Methods in molecular biology最新文献

In vitro skin aging models represent a valuable tool for the study of age-related pathologies and potential treatments. However, the currently available models do not adequately represent the complex microenvironment of the dermis since they generally focus on cutaneous cellular senescence, rather than the full range of factors that contribute to the aging process, such as structural and compositional alteration of the dermal extracellular matrix. The following protocol describes the extraction and characterization of human adult extracellular matrix scaffolds for use in in vitro aging models.

Aging adversely affects the self-renewal and differentiation capabilities of stem cells, which impairs tissue regeneration as well as the homeostasis. Epigenetic mechanisms, specifically DNA methylation, play a key role in the maintenance of pluripotency in stem cells and regulation of pluripotency-related gene expression. Age-related modifications in methylation patterns could influence the expression of genes critical for stem cell potency maintenance, including transcription factors Nanog and Sox2. The following chapter describes a step-by-step bisulfite sequencing protocol for detection of methylation changes in the aging stem cells and provides valuable insights into the stem cells epigenetic profile. Further, the methodology describes the steps of genomic DNA extraction, bisulfite conversion, real-time PCR amplification, and sequencing for an in-depth view of the epigenetic profile derived from aging stem cells.

Human brain organoids (HBOs) derived from pluripotent stem cells hold great potential for disease modeling and high-throughput compound screening, given their structural and functional resemblance to fetal brain tissues. These organoids can mimic early stages of brain development, offering a valuable in vitro model to study both normal and disordered neurodevelopment. However, current methods of generating HBOs are often low throughput and variable in organoid differentiation and involve lengthy, labor-intensive processes, limiting their broader application in both academic and industrial research. Key challenges include high costs of growth factors, variability in organoid size and function, suboptimal maturation, and manual handling that reduces throughput. Here, we present a standard operating procedure (SOP) for the scalable production of HBOs using a novel pillar plate system that simplifies the spheroid transfer process and allows miniature organoid culture. This method enables the reproducible generation of HBOs without the need for extensive manual intervention, providing a streamlined solution for high-throughput screening (HTS). The resulting assay-ready pillar plate with HBOs is optimized for compound testing, in situ staining, and analysis, offering an efficient platform to advance neurodevelopmental research and therapeutic screening.

Stem cell nanotechnology (SCN) is an important scientific field to guide stem cell-based research of nanoparticles. Currently, nanoparticles (NPs) have a rich spectrum regarding the sources from which they are obtained (metallic, polymeric, etc.), the methods of obtaining them (physical, chemical, biological), and their shape, size, electrical charge, etc. properties. It is also essential to expand green synthesis applications for the use of NPs in the field of biomedical sciences. For this purpose, there is a need to produce NPs using biological sources (plant, microorganism, algae, yeast etc.…), characterization and investigation of their effects on biological activities of stem cells. This process involves long and laborious procedures, and there may be differences in methods between individual laboratories.In this protocol, biofabrication and characterization of ZnO NPs using dried leaves of Camellia sinensis is described. This experimental setup includes conventional and novel methods that can be applied to biofabricate and characterize the NPs and to examine the viability, apoptotic, and necrotic effects on human adipose tissue-derived mesenchymal stem cells (ADMSCs) in vitro.

Spheroid culture systems have been extensively used to model the three-dimensional (3D) behavior of cells in vitro. Traditionally, spheroids consist of a single cell type, limiting their ability to fully recapitulate the complex inter-cellular interactions observed in vivo. Here we describe a protocol for generating cocultured spheroids composed of two distinct cell types, embedded within a 3D extracellular matrix (ECM) to better study cellular interactions. Fluorescent labeling of each cell type enables clear distinction and visualization, facilitating the analysis of cell invasion, proliferation, and behavior within the matrix. This method is particularly suited for studying matrix invasion, an essential process in cancer metastasis, using both fixed and live cell microscopy. The protocol is versatile and can be adapted for various cell types, providing a robust platform for investigating cell-cell interactions in cancer research, tissue remodeling, and drug screening.

In this chapter, we provide a method for silencing target genes in epidermal cells via RNA interference. Specifically, we describe a protocol for transfection-mediated delivery of small interfering RNA oligonucleotides (siRNA). Functional assays are indispensable to characterize the biological consequences of gene knockdowns, and we also provide a method to analyze alterations in cell adhesion properties, consequent to knockdown of genes involved in this process.

Human liver organoids (HLOs) derived from pluripotent stem cells hold potential for disease modeling and high-throughput compound screening due to their architectural and functional resemblance to human liver tissues. However, reproducible, scale-up production of HLOs for high-throughput screening (HTS) presents challenges. These include the high costs of additives and growth factors required for cell differentiation, variability in organoid size and function from batch to batch, suboptimal maturity of HLOs compared to primary hepatocytes, and low assay throughput due to excessive manual processes and the absence of assay-ready plates with HLOs. To address some of these issues, here we present standard operating procedures (SOPs) for the scale-up production of HLOs using a pillar plate through microarray 3D bioprinting. This technology facilitates the rapid, uniform seeding of foregut cells onto the pillar plate, maintaining cell viability and enabling the scale-up generation of HLOs. The assay-ready pillar plate with HLOs is suitable for compound testing, as well as in situ organoid staining and analysis.

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.