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PriCells: Introduction of Isolation and Culture of Human Primary Hepatocytes

時間:2021-10-22 閱讀:240
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PriCells: Introduction of Isolation and Culture of Human Primary Hepatocytes 


The liver performs a wide range of physiologically important metabolic functions including the synthesis and secretion of albumin, fibrinogen, and other plasma proteins, the synthesis of cholesterol and bile acids, and the metabolism of drugs, steroids, and amino acids.  In addition, the liver has a central role in energy metabolism as the major store of glycogen and the site of gluconeogenesis and synthesis of fatty acids and triglycerides.  The liver is, therefore, a vital organ; however, it is sometimes difficult to study specific liver functions in vivo owing to interfering influences from other organs, e.g., the kidney, gut, and lungs, which metabolize drugs and the muscle involvement in glucose homeostasis.  A number of in vitro preparations such as tissue homogenates or subcellular fractions are available to study tissue-specific functions, although experiments in intact cells, with cell membrane and cellular organelles are postulated to reflect the in vivo situation more closely.  Hepatocytes represent 60–65% of the number of cells in the liver but occupy some 80% of the liver volume because of their large volume.  Nonparenchymal cells such as Kuppfer cells, lipocytes, and endothelial cells (6%) make up the remaining tissue. Intercellular spaces account for approx 14% of the liver volume.  Isolated human hepatocytes have been used extensively for a variety of techniques investigating cellular metabolism and the induction, metabolism, and toxicology of xenobiotics.


The preparation of isolated hepatocytes from a heterogeneous tissue such as the liver involves several steps.  Initially, the cells must be dissociated from the fibroconnective skeleton. Tissue dispersion requires the disruption of reticulin fibrils along with adhesion proteins such as fibrinonectin and laminin, which constitute the framework of the liver lobule.  Hepatocyte isolation also requires the disruption of cell–cell adhesions, i.e., the junctional complexes, and finally, the recovery of the hepatocytes and their preparation for further use.

Enzymatic hepatocyte isolation was first introduced in 1967. Prior to this time, mechanical and chemical dissociation of the liver parenchyma had been used. This latter isolation yielded very poor quality hepatocytes.  The chemical and mechanical dissociation methods are discussed at length in a review. The development of a technique utilizing enzymes to digest the liver providing a high yield of functionally active rat hepatocytes led to a rapid increase in the utility of the isolated hepatocyte as a model system for the study of liver functions.  The perfusion technique originally developed for the preparation of hepatocytes from small animal species has, over the last 20 yr, been adapted and applied to human material.


Human hepatocytes have been prepared from a variety of liver samples, including a whole liver, a portion of whole liver, end of lobe wedge biopsy samples, and small biopsy fragments.  The majority of human liver made available for research is in the form of large biopsies, which are usually obtained from patients undergoing partial hepatectomy. This material is usually 5–10 g in size and normally has only one cut surface.  Therefore, the wedge biopsy perfusion method for preparing hepatocytes appears to be the most suitable method for utilizing the available material.  The preparation of human hepatocytes using this method has produced yields of between 0.1–30 × 106 hepatocytes/g wet liver with cell viability in the range of 70–95% as determined by trypan blue exclusion.  However, cell yields and viability vary depending on the size of the biopsy, the ability of the operator to cannulate the exposed vessels, and the resultant perfusion of the tissue.


The lack of human liver tissue has prompted developments in maximizing the use of human hepatocytes by developing isolation, culture, co-culture, and cyropreservation techniques.  The limited availability of human material requires a system that can provide a high yield of functional isolated hepatocytes rapidly utilizing all the available material.


A major consideration when using human material to prepare hepatocytes is the variation in the functional activity between cell preparations.  This variability may be caused in part to the length of time taken in obtaining the sample, sample storage conditions, or the sample history, as well as the genetic and environmental factors affecting the donor. Information regarding the patient history, social history, dietary habits, and genetic polymorphism are important for interpreting results.  For example, a liver sample removed from a patient who had taken a barbiturate overdose can have drug-metabolizing enzymes that are significantly induced.  These enzymes remain elevated in isolated hepatocytes, which are cultured for several days.  Furthermore, studies have also suggested that variable intracellular ATP and glycogen content in hepatocyte populations is attributed to the nutritional status of the donor.


Isolated human hepatocytes used in suspension with no attachment to any form of extracellular matrix are viable for only a few hours.  Hepatocyte survival and functionality can be extended considerably when cells are maintained in monolayer culture conditions in the presence of an extracellular matrix and/or a supplemented media.  Human hepatocytes in monolayer culture have been shown to maintain plasma protein production, glycolysis, and urea synthesis for extended periods of time from periods of several days up to 35 d.


Drug metabolizing enzymes, on the other hand, particularly the cytochromes P450, are maintained during the initial stages of culture, but decline with time.  However, these enzymes can be elevated in culture to some extent by the use of inducing agents such as phenobarbital, 3-methycholanthrene, and rifampicin.


A number of factors including the extracellular matrix used, seeding densities, culture media, media supplements, and cell–cell interactions need to be considered when culturing human hepatocytes.  Culture configurations that allow greater cell–cell contacts have been suggested to have more influence over the quality of the resultant hepatocytes than the constitution of the medium.  The encapsulation of hepatocytes in gel matrices (sandwich configuration) or production of cell aggregates (spheroids) has been suggested.  The isolation and culture of hepatocytes and the evaluation of many of the above factors has been discussed and reviewed by a number of workers.


It should be emphasized that a number of different conditions for isolating and culturing human hepatocytes have been published and are dependent on the type of studies to be carried out with the resultant cell preparation.  This chapter represents one of the accepted practices at the time of writing.

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