Subsequently, the interpretation of the heterogenous single-cell transcriptome's role in generating the single-cell secretome and communicatome (cellular discourse) remains largely unexplored. We present, in this chapter, a detailed account of the modified enzyme-linked immunosorbent spot (ELISpot) methodology for studying collagen type 1 secretion by individual hepatic stellate cells (HSCs), with a view to improving our comprehension of the HSC secretome. A future integrated platform will be developed to examine the secretome of specific cells, distinguished by immunostaining-based fluorescence-activated cell sorting, extracted from both healthy and diseased liver tissue. Employing the VyCAP 6400-microwell chip and its integrated puncher device, our objective is to characterize single cell phenomics through the analysis and correlation of cellular phenotype, secretome, transcriptome, and genome.

Hematoxylin-eosin and Sirius red tissue staining, along with immunostaining techniques, remain the definitive approaches for diagnostic and phenotypic analysis in liver disease research and clinical practice. Tissue sections yield more information thanks to advancements in -omics technologies. We outline a sequential immunostaining process, employing repeated cycles of immunostaining and chemically-induced antibody removal, adaptable to a range of formalin-fixed tissues, including liver and other organs from both mice and humans. This method avoids the need for specialized equipment or commercially available kits. The configurable nature of antibody pairings allows for adaptation to individual clinical or scientific exigencies.

The burgeoning global rate of liver disease is driving an increasing number of patients to present with significant hepatic fibrosis and substantial mortality risk. Transplantation capacities fall dramatically short of the high demand, hence the critical drive to discover innovative pharmaceutical agents capable of halting or reversing the progression of liver damage, particularly hepatic scarring. The recent failures of advanced-stage lead compounds highlight the formidable challenges in overcoming fibrosis, a condition that has evolved and entrenched itself over a considerable timeframe and displays substantial individual differences in its type and makeup. Subsequently, tools for preclinical research are being developed in the hepatology and tissue engineering communities to clarify the makeup, components, and cellular relationships within the liver's extracellular matrix, both in healthy and diseased states. Using this protocol, decellularization strategies for cirrhotic and healthy human liver specimens are outlined and subsequently applied in basic functional tests, measuring the effect on 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 activation of hepatic stellate cells (HSCs) into collagen type I-secreting myofibroblasts is a defining feature of liver fibrosis of various origins. These myofibroblasts form a fibrous scar, thus establishing the fibrotic condition of the liver. Anti-fibrotic treatments should prioritize aHSCs as the principal source of myofibroblasts and, consequently, their key targets. Sulfate-reducing bioreactor While extensive investigations have been undertaken, targeting aHSCs in patients proves problematic. Translational research is essential for anti-fibrotic drug development, but primary human hepatic stellate cells are not readily accessible. 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.

Hepatic stellate cells (HSCs) are deeply involved in the overall course and nature of liver disease progression. Cell-specific genetic marking, gene knockout techniques, and gene depletion are instrumental in understanding the function of hematopoietic stem cells (HSCs) in the context of homeostasis and a wide spectrum of diseases, encompassing acute liver injury and regeneration, non-alcoholic fatty liver disease, and cancer. A comprehensive evaluation of Cre-dependent and Cre-independent strategies for genetic marking, gene disruption, hematopoietic stem cell tracking and depletion will be presented, including a discussion of their applications in diverse disease models. Detailed protocols for every method are included, ensuring methods for verifying successful and efficient HSC targeting.

In vitro models of liver fibrosis have transformed from utilizing isolated rodent hepatic stellate cell cultures and cell lines to more elaborate co-cultures incorporating primary liver cells, or cells sourced from stem cells. Significant progress has been made in the cultivation of stem cell-based liver tissues; yet, the liver cells generated from stem cells do not completely mirror the characteristics of their naturally occurring counterparts. In in vitro cultivation, freshly isolated rodent cells remain the most exemplary cellular model. Hepatocyte and stellate cell co-cultures serve as a valuable, minimal model for exploring liver injury-induced fibrosis. Temsirolimus cost We describe a technique for isolating hepatocytes and hepatic stellate cells from a single mouse organism, emphasizing the method of subsequently culturing these cells as free-floating spheroids.

Liver fibrosis, a global health concern of mounting severity, is becoming increasingly prevalent. Nonetheless, pharmaceutical interventions specifically addressing hepatic fibrosis remain unavailable at present. For this reason, a significant need is apparent for extensive basic research, which includes the necessity of employing animal models to evaluate new anti-fibrotic treatment concepts. Studies have unveiled numerous mouse models designed to study liver fibrogenesis. Ponto-medullary junction infraction Activation of hepatic stellate cells (HSCs) is a crucial component of chemical, nutritional, surgical, and genetic mouse models. Despite its importance, choosing the ideal model for a given inquiry regarding liver fibrosis research might prove difficult for numerous investigators. This chapter offers a concise summary of prevalent mouse models for HSC activation and liver fibrogenesis, followed by detailed, step-by-step protocols for two exemplary fibrosis models, selected based on personal experience and deemed optimal for addressing contemporary scientific inquiries. A cornerstone of toxic liver fibrogenesis research is the carbon tetrachloride (CCl4) model, which, on one hand, continues to be a highly suitable and replicable model for the basic elements of hepatic fibrogenesis. Conversely, our laboratory has developed a novel DUAL model, combining alcohol with metabolic/alcoholic fatty liver disease. This model accurately reflects all histological, metabolic, and transcriptomic gene signatures of advanced human steatohepatitis and associated liver fibrosis. We furnish a comprehensive list of the necessary details for proper preparation and implementation of both models, incorporating animal welfare standards, and thus creating a valuable resource for laboratory mouse experimentation in liver fibrosis research.

Rodent models employing experimental bile duct ligation (BDL) manifest cholestatic liver damage, exhibiting structural and functional changes, prominently including periportal biliary fibrosis. The progression of these alterations hinges on the extended build-up of excess bile acids inside the liver. This ultimately causes damage to the hepatocytes and results in a loss of their functions, leading to the recruitment of inflammatory cells. The extracellular matrix's synthesis and remodeling process is supported by pro-fibrogenic cells native to the liver. The increase in bile duct epithelial cells leads to a ductular reaction, manifesting as bile duct hyperplasia. The technical simplicity and rapid execution of experimental BDL surgery consistently produce predictable progressive liver damage with a clear, demonstrable kinetic profile. This model's cellular, structural, and functional changes mirror those seen in human patients with diverse forms of cholestasis, including the specific instances of primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). In this vein, this extrahepatic biliary obstruction model is commonly used across laboratories worldwide. In spite of its potential uses, BDL-related surgeries, executed by unqualified or inexperienced personnel, may still produce substantial discrepancies in patient outcomes and unfortunately high mortality rates. A comprehensive and detailed protocol for a sturdy experimental obstructive cholestasis model in mice is presented.

Hepatic stellate cells (HSCs) stand out as the principal cellular source for generating extracellular matrix within the liver's structure. In consequence, this liver cell population has been the subject of much focused investigation to determine the foundational principles of hepatic fibrosis. Nonetheless, the constrained supply and the consistently growing demand for these cells, joined with the added strictness in animal welfare guidelines, renders the employment of these primary cells increasingly cumbersome. Furthermore, biomedical researchers face the challenge of incorporating the 3R principle of replacement, reduction, and refinement into their research practices. Widely endorsed by legislators and regulatory bodies in numerous countries, the 1959 principle proposed by William M. S. Russell and Rex L. Burch now guides the ethical considerations associated with animal experimentation. Hence, working with immortalized HSC cell lines constitutes a worthwhile alternative for limiting animal participation and their suffering in the context of biomedical research. A comprehensive overview of factors to consider when working with pre-existing hematopoietic stem cell lines (HSC), including guidelines for maintaining and preserving HSC cultures from mice, rats, and humans, is presented in this article.