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 biosynthetic 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 suspensions 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 biosynthetic 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 experiments. 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 flaskswer e 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. To test the effectiveness of these antibacterial measures, after 2 h of incubation 025-ml aliquots from the incubation mixture were streaked on blood agar plates which were then incubated at 37 °C for 48 h. Cells isolated by the TPB method of Rappaport & Howze (1966), which involves passage of tissue fragments through a pipette, may be contaminated by salivary bacteria. 14C-amino acid incorporation was inordinately rapid in contaminated cells and numerous colonies appeared on the agar plates. When tissue fragments were instead passed through a syringe, lower but reproducible rates for amino acid incorporation were found and there was no bacterial growth. The magnitude of this effect is illustrated by an experiment in which amino acid incorporation was increased from 627 to 25000 dpm by the addition of o-i ml of saliva to TPB cells. 170 L. G. Lipson, D, M. Capuzzi and S. Margolis

RESULTS Cell yield and morphological characteristics The data in Table 1 show that the yield of cells by the TPB procedure (34 x io6 cells/g liver) was about 10-fold greater than that obtained by either enzyme method and about 3 times that produced by mechanical treatment. Since 4-7 x io6 cells were used in each incubation, as many as 50 incubations can be carried out with TPB cells prepared from a single liver.

Table 1. Yield and morphological characteristics of mechanical, TPB and enzyme cells

Mechanical TPB Enzyme Modified enzyme Cell yield* 10 34 32 4-5 Appearance by Moderate cellular Severe cellular Moderate cellular Intact electron damage damage damage microscopy Trypan blue Entered cells Entered cells Entered cells Excluded from cells dye • Results are from a single representative experiment and are expressed as io" cells/g liver.

In good preparations, cells were largely single, slightly ovoid, and grossly intact. Some cell fragments and free nuclei were present after all methods of preparation, but this was most marked in the original enzyme procedure. The cytoplasm of unstained cells from the mechanical and enzyme methods was distinctly granular while that of TPB cells was pale. Almost every cell took up new methylene blue or trypan blue dye, except for cells prepared by the modified enzyme method. In these latter preparations 50-70 % of the cells were impermeable to the dye, depending largely on the source of the collagenase. Preparations with high collagenase and low proteinase activity were the most effective. Those cells that were not stained by the dye had a distinct cellular membrane under the microscope. The stain with new methylene blue was ortho- chromatic with mechanical and enzyme cells but metachromatic (green) with TPB cells. Over a wide pH range new methylene blue did not turn green in the presence of sodium TPB alone. Electron-microscopic studies demonstrated ultrastructural modifications in the mechanical, TPB, and original enzyme cells (methods 1-3). These changes consisted of breaks in the plasma membrane, dilatation of the cisternae of the endoplasmic reticulum, and mitochondrial swelling. However, intracellular membranes were intact in each case. In contrast, at least 50% of the hepatocytes prepared by the modified enzyme method (method 4) were ultrastructurally intact with no evidence of subcellular or plasma membrane damage. Fig. 1 depicts a typical intact rat hepatocyte as viewed under the electron microscope. The plasma membrane as well as membranes of the nuclear envelope and endoplasmic reticulum (ER) are intact. Ribosomes are studded along the ER, and mitochondrial morphology is normal. The conditions of cell preparation have a profound effect on the yield, appearance, Isolation method and rat liver cell metabolism 171 and metabolic activity of cells. In methods (1-3), perfusion at 4 °C instead of at 37 °C, failure of the liver to distend and blanche during perfusion, or perfusion with isotonic saline rather than with Locke's citrate solution all reduced the yield of cells and increased the number of cell clumps. The mechanical and both enzyme methods were particularly susceptible to changes in perfusion technique, whereas the TPB technique was less affected.

Amino acid incorporation Under the same incubation conditions, the incorporation of amino acids into cellular proteins was 5 times greater in mechanical and the original enzyme cells than in TPB cells (Table 2). In these 3 cell types 3-10% of the labelled protein was found in the incubation medium. Cells isolated by the modified enzyme method gave 2-5 times greater incorporation than did mechanical cells. Mg2+, phosphate, succinate, and nicotinamide were routinely included in the incubation mixtures since this cofactor combination maximized amino acid incorporation by liver cells (Capuzzi & Margolis, 1971a). The low rate of amino acid incorporation by TPB cells did not result from cell damage incurred by prolonged exposure to TPB since incorporation was even lower in cells exposed to TPB for shorter periods. Table 2. Effect 0/ isolation procedure on incorporation of amino acids into proteins and acetate into lipids by rat liver cells

Substrate incorporation*

Amino acid Isolation procedure mixture Acetate Mechanical 750 1300 TPB 150 o Enzymef 750 o Modified enzyme 1850 2700 In each experiment cells were prepared from a single rat liver by both the mechanical method and one of the other techniques. Cells were incubated at 37 °C under air in stoppered 25-ml Erlenmeyer flasks at 130 oscillations/min. Cell concentrations were 1-5-1-8 x ioe cells/ml in amino acid experiments and 1-8-2-2 x 10* cells/ml in acetate studies. Incubation conditions for acetate and amino acid incorporation are as described by Capuzzi & Margolis (1971a, b). For acetate incorporation, each flask contained 1-5 /tCi sodium [i-14]acetate; potassium penicillin G (1 mg/ml); KC1 (100 mM); MgCl, (3-3 ITIM); MnCl, (o-i nw); sodium succinate (iorain); Coenzyme A (0-03 tnM); sodium citrate (3-3 DIM); nicotinamide (6-7 min); NADP (0-23 ITIM); and gluco8e-6-phosphate (1-7 mM) in a total volume of 3 ml. For amino acid incorporation, each flask contained 14C-amino acid mixture (1 /tCi), the same concentrations of antibiotics; Tris-HCl (20 mM), pH 7-3; MgCla (7-5 mM); potassium phosphate (10 DIM); nicotinamide (7-5 DIM) ; and sodium succinate (12 mM) in a total volume of 4 ml. • All results are expressed as cpm per 4x10' cells. f CaCl, (3 mM) was added to prevent clumping of cells.

Bacterial growth was prevented by the routine use of antibiotics, which did not inhibit amino acid or acetate incorporation in mechanical, enzyme, or TPB cells. In fact, there was no inhibition of amino acid incorporation even when antibiotic concentrations were increased 4-fold. 172 L. G. Lipsott, D. M. Capuzzi and S. Margolis

Acetate incorporation As indicated in Table 2, a combination of lipogenic cofactors was included for incubation of liver cells with 14C-acetate since a separate group of experiments demonstrated that lipid synthesis in mechanical cells was optimal in their presence. As further shown, incorporation of 14C-acetate into cellular lipid was active in both the mechanical and the modified enzyme cells, but no incorporation was found in either the original enzyme or TPB cells. Cells isolated by the modified enzyme procedure required no cofactor supplementation for active acetate incorporation into lipids. In fact, acetate incorporation was decreased by cofactor additions in the case of modified enzyme cells. Attempts to modify the procedure for isolation of TPB cells so that these would incorporate acetate into lipids were unsuccessful. Acetate incorporation was not promoted by changes in cofactors or by a decrease in cell preparation time. Thus, cells isolated after 15, 30 and 45 min of incubation with TPB failed to generate cells capable of incorporation of acetate into lipids.

Table 3. Distribution of cellular radioactivity into lipid classes in enzyme and mechanical cells and liver slices

Modified. Mechanical* enzyme Slices Lipid class (%) (%) (%)

Cholesterol 9 60 43 Triglycerides 9 13 20 Fatty acids 23 6 5 i ,2-Diglycerides 6 5 5 Monoglycerides 17 3 0 Cholesterol ester 2 5 4 Phospholipids 35 8 23 Extracted lipids were resolved by thin-layer chromatography as described in Materials and Methods. • Values are those of Capuzzi & Margolis (1971).

Distribution of label among lipid classes The results in Table 3 demonstrate that there were marked differences in the labelling pattern of lipids synthesized by mechanical versus modified enzyme cells. The latter incorporated a strikingly greater proportion of labelled acetate into cholesterol than did mechanical cells. As further shown in the table, the distribution of label was quite similar in modified enzyme cells and hepatic slices. This similarity may reflect the greater retention of biochemical and morphological integrity by the modified enzyme cells.

Effects of preincubation The different times required for preparation of the 4 cell types, which were 30 min for mechanical cells, 75 min for TPB cells, and 90 min for both types of enzyme cells, may influence amino acid and acetate incorporation by each cell type. To investigate Isolation method and rat liver cell metabolism 173 this question, amino acid (Table 4) and acetate incorporation were compared before and after preincubation for 1 h at either o or 37 °C. Amino acid incorporation by TPB cells was not impaired after preincubation at either temperature, but incorporation was reduced by preincubation of both other cell types. The reduction was greater at 37 °C, and the enzyme cells were more sensitive to preincubation. Acetate incorporation was decreased about 50% by preincubation of mechanical cells for 1 h at o °C; no incorporation was found after preincubation at 37 °C.

Table 4. Effect of pre-incubation of the 3 cell types on the incorporation of xxC-amino acids into cellular protein

Relative incorporation* Pre-incubation conditions Mechanical TPB Enzyme

No pre-incubation 100 (356)! 100(174) 100 (524) 1 h at 0 °C 87 100 63 Shaken 1 h at 37 °C Si 100 40 • Results are calculated as % of incorporation compared with cells that were not pre- incubated. f Numbers in parentheses indicate 14C-amino acid incorporation into cellular protein, expressed as cpm per 4x10" cells.

Table 5. Effect of washing procedure on acetate and amino acid incorporation in mechanical cells

Incorporation of label*

None 2720 1070 Regular 1020 900 Wash and resuspension 1180 961 Regular wash procedure is described in methods. Unwashed cells were separated from the Locke's citrate solution but not washed with Tris-KCl. The partial wash was identical with that of the regular wash except that the washed cells were then resuspended in an equal volume of the wash solution. • Expressed as cpm per 4x10* cells.

Effect of cell washes Mechanical cell preparations were routinely washed to reduce tissue debris and thus minimize the possibility of amino acid or acetate incorporation by extracellular enzymes or free subcellular particles. The effect of the wash procedure on amino acid and acetate incorporation into mechanical cells is shown in Table 5. Amino acid incorporation was reduced 2-5-fold after the cells were washed and resuspended once in fresh buffer. This fall in incorporation was not due to removal of subcellular particles or soluble enzymes since the same reduction was found when the washed cells were 174 L. G. Lipson, D. M. Capuzzi and S. Margolis resuspended in the wash fluid (Table 5). Acetate incorporation was little affected when the cells were washed and resuspended in fresh buffer before incubation.

Effects of calcium Initial attempts to utilize enzyme cells were unsuccessful because the cells clumped as soon as they were washed in Tris-KCl buffer. Microscopically many cells became fragmented and masses of cells were bound together. The addition of 1 % albumin, 1 mM MgCl2, or 1 % PVP did not prevent cell aggregation. Although mechanical and TPB cells did not clump immediately when washed in Tris-KCl buffer, in about one quarter of the preparations gross aggregation of both cell types occurred after 30 min of incubation.

It was found that clumping of enzyme cells was prevented by the addition of CaCl2 to the wash and incubation buffers. Clumping of mechanical and TPB cells was also prevented by addition of CaCl2 to the incubation medium in concentrations of 1 mM or greater. The addition of CaCl2 (3 mM) to the wash solution alone did not protect enzyme cells against aggregation, which usually occurred after 30 min of incubation at 37 °C in calcium-free, Tris-KCl solution. Aggregation was less of a problem with modified enzyme cells, but CaCl2 addition was necessary for complete prevention of clumping by these cells.

Table 6. Effects of calcium concentration on incorporation of ^C-amino acids and 1AC-acetate

Calcium 14C-amino acid incorporation* uC-acetate concentration, A incorporation:* mM Mechanical TPB mechanical

0 555 i55 582 0-3 283 (49)t 129(17) 146 (75) 10 194(65) 92 (41) 0 (100) 30 211 (62) 149 (3) 0 (100) • Results are expressed as cpm per 4x10' cells. f Numbers in parentheses indicate % inhibition compared with cells incubated in the absence of calcium.

As shown in Table 6, however, protein synthesis in mechanical cells was inhibited when CaCl2 was added in sufficient concentrations to prevent clumping of enzyme cells. This inhibition increases as the calcium concentration was raised to 1 mM, but there was no further inhibition at a calcium concentration 3-fold higher. CaCl2 also inhibited amino acid incorporation slightly in TPB cells. In contrast, amino acid incorporation was nearly doubled by the addition of CaCl2 (i-omM) to modified enzyme cells. Calcium ions also inhibited lipid synthesis in mechanical cells. At calcium concentrations of 0-3 mM, synthesis fell to 25 % of the level found in cells incubated in the absence of calcium. No acetate incorporation occurred at calcium concentrations greater than 1 mM. These inhibitory effects of calcium on lipid synthesis suggested Isolation method and rat liver cell metabolism 175 the possibility that small concentrations of calcium might account for the failure of TPB cells to incorporate acetate. However, the addition of sodium citrate (20 mM) as a chelator of divalent cations did not promote acetate incorporation in TPB cells. In the modified enzyme cells, acetate incorporation was increased 3-fold when 1 mM calcium chloride was added to the incubation medium.

DISCUSSION Both amino acid and acetate incorporation have been studied in rat liver cell suspensions, but in the past there has been little effort to compare the biosynthetic capacity of cells prepared by different techniques. In no reported instance has the metabolic activity of cells prepared by all 3 major methods (perfused mechanical, TPB, enzymic digestion) been systematically compared under the same incubation conditions. Friedmann & Epstein (1967) reported that both TPB and mechanical cells incorporated pHJleucine into peptide linkage at comparable rates. However, the report does not specify whether their crude cell preparations were washed free of subcellular debris before incorporation studies were begun. Moreover, the rapid rate of amino acid incorporation that they observed with the TPB cells may have represented bacterial contamination in view of our experience with the original Rappaport & Howze (1966) method and since very low concentrations of streptomycin reduced the incorporation by two-thirds according to their report. Jezyk & Liberti (1969) compared the synthesis of lipid, protein, and RNA from labelled precursors in mechanically and enzymically prepared rat hepatocytes. They found the latter preparation superior in all respects. However, no morphological correlations were reported in the study and TPB cells were not included. Moreover, in most of their metabolic comparisons, the incubation media employed for the mechanical cells differed from that used for the enzyme cells, although we have found that the composition of the incubation medium has a profound effect on the biosynthetic capacity of suspended cells. In addition, their mechanical cells were prepared without prior calcium chelation, and avoidance of this step in our studies has resulted in numerous cell clumps with reduction in cell yield, visibly damaged cells and much subcellular debris. Preliminary chelation of calcium also leads to a marked increase in the respiratory capacity of the isolated cells (Suzangar & Dickson, 1970). In the present study, we compared the metabolic activity of cells prepared by all 3 principal methods; tested the cell preparations under the incubation conditions found optimal for labelled acetate and amino acid incorporation; and performed the com- parative biosynthetic studies under identical incubation conditions. Despite considerable effort to maximize the biosynthetic activity of the other cell preparations by modification of the isolation method and incubation medium, the metabolic activity of modified enzyme cells was clearly superior to that of the other hepatocyte preparations. The rate of amino acid incorporation by enzyme cells was more than twice that of mechanical cells, and cofactors were not required. Mechanical and the original enzyme cells incorporated labelled amino acids at the same rate, which was 5 times greater than TPB cells. Modified enzyme cells were also more effective than the other cell types for pro- 176 L. G. Lipson, D. M. Capuzzi and S. Margolis motion of acetate incorporation into lipids even in unfortified media. This indepen- dence of modified enzyme cells on added cofactors for lipid synthesis probably reflects the ultrastructural intactness of plasma membrane and consequent retention of intracellular lipogenic factors. Conversely, the lipid cofactor requirements of cells prepared by the other methods correlates with their plasma membrane breaks and cofactor leakage. The integrity of the cell membrane may be particularly crucial for lipid synthesis, which occurs predominantly in the cytosol. Variable leakage of soluble cyto- plasmic enzymes may explain the greater sensitivity of acetate incorporation to changes in incubation conditions and to cell isolation procedures when compared with amino acid incorporation. These factors may also account for the disparity between acetate and amino acid incorporation when the synthetic capacities of different hepatocyte preparations are compared. Howard et al. (1967) observed intact plasma membranes in electron-microscopic studies of enzyme cells. In the present work, the majority of modified enzyme cells excluded vital dyes and showed intact plasma membranes under electron microscopy. In contrast, cells isolated by the other methods all stained with vital dyes and showed breaks in the plasma membranes. The major difference between the original and modified enzyme cell methods is the gentler mechanical manipulation of the liver entailed in the modified procedure. This suggests that gentle treatment of the liver is essential to maintain membrane integrity and cellular biosynthetic capacity. The large percentage of damaged hepatocytes obtained by Gallai-Hatchard & Gray (1971), using collagenase and hyaluronidase perfusion, and the improved cells obtained when trypsin was added to the perfusate may reflect the fact that gentler mechanical manipulation was also employed when trypsin was incubated. The cellular damage observed by the workers with the use of chelating agents or TPB correlates well with the present observations. Workers in other laboratories have shown that mechanical cells (Inoue, Hosokawa & Takeda, 1965), TPB cells (Friedmann & Epstein, 1967) and cells isolated after tryptic treatment (Scarpinato, 1963) are able to incorporate labelled amino acids into proteins. It is difficult however, to compare the present results with those of others since different cell concentrations, buffers, and incubation conditions were employed in each study. In preincubation experiments with mechanical cells, under conditions similar to those used for TPB cell isolation, the rate of acetate incorporation was reduced in proportion to duration of preincubation. These results suggest that the failure to obtain acetate incorporation in TPB and the original enzyme cells resulted either from a loss of intracellular material or from inactivation of lipogenic enzymes during cell preparation. It is also possible that TPB not only chelates potassium but also acts as a metabolic poison. When concentrations of TPB greater than 3 mM were used to prepare isolated liver cells, Rappaport & Howze (1966) noted gross cellular damage and an increased number of free nuclei. Electron-microscopic studies have shown that the addition of TPB to mouse liver or isolation of cells by the TPB method caused considerable ultrastructural damage to the cells (Harris & Leone, 1966). Utsumi & Packer (1967) Isolation method and rat liver cell metabolism 177 found that TPB was bound to mitochondrial membranes where it uncoupled oxidative phosphorylation. These observations of others, coupled with the low rate of protein synthesis, the lack of acetate incorporation, and the metachromatic staining of TPB cells, suggest that this agent may interfere with basic cellular biosynthetic processes. The inhibition of potassium accumulation observed in liver cells and liver slices incubated in extremely low concentrations of TPB (Murthy & Petering, 1969) have been emphasized as evidence of its toxicity to metabolic processes. However, it may be premature to discard the TPB method since there are reports that cells prepared with this agent can repair their initial metabolic damage after several days of growth in suspension culture (Casanello & Gerschenson, 1970). Both acetate and amino acid incorporation by mechanical cells were severely reduced by low concentrations of Ca2+. In contrast, Ca2+ stimulated both of these parameters of hepatocyte metabolism in the modified enzyme cells. These results are consistent with the data reported by others. Thus, ionized calcium inhibits cellular respiration in mechanical cells (Exton, 1964). In isolated mitochondria, calcium ions have produced mitochondrial swelling and uncoupling of oxidative phosphorylation (Hunter & Ford, 1955). These adverse affects of calcium in mechanical cells probably result from their abnormal entry into the cell because of loss of selective permeability of the plasma membranes. The stimulatory effect of Ca2+ on acetate and amino acid incorporation in modified enzyme cells may then provide further evidence for the intactness of the plasma membrane in this cell type. This differential effect of Ca2+ on mechanical versus enzyme cells has also been observed by Thimmappayya, Reddy & Bhargava (1970), who also point out that the average dry weight of a hepatocyte obtained by enzymic digestion is 2-2 times that obtained by the mechanical method. These data are consistent with a loss of soluble protein and entry of extracellular Ca2+ through plasma membrane interruptions in mechanical cells. Clearly, hepatocytes isolated by perfusion with enzymes provide the most suitable preparations of liver cells for metabolic studies. Both morphological and biochemical criteria attest to this conclusion in the present study. Experiments currently in progress in this laboratory indicate that the total recovery of morphologically intact cells can be increased several-fold by prolongation of the perfusion period. Thus, even the cell- yield advantage obtained with mechanical and TPB cells will probably be equalled with enzymically prepared liver cells.

This study was supported in part by Grant HE-09596 from the National Institutes of Health, United States Public Health Service, and by United States Public Health Service Training Grant 5-Fo3-GM-35,S7i, from the National Institute of General Medical Sciences. The authors wish to express their appreciation to Dr John W. Greenawalt and Mr Glenn Decker for their assistance in the electron-microscopic studies of isolated liver cells.

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[Received 2 July 1971) Isolation method and rat liver cell metabolism

Fig. i. Isolated rat liver cell prepared by method 4. x 10 000.