Subsequently, the interpretation of the heterogenous single-cell transcriptome's role in generating the single-cell secretome and communicatome (cellular discourse) remains largely unexplored. The modified enzyme-linked immunosorbent spot (ELISpot) technique is presented in this chapter to characterize the collagen type 1 secretion from individual hepatic stellate cells (HSCs), enabling a more thorough analysis of the HSC secretome. For the imminent future, we intend to construct a unified platform for scrutinizing the secretome of uniquely identified cells, isolated from healthy and diseased liver tissue by immunostaining-based fluorescence-activated cell sorting. Using the VyCAP 6400-microwell chip and its associated puncher apparatus, we seek to perform a comprehensive analysis of single cell phenomics, encompassing the study and correlation of phenotype, secretome, transcriptome, and genome.
In the investigation of liver disease, whether in research or clinical settings, the use of hematoxylin-eosin, Sirius red staining, and immunostaining for histological analysis remains the gold standard. Thanks to the development of -omics technologies, tissue sections provide more detailed insights. A method for immunostaining is detailed that employs repeated cycles of staining and antibody removal using chemical solutions. This procedure is readily applicable to various formalin-fixed tissues, including those from the liver, other organs, mice, or humans, and is not contingent on unique equipment or commercial products. Crucially, the tailoring of antibody combinations can be adjusted to meet specific clinical or scientific requirements.
The global increase in cases of liver disease is reflected in the rising number of patients with advanced hepatic fibrosis and a substantial mortality risk. Liver transplantation capacity is demonstrably unable to cope with the excessive demand, leading to a concentrated effort to develop novel pharmacological therapies aimed at preventing or reversing the advancement of liver scarring. The recent failure of lead-based compounds in advanced stages emphasizes the complexities of resolving fibrosis, a condition that has established itself and remained stable for years, showing substantial differences in makeup and composition from individual to individual. Consequently, preclinical instruments are being created within the hepatology and tissue engineering spheres to unravel the characteristics, composition, and cellular interplays of the hepatic extracellular environment in both wellness and illness. This document details procedures for decellularizing human liver samples, both cirrhotic and healthy, and illustrates their subsequent use in basic functional assays evaluating stellate cell function. Employing a straightforward, small-scale technique allows for adaptation across diverse laboratory contexts, resulting in cell-free substances suitable for numerous in vitro procedures and acting as a scaffold to repopulate with crucial liver cell types.
The process of liver fibrosis, irrespective of its cause, involves the activation of hepatic stellate cells (HSCs). These activated cells then produce collagen type I, ultimately leading to the accumulation of fibrous scar tissue and the fibrotic nature of the liver. Anti-fibrotic therapies should primarily focus on aHSCs, the principal originators of myofibroblasts. SW-100 mouse Despite exhaustive studies into this matter, aHSCs in patients remain difficult to target effectively. The advancement of anti-fibrotic drug therapies is predicated on the implementation of translational studies, but restricted by the availability of primary human hepatic stellate cells. For the large-scale isolation of highly purified and viable human hematopoietic stem cells (hHSCs) from both diseased and healthy human livers, a perfusion/gradient centrifugation-based method is presented, encompassing cryopreservation strategies for hHSCs.
In the establishment of liver disease, hepatic stellate cells (HSCs) assume a vital role. Understanding hematopoietic stem cells (HSCs) in their homeostatic state and disease contexts, from acute liver injury and regeneration to non-alcoholic liver disease and cancer, necessitates techniques like cell-specific genetic labeling, gene knockout, and depletion. Different Cre-dependent and Cre-independent approaches for genetic tagging, gene ablation, hematopoietic stem cell tracking and elimination will be reviewed and contrasted in their application to various disease models. We furnish comprehensive protocols for each method, encompassing procedures to verify the precise and effective targeting of HSCs.
Moving beyond the initial mono-cultures of primary rodent hepatic stellate cells and cell lines, in vitro models of liver fibrosis now often feature more complex co-cultures including primary or stem cell-derived liver cells. Stem cell-derived liver cultures have experienced notable progress; nevertheless, the liver cells produced from these stem cells are not yet fully equivalent to the phenotypes observed in naturally occurring liver tissue. Freshly isolated rodent cells, in their role as the most representative cellular specimen, are still the choice for in vitro culture. Hepatocyte and stellate cell co-cultures serve as a valuable, minimal model for exploring liver injury-induced fibrosis. Bone morphogenetic protein A dependable protocol for the isolation of hepatocytes and hepatic stellate cells from a single mouse, followed by methods for their subsequent seeding and culture as free-floating spheroids, is presented.
A severe health problem, liver fibrosis, is experiencing a rising incidence across the world. Despite this, the pharmaceutical market lacks effective medications for hepatic fibrosis. Subsequently, a critical demand emerges for rigorous foundational research, including the utilization of animal models in the assessment of new anti-fibrotic therapeutic methodologies. A substantial number of mouse models focused on liver fibrogenesis have been described. Knee biomechanics Chemical, nutritional, surgical, and genetic mouse models are employed, along with the activation of hepatic stellate cells (HSCs). The selection of a suitable model for a specific liver fibrosis research question, however, can be demanding for many investigators. To initiate, this chapter presents a brief overview of the most frequent mouse models used for exploring hematopoietic stem cell activation and liver fibrogenesis. Then detailed step-by-step protocols are offered for two specific mouse fibrosis models. Our selection of these models is based on practical experience and their potential to effectively address various current research topics. In the study of toxic liver fibrogenesis, the carbon tetrachloride (CCl4) model, on one hand, continues to be one of the best-suited and most reproducibly successful models for understanding the basic mechanisms of hepatic fibrogenesis. Instead, our laboratory's innovative DUAL model incorporates both alcohol and metabolic/alcoholic fatty liver disease. This model accurately mimics the histological, metabolic, and transcriptomic gene signatures of advanced human steatohepatitis and related liver fibrosis. In this laboratory guide for mouse experimentation in liver fibrosis research, we present the required information for the accurate preparation and detailed implementation of both models, including all relevant animal welfare considerations.
The experimental bile duct ligation (BDL) procedure in rodents produces cholestatic liver injury, where periportal biliary fibrosis is a prominent structural and functional consequence. The liver's excess accumulation of bile acids is the basis for these time-sensitive changes. Consequently, hepatocyte damage and functional impairment occur, prompting the influx of inflammatory cells. The extracellular matrix's formation and alteration are critically dependent on the actions of pro-fibrogenic liver-resident cells. Bile duct epithelial cell proliferation induces a ductular response, marked by an increase in bile duct hyperplasia. Experimental BDL surgery, despite its technical ease and quick execution, reliably produces predictable progressive liver damage with a clear kinetic profile. The model demonstrates cellular, structural, and functional modifications akin to those present in human sufferers of diverse cholestatic conditions, for example, primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). This extrahepatic biliary obstruction model is, thus, commonly employed in laboratories across the world. Undeniably, BDL-related surgical interventions, when executed by personnel who lack sufficient training or experience, can result in substantial variations in patient outcomes, and unfortunately, elevated mortality rates. We describe a robust protocol for creating an experimental obstructive cholestasis in mice.
In the liver, hepatic stellate cells (HSCs) are the key cellular producers of extracellular matrix. For this reason, this particular liver cell population has received intensive scrutiny in studies exploring the fundamental characteristics of hepatic fibrosis. Yet, the scarcity and escalating need for these cells, in addition to the stricter adherence to animal welfare regulations, make the process of working with these primary cells more challenging. Subsequently, biomedical researchers encounter the need to integrate the 3R approach of replacement, reduction, and refinement into their research methodologies. The ethical dilemma of animal experimentation is being addressed globally by legislators and regulatory bodies who largely rely on the 1959 guideline proposed by William M. S. Russell and Rex L. Burch. Consequently, the employment of immortalized hematopoietic stem cell lines offers a viable alternative to reduce animal use and suffering in biomedical research. For those working with pre-existing hematopoietic stem cell (HSC) lines, this article details essential factors and offers standard procedures for maintaining and preserving HSC lines from murine, rat, and human sources.