The serum dependence of baby hamster kidney cell attachment to a substratum

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Experimental

THE SERUM

Cell Research 97 (1976) 265-274

DEPENDENCE

KIDNEY

CELL

OF BABY

HAMSTER

ATTACHMENT

TO A SUBSTRATUM F. GRINNELL Department

of Cell Biology,

University of Texas Southwestern Dallas, TX 75235, USA

Medical School,

SUMMARY Normal attachment and spreading of baby hamster kidney cells onto a non-living substratum requires the presence of a specific serum component adsorbed to the substratum surface and Ca*+ ions in the medium. In the absence of the adsorbed serum factor or Ca*+ ions cells attach but do not spread. Thus, although the initial rate of BHK cell attachment is faster in serum-free medium than serum-containing medium, no cell spreading occurs in serum-free medium. Adsorption of serum onto the substratum results in a lag phase in the time course of cell attachment which can be eliminated by blocking the negatively charged groups of the serum components adsorbed to the substratum surface; blocking positively charged groups or free sulfhydryl groups of the adsorbed setum components is without effect. The requirement for serum components can be substituted for by adsorbing molecules such as concanavalin A or polycationic ferritin to the substratum surface; however, only ConA results in normal morphology of cell spreading. The data are discussed in terms of a non-electrostatic direct cell-substratum binding model of cell attachment.

The molecular mechanism of cell attachment to substrata has yet to be elucidated. Curtis has recently reviewed the subject of cell adhesion and the various models that have been postulated to account for cellular adhesive properties [l]. It is likely that a major drawback in our attaining a better knowledge of the mechanism of cell attachment has been the lack of understanding concerning the role of serum in this process. Part of the problem in determining the serum requirement is that cells require serum factors in the medium in order to survive [2, 3, 41. A second difficulty is that most cells are grown attached to a substratum and must be separated from the substratum prior to use. This separation is often accomplished by trypsinization and 18-731X18

serum factors may then be required to reverse damage caused by trypsin or neutralize trypsin adsorbed to the cell surface [5, 61. Thus, it is difficult to evaluate the possible roles of some of the factors which have been reported to promote cell attachment [7-lo]. Evidence from this and other laboratories has revealed that cell attachment to substrata occurs by different mechanisms in the presence and absence of serum in the culture medium [I l-141. It has been postulated that the difference depends upon the adsorption of serum proteins onto the substratum surface in which case the cells become attached to adsorbed serum proteins and not directly to the substratum [I I, 12, 131. Indeed, electron microscopic evidence Exprl Cell

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Fig. I. Abscissa: time (min); ordinate: % attached. Effect of serum concentration on attachment of BHK-2 1-13scells. Fetal calf serum was added to the incubations as indicated. Other details are given in Methods.

has been presented indicating that cells are attached to substances adsorbed to the substratum [15, 16, 171. These considerations have led us to investigate some of the properties of cell attachment to substrata coated with adsorbed serum. MATERIALS

AND METHODS

Baby hamster kidney cells which were suspension culture adapted were the gift of Dr Adrian Chappel, Communicable Disease Center, Atlanta, GA. Eagle’s minimal essential medium (‘spinner modified’), MEM amino acids, MEM vitamins and fetal calf serum were obtained from GlElCo, Grand Island, NY. Hepes buffer, N-ethylmaleimide and bovine albumin were purchased from Sigma Chemical Co., St Louis, MO. I -Ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCI (EDC) and glycine methyl ester HCI (GME) were obtained from Pierce Chemical Co., Rockford, Ill. Polycationic ferritin and ConA were purchased from Miles Laboratories. DE-52 was the product of Reeve Angel, Clifton, NJ. Other reagent grade chemicals were purchased from Fisher Scientific, Houston, Tex. BHK-21-13s cells were grown in suspension culture. The culture medium was Eagle’s MEM (‘spinner modified’) with double the concentration of amino acids (except I x glutamine) and vitamins and supplemented with Hepes buffer (final conc.=20 mM), 0.1 g/l ferric nitrate, 2.0 g/l dextrose, 10% tryptose phosphate broth, and 10% fetal calf serum. The final sodium bicarbonate concentration in the medium was 0.5 g/l. Cellular adhesiveness was measured by a previously used technique [I I]. Suspension culture cells in the logarithmic growth phase were collected by centrifugation at 500 g for 2 min. The cells were resuspended in 3.0 ml of attachment salts (0.8 mM MgSO,.7 H,O; I I6 mM NaCI; 5.4 mM KCI; 10.6 mM Na,HPO,; 5.6 mM D-ghCOSe; 20 mM Hepes; final Exptl Cell Res 97 (1976)

pH=7.0) containing fetal calf serum as indicated. Incubations were carried out in ‘Falcon’ 3013 polystyrene culture flasks (50 ml capacity). 1.0-1.5x lo6 cells were used in each experiment. The flasks were incubated for the time periods indicated at 37°C. At the end of the incubations the flasks were subjected to shaking at I50 rpm on a New Brunswick R-2 reciprocating shaker (I in. stroke) for IO set at room temperature and the cells which were resuspended by this procedure (considered to be non-attached) were removed with a pipet. The turbidities of the starting cell suspensions and non-attached cell suspensions were determined at 640 nm with a Bausch & Lomb Spectronic 70 equipped with digital readout. Cell concentrations were calculated from a previously determined linear relationship between cell number and absorbancy. The per cent.of cells attached in an experiment was calculated as the starting number of cells in an incubation minus the number of non-attached cells divided by the starting number of cells. Control experiments in which the attached cells were recovered indicated the reliability of this technique. Serum-coated substrata were prepared by incubating Falcon dishes for 2 min at room temperature with 2.0 ml of adhesion salts containing fetal calf serum or other proteins as indicated. Subsequently, the proteincoated substrata (designated SUB+) were washed with physiological saline or deionized H,O. Little reversal of rotein adsorption occurred as a result of washing

Pd.

RESULTS AND DISCUSSION Fig. 1 illustrates the time course of cell attachment to substrata with various concentrations of serum added to the attachment incubation medium. The addition of serum resulted in a lag phase in attachment which was more pronounced with increasing serum concentrations. Nevertheless, the f ma1 extent of cell attachment in serumcontaining medium reached that level observed in serum-free medium. In the absence of serum in the medium, cells were never observed at any time to flatten onto the substratum (fig. 2A), whereas by 30 min, many cells attained a flattened morphology in 1% or higher serum-containing medium (fig. 2B). After longer incubations of 45 min to 1 h, almost all cells became attached and flattened in serum-containing medium (fig. 2C). It is important to note that our studies were carried out using suspension-cultured

Serum dependence of cell attachment

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Fig. 2. Morphology of cell attachment to Falcon poly-

styrene. Experiments anaiagous to those described in fig. 1 were carried out in Falcon 35 x 10 mm tissue culture dishes. Using the dishes instead of flasks permitted better photography; however, the results were the same in either case. (A) 30 min attachment in serum-free medium; (B) 30 mm attachment in medium containing 5 % serum; (C) 45 min attachment in medium containing 5% serum. Other details appear in Methods. x280.

cells. We have avoided the use of station- that in simple solutions, single species of ary-cultured cells because cell damage can serum proteins adsorb to the substratum as occur during separation of cells from their a monolayer of molecules [19]; however, substratum by treatment with proteases or in a complex system such as serum, a multichelating reagents. However, we did carry layered heterogenous mosaic of adsorbed out some control experiments with sta- proteins may form [20]. The time course of cell attachment to tionary-cultured cells that were separated from their substratum with 0.025% trypsin SUB+ prepared with various concentra(GIBCO). Using these cells, we observed tions of serum is shown in fig. 3. In these results essentially the same as those re- experiments no additional serum was added to the attachment incubation medium. With ported in fig. 1. The properties of cell attachment to sub- increasing serum concentrations there was strata coated with serum (SUB+) were then a decrease in the rate and extent of cell atstudied. SUB+ were prepared by exposing tachment, and in no case was cell spreadFalcon culture dishes to serum-containing ing onto the substratum observed. The results presented in figs 1, 2, and 3 medium for 2 min at room temperature and then washing the dishes with deionized suggest that the adsorption of serum proHzO. We have shown elsewhere [18] that teins onto the substratum can account for adsorption of serum components occurs the lag period in the time course of cell atvery rapidly, within seconds. It is known tachment observed in serum-containing ExptlCeNRes 9711976)

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F. Grinnell

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5 15 30 Figs 3-5. Abscissa: time (min); ordinate: % attached. Fig. 3. Effect of serum adsorption on attachment of

BHK-21-13s cells. Falcon flasks were treated with attachment salts containing fetal calf serum at the concentrations indicated. Subsequently, attachment was carried out in attachment salts. Other details are described in Methods.

medium (fig. l), but that for the cells to attain the full extent of attachment and flattened morphology there is a requirement for additional serum components in the medium. This point was further investigated by experiments- in which substrata were treated with medium containing 10% serum, and then these SUB+ were used as substrata for incubations in which additional serum was added to the medium at various concentrations. As shown in fig. 4, the final extent of cell attachment to SUB+ (10 %) increased with increasing serum concentration in the medium and within 30 min spreading of cells onto the substrata occurred when the serum level in the medium was 1% or higher. We then attempted to test what reactive groups of adsorbed serum proteins were required for attachment and flattening of cells to their substratum. Following preparation of SUB+ (made with 5% serum) the substrata were subjected to treatment with 1.5 % formaldehyde to block free -NH, groups, 0.01 M IV-ethylmaleimide to block free -SH groups, or a soluble carbodiimide (EDC) in the presence of glycine methyl ester (GME) to block free COOH groups Exptl CeNRes 97(1976)

Fig. 4. Effect of serum in the medium on attachment

of BHK-21-13s cells to serum-coated substrata. Substrata were pretreated with medium containing 10% fetal calf serum (SUB+ 10%). Subsequently, attachment was carried out in attachment salts containing fetal calf serum at the concentrations indicated. Other details are described in Methods.

[2 I]. Neither formaldehyde nor N-ethyl maleimide had any effect on subsequent attachment or spreading of cells to SUB +: however, there was a substantial stimulation of cell. attachment to SUB+ treated with EDC, GME as shown in fig. 5. Both the rate and extent of attachment were

60

5

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Fig. 5. Effect of treating SUB+ with -COOH block-

ing reagent on subsequent cell attachment to the substrata. Substrata were prepared with medium containing 5% fetal calf serum (SUB+ 5%). The SUB+ (5%) were then treated with a solution of 0.25 M l-ethyl-3-(3dimethylaminopropyl)carbodiimide HCl (EDC) and 1.0 M glycine methyi ester HCI (GME, Pierce Chem. Co.) (final pH 4.75). In control experiments, SUB+ (5 %) only were treated with deionized H,O adjusted to pH 4.75. The incubations were carried out for I5 min at room temperature after which the substrata were washed with deionized H,O and used to assay cell attachment. Serum was added to the attachment incubation medium as indicated. Other details are described in Methods.

Serum dependence of cell attachment

Fig. 6. Morphology of cell attachment to neutralized

substrata. The experiments are analogous to those described in the leg&d to fig. 5. (A) SUB + (5 %) treated with EDC, GME; 30 min attachment in serum-free medium. @) SUB+ (5%) treated with EDC, GME; 30 min attachment in medium containing 1% serum. Other details are described in the captions to figs 2 and 5 and in Methods. x280.

markedly enhanced; however, in the absence of added serum to the attachment incubation medium there was still no cell spreading (fig. 6A). When serum was added to the incubation medium, spreading did occur beginning about 15 min (fig. 6B) and there was no decrease in the rate or extent of cell attachment. It is well known that the surfaces of both the cells and substrata carry a net negative charge and various investigators have suggested that to overcome the mutual charge repulsion, cells might make initial contacts with their substratum by microextensions

269

of the cell surface [I 1, 22, 23, 241. Therefore, by removing the negative charges from the surface of the substratum, it is reasonable that adhesion would be enhanced, as was observed in fig. 5. In this case, there would also be the possibility of direct electrostatic attraction between the negatively charged cell surface and remaining positive charges on the substratum; however, treatment of SUB + with formaldehyde in addition to EDC, GME did not alter the kinetics or extent of cell attachment. Thus, positively charged groups on the substratum are probably not involved. We attempted to investigate the specificity of the requirement for adsorbed serum protein on the substratum surface. Heatdenatured serum has been reported to inhibit cell attachment [25]. As shown in fig. 7, no cell attachment occurred in medium containing serum that had been heated for 10 min at 7O”C, whereas there was some cell attachment with 60°C heated serum and normal cell attachment with 50°C heated serum. After the substratum was pretreated with 70°C serum at concentrations as low as 0.2% in adhesion salts (ap-

60

Fig. 7. Abscissa:

time (min); ordinate: % attached. Attachment of cells in medium containing heated fetal calf serum. Fetal calf serum was heated for 10 min at 50°C. 60°C. or 70°C. Attachment was then carried out in medium containing 5 % heat-treated serum as indicated. Otherdetails are described in Methods. Exprl Cell Res 97 (1976)

270

F. Grinned

0.80.6 04

-

20

40

60 a0 loo 120 140 I60

Fig. 8. Ab&issa: fraction no.; ordinate: OD 280. Chromatography of fetal calf serum on DEAEcellulose. 10 ml of fetal calf serum was exhaustivelv

diGsed against0.0) M pot=sium phosphate(PH &

buffer and loaded onto a 2.5~45 cm DE-52 column previously equilibrated with the same buffer. The serum was eluted from the column first with a linear gradient of 500 ml 0.01 M potassium phosphate pH 8 and 500 ml 0.1 M notassium phosphate (nH 8), then with 500 ml 0.3 M potassium phosphate (pH 8) which was started at fraction no. 118. The fractions contained approx. 9.0 ml (150 drops) and were collected with an LKJSultrarac fraction collector. Peak fractions were pooled and concentrated to approx. 4 mg/ml with a 50 ml Amicon Filtration unit, P-IO filter. Other details appear in Methods.

prox. 100 pglml), cells were no longer able to attach to that substratum regardless of subsequent additions to the attachment incubation medium. On the contrary, after the substratum was pretreated with medium containing native serum, cell attachment occurred normally regardless of the addition of 70°C heated serum. Therefore, the inhibition of cell attachment by heat-treated serum depends upon its adsorption onto the substratum surface and inability to function as a target for cell attachment. In other experiments we measured the attachment of BHK cells to substrata coated with bovine albumin. Cells attached to these substrata but were unable to soread even though 5 % fetal calf serum was added to the attachmerit incubation medium (fig. 9A). These results are consistent with the notion that adsorbed serum proteins in general may function as targets for cell attachment as long as they are not “inactivated”, for inE.rprl Cell Res 97 11976)

stance by heat treatment. On the other hand, there appeared to be a specific requirement for a particular serum protein adsorbed on the substratum in order that cell spreading take place. To further test the possibility of a specific serum-spreading factor, fetal calf serum was fractionated on a DEAE column as shown in fig. 8. Activity was tested for by pretreating the substrata with various column fractions and then carrying out cell attachment in medium containing 5 % fetal calf serum. By this analysis, all of the spreading activity was associated with peak IV (fig. 9B). Although cells became at-

Fig. 9. Morphology of cell attachment to substrata pretreated with bovine albumin or serum spreading factor. Substrata (35 mm Falcon tissue culture dishes) were pretreated with attachment salts containing (A) bovine albumin (I mglml) or (B) serum spreading factor (0. I mglml) for IO min at room temperature. Subsequently, attachment was carried out for 45 min in attachment salts containing 5 ‘% fevdl calf serum. Other details are described in caption to fig. 2 and in Methods. ~280.

Serum

5

15

Figs 10, II. Abscissa:

20

time (min);

ordinate: % attached . Fig. IO. Effect of Ca*+ ions on cell attachment. Substrata were pretreated with medium containing 10% fetal calf serum. Subsequently, attachment was carried out in attachment salts containing CaCI, as indicated. Other details are described in Methods.

tached to substrata that had been pretreated with material from peaks I, II, or III, there was no cell spreading. Furthermore, pretreating the substratum with bovine albumin, or peaks I, II, or III, prevented the subsequent ability of peak IV to induce spreading. These findings confirmed the idea that there is a specific serum protein (or class of serum proteins) which adsorbs to the substrata and is required for the

5

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Fig. Il. Effect of treating SUB+ with polycationic fer-

ritin or ConA on subsequent cell attachment to the substrata. SUB+ (5%) were subjected to treatment with 0.5 mg/ml polycationic ferritin (Miles) or I .O mg/ ml ConA (Miles) in 0.15 M NaCI. In control experiments, SUB+ (5%) were treated only with 0. I5 M NaCI. The incubations were carried out for 5 min at room temperature after which the substrata were washed with deionized H,O and used to assay cell attachment. No serum was added to the attachment incubation medium. Other details appear in Methods.

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spreading activity of BHK cells. Whereas approx. 640 pg/ml of whole serum was required in the incubation medium for complete cell spreading to occur during 45 min of attachment, only about 32 pg/ml of peak IV material was required, representing a more than 20-fold purification. Fetuin, at one time thought to be a protein required for cell attachment [7], was located in peak II. Further studies on the purification and properties of the spreading factor are in progress. During the experiments on the isolation of the serum-spreading factor, we became aware that the soluble serum factor required for cell spreading was dialysable. This factor could be replaced by the addition of Ca*+ ions to the medium and the effect of various concentrations of Cal+ on cell attachment to SUB+ (10 %) is shown in fig. 10. There was a stimulation of the final extent of attachment by the addition of Ca*+ (compare with fig. 4) and normal cell spreading occurred at a concentration of 1.OmM. It should be noted that Mg*+ is normally present in adhesion salts at a concentration of 0.8 mM; therefore, cell spreading is apparently independent of Mg*+. This result was unexpected and we then retested the attachment and spreading of cells onto substrata in the complete absence of serum, but with Ca*+ in the medium. No spreading of cells was observed. Thus, unless either serum or the partially purified spreading factor had been adsorbed to the substratum, Ca*+ did not induce cell spreading. The evidence presented in this manuscript is consistent with a model of cell attachment and spreading in which appropriate receptors on the cell surface directly bind to adsorbed spreading factor on the substratum surface. Ca*+ ions are probably required for activation of the receptors on Exptl Cell Res 97 (1976)

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F. Grinnell

plished by treating SUB+ (5 %) with polycationic ferritin or ConA. Polycationic ferritin has the capacity to bind to many anionic sites [26] and ConA is tetrameric for binding to certain terminal galactose and mannose groups [27]. We observed a dramatic increase in the rate and extent of cell attachment to these substrata (fig. 11). Furthermore, spreading of cells onto the substrata occurred within 15 min in the absence of added serum or Ca2+ in the incubation medium (fig. 12A, B). It is interesting to note that cells which spread onto polycationic ferritin-treated substrata were generally of circular shape, whereas cells spread onto ConA-treated substrata were triangular in shape, similar to the shape of spread cells under normal conditions. These observations further support the non-electrostatic model of cell attachment and spreading discussed above and imply that the sites on the cell surface to which ConA is binding may be topographiFig. 12. Morphology of cell attachment to substrata pretreated with ConA or polyc,tionic ferritin. The ex- tally related to the normal cell surface adheperiments are analogous to those described in the sion receptors. legend to fig. 11. (A) SUB+ (5%) treated with polyThe model of cell attachment and spreadcationic fenitin, attachment in serum-free medium; (B) SUB+ (5 %) treated with ConA, attachment in serum- ing we are proposing is diagrammatically free medium. Other details are described in the presented in fig. 13A. It is important to note captions to figs 2 and 11 and in Methods. x280. that although some cell lines such as BHK [18, 271, HeLa [13], and L cells [28] rethe cell surface. Although we cannot con- quire serum factors for cell spreading, other clusively rule out the possibility that Ca2+ cell lines such as WI38 [29]; MCR-5 [30] and ions form an electrostatic bridge between human conjuctiva cells [12] have no serum the cell and substratum surfaces, this idea requirement. Culp and his collaborators [3 l] seems less attractive for several reasons: have described the cellular production and (1) the apparent specificity of the protein secretion of a glycoprotein which becomes which must be adsorbed to the substratum adsorbed to the substratum (SAM), that surface; and (2) the ability of cells to attach they believe is the target protein on the suband spread onto substrata which have had stratum to which 3T3 cells attach and spread. Significantly, spreading required their negative charges neutralized. In model system studies we produced only Ca2+ ions in the medium along with substrata activated for direct binding to the SAM adsorbed to the substratum surface. cell surface either by electrostatic or non- This model is shown in fig. 13B and is forelectrostatic interactions. This was accom- mally analogous to the model we have Exprl CellRes 97(1976)

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that upon adsorption to substrata, serum proteins undergo conformational changes RECEPTOR and that these changes are dependent upon kz the chemical nature of the surface to which they adsorb [30, 381. Also, there are examples of proteins which are activated by SUBSTRATUM their adsorption onto appropriate substrata, such as factor XII (Hagemen factor) which is involved in thrombogenesis [38, 391. We have previously shown that cell attachment occurs preferentially on high energy substrata, such as Falcon polystyrene or glass, as compared with low energy substrata, such as teflon or polyethylene [40]. This Fig. 13. Models of cells and platelet attachment. might be accounted for by adsorption of the spreading factor in a certain conformation induced by the high energy substrata. presented. Perhaps the difference between Another important question arising from cells not requiring serum or requiring serum this work is why cells normally attach but for cell spreading depends upon whether or do not spread onto serum-free substrata or not the cells are capable of synthesizing the substrata coated with serum proteins other than the spreading factor. One explanation appropriate spreading factor. An analogous model of cell attachment may relate to a physical requirement to has also been proposed for platelet attach- generate a certain strength of adhesion per ment to collagen [32] as shown in fig. 13C. unit surface area in order for cell rigidity to This is based upon the interaction between be overcome, thus permitting the deformaplatelet surface glucosyl transferases and tion of the cell surface that is attendant appropriate carbohydrate receptors on the upon spreading. It may be that cell surface collagen. In this regard it is significant that receptors mediating non-specific (without BHK cell attachment to serum-coated, non- spreading) cell attachment are less dense living substrata and the attachment of plate- than the specific receptors for both cell atlets to collagen are similar processes in tachment and spreading and therefore the many ways. For instance, they are both former might not generate an adequate unit sensitive to inhibition by sulfhydryl-bindstrength of attachment to result in spreading reagents [II, 33, 341, trypsin [33, 351, ing. Clearly, much more work is required tranquilizers [24, 361, or cytochalasin B [24, to begin to adequately resolve this problem. 371. In summary, we have presented evidence In consideration of the non-electrostatic that the attachment and spreading of BHK direct interaction model, there is the ques- cells requires a specific serum component tion whether molecular complexes form in to be adsorbed to the substratum surface solution between the spreading factor and and Caz+ ions in the attachment incubation the cell surface. We suggest that the spread- medium. In the absence of either adsorbed ing factor is not functional until after it is serum protein or Ca”+ ions there is cell atadsorbed onto the substratum. It is known tachment but no cell spreading. A

BHK CELL

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MS Linda Forsyth provided expert technical assistance and Dr Richard Anderson participated in many helpful discussions during this work. MS Paulette Saddler and MS Roselyn Powell provided expert editorial assistance. This research was supported by a grant, CA 14609, from the NIH. NCI.

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