Abstract
BackgroundPrevious methods for Kupffer cells (KCs) isolation require sophisticated skills and tedious procedures. Few studies have attempted to explore the self-renewal capacity of KCs in vitro. Therefore, the aim of this study was to establish a simple method for rat KCs isolation and further investigate the mitotic potential of KCs in vitro.MethodsKCs were obtained by performing one-step perfusion, enzymatic tissue treatment, differential centrifugation and selective adherence. The proliferation ability of cultured KCs was determined by MTT assay and Propidium Iodide FACS analysis. Phagocytic assay and ED-1, ED-2 immunofluorescence were used to identify cell phenotype. After stimulation with LPS, the expression of surface antigens (MHCII, CD40, CD80, and CD86) and the production of cytokines (NF-κB, TNF-α, IL-6 and IL-10) were measured for cell function identification.ResultsKCs were isolated with certain numbers and reasonable purities. The KCs were able to survive until at least passage 5 (P5), and at P3 showed equally strong phagocytic activity as primary KCs (P0). After stimulation with LPS, the change in the expression of surface antigens and the production of cytokines for P3 cells was similar to that for P0 cells.ConclusionsOur study provides a simple and efficient method for KCs isolation, and reveals that self-renewing KCs have the same phagocytic activity and functions as primary KCs.
Highlights
Kupffer cells (KCs), named after the pathologist C. von Kupffer, are resident hepatic macrophages that account for 80–90% of total fixed tissue macrophages in the body [1]
Cytokines act as protective mediators for recovery of normal liver function [9], in some instances, excessive activation of KCs may result in exacerbation of the damage [10]
A cut was made in the right atrium wall, simultaneously, the portal vein was cannulated with a 22G catheter and the liver was perfused in situ with phosphate-buffered saline (PBS) at a rate of 10 ml/min for 5 min
Summary
Kupffer cells (KCs), named after the pathologist C. von Kupffer, are resident hepatic macrophages that account for 80–90% of total fixed tissue macrophages in the body [1]. KCs play an important role in liver physiological homeostasis and are intimately involved in the liver’s response to infection, toxins, transient ischemia, and various other stresses [3] through the expression and secretion of soluble inflammatory mediators [4,5]. Cytokines act as protective mediators for recovery of normal liver function [9], in some instances, excessive activation of KCs may result in exacerbation of the damage [10]. Proper therapeutic modulation of the inflammatory activities of KCs provides opportunities for new treatment approaches toward liver disease, and primary cell culture is indispensable for further studies in this area. Previous methods for Kupffer cells (KCs) isolation require sophisticated skills and tedious procedures. The aim of this study was to establish a simple method for rat KCs isolation and further investigate the mitotic potential of KCs in vitro
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