ATTACHMENT

OF RAT HEPATOCYTES

TO SUBSTRATA COLLAGEN,

OF SERUM

IN VITRO

PROTEIN,

OR CONCANAVALIN

A

PER 0. SEGLEN and JON FOSSA Department of Tissue Culture, Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3, Norway

SUMMARY Isolated rat hepatocytes attach to, and spread on, the surface of polystyrene tissue culture dishes in the presence of serum. The attachment is essentially complete in 30 min at 37”C, whereas no attachment occurs at 0°C. Dead (trypan blue-stainable) cells do not attach; hence the plating efficiency (percentage of cells attaching) is close to the percentage of intact cells in the hepatocyte suspension. Attachment in the presence of serum is relatively independent of pH, but requires divalent cations. Mg2+ stimulates attachment more effectively than Ca2+, and a combination of both cations gives maximal attachment. Cells do not attach readily to untreated dishes in the absence of serum, but attach to and spread on dishes precoated with adsorbed serum protein, concanavalin A (ConA), or a film of collagen. The attachment-promoting activity in serum is destroyed by acid treatment, by heating to 7O”C, and by protease treatment. It is therefore most probably a protein, which, like collagen and ConA, can bind to receptors on the hepatocyte surface.

Normal cells require attachment to a solid substratum in order to be able to proliferate in vitro, whereas the growth of malignant cells is anchorage-independent [31, 331. The mechanisms of cell attachment are therefore of sufficiently great interest to fully justify the large amount of work devoted to the study of this process [6-11, 13, 15-17, 22, 24, 25, 32, 35, 411. However, practically all previous work on cell attachment has been done with mesenchymal cells (fibroblasts or fibroblastoid cell lines), although epithelial cells are in fact responsible for the majority of human cancers. The recent development of methodologies for the preparation of intact single-cell suspensions from the rat liver [29] has now made it possible to study the in vitro attachment of a normal, epithelial cell

type, i.e. the rat hepatocyte [3-5, 13, 14, 18-21, 25, 371. In the present communication some of the basic requirements for hepatocyte attachment will be described. MATERIALS

AND METHODS

Isolated hepatocytes were prepared from the livers of I6 h fasted-male Wistar rats (ZSO-300g) by the method of collagenase perfusion 128. 291. Under standard conditions,the cells were incubated at 37°C in 6 cm polystyrene tissue culture dishes (Falcon) at a cell concentration of 3 mglml(4X lo5 cells/ml; I4000 cells/cm*) in an incubation volume of 3.4 ml (3.1 ml of suspension buffer [29] plus 0.3 ml fetal calf serum (FCS), i.e. 8.8 % serum, conventionally designated ” 10% serum” in the text), containing various additives as indicated. Attachment was measured essentiallv as described by Ballard & Tomkins [I]. The incubated culture dishes were placed on a shaker platform (Cenco) at an axial rotation frequency of 50 r$m for I min; the absorbance of the medium containing the suspended (i.e. non-attaching) cells was then measured at 650 nm. The absorbance was a linear function of the cell concentration up to 5 mg cells/ml (Assa=l.O). The reduction in E.rp

Cell

Res

/I6

(1978)

200

Seglen and Fossd

Fig. 1. Morphology of rat hepatocyte 5 in primary culture. The cells were incubated in PC slystyrene tissue culture dishes with a growth mediun1 and 10% FCS. (A, D, E, F) Photographed with or (B, C) without phase contrast. x325. (A) 3 h in culture; moderately den se seeding. Most cells attached and spread; (B) 4 h in culture, densely seeded. Continuous monolayer of jwell-spread cells (pale) at the dish surface; clumps (If non-attaching, rounded cells (dark) above the surface:; (C) 20 h in cul-

ture, then 30 min shaking in collagenase-buffer [29] The cells have rouinded up and detached from the dish surface as cell cl1umps. (D) 48 h in culture. Notice phase-clear clefts t:bile canaliculi) between some of the cells; (E) 72 h in ,culture. Periphery of monolayer reeion with extreme spreading (flattening) of cells. (F) 6 days in culture. Solitary hepatocyte with morphological polarizationI; notice ruffling membrane activity at both ends.

Attachment

of hepatocytes

absorbance in incubated vs non-incubated (zero-time) culture dishes was taken as a measure of attachment, and expressed in % of the zero time absorbance. With freshly prepared cell suspensions, the percentage of cells attaching was only-slightly lower than the percentage of intact cells, i.e. 8540%. However, for reasons extrinsic to the experiments, most of the work in this paper had to be done with cells which had been stored on ice for half a day, and which therefore exhibited somewhat lower attachment efftciencies. Adsorption of serum, ConA or gelatin to tissue culture dishes was achieved by leaving the dishes for 10 min at room temperature with the surface covered by FCS (undiluted); a solution of ConA (1% w/v in suspension buffer); or gelatin (I % w/v in suspension buffer), respectively. The dishes were then washed 5~ with water and air-dried. Coating of the dishes with a layer of gelatin was done by wetting the surface with 1% gelatin solution, then air-drying without washing. Coating with bovine tendon collagen (Sigma type I) was performed essentially as described by Lin & Snodgrass [20]: collagen powder was boiled in water overnight, and the saturated solution was filtered. 0.5 ml of filtrate was distributed evenly over the surface of a 6 cm dish, and evaporated to dryness in an incubator at 37°C. For long-term culturing of hepatocytes (up to 7-g days), the cells were incubated in 6 cm dishes with 5 ml of growth medium. The medium consisted of suspension buffer [29] fortified with 10x normal concentration of the amino acid mixture previously described [30], plus glucose, vitamins and trace elements essentially as in Ham’s FlO medium r121. The henatocvtes were better maintained in serum-pretreated dishes with a serum-free medium than in dishes containing 10% FCS, because tibroblastic overgrowth was prevented in the absence of serum. FCS was purchased from Grand Island Biol. Co., and gelatin granulate from Matheson Coleman & Bell; other biochemicals were from Sigma.

RESULTS AND DISCUSSION Morphology of hepatocytes tissue culture

in

Isolated hepatocytes readily attach to and spread on the surface of polystyrene tissue culture dishes in the presence of serum [3, 4, 14, 18, 19, 371. The attachment is virtually completed in 20-30 min, whereas spreading beyond the initial circumference of the cell, i.e. morphologically visible cell flattening, is evident only after 24-3 h (fig. 1A). Where the margins of spreading cells meet, they form cell contacts [5, 371, and when the cells are seeded at a high density,

to protein,

collagen, or ConA

201

a continuous monolayer can be formed in 3-4 h (fig. 1B). Standard trypsinization procedures cannot be used for the detachment of hepatocytes in culture, because these cells are particularly easily destroyed by proteolytic enzymes [2, 231. However, incubation with collagenase readily dislodges the cells from the surface (fig. 1C). Cell-to-cell contacts are preserved, however, as also observed by Rubin et al. [25], resulting in the detachment of cell clumps rather than single cells. In contrast to the procedure used for dissociation of the whole liver [26-291, preliminary Caz+ removal before collagenase treatment is not necessary in the hepatocyte cultures, indicating that desmosomal elements (hemidesmosomes) do not play a major role in attachment of the cells to the surface. Hepatocyte monolayers can be maintained for several days without undergoing any dramatic morphological changes (fig. lD-E). The cells in central monolayer regions retain round nuclei and polygonal shapes, and wide intercellular clefts reminiscent of bile canaliculi [4, 5, 371 are frequent (fig. 1D). In the periphery of cell islands the hepatocytes (including their nuclei) may sometimes stretch out very thinly (fig. IE), and solitary hepatocytes may move about and acquire distinctly polarized shapes (fig. 1F). Hepatocytes are at all times easily distinguished from macrophages and mesenchymal cells (“fibroblasts”) on basis of their size, yellow colour and the shape of their nucleus, which is nearly always rounded (except in cases of extreme unidirectional stretching, as in fig. IF). In contrast to the hepatocytes, which do not divide, mesenchymal cells divide rapidly in culture, and can be observed in increasing numbers after 3-4 days unless preEA-p Cdl Res 116 (1978~

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Seglen and FossH

5

IO

15

20

25

30

180

Figs 2-7. Each point represents a single dish. Fig. 2. Abscissa: incubation time (min); ordinute: % attachment. Time-course of attachment. Isolated hepatocytes were incubated in nolvstvrene dishes with 10% serum at 37°C (0, 0, & three different experiments are shown in order to illustrate the variabilitv) or at 0°C (0, no attachment occurring). Dead cells (A, no attachment) were obtained by freezing (in liquid N,) and thawing a sample of the cell suspension.

(e.g. settling on top of other cells, or being the non-attaching member of a cell pair). The viability of the non-attaching cells (as measured by trypan blue exclusion) was only lo-15%; i.e. all living cells seemed to be capable of attaching, whereas dead cells were unable to do so [22]. This was confirmed by killing all the cells by freezing (brief immersion in liquid nitrogen) and thawing; these dead cells showed no attachment (fig. 2). The attachment process was temperature-dependent, with no attachment at 0°C (fig. 2). The attachment of other cell types has also been shown to be highly temperature-sensitive [ 15, 351. Attachment and spreading occurred normally in the presence of cycloheximide (1 mmol/l), i.e. new protein synthesis was not required for these processes. The attachment was also unaffected by the microtubule poison colchicine. Other cell types have similarly been found to attach relatively unaffected by cycloheximide [ 11, 391 or colchicine [7, 15, 381.

cautions are taken to prevent their growth. One such precaution is to keep the cultures in serum-free medium. Provided the hepatocytes are offered a suitable attachment surface such as collagen [20, 251 or preadsorbed serum protein, they can be maintained in serum-free medium at least for a week, under which conditions no growth of Effect of pH fibroblasts occurs. Attachment was relatively independent of pH in the range 6.545 (fig. 3). Higher or Time course of attachment-effect of lower pH values may reduce attachment by temperature, cell viability killing the cells [ 17, 321. and inhibitors Fig. 2 shows the time course of hepatocyte , 100 attachment to polystyrene tissue culture dishes in a balanced salt solution (suspension buffer [29]) with 10% FCS. Attachment at 37°C exhibited a variable initial lag, ‘0 but was essentially completed in 20-30 min 20 in all cases, with, W-90% of the cells ati 55 70 75 80 tached. Incubation for several hours in3. Abscissa: pH in the medium; ordinate: % creased the percentage of attached cells Fig. attachment. Effect of medium pH on attachment. Isolated hepaslightly; presumably the increase reprewere incubated at 37°C for 30 min in the pressented cells which were prevented from at- tocytes ence of 10% serum. The medium pH (measured at taching earlier because of steric hindrances 3°C) was varied by the addition of HCI or NaOH. Exp Cdl RPS 116 (1978)

Attachment of hepatocytes to protein, collagen, or ConA Table 1. Effects of Ca2+, Mg2+ and divalent ion chelators on hepatocyte attachment in the presence of serum Isolated hepatocytes were incubated for 30 min at 37°C with 10% serum (contributing approx. 0.22 mmol/l Ca*+ and 0.08 mmol/l Mg2+) and added CaCI, (2 mmol/l), MgCl, (2 mmol/l), EGTA (1 mmol/l) or EDTA (I mmol/l) as indicated. Each value is the mean of 2 dishes % Attachment Additions

+EGTA

None Ca*+ Mg*+ CaZ++Mg2+

4.6 45.2

51.0 78.9

8.2 65.1 71.8 84.0

60 10 20 01

06

08

10 -

+EDTA

100 80l-7

02

r

20 -

Effect of divalent cations Attachment and spreading is, in most cases studied, dependent on the presence of divalent cations. A number of different cations have been shown to be capable of promoting attachment, most notably Mn2+ [7-9, 16, 17, 22, 24, 32, 411. Under the standard conditions used in our experiments, Ca2+ and Mg2+ are present at concentrations of 1.4 and 0.7 mmol/l, respectively (including the amounts contributed by 10% serum). Incubation with the divalent ion chelators EGTA and EDTA largely prevented the attachment of hepatocytes (table 1). Fig. 4

:

100

203

10

Fig. 4. Abscissa: EGTA cont. (mmol/l); ordinate: % attachment. Effect of EGTA on attachment. Isolated hepatocytes were incubated at 37°C for 30 min in Ca’-‘+-and Mg*+-free suspension buffer with 10% serum and the concentration of EGTA indicated.

5

IO

n I5

20

25

30

Fig. 5. Abscissu: incubation time (min); ordinare: % attachment. Effect of serum concentration on attachment. Isolated hepatocytes were incubated at 37°C for the time period indicated, in the absence of 0, serum or in the presence of l ,O.I; A, I; A, IO or 0, 20% FCS.

demonstrates that upon inclusion of increasing amounts of EGTA in the dishes, a rather sharp transition from maximal attachment (>90%) to no attachment can be seen. In HeLa cells, it has been shown that incubation with a large excess of EGTA is capable of removing all Ca2+ from the cell surface, while leaving the intracellular Ca2+ intact [34]. Addition of Ca2+ and Mg2+ to EDTA or EDTA-containing dishes restored the capacity for cell attachment (table 1). Mg2+ was somewhat more effective than Ca2+, but the best result was obtained with both cations present, as reported with other cell types [7, 221. Serum dependence of attachment As shown in fig. 5, attachment was strongly dependent on serum. A maximal rate of attachment was observed at 1% FCS; at 20 % serum the attachment rate was slightly reduced, possibly due to attachment inhibitors in serum [lo]. Other sera (newborn or adult calf serum, rat serum) could substitute for FCS to some extent, but the attachmentE-\p Cd Rrs I16 11978)

204

Seglen and FossB

Table 2. Effects of heat, acid and protease treatment on the attachment-promoting activity of serum

proteins. Since several serum proteins are known to adsorb to polystyrene tissue culture dishes [6], an attempt was made to preIsolated hepatocytes were incubated for 30 min at 37°C treat the dishes with serum (10 min at room with 10% FCS treated in various ways: heated to 70°C; acidified to pH 2.2 with PCA and then neu- temperature with undiluted FCS, then extensive washing with water) to see if the attralized with KOH (precipitated potassium perchlorate removed by centrifugation); or added to the cells together with a protease mixture (Sigma type I col- tachment factor(s) could be adsorbed. As lagenase, 0.5 g/l). Each value is the mean of 2-5 dishes shown in table 3, serum pretreatment of the dishes followed by incubation in serum-free Treatment % Attachment medium gave the same attachment effiUntreated serum 76.1 ciency as incubation in the presence of seNo serum 0.6 rum. The adsorbed attachment factor(s) Heated to 70°C 0.0 could be inactivated by treatment with Acidified/neutralized 7.0 Protease present 3.2 crude collagenase, confirming that this proteolytic enzyme mixture acted on the factor(s) rather than on the cells. Preliminary experiments indicate that the inactivation is promoting effect of the latter was generally due to proteases other than the collagenase better, despite some variability. itself. During prolonged incubation (24 h) considerable numbers of cells attached even in Pretreatment of tissue culture dishes the absence of serum, as previously re- with collagen or ConA ported [4], but there was more cell clumping Since it has been demonstrated that hepatoand less regular monolayer formation than cytes are capable of attaching and spreading in the presence of serum. on collagen substrata [20, 21, 251, a comThe activity of the attachment factor(s) in parison was made between the efficiencies serum was destroyed upon heating to 70°C of attachment to serum-pretreated and (table 2), in agreement with Grinnell [8], collagen-coated tissue culture dishes. As who studied the attachment of BHK cells, a shown in fig. 6, dishes coated with a thin fibroblastoid cell line. Acidification of the layer of bovine tendon collagen provided an serum with perchloric acid (PCA) to pH 2.2 and subsequent neutralization likewise deTable 3. Effect of protease on attachment stroyed most of the attachment activity (table 2). Attachment was also prevented if to pre-adsorbed Serum Isolated hepatocytes were incubated for 30 mm at 37°C proteases in the form of a crude collagenase with 10% serum, or in a serum-free medium in dishes preparation was present in the system (table pre-coated with adsorbed serum. Some of the precoated dishes were pretreated with a protease mixture 2). This mixture of proteolytic enzymes (s’tgma type I collagenase, 0.5 g/l) for I h at 37”C, then would be expected to affect the serum fac- extensively washed before incubation with cells. Each value is the mean of 2-3 dishes tar(s) rather than the cells, since the latter % Attachment have already been exposed to the enzymes Attachmentconditions during the cell preparation procedure. 81.2 Serum present dishes The heat, acid and protease sensitivity of Serum-coated 79.4 38 the attachment-promoting serum factor(s) Serum-coated+treared with protease indicates the involvement of one or more Exp Cd RPS 116 (1978)

Attachment of hepatocytes to protein, collagen, or ConA

205

30

7. Abscissa: incubation time (min); or¬e: % attachment. Attachment of hepatocytes to ConA or adsorbed serum. Isolated hepatocytes were incubated at 37°C in a serum-free medium for the time period indicated. The polystyrene dishes were either A, untreated; 0, coated with adsorbed serum protein: or, 0, coated with adsorbed ConA.

Fig. 6. Abscissu: incubation time (min); ordinare: % attachment. Attachment of hepatocytes to collagen, gelatin and adsorbed serum. Isolated hepatocytes were incubated at 37°C in a serum-free medium for the time period indicated. The polystyrene dishes contained either adsorbed serum protein (0, 0, two different experiments); A, adsorbed gelatin; A, a film of gelatin; or, 0, a film of bovine tendon collagen.

Fig.

equally good substratum for attachment as dishes to which serum factor had been adsorbed. Dishes coated or pretreated with collagen in form of gelatin, however, were unsuitable for attachment (fig. 6). Gelatin, which is a highly degraded and denatured, small molecular weight form of collagen, may not have the three-dimensional structure required for binding to cellular receptors. Cell spreading (examined after 3.5 h) took place equally well on collagen and on adsorbed serum, but the few cells attaching to gelatin did not spread. Cells in culture have also been reported to attach to adsorbed ConA [8], and fig. 7 shows that hepatocytes attach with the same efficiency to dishes pretreated either with serum or with the lectin. Cell spreading also appeared to occur to a similar extent on both substrata. It is clear from our results that hepatocytes can attach and spread on several different types of substratum. Recent work by Grinnell et al. [lo] and Hook et al. [13] has

indicated that the serum factor mediating attachment of BHK cells and hepatocytes may be identical to cold-insoluble globulin, also known as fibronectin or LETS protein [36, 401. It would obviously be of great interest to identify the cellular structures (receptors) involved in this binding, and to establish their relationship to the other types of cell anchorage in vitro and in vivo. We wish to thank Frances Dodman for technical assistance and Rolv Gjessing for experimental cooperation. The work was supported by a grant from The Norwegian Cancer Society.

REFERENCES I. Ballard, P L & Tomkins, G M, J cell bio147 (1970) 222. 2. Berg, T & Boman, D, Biochim biophys acta 321

(1973) 585. 3. Bissell, D M, Hammaker, L E & Meyer, U A, J

cell biol 59 (1973) 722. 4. Bonney, R J, Becker, J E, Walker, P R &Potter, V

R, In vitro 9 (1974) 399. 5. Chapman, G S, Jones, A L, Meyer, U A & Bissell,

D M, J cell biol59 (1973) 735. 6. Gulp, L A & Buniel, J F, J cell physio188 (1976) 89. 7. Grinnell, F, Arch biochem biophys I65 (1974) 524. 8. - Exp cell res 97 (1976) 265. Exp Cell Res II6 f 1978)

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9. -Ibid 102(1976) 51. 28. -Ibid 82 (1973) 391. IO. Grinnell, F, Hays, D G & Minter, D, Exp ceil res 29. - Methods in cell biology (ed D M Prescott) vol. llO(l977) 17.5. 13, p. 29. Academic Press, New York (1976). Il. Grinnell, F, Milam, M & Srere, P A, J cell biol 56 30. - Biochim biophys acta 442 (1976) 391. (1973) 659. 31. Shin, S-I, Freedman, V H, Risser, R & Pollack, R, 12. Ham: R G, Exp cell res 29 (1963) 515. Proc natl acad sci US 72 (1975) 4435. 13. Hook, M, Rubin, K, Oldberg, A, Gbrink, B & 32. Takeichi, M & Okada, T S, Exp cell res 74 (1972) Vaheri, A, Biochem biophys res commun 79 (1977) 51. 726. 33. Tucker, R W, Sanford, K K, Handleman, S L & 14. type, P T, J cell physiol 78 (1971) 281. Jones, G M, Cancer res 37 (1977) 1571. 15. Juliano, R L & Gagalang, E, J cell physiol 92 34. Tupper, J T & Zorgniotti, F, J cell biol 75 (1977) ( 1977)209. 12. 16. Klebe, R J, Nature 250 (1974) 248. 35. Ueda, M J, Iota, T, Okada, T S & Ohnishi, S-I, J cell biol 71 (1976) 670. 17. Klebe, R J, Hall, J R, Rosenberger, P&Dickey, W D, Exp cell res I10 (1977) 419. 36. Vaheri, A, Mosher, D, Wartiovaara, J, Keski-Oja, 18. Laishes, B A & Williams. G M, In vitro I2 (1976) J, Kurkinen, M & Stenman, S, Ceil interactions in differentiation (ed M Karkinen-Jaaskelainen, L 521. 19. -Ibid I2 (1976) 821. Sax&r & L Weiss), p. 31 I. Academic Press, 20. Lin, R C & Snodgrass, P J, Biochem biophys res London ( 1977). commun 64 (1975) 725. 37. Wanson, J-C, Drochmans, P., Mosselmans, P & Ronveaux, M-F, J cell biol74 (1977) 858. 21. Michalopoulos, G & Pitot, H C, Exp cell res 94 38. Weiss, L, Exp cell res 74 (1972) 21. (1975) 70. 39. Weiss, L & Chang, M K, J cell sci I2 (1973) 655. 22. Moore, E G, J cell biol70 (1976) 634. 23. Munthe-Kaas, A C, Berg, T, Seglen, P 0 & Sel- 40. Yamada, K M, Yamada, S S & Pastan, I, Proc natl acad sci US 73 (1976) 1217. ielid, R. J exp med I41 (1975) 1. 24. Rabinovitch,‘M &De Stefano, M J, Exp cell res 79 41. Yasuda, K, J cell sci I5 (1974) 269. (1973) 423. 2s. Rubin, K, Kjellen, L & Obrink, B, Exp cell res 109 Received February 28, 1978 (1977) 413. Revised version received April 2 I, 1978 26. Seglen, P 0, Exp cell res 74 (1972) 450. Accepted April 28, 1978 27. - Ibid 76 (1973) 25.

E.rp Cdl Res I16 11978)

Attachment of rat hepatocytes in vitro to substrata of serum protein, collagen, or concanavalin A.

ATTACHMENT OF RAT HEPATOCYTES TO SUBSTRATA COLLAGEN, OF SERUM IN VITRO PROTEIN, OR CONCANAVALIN A PER 0. SEGLEN and JON FOSSA Department of Ti...
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