Mechanisms of Ageing and Development, 10 (1979) 405-443

405

© Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

F I N E S T R U C T U R E O F I M R - 9 0 C E L L S IN C U L T U R E AS E X A M I N E D BY SCANNING AND TRANSMISSION ELECTRON MICROSCOPY

JOHN E. JOHNSON, Jr National lnstitute on Aging, N.LH., Gerontology Research Center, Baltimore Oty Hospitals, Baltimore, Maryland 21224 (U.S.A.)

(Received November5, 1978; in revised form January 22, 1979)

SUMMARY Cells from the new strain IMR-90 were examined by scanning (SEM) and transmission (TEM) electron microscopy at early, middle, and late population doubling levels. The cells are characteristically flattened and elongated and arranged in clusters from 1 to several cells thick. Long thin processes extend from the poles and sides of the cells. The number of blebs and microvilli on the cell surface varies. In later population doubling level (PDL) cultures, a larger number of cells have greater quantities of microvilli on their surface. It is suggested that the increased number of microvilli might represent an increased level of differentiation. By TEM the cells typically have elongated to oval shaped nuclei which are sometimes deeply invaginated, The cytoplasm contains a well developed Golgi region, elongated mitochondria, microtubules, fdaments, a variety of vesicles, vacuoles and dense bodies and large amounts of RNA in the form of granular endoplasmic reticulum and ribosomes. Cytoplasmic appearance, particularly the number of dense bodies, varies widely at all PDL. With increasing PDL, cells tend to have nuclei with more condensed chromatin, and a cytoplasm containing less mitochondria and granular endoplasmic reticulum and more dense bodies. Also at later PDL there is a higher frequency of cells containing long, thin dense mitochondria as well as bizarre shaped mitochondria. In older populations there are many cells in a state of filamentous degeneration. Ceils with large numbers of surface projections (microviUi) tend to be correlated with an osmiophilic cytoplasm containing many filaments and numerous dense bodies.

INTRODUCTION Although an enormous amount of literature on cells grown in culture is available in several scientific disciplines, comparatively little is known about aging in vitro. In gerontology, most of the in vitro work has been at the biochemical and genetic levels. Anatomical studies have been performed on WI-38 human fibroblasts [1] as well as other types of

406 cells [2]. The WI-38 human strain has been depleted over the years, and the IMR-90 strain has been developed to replace it [3, 4]. The present report describes the external and internal morphology of the IMR-90 cell strain as examined by scanning (SEM) and transmission (TEM) electron microscopy in order to obtain baseline anatomic data. A preliminary communication has already been published [5].

MATERIALS AND METHODS Human fetal lung fibroblasts (IMR-90) were obtained from the Institute for Medical Research, Camden, N.J. at early (18-19), middle (29-30), and late (49-50) population doubling levels (PDL). Cells were cultured in Eagle's minimum essential medium supplemented with glutamine, non-essential amino acids, 20% fetal calf serum (Flow Labs) and 50 /ag/ml chlorotetracycline (Aureomycin, Gibco) according to previously published methods [6]. Cultures were screened for mycoplasma contamination by the Ur/U biochemical technique [7]. To ensure the accuracy of population doubling levels, several cultures of each cell strain (each PDL) were carried to senescence. Senescence was defined when 250,000 cells were plated into a 75 cm 2 flask and confluency was not reached within one month. Senescence occurred between 51 and 55 cumulative population doublings. At various points, cells were plated into Leighton tubes and fixed as described below. Other cells were plated into the Leighton tubes and fixed at the PDL received from IMR.

The cells for the present study were plated at an average density of 150,000 cells into Costar 3393 Leighton tubes containing 3 ml of medium. In certain cases, cells were plated at heavier densities. For cell classification (see below) the cells were fed every "24 hours for 2 days and then fixed. This was done in order to keep the pH of the medium under more strict control (pH 7.3-7.4). Preliminary studies indicate that if the cells are plated too heavily and are allowed to grow to a thick confluent culture without daily feeding, many cells degenerate and morphological data are widely variable (Johnson, in progress). In the remaining cases the cells were fed upon initial inoculation into the Leighton tube and incubated for 3 4 days without further feeding, and then fixed. The 3 4 day schedule is in keeping with feeding and subcultivation schedules of other laboratories. On the date of fixation, the Leighton tubes were removed from the incubator and examined in a culture microscope. The incubation medium was decanted and room temperature fixative was added. The fixative consisted of 1% paraformaldehyde and 1.5% glutaraldehyde in 0.12 M phosphate buffer (pH 7.4-7.5). The tubes were placed in ice for about I0 min to 1 h with fixative. This solution was replaced with 2% OsO4 in 0.12 M phosphate buffer (pH 7.4-7.5) for 30 min to 1 h, also in ice. Following the osmium solution, the tubes were rinsed in 0.1 M acetate buffer (pH 5.0) followed by a 30 min treatment with 1-2% uranyl acetate in 0.1 M acetate buffer (pH 5.0). This was followed by treatment with 70% ethyl alcohol. The acetate rinses and uranyl acetate

407 treatment were in ice. A portion of the coverslip in each tube was then cut with small scissors into a Petri dish f'dled with 70% ethyl alcohol, for several minutes, followed by a transfer to 95% ethyl alcohol and three changes of absolute ethyl alcohol for 5 - 1 0 min each. These cut coverslip specimens (SEM specimens) were then critical point dried with CO2, mounted on stubs with silver adhesive, and sputter coated with gold at 2000 V, 10mA, at a target to specimen distance of 3 cm for 1.5 min under 2 0 - 3 0 mTorr vacuum. In some cases, the coverslips were not treated with uranyl acetate after osmium but were transferred from the osmium to saline and then dehydrated as above. Also, some SEM coverslips were treated with the osmium solution, then rinsed in 0.12 M phosphate buffer, then treated with 1% thiocarbohydrazide in 0.12 M phosphate buffer for 30 min, rinsed in 0.12 M phosphate buffer, then fresh osmium solution for 30 min followed by saline, dehydration and critical point drying. In this case, only 30--45 s of sputter coating was necessary. The remaining coverslips were dehydrated as above and then treated with 50% epoxy (Quetol)/50% absolute ethyl alcohol for 30 min followed by two changes of 100% epoxy and final embedding in Petri dishes according to previously described methods [8]. Thin sections were stained with uranyl acetate followed by lead citrate and occasionally followed by a third staining with bismuth and examined in a Zeiss 9S-2 transmission electron microscope. The SEM coverslips were examined in an Hitachi HFS-2 field emission, ion-pumped scanning electron microscope at 25 kV and 5/aA emission. Photographs were made on Polaroid type 52 film. For the cell classification study, cells were examined for the presence ofblebs and microvilli on their surface. For this purpose, areas 10,800 tan 2 (the area of the CRT on the SEM at 1000× magnification)were selcted at random and then several smaller regions 108/an 2 were examined (the area of the CRT at 10,000X magnification). The cell occupying the major portion of the field was then classified as " - " if it had an essentially smooth surface, as "+" if it had only a few microvilli or blebs (perhaps 5), as "++" if the microvilli were rather numerous, and as "+++" if the surface was covered with them. One hundred cells were classified at each of three population doubling levels (18, 30, and 49). RESULTS

Scanning electron microscopy Cultures at early (18), middle (30), and late (49) population doubling levels (PDL), when plated as described and fixed after 2 days of incubation, would be classified as approaching confluency if observed in a culture microscope under phase contrast. By SEM, the cells are observed to be arranged in small clusters with, perhaps, 50% of the coverslip surface being unoccupied (Figs. 1, 2 and 3). The later PDL cultures (49 in this case) appear slightly less densely populated (Fig. 3). Upon closer examination, the cell clusters are observed to consist of a net-like arrangement of flattened cells, 1 to perhaps 3 cells thick in places (Figs. 4, 5 and 6).

408

Figs. 1, 2 and 3. These three figures show the IMR-90 cells plated at 50,000 cells per ml, fed each day for two days and then fixed. The cells in Fig. 1 are at 18 PDL, those in Fig. 2 at 30 PDL, while the cells in Fig. 3 are at 49 PDL. Notice that in all three eases, about half the substrate is free of cells, but the largest clear spaces are in 49 PDL. All three Figs. × 60.

409

Fig. 4. In this 19 PDL culture, cells are seen to overlap (arrow). × 1065. Fig. 5. Large solid arrows (without dots) point out holes in smoothly surfaced cells overlaying areas of degenerating cells. Scores of small holes are seen on the degenerating cells (thin arrows). In some regions, a reticulated pattern is seen (arrow with one dot) suggesting the presence of collagen or filaments. In another area, apparent intercellular gaps are found (arrow with two dots). A long process (hollow arrow) extends from the end of one of the smooth cells over the surface of the degenerating cells. 19 PDL. × 5315. Fig. 6. At least three layers of cells exist in this region of a 49 PDL culture (solid arrows with dots). Overlaying the three layers is a cell with blebs (hollow arrow) and numerous processes (thin arrows). × 5315.

410 Delineation of cell boundaries is difficult in some instances, but easier in cases where surface morphology of the various cells is obviously different and where overlap of two cell layers or cell processes is evident (Figs. 5 and 6). Small pits or "holes" 50-150 nm in size are seen on the surface of most cells (Fig. 5). Some regions have very large numbers of holes, and a reticulated appearance, suggesting cell degeneration and, perhaps, the presence of collagen or filaments (Fig. 5). It is difficult to assess the nature of the holes at present. They may be sites of pinocytosis, exocytosis, artifacts, or, in some cases, regions of intercellular attachment. Small holes are seen on the surface of apparently healthy cells (Fig. 5). These holes are, however, much less extensive than on the reticulated cells. The possibility that the holes are artifacts is mitigated by the fact that such holes are not seen in all tissue types examined in our laboratory. Processes of varying length extend from all parts of the fibroblasts. They may be seen emanating from middle regions of the cells coursing over the surface (Fig. 7) or from the edges and ends of the cells (Figs. 5 and 8). The processes are usually about 100-150 nm in width and 10/an or more in length. The short processes (approximately 1 ~m or less) in abundance on the surface of some cells, are more appropriately called microviUi. Blebs, irregular ridges, and microvilli were observed in varying numbers on the cells (Figs. 9 - 1 6 ) to an extent that classification could be made (see method section and below). A few blebs may actually be mycoplasma. According to previous literature and personal communications, mycoplasma appears to be an almost unavoidable addition to cultures which have been subcultivated numerous times. However, in the present study, examination of our cultures by SEM and TEM indicate that, if mycoplasma is present, it is in extremely low amounts. The cells are almost uniformly seen in flattened arrays. Rarely, a microcolony of cells is observed, arranged in a rounded, raised configuration (Fig. 17). In this particular example, the rounded mass, thought to consist of several cells because of its size, is covered with microvilli. The greatest concentration of microvilli appears to be at the apex, perhaps on one or two cells (Fig. 18). For the cell classification according to amount of blebs and microvilli, 100 cells were examined at 10,000× magnification from each of three population doubling levels; 18 PDL, 30 PDL, and 49 PDL. These data are shown in Table I. It can be seen that with increased PDL, there are more cells which have large numbers of microvilli on their sur-

TABLE I Population doubling level

Classification* --

+

++

18

30 16 3

60 54 46

7

3

27 39

3 12

30 49

+++

*Numbers in the different classifications are out of 1O0 cells examined in each population doubling level. These data were found to be statistically significant at the 0.01 level by X~ analysis.

411

Fig. 7. Long processes course over the surface of this cell from a 49 PDL culture. The origin of one or two of the long processes may be seen (hollow arrow). Several microvllli (solid arrows) are also present. × 11,220. Fig. 8. A heavily populated region is illustrated from a 19 PDL culture. Long stringy material, some of which may be collagen or filaments, pervades the area. Several processes can be seen emanating from the edge of one of the cells (arrow). × 5610.

412

© j

Fig. 9. A cell elas~fied as " - " is shown from an 18 PDL culture. × 7545. Fig. 10. This cell from a 30 PDL culture was classified as " - " . X 7545. Fig. 11. Several microviUi (arrows) extend from the surface of this cell classified as "+" from a 49 PDL culture. × 7545. Fig. 12. This "+" cell, having several microvilli (arrows) is from a 30 PDL culture. X 7 5 4 5 .

413

Fig. 13. Numerous microvilli (arrow without dot) and blebs (arrow with dot) can be seen on the surface of this cell classified as "++" from an 18 PDL culture. X7120. Fig. 14. Microvilli (arrow without dots), blebs (arrow with one dot) and surface ridges (arrow with two dots) are shown on this "++" cell from a 49 PDL culture. X 7120. Fig. 15. Blebs, microvilli and surface ridges are present in extensive amounts on this cell classified as "+++" from a 49 PDL culture. X 7120. Fig. 16. Many processes and extensive surface ridges cover the surface o f this "+++" cell from a 30 PDL culture. × 7120.

414

Fig. 17. A microcolony of cells is shown from a 19 PDL culture. The colony is oval shaped and perhaps half a dozen cells are thought to be present. Possible cell boundaries are pointed out by arrows. X 1150. Fig. 18. A higher magnification view of the microcolony in Fig. 17 is illustrated. The area to the lower left is thought to be the apex and has more microvilli than the region to the upper right. × 5750.

415 face. Cells classified as "+++" are few in the early and middle PDL, while " - " cells represent about one third of the early population. In the late PDL (49) the " - " ceils are very sparse while "++" and "+++" cells have increased substantially in number.

Transmission electron microscopy One of the most striking observations in the present study is the wide variety of morphological appearances of the cells at all population doublings. Relatively "healthy" appearing IMR-90 cells are seen at all PDL. Healthy appearing is defined as relative lack of dense bodies and f'dament masses, and by the presence of extensive arrays of parallel granular endoplasmic reticulum cisternae (Figs. 19 and 20). The "healthy" cell is less frequent at later PDL. The typical young (18 PDL) cell has an elongated to oval shaped nucleus with from one to several nucleoli (Fig. 19). Some cells have deeply invaginated nuclei (Fig. 21). The nuclei of older cells tend to have more invaginations and condensed chromatin patches at the periphery, compared to a more evenly distributed chromatin pattern in cells at earlier PDL (Figs. 19 and 20). The cytoplasm contains numerous elongated mitochondria, a well developed Golgi region, extensive arrays (often parallel) of granular endoplasmic reticulum, various sorts of vesicles (both coated and uncoated) and dense bodies (Figs. 22 and 23). Overall number of mitochondria and arrays of granular endoplasmic reticulum appear to decrease by 49 PDL over the number at 18 PDL (Figs. 19 and 20). However, morphometric analysis was not performed, and this opinion is based on visual comparison of electron micrographs. Occasional young cells have numerous dense bodies and vacuolated regions (Figs. 24 and 25). The number of dense bodies in some cells is increased at later PDL (Fig. 26). However, some cells at 49 PDL only have a few dense bodies (Fig. 20). The dense bodies observed in IMR-90 cells have a wide variety of appearances. Some are rather granular with vacuoles (Fig. 26) while others consist of numerous membranes often arranged in a concentric or parallel fashion (Fig. 26). The dense bodies vary in size from 0.25 to 2/ma or more. Much larger bodies are also seen but these are probably aggregates of smaller dense bodies. Vacuolar bodies are often observed in the cytoplasm of IMR-90 cells (Figs. 24 and 26). Usually there is a certain amount of dense material at the edge of these vacuolar bodies but the major portion of the body is electron lucent. Other inclusions appear to be lipid droplets which only occasionally have an extremely electron dense periphery (Fig. 27). The mitochondria are usually long and thin, varying in length up to several microns (Fig. 28). There are two distinct types of mitochondria in these cells. One type is much longer, thinner and denser than the other type (Fig. 28). In the long thin type, longitudinal cristae are common (Fig. 28). The long thin type, designated type 1 in the present study, has an appearance sometimes referred to as "condensed", while the shorter, wider, less dense form, designated type 2, has an "ortho" appearance. Cells containing both types of mitochondria were n o t found in the present study. Cells with the type 1 mitochondria are present, although rarely, even at 18-19 PDL, and are found with increasing

416

Fig. 19. An IMR-90 cell at an early PDL (in this case 18 PDL) typically has an oval nucleus free of deep invaginations. This cell has two nucleoli, and the nuclear periphery has only small amounts of condensed chromatin (solid arrows). The cytoplasm contains a well developed Golgi (solid arrows with one dot), parallel arrays of granular endoplasmie retieulum (hollow arrows), vesicles (often concentrated at the cell surface suggesting pinocytosis; hollow arrow with asterisk), dense bodies (solid arrow with two dots) and numerous mitoehondria (small thin arrow). X 7295.

417

Fig. 20. In this 49 PDL IMR-90 cell, the condensed peripheral nuclear ehromatin is more patchy (solid arrows) than at earlier PDL (Fig. 19). The cytoplasm contains extensive Golgi regions (solid arrows with one dot) and in spite of being a later PDL cell, parallel arrays of granular endoplasmic reticulum (hollow arrows) and only a few dense bodies (solid arrow with two dots). The number of mitochondria (small thin arrow) appears to be reduced over cells at earfier PDL. × 7180.

418

Fig. 21. With increasing PDL the amount of nuclear invagination (thin arrows) increases. Adjacent to this 36 PDL cell is an array of reticulated membranes (short solid arrows). X 15,750.

419

Fig. 22. This electron micrograph illustrates an 18 PDL IMR-90 cell cytoplasm to show the parallel arrays of granular endoplasmic reticulum cut perpendicular to the cisternal wall (hollow arrows) and ribosomal rosettes when cut tangentially (hollow arrow with asterisk). A Golgi region (solid arrow with dot) and a portion of the nucleus (N) are also seen. × 15,295.

420

Fig. 23. A very extensive Golgi (Go) pervades the cytoplasm of this 36 PDL cell. Associated with the Golgi are coated (solid arrowhead) and uncoated (solid arrowhead with dot) vesicles. Also associated with the Golgi are numerous larger vesicles with a small dark center (hollow arrows). These dark centered vesicles may be a Golgi product. × 15,220.

421

Fig. 24. Although this cell is only at 18 PDL, it has an invaginated nucleus and numerous cytoplasmic dense bodies (small thin arrows) and vacuolar bodies (asterisks). × 6235.

422

?

V

j

ii!ii ¸ ?~,

\

® Fig. 25. This 18 PDL cell contains several large vacuoles (V) some of which have electron dense borders (arrows) suggesting that the large vacuoles are related to the vacuolar bodies (see text and Fig. 8). × 6135.

423

Fig. 26. Dense bodies and vacuoles are illustrated in cells at several PDL. In 26A (36 PDL) vacuoles (V) are seen, defined as having no dense aggregations at their periphery, whereas vacuolar bodies (VB) have such aggregations. ×" 13,985. An unusual dense body is shown in 26B (36 PDL). It has numerous vesiculated regions. X 13,985. The cytoplasm of the 19 PDL cell in 26C has several laminated inclusion bodies (solid arrows). Note the similarity in size and shape of these laminated bodies to nearby mitochondria (solid arrow with two dots). Also seen at this magnification are microtubules (hollow arrow), filaments (thin arrow) and a dark centered vesicle (solid arrow with one dot). x 27,675. The dense body is 26D (49 PDL) has several electron lucent vesicles. X 27,675.

424

Fig. 27. Cells containing large numbers of droplets thought to be lipid (L) such as illustrated in this 36 PDL cell, are not common. The droplets have an electron dense periphery (hollow arrow), thought to be regions of auto-oxidation. Occasional droplets have a very dense periphery (solid arrow). x 15,245.

425

Fig. 28. IMR-90 cells are observed to contain two types of mitochondria. Type 1 is long, thin and dense while type 2 is wider and not quite as dense. The cristae of type 1 may be transverse or at an angle with the long axis of the mitochondria as shown in the 36 PDL cell in 28A (thin arrow), or longitudinally situated as shown in the 49 PDL cell in 28B (thin arrow). Both Figs. x 15,245.

426 frequency with increasing PDL. Occasional mitochondria have bizarre shapes including "T" forms and ballooned forms, some of which are enlarged only at one end (Figs. 29 and 30). A few dense bodies have the appearance of altered mitochondria (Fig. 31), but because of the uncertain membrane structure, interpretation is difficult. Cells containing the bizarre shaped mitochondria are found in larger numbers at later PDL. The RNA in IMR-90 is seen in large amounts as granular endoplasmic reticulum (GER). Usually, a finely granulated material can be observed within the cisternae of the GER (Fig. 29). Occasionally, the GER is quite dilated, and the enormous amount of product inside would suggest that these particular cells are very active (Fig. 29). At the higher magnifications, one can observe not only the ribosomal patterns of the GER, but also small vesicles, filaments, and microtubules contained in the cytoplasm (Figs. 22 and 26). Some of the vesicles appear to be pinching off from the GER (Fig. 31) and may be "coated". Golgi areas also have numerous vesicles (Fig. 32). Many of the vesicles located here are coated. The Golgi region of cells at later PDL appears to have more vesicles associated with it than cells of earlier PDL (Fig. 32). The GER of cells at later PDL appears, in some cases, to be disarrayed (Fig. 33). This includes a shortening of the cisternal segxnent, less aggregation of cisternae into three or more semi-parallel groups, and a loss of ribosomes on the cisternal surface (Fig. 33). Smooth endoplasmic reticulum is seen in some cells (Fig. 34) but this is much different than the denuded GER observed in older populations. Along the inside edge of the fibroblasts may be seen a compact bundle of fine filaments (Fig. 35). These filaments are arranged in parallel rather than in random directions such as those seen in the cytoplasm (Fig. 26). The individual filaments are like those seen in many other cell types and are approximately 8 nm in diameter. Adjacent to some cells are arrays of degenerating cell debris consisting, to a large degree, of reticulated membranes (Fig. 21). Filamentous degeneration is particularly prominent in cells of older populations. The typical profile consists of a cytoplasm filled with fdaments and dense bodies (Figs. 36 and 37). Sometimes there is a core of filaments surrounded by dense bodies (Fig. 36A). Cells containing these massive numbers of filaments appear somewhat more electron dense than normal cells. Some of these dense cells, not all of which contain filaments, have a very large number ofblebs and microvilli on their surface (Figs. 36 and 37).

DISCUSSION It has been shown in the present study that IMR-90 cells in culture are generally flattened and elongated, with varying numbers of blebs and microvilli which appear to increase in number with increasing population doublings. Several laboratories [9, 10] have observed alterations on the surface of aging cells in vitro, and Kelley et al. [ 11 ] have suggested that variability in concanavalin A binding may be due, in part, to these surface irregularities.

427

Fig. 29. In 29A (36 PDL) g~anular endoplasmic reticulum (GER) is seen to be Efiled with f'mely granulated material, probably a proteinaceous product. Also seen is a mitochondrion (arrow without dot) enlarged at one end (arrow with dot). X 29,425. In 29B (36 PDL) numerous mitochondria are seen, one of which has an enlarged region (arrow). A very large dense body (DB) undoubtedly consisting of aggregates of smaller dense bodies, is present. X 14,870.

428

Fig. 30. The electron micrograph in 30A shows the cytoplasm of two cells (36 PDL) although the cell boundaries are indistinct. Mitochondrion M1 (type 1) is in one cell while the second cell contains several type 2 mitochondria one of which is "T" shaped (M2). X 28,675. The cell in 30B (36 PDL) contains several mitochondda, one of Which is enlarged along a portion of it (arrow). × 14,490. An enlarged ("ballooned") mitochondrion from a 36 PDL cell is shown in 30C. Most of the mitochondrion contains fine granules and some filaments, and at the right end is the boundary between a cristae containing area and the granulated area (arrow). × 28,675.

429

Fig. 31. In 31A, the cytoplasm of a 36 PDL cell contains granular endoplasmic reticulum (G) with fine granular material inside. At one end of a cisternal segment, a coated vesicle appears to be connected, perhaps pinching off (arrow). A nearby dense body (D) appears to have remnants of mitochondrial cristae at one end (small thin arrow). X 28,625.Two enlarged mitochondria are shown in 31B (36 PDL). Remnants of cristae are at one end (arrows). The dense body in 31A is thought to be at a later stage than those in 31B in the development of mitochondria into dense bodies. X 28,625.

430

Fig. 32. The Golgi regions of an 18 PDL cell (32A) and a 49 PDL ceil (32B) are shown at 30,920 × More vesicles, principally coated (arrows) are present in the 49 PDL cell than in the 18 PDL cell

431

Fig. 33. Cells such as illustrated here are not uncommon at later PDL cultures. The nucleus (N) is tortuous with a great deal of condensed chromatin. The granular endoplasmic reticulum is no longer arranged in parallel arrays and the segments are shortened. Segments have ribosomes on part of their length (solid arrow) and are bare (smooth) along other areas (arrow with dot). × 14,440.~ The inset 33B is a higher magnification (× 28,575) of another region of this same cell. Notice the absence of ribosomes on several areas of the granular endoplasmic reticulum (arrows). 49 PDL.

432

Fig. 34. Large amounts of smooth endoplasmic reticulum (SER) are present in this 18 PDL cell. X 15,155. Fig. 35. A parallel row of filaments immediately beneath the cell surface (solid arrow) is characteristic of IMR-90. Several cell processes (hollow arrow), perhaps part of this 18 PDL cell, lie on the surface. X 15,155.

433

A

Fig. 36. Parts of two electron dense cells from a 49 PDL culture are illustrated. In 36A the cell has a ftlamentous core (F) which is surrounded by dense bodies (arrows). The surface of this cell is free of blebs and microvilli. X 11,830. The dense cell in 36B is f'dled with ~aments and dense bodies. The surface is covered with blebs and microvilli. Cells with a more normal appearing cytoplasm usually do not have such an irregular surface. X 11,830.

434

Fig. 37. A higher magnification of the cell in 36B is shown in 37A. The massive array of filaments can be seen. X 30,545. Cell 1 (C1) in 37B (49 PDL) is electron dense and has numerous blebs (hollow arrows). Cell 2 (C2) is electron lucent and is estimated to be much healthier than cell 1. X 15,435.

435 The question of the meaning of the increase in number of blebs and microvilli on ceils of populations at higher PDL and the relationship to aging is complicated by the fact that the cells, and all cultures for that matter, are growing in an abnormal environment, lacking in a variety of stimuli from hormones and other substances present in the blood and interstitial fluids but not present in culture media. The cells also have lost contact by other tissues, unless the culture environment (One important factor being pH) is strictly controlled, consistent morphological data cannot be obtained. Many cell culture laboratories feed their cells every few days, allowing the cells to grow to confluency and the pH to drop. In our own tests, the pH of the medium, which is normally 7.4, may drop to 6.8 in three days if the cells are plated heavily. This results in a slowing down of cell division, and an increase in cell degeneration. The biochemical changes which accompany these cell alterations undoubtedly have contributed to the variability in results in not only in vitro aging studies but in cell culture experiments of other disciplines as well. In electron microscopic studies this problem becomes critical when single cells are being examined. It is a fact that normal cells from vertebrates and some invertebrates can be cultured. The cells which divide and remain "normal", that is to say, do not become "transformed", will produce about 50 population doublings after which the culture senesces, or dies out [ 12]. Several phases are associated with the life of a normal culture with phase III being taken as the senescent phase. The curve which defines the three phases is not a straight line during phase III but is represented by an increasing negative slope [13]. This suggests that the cells are gradually dying out over a period of several successive population doublings. It is probable that the reticulated cells illustrated in the present study may be degenerating and represent some of the cells which die throughout the life of the culture. However, the number of these reticulated cells is quite variable, and until the factors which affect their numbers can be critically studied, interpretation, at present, is limited. It has been stated that the finite capacity for replication of a normal cell is reached in vitro but is rarely reached when the cell remains in rive [13]. It was further suggested that the functional losses that occur in cells in vivo long before their proliferative capacity is reached, are at the foundation of the aging process [13]. On the other hand, data at the in rive as well as in vitro level suggest that cell senescence is governed by the number of times the cell has divided rather than the passage of chronological time the cell has existed [14, 15]. It is generally felt that there is an inverse relationship between rates of cell division and level of cell differentiation [16]. This would imply that some cells which normally divide slowly in rive and are moderately differentiated would de-differentiate to a certain degree when they are placed into an in vitro environment and begin dividing rapidly. During the course of proliferative capacity, cells would become differentiated to the point where a few microvilli are present (perhaps the "+" classification). We might expect to see a cell like this from in rive preparations. We might also expect these differentiated cells to divide at a slower rate. As the cells continue to divide, they might become "overdifferentiated", with extremely long and numerous processes. This is a stage which pro-

436 bably is not reached in vivo since fibroblasts normally would not go through as many cell divisions as they do in vitro. At this point (the "+++" stage), the cell probably would stop dividing and die sometime during the next few subcultivations. That some cells might become differentiated at an earlier population doubling level is evidenced in the present report by the presence of some "++" and "+++" cells, as well as degenerating cells at 18 PDL. The reticulated debris seen in Fig. 21 of the present report probably represents the TEM correlates of some of the reticulated regions shown at the SEM level in Fig. 5. Previous thought has centered on the idea that cell populations in vitro have some "young" and some "old" cells and that later PDL cultures merely have more of the latter and less of the former [1]. In accordance with present observations, it is felt that the "young" cells are actually de-differentiated or, at least, not fully differentiated cells. As long as a rapidly dividing undifferentiated state is maintained, a cell would be classified as "young". This type of cell, in the present case, IMR-90, would have few dense bodies, and large numbers of parallel arrays of granular endoplasmic reticulum. The surface of such a cell would probably be classified as " - " , "+" and perhaps at a certain point, "++". Therefore, it is suggested that the cells in this presumably normal strain, become increasingly differentiated with successive population doublings and that this increased level of differentiation would be correlated structurally with increased numbers of cell processes and functionally with a decreased rate of cell division. This is in keeping with previous literature showing that in vitro populations as a whole have an increased population doubling time at later PDL [17, 18]. In the present instance, of course, we are speaking of individual cells. The concept of the formation of neoplasms in vivo through the de-differentiation of mature cells has been controversial and challenged. It is currently felt that the undifferentiated appearance of malignant tumor cells in vivo is a result of neoplasia of undifferentiated reserve or stem cells to begin with rather than mature differentiated cells becoming more primitive [ 19]. However, a different situation may exist in regards to de-differentiation in vitro where cells have been removed from their normal surrounding environment. The sudden lack of parenchymal cells, substances normally present in the interstitial fluids, these factors may indeed result in de-differentiation. This speculation raises important questions concerning differences in species cell types when we observe the fact that mouse cells in vitro will quite often transform from a cell strain (finite number of cell divisions before senescence) to a cell line (cells which continue to divide indefinitely), whereas chick cells in vitro do not transform into cell lines [13]. The following example has been selected to illustrate the relationship of the present hypothesis at the in vitro level to an in vivo situation regarding increases in microvilli and states of differentiation. In the dudodenal crypts of Lieberkuhn of the small intestine, are undifferentiated stem cells which divide approximately once or twice each day [20, 21 ]. The new cells then move up the walls of the crypts, and the duodenal villi, to be finally shed from the villus tip. The time from cell division to shedding from the villus tip appears

437 to be about three days depending on the species [22, 23]. The newly divided cell in the crypt has only a few microvilli (Fig. 38). As the cell ascends the crypt and moves onto the villus, it differentiates and develops an enormous number of microvilli on the external surface, sometimes referred to as the striated border. Thus the structural correlates of differentiating duodenal cells might support a "differentiating" hypotheses of fibroblasts in vitro over a series of cell divisions. In the present study the IMR-90 strain of cells are seen to have a wide variety of structural appearances at all population doubling levels not only by SEM, but by TEM as well. To be sure, there are certain structural characteristics which are more common in the older cells, such as dense bodies and disarrayed granular endoplasmic reticulum. That a population of cells at a given population doubling level is heterogeneous biochemically as well as morphologically has been suggested before [24]. Table II presents a summary of similarities of the present findings to those seen in WI-38 cells by Lipetz and Cristofalo [1] and in chick fibroblasts by Brock and Hay [2]. It can be seen that, in general, IMR-90 cells are similar to WI-38 cells in structural changes with increasing PDL. The increase in dense bodies and bizarre shaped mitochondria seen in chick fibroblasts is similar to findings in the human ceils but other similarities remain to be established. Loss of ribosomes in aging cells has previously been reported at the in vitro [25] and at the in vivo [26-28] level. These data conflict with a report of increased RNA synthesis in older cell cultures [29] and, thus, the point of RNA changes and aging is still in dispute. In the present study, the disarray and shortening of GER segments in older cells, plus the lack of ribosomes on portions of the segments, would suggest a loss of RNA in many old cells. However, many other old cells appear normal. (See a recent review for a discussion of RNA and aging [30] .) The present observations that the Golgi region of older cells has more associated vesicles is in agreement with studies on WI-38 cells by Lipetz and Cristofalo [1]. As shown in the present report, some of the coated vesicles may pinch off from endoplasmic reticulum. It is possible that these coated vesicles contain hydrolytic enzymes as Holtzrnan et al. [31] have shown to be true in a histochemical examination of the rat nodose ganglion. Secondly, hydrolytic enzyme content has been reported to increase with age in vitro [32]. Dense bodies, often referred to as age pigment granules or lipofuscin [33] are, perhaps, the most frequently reported observation in aged cells. In spite of the scores of articles on the subject, some of which date to the 19th century, the origin of dense bodies is still controversial. The bodies have been suggested to come from lysosomes [34] and mitochondria [35-38]. The fine structure and biochemistry of age pigment has been thoroughly examined. Samorajski et al. [39] described lipofuscin accumulation in dorsal root ganglia and cerebellum in aging mice. Sekhon and Maxwell [40] clearly illustrated the development of lipofuscin granules in the anterior horn of the mouse cervical spinal cord. Their illustrations are typical of age pigment in healthy animals. Siakotos and Armstrong [41 ] have distinguished between two types of pigment, ceroid and lipofuscin, the former having a

438

Fig. 38. Newly divided duodenal stem cells of the mouse, as illustrated here in telophase, have occasional microvflli (hollow arrow) while other ceils, which are moving up the crypt to the villus, have more microviili (solid arrow). X 11,125.

IMR-90

Increases

Chick fibroblasts [2]

Decreases

No change

Increase in number

increase in number

Increase in number

Dense bodies

Bizarre shaped, more dense. *Longitudinal cristae

No change in number, *No longitudinal cristae

May decrease in number, Bizarre shaped, *Long thin type have longitudinal cristae

Mitochondria

*The cristae orientation refers to their presence or absence rather than age related change per se.

Increases

Increases

increases

WI-38 [11

(present study)

Nuclear chromatin condensation

Nuclear invagination

No change

Becomes constricted and empty

Decreases in amount and becomes disarrayed

Granular endoplasmic reticulum

COMPARISON OF I N VITRO ELECTRON MICROSCOPIC FINDINGS WITH INCREASING PDL IN THREE CELL TYPES

T A B L E II

No change

Has more vesicles associated with it

Has more vesicles associated with it

Golgi

Not reported

amount

in

Increases

Appears in degenerating cells

Filaments

~o

.b.

440

i ¸

Fig. 39. Part of a pericapillary cell from a one month old rat cerebellum (perfusion f'Lxed) is shown in 39A. The dense bodies (arrow) are characteristic of brain and other tissues. × 9940. An astrocyte dense body from a 7 year old squirrel monkey superior coiliculus (perfusion f'Lxed) is shown in 39B. × 9940. In 39C a glial cell is illustrated from a newborn rat eerebellar explant grown for one month in culture. Notice the ringed laminated inclusion bodies (arrows). X 22,420. In 39D, an electron mierograph (courtesy of Dr. M. Herman and Dr. A. Dekaban of Stanford University) is shown from a patient with Tay Sachs disease. There is a definite similarity between the laminated inclusions (arrow) in this and other storage diseases and the inclusions shown in 39C. × 23,670.

441

spectral emission maximum at 435 nm and the latter at 450 nm. There is general disagreement, however, as to how many types there really are [33]. Although the dense bodies seen in vitro have generally been referred to as lipofuscin and age pigment, with indications of possible biochemical similarities to lipofuscin accumulated in vivo [42], observations in the present author's laboratory indicate that their (in vitro) fine structure is more like that of the bodies seen in storage diseases such as metachromatic leucodystrophy [43], Tay-Sachs [44], Nieman-Pick disease [45], and other pathological processes [46] (see also Glees and Hasan [47] and Collins and Brunk [48]). Figure 39 illustrates a comparison of normal brain material from a perfusion fixed preparation with a newborn rat brain explant grown for one month in culture. Granules and lipid droplets are characteristic of "normal" in vivo age pigment while the lamellar or ringed inclusion body is more characteristic of many dense bodies from cells grown in vitro and from patients with storage diseases (Fig. 39). Not all in vitro pigment bodies have the laminated appearance, but the fact that many of them do raises serious questions regarding the adequacy of typical cell culture media. The presence of two distinct types of mitochondria in IMR-90 cells suggests the possibility of a mutation at some point in the development of the culture. The shorter and wider mitochondria appear more like those seen in WI-38 cells. The long, thin, dense type also have a counterpart in WI-38 cells except for the longitudinal cristae [1 ]. In one other human lung fibroblast strain, GM-1380, the mitochondria are slightly different than the two types seen in IMR-90. The GM-1380 mitochondria have less distinct cristae membranes. However, mitochondrial differences may reflect only metabolic state and further research into this question is necessary. In conclusion, the IMR-90 strain of human lung fibroblasts has been examined by scanning and transmission electron microscopy. It appears that the cells are similar to WI-38 fibroblasts, and are equally suitable for studies of aging in vitro. The results of the present study as well as those from other laboratories [49, 50] suggest that states of differentiation will play an important role in the examination of cultured cells.

ACKNOWLEDGEMENTS The author's gratitude is expressed to Dr. C. H. Barrows and Dr. J. Miquel for critical reading of this manuscript. He is also indebted to Dr. E. L. Schneider and Mr. R. Monticone for culturing the IMR-90 cells at the various PDL, and to his technical assistants Mr. W. H. Parson, Mr. G. H. Wharran and Ms. Faye DoweR. The secretarial assistance of Ms. Pat Pemn is always appreciated.

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442 2 M. A. Brock and R. J. Hay, Comparative ultrastructure of chick fibroblasts in vitro at early and late stages during their growth span, J. Ultrastr. Res., 36 (1971) 291-311. 3 W. W. Nichols, D. G. Murphy, V. J. Cristofalo, L. H. Toji, A. E. Greene and S. A. Dwight, Characterization of a new human diploid cell strain, IMR-90, Science, 196 (1977) 6 0 - 6 3 . 4 Public Health Rep., 93 (1978) 398. 5 J. E. Johnson, Jr., Fine structure of IMR-90 cells in culture, Proc. Gerontol. Soc., Dallas, 1978, p. 86. 6 E. L. Schneider and Y. Mitsui, The relationship between in vitro cellular aging and in vivo human age, Proc. Nat. Acad. Sci. U.S.A., 73 (1976) 3584-3588. 7 E. L. Schneider, E. J. Stanbridge and C. J. Epstein, Incorporation of [3H] uridine and [3H] uracil into RNA: a simple technique for the detection of mycoplasma contamination of cultured ceils, Exp. Cell R es.; 84 ( 1974) 311 - 318. 8 J. E. Johnson, Jr., Transmission and scanning electron microscope preparations of the same cell culture, Stain Technol., 53 (1978) 2 7 3 - 2 8 0 . 9 P. D. Bowman and C. D. Daniel, Aging of human fibroblasts in vitro: surface features and behavior of aging WI-38 cells, Mech. AgeingDev., 4 (1975) 147-158. 10 E. Blomquist, U. Brunk, B. Westermark and E. Arro, Surface characteristics of phase II and III cells in culture, in O. Johari (ed.), Scanning Electron Microscopy, III Research Institute, Chicago, 1977, pp. 1 3 - 2 0 . 11 R. O. Kelley, R. Azad and K. G. Vogel, Development of the aging cell surface: concanavalin Amediated intercellular binding and the distribution of binding sites with progressive subcultivation of human embryo fibroblasts, Mech. AgeingDev., 8 (1978) 2 0 3 - 2 1 7 . 12 L. Hayflick, The limited in vitro lifetime of human diploid call strains, Exp. Cell Res., 37 (1965) 614-636. 13 L. Hayflick, The cellular basis for biological aging, in C. E. Finch and L. Hayflick (eds.), The Handbook of the Biology o f Aging, Van Nostrand/Reinhold, New York, 1977, pp. 159-186. 14 R. T. Dell'Orco, J. G. Mertens and P. F. Kruse, Jr., Doubling potential, calendar time, and donor age of human diploid cells in culture, Exp. Cell Res., 84 (1974) 3 6 3 - 3 6 6 . 15 C. W. Daniel and L. J. T. Young, Influence of cell division on an aging process, Exp. Cell Res., 65 (1971) 2 7 - 3 2 . 16 E. D. P. DeRobertis, F. A. Saez and E. M. F. DeRobertis, Cell Biology, W. B. Saunders, Philadelphia, 1975. 17 L. Hayflick and P. S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25 (1961) 5 8 5 - 6 2 1 . 18 M. Hayakawa, Progressive changes of the growth characteristics of human diploid cells in serial cultivation in vitro, Tohoku J. Exp. Med., 98 (1969) 171-179. 19 S. L. Robbins, Pathologic Basis of Disease, W. B. Saunders Co., Philadelphia, 1974. 20 S. Lesher, Chronic irradiation and aging in mice and rats, in: P. J. Lindopp and G. A. Sacher (eds.), Radiation and Aging, Taylor and Francis, London, 1966, pp. 183-206. 21 J. D. Thrasher and R. C. Greulich, The duodenal progenitor population, J. Exp. Zool., 159 (1965) 39-46. 22 W. Bloom and D. W. Fawcett, A Textbook of Histology, W. B. Saunders, Philadelphia, 1975. 23 S. Lesher, R. J. M. Fry and H. I. Kohn, Influence of age on the transit time of cells of the mouse intestinal epithelium. I. Duodenum, Lab. Invest., 10 (1961) 2 9 1 - 3 0 0 . 24 G. S. Merz and J. D. Ross, Viability of human diploid cells as a function of in vitro age, J. Cell Physiol., 74 (1969) 2 1 9 - 2 2 2 . 25 R. Robbins, E. M. Levine and H. Eagle, Morphological changes accompanying senescence of cultured human diploid ceUs, J. Exp. Med., 131 (1970) 1211-1222. 26 J. E. Johnson, Jr. and J. Miquel, Fine structural changes in the lateral vestibular nucleus of aging rats, Mech. Ageing Dev., 3 ( 1 9 7 4 ) 2 0 3 - 2 2 4 . 27 W. Andrew, The Anatomy of Aging in Man and Animals, Grune and Stratton, New York, 1971. 28 D. N. A. Mann and P. O. Yates, Lipoprotein pigments - their relationship to ageing in the human nervous system. I. The lipofuscin content of nerve cells, Brain, 97 (1974) 4 8 1 - 4 8 8 . 29 E. L. Schneider and S. S. Short, Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts, Cell, 6 (1975) 179-184.

443 30 J. Miquel and J. E. Johnson, Jr., Senescent changes in the ribosomes of animal cells in vivo and in vitro, Mech. Ageing Dev., 9 (1979) 2 4 7 - 2 6 6 . 31 E. Holtzman, A. B. Novikoff and H.Villaverde, Lysosomes and GERL in normal and chromatelyric neurons in the rat ganglion nodosum, J. Cell Biol., 33 (1967) 419-435. 32 V. J. Cristofalo and J. Kabakjian, Lysosomal enzymes and aging in vitro: subceUular enzyme distribution and effect of hydrocortisone on cell life span,Mech. AgeingDev., 4 (1975) 1 9 - 2 8 . 33 J. Miquel, J. Ore, K. G. Bensch and J. E. Johnson, Jr., Lipofuscin: fine structural and biochemical studies, in W. A. Pryor (ed.), Free Radicals in Biology, Academic Press, New York, 1977, pp. 133-182. 34 E. Essner and A. B. Novik0ff, Human hepatoceUular pigments and lysosomes, J. Ultrastr. Res., 3 (1960) 3 7 4 - 3 9 1 . 35 A. Hess, The fine structure of young and old spinal ganglia, Anat. Rec., 123 (1955) 399-424. 36 T. Kummnoto and G. H. Bourne, Histochemical localization of respiratory and other hydrolytic enzymes in neuronal lipopigment (lipofuscin) in old guinea pigs., Acta Histochem., 16 (1963) 87-100. 37 J. Miquel, P. R. Lundgren and J. E. Johnson, Jr., Spectrophotofluorometric and electron microscopic study of lipofuscin accumulation in the testis of aging mice, J. Gerontol., 33 (1978) 5 - 1 9 . 38 P. E. Spoerri and P. Glees, Neuronal aging in cultures: an electron microscopic study,Exp. Gerontoi., 8 (1973) 2 5 9 - 2 6 4 . 39 T. Samorajski, J. M. Ordy and P. Rady-Reimer, Lipofuscin pigment accumulation in the nervous system of aging mice,Anat. Rec., 160 (1968) 5 5 5 - 5 7 4 . 40 S. S. Sekhon and D. S. Maxwell, Ultrastructural changes in neurons of the spinal anterior horn of ageing mice with particular reference to the accumulation of lipofuscin pigment, J. Neurocytol., 3 (1974) 5 9 - 7 2 . 41 A. N. Siakotos and D. Armstrong, Age pigment, a biochemical indicator of intracellular aging, in J. M. Ordy and K. R. Brizzee (eds.),Neurobiology of Aging. An Interdisciplinary Lifespan Approach, Plenum, New York, 1975, pp. 3 6 9 - 3 9 9 . 42 V. P. Collins and U. T. Brunk, Characterization of residual bodies formed in phase II cultivated human glial cells, Mech. AgeingDev., 5 (1976) 193-207. 43 C. Meier and A. Bischoff, Sequence of morphological alterations in the nervous system of metachromatic leucodystrophy. Light and electron-microscopic observations in the central and peripheral nervous system in a prenatally diagnosed fetus of 22 weeks, ActaNeuropathol., 36 (1976) 369 -379. 44 G. Fontaine, A. Resibois, M. Tondeur, G. Jonniaux, J. P. Farriaux, W. Veer, E. Maillard and H. Loeb, Gangliosidosis with total hexosaminidase deficiency: clinical, biochemical and ultrastructural studies and comparison with conventional cases of Tay-Sachs disease, Acta Neuropathol., 23 (1973) 118-132. 45 K. Harzer, W. Scholte, J. Peiffer, H. U. Benz and A. P. Anzil, Neurovisceral lipidosis compatible with Nieman-Pick Disease Type C: morphological and biochemical studies of a late infantile case and enzyme and lipid assays in a prenatal case of the same famfly,ActaNeuropathol., 43 (1978) 97-104. 46 L. Roizin, M. Kaufman, N. Willson, S. Stenar and J. C. Liu, Organdie membrane diversity in cerebrum of a patient with psychopathic behavior, Prec. VIIth Int. Congr. Neuropathol., Budapest, III (1975) 155-161. 47 P. Glees and M. Hasan, Lipofuscin in neuronal aging and diseases, Normal Pathol. Anat., 32 (1976) 1-67. 48 V. P. Collins and U. T. Brunk, Quantitation of residual bodies in cultured human glial cells during stationary and logarithmic growth phases, Mech. AgeingDev., 8 (1978) 139-152. 49 E. Bell, L. F. Marek, D. S. Levinstone, C. Merrill, S. Sher, I. T. Young and M. Eden, Loss of division potential in vitro: aging or differentiation? Science, 202 (1978) 1158-1163. 50 G. M. Martin, C. A. Sprague, T. H. Norwood and W. R. Pendergrass, Clonal selection attenuation and differentiation in an in vitro model of hyperplasia, Am. J. Pathol., 74 (1974) 137-154.

Fine structure of IMR-90 cells in culture as examined by scanning and transmission electron microscopy.

Mechanisms of Ageing and Development, 10 (1979) 405-443 405 © Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands F I N E S T R U C T U R...
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