Cell Biology

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inconsistent or alter the physiological properties of the cells. Here, we describe an optimized method that overcomes these limitations. By using electroporation to deliver CRISPR constructs that target the Tp53 gene, the method reliably generates immortalized MEFs (iMEFs) within three weeks. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted broadly to immortalize other mouse primary cell types.

Recurrent hormone receptor-positive (HR+) breast cancer is a leading cause of cancer mortality in women. Recurrence and resistance to targeted therapies have been difficult to study due to the long clinical course of the disease, the complex nature of resistance, and the lack of clinically relevant model systems. Existing models are limited to a few HR+ cell lines, organoid models, and patient-derived xenograft models, all lacking components of the human tumor microenvironment. Furthermore, the low take rate and loss of estrogen receptor (ER) expression in patient-derived organoids (PDOs) has been challenging. Our protocol allows simultaneous isolation of PDOs and matching cancer-associated fibroblasts (CAFs) from primary and metastatic HR+ breast cancers. Importantly, our protocol has a higher take rate and enables long-term culturing of PDOs that retain ER expression. Our matching PDOs and CAFs will provide researchers with a new resource to study the influence of the tumor microenvironment on various aspects of cancer biology such as cell growth and drug resistance in HR+ breast cancer.

Primary neuronal culture and transient transfection offer a pair of crucial tools for neuroscience research, providing a controlled environment to study the behavior, function, and interactions of neurons in vitro. These cultures can be used to investigate fundamental aspects of neuronal development and plasticity, as well as disease mechanisms. There are numerous methods of transient transfection, such as electroporation, calcium phosphate precipitation, or cationic lipid transfection. In this protocol, we used electroporation for neurons immediately before plating and cationic lipid transfection for neurons that have been cultured for a few days in vitro. In our experience, the transfection efficiency of electroporation can be as high as 30%, and cationic lipid transfection has an efficiency of 1%–2%. While cationic lipid transfection has much lower efficiency than electroporation, it does offer the advantage of a higher expression level. Therefore, these transfection methods are suitable for different stages of neurons and different expression requirements.

Histological techniques to study muscle are crucial for assessing skeletal muscle health. To preserve tissue morphology, samples are usually fixed in formaldehyde or cryopreserved immediately after excision from the body. Freezing samples in liquid nitrogen, using isopentane as a mediator for efficient cooling, preserves the tissue in its natural state. However, this method is highly susceptible to freeze-fracture artifacts, which alter or destroy tissue architecture. Isopentane is most commonly used in a semi-frozen/liquid state that is visually assessed by the experimenter, which can pose a challenge when freezing multiple tissues at a time or maintaining a consistent temperature. Furthermore, tissue size is also a confounding factor; depending on the size, freezing times can vary. In this study, we compare two different options for using isopentane while cryopreserving tissue. We also present an easy and reproducible method of freezing the soleus tissue of mice using frozen isopentane. This method decreased the occurrence of freeze-fractures by an order of magnitude, to ~4%, whereas the traditional method of cryopreservation resulted in ~56% freeze-fracturing.

Neurons are highly polarized cells, with axons that may innervate distant target regions. In the brain, basal forebrain cholinergic neurons (BFCNs) possess extensive axons that project to several target regions such as the cortex, hippocampus, and amygdala, and may be exposed to a specific microenvironment in their axon targets that may have retrograde effects on neuronal health. Interestingly, BFCNs express the pan-neurotrophin receptor p75NTR throughout life while also concomitantly co-expressing all Trk receptors, making them capable of responding to both mature and precursor neurotrophins to promote survival or apoptosis, respectively. Levels of these trophic factors may be modulated in the BFCN axon or soma microenvironment under neurodegenerative conditions such as seizure and brain injury. In this protocol, BFCNs are established in microfluidic devices for compartmental culture, with the aim of studying the effects of axon- or soma-specific stimulation of BFCNs for an in vitro representation of distal axon vs. soma environments as seen in vivo. This study further establishes a novel method of tracing and imaging live BFCNs exposed to stimuli in their distal axons with the aim of assessing retrograde cell death. The in vitro compartmental culture system of BFCNs that allows live imaging may be applied to investigate various effects of axon- or soma-specific stimuli that affect BFCN health, maintenance, and death, to model events that occur in the context of brain injury and neurodegenerative disorders.

The human intestine plays a pivotal role in nutrient absorption and immune system regulation. Along the longitudinal axis, cell-type composition changes to meet the varying functional requirements. Therefore, our protocol focuses on the processing of the whole human intestine to facilitate the analysis of region-specific characteristics such as tissue architecture and changes in cell populations. We describe how to generate a biobank that can be used to isolate specific immune cell subtypes, generate organoid lines, and establish autologous immune cell-organoid co-cultures.

The human T-lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus that predominantly spreads through cell-to-cell contact due to the limited infectivity of cell-free viruses. Among various modes of intercellular transmission, HTLV-1 biofilms emerge as adhesive structures, polarized at the cell surface, which encapsulate virions within a protective matrix. This biofilm is supposed to facilitate simultaneous virion delivery during infection. Yet, the molecular and functional intricacies of viral biofilms remain largely unexplored, despite their pivotal role in understanding retroviral pathogenesis. In this study, we optimized a protocol to isolate HTLV-1 biofilms from chronically infected T cells, facilitating their structural and molecular characterization using proteomic and super-resolution microscopy analyses. This protocol involves cultivating HTLV-1 chronically infected T cells at high density to facilitate the natural detachment of viral biofilms into the supernatant. Then, employing successive centrifugations, the cells are separated from the detached biofilms, and these structures are pelleted at medium speed (10,000× g). This method circumvents the need for mechanical, chemical, or enzymatic biofilm detachment, bypasses the use of ultracentrifugation, and enables us to resuspend the biofilms in the appropriate buffer for subsequent analyses such as western blotting or super-resolution microscopy imaging as presented.

Developing a physiologically relevant in vitro model of the respiratory epithelium is critical for understanding lung development and respiratory diseases. Here, we describe a detailed protocol in which the fetal mouse proximal epithelial progenitors were differentiated into 3D airway organoids, which contain terminal-differentiated ciliated cells and basal stem cells. These differentiated airway organoids could constitute an excellent experimental model to elucidate the molecular mechanisms of airway development and epithelial cell fate determination and offer an important tool for establishing pulmonary dysplasia disease in vitro.

This study explores the novel application of pyronin Y for fluorescently labeling extracellular matrices (ECMs) and gelatin cryogels, providing a simple and reliable method for laser scanning confocal microscopy. Pyronin Y exhibited remarkable staining ability of the porous structures of gelatin cryogels, indicating its potential as a reliable tool for evaluating such biomaterials. Confocal imaging of pyronin Y–stained cryogels produced high signal-to-noise ratio images suitable for quantifying pores using Fiji/Image J. Importantly, pyronin Y enabled effective dual-color imaging of cryogel-labeled mesenchymal stem cells, expanding its utility beyond traditional RNA assays. Traditional staining methods like Mason’s trichrome and Sirius Red have limitations in cryogel applications. Pyronin Y emerges as a powerful alternative due to its water solubility, minimal toxicity, and stability. Our results demonstrate pyronin Y’s ability to specifically stain gelatin cryogel's porous structures, surpassing its weak staining of ECMs in 2D. Confocal imaging revealed enduring staining even under rigorous scanning, with no notable photobleaching observed. Furthermore, pyronin Y's combination with Alexa Fluor 647 for dual-color imaging showed promising results, validating its versatility in fluorescence microscopy. In conclusion, this study establishes pyronin Y as a cost-effective and rapid option for fluorescent staining of gelatin cryogels. Its simplicity, efficacy, and compatibility with confocal microscopy make it a valuable tool for characterizing and evaluating gelatin-based biomaterials, contributing significantly to the field of cryogel imaging. The study opens new avenues for dual-color imaging in biomaterial research and tissue engineering, advancing our understanding of cellular interactions within scaffolds.

Osteoclasts are terminally differentiated multinucleated giant cells that mediate bone resorption and regulate skeletal homeostasis under physiological and pathological states. Excessive osteoclast activity will give rise to enhanced bone resorption, being responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro models of osteoclast-mediated bone resorption is helpful to better understand the functional status of osteoclasts under (patho)physiological conditions. Notably, it is essential to provide an in vivo–relevant bone substrate that induces osteoclasts to generate authentic resorption lacunae and excavate bone. Here, we summarize the experimental design of a reproducible and cost-effective method, which is suitable for evaluating the regulatory mechanisms and influence of molecular agonists and antagonists as well as therapeutics on osteoclast-mediated bone-resorbing activity.