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Doubling potential, calendar time, and donor age of human diploid cells in culture
Printed in Sweden Copyright 0 1974 by Academic Press, Inc. AN rights of reproduction in any form reserved
Experimental Cell Research84 (1974) 363-366
DOUBLING
POTENTIAL, CALENDAR TIME, AND DONOR AGE OF HUMAN DIPLOID CELLS IN CULTURE R. T. DELL’ORCO,
Biomedical
Division,
J. G. MERTENS,l and P. F. KRUSE, Jrt2
The Samuel Roberts and Noble Foundation,
Inc., Ardmore,
Okla. 73401, USA
SUMMARY Human diploid fibroblasts from an embryonic and an adult donor were maintained in an essentially nonmitotic state for extended periods of time by reducing the serum concentration of the growth medium from 10 to 0.5 %. These cells could be returned to the rapidly proliferating state by subcultivation with medium containing 10 % serum. Cells treated in such a manner took a proportionately longer calendar time to reach phase III than did controls that had been continuously cultured on growth medium. The division potential of cells from the adult donor was unaffected by the arrested state; however, that of cells from the embryonic donor was extended beyond that of growth controls. The extension of division potential in cells from the embryonic donor never exceeded the maximum passage level reported for these cells. During 21 days of cultivation with medium containing 0.5 % serum, cell losses ranged from 15 to 35 % and the protein content of the remaining cells decreased by 15 to 20 %. It was concluded that division potential and not total calendar time was the primary determinant of the in vitro lifespan of these human diploid cells and that arresting cells from younger donors increased their ability to attain the maximum limit of their lifespan.
Hayflick [l] originally proposed that the limited in vitro lifespan exhibited by human diploid fibroblasts (HDF) in culture was a function of the number of population doublings undergone and not of the length of time that the cells were maintained in vitro. This has subsequently been directly established by Dell’Orco et al. [2, 31 through the use of arrested HDF populations and has been supported by the observations of Brunk et al. [4] in a glia cell system, Goldstein & Singal [5] with adult HDF, and Daniel & Young [6] in vivo with a mouse mammary epithelium model. In the present study, cell strains from an embryonic and an adult donor were main1 Present address: 506 City View Drive, Oklahoma City, Okla. 73149, USA. t Deceased, January 31, 1973.
tained in an essentially nonmitotic state by reducing the serum concentration of the growth medium from 10 to 0.5 %. The cell strains were compared for their ability to be maintained in an arrested state, to be recovered to a proliferative state, and subsequently to complete their in vitro lifespan. The data revealed that there was a difference in the ability of the cell strains to be maintained with medium containing 0.5 % serum. Although calendar time was extended in each case, the capacity of these cells to complete their lifespan following releasefrom the arrested state appeared to be related to donor age. MATERIALS AND METHODS The cells used in the experiments to be described were normal HDF stains obtained from an embryonic and Exptl Cell Res 84 (1974)
364 Dell’Orco, Mertens and Kruse Table 1. WI-38 and WL W cells incubated with medium 7a containing 0.5 % seruma Cell count
Total protein
pg protein lo6 cells
100.0
100.0
100.0
1: 21 0
89.Ok4.4 68.1 22.5 65523.9 100.0
68.4?3.6 53.Ok4.9 53.3k5.1 100.0
76.426.3 77.1 k6.9 80.6k5.3 100.0
1: 21
95.626.0 92.2 k2.9 86.5 k 5.8
85.Ok3.0 79.1 k2.5 7l.lk4.0
Cell strain
JW
WI-38
0
WLW
84.4 i& 3.7 92.4 3.4 84.1 k 5.2
a Taking 0 time as 100 %, values are percentages at 0 time (mean i S.E.M.). Data for WI-38 were taken from 5 experiments placed on 0.5 % serum medium at passage numbers ranging from 28 to 37. Data for WLW were from 8 experiments at passage numbers ranging from 6 to 15.
an adult donor. Cell strain WI-38, derived from embryonic lung as described by Hayflick [l], under our conditions enters nhase III after 45 i-5 nonulation doublings in vitro. Cells from adult for&kin, obtained from an individual at age 35 and designated WLW. enter phase III after 15to 20 popula&n doublings: Entry into phase III was arbitrarily designated as that time at which the cells would not undergo one population doubling in one week following transfer at a 1 : 2 split ratio. Population doublings were calculated from the number of transfer manipulations. Cells were routinely subcultured at confluence at a 1 :4 split ratio in-McCoy’s 7a Medium [7] supplemented with 10 % fetal bovine serum but without antibiotics. The medium was replaced every 48 h until confluency was reached. Routine procedures [8] were used to determine that the cells were free of mycoplasma contamination. For the induction of the arrested state, cultures were grown to confluency and the growth medium was replaced with McCoy’s 7a Medium supplemented with 0.5 % fetal bovine serum and 25 yg/ml of both penicillin and streptomycin. This medium was replaced every 3 or 4 days for the duration of the experimental period. Cells maintained in this manner have been shown to be essentially nomnitotic by the determination of mitotic indices [2]. Recovery to the proliferative state after 3 to 14 weeks of exposure to the low serum medium was accomplished by transferring the cells at a 1 : 2 split ratio into medium containing 10 % serum. Cell numbers were determined either by counting nuclei according to the citric acid-crystal violet method of Paul [9] or by counting the cells in situ after staining with Giemsa (Fisher SO-G-28). Cell protein was determined by a modified Lowry procedure [lo].
RESULTS Table 1 contains results for cell counts and protein determinations from a seriesof experiExp tl Cell Res 84 (1974)
ments with WI-38 and WLW cells that were exposed to a low serum medium for 21 days. The passage numbers at which these cells were placed on 0.5 % serum medium ranged from 28 to 37 for the WI-38 and 6 to 15 for the WLW. Passagelevel had no effect on the results from either cell strain. There was a 30 to 40 % loss of cells in the WI-38 cultures and a 10 to 20% loss with the WLW cells during the 21-day experimental period. The cell loss was accompanied by a loss of total protein which amounted to approx. 50 % for the WI-38 cells and 30 % for the WLW cells. In both cases, one-half of this loss occurred during the first 7 days of the experimental period. When protein losses were calculated on a per cell basis, it was found that at the end of the experimental time WI-38 cells contained an average of 20 % less protein than they did at 0 time and WLW cells contained 15% less. Table 2 shows results from a series of experiments in which cells maintained on low serum medium for varying lengths of time exhibited an extention of in vitro calendar time. The cells for the individual experiments were derived from separate sets of controls that were revived from stocks frozen in liquid nitrogen. In every experiment, cells
Senescence in vitro
365
Table 2. Extension of in vitro calendar time of WI-38 and WLW cells after incubation with low serum medium Cell strain
Expt
WI-38
1 2
WLW
1 2 3
P at phase III’
Days in cultured
Pa
A-timeb (days)
Arrested
Control
Arrested
Control
23 27 25 25 27 8 15 8 13 11 11 11
21 21 39 66 102 21 21 21 21 21 59 99
42 50 50 49 51 16 17 18 18 20 18 16
41
96 130 116 133 175 64 36 80 56 79 109 133
75
41
17 19 18
22 49 45 37 9 50 31 42 42 42
a Passage number of cells when exposed to medium containing 0.5 % serum. b Days of incubation with medium containing 0.5 % serum. ’ Passage number at entrance into phase III. d Days in culture from indicated passage numbera until entrance into phase III.
exposed to low serum medium could be recovered to the proliferative state by subcultivation with growth medium containing 10 % serum. In all cases,cells incubated in the presence of 0.5 % serum were maintained in culture for a proportionately longer time than control cells that were continuously subcultured under the normal regime in growth medium. This result was independent of cell strain, passagenumber, and length of exposure to medium containing 0.5 % serum. For example, in two experiments in which WI-38 and WLW cells were maintained under the experimental conditions for 102 and 99 days, respectively, the in vitro lifespan of the WI-38 cells was increased by 130 days and that of the WLW cells by 91 days. These results were similar to those reported for the HDF strain CF-1 obtained from newborn foreskin material [2]. Although no difference between cell strains was detected with respect to the extention of calendar time, a notable difference was apparent in the ultimate passagelevel achieved
by experimental cells as compared with growth controls. Arrested WLW cells from the adult donor attained passage numbers that were essentially equivalent to their controls; on the other hand, WI-38 cells from the embryonic donor exceededthe passagenumber of comparable controls by at least 8 in all but one case. When the differences in passagelevel achievement for these two cell strains were compared with those previously reported for HDF strain CF-1 from a new Table 3. Change in achieved population doublings Cell strain’
Donor age
APb
SignificanceC
Embryonic Newborn Adult (35 yr)
7.4 3.1 -0.4
PcO.02 PCO.01
a Numbers in parentheses are numbers of observations. b Mean change in achieved population doublings (control minus experimental). c P values obtained from Student r-test. ’ Values obtained from data reported by Dell’Orco et al., Exptl cell res 77 (1973) 356. Exptl Cell Res 84 (1974)
366 Dell’Orco, Mertens and Kruse born donor [2], a gradient of attained passage number according to donor age was established. Table 3 shows that when compared to growth controls, arrested cells from younger donors have the ability to reach a significantly higher number of population doublings than do those from older donors.
capability of undergoing more population doublings when compared to growth controls than did the WLW strain which lost fewer cells. No definitive explanation for this observation can be given now. The reason(s) for the observed gradient of decreased achieved passage level with increaseddonor age is open to speculation. One explanation couId be the expression of intrinDISCUSSION sic controls which are effective only when The data reported in this investigation con- division has been inhibited. Regardlessof the cerning the extention of in vitro calendar mechanism, it has been shown that by placing time confirm earlier studies relating to this cells in an arrested, essentially nonmitotic question [2-51. These reports offer convincing state the division potential of populations evidence that the primary determinant of in derived from younger donors is effected to a vitro lifespan in HDF is related to the cumu- greater extent than it is in populations derived lative number of population doublings (mitotic from older donors. It should be noted, howevents) that have taken place and not the ever, that in no case did the passage level length of time that these cells have been main- attained by arrested populations exceed that tained in culture. These in vitro results [2-51 which has been reported as the maximum for can be directly compared to data obtained in thesecell strains, i.e., WI-38, 50& 10 [l]; CF-1, vivo in the mouse mammary epithelium 35_f 5 [2]; WLW, 15-20 [I 11. Therefore, by model of Daniel & Young [6] and, within the arresting the populations we may be improvlimits of experimentation, are independent of ing their ability to reach the limit of their in tissue of origin, donor age, passagelevel, and vitro division potential but, thus far, this procedure has not allowed cells to exceedthat culture conditions. A comparable loss of protein on a per cell limit. basis was observed during the arrested state REFERENCES for both WI-38 and WLW cells, 20 and 15 %, respectively. While the loss of protein may 1. Hayflick, L, Exptl cell res 37 (1965) 614. 2. Dell’Orco, R T, Mertens, J G & Kruse, P F Jr, reflect a nutritional deficiency brought about Exotl cell res 77 (1973) 356. by the experimental conditions, it could also 3. ---Tissue culture; methods and applications (ed P F Kruse, Jr & M K Patterson, Jr) p. 231. be explained as a normal cellular response to Academic Press, New York (1973). the temporary inhibition of the protein 4. Brunk, U, Ericsson, J L E, Ponten, J & Westermark, B, Exptl cell res 79 (1973) 1. synthesis necessaryfor cellular division. 5. Goldstein, S & Singal, D P, In vitro 8 (1973) 430. Through 21 days of exposure to medium 6. Daniel, C W & Young, L J T, Exptl cell res 65 (1971) 27. containing 0.5 % serum, WLW cultures lost 7. Kruse, P F Jr, Whittle, W & Miedema E, J cell approx. 15 % of their cell population. This bio142 (1969) 113. 8. Hayflick, L, Tex rep biol med 23 (1965) 285. loss was similar to that found with HDF 9. Paul, J, Cell & tissue culture, 3rd edn, p. 350. strain CF-1 [2] and has been tentatively attribWilliams & Wilkins, Baltimore, Md (1965). Lowry, 0 H, Rosebrough, N J, Farr, A L & uted to culture manipulation, i.e., medium 10. Randall, R J, J biol them 193 (1951) 265. replacement and sample preparation. Al- 11. Dell’Orco, R. T. Unpublished observations. though WI-38 cells had greater losses (35 %) during the arrested state, these cells had the Received September 27, 1973 Exptl Cell Res 84 (1974)
Experimental Cell Research84 (1974) 363-366
DOUBLING
POTENTIAL, CALENDAR TIME, AND DONOR AGE OF HUMAN DIPLOID CELLS IN CULTURE R. T. DELL’ORCO,
Biomedical
Division,
J. G. MERTENS,l and P. F. KRUSE, Jrt2
The Samuel Roberts and Noble Foundation,
Inc., Ardmore,
Okla. 73401, USA
SUMMARY Human diploid fibroblasts from an embryonic and an adult donor were maintained in an essentially nonmitotic state for extended periods of time by reducing the serum concentration of the growth medium from 10 to 0.5 %. These cells could be returned to the rapidly proliferating state by subcultivation with medium containing 10 % serum. Cells treated in such a manner took a proportionately longer calendar time to reach phase III than did controls that had been continuously cultured on growth medium. The division potential of cells from the adult donor was unaffected by the arrested state; however, that of cells from the embryonic donor was extended beyond that of growth controls. The extension of division potential in cells from the embryonic donor never exceeded the maximum passage level reported for these cells. During 21 days of cultivation with medium containing 0.5 % serum, cell losses ranged from 15 to 35 % and the protein content of the remaining cells decreased by 15 to 20 %. It was concluded that division potential and not total calendar time was the primary determinant of the in vitro lifespan of these human diploid cells and that arresting cells from younger donors increased their ability to attain the maximum limit of their lifespan.
Hayflick [l] originally proposed that the limited in vitro lifespan exhibited by human diploid fibroblasts (HDF) in culture was a function of the number of population doublings undergone and not of the length of time that the cells were maintained in vitro. This has subsequently been directly established by Dell’Orco et al. [2, 31 through the use of arrested HDF populations and has been supported by the observations of Brunk et al. [4] in a glia cell system, Goldstein & Singal [5] with adult HDF, and Daniel & Young [6] in vivo with a mouse mammary epithelium model. In the present study, cell strains from an embryonic and an adult donor were main1 Present address: 506 City View Drive, Oklahoma City, Okla. 73149, USA. t Deceased, January 31, 1973.
tained in an essentially nonmitotic state by reducing the serum concentration of the growth medium from 10 to 0.5 %. The cell strains were compared for their ability to be maintained in an arrested state, to be recovered to a proliferative state, and subsequently to complete their in vitro lifespan. The data revealed that there was a difference in the ability of the cell strains to be maintained with medium containing 0.5 % serum. Although calendar time was extended in each case, the capacity of these cells to complete their lifespan following releasefrom the arrested state appeared to be related to donor age. MATERIALS AND METHODS The cells used in the experiments to be described were normal HDF stains obtained from an embryonic and Exptl Cell Res 84 (1974)
364 Dell’Orco, Mertens and Kruse Table 1. WI-38 and WL W cells incubated with medium 7a containing 0.5 % seruma Cell count
Total protein
pg protein lo6 cells
100.0
100.0
100.0
1: 21 0
89.Ok4.4 68.1 22.5 65523.9 100.0
68.4?3.6 53.Ok4.9 53.3k5.1 100.0
76.426.3 77.1 k6.9 80.6k5.3 100.0
1: 21
95.626.0 92.2 k2.9 86.5 k 5.8
85.Ok3.0 79.1 k2.5 7l.lk4.0
Cell strain
JW
WI-38
0
WLW
84.4 i& 3.7 92.4 3.4 84.1 k 5.2
a Taking 0 time as 100 %, values are percentages at 0 time (mean i S.E.M.). Data for WI-38 were taken from 5 experiments placed on 0.5 % serum medium at passage numbers ranging from 28 to 37. Data for WLW were from 8 experiments at passage numbers ranging from 6 to 15.
an adult donor. Cell strain WI-38, derived from embryonic lung as described by Hayflick [l], under our conditions enters nhase III after 45 i-5 nonulation doublings in vitro. Cells from adult for&kin, obtained from an individual at age 35 and designated WLW. enter phase III after 15to 20 popula&n doublings: Entry into phase III was arbitrarily designated as that time at which the cells would not undergo one population doubling in one week following transfer at a 1 : 2 split ratio. Population doublings were calculated from the number of transfer manipulations. Cells were routinely subcultured at confluence at a 1 :4 split ratio in-McCoy’s 7a Medium [7] supplemented with 10 % fetal bovine serum but without antibiotics. The medium was replaced every 48 h until confluency was reached. Routine procedures [8] were used to determine that the cells were free of mycoplasma contamination. For the induction of the arrested state, cultures were grown to confluency and the growth medium was replaced with McCoy’s 7a Medium supplemented with 0.5 % fetal bovine serum and 25 yg/ml of both penicillin and streptomycin. This medium was replaced every 3 or 4 days for the duration of the experimental period. Cells maintained in this manner have been shown to be essentially nomnitotic by the determination of mitotic indices [2]. Recovery to the proliferative state after 3 to 14 weeks of exposure to the low serum medium was accomplished by transferring the cells at a 1 : 2 split ratio into medium containing 10 % serum. Cell numbers were determined either by counting nuclei according to the citric acid-crystal violet method of Paul [9] or by counting the cells in situ after staining with Giemsa (Fisher SO-G-28). Cell protein was determined by a modified Lowry procedure [lo].
RESULTS Table 1 contains results for cell counts and protein determinations from a seriesof experiExp tl Cell Res 84 (1974)
ments with WI-38 and WLW cells that were exposed to a low serum medium for 21 days. The passage numbers at which these cells were placed on 0.5 % serum medium ranged from 28 to 37 for the WI-38 and 6 to 15 for the WLW. Passagelevel had no effect on the results from either cell strain. There was a 30 to 40 % loss of cells in the WI-38 cultures and a 10 to 20% loss with the WLW cells during the 21-day experimental period. The cell loss was accompanied by a loss of total protein which amounted to approx. 50 % for the WI-38 cells and 30 % for the WLW cells. In both cases, one-half of this loss occurred during the first 7 days of the experimental period. When protein losses were calculated on a per cell basis, it was found that at the end of the experimental time WI-38 cells contained an average of 20 % less protein than they did at 0 time and WLW cells contained 15% less. Table 2 shows results from a series of experiments in which cells maintained on low serum medium for varying lengths of time exhibited an extention of in vitro calendar time. The cells for the individual experiments were derived from separate sets of controls that were revived from stocks frozen in liquid nitrogen. In every experiment, cells
Senescence in vitro
365
Table 2. Extension of in vitro calendar time of WI-38 and WLW cells after incubation with low serum medium Cell strain
Expt
WI-38
1 2
WLW
1 2 3
P at phase III’
Days in cultured
Pa
A-timeb (days)
Arrested
Control
Arrested
Control
23 27 25 25 27 8 15 8 13 11 11 11
21 21 39 66 102 21 21 21 21 21 59 99
42 50 50 49 51 16 17 18 18 20 18 16
41
96 130 116 133 175 64 36 80 56 79 109 133
75
41
17 19 18
22 49 45 37 9 50 31 42 42 42
a Passage number of cells when exposed to medium containing 0.5 % serum. b Days of incubation with medium containing 0.5 % serum. ’ Passage number at entrance into phase III. d Days in culture from indicated passage numbera until entrance into phase III.
exposed to low serum medium could be recovered to the proliferative state by subcultivation with growth medium containing 10 % serum. In all cases,cells incubated in the presence of 0.5 % serum were maintained in culture for a proportionately longer time than control cells that were continuously subcultured under the normal regime in growth medium. This result was independent of cell strain, passagenumber, and length of exposure to medium containing 0.5 % serum. For example, in two experiments in which WI-38 and WLW cells were maintained under the experimental conditions for 102 and 99 days, respectively, the in vitro lifespan of the WI-38 cells was increased by 130 days and that of the WLW cells by 91 days. These results were similar to those reported for the HDF strain CF-1 obtained from newborn foreskin material [2]. Although no difference between cell strains was detected with respect to the extention of calendar time, a notable difference was apparent in the ultimate passagelevel achieved
by experimental cells as compared with growth controls. Arrested WLW cells from the adult donor attained passage numbers that were essentially equivalent to their controls; on the other hand, WI-38 cells from the embryonic donor exceededthe passagenumber of comparable controls by at least 8 in all but one case. When the differences in passagelevel achievement for these two cell strains were compared with those previously reported for HDF strain CF-1 from a new Table 3. Change in achieved population doublings Cell strain’
Donor age
APb
SignificanceC
Embryonic Newborn Adult (35 yr)
7.4 3.1 -0.4
PcO.02 PCO.01
a Numbers in parentheses are numbers of observations. b Mean change in achieved population doublings (control minus experimental). c P values obtained from Student r-test. ’ Values obtained from data reported by Dell’Orco et al., Exptl cell res 77 (1973) 356. Exptl Cell Res 84 (1974)
366 Dell’Orco, Mertens and Kruse born donor [2], a gradient of attained passage number according to donor age was established. Table 3 shows that when compared to growth controls, arrested cells from younger donors have the ability to reach a significantly higher number of population doublings than do those from older donors.
capability of undergoing more population doublings when compared to growth controls than did the WLW strain which lost fewer cells. No definitive explanation for this observation can be given now. The reason(s) for the observed gradient of decreased achieved passage level with increaseddonor age is open to speculation. One explanation couId be the expression of intrinDISCUSSION sic controls which are effective only when The data reported in this investigation con- division has been inhibited. Regardlessof the cerning the extention of in vitro calendar mechanism, it has been shown that by placing time confirm earlier studies relating to this cells in an arrested, essentially nonmitotic question [2-51. These reports offer convincing state the division potential of populations evidence that the primary determinant of in derived from younger donors is effected to a vitro lifespan in HDF is related to the cumu- greater extent than it is in populations derived lative number of population doublings (mitotic from older donors. It should be noted, howevents) that have taken place and not the ever, that in no case did the passage level length of time that these cells have been main- attained by arrested populations exceed that tained in culture. These in vitro results [2-51 which has been reported as the maximum for can be directly compared to data obtained in thesecell strains, i.e., WI-38, 50& 10 [l]; CF-1, vivo in the mouse mammary epithelium 35_f 5 [2]; WLW, 15-20 [I 11. Therefore, by model of Daniel & Young [6] and, within the arresting the populations we may be improvlimits of experimentation, are independent of ing their ability to reach the limit of their in tissue of origin, donor age, passagelevel, and vitro division potential but, thus far, this procedure has not allowed cells to exceedthat culture conditions. A comparable loss of protein on a per cell limit. basis was observed during the arrested state REFERENCES for both WI-38 and WLW cells, 20 and 15 %, respectively. While the loss of protein may 1. Hayflick, L, Exptl cell res 37 (1965) 614. 2. Dell’Orco, R T, Mertens, J G & Kruse, P F Jr, reflect a nutritional deficiency brought about Exotl cell res 77 (1973) 356. by the experimental conditions, it could also 3. ---Tissue culture; methods and applications (ed P F Kruse, Jr & M K Patterson, Jr) p. 231. be explained as a normal cellular response to Academic Press, New York (1973). the temporary inhibition of the protein 4. Brunk, U, Ericsson, J L E, Ponten, J & Westermark, B, Exptl cell res 79 (1973) 1. synthesis necessaryfor cellular division. 5. Goldstein, S & Singal, D P, In vitro 8 (1973) 430. Through 21 days of exposure to medium 6. Daniel, C W & Young, L J T, Exptl cell res 65 (1971) 27. containing 0.5 % serum, WLW cultures lost 7. Kruse, P F Jr, Whittle, W & Miedema E, J cell approx. 15 % of their cell population. This bio142 (1969) 113. 8. Hayflick, L, Tex rep biol med 23 (1965) 285. loss was similar to that found with HDF 9. Paul, J, Cell & tissue culture, 3rd edn, p. 350. strain CF-1 [2] and has been tentatively attribWilliams & Wilkins, Baltimore, Md (1965). Lowry, 0 H, Rosebrough, N J, Farr, A L & uted to culture manipulation, i.e., medium 10. Randall, R J, J biol them 193 (1951) 265. replacement and sample preparation. Al- 11. Dell’Orco, R. T. Unpublished observations. though WI-38 cells had greater losses (35 %) during the arrested state, these cells had the Received September 27, 1973 Exptl Cell Res 84 (1974)