Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting

CopFright C 1982 hy Acedemic Pros?. lsc. Al! rights of reproduction in any form reserved M)I4-4827:8?:0-10~C9-09fOi.~~~D

Experimental Cell Research 138 (1982) 409-417 ISOLATION

OF AUTOFLUORESCENT FIBROBLASTS

BY FLOW

MOI-phologp, Enzyme Activity J. F. JONGKIKD. Department

of Cell

A. VERKERK,

Biology

‘AGED’

HUMAN

SORTING

and Pr-oliferntive

Capncit?

W. J. VISSER and J. M. VAN DONGEN

and Genetics, Erasmus University,

Rotterdam,

The Netherla~~ds

SUMMARY In cultures of human fibroblasts the percentage of bright autofluorescent (AF) cells increases with increasing passage number. These autofluorescent cells were isolated using a FACS II cell sorter and compared with sorted non-fluorescent (NF) cells. The AF cells showed an increase in population doubling time (2.3-fold): cell protein (l.Pfold), and in specific activities of the lysosomal enzymes: p-hexosaminidase (4.2-fold). P-galactosidase (3%fold) and acid phosphatase (2.5 fold). The specific activities of two non-lysosomat enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase had increased only slightly (1. l-fold) respectively (1.S-fold). The autofluorescence in the AF cells was restricted to small round organelles. The distribution and size of these autofluorescence granules were similar to the acid phosphatase-containing granules in the cytochemically stained cells. Electronmicroscopical examination showed that these AF cells contained a large amount of small electron-dense granules containing amorphosmophilic material. These granules which were positive for the acid phosphatase reaction, were classified as secondary lysosomes. The low percentage of the sorted AF cells which incorporate [3H]thymidine during a 24 h test period (19 5%)as compared with the labelling percentage of sorted NF ceils (73 a/,! fromthe same cilture, indicate that ihe autofluorescent cells in a ‘young’ culture have a very limited remaining proliferative capacity. The results imply, that by flow sorting it is possible to isolate ‘aged’ cells with characteristics of ‘phase III’ cells out of non-aged fibroblast cultures.

In cultures of human fibroblasts, the content of bright autofluorescent (Al?) cells is high in late passage cultures, while it is also found, that these AF cells are unable to incorporate [“Hlthymidine into DNA [l]. Although the nature of the autofluorescent material is unknown these results suggest a correlation between the lysosomal accumulation of fluorescent material and in vitro aging. In the last few years preparative cell sorters have been developed, which can separate cells on basis of fluorescence [2, 31. In principle this makes it possible to isolate large numbers of autofluorescent cells from a mixed cell population. In the present study we describe the

isolation of bright AF cells with a FACS II cell sorter from a relatively young in vitro culture of control human fibroblasts. On this population of sorted cells we determined cell kinetic, biochemical and morphological parameters of in vitro aging.

MATERIALS

,4ND METHODS

Cells Control diuloid human skin fibroblasts (strain: Trin& were cultired in Ham’s FlO medium’supplemented with 10% fetal calf serum (FCS) and antibiotics (l(m pg/ml streptomycin and lb0 U/ml penicillin). ConI fluent cultures were subcultivated by 1: 3 splits, each split being counted as a passage number. Just ‘before cell sorting the cells were harvested by trypsinization and, after washing, resuspended in medium with 10% FCS. ‘-,

Exp Ceil Res !38 (1982)

410

Jongkind et al.

Cell separation Flow-cytometry and sorting were carried out using a FACSII cell sorter (Becton & Dickinson) equipped with an argon-ion laser (Spectraphysics 164-05) at 457 nm using a 70 pm nozzle. Fluorescence was measured above 520 nm by using a combination of K510 (Schott) and LP520 (Ditric Optics) filters. The instrument alignment was optimalized by using bright fluorescent microspheres (Polysciences: I.51 pm; no. 9719, lot no. 3-0826) till a coefficient of variation (cv), of 1.7%. Small blue-green weak fluorescent latex microspheres (Polysciences: 0.83 pm, no. 7766, lot no. 41196; cv 11%) were used to set the windows of the cell sorter. The reproducibility of this setting, from run to run, was tested by determining the variability of the modal fluorescence- and scatter-channel numbers of the 1.51 ym (no. 9719) green fluorescent microspheres, after disarrangements and readjustments of the instrument setting with 0.83 r*m (no. 7766) test microspheres. In five readjustments, the coefficients of variation were calculated to be 7.8 and 4.6% for the fluorescence respectively scatter modal channel numbers of the 1.51 pm microspheres. Fibroblasts which had more than 3x the fluorescence of the 0.83 pm beads were sorted as AF cells. Cells were sorted with a velocity of 2000 cellslsec into sterile tubes containing medium at 4°C. These cells were either distributed over the wells of culture plates in the desired cell concentration, or washed with saline (4°C) and kept as a pellet at -70°C for the enzyme activity determinations.

Doubling time and labelling index Sorted cells from a culture in logarithmic phase were distributed over the 16 mm wells of a 24 multiwell cluster plate (Costar). Each well contained 5 000 cells. After 1.; 2, 3, 4 and 7 days, individual cultures were trypsimzed in triplicate, and were counted with a hemocytometer. The determination of the labelling index was done essentially according to James & Veomett [4] on cells which were seeded on coverslips in the 16 mm wells (5000 cells/well). Three days after seeding, the medium was replaced by medium containing [3H]thymidine (0.1 pCi/ml; 20 Cilmmol). In this radioactive medium the cells were incubated for 24 h. After washing and fixing, the cells were subjected to autoradiography (Ilford K2 emulsion). In three separate experiments the percentage of cells with labelled nuclei was determined bv counting at least 200 cells in each exoeriment. The signific&ce of the difference between-percentages of labelled AF cells and labelled NF cells was tested with Student’s t-test.

Cell homogenization Sorted cells were washed twice by centrifugation. The cell pellets were stored overnight at -70°C. The pellets were suspended in cold Triton X-100 (0.25% in water) and subjected to a freezing and thawing procedure. The cell debris were centrifuged down in an Exp Cell Res 138 (1982)

Eppendorf 5412 centrifuge (3 min at 6 400 g)! and protein and enzyme assays were then carried out using the supernatant. The homogenization procedure was carried out at 4°C.

Protein assay Total protein was determined in the Triton extract by a micro modification of the fluorescamine method r51. Three ~1 of the orotein solution were mixed with ii pl borate buffer- (250 mM; pH 9) in polythene microtest tubes (Brand). Subseauentlv 25 ul of a fresh fluorescamine solution (0.2 mi/ml d;y acetone) was added under vigorous stirring on a Vortex mixer. The mixture was diluted with 200 ~1 of water and the fluorescence of the sample was read in 200 ~1 glass cuvettes in a spectrofluorimeter (Perkin Elmer fluorescence spectrophotometer MPF 2A; excitation 395 nm, emission 470 nm).

Enzyme activity determination The conditions for the microchemical enzyme determinations on the Triton extracts are listed in table 1. All incubations were carried out in the wells of Terasaki microtest ulates (Greiner). The samnles were covered with oil (n-hexadecane-paraffin 4: 6) to prevent evauoration T61 during incubation at 37°C on a Fischer isotemp. h;y bath: Lactate dehydrogenase (LDH) was measured in an end volume of 200 pl in the spectrofluorimeter. Acid phosphatase (AP), /3hexosaminidase @-hex), /3-galaitosidase (P-gal) and glucose-6-phosphate dehydrogenase (G6PD) were determined in an end volume of 13 pl in the original Terasaki trav using a Leitz microfluorimeter r7, 81. Substrates aid co&tors were from Boerhinger except the 4-methylumbelliferyl(4 MU) substrates which were from Koch-Light Laboratories and cr-naphthol phosphate which was from BDH.

Ultrastructure Sorted fibroblasts were seeded in 16 mm wells of a 24 multiwell (100000 cells/well) and cultured overnight. After washing with saline,. the attached cells were fixed for 1 h in 3 % glutaraldehyde in 0.1 M cacodylate buffer pH 7.3 containing 6.1 M sucrose. After a 30 min wash in 0.1 M sucrose in 0.1 M cacodylate the cells were post-fixed for 1 h in 1% 0~0, in 0.1 M cacodylate pH 7.3 containing 0.1 M sucrose. All steps in the fixation procedure were carried out at room temperature. After washing with 0.1 M cacodylate buffer (pH 7.3) the cells w&-e incubated with tannic acid according to Simionescu r91. Dehvdration and Epon embeddhg was carried out v’ia an ethanol series. In order to release the polymerized Epon (with cells) from the plastic, the dishes were alternately cooled in liquid nitrogen and heated in hot water. Small cylinders were drilled from the Epon and mounted on dummy Epon blocks. Ultrathin sections were cut on a Reichert OMU2microtome and stained with a saturated solution of uranyl-acetate in water and lead-citrate according to Venable [lo].

Flo~v sorting ofagedfibroblasts

411

Table 1. Conditions of incubation and analyiP

Enzyme*

Buffer

Substrate

cofactor

LDH

0.15 M Tris, pH 7.1

1.5 mM sodium pyruvate

1.5 mM NADH2

0.15 ~~ AMP.” pH 9.3

18 mM glucose-6phosphate

1.4 mM NADP+

0.15 M Tris-maleate, pH 4.2

15 mM oc-naphthyl phosphate 1 mM 4 MU-

iI31

G6PD [I31

AP D41

.&Gal [15] &Hex [15]

0.2 M phosphate 0.1 M citrate, pH 4.3 0.2 M phosphate 0.012 M citrate, pH 4.4

Other

Incubatian time at 37°C (hours)

30 mM

1. +1opi 0.3 NHCI 2. (lo&” 100/.Ll 7.5 NaOH 10 min, 6OY 3. + 100pl H,O ilO~lO.3 M phosphate, pH 11.5 15 min, 60°C +lO /J 0.25 N NaOH

210

1

+I0 plO.5 M carbonate. pH 10.7

13

*

+10~10.5 M carbonate. pH 10.7

13

nicotinamide 0.075 76 BSA”

1.5 mM EDTA 15 mM MgClL 0.075% BSA 0.075 9%BS.4

-

P-D-gakiCt0

pyranoside 5mM4MU2 acetamide2 deoxy-p-oglycopyranoside

0.1 M NaCl 0.15 % Tritonx-100 0.1% BSA 0.2 5%BSA

stop

End WA W

13

14

D Volume of complete substrate solutions 2 ~1. Sample size and cell protein content were /3-ga! and GSPD 1~1 100 pg/ml, LDH and &hex 1~1 10 pg/ml, acid phosphatase 2 ~1 150pg/ml. b Lactate dehydrogenase; glucose-6-phosphate dehydrogenase; acid phosphatase: fi-galactosidase; ,&hexosaminidase. c Bovine serum albumin, heat-inactivated: 1 h 50°C. ’ 2-Amino-2-methyl- 1,3 propanediol.

The sections were viewed in a Philips EM 400 microscope.

Cytochemistry Sorted cells were seeded on coverslips and cultured overnight. The attached cells were fixed in 4 !%formolmacrodex for 10 min at room temperature, washed in saline. and incubated at 37°C during 1 h in a medium containing ,Naphthol-ASB l-phosphate as a substrate and hexazotized p-rosaniline as a coupling agent [ 111. After incubation the coverslips with attached cells were washed in saline, post-fixed in 4% formolrnacrodex for 2 h, washed in tap water. and mounted with glycerin-jelly for observation with the light microscope. For the electronmicroscopical localization of ‘acid phosphatase the attached cells. Cere fixed and incubated as described by Barka & Anderson [12] using fi-glycerophosphate and lead nitrate. Fibroblasts which were heated during 5 min at 90°C were used as a control. Other controls were assayed by incubating

cells in a medium lacking /3-glycerophosphate. Dehydration, embedding and sectioning were performed as described above (Ultrastructure). The cytochemitally stained sections were studied without additiona! staining at 60 kV in a Philips EM 300.

RESULTS Flo,z~cytofluorimetry In cultures of human fibroblasts, autofluorescent cells were observed by fluorescence microscopy (fig. 1a). FlGv cytofluorimetric examination of the cell fluorescence (Yaxis) of non-confluent passage 15 cells revealed, that approx. 30 5%-of the cells was non-fluorescent (NF cells) (fig. 2a, windo-w

412

Jongkind et al.

Fig. I. Sorted autofluorescent fibroblasts (passage 26). (a) Fluorescence of living fibroblasts (unstained; Leitz orthoplan with Ploemopak illumination; excitation:

450-490 nm); (b) distribution of acid phosphatase activity in formol-fixed fibroblasts. x 1600.

A), while 1.7% (SD, 0.45; IZ, 7) of the cells cells increase with increasing passage numfell within the category bright autofluober. The NF cells constitute still approx. rescent cells (AF cells) (fig. 2a, window B). 30% in passages 19 and 27. In between these categories, cells with The distribution of the forward light intermediate fluorescence could be ob- scatter of the cells shows that a large size served. Without perturbation of the instruvariability exists in both the NF and AF ment- and window-setting, the percentage cell samples (fig. 2a, b). The modal channel of AF cells was determined immediately number of the AF cells (channel no. 184) afterwards in both a suspension of passage was much higher than that of the NF cells 19 and passage 27 cells. The percentage of (channel no. 108) which is indicative for a AF cells in these passages increased from large difference in modal size between the 3.7% (SD, 0.34; n, 10) till 6.2% (SD, 0.91; two classes of cells. n, 10). In this experiment, these three percentages are significantly different at the Doubling time and labelling index 1% level (Student t). These flowcytometric From passage 26 fibroblasts the AF cells data indicate that the percentage of AF (fig. 26, window B) were separated from E.xp Cd

Res 138 (1982)

F~GWsorting of agedfibroblasts

Intensity

of forward

scatter/cell

(arbitrary

413

units)

Fig. 2. Flow-fluorocytometric distribution of autofluorescent fibroblasts from (n) passage 15; (b) passage 27

cultures. Window A, NF cells: window 3, Bright AF cells.

the NF cells (fig. 2 b, window L4) by flow sorting. Trypsinization and sorting did not preferentially influence the viability of one group of fibroblasts. In both AF as well as NF cells, the plasma membrane of 99% of the sorted cells was intact as evidenced by accumulation of fluoresceine during incubation of the cells with fluoresceine diacetate by the method of Rotman & Papermaster [28]. Moreover, after culturing of sorted cells both classes of cells adhered to the culture vessel without observable cell loss. Both populations were collected in tubes and distributed in 16 mm wells of a 24 well titerplate (5 000 cells/well). After varying time intervals the increase in cell number of NF cells was compared with that of AF cells (fig. 3). The doubling time of the AF cells studied from 3 to 7 days in culture after sorting was estimated to be 3 days compared with 1.3 days for NF cells studied for the same period. The percentage of labelled cells after 24 h incubation in a [3H]thymidine-containing medium was 19 (SD, 5.57; n, 3) for the AF sample and 73 (SD? 14.19; n, 3) for the NF cells. These percentages were significantly different.

Cellular protein and enzyme activiQ In order to compare the cellular protein of AF and NF cells, total protein was determined on a cell sample containing a counted number of cells. In two separate experiments the sorted NF cells had a mean pro-

number

;f C~IIS

,oo ‘lo i 90-1 8. -I 70

r I / non fb3reSCFrlt

Cells

i

60 -1 50 i 1 LO3.

1

20-

,o4 ’ b

auto fluorescent ;

;

;

days

after

1

;

;

b

subcthxe

3. Growth curve of sorted O-O, NF cei!s (passage 26).

Fig.

;

AF; X-X,

414 Jongkind et al.

Uh :rast:ruczture of a sorted AF cell (Passage 26). x 14200.

Fig.4.

Fig. 5. Ult rast ru(:ture of a

sorted NF cell (Passage 26). x 14200. E.xp Cell Res 138 (1982)

Flow sorting qf agedfibroblasts

415

containing structures in cytochemically stained cells (fig. lb). Electronmicroscopical observation of Increase NF AF sorted fibroblasts from passage 26 (50 AF Enzyme* ceW cellsC (c;rc) cells and 100 NF cells) indicated, that 90% LDH 50 100’ 74 200 48 of the AF cells did contain a large number G6PD 1s 200 19 300 6 of membrane-bound electron-dense granAP 2 410 150 950 P-Gal 560 2 150 280 ules (fii. 4), whereas 80% of the NF cells 63 100 P-Hex 15 100 320 possessed only a few or none of these cytou From a passage 26 culture. Cells were subcultivated plasmic inclusions (fig. 5). With electronfor 3 days before cell sorting. microscopical cytochemistry it could be b For abbreviations, see table 1. c Gated within deflection window 4 (fig. Zb). shown that most of these granules in the d Gated within deflection window B (fig. 26). e Activities are expressed as pmoles substrate AF cells contained acid phosphatase acconverted. pg protein’. h-r. Each value is the mean tivity (fig. 6). of two independent determinations on the same cell

Table 2. Enzyme activit?; in sorted Jibroblasts”

sample.

DISCUSSION tein content of 0.27 rig/cell (range 0.2?0.28), whereas the mean cellular protein for the AF cells was 0.51 nglcell (range 0.46 0.56). The specific activity of the lysosomal enzymes in AF cells increased more than 100% (1%320%) over the activity of NF cells. The specific activity of LDH in AF cells was ca 50% higher than in NF cells, whereas there was no difference in G6PD activity (table 2). Since the protein content of the AF cells is ca twice that of NF cells, the lysosomal enzyme activity of AF ceils is more than 4x that of NF cells on a cell basis. In a fibroblast culture which was kept confluent for 5 days, the specific activities of lysosomal enzymes, i.e. acid phosphatase P-galactosidase and p-hexosaminidase, for AF cells were again 3-4-fold the activity of the NF cells. Morphology and cytochemistry In sorted AF cells (passage 26), the distribution of autofluorescent granules (fig. 1a) corresponds with that of acid phosphatase

The similar size and distribution of acid phosphatase containing granules in the AF cells and of autofluorescent granules in the AF cells (fig. la? b) points to a lysosomelike nature of the autofluorescent granules” The direct observation of acid phosphatase activity in autofluorescent granules of WI38 fibroblasts [l] makes it also very likely thar

Fig. 6. Ultrastructural localization of acid phosphatase activity in a sorted AF cell (passage 26). x 1330.

416

Jongkind et al.

the autofluorescent structures observed are of lysosomal origin. At the ultrastructural level, the. presence of large membranebound osmiophilic structures correlates well with the autofluorescent state of the cell. These structures, which are also acid phosphatase positive (fig. 6), resemble the acid phosphatase-containing secondary lysosomes in aging glia cells [16] and residual bodies observed in late-passage tibroblasts [17]. As for the nature of the autofluorescent material a number of possibilities coexist. Autofluorescence can be due to an increase in lysosomal lipofuscin which could accumulate as a result of supposed free radical lipid damage [18]. Other authors [ 1, 191 point out that the autofluorescence of fibroblasts could be caused by flavins. It is even possible that an increased uptake of fluorescent substances normally present in the culture medium is the cause of the autofluorescence in the (non-cycling) AF ceils. This suggestion is supported by the observations that in wounded confluent fibroblast cultures the uptake of the fluorescent cyanine dye 3,3’-diheptyloxycarbocyanine seems to be higher in the confluent (noncycling) cells than in the proliferating cells [29]. However, we never observed an increase in cell autofluorescence in cultures of sorted NF cells which were kept confluent in our culture medium for a period of 5 days. Thus, in general, autofluorescence is not due to an increased accumulation of substances from our medium in confluent (non-cycling) cells. No definite conclusion about the nature of the autofluorescent material in fibroblasts can yet be drawn. In our study the amount of AF cells was correlated with the passage number, the older cultures showing a higher percentage of autofluorescent cells. This correlation E.rp CellRes 138 11982)

was already observed by Deamer & Gonzales [l] who also reported a decline in proliferation rate of autofluorescent cells using the method of r3H]thymidine incorporation. This coupling between proliferation and autofluorescence could be confirmed in our study using sorted autofluorescent cells. Cultured glial cells, which were kept contact-inhibited for several months, showed a continuous increase in acid phosphatase containing secondary lysosomes [ 161. These findings could suggest that the accumulation of autofluorescent granules in our study is a result of insufficient dilution due to a limited potential for division. Since the lysosomal enzyme activity of cultured fibroblasts is dependent on the culture conditions at the moment of subculture and the period after subculture [20], the observed differences in lysosomal enzyme activity (table 2) could be the result of the differences in proliferation rate between AF and NF cells. Sorted cells from a 7-day confluent culture, however, gave identical results, so the vast distribution of autofluorescent granules is correlated with a high lysosomal enzyme activity. This correlation points again to a lysosomal identity of the granules. In this study using flow sorted AF cells, a correlation could be found between autofluorescence and parameters for in vitro ‘aging’ like proliferative capacity [21], ultrastructural changes [16, 17, 221 and the activity of lysosomal enzymes [23-251. Furthermore, the sorted AF cells showed an increase in cell protein which is also indicative for cellular aging [26]. Since the labelling index of a population of cells after 24 h period of [“Hlthymidine incorporation has been shown to be a measure of remaining proliferative capacity in three different human Iibroblast strains [4, 271, we employed the same experimental conditions to

Flow sorting of agedfibroblmrs extrapolate the labelling index to the expected remaining lifespan in the sorted AF and NF fibroblasts. This extrapolation may indicate that the population of sorted AF cells from passage 26 have nearly completed their proliferative lifespan. Determinations of cell protein, /3-galactosidase activity and population doubling time on sorted cells of another control tibroblast strain (JJ) gave increases in AF cells, as compared with NF cells, which were identical with the results obtained with our control fibroblast strain (Trinet) described in this study. Whether these sorted AF cells at the end of their proliferative lifetime are senescent or terminally differentiated depends on the interpretation of the increase in lysosomal enzyme activity which accompanies this stage, Some authors [30] consider this increase an “adaptation of the cell to accumulated senescent damage or errors” whereas others [31] state that these lysosomal enzyme activity changes “reflect differences of differentiation”. In this respect our study on flow-sorted AF cells does not favour one theory over the other. Altogether the results indicate that it is possible to isolate autofluorescent in vitro ‘aged’ cells from a relatively young fibroblast culture by flow sorting. We thank Professor Dr I. Molenaar and Mr R. Kalicharan for their advice and assistance in the ultrastructural study. Mr T. van OS is acknowledged for his excellent photographical work, and we are grateful to Mrs Rita Boucke for her excellent secretarial help.

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417

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