Seven Steps to Stellate Cells

Summary

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Abstract

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes^1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A^{1, 2}. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis³. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension⁴. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes⁵.

Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets.

Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Protocol

C57BL/6 mice should be used at ~20 weeks of age or older. The use of male mice weighing 25-30g is recommended. The yield of stellate cells can be increased by feeding mice a vitamin A-enriched diet for 2 months prior to stellate cell isolation. Approximately 2x10⁵ hepatic stellate cells can be purified from the liver of one C57BL/6 mouse, whereas the yield of stellate cells from Balb/c mice is considerably higher. The following protocol is adjusted to 5 mice. Animal care and experimentation were performed in accordance with approved Institutional Animal Care and Use Committee protocols.

1. In situ perfusion of mouse liv....

Discussion

Hepatic stellate cells regulate essential physiological and pathophysiological processes in the liver. Moreover, stellate cells possess antigen presenting properties, rendering them an important component of the hepatic immune response. Although hepatic stellate cells comprise 10-15% of the total cell number in the liver, isolation of these cells is challenging due to their localization in the perisinusoidal space of Disse.

Here we present a straightforward method to isolate hepatic .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70μm Cell Strainer	BD Biosciences	352350
CaCL2 x 2H2O	Sigma-Aldrich	C3306
Collagenase P	Roche Group	11249002001
DMEM	GIBCO, by Life Technologies	11960
DNase I	Roche Group	10104159001
DPBS	Cellgro	21-031-cv
EGTA	Fluka	3777
Fetal Bovine Serum	GIBCO, by Life Technologies	16000
Glucose	Sigma-Aldrich	G7528
Gradient Centrifugation Tubes	Greiner Bio-One	163160
HEPES	GIBCO, by Life Technologies	15630
KCL	Sigma-Aldrich	P9541
KH2PO4	Sigma-Aldrich	P9791
L-Glutamine	GIBCO, by Life Technologies	25030
MgCl2 x 6H2O	Fluka	63068
MgSO4 x 7H2O	Sigma-Aldrich	M2773
Na2HPO4 x 2H2O	Fluka	71643
NaCl	Sigma-Aldrich	S3014
NaH2PO4 x H2O	Fluka	71507
NaHCO3	Fluka	71628
Nycodenz	Accudenz AG	AN7050/BLK
Penicillin/Streptomycin	GIBCO, by Life Technologies	15140
Peristaltic Pump	Cole-Parmer	HV-7523-70
Phenol Red	Sigma-Aldrich	P4633
Pronase E	Calbiochem	7433-2
Silicone Tube	Masterflex (Cole Palmer)	HV-96440-14
Sodium Pyruvate	GIBCO, by Life Technologies	11360
Tissue culture flask (25cm2)	BD Biosciences	353108
Winged Infusion Set	Terumo Medical Corp.	1SV27EL