Effect of Method of Cell Isolation on the Metabolic Activity of Isolated Rat Liver Cells

J. Cell Sci. 10, 167-179 (i972) 167 Printed in Great Britain EFFECT OF METHOD OF CELL ISOLATION ON THE METABOLIC ACTIVITY OF ISOLATED RAT LIVER CELLS L. G. LIPSON, D. M. CAPUZZI AND S. MARGOLIS Clayton Laboratories, Department of Medicine, and the Department of Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A. SUMMARY Rat liver cells isolated with a tissue press, by tetraphenylboron (TPB) chelation, or by hyaluronidase and collagenase digestion were compared as to morphology, cell yield, and bio- synthetic activity. The cells were intact by light microscopy; ultrastructural changes were present on electron-microscopic examination of all cell types except those prepared by a modified enzyme incubation method. TPB chelation gave the largest and enzyme techniques the smallest yield of cells. All cell types incorporated labelled amino acids into cellular protein; however, amino acid incorporation was greatest in cells isolated by the revised enzyme technique. Only enzyme and mechanical cells incorporated acetate into cellular lipid. Cofactor supplementation was not required in the modified enzyme cells. Acetate incorporation was more sensitive to preincubation than was amino acid incorporation. Calcium, which was required to prevent aggregation of enzyme cells, inhibited amino acid incorporation moderately and acetate incorporation completely in mechanical cells. INTRODUCTION Various techniques have been employed for the preparation of hepatocyte sus- pensions from rat liver. Because cells from each preparative method were reported to give different results in metabolic studies (Ontko, 1967; Rappaport & Howze, 1966; Howard, Christensen, Gibbs & Pesch, 1967), we undertook a systematic comparison of the effect of preparative technique on cell morphology, cell yield, and two bio- synthetic parameters of the isolated cells. A preliminary report of this work has been presented (Lipson, Margolis & Capuzzi, 1969). Initially 3 widely different methods for the isolation of individual liver cells were selected for study. These included extrusion through a tissue press (Ontko, 1967), potassium chelation by tetraphenylboron (TPB) (Rappaport & Howze, 1966), and enzymic digestion with hyaluronidase-collagenase (Howard et al. 1967). The incorporation of 14C-amino acids into cellular proteins and of 14C-acetate into cellular lipids were chosen as indicators of liver cell synthetic activity. Later in the course of this work a modification of the enzymic method was tested (Howard & Pesh, 1968), and the synthetic capacity of these cells was then compared with that of mechanical cells. All cell preparations incorporated uC-amino acids into proteins; however, 14C- acetate was incorporated into cellular lipids only by hepatocytes isolated by the per- 168 L. G. Lipson, D. M. Capuzzi and S. Margolis fused mechanical or modified collagenase-hyaluronidase methods. Cells isolated by the modified enzyme procedure are best suited for study of the biosynthesis of cellular protein and lipids. MATERIALS AND METHODS Uniformly labelled 14C-mixed amino acids and sodium [i-14C]acetate were obtained from New England Nuclear Corporation; sodium tetraphenylboron (TPB) from Sigma Chemical Company; and collagenase and hyaluronidase from Sigma Chemical Company and Nutritional Biochemical Corp. Bovine serum albumin (BSA), powder fraction V, was supplied by the Armour Pharmaceutical Co.; polyvinylpyrrolidone (PVP) by the Arthur Thomas Co. and new methylene blue and nigrosin by Fisher Scientific Co. All other reagents were products of the J. T. Baker Chemical Co. Methods of cell preparation Male Sprague-Dawley rats weighing about 300 g, fed ad libitum, were used in all experi- ments. Under light ether anaesthesia, the liver was perfused through the portal vein with 50 ml of Cai+-free Locke's solution (Ontko, 1967) containing 0-027 M sodium citrate (Locke's citrate solution). The liver was removed and rinsed twice in iced physiological saline. Following this stage, 3 different methods were used to isolate cells. Method 1: mechanical, tissue-press extrusion. Mechanical liver cells were prepared by a modification of the method of Ontko (1967). The washed liver was extruded through a tissue press into 50 ml of cold Locke's citrate solution. This mixture was filtered through a 100-mesh silk cloth, and the filtrate was centrifuged at 100 g for 5 min in a clinical centrifuge at 4 °C. The supernatant was discarded, and the cellular pellet was dispersed in 3 volumes of 0-02 M Tris- o-i M KC1 solution, pH 76. Centrifugation was repeated as outlined above. The supernatant was removed and the packed cells were dispersed in an equal volume of cold Tris-KCl solution to form the concentrated cell suspension used for metabolic studies. Method 2: tetraphenylboron. TPB cells were prepared by a modification of the method of Rappaport & Howze (1966). The liver was finely minced with scissors in 5 ml of 'sucrose-salt' solution containing 2 mM sodium TPB. The mince was diluted with an additional 70 ml of this solution, stirred magnetically for 40 min at 4 °C, and twice passed through a 30-ml syringe (inner diameter of 1 mm). After the mixture was filtered through a silk cloth, cells were washed as described for the mechanical cells. Cells were dispersed in 2 volumes of cold Tris-KCl buffer. Method 3: collagenase and hyaluronidase digestion, syringe extrusion. Enzyme cells were isolated by a slight variation of the method of Howard et al. (1967). The liver was minced in 3 ml of cold, calcium-free Hanks's solution (Hanks & Wallace, 1949) that contained 0-05% collagenase and 015% hyaluronidase and transferred to two 250-ml Erlenmeyer flasks which contained 85 ml of additional enzyme solution. The flasks were shaken (140 oscillations/min) in a waterbath shaker at 37 °C for 1 h. The contents of the flasks were then combined and diluted to 40 ml with cold, calcium-free Hanks's solution. The supernatant was discarded; the re- maining pieces of tissue were suspended in 30 ml of cold calcium-free Hanks's solution and twice extruded through a 30-ml syringe. The cells were filtered through the silk cloth and washed as in the mechanical method, except that 3 mM CaCl, was added to the Tris-KCl buffer to prevent cell clumping. Method 4: collagenase and hyaluronidase digestion, gentle mincing (modified enzyme cells). Cells were isolated by a modification of the method of Howard & Pesch (1968). In this procedure the liver was first perfused with 15 ml of 0-05 % collagenase and o-io% hyaluronidase in cold calcium-free Hanks's solution. The liver was minced in 3 ml of this solution and then placed in a 250-ml Erlenmeyer flask with 20 ml of additional enzyme solution. After 60 min of incubation the flask contents were diluted with 40 ml of cold calcium-free Hanks's solution, filtered and then washed as above. No mechanical procedures were performed on tissue fragments. Plastic containers were used for all steps except the incorporations. Isolation method and rat liver cell metabolism 169 Cell morphology and cell counts Morphology of unstained cells and of cells stained with haematoxylin and eosin was examined grossly by light. Intactness of cellular membranes was assessed by staining with new methylene blue, nigrosin, or trypan blue. Exclusion of these dyes was considered an index of viability. Cells were routinely counted in a haemocytometer. Electron microscopy Cells were fixed in suspension in 3 % glutaraldehyde in o-1 M phosphate buffer, pH 7-2, which contained 0-5 mg of CaCl2 per 10 ml of buffer. The pellet was washed 3 times in phosphate (01 M) sucrose (0-25 M) buffer and allowed to remain for 18 h in this solution. The pellet was treated for 2 h with 2 % osmium tetroxide in the same buffer and then dehydrated in a series of 50-100 % ethanols. The preparation was immersed in propylene oxide and embedded in Epon 812. Sections 50-60 nm thick were made on an LKB ultramicrotome, stained with a saturated solution of uranyl acetate in 1 % sodium borate solution, and stained again with Reynolds lead citrate. Preparations were viewed in an Elmiskop I electron microscope at 80 kV. Isolation of labelled lipids After incubation with "C-acetate at 37 °C, liver cells were separated from the medium by centrifugation at 2ooog and the labelled lipids were extracted and washed by the method of Folch, Lees & Sloane Stanley (1957). Dried labelled lipids were then dissolved in 0-5 ml of chloroform-methanol (2:1), and applied along with standards to glass plates coated with silica gel G. The lipids were separated by thin-layer chromatography (TLC) using hexane-diethyl ether-glacial acetic acid (70:20:1 by volume) as the developing solvent. The spots were identi- fied, scraped from the plates, and counted in an Ansitron liquid scintillation spectrometer which had an efficiency of 58 % for 14C. Isolation of labelled protein After incubation of cells with a 14C-amino acid mixture at 37 °C, the flasks were placed on ice, 0-2 ml of 10 % casein hydrolysate was added, and cells were separated from the medium as described. Cellular protein was precipitated by the addition of 3 ml of cold 10 % trichloroacetic acid (TCA). The labelled proteins were washed by the method of Manganiello & Phillips (1965), dissolved in 0-5 ml of 90 % formic acid, applied to 2'3-cm filter paper disks, and dried with a hair dryer. Disks were then placed in vials which contained scintillation fluid (100 mg i, 4-bis- 2(phenyloxazolyl)-benzene and 4 g diphenyloxazole per 1. of toluene) for counting in the scintillation spectrometer. Prevention of bacterial contamination Before use all solutions were passed through o-45-/tm Millipore filters into vessels which had been sterilized by heating at 130 CC for 20 min; 50 /tl of an antibiotic solution, which contained penicillin G (80 mg/ml) and streptomycin sulphate (2^5 mg/ml), was added to each incubation flask.

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