NNRTIs) threatens the long-term success of antiretroviral therapy. In 2019, antimicrobial resistance directly accounted for an estimated 1.27 million deaths globally. This study employed an in-silico approach to investigate NNTRI drugs and their derivatives. Techniques used included density functional theory calculations, molecular docking, enumeration, quantitative structure-activity relationship (QSAR) analysis, molecular dynamics simulation (MDS), and molecular mechanics with generalized Born and surface area methods. The analysis focused on various pyrimidine derivatives and six NNRTI drugs, examining their interactions with the HIV-1 protein (PDB code 1HQU). A QSAR model was developed to predict the biological activity of the six NNRTIs under investigation. Using 94 pyrimidine derivatives, the QSAR model achieved an R² OF 0.822 and a Q² of 0.815, indicating a high level of predictive accuracy. MDS were conducted to assess the stability of various ligands and their newly developed alternatives, ensuring they remained bound to the protein's active site over a 200-nanosecond simulation timeframe. Etravirine exhibited root mean square deviation (RMSD) fluctuations of approximately 4.5 Å, while its enumerated derivatives showed RMSD fluctuations of 3.5 Å. Through molecular docking, MDS, and free energy computations, enumerated Etravirine demonstrated the best performance, with an activity value of 7.373 and a docking score of -10.517 kcal/mol. Furthermore, the calculated free energy of binding for enumerated Etravirine was -89.684 kcal/mol, outperforming other ligands under investigation. The significant improvement suggests that the modified Etravirine holds promising potential as a novel agent in antiretroviral therapy. The obtained lower RMSD value, the enhanced amino acid interactions, and the highest binding free energy indicate that enumerated Etravirine could serve as a viable alternative for HIV/AIDS treatment.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152483","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Protocol for Explant Cultures of IDH1-mutant Diffuse Low-grade Gliomas.","authors":"Kasandra Aguilar-Cázarez, Donovan Pineau, Leonor Garcia, Guo-Hao Huang, Sheng-Qing Lv, Hugues Duffau, Jean-Philippe Hugnot","doi":"10.3791/67260","DOIUrl":"https://doi.org/10.3791/67260","url":null,"abstract":"<p><p>Diffuse gliomas, the most prevalent primary brain tumors, are categorized into low and high grades based on histopathological criteria and genetic mutations. Diffuse low-grade gliomas constitute a subset, predominantly afflicting young adults and predisposing to high-grade gliomas. There are two main types of diffuse low-grade gliomas: astrocytomas and oligodendrogliomas, each defined by distinct genetic and histological features. These tumors characteristically exhibit a mutated variant of the metabolic enzyme IDH1, disrupting cellular differentiation. They are heterogeneous, comprising both proliferative stem cells and more differentiated, quiescent glial cells. Patient-specific, in vitro tumor models hold significant potential for unraveling oncogenic mechanisms and tailoring drug selection for personalized therapy. However, the absence of reliable in vitro models specifically for IDH1-mutant low-grade gliomas has hindered fundamental and translational research. Here, we present a simple method for culturing IDH1-mutant tumors as explants in defined media without needing enzymatic dissociation. These explants can be sustained for extended periods, maintaining IDH1-mutant protein expression as well as markers for oligodendrocyte progenitor cells such as ASCL1 (MASH1), OLIG1, and OLIG2 and for astrocytes (GFAP, SOX9). They can also be established from frozen tumors in DMSO. These cultures can serve as a valuable platform for drug screening, videomicroscopy, and gene studies, thus facilitating advancements in understanding and treating these challenging tumors. They also offer the potential to derive IDH1-mutant cell lines.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Laparoscopic S7 Hepatectomy with Positive Fluorescence Staining.","authors":"Fa Luo, Xianbo Wu, Wei Li, Haixiong Zhang","doi":"10.3791/67493","DOIUrl":"https://doi.org/10.3791/67493","url":null,"abstract":"<p><p>Laparoscopic liver resection for tumors located in the S7 segment of the liver typically adopts a traditional surgical approach. The main goal of this procedure is to accurately dissect the liver pedicle of the S7 segment. Dissecting the hepatic pedicle of the S7 segment along the hepatic hilum requires a relatively long path within the liver, which increases the risk of losing orientation and potentially injuring the adjacent hepatic pedicle of the S5 and S6 segments, thereby compromising the liver resection plane. A positive staining method was used to directly puncture the corresponding portal vein under ultrasound guidance (commonly for S7 and S8 segments) to specifically color the target liver segment, thereby avoiding extensive resection of the liver parenchyma, and reducing damage to the surrounding healthy liver tissue. However, the positive staining method requires a specific foundation in intraoperative procedures, which can be challenging for surgeons and has a certain learning curve. Currently, technologies such as three-dimensional reconstruction territory analysis, intraoperative ultrasound, and indocyanine green fluorescence imaging are popular and commonly used in laparoscopic liver resection. In this protocol, under laparoscopic ultrasound guidance, the tumor basin was punctured through the visceral and diaphragmatic surfaces of the liver to stain segment S7. Laparoscopic resection of segment S7 within the portal territory anatomic liver was successfully performed, further confirming the feasibility and advantages of positive fluorescence staining in laparoscopic liver resection at this stage.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluid-cell Raman Spectroscopy for operando Studies of Reaction and Transport Phenomena during Silicate Glass Corrosion.","authors":"Mara I Lönartz, Lasse Stausberg, Gerrit Trapp-Müller, Lars Dohmen, Christoph Lenting, Moritz B K Fritzsche, Thorsten Geisler","doi":"10.3791/67763","DOIUrl":"https://doi.org/10.3791/67763","url":null,"abstract":"<p><p>Fluid-cell Raman Spectroscopy (FCRS) enables the real-time and space-resolved (operando) study of reaction mechanisms, kinetics, and their mutual interactions with transport processes during silicate glass corrosion at the micrometer scale and at elevated temperatures. This manuscript provides a detailed protocol for setting up an FCRS experiment, exemplified by a corrosion experiment with a ternary Na borosilicate glass and a 0.5 M NaHCO3 solution at a temperature of 86 ± 1 °C. The protocol involves (i) sample preparation, (ii) assembly of the fluid cell, and (iii) setting of Raman measurement parameters for collecting Raman spectra across the sample/solution interface in regular time intervals. The results from the experiment show the formation of a water-rich zone between a silica-based surface alteration layer (SAL) and the pristine glass, which is an intrinsic feature of an interface-coupled dissolution-precipitation model for the formation of a SAL during silicate glass corrosion. The ability to track the reaction and transport processes during the corrosion of silicate glasses and potentially of other transparent materials, spatially resolved and in real-time, represents a unique strength of this technique, overcoming the disadvantages of conventional analysis of multi-step quenching experiments. The corrosion of the top side of the glass sample represents a current issue, reducing spatial resolution at depth due to precipitation within the laser pathway. This is caused by a solution-filled gap between the sapphire window of the fluid cell lid and the top side of the monolith, which is difficult to avoid during the experimental setup. This must be taken into account when choosing the depth at which the measurement should be made. In a few cases, the formation of air bubbles was observed, which disrupted or even led to the termination of the experiment. However, this can be avoided by carefully setting up the experiment, which requires little practice.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152465","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation of Primary Human Proximal Tubule Epithelial Cells and Their Use in Creating a Microphysiological Model of the Renal Proximal Tubule.","authors":"Brooke Chalker, Yik Pui Tsang, Anish Mahadeo, Catherine K Yeung, Jonathan Himmelfarb, Edward J Kelly","doi":"10.3791/67997","DOIUrl":"https://doi.org/10.3791/67997","url":null,"abstract":"<p><p>Kidney disease affects over 850 million people globally, including 37 million Americans. Risk factors for chronic kidney disease include environmental influences, genetic predispositions, co-existing medical conditions, and a history of acute kidney injury. These factors often take months or years to develop, complicating longitudinal studies of disease etiology and pathophysiology. Advanced kidney models are needed to improve our understanding of disease mechanisms and enhance nephrotoxicity prediction in drug development. Proximal tubule epithelial cells (PTECs) in the kidney play a critical role in xenobiotic and toxin clearance as well as the reabsorption of essential nutrients. We have previously demonstrated that three-dimensional (3D) microphysiological system (MPS) platforms, populated with isolated primary PTECs, can be used to investigate renal drug interactions, assess nephrotoxicity of compounds, and predict drug clearance. Here, we present protocols for isolating and culturing primary PTECs from whole human kidneys and for seeding them into a 3D MPS platform that mimics in vivo renal physiology. This protocol enables long-term studies supporting PTEC viability, physiological morphology, and functional polarization of key transporter proteins in MPS devices for up to 6 months.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152467","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Plasmonic Photothermal Cancer Therapy: Nanoparticle-embedded Tumor-tissue-mimicking Phantoms for Visualizing Photothermal Temperature Distribution.","authors":"Amit Kumar Shaw, Divya Khurana, Sanjeev Soni","doi":"10.3791/67842","DOIUrl":"https://doi.org/10.3791/67842","url":null,"abstract":"<p><p>Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) irradiation to generate localized heat that destroys cancer cells. Before administering PPTT, the therapeutic parameters -- NP concentration, irradiation intensity, and duration -- need to be estimated. For this, numerical simulations are performed. However, to ensure robust computation, these simulations must be validated through photothermal experiments on tumor-tissue-mimicking phantoms replicating the optical properties of tumor tissue. For PPTT, therapeutic parameters are governed by the scattering and absorption of incident radiation by the tissue and NPs. Therefore, validation experiments can be conducted on phantoms mimicking the reduced scattering coefficient (µs') and absorption coefficient (µa) of the target tumor/tissue. Specifically, this protocol provides instructions for preparing phantoms mimicking µs' and µa of breast tumor injected with gold nanorods, surrounded by normal breast tissue. The protocol also details NIR irradiation, temperature monitoring, and validation of numerical results by comparing spatiotemporal temperatures with those measured using thermocouples. The protocols presented in this study facilitated the preparation of hydrogel-based cylindrical breast tumor-tissue phantoms with dimensions (ϕ40 x 12 mm) and a central tumor region (ϕ20 x 6 mm), comprising 1% agarose as the base matrix and intralipid as the scattering constituent and tumor region embedded with gold nanorods at 25 µg/mL concentration. Representative results from a case study illustrate the application of fabricated phantoms for validating numerical simulations for PPTT. The study concludes that the demonstrated protocols are valuable for conducting photothermal experiments aimed at optimizing and planning therapeutic parameters prior to in vivo experiments and validating numerical simulations for PPTT.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid Depletion of Renal Macrophages using Human CD59/Intermedilysin Cell Ablation Tool.","authors":"Mohammad Islamuddin, Jefferson Evangelista, Mohammad Enamul Kabir, Ana Karina Nisperuza Vidal, Shumei Liu, Xuebin Qin","doi":"10.3791/67957","DOIUrl":"https://doi.org/10.3791/67957","url":null,"abstract":"<p><p>Renal macrophages (RMs) are essential for kidney health, orchestrating immune surveillance, tissue homeostasis, and responses to injury. Previously, we reported the use of a human CD59 (hCD59)/intermedilysin (ILY) cell ablation tool to study the distinct fate, dynamics, and niches of RMs of bone marrow or embryonic origin. RMs originate from yolk sac-derived macrophages, fetal liver monocytes, and bone marrow-derived monocytes and are maintained in adulthood through local proliferation and recruitment of circulating monocytes. Here, we report a detailed protocol for the selective ablation of RMs to study their regeneration, including 1) generation and characterization of the Cre-inducible expression of hCD59 in mouse RMs, 2) purification of ILY and characterization of ILY activity, 3) induction of hCD59 expression on RMs in compound mice, and 4) characterization of regeneration after ILY-mediated RM ablation. ILY specifically and rapidly depletes RMs in compound mice, with efficient macrophage ablation within 1 day of ILY administration. Renal macrophage regeneration began by day 3 post-ablation, with ~88% recovery by day 7. This model offers a powerful tool for studying macrophage biology and can be used for selectively ablating other cell populations in the kidney, liver, and fatty tissues to investigate their function and regeneration.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}