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May 4, 1978 - collagen, concanavalin A and galacto(asialo-)proteins (Lin & Snodgrass, 1975;. Michalopoulos & Pitot, 1975; Rubin, Kjelle'n & Obrink, 1977; ...
J. Cell Sci. 34, 117-131 (1978) Printed in Great Britain © Company of Biologists Limited 1978

EFFECT OF TEMPERATURE AND DIVALENT CATIONS ON THE SUBSTRATUM ATTACHMENT OF RAT HEPATOCYTES IN VITRO PER O. SEGLEN AND ROLV GJESSING Department of Tissue Culture, Norsk Hydro's Institute for Cancer Researcli, Tlie Norwegian Radium Hospital, Montebello, Oslo 3, Nortoay

SUMMARY The attachment of rat hepatocytes to polystyrene-adsorbed serum protein is relatively insensitive to inhibitors such as dextran sulphate, cycloheximide, colchicine and cytochalasin B, and enzymes like trypsin and neuraminidase, but it is strongly dependent on divalent cations. Mg a + supports attachment better than Ca s+ , but a combination of both is required for maximal attachment. The attachment is very temperature-sensitive, with a biphasic Arrhenius plot indicating an activation energy of 123 kj/mol above 34 °C and 374 kj/mol below 34 ° C The adsorbed attachment-promoting serum factor is inactivated by trypsin, or by Ca 1+ dependent proteases which contaminate commercial preparations of collagenase. The adsorbed factor is resistant to treatment with glutaraldehyde, neuraminidase and heating to 90 °C, whereas the same factor in the unadsorbed state (in serum) is destroyed by heating to 70 °C. The factor in serum is unable to compete with the adsorbed factor for cell binding, hence it would appear that adsorption to polystyrene induces the active, heat-resistant conformation of the factor.

INTRODUCTION

The attachment and spreading of cells in tissue culture has been subject to intensive investigation (Takeichi & Okada, 1972; Grinnell, 1974, 19760,6; Grinnell, Hays & Minter, 1977; Moore, 1976; Ueda, Ito, Okada & Ohnishi, 1976; Juliano & Gagalang, 1977), but very few studies have dealt with the attachment properties of normal epithelial cell types. With the development of the collagenase perfusion techniques for mass isolation (Berry & Friend, 1969; Seglen, 1976a) and in vitro maintenance (Bissell, Hammaker & Meyer, 1973; Bonney, Becker, Walker & Potter, 1974) of normal rat hepatocytes, it has become possible to investigate the attachment of these typically epithelial cells. It has previously been shown that rat hepatocytes can attach to several different protein substrata, such as polystyrene-adsorbed serum piotein, collagen, concanavalin A and galacto(asialo-)proteins (Lin & Snodgrass, 1975; Michalopoulos & Pitot, 1975; Rubin, Kjelle'n & Obrink, 1977; Hook et al. 1977; Seglen & Fossa, 1978). In the present paper the divalent cation requirements for attachment to adsorbed serum protein and the effect of temperature on attachment have been investigated.

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MATERIALS AND METHODS Isolation of hepatocytes Isolated hepatocytes were prepared from the livers of 18 h-fasted male Wistar rats (250300 g) by the method of collagenase perfusion (Seglen, 1973, 1976a). Serum pretreatment of culture dishes Six-centimetre polystyrene tissue culture dishes (Falcon cat. no. 3002) were pretreated with serum on the day of the experiment. The dishes were left for 10 min at room temperature with the bottom covered with foetal calf serum; then washed 3 times with distilled water and airdried at room temperature. This treatment leaves a layer of serum protein adsorbed to the polystyrene surface. Measurement of attachment One hundred microlitres of suspended hepatocytes were incubated in the serum-pretreated 6-cm culture dishes with 2 ml of suspension buffer or perfusion buffer fortified with Ca I+ and Mg1+ (Seglen, 1973), and 1 ml of 0-9% NaCl or various additives in isotonic solution. The final standard concentrations of Ca1+ and Mg1+ were 2 mM unless otherwise indicated. The standard cell concentration in the incubate was 3 mg/ml (4 x io 5 cells/ml; 14000 cells/cm1). Attachment was measured as previously described (Seglen & Fossa, 1978), i.e. as the reduction (in %) in the initial optical density (at 650 nm) of the cell suspension. For the measurement of final optical density, non-attached cells were uniformly suspended by shaking the culture dishes on a shaker platform (Cenco) at 50 rev /min for 1 min. Scanning electron microscopy Cells to be prepared for scanning electron microscopy were allowed to attach to serumpretreated i-cm circular glass coverslips (polystyrene tended to melt during critical point drying) for various lengths of time; then fixed in 1 % glutaraldehyde/o-i M cacodylate buffer, pH 7-4, 305 mosM, for at least 1 h. The fixed cells were dehydrated in ethanol, transferred to amyl acetate and critical point dried (Balzers union drier) with carbon dioxide. The specimens were coated with gold (Polaron SEM coating unit E 5000) and examined with a high-resolution scanning electron microscope (Philips PSEM 500) at 50 kV. Pictures were taken on Polaroid film (Polapan type 52). Measurement of proteolytic activity Collagenolytic activity was measured with azocoll as substrate: 0-5 ml of perfusion buffer, pH 7-4 (Seglen, 1973) containing azocoll (30 mg/ml) and Ca1+ (1 min) was incubated with 25 fli of enzyme solution (Sigma collagenase type I or III at 0-2 mg/ml in perfusion buffer) for 5 min at 37 °C. The reaction was stopped by the addition of 3 ml ice-cold H,O; the supernatant was filtered through a cotton-wool plug in a 5-ml disposable pipettor tip, and the absorbance measured at 520 nm. Caseinolytic activity (taken to represent general proteolytic activity) was measured with azocasein (Sigma) as substrate: 1 •$ ml of perfusion buffer containing azocasein (20 mg/ml) and Ca1+ (2 mM) was incubated with 100 fil of enzyme (Sigma collagenase type I or III at o-z mg/ ml in perfusion buffer) for 30 min at 37 °C. The reaction was stopped by the addition of 2 ml ice-cold perchloric acid (2% w/v). After 10 min at o °C the tubes were centrifuged for 5 min at 6000 rev/min, and the absorbance measured in the supernatants at 366 nm. Chemicals Foetal calf serum was from Grand Island Biological Company, Grand Island, N.Y., U.S.A. Glutaraldehyde (25 % solution) was from Serva Fine Biochemicals, Heidelberg, Germany, and cytochalasin B from Aldrich Chem. Co. Trypsin (Difco 1 : 250) was from Difco Laboratories,

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Detroit, Michigan. Dextran sulphate (made from dextran with mol. wt 5 x io Daltons) from Pharmacia, Uppsala, Sweden; and Azocoll (50-100 mesh) from Calbiochem, Los Angeles, California. Other enzymes and biochemicals were purchased from Sigma Chem. Co., St Louis, Missouri, U.S.A. Abbreviations EDTA ethylenediamine tetra-acetic acid. EGTA ethyleneglycol-4« (/?-aminoethyl ether) iV.iV'-tetra-acetic acid. NEM N-ethyl-maleimide. RESULTS Kinetics of attachment Hepatocytes attach to serum-pretreated dishes with a somewhat variable time lag, generally of 5-10 min duration (Fig. 1). This may be the minimum time required for development of an attachment strength sufficient to withstand the subsequent shaking, routinely used to stop further attachment and to suspend the non-attaching cells uniformly. Attachment was essentially completed in 30 min, as previously found for incubation in the presence of serum (Seglen & Fossa, 1978). Most of the intact cells attach, giving plating efficiencies of 80-85 %. i

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-

60 E

/

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-

7 20

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r ^i—n—oJLri c\^\

i 1 . 15 20 Incubation time, min .

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Fig. 1. Time-course of attachment. Hepatocytes were incubated with i a mM Ca t+ and 06 mM Mg i+ in serum-pretreated culture dishes at 37 °C, and the percentage of attached cells was measured at the time points indicated. Each point represents a single dish.

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P. O. Seglen and R. Gjessing

Fig. 2. Morphology of attaching and spreading cells. Hepatocytes were allowed to attach to serum-pretreated glass coverslips for 30 min (A, B), 25 h (c, D) and 5 h (E, F). The cells were then prepared for scanning electron microscopy, and photographed at magnifications of x 2500 (A, C, E) or x 10000 (B, D, F).

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Morphology of attaching and spreading cells Hepatocytes settling on the bottom surface of the culture dish immediately attach and begin to spread. Anchoring filopodia can be observed (by scanning electron microscopy) as early as 5 min, and by 30 min the cells are firmly attached to the surface by a cytoplasmic stem from which filopodia and thin, lamellar cytoplasm spread radially (Fig. 2A, B). By 2-5 h the main cell body as well as the lamellar cytoplasm is beginning to spread beyond the initial periphery of the cells (Fig. 2C,D), and cell spreading is now also observable by light microscopy (Seglen & Fossa, 1978). By 4-5 h the cells are well spread and flattened (Fig. 2E,F). Table 1. Effects of various pretreatments on the ability of adsorbed serum protein to support hepatocyte attachment % attachment Expt. i Control Glutaraldehyde Neuraminidase Heating to 70 °C Expt. 2 Control Trypsin Expt. 3 Control Crude collagenase + Ca1+ Crude collagenase + EGTA Crude collagenase + Ca*++ NEM Expt. 4 Control Purified collagenase + Ca1+ Purified collagenase + EGTA Purified collagenase + Ca t+ + NEM

92 90 93 90

88 18 87 2

87 2

84 51 83 77

Serum-pretreated culture dishes, containing a layer of adsorbed serum protein, were subjected to one of the following treatments: incubation for 10 min at room temperature with glutaraldehyde (3%); incubation for 2 h at 37 °C with neuraminidase (Sigma type VIII, 0-25 units/ml); heating to 70 CC for 5 min in H,O; incubation for 1 h at 37 °C with trypsin (Sigma type III, o-i mg/ml); or incubation for 90 min at 37 °C with either crude (Sigma type I, 0-5 mg/ml) or purified (Sigma type III, 05 mg/ml) collagenase in the presence of Ca1+ (1 ITIM), EGTA (1-6 HIM) or NEM (25 min). After the treatment, the dishes were washed 3-5 times in distilled water and dried. For measurement of attachment, the dishes were incubated with hepatocytes for 30 min at 37 °C. Each value is the mean of 2 dishes.

Properties of the substratum-adsorbed attachment factor from serum To characterize the substratum-adsorbed serum factor(s) required for hepatocyte attachment, the serum-pretreated dishes were subjected to various forms of treatment prior to incubation with cells (Table 1). Neither the fixative glutaraldehyde nor

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the sialic acid-removing enzyme neuraminidase affected the ability of the adsorbed serum factor to support attachment, and surprisingly heating to 70 °C, which destroys the ability of fluid serum to support attachment and spreading (Grinnell, 1976 a; Seglen & Fossa, 1978), was also ineffective. Trypsin treatment reduced the extent of attachment considerably, but part of this effect may have been due to adsorption of trypsin and subsequent infliction of damage upon the hepatocytes, as evidenced by a reduction in cellular viability (from 96 to 80 %) and extensive cell clumping.

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50 60 70 Temperature, °C

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Fig. 3. Heat sensitivity of adsorbed and soluble serum attachment factor. Hepatocytes were incubated in culture dishes which had been pretreated with heated serum (iomin heating at the temperature indicated (•)), or in dishes which had been similarly heated after adsorption of untreated serum (O). Attachment was measured after 30 min incubation at 37 CC; each point is the mean of 2 dishes.

Treatment with a crude collagenase preparation (Sigma type I) destroyed the attachment-promoting activity of the adsorbed factor (Table 1, expt. 3). This proteolytic destruction was prevented by EGTA but not by iV-ethyl-maleimide, consistent with collagenase being the active enzyme (Seifter & Harper, 1970; Peterkofsky & Diegelmann, 1971). However, a more purified collagenase (Sigma type III), which had a high collagenolytic activity as tested by the azocoll assay, affected the attachment factor much less than did the crude collagenase (Table 1, expt. 4). Moreover, the effect (of the purified collagenase preparation) was completely prevented by iV-ethyl maleimide, which does not affect collagenase itself (Peterkofsky & Diegelmann, 1971). Inactivation of the attachment factor is therefore not due to collagenase, but to contaminating proteases known to be present even in chromatographically purified collagenase preparations (Peterkofsky & Diegelmann, 1971). Although the active protease con-

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taminants in both types of collagenase preparation appear to be Ca2+- dependent, the contaminants in the crude collagenase preparation (which has at least 10 times higher general proteolytic activity than the purified collagenase preparation, as measured by a caseinolytic assay) are NEM-resistant whereas the contaminating protease in purified collagenase is NEM-sensitive (Peterkofsky & Diegelmann, 1971), showing that the attachment factor is susceptible to attack by several types of proteolytic enzyme. To study further the surprising heat stability of the adsorbed serum factor, the attachment-supporting properties of whole serum and polystyrene-adsorbed serum were compared after heating to various temperatures (Fig. 3). The activity in serum was destroyed by heating to 70 °C, as previously reported (Seglen & Fossa, 1978), whereas the adsorbed activity was unaffected even by heating to 90 °C, at which point melting deformation of the polystyrene dish was considerable. Apparently the adsorbed factor is immobilized in its active conformation with such strength as to resist denaturation both by heat and glutaraldehyde. Effects of inhibitors

In an attempt to uncover some of the functional requirements for attachment, a number of inhibitors were tried. Dextran sulphate, reported to prevent the attachment of tissue culture cells (Bremerskov, 1973) had very little effect on hepatocytes (less than 20 % inhibition of attachment at 1 mg/ml). Cycloheximide, at a concentration (1 HIM) which blocks hepatocytic protein synthesis (Seglen, 1977) had no effect, i.e protein synthesis is not required for attachment. The very moderate effects of colchicine, cytochalasin B and NH4C1 (4, 25 and 13% inhibition, respectively), which interfere with cellular motility and/or hepatocytic protein secretion (Seglen & Reith, 1977), indicate that neither of these processes play any major role in cell attachment. Even cytochalasin-treated hepatocytes attach with 75 % of the efficiency of the control, despite the extensive morphological alterations (disappearance of microvilli, appearance of large cytoplasmic blebs) undergone by these cells (Seglen, 19766). Pretreatment of the hepatocytes with neuraminidase (Sigma type V, 2 mg/ml, for 1 min at 37 °C), which removes terminal sialic acid from a number of cell-surface receptors (Ashwell & Morell, 1977), had no effect on attachment to adsorbed serum protein. Such enzyme treatment did, however, prevent attachment to adsorbed galactoprotein (asialo-fetuin). The binding to serum protein would therefore seem to be sialic acid-independent, not involving the hepatic galactoprotein receptor (Ashwell & Morell, 1977). Trypsin pretreatment (Difco crude trypsin, 2-5 mg/ml, for 1 min at 37 °C) also had little effect; the slight inhibition observed (20%) can probably be ascribed to cell damage (the viability of the preparation was reduced from 95 to 78%) and clumping. It is therefore unlikely that trypsin-sensitive receptors on the cell surface play any role in attachment. Fixation of the cells with glutaraldehyde, on the other hand, inhibited attachment completely, suggesting that a certain mobility of cell surface structures is necessary for attachment.

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Effects of divalent cations It was previously observed that attachment of hepatocytes in the presence of serum required divalent cations, most notably Mg2+ (Seglen & Fossa, 1978). Attachment to adsorbed serum protein exhibits a very similar cation dependence (Table 2). Mg2+ stimulates attachment somewhat better than Ca2+, but both ions are required for maximal attachment, as in the presence of serum. It should be noted that cells attach with the same efficiency in the presence and absence of serum, i.e. there is no apparent competition between adsorbed and soluble serum factors for hepatocyte binding. Table 2. Divalent cation requirement of attachment o attachment None

Ca»+

Mg 1 +

5 8 3

45

Si 72 77

Serum+ EGTA Serum+ EDTA Adsorbed serum

6o

C 79 84 86

Hepatocytes were incubated for 30 min at 37 °C either in the presence of 10 % foetal calf serum, or with serum-pretreated culture dishes in the absence of serum (i.e. with adsorbed serum). EDTA (1 ITIM), EGTA ( I HIM), CaCl, (2 ITIM) or MgCl, (2 miu) were present as indicated. Each value is the mean of 2dishes.

Table 3. Effect of divalent cations on attachment stability % attachment Shaking time, min 5 10 15 20 25

Ca 1+

Ca 1+ + Mg 1 +

7i 48 52 48 42

81 79 78 73 77

Hepatocytes were allowed to attach to preadsorbed serum for 30 min at 37 °C in the presence of either Ca 1+ alone (o-8 mM) or Ca 1+ (08 HIM) + Mg«+ (0-4 I M ) . The culture dishes were then shaken on a platform at 50 rev/min for the length of time indicated, and the percentage of cells remaining attached was determined. Each value represents a single dish.

Fig. 4 shows the dose-response relationship for the two cations alone and in combination. Mg2+ supports attachment better than Ca2+ at all concentrations; the effectiveness of the latter being somewhat variable (cf. Tables 2 and 3). Attachment with Ca2+ alone is apparently not as firm as when both ions are present, since the Ca2+attached cells are more easily detached by shaking (Table 3). Effect of temperature The attachment of fibroblastoid cells in vitro has been shown to be extremely temperature-sensitive (Ueda et al. 1976; Juliano & Gagalang, 1977). This was also

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found to be the case with rat hepatocytes: in the experiment shown in Fig. 5, very little attachment occurred within 30 min at 22 °C. Although the temperature-sensitivity varied somewhat from experiment to experiment, significant inhibition of attachment could usually be observed already below 30 °C (Fig. 6). However, at long incubation times (30-40 min) maximal attachment could be achieved at suboptimal temperatures. For a more precise characterization of the temperature effect a short incubation period (10 min), during which attachment proceeded at a maximal rate, had to be chosen. Fig. 7 shows an Arrhenius plot of the data from such an

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Divalent cation concentration, mM

Fig. 4. Dose-response curves for the effects of Ca1+ and Mg1+ alone and in combination on hepatocyte attachment. The cells were incubated for 30 min at 37 °C in the presence of Ca1+ (O) or Mg*+(#) alone at the concentration indicated, or with 4 mM Mg l+ and the concentration of Ca t+ indicated (A). Each value represents a single culture dish.

experiment. The curve is biphasic with a break point in the region around 34 °C, possibly corresponding to a phase transition in the membrane lipids at this temperature (Overath, Thilo & Trauble, 1976; Seglen & Solheim, 1978). The activation energy in the physiological temperature range (above 34 °C), as calculated from the regression line, is 123 kj/mol; below 34 °C it is 374 kj/mol. For comparison, we have previously found that the activation energies for protein synthesis and protein degradation are also of the order of 100 kj/mol at physiological temperatures (Seglen & Solheim, 1978).

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Fig. 5. Time-course of attachment at high and low temperature. Hepatocytes were incubated with o-8 miu Ca1+ and 0-4 HIM Mg1+ at 37 °C (O) or 22 °C ( • ) for the period of time indicated; each value represents a single culture dish. DISCUSSION

The basic requirements for hepatocyte attachment as established in the present and previous papers (Rubin et al. 1977; Hook et al. 1977; Seglen & Fossa, 1978) are similar to those defined for attachment and spreading of other cell types (Weiss, 1972; Weiss & Chang, 1973; Takeichi & Okada, 1972; Grinnell, 1974, 19760,6; Grinnell et al. 1977; Moore, 1976; Ueda et al. 1976; Juliano & Gagalang, 1977). The initial attachment of hepatocytes is probably largely due to the formation of stable contacts between cell surface microvilli and the substratum, but it may be aided by short anchoring filopodia (attachment microextensions) (Witkowski & Brighton, 1972; Albrecht-Buehler, 1976), observable by scanning electron microscopy as early as s min after seeding the hepatocytes. Spreading of lamellar cytoplasm at the cell base (Fig. 2B) also takes place so early that it well may be an integral part of the attachment reaction. Subsequent spreading occurs by the gradual peripheral extension of the lamellar cytoplasm eventually followed by a general cell flattening. A similarly uniform spreading of the peripheral cytoplasm in all directions, maintaining the circular cell outline (except in regions of cell contact), has been observed, e.g. in Chang cells (Dalen & Scheie, 1969). Attachment of hepatocytes to adsorbed serum protein requires divalent cations, with Mg 2+ being more effective than Ca2+, and the combination of both giving maximal attachment. A similar relationship has been found for the attachment of rat

Attachment of rat hepatocytes

| 40 -

20 -

20 Temperature, °C

Fig. 6. Effect of temperature on attachment. Hepatocytes were incubated with i-2 mM Ca t+ and o-6 mM Mg1+ for 45 min at the temperature indicated; 2 different experiments (O, • ) are shown. Each value is the mean of 3-4 culture dishes. 1

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