Stages of spreading of human diploid cells on glass surfaces

E,uperitnnental Cdl Research 68 ( I97 I ) 372-380

STAGES OF SPREADING

OF HUMAN

DIPLOID

CELLS

ON GLASS SURFACES J. A. WITKOWSKI Division

of

Imrmmologicul

Products

(Hampstead

and W. D. BRIGHTON

Control,

Nrrtionol

Laboratories),

London,

Institute NW

for

Mrdicul

Rrsrrrrch,

3, UK

SUMMARY Interference contrast and scanning electron microscopy have been used to study the spreading of the human diploid cell MRC-5 on glass. After trypsinization the cells were rounded-up and had a folded surface. Possible functions for these surface folds are discussed and related to the microvilli oresent on other cells. Microextensions developed very early after attachment to the glass surface and appeared to guide the spreading cytoplasm. During spreading, two intermediate cell shapes could be recognised before the fully spread fibroblast form was achieved. The rate at which the cells spread was estimated by measuring the proportions of these cell forms present.

It has been suggested that the ability of cells to grow in culture is determined in part by the interaction between cell surface and substratum [l, 2, 31 and the importance of the cell surface in controlling cell behaviour has often been emphasised [l, 2, 41. Taylor [5] studied the effects of various media on cell adhesion by phase contrast microscopy, but the development of more sophisticated techniques has made possible a more detailed study. We have used interference contrast microscopy and the scanning electron microscope to study the stages and rate of spreading of human diploid cells on a glass surface. MATERIALS

AND METHODS

Cultures of the human diploid cell strain MRC-5, derived from foetal lung by Jacobs 161, were used throughout this study. The cells were grown routinely in Eagle Basal Medium (Gibco, New York), with 10% calf serum. (Fraburg, Maidenhead, UK). Penicillin (100 II-t/ml) and streptomycin (100 /lg/ml) were also added. Cells for use in experiments were detached from the glass surface on which they were growing by a Exptl

Ceil

Res 68

short treatment with a 0.25”,, trypsin solution (Difco I :250) and were then washed three times with warm Hanks salt solution. After washing they were suspended in growth medium and plated in 60 mm plastic Petri dishes (Falcon Plastics) containing IX mm coverslips (Chance No. I). The coverslips were previously cleaned by soaking in chromic acid, followed by a thorough washing with distilled water [3]. The cultures were incubated at 37-C and at various times after plating were fixed for 15 min with 1 “b glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. After fixing, the preparations were washed with distilled water, dehydrated with increasing concentrations of alcohol. and air-dried. light microscopy was carried out using a Zeiss Photomicroscooe II with Nomarski differential interference contrast optics [7]. Preparations were mounted in distilled water and photographed on Recordak Microfile film (Kodak). For examination in the scanning electron microscope, the dried specimens were coated with an even layer of gold/palladium, about 500 A thick, by vacuum evaporation. A Cambridge Stereoscan microscope Mk I, operating voltage 20 kV, was used. Preparations were tilted at an angle of 45 to the electron beam and photographed on llford FP4 film.

RESULTS Stages of spreading

MRC-5 cells were considerably rounded 10 min after plating in medium containing serum

Fig. /. Cells that have been spreading on glass for 10 min. (a) A rounded cell with a folded surface; (h) detail of a cell showing extensions to the glass. The extensions end in swellings; (c) A rounded cell with long extensions; (d) detail of(c) showing movement of cytoplasm from the raised cell mass on to theglass surface. (a-d)Stereoscan.

(fig. I CI, c). Their surfaces were “bubbly” but without the short microextensions seen on BHK21/C13 cells by Follett & Goldman [8]. Cells at a very early stage of attachment rested on a broad base of cytoplasm (fig. I a) and later extensions from cell to the glass surface developed (fig. 16). These extensions often ended in swellings (fig. 1b) [9, lo] but

such enlarged endings were not common to all extensions. At a later stage these had elongated, and had given rise to long microextensions (N 12 ,um long) that radiated from the cell (fig. I c). Such extensions that play a part in attachment and spreading of cells will be called attachment microextensions (AMs). They thickened as cytoplasm Exptl Cell Rrs 68

374

J. A. Witkowski & W. D. Brighton

Fig. 2. Cells that have been spreading on glass for I h. (u) A cell in which cytoplasm has begun to spread over the glass surface; (b) cytoplasm continuing to spread. The bend/kink present in the AMs is a fixation artefact; (c) detail of the perimeter of a cell. Some AMs end in swellings, and local thickenings occur at the edge of the cytoplasm; (d) a cell at a later stage with short AMs. (n-d) Interference contrast.

began to move down from the bulk of the cell (fig. 1d). Many cells after I h in cultures were still almost spherical, but most of those that had attached to the glass surface had begun to spread (fig. 20, b). At this stage much of the cell mass was raised, and was surrounded by cytoplasm that was spreading outwards, following the AMs (fig. 2a, c). The regularity Expil

Cell Rrs 68

of this spreading cytoplasm contrasted with the initial, localised outgrowth of the AMs although around the perimeter of the cell, the cytoplasm was locally thickened (fig. 26, c). These thickenings may have been a form membrane” observed by of the “ruffled Abercrombie & Ambrose [12] as they occur where the edge of the cell is particularly involved in movement [13]. The cytoplasm

Fig. 3. Cells that have been spreading on glass for 3 h. (a) A cell that is sufficiently well spread to show nucleoli, the number of AMs is reduced; (b) cell with nucleus showing as smooth area; (c) a polygonal shaped cell; (d) detail of a corner of a polygonal cell. The AM ends within the cell in a swelling. (U--C) interference contrast; (d) Stereoscan.

continued to move outwards until the AMs were very short (fig. 24. After 3 11 in culture, the cells were sufficiently well spread for nucleoli to be seen (fig. 3~). At a later stage the smooth area of the nucleus became visible (fig. 36) and it was evident that the pattern of spreading had begun to change. The outline of the

cells had altered from round (fig. 3a), through an intermediate form (fig. 3h). to polygonal (fig. 3~). This change seemed to be determined by the position of the AMs, which were reduced in number, and those remaining were found chiefly at the angles of the cells (fig. 34. It seems that AMs end within the cell in some definite structure

376

J. A. Witkowski & W. D. Brighton

Fig. 4. Cells that have been spreading before the cell becomes fibroblast-like. Interference contrast

on glass for 6 h. (a) A cell that shows the transitional angular shape Ruffled membrane (RM) has formed; (h) a moving fibroblast (LI--b).

(fig. 3d), which may be analogous to the basal bodies of cilia. By the time 6 h had elapsed, almost all the cells had flattened enough to show nucleoli, but the change of cell shape continued (fig. 40). Many cells had become fibroblast like (fig. 4h), and, because of the presence of “ruffled membrane”, appeared to be moving. After 30 h in culture, all cells that were not dividing were fully spread (fig. 50). Although AMs were not apparent along the sides of the cells, they and the localised thickenings of cytoplasm present at earlier stages of spreading occurred at the ends (fig. 5h, c). The areas above the nuclei were smooth, except where the nucleoli caused bulging of the surface, but the membrane over the main part of the cells was pitted (fig. 5d). These pits may mark areas of pinocytotic activity.

possible to measure the rate of spreading. The method used by Taylor [5] was followed, but four forms of MRC-5 cells were recognised: (i) spherical cells with little or no spreading; (ii) spreading cells, but still with a raised cell mass; (iii) cells either circular or polygonal in outline, that are sufficiently spread to show nucleoli; (iv) angular and fully spread cells. The numbers of each of these cell forms present in each preparation were counted, and expressed as percentages of the total. The results (table I) show that the fully rounded cells gradually disappear, and the proportion of those which were fully spread increased. It can be seen that types B and C were the predominant forms at 1 and 3 h respectively.

Measurement of rate of cell spreading

Cells of the human diploid cell strain MRC-5, spreading on glass in normal culture medium, change in shape from a spherical to an

After the changes that take place during cell spreading had become familiar, it became Expil

Cell Res 68

DISCUSSLON

Spwading

Fig. 5. Cells that have been in culture AMs at the edge; (c) thickenings of area of the nucleus is smooth, and is above the nucleus. (n-b) Interference

of‘ cells on glass

377

for 30 h. (a) Fully spread cells; (b) detail of the end of cell showing short the cytoplasm present on a spread cell; td) detail of the cell surface. The surrounded by a pitted surface. The nucleoli show as bumps in the surface contrast; (c-d) Stereoscan.

elongated, fibroblast form. After initial contact with the glass surface is made, extensions develop between cell and glass, and these then elongate until they are often longer than the diameter of the cell. Cytoplasm then begins to spread over the glass surface giving the cell a circular shape, and at a later stage polygonal and angular forms arise before the fibroblast shape is assumed. A similar

sequence of events has been suggested for mouse macrophages [ 141. The cells used in this study were removed from the glass surface on which they were growing by treatment with trypsin. Although the cells were washed before use their surfaces may have been affected by the trypsin. Price [20], however, states that trypsin produced no changes in the form or Exptl Cell Res 68

378

J. A. Witkowski & W. D. Brighton

Table 1. Percentages of four cell forms present in cultures of spreading A4RC15 cells Time

“b of

cell

cultures fixed

Type A

IO min.

100

I h

3h 6h

40.1 3.4

0

types present Type B

Type C

Type D

0

0

0

54.1 22.2 4.2

10.3 64. I

0.9 10.3

33.6

62.2

numbers of the microvilli of L cells, and that the surface appeared to be unaltered. Moreover, we are interested in the behaviour of cells under the normal conditions used in tissue culture and trypsin is used routinely in the subculturing of MRC-5 and many other cells. Mechanical disaggregation of MRC-5 cells is unsatisfactory, producing few single cells and considerable cell death. The transition from one shape to another is clearly illustrated by the measurement of the rate of change of cell shape (table I) where it can be seen that there is a progressive decrease in the number of spherical cells and a corresponding increase in the number of spread cells. The proportion of the intermediate form B rises to a maximum at I h, and then falls as it changes to form C. Taylor [5] showed a similar series of changes in human conjunctival cells undergoing spreading, but MRC-5 cells appear to spread more rapidly than human conjunctival cells. The latter showed no form corresponding to B until 2 h after plating, and only at 4 h did they make up 50 oo of the cell population. The difference in the rates of spreading may result from the different morphologies of the fully spread cells. Conjunctival cells after 20 h in culture were polygonal in shape, compared with the fibroblast-like MRC-5 cells. Mouse macrophages are also fibroblast in form when fully spread and appear to spread at a rate similar to MRC-5 [14]. It is difficult to Exptl Cell Res 68

attribute the difference in rate of spreading solely to the use of cell types with different surface properties, because it might also result from experimental differences, e.g. the type of glass used in the experiments. A number of studies of cell surface structure have been made using light microscopy [ 13, 15, 161 and the transmission electron microscope [l3-201, and more recently the surface replica technique [8, IO, 15, 19-211 and scanning electron microscope [9. I I, 14, 21. 221 have been used. Perhaps because of this profusion of data, the nomenclature for surface structures is confused [9]. Our observations suggest that microextensions are of two kinds [I I], attachment microextensions (AMs) which are long, thin and arise at the base of the cell, and short microextensions (SMs) which are found over the whole of the upper surface of the cell. The form of the short microextensions depends both on ccl1 type and the degree of cell spreading. The AMs are produced very shortly after contact between cell and glass and are initially short (- 1 ,um) and thick, but it seems unlikely that they “anchor” the cell to the glass. The cell appears to deform on the surface so that it rests on a fiat base of cytoplasm. A more likely function of the AMs is that they explore the surface about the cell, as they lengthen rapidly (- IO ,~m) and arc directed radially from the cell. This triples the effective diameter of the cell and increases by 9 the area available to the cell. It seems likely that they explore this area for local variations in the glass surface [l6, 171 or follow surface detail such as stress lines, in a manner similar to the contact guidance shown by whole cells [23]. It has been suggested that AMs may prepare the surface for the cell to spread [9], but it appears that they also act as guidelines for the spreading cytoplasm [9, 171. The cytoplasm spreads as

Spreading

a thin web between the AMs, clearly shown in the early stages of spreading (fig. 2b). At a later stage the AMs still seem to be directing cytoplasmic spreading and by their position determine cell shape. Initially when the AMs are distributed regularly around the cell, the cytoplasm spreads evenly, but at the time when the cell changes in shape from circular to polygonal, the AMs are found mainly at the angles of the cell. This is also true of the fibroblast where AMs are restricted to the ends of the cell, and are absent from the sides of the cell. A feature of AMs is their terminal swelling (8.9.20). This swelling probably does not arise as a specialised structure for attachment to the substratum because such contacts occur at many points along an AM [18] and bulbous swellings do not arise from these contacts. We believe that, as they are restricted to the ends of the AMs, they may be involved in elongation of the AMs. The AMs of MRC-5 cells do not appear to be branched, unlike mouse embryo [16] and mitotic Chang liver cells [9]. We have not examined MRC-5 cells that have rounded up prior to division, but why only such cells should have branched AMs is not clear. The short microextensions vary considerably in appearance. MRC-5 cells have a folded or “bubbly” surface without clearly defined structures, and Chang liver cells [9] and mouse macrophages [14] have similar surfaces. A second form of SM that closely resembles the microvilli of intestinal cells occurs in other cells [lo, 21, 221 whilst both forms have been found on BHK21/C13 cells [S]. Whether or not SMs are found also depends on the degree of cell spreading, since it appears that all rounded cells have SMs, but when some cells spread their SMs are lost (8. 9.14). As MRC-5 cells spread the folding becomes reduced and by the time the nucleoli become visible the folds have dis-

of‘ cells on glass

379

appeared. It seems likely that cell surface is conserved in these “bubbles” and that the membrane required for the increase in surface area that occurs when round cells spread comes from this store. Similar suggestions have been made to account for the increase in surface area without synthesis of new membrane that occurs when cells divide. The apparently simple folds of the surface of MRC-5 cells appear to be an efficient means of conserving cell surface. Follett & Goldman [8] have suggested the microvilli-like SMs of BHK21/C I3 cells have a similar function, that is they conserve membrane when the cell is round and that this membrane is used during spreading. Recently O’Neill & Follett [24] have demonstrated an inverse relationship between number of microvilli and cell density, and suggest that the disappearance of microvilli results directly from cell contact. We believe, however, that conservation of cell surface is a secondary function of the microvilli-like SMs of BHK2l/C13 and other cells, and that is the function of the surface folds and “bubbles”. These microvilli have been shown to possess a complex internal structure [8] which is identical with that of the microvilli of duodenal epithelial cells [25]. The microvilli of these cells serve only to increase the area of cell surface available for uptake of substances from the medium. We suggest that the microvilli present on rounded BHK 21/C 13 cells increase the surface area of the round cells so that they can absorb substances efficiently from the medium [16]. When the cells are spread, their flattened surface area is sufficiently large for absorption to take place in the absence of microvilli. Follett & Goldman [8] have argued that because microvilli are not permanent features of the cell surface, they cannot function in

380

J. A. Witkowski & W. D. Brighton

the same way as the microvilli of duodenal cells. However, it is significant that microvilli are not labile structures of other cells. HeLa [IO, 211, L cells [20] and a transformed mouse kidney cell line [22] possess microvilli even when fully spread, and their presence on these cells may be related to the high metabolic activity of such cells. Nor does the sparse distribution of the microvilli on tissue culture cells compared with the large numbers on duodenal cells preclude their functioning as organelles for absorption. It is very unlikely that a cell selected for growth in vitro could form microvilli as efficiently as the highly differentiated duodenal cell. The difference in the density of the microvilli is more likely to reflect a difference in the degree of specialisation of the two cell types rather than a difference in function of their microvilli. We believe that the bubble-like and microvilli-like SMs have different functions. The former act as stores of surface material and disappear when the cell surface area is enlarged. They are common to rounded MRC-5, BHK21/Cl3 [8], mouse macrophages [14] and L cells [20]. The microvillilike SMs are found only on certain rounded cells, and provide a large surface for absorption from the medium. They may persist on fully spread cells, and this may be related to the metabolic activity of these cells. The surfaces of spread cells differ. The membrane above the nucleus of a cell of the MRC-5 line is very smooth, but the rest of the exposed cell surface is heavily pitted. Similar features can be seen in mouse kidney cells [22], whilst fully spread BHK21 /C 13 cells [8] have a smooth surface over the whole of the cell. with the nucleus showing as a shallow depression. Under the conditions of culture used here, MRC-5 cells spread in an ordered sequence of steps, but it is far from clear how the Exptl Cell Res 68

sequence is controlled. Although it might be expected that a cell would spread from all points of contact with the glass surface, the initial reaction is limited to areas that give rise to AMs. In addition the elongation of the AMs is controlled so that they are directed away from the cell, and at some stage after this elongation the mass of cytoplasm begins to follow the AMs. It is also necessary to account for the changes in cell shape. Knowledge of these control mechanisms would be of great value in understanding these aspects of cell behaviour that are determined by the surface properties of the cell. REFERENCES :: 3. 4. 5. 6 7. 8. 9. 10. I I. 12. 13. 14. 15. 16. 17. IS. 19. 20. 21. 22. 23. 24.

Abercrombie, M, Europ j cancer 6 (1970) 7. Auersperg, N, J natl cancer inst 43 (1969) 175. Nordling, S, Acta pathol microbial Stand, suppl. 192 (1967) I. Vasiliev, Ju M & Gelfand, I M, Currents modern biol 2 (1968) 43. Taylor, A C, Exptl cell res, suppl. 8 (1961) 154. Jacobs, J P, Jones, C M & Baillie, J P, Nature 227 (1970) 168. Allen, R D, David, G. B & Nomarski, G, Zwiss Mikroskop 69 (1969) 193. Follett, E A C & Goldman, R D, Exptl cell res 59 (1970) 124. Dalen, H & Scheie, P, Exptl cell res 57 (I 969) 351. Overman, J R & Eiring, A G, Proc sot exptl biol med 107 (1961) 812. Dalen, H & Scheie, P, Exptl cell res 53 (I 969) 670. Abercrombie, M & Ambrose, E J, Exptl cell res 15 (1958) 332. Abercrombie, M, Heaysman, J & Pegrum, S M, Exptl cell res 59 (1970) 393. Cart-, K & Carr, I, Z Zellforsch 105 (1970) 234. Goldman, R D & Follett, E A C, Exptl cell res 57 (1969) 263. Cornell, ‘R, Exptl cell res 57 ( 1969) 86. Tavlor. A C & Robbins. E. Develop biol 7 (1963) 660. Cornell, R, Exptl cell res 58 (1969) 289. Cooper, T W & Fisher H W, J natl cancer inst 41 (1968) 789. Price P. G, J mem biol 2 (1970) 300. Pugh-Humphreys, R G P & Sinclair W, J cell sci 6 ( 1970) 477. Hodges, G’ M, Europ j cancer 6 (1970) 235. Weiss. P. Exntl cell t-es. suoul. 8 (1961) 260 ~;;eifl, C H’ & Follett; E ‘A C, J cell &i 7 ( 1970)

25. Sandborn, E. B, Cells and tissues by light and electron microscopy, vol. I (1970). Received March 8, 1971 Revised version received May 13, 1971