<em>In vivo</em> Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Abstract

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of . These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4 . Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6 . For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7 .With the exception of one report 8 , there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1 . Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11 .In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SECspecific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis. Video LinkThe video component of this article can be found at https://www.jove.com/video/3138/ Protocol 1. Excision of the liver (adopted from P.O. Seglen 12 and R. Blomhoff 13 with modifications 14,15 )1. Add 10 mL 30% isoflurane in polyethylene glycol to a petri dish in a closed 5.5 L chamber. It is optimal to wait 10-15 min after addition of isoflurane to create a stabilized atmosphere within the chamber. 2. Place a rat in the sealed chamber and allow it to become anesthetized. Full effects of the anesthesia are apparent when the animal becomes limp, unresponsive, and exhibits deep breathing. 3. Place 2 mL of isoflurane in po...

Respective roles of hyaluronidases 1 and 2 in endogenous hyaluronan turnover

“…The liver was then removed, minced, and incubated with 1 mg/ml collagenase for 45 min at 37°C. As described previously (27), liver parenchymal cells (PCs) and nonparenchymal cells (NPCs) were separated by successive differential centrifugations. This method allows a ≥97% hepatocyte‐pure PC preparation.…”

Alcoholic vs non-alcoholic fatty liver in rats: distinct differences in endocytosis and vesicle trafficking despite similar pathology

“…Blood samples were collected via the axillary artery, and serum used for analysis. A piece of whole liver was obtained for histological observation (after knotting a small portion of a small liver lobe with surgical polyester thread); the remaining liver was perfused with isotonic buffers containing collagenase (Type 1 V, Sigma #C5138) as described previously [ 14 , 15 ].…”

Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

“…Primary hepatocytes were isolated from 129S1/SvImJ (002448, Jackson laboratory) wild type mice (10–12 weeks) as described [10]. Prior to sacrifice animals were housed in the AAALAC approved facility at the University of Nebraska – Lincoln (UNL) in ventilated cages at 22 °C with a 14/10 hour day/night cycle and were allowed free access to water and standard laboratory chow (2016 Teklad Global 16 % Protein Rodent Diet (Harlan Laboratories)).…”