Methods in molecular biology最新文献

Human nasal epithelium (HNE) organoid models of SARS-CoV-2 infection were adopted globally during the COVID-19 pandemic once it was recognized that the Vero cell line commonly used by virologists did not recapitulate human infection. However, the widespread use of HNE organoid infection models was hindered by the high cost of media and consumables, and the inherent limitation of basal cells as a scalable continuous source of cells. The human Calu-3 cell line, generated from a lung adenocarcinoma, was shown to largely recapitulate infection of the human epithelium and to preserve the SARS-CoV-2 genomic fidelity. We have previously shown that continuous cancer cell lines can polarize along the apical-basal axis when embedded in matrix and to more closely mimic infection of human cells when compared to their non-polarized, simple monolayer state. We have established and demonstrated that polarized Calu-3 cells constitute a robust SARS-CoV-2 infection model. The polarized Calu-3 cells are implemented in our respiratory virus isolation and amplification pipeline as an inexpensive, scalable, intermediary culture system to complement the HNE organoid model against which all respiratory culture models are benchmarked.

The choroid plexus (ChP) is a vital brain structure that produces cerebrospinal fluid (CSF) and forms a selective barrier between the blood and CSF, essential for brain homeostasis. Composed of secretory epithelial cells, connective stroma, and a fenestrated vascular network, the ChP supports nutrient transport, immune surveillance, and the clearance of toxic by-products. Despite its significance in maintaining cerebral function, the mechanisms underlying its development and maturation remain poorly understood. Recent advancements, such as the creation of stem cell-derived three-dimensional (3D) ChP organoid model, provide a promising platform for studying these processes. The ChP organoid model replicates key developmental stages and functions of the ChP, including CSF secretion and barrier formation. Additionally, they offer unique opportunities to investigate the impacts of drugs, pathogens, and toxins on the blood-CSF barrier. This study highlights imaging techniques critical for the characterization and utilization of ChP organoids, illustrating their value in advancing our understanding of ChP biology and its role in health and disease.

Muscle stem cells (MuSCs) lose a large proportion of their characteristics when removed from their niche, hampering the analysis of muscle stem cell functionality. However, the isolation and culture of single floating myofibers with their adjacent muscle stem cells allow the short-term culture and manipulation of muscle stem cells in conditions as close as possible to the endogenous niche. Here, the isolation, culture and transfection with siRNA of muscle stem cells on their adjacent myofibers from young as well as old mice are described.

Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer. The current standard for treating primary OSCC is surgical resection combined with radiotherapy and chemotherapy. Despite improved therapeutic strategies, OSCC has high rates of metastasis and mortality, with one in two patients dying of the disease. Patient-derived organoids have become promising cell culture systems for disease modeling and precision medicine. Here we describe the high-efficiency generation of organoids from OSCC patients, which can be maintained in the culture for the long term. We further provide protocols for characterizing OSCC organoids using histology and immunofluorescence staining.

While traditional assay methods face challenges in detecting specific proteins, aptamers, known for their high specificity and affinity, are emerging as a valuable biomarker detection tool. Aurora kinase A (AURKA) plays a role in cell division and influences stem cell reprogramming. In this study, an in silico approach method was conducted for a random ssDNA aptamer sequence selection and its binding with AURKA. The aptamer was designed based on AURKA's structure and nucleic acid sequence, obtained from PDB RCSB. Using RNAfold and RNA composer, we predicted the aptamer's secondary and tertiary structures. Protein-aptamer binding was analyzed via HDOCK and HADDOCK, with 2D interactions visualized in LIGPLOT+ v1.4. Autodock 4 and NAMD 2.3 tools were used to conduct docking and MD simulation studies.

The discovery of leucine-rich-containing G-protein-coupled receptor 5 (Lgr5) as an intestinal adult stem cell marker had blazed a trail in stem cell biology and laid the foundations of modern organoid technology. Up to date, several well-established intestinal organoid protocols have been reported in the literature from different sources, including adult and induced pluripotent stem cells. Here, we demonstrate a BALB/c mouse-derived intestinal organoid culture protocol, passaging, and cryopreservation procedures.

This study describes an intramolecular quenching assay to evaluate gamma-secretase (GS) enzyme activity in human dermal cells. The method utilizes a fluorogenic peptide substrate, mimicking a fragment of amyloid precursor protein (APP), in which a quencher suppresses the fluorescence of a fluorophore until enzymatic cleavage occurs, resulting in a measurable increase in fluorescence. This real-time, direct measurement of GS activity allows for precise kinetic analysis using Michaelis-Menten modeling to define Kd and Vmax. The assay is designed to quantify GS activity in human dermal fibroblasts and keratinocytes, enabling comparison between samples derived from hidradenitis suppurativa (HS) patients and healthy controls, as well as investigating the effects of subunit knockdown, such as nicastrin, on GS function. The method offers several advantages, including simplicity, cost-effectiveness, and adaptability for high-throughput screening for GS enzyme inhibitors.

Negative magnetophoresis is employed to levitate cells in a paramagnetic medium without the need for magnetic labeling, preserving their natural state and minimizing toxicity. The single-ring magnet configuration that provides an open space in the levitation chamber enhances culture accessibility and scalability, enabling the formation of millimeter-sized 3D structures through cellular self-assembly. This system provides a versatile and cost-effective approach for diverse applications, including tissue engineering and biofabrication. This protocol outlines a method for biofabrication and maintenance of 3D cellular structures using magnetic levitation with a ring magnet-based setup.

In this chapter, we present a detailed protocol for establishing a three-dimensional (3D) multicellular tumor spheroids (MCTSs) model to simulate the tumor microenvironment (ME) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) for the study of hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cell aggressiveness, growth, and metastasis potential. The MASLD microenvironment (MASLD-ME) is recreated by embedding hepatic stellate cells in a collagen I matrix within a Boyden chamber system. The metabolic medium mimics MASLD conditions, enriched with high glucose, fructose, insulin, and fatty acids, to simulate metabolic stresses associated with the disease.In the protocol, cancer cells are loaded in the upper compartment to analyze their migration toward the MASLD-ME, thereby facilitating studies on cancer cell invasiveness and metastatic capacity. This method offers an adaptable, reproducible model to research disease progression and investigate therapeutic interventions, contributing to preclinical research on MASLD-related liver cancer pathophysiology and potential drug responses.

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen.