细胞生物学

The study of whole organs or tissues and their cellular components and structures has been historically limited by their natural opacity, which is caused by the optical heterogeneity of the tissue components that scatter light as it traverses through the tissue, making 3D tissue imaging highly challenging. In recent years, tissue clearing techniques have received widespread attention and undergone rapid development. We recently demonstrated the synthesis of a 2-hydroxyethyl methacrylate (HEMA)-acrylamide (AAm) copolymer. This was achieved using antipyrine (ATP) and 2,2′-thiodiethanol (TDE) as solvents. The resulting solution rapidly embedded tissue samples with a high degree of transparency and is compatible with multiple fluorescence labeling techniques. The method exhibits significant transparency effects across a range of organs, comprising the heart, liver, spleen, lung, kidney, brain (whole and sectioned), esophagus, and small intestine. It can enable volumetric imaging of tissue up to the scale of mouse organs, decrease the duration of the clearing, and preserve emission from fluorescent proteins and dyes. To facilitate the use of this powerful tool, we have provided here a detailed step-by-step protocol that should allow any laboratory to use tissue transparency technology to achieve transparency of tissues and organs.

Intestinal glucose absorption has been studied for several decades. However, the different methods available for investigating absorption are often the reason for variability in the results, and it is difficult to measure the relative contribution of paracellular absorption using existing methods. Thus, we have established a new model for measuring glucose absorption. In the isolated in situ vascularly perfused small intestine, the intestinal epithelium is completely preserved, and the entire transport pathway is intact. In the present model, we use radioactive labeled ¹⁴C-d-glucose, which allows for sensitive quantification of glucose absorption even with low luminal concentrations. The described method is optimized for intestinal glucose absorption but can be applied to other macro/micronutrients that can be radioactively labeled. The described procedure is a novel approach for measurements of intestinal nutrient absorption and gut permeability in which luminal nutrient concentrations resemble physiological concentrations.

Cell transplantation is a promising strategy for treating age-related muscle atrophy, but its critical application remains limited. Cultured myoblasts, unlike freshly isolated muscle stem cells, show poor engraftment efficiency and fail to contribute effectively to muscle regeneration. Moreover, successful engraftment generally requires prior muscle injury, as skeletal muscle regeneration is typically triggered by a damaged microenvironment. These limitations present major obstacles for applying cell therapy to sarcopenia, where muscle degeneration occurs without injury. In this protocol, we describe a novel approach that enables the transplantation of cultured myoblasts into intact skeletal muscle without the need for preexisting injuries or genetic modification. By combining myoblasts with extracellular matrices (ECM), such as Matrigel, which mimic the native muscle niche and support cell survival, adhesion, proliferation, and differentiation, we achieve efficient engraftment and increased muscle mass without the need for preexisting injury. The ECM also provides a scaffold and retains bioactive factors that enhance the regenerative capacity of transplanted cells. This is the first protocol that enables robust myoblast engraftment in non-injury muscle conditions, offering a practical tool for studying and potentially treating sarcopenia.

Proper brain function depends on the integrity of the blood–brain barrier (BBB), which is formed by a specialized network of microvessels in the brain. Reliable isolation of these microvessels is crucial for studying BBB composition and function in both health and disease. Here, we describe a protocol for the mechanical dissociation and density-based separation of microvessels from fresh or frozen human and murine brain tissue. The isolated microvessels retain their molecular integrity and are compatible with downstream applications, including fluorescence imaging and biochemical analyses. This method enables direct comparisons across species and disease states using the same workflow, facilitating translational research on BBB biology.

Osteoarthritis (OA) is the primary cause of joint impairment, particularly in the knee. The prevalence of OA has significantly increased, with knee OA being a major contributor whose pathogenesis remains unknown. Articular cartilage and the synovium play critical roles in OA, but extracting high-quality RNA from these tissues is challenging because of the high extracellular matrix content and low cellularity. This study aimed to identify the most suitable RNA isolation method for obtaining high-quality RNA from microquantities of guinea pig cartilage and synovial tissues, a relevant model for idiopathic OA. We compared the traditional TRIzol^® method with modifications to spin column–based methods (TRIspin-TRIzol^®/RNeasy^TM, RNeasy^TM kit, RNAqueous^TM kit, and Quick-RNA^TM Miniprep Plus kit), and an optimized RNA isolation protocol was developed to increase RNA yield and purity. The procedure involved meticulous sample collection, specialized tissue processing, and measures to minimize RNA degradation. RNA quality was assessed via spectrophotometry and RT–qPCR. The results demonstrated that among the tested methods, the Quick-RNA^TM Miniprep Plus kit with proteinase K treatment yielded the highest RNA purity, with A_260:280 ratios ranging from 1.9 to 2.0 and A_260:230 ratios between 1.6 and 2.0, indicating minimal to no salt contamination and RNA concentrations up to 240 ng/μL from ⁓20 mg of tissue. The preparation, storage, homogenization process, and choice of RNA isolation method are all critical factors in obtaining high-purity RNA from guinea pig cartilage and synovial tissues. Our developed protocol significantly enhances RNA quality and purity from micro-quantities of tissue, making it particularly effective for RTqPCR in resource-limited settings. Further refinements can potentially increase RNA yield and purity, but this protocol facilitates accurate gene expression analyses, contributing to a better understanding of OA pathogenesis and the development of therapeutic strategies.

Single-cell RNA sequencing has revolutionized molecular cell biology by enabling the identification of unique transcription profiles and cell transcription states within the same tissue. However, tissue dissociation presents a challenge for non-model organisms, as commercial kits are often incompatible, and current protocols rely on tissue enzymatic digestion for extended periods. Tissue digestion can alter cell transcription in response to temperature and the stress caused by enzymatic treatment. Here, we propose a protocol to stabilize RNA using a deep eutectic solvent (Vivophix, Rapid Labs) prior to tissue dissociation, thereby avoiding transcription changes induced by the process and preventing RNase activity during incubation. We validated this methodology for three medically important insect vectors: Anopheles gambiae, Aedes aegypti, and Lutzomyia longipalpis. Single-cell RNA sequencing using our insect midgut dissociation protocol yielded high-quality sequencing results, with a high number of cells recovered, a low percentage of mitochondrial reads, and a low percentage of ambient RNA—two hallmark standards of cell quality.

The neuromuscular junction (NMJ) is critical for muscle function, and its dysfunction underlies conditions such as sarcopenia and motor neuron diseases. Current protocols for assessing NMJ function often lack standardized stimulation parameters, limiting reproducibility. This study presents an optimized ex vivo method to evaluate skeletal muscle and NMJ function using the Aurora Scientific system, incorporating validated stimulation protocols for both nerve and muscle to ensure consistency. Key steps include tissue preparation in a low-calcium, high-magnesium solution to preserve NMJ integrity, determination of optimal muscle length, and sequential stimulation protocols to quantify neurotransmission failure and intratetanic fatigue. By integrating rigorous standardization, this approach enhances reproducibility and precision, providing a robust framework for investigating NMJ pathophysiology in aging and disease models.

Skeletal muscle–specific stem cells are responsible for regenerating damaged muscle tissue following strenuous physical activity. These muscle stem cells, also known as satellite cells (SCs), can activate, proliferate, and differentiate to form new skeletal muscle cells. SCs can be identified and visualized utilizing optical and electron microscopy techniques. However, studies identifying SCs using fluorescent imaging techniques vary significantly within their methodology and lack fundamental aspects of the guidelines for rigor and reproducibility that must be included within immunohistochemical studies. Therefore, a standardized method for identifying human skeletal muscle stem cells is warranted, which will improve the reproducibility of future studies investigating satellite activity. Additionally, although it has been suggested that SC shape can change after exercise, there are currently no methods for examining SC morphology. Thus, we present an integrated workflow for three-dimensional visualization of satellite cell nuclei, validated by the spatial context of the fluorescent labeling and multichannel signal overlap. Our protocol includes, from start to finish, post-biopsy extraction and embedding, tissue sectioning, immunofluorescence, imaging steps and acquisition, and three-dimensional data post-processing. Because of the depth volume generated from the confocal microscope z-stacks, this will allow future studies to investigate the morphology of SC nuclei and their activity, instead of traditionally observing them in two-dimensional space (x, y).

The osteocyte lacuno-canalicular system (LCS) plays a crucial role in maintaining bone homeostasis and mediating cellular mechanotransduction. Current histological techniques, particularly the Ploton silver nitrate staining method, face challenges such as variations in solution concentrations and types as well as a lack of standardization, which limits their broader application in osteocyte research. In this study, we present a simplified and more effective silver nitrate staining protocol designed to address these issues. Our method utilizes a 1 mol/L silver nitrate solution combined with optimized gelatin-formic acid solutions at varying concentrations (0.05%–0.5% type-B gelatin and 0.05%–5% formic acid, or 1%–2% type-B gelatin and 0.1%–2% formic acid). Staining is performed for 1 h under 254 nm ultraviolet light or 90 min under room light, followed by washing with Milli-Q water to terminate staining. This novel optimized method yields consistent and distinct staining of the osteocyte LCS across multiple species, demonstrating superior efficiency and reliability compared to the Ploton method. It will significantly advance research in osteocyte biology and provide a valuable tool for exploring the adaptive evolution of osteocyte LCS morphology and function across various taxa.

Fuchs endothelial corneal dystrophy (FECD) is a rare and multifactorial disorder leading to cell death in the innermost layer of the cornea, i.e., the endothelium; UV radiation is reported as the major environmental risk for the disease. Establishing an animal model for this disease has remained challenging in FECD research. We have developed a detailed protocol for the establishment of a UVA-induced FECD mouse model and removal of corneal endothelium from the eye for further molecular and histological studies by taking references from previous studies. UVA light of 500 J/cm² was focused on the C57BL/6J female mouse cornea and kept for an observation period of 90 days. The animal developed corneal scarring by the end of three months. Slit-lamp microscopy and alizarin red–trypan blue staining confirmed endothelial cell death and formation of corneal guttae in the endothelium. Surgical removal of the endothelial layer was successfully done in the diseased mouse, and the result was confirmed by immunofluorescence. This study is relevant for in-depth research using a FECD mouse model, which will surpass the limitation of human tissue scarcity and can be used for in vivo drug targeting to develop therapeutics to cure FECD.