Printed in Sweden Copyright 8 1975 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental Cell Research 92 (1975) 383-393

ELECTRON

MICROSCOPY

IN SITU DURING A. W. LUCKY,l

M. J. MAHONEY,

OF HUMAN

SKIN FIBROBLASTS

GROWTH IN CULTURE R. J. BARRNETT

and L. E. ROSENBERG2

The Departments of Human Genetics and Anatomy, Yale University School of Medicine, New Haven, Corm. 06510, USA

SUMMARY We have studied the electron microscopic (EM) appearance of cultured diploid human skin fibroblasts during logarithmic and confluent stages of growth using a simple technique which permits in situ visualization of individual cells in monolayer. By comparison, cells disrupted from a monolayer and pelleted showed drastic distortion of the cell surface and appearance of organelles: mechanical scraping produced massive dilatation of the rough endoplasmic reticulum (RER); and trypsin-produced multiple blebs of the plasma membrane and cytoplasmic vacuoles. In situ, these changes in trypsinized cells disappeared within 1 h after plating. Six hours later, microtubules and microfilaments had surrounded the nucleus and were oriented in longitudinal bundles beneath the plasma membrane during rapid growth. At confluence these cytoskeletal elements seemed to extend beyond the plasma membrane at intercellular junctions. Pinocytotic vesicles were abundant at those surfaces devoid of filaments. Mitochondria, aligned with the long axis of the cell, were extremely long and narrow. During logarithmic growth there were many free ribosomes, polysomes, and some flat cistemae of RER. At confluence most ribosomes were found in spirals on dilated saccules of RER which contained electron-dense material. Lysosomes of several types were present during all phases of growth and varied in number from cell to cell. Late in culture the lysosomes tended to be larger, often occupying whole areas of cytoplasm or even extruding from the cell, and resembled those seen in lysosomal storage diseases. Understanding the in situ ultrastructure of normal human fibroblasts during growth in culture will permit systematic examination of such cells in a variety of pathological states.

Many recent advances in our understanding of human gene expression have come from studies of cultured diploid skin fibroblasts. Such studies have concentrated on measurements of enzyme activities in a variety of metabolic disorders, under diverse culture conditions, and during different phases of the growth cycle. Increasingly, however, light and electron microscopic (EM) abnormalities are being reported in fibroblasts expressing 1 Present address: Reproduction Research Branch, National Institute of Child Health and Human De velopment, National Institutes of Health, Bethesda, Md 20014, USA. * To whom reprint requests should be sent at the Department of Human Genetics.

metabolic defects [l-6]. It is surprising that normal human fibroblasts in culture have not been extensively studied by EM. Comings & Okada [7] examined one normal cell line during logarithmic and confluent stages of growth using trypsinized pelleted material. Robbins et al. [8] studied the appearance of cells undergoing senescence after multiple passagesin culture. To our knowledge, there are no systematic reports of the in situ appearance of human fibroblasts in monolayer during growth. Using a simple method for the in situ fixation, embedding and light microscopic localization of cells in monolayer, we have studied the ultrastructure of Exptl Cell Res 92 (1975)

384 Lucky et al.

Fig. 1. Light micrographs of eponembedded human skin fibroblasts in situ stained with toluidine blue 0. (a) 1 h; (b) 6 h; (c) 24 h; (d)7 days in culture. All x 40.

normal human skin fibroblasts from 1 h through 8 days in culture. We have also compared the appearance of the cells using our in situ technique with that obtained by trypsinization or mechanical scraping followed by pelleting. MATERIALS AND METHODS Tissue culture Skin fibroblasts in the eighth or ninth passage in culture from three normal males aged 1, 2, and 24 years were used for timed studies. Several other confluent control lines were also examined. Previous freezing in liouid nitrogen and subseauent thawinc of celllines did not affect growth or morphology ii culture. Cells were grown in Minimal Essential Medium (MEM) (GIBCo, New York) plus nonessential amino acids, 10% fetal calf serum, and Kanamycin (100 pg/ml). Equal aliquots of a suspension of cells which had been trypsinized from a confluent monolayer were sparsely plated on multiple Petri dishes (Lux or Falcon plastic tissue culture dishes). Growth was followed by phase microscopy of living cells, and by cell counts and total protein determinations in parallel dishes at each time of fixation. Cells were fixed at 1 and 6 h and daily from 1 to 8 days after plating. Cells were not re-fed during this interval.

layer at its surface was separated from the Petri dish with gentle pressure. Confluent monolayers of the same cell lines were either scraped from the mastic surface of the Petri dish with a rubber policeman or trypsinized for 2-5 min in 0.125 % trvnsin at 37°C. These susnensions were centrifuged i&io pellets, fixed in 3 % glutaraldehyde, post-fixed in osmium tetroxide, dehydrated through ethanols to propylene oxide, and embedded in capsules of Epon.

Microscopy To localize individual cells for electron microscopy, the polymerized block of Epon was stained by floating it, monolayer side down, on a warm solution of 0.2 % toluidine blue 0 in 1 % sodium bicarbonate for 2 to 8 min. The entire monolayer was viewed with a Zeiss light microscope. With the aid of a Leitz diamond microscope marker, specific cells were encircled and photographed (fig. 1). These same cells were removed on plugs of Epon bored from the plate with a 3 mm skin biopsy punch and remounted on Epon blocks for sectioning. Thin sections were cut from the face of the monolayer with a DuPont diamond knife. Toluidine blue 0 stained cells were visible on the face of the block during sectioning. The sections were mounted on uncoated copner arids and stained with a saturated solution of &&ylacetate followed by lead citrate [lo]. Cells were viewed and photographed with a Hitachi HU 11B electron microscope

RESULTS Fixation, dehydration and embedding All procedures were done in situ in the original dish on which cells were grown. After a single brief rinse in phosphate buffered saline (PBS), monolayers were fixed in either 1.5 or 3 % glutaraldehyde buffered with 0.05 M sodium cacodylate buffer at pH 7.2 for 1 h on ice. The fixed cells were stored at 4°C in buffer for up to two weeks. They were then post-fixed in 1% osmium textroxide (diluted from 2 % with sodium cacodvlate buffer) and dehvdrated in a series of ethanols,-25 to 95 % for approx. 2 min each. Following three changes of 100% ethanol for 15 min, the cells were infiltrated with 50% Epon 812 [9] in absolute ethanol for at least 1 h and three changes of pure Epon for at least 1 h each. A 3 mm layer of Epon was left in the dish and polymerized overnight in a 60°C oven that was leveled in all directions. When hardened, the Epon cast with the cell monoExptl Cell Res 92 (1975)

The change in light microscopic appearance of normal diploid human fibroblasts in tissue culture at various times is illustrated in fig. 1. These are photomicrographs of the actual cells embedded in Epon which are subsequently viewed by electron microscopy, and illustrate the sequence of events which occurs after trypsinized cells are plated: at 1 h cells are rounded and isolated (fig. 1a); by 6 h broad thin processeshave spread out in all directions as cells move across the plastic surface (fig. 1b); by 24 h most cells are

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385

tubules r& filaments not only surround the nucleus but radiate from the nuclear membrane. In the upper half of this figure a distal process of an adjacent cell is also shown demonstrating the paucity of cytoplasmic Pelleted cells organelles at this site distant from the nuThe effect of gentle scraping with a rubber cleus. By 24 h in culture the cells have assumed policeman and pelleting are apparent in fig. 2. The cells are rounded with an irregular their characteristic spindle shape (figs 8, 9). nucleus and, most conspicuously, the rough Organelles are lined up with the long axis of endoplasmic reticulum (RER) is greatly di- the cell. Mitochondria are extremely long lated. Mitochondria and lysosomes are seen and thin with transverse cristae, electron as small profiles in the cytoplasm. Trypsiniza- lucent matrix, and dense granules (fig. 9a). tion (figs 3, 4) also causescells and nuclei to Cisternae of RER are relatively flat. Perinuround up. It has profound effects on the cell clear Golgi complexes have flattened vesicles surface, creating blebs of the plasma mem- aligned with the long axis of the cell. The more brane which may appear as isolated islands distal cell process shown in fig. 9 at higher of cytoplasm when cut in cross section (fig. magnification demonstratesthe densebundles 4). Within the blebs there are single mem- of microtubules and microfilaments running brane bounded vesicles resembling pinocy- under the cell surface. A tangential section totic vesicles, but these are larger, more of a saccule of RER illustrates the spiral patnumerous and extend far into the cytoplasm. tern of ribosomes on its surface which is There are random arrays of filaments characteristic of this cell type. throughout the cytoplasm. Short profiles of By confluence, 7 and 8 days in culture, cells mitochondria and lysosomes are apparent. are extremely elongated (figs 10-12). Fig. 10 (at approximately the same magnification as In situ monolayers figs 2, 3, 5) demonstrates the typical ovoid Within 1 h after plating trypsinized cells, the nuclear shape. The cytoplasm is now packed plasma membrane is again smooth and the with dilated saccules of RER which contain nucleus regular with an ovoid contour (fig. 5). an electron-dense material (fig. 11). The ends Microtubules (250 A diameter) and smaller of cells in contact with each other possess microfilaments (approx. 50 and 100 A dia- dense massesof microfilaments which seem meter) course at random throughout the to extend beyond the plasma membrane into cytoplasm (fig. 6). There are short segments the intracellular spaces (fig. 12). There are of rough endoplasmic reticulum and many many more lysosomes in the cytoplasm seen free ribosomes and polysomes in the cyto- as membrane-bounded vacuoles containing plasm. Mitochondria and lysosomes appear electron-dense material, myelin figures, or as small rounded profiles throughout the cell. even fragments of organelles. At times these The most striking change in the cells oc- vacuoles may be quite large and they often curring 6 h after plating is the proliferation occupy whole areas of cytoplasm (fig. 13) or of cytoskeletal elements, microtubules and are found extruding from the cell (fig. 14d). microfilaments, and their orientation in the However, lysosomes are present during all long axis of the cell and around the nucleus stages of cell growth (fig. 14a-c). The pino(fig. 7). In some sections it appearsthat micro- cytotic vesicles illustrated in fig. 13 are also elongated with lengthy processes (fig. 1c); and in the confluent (7 day) culture the elongated cells are aligned side by side (fig. 1d).

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386 Lucky et al.

Fig. 2. Human skin fibroblasts scraped from a confluent monolayer with a rubber policeman and pelleted. Cells are irregular, the nucleus (N) convoluted, and the rough endoplasmic reticulum (rer) markedly dilated.

(m) Mitochondria; (L) lysosome. x 3 900.

Fig. 3. Fibroblasts trypsinized and pelleted from a confluent monolayer are rounded up and the nucleus (N) is convoluted. Flat cisternae of rough endoplasmic reticulum (rer), short profiles of mitochondria and lyso-

somes and random fine filaments (f) are dispersed throughout the cytoplasm. The cell surface is distorted. x3900. Fig. 4. Higher magnification detail of the cell surface of a trypsinized, pelleted cell showing pleomorphic blebs continuous with the plasma membrane (pm) and numerous clear vesicles (u). Intercellular space (is). x 17 800. Fig. 5. One h after plating trypsinized cells they remain rounded up but they are adherent to the growing surface. The plasma membrane is regular, and the nucleus (N) no longer convoluted. Organelles are randomly dispersed in the cytoplasm. Four nucleoli are present. x 3 600. ExptI Cell Res 92 (1975)

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Fig. 6. Detail of cell 1 h after plating showing microfilaments and microtubules in random array throughout

the cytoplasm. Short ovoid segments of mitochondria (m), lysosomes (L) and fragments of rough endoplasmic reticulum (rer) are seen as are many free ribosomes and polysomes. (N) nucleus. x 11 000. Fig. 7. After 6 h in culture, cells contain many microfilaments (F) and microtubules (arrows) aligned in the long axis of the cell and encircling the nucleus. Some tubules radiate from the nuclear membrane. A distal cell process (cp) of an adjacent cell contains few organelles except for sparse free ribosomes. Intercellular space (is). x 15 000. 26-751808

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Lucky et al.

Fig. 8. After 24 h in culture the cell is further elongated and polarized and the nucleus (N) is ovoid in longitudinal section. Microfilaments and microtubules are aligned with the long axis of the cell as are long thin mitochondria (m). A juxtanuclear Golgi apparatus (G) is present. x 7 800. Fig. 9. A distal cell process at 24 h in culture. Bundles of microfilaments (F) accumulate in the cytoplasm and beneath the cell surface obscuring the plasma membrane. Many ribosomes are arranged in spirals on saccules of rough endoplasmic reticulum (rer) which contain an electron-dense substance. There are fewer cytoplasmic organelles further from the nucleus. The elongated nature of mitochondria after 24 h is shown in fig. 9~. x 14 000. Exptl Cell Res 92 (1975)

Cultured&nan

fibroblasts in situ

389

Fig. 10. Fully confluent cell at 8 days in culture maintains its elongated structure. There are fewer free ribosomes, and more dilatation and accumulation of electron dense material in the cisternae of the rough endoplasmic reticulum. (N) Nucleus. x 3 900. Fig. II. Higher magnification view of a dilated saccule of rough endoplasmic reticulum (rer) with spirals of ribosomes on the surface of a confluent cell. x 15 000. Fig. 12. The distal ends of confluent cells 8 days in culture possess abundant filaments which appear to be extracellular at areas of cell to cell contact. Q Filaments; (N) nucleus; (is) intercellular space. x 8 700.

390 Lucky et al.

Exptl Cell Res 92 (1975)

Cultured human fibroblasts in situ

found during earlier stages irf growth. They are characteristically more abundant at those surfacesdevoid of microfilaments and appear just beneath the plasma membrane. DISCUSSION We suspect that many tissue culture laboratories have avoided using in situ methods for EM visualization of monolayers in culture because of the complex techniques which have been described in the literature. We would like to emphasize that the in situ fixation and preparation of monolayers for EM as described in this paper are simple procedures which require no special materials. Cells from a parallel biochemical experiment or a line of particular interest can be fixed and stored at any time without prior preparation. Although these methods are not original in our laboratory [11-151, we and our colleagues [16] have found that the addition of en bloc staining with toluidine blue 0 to in situ fixation and embedding in the dish or flask maximizes both the simplicity of preparation and the accurate localization of individual cells in monolayer. With this method, we have been able to characterize the appearance of cultured cells during logarithmic and confluent stages of growth in situ. One of the most striking changes we observed in fibroblasts during their growth in culture is in the number and arrangement of microtubules and microfilaments. They proliferate rapidly, starting 6 h after plating and accumulate predominantly beneath the plasma membrane in longitudi-

391

nally oriented bundles. It is thought that these cytoskeletal elements are important determinants of cell structure and motility [1721], and play an essential role in contact inhibition [22-251 and transport [26-281. It is of interest that Perdue [21], working with chick fibroblasts, noted microfilaments that seemed to be extracellular, and appeared similar to those we observe in human cells. Further studies are needed to determine whether these structures truly extend beyond the plasma membrane, and to define their role in cell structure and transport. Pinocytotic vesiclesare surprisingly numerous in cultured human skin fibroblasts. They contain no electron dense material and are rarely found far from the plasma membrane, often invaginating from it. This appearance suggests an endocytotic rather than exocytotic function. Uptake studies of electron dense particles such as ferritin would be of interest to confirm this role, and indeed it would be feasible to study the uptake of a variety of tagged macromolecules in vitro in this system. It is of interest that the plasma membrane appears to have specialized functional areas, as most pinocytotic vesicles are found at those surfaces devoid of microfilaments. Ribosomes and rough endoplasmic reticulum (RER) occupy a major portion of the cell cytoplasm. The change in distribution of ribosomes with larger, more dilated cisternae of RER late in culture confirms the observations previously made in this cell type [7] and correlates well with the available biochemical data showing decreasednumber and

Fig. 13. A distal cell process at 8 days in culture with numerous pinocytotic vesicles (Pu) at the plasma membrane. Lysosomes (L) occupy a large portion of the cytoplasm. x 15 200. Fig. 14. A variety of lysosomes are seen at various time-s in cuhure. (a) One hour. Lamehated myehn figure x 19 800; (6) 6 h. Multivesicular body and lamellated myelin figure adjacent to a mitochondrion (m). x 26 100; (c) 24 h. Dense myelin figures and single-membrane bounded vesicles with homogeneous material. x 14 Ooo; (d) 7 days. Extruded lysosomal body in the intercellular space (is) containing ribosomes and electron-dense vesicles. x 15 200. Exptl Cell Res 92 (1975)

392 Lucky et al.

aggregation of polysomes with serum starvation [29] or age in culture [30]. Although there are no known inborn errors of metabolism specifically affecting the structure of the RER, changesin RER morphology can be correlated with pathological conditions. For example, the addition of galactoseto galactosemicfibroblasts in culture causesswelling of the RER and eventual cell death [31]. Deficiency of ascorbic acid in cultured cells also causesthe RER to dilate [32] and may in part be responsible for the changes we observed late in culture. The large quantity of RER and number of ribosomes in these cells imply that protein synthesis is occurring but the nature of the intracisternal material is undefined. Likewise, the significance of the spiral pattern of ribosomes on the RER membranes is not known. Inclusion bodies, presumably lysosomes, accumulate in cells with specific deficiencies of lysosomal hydrolases in vivo and in vitro [l-6]. They have been seen as well in some non-lysosomal disorders [33, 341.It has been inferred that an increased number and/or an unusual type of lysosome reflects these pathological states. Lyon [3], in his review of storage diseases, noted that not all cultured skin fibroblasts demonstrated such pathognomonic inclusions. There is evidence, for instance, that only with the addition of sulfatides could the typical inclusion bodies of metachromatic leukodystrophy be reproduced [4]. An increasein number of lysosomes with time in culture [7], senescence[8, 35, 361 and decreasing pH of the culture medium [37] have been described by others. From our studies, it is apparent that several types of lysosomes are present at all stages of growth in culture-the newly plated cell was, after all, previously part of a confluent monolayer. However, lysosomes appear larger and more numerous at later times in culture. In some confluent cells we saw such an acExptl Cell Res 92 (1975)

cumulation of lysosomes in the cytoplasm and even extruding from the cell that the appearance was identical to that reported in storage diseases.We wish to underscore the importance of careful, controlled observations on normal as well as pathological material in well defined culture conditions before the diagnosis of an abnormality of lysosomal function is made on morphologic grounds. We have noted few alterations in the nuclear shape during growth in culture. Mitochondria in growing and confluent cells do not differ in appearance, but are remarkable for their long narrow contour and conformity to the elongated shape of the cell. The proximity of mitochondria to microfilaments and microtubules may indicate a functional relationship in which the mitochondrion is physically supported by these cytoskeletal elements. The occasional swelling of a part of a mitochondrion noted previously [7] seems to us to be an inevitable fixation artifact which we have also found in our cells. Finally, we wish to emphasize the differences between the in situ and the pelleted appearance of monolayers of cells. Although mechanical scraping of monolayers with a rubber policeman does not cause great losses of intracellular metabolites, regrowth in culture is severely limited after this procedure [38]. The prominent dilatation of RER which we observed under these conditions is never seenin the in situ cell and may be a correlate of the non-viability. In the trypsinized, pelleted cell we have observed contraction of the plasma membrane, convolution of the nucleus, random orientation of microtubules and microfilaments, pleomorphic cell surface projections and multiple clear cytoplasmic vesicles. This appearance is similar to that described by Comings & Okada [7]. These morphological features were never seen in monolayers of cells viewed in situ and must,

Cultured human fibroblasts in situ

therefore, be attributed to the effects of trypsin and pelleting on the cells. Trypsin renders cultured fibroblasts more permeable to a variety of macromolecules [38], yet cell viability is preserved [38]. Although the relatively normal in situ appearance of the plasma membrane as early as 1 h after plating indicates rapid recovery, our results show that trypsinized, pelleted material should not be used to examine the morphology of normal or pathologic human cells in culture. We must, instead, look to the in situ fibroblast in monolayer to understand its changeswith culture conditions, age in culture, and intrinsic pathological states. We are grateful to Dr G. Moellman for many helpful discussions. This work was supported by a training grant (AM 05708-01) and a research grant (AM 12579) from the National Institute of Arthritis, Metabolic and Digestive Diseases of the NIH, Bethesda, Md.

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12. Nelson. B K & Flaxman. B A. J microsc 97 (1972) 377. 13. Brinkley, R R & Chang, J P, Tissue culture, methods and aunlication (ed P F Kruse Jr & Y M K Patterson) p. 438. Academic Press, New York (1973). 14. Brinkley, B R, Murphy, P & Richardson, L L, J cell biol 35 (1967) 279. 15. F60uglas, W H S & Elser, J E, In vitro 8 (1972) I

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16. Moellman, G. Personal communication. 17. Abercrombie, M, Heaysman, J E M & Pegrum, S M, Exptl cell res 67 (1971) 359. 18. Goldman, R D & Follett, E A C, Exptl cell res 57 (1969) 263. 19. Revel, J P & Wolken, K, Exptl cell res 78 (1973) 1. 20. Revel, J P, Hoch, P & Ho, D, Exptl cell res 84 (1974) 207. 21. Perdue, J F, J cell biol 58 (1973) 265. 22. Courington, D & Knogt, P, J virol 1 (1967) 400. 23. Heaysman, J E M & Pegrum, S M, Exptl cell res 78 (1973) 71. 24. - Ibid 78 (1973) 479. 25. McNutt, N‘ S, Wip, L A & Black, P H, J cell biol 50 (1971) 691. 26. Estensen, R D & Plagemann, P G W, Proc natl acad sci US 69 (1972) 1430. 27. Kletzien, R F, Perdue, J F & Springer, A, J biol them 247 (1972) 2964. 28. El;l, S B & Wilson, L, J biol them 247 (1972) 29. Evans, R B, Morherm, V, Jones, A L & Tomkins, G, J cell biol61 (1974) 95. 30. Levine, E M, Becker, Y, Boon, C W & Eagle, H, Proc natl acad sci US 53 (1968) 350. 31. Miller, L R, Gordon, G B & Bensch, K G, Lab invest 19 (1968) 428. 32. Schafer, I‘ A, Silverman, L, Sullivan, J C & Roberton, W B, J cell biol 34 (1967) 83. 33. Bartman, J,. Wiesmann, V & Blanc, W A, J pediat 76 (1970) 430. 34. Laxova, R, Ohara, P T, Ridler, M A C & Timothy, J A D, Arch dis child 48 (1973) 212. 35. Brock, M A & Hay, R J, J ultrastruct res 36 (1971) 291. 36. Gordon, G B, Miller, L R & Bensch, K G, J cell biol 25 (1965) 41. 37. Lie, S 0, Schofield, B H, Taylor, H A & Doty, S B, Pediat res 7 (1973) 13. 38. Magee, W E, Sheek, M R & Sagi, K B P, Proc sot exptl biol med 99 (1958) 390. Received October 1, 1974

Exptl Cell Res 92 (1975)

Electron microscopy of human skin fibroblasts in situ during growth in culture.

Printed in Sweden Copyright 8 1975 by Academic Press, Inc. All rights of reproduction in any form reserved Experimental Cell Research 92 (1975) 383-3...
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