Heilbrigðisskýrslur - 01.12.1990, Blaðsíða 137

Primary Culture of Rat Hepatocytes
A Technique for Studying Long -Term Effects of Extracellular Mediators
on Hepatic VLDL Metabolism
O* G. Bjömsson, S. M. Bartlett University of Oxford, J. M. Duerden, and G. F.
Gibbons. Metabolic Research Laboratory, Radcliffe Infírmary,
Woodstock Road, Oxford 0X2 6HE, EnglandL
•ntroduction
In 1969, Berry and Friend described a technique which for the fírst time made it
Possible to isolate high-quality hepatic parenchymal cells (hepatocytes) in high
yield [1]. Hepatocytes were isolated by continuously perfusing a rat liver in situ in a
recirculating system with a buffer containing collagenase. Variants of the
technique [2 - 4] are now used extensively, and fresh isolated hepatocytes have
feplaced liver slices and, to some extent, whole-liver perfusions for metabolic
studies. During the past two decades this technique has also been widely used to
prepare cells for cell culture L3-7J. This short technical note describes modifications
°f these techniques that are suited for studying long-term effects of extracellular
^ediators on hepatic very low density lipoprotein (VLDL) metabolism.
Methods
Isolation of hepatocytes: Male Wistar rats were housed in a windowless room
uluminated with an artificial light (from 8 a.m. to 8 p.m.). The animals had free
^ccess to water and were fed ad libitum on a commercially available, pelleted
diet (Diet 41B; E. Dixon and Sons, Ware, Herts., U.K) containing (w/w) 47%
^urbohydrate (mainly starch), 15% protein, 3% fat and 4% crude fibre.
ttepatocytes were prepared between 10 and 12 a.m. under sterile conditions by a
r^odified two-step collagenase perfusion technique [2-5]. Rats (200-250 g) were
ar>aesthetized with pentobarbital and heparinized, the abdomen was opened, and
he portal vein was cannulated with a #18 Argyle Medicut Luer-Lok needle (Fig.
^ The inferior vena cava was ligated just above the level of the renal veins, the
chest was opened, and the thoracic portion of the inferior vena cava was
cannulated via the right atrium with a #16 Luer-Lok needle (Fig. 1). The liver
^as perfused at the rate of 50 - 60 ml min'1 with a modified Krebs-Henseleit buffer
JNaCl, 121.5 mM; KCl, 4.8 mM; KH2P04, 1.2 mM; MgS04 . 7H20, 1.3 mM;
aHC03, 25.6 mM; EGTA, 0.5 mM; glucose, 26.0 mM) at 37° C using a Watson-
^larlow (Type MHRE) peristaltic pump. With the exception of the first blood-
stained 50 ml, which was discarded, the perfusate was returned to the reservoir
ar>d recirculated. The buffer was oxygenated with 02 (95%) + C02 (5%) for 1 h
a ” kafore initiation of perfusion as well as during the entire perfusion period.
■^Vter = 5 mjn 0f perfusion, collagenase (Collagenase A, 103 586, Boehringer