Some Practical Aspects of Storing Mammalian Cells
in the Dry-Ice Chest*
H. E. SWIM,R. F. HAFF,| ANDR. F. PARKER
(Department of Microbiology, Western Reserve University, School 0} Medicine, Cleveland, Ohio)
Studies with mammalian cells cultured in vitro
frequently involve serial propagation of many
strains of cells. The maintenance of such cultures
when not in use constitutes a practical problem
of considerable magnitude. Of equal importance
in maintaining stock cultures is the ever-present
threat of loss as a result of microbial contamina
tion. A further difficulty which may be encoun
tered in the use of cultured cells for long-term
experiments stems from the fact that various
alterations may occur in vitro (7, 9, 21) or that
variants which are present in relatively small
numbers in a heterogeneous population may be
come predominant as a result of selection (12,
28, 29). A means of storing cells for extended
periods would aid greatly in circumventing these
difficulties. The results of earlier studies (26) dem
onstrated that a number of permanent strains
of cells can be stored at 4°C. for periods of
6-9 weeks. This method eliminates some of the
overhead in maintaining cultures when not in
use and affords considerable protection against
microbial contamination, but is of little value
in maintaining strains of constant physiological
characteristics for long-term experimentation. The
feasibility of storing cells for longer periods in
the frozen state was indicated in a report by
Scherer and Hoogasian (23) on the storage of
strains L and HeLa at —70°
C. and by the success
achieved by numerous investigators with normal
tissues (1, 15, 18, 24), tumor tissues (4), erythrocytes (3), and spermatozoa (22). This study was
undertaken to investigate some of the practical
aspects of storing cells in solutions containing
glycerol at -70°C. (dry-ice chest). The data
*Aided by grants from The National Foundation for In
fantile Paralysis; and from the Institute of Allergy and In
fectious Diseases, National Institutes of Health, United States
Public Health Service (No. E-1547).
t Former United States Public Health Service Research
Fellow of the National Microbiological Institute. Present ad
dress: E. I. duPont deNemours and Co., Inc., Stine Laboratory,
Newark, Delaware.
Received for publication February 7, 1958.
demonstrate that a variety of cultured cells can
be stored for periods of at least 3 years and
that the success of the procedure is a function
of the glycerol concentration, the method of freez
ing, and the strain of cells employed.
MATERIALS
AND METHODS
Strains of cells and their serial propagation.—Thefollowing
permanent strains of cells were propagated serially as described
previously (11, 26, 27): human fibroblasts, strain U12-705
and FS4-705 (27): rabbit fibroblasts, strains RM3-F17, RSlF17, and RT6-F17 (12): mouse fibroblasts, strain L-705 (7);
human epidermoid carcinoma cells, strain HeLa-715 (10) and
strain MB-13 (20), derived from mouse lymphosarcoma (2).
Strains CM, FS, and HS were isolated according to the pro
cedure of Swim and Parker (29) from chick embryo muscle,
foreskin of neonatal infants, and human embryo skin muscle,
respectively, and were used in experiments after one to five
passages in vitro. Since it has been demonstrated that the
nutritional characteristics of a particular strain of cells is
a function of the medium in which it propagated (11, 28, 29),
the practice has been adopted in this laboratory of referring
to a strain of cells according to its origin as well as the medium
employed for growth. For example, U12-705 indicates strain
number 12 of uterine fibroblasts which is propagated only
in medium 705 (Table 1). Additional strains of U12 such
as U12-79 and U12-80 are propagated only in the corresponding
media (Table 1), and each differs nutritionally from strain
U12-705 (28).
Methods of storage.—Cellswere suspended in the appropriate
medium (see Tables 1, 3, and 4) at a concentration of 2-6 X 10*
cells/ml as determined by the nuclear counting procedure
(13), and 0.5 ml. was added to each of a series of 13 X 100-mm.
Pyrex tubes. The tubes were gassed with a mixture containing
5 per cent CÛ2,50 per cent O2, and 45 per cent Nz to insure
the proper pH, and were then sealed immediately about
1 cm. from the end with the aid of an oxygen-gas torch.
The cell suspension was then frozen according to one of the
following procedures: (a) the tubes were placed immediately
into one of the metal compartments of the dry-ice chest;
(b) the tubes were stored at 4°C. for 18 hours and then
placed in the dry-ice chest; (c) the contents of the tube
were frozen rapidly by rotating the tubes in a mixture of
dry-ice and ethanol prior to storage in the dry-ice chest.
To test for survival of cells after storage.—-The
tubes were
removed from the dry-ice chest and immediately immersed
in the water bath at 37°C. and rotated rapidly until thawing
was complete. The tubes were opened immediately, and the
cells were washed twice in 10 ml. of the medium in which they
were propagated (Table 1) and were finally planted in T-flasks
(17). The flasks were incubated at 37°C. for 18-24 hours,
the fluid was removed, and the cells which adhered to the
711
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712
Cancer Research
glass after the flasks were rinsed once with fresh medium
were enumerated by the nuclear counting procedure used
in this laboratory (13). The per cent survival (see Tables 2-4)
after storage was calculated on the basis of the recovery of
cells from the same experiment which were not frozen but
otherwise treated in the same fashion.
Vol. 18, July, 1958
after storage at -70° C. for 2-3 years (Table 4),
a high proportion
to the glass were
number of viable
of growth during
of the cells which attached
obviously necrotic, and the
cells as determined
by rate
the 1st week at 37°C. was
very small. Similarly, a high proportion of cells
had been stored under adverse conditions
Viability of cells after storage at —70°
C.—Data which
(Tables 2-4) might attach to glass at 37°C., but
RESULTS
on the proportion of cells which survived storage
at -70°C. (Tables 2-4) as determined by the
number of cells which were attached to the glass
after incubation at 37°C. correlated well with
the general appearance of the cells. Since neither
TABLEl
COMPOSITION
OFMEDIA
MEDIUM
F15
F17
19
704
705
715
18
63
20
56
73
79
80
83
87
89
(V/V)EmbryoCOMPOSITIONPER CENT
extract5
solutions85
BEE10
CEE1.5
BEE10
CEE5
CEE5
BEE5
NHS20
NHS20
NHS20
the cultures frequently recovered slowly, and mi
croscopic examination revealed cytologie changes
in the cells which varied in intensity with the
strain of cells and conditions employed. In general,
it was found that, when the number of cells
per tube was reduced from 1 to 3 X IO6 to about
IO4 as a result of freezing and thawing or by
gradual deterioration
at —70°
C., a large pro
portion of the cultures failed to survive
tinued incubation at 37°C.
on con
V-61470
V-61478.5
The data on cell survival which are presented
in Tables 2-4 are average values for the number
70390703757037570390703807036070385
NHS20
HuS10
NHS20
NHS40
NHS10
CEEserum10 NHS2
898S1695
SI
DHS5
DHSSDKS5 S10395
S103A95
DHSSDKSSDKSDefined
S103B95
S103C95
S103D
The following abbreviations are employed: BEE = beef
embryo extract; CEE = chick embryo extract; NHS = normal
horse serum ; HuS = normal human serum; DHS = dialyzed
horse serum; V-614 = solution described by Fischer et al. (8);
703 = solution of Healy et al. (14); S16 and S18 = solutions de
scribed
by Haff
(13);
is similarofinEarle's
composition
to S16 and
S18 and
and Swim
contains
theS103
ingredients
saline
(6) plus amino acids and vitamins; S103A, S103B, S103C, and
S103D are similar to S103 but lack meso-inositol, choline,
pyridoxal, and pyridoxal and inositol, respectively.
of these criteria established the viability of cells,
the growth rate of a number of strains was deter
mined for the first 7-day interval after storage
at —70°
C. The average increase in cells during
this period was usually about one-half that ob
tained with the corresponding stock culture, which
indicates that at least 50 per cent of the cells
originally attached to the glass were viable. Al
though mitotic figures were frequently observed
within 24 hours, there was an increased lag period
over that observed when the corresponding stock
cultures were subcultured. In view of this delay
in multiplication
after storage, the proportion of
cells attached
to the glass which were viable
was actually considerably
greater than 50 per
cent. There were, however, a number of exceptions
in the correlation between viability and ability
of the cells to attach to the glass. Frequently
of experiments
indicated.
It should be noted,
however, that there was considerable variation
among experiments employing a single strain of
cells under a given set of circumstances.
The
following results obtained with FS4-705 are typical
for most of the strains examined. In five experi
ments employing the same medium and conducted
at intervals over a 6-month period, 96, 85, 72,
63, and 44 per cent of the cells survived after
1 month at —70°
C. On the other hand, variations
among duplicate tubes in the same experiment
fell within the limits of accuracy of the nuclear
counting procedure. Similarly, the degree of cor
respondence between duplicate experiments with
regard to the rate at which the cells deteriorated
with time (Table 4) was usually within the limits
of the errors in counting. Until recently little
attention was directed to the age of the culture
as a factor in cell storage at —70°
C. On the
basis of studies, as yet incomplete,
it appears
that the proportion of cells which survive is mark
edly reduced when cultures are employed which
have reached maximum growth and are beginning
to show cytologie symptoms characteristic of over
crowding. When young, actively growing cells
have been employed routinely, the inconsistencies
in the initial percentage survival between dupli
cate experiments have been reduced considerably.
It is emphasized, however, that additional factors
which have not yet been recognized may also
be involved.
Survival of cells as a function of the method
of freezing and thawing.—The data presented in
Table 2 indicate that the ability of cultured cells
to survive freezing was largely dependent on the
method employed. When sealed tubes containing
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TABLE 2
SURVIVAL
OFCELLSASAFUNCTIONOFMETHODOFFREEZING
PER
CELL
»TIMIN
STORAGE
«niniM
No.
EXPEBIUENT8
Dry-ice
chert
directly
CENT SURVIVAL,
4°C. prior
to dry-ice
cheat
1 MONTH*
Dry-ice
ethanol
CM-56FS4-70SHeLa-715HeLa-19L-705MB1S-705RMS-56RM3-F17RSI
-56RT6-56U12-705U12-79U12-80U12-82U12-8756-lOf18-1018-1018-1018-1020-1056-10F17-1056-1056-1018-1079-580-579-579-53434SB6t2S
* Average values for number of experiments indicated in column 3. Tests for survival
of cells were performed at intervals ranging from a few days to 1 month.
t Number following medium in column 2 indicates concentration of glycerol em
ployed (see Tables 3 and 4).
ÃŽ
FEC = too few extended cells to make counting feasible; only very small propor
tion of cultures survived on continued incubation at 37°C.
TABLE 3
SURVIVAL
OFCELLSFORi-s MONTHSASAFUNCTIONOFGLYCEROL
CONCENTRATION
CELL
BASAL
No.
STRAIN
MEDIUM*
EXPERIMENTS
GLYCEROLCONCENTRATION,
PER CENT(V/V)
0
5
10
20
CM-56
56
4
83
45
FS-F15
18
3
50
55
30
FS4-705
18or705f
5f
73
72
20
HeLa-715
18or715f
3
5
22
65
71
HeLa-19
18
4ÃŽ
27
87
82
HS-F15
18
2
22
37
L-705
18or705f
4ÃŽ
87
80
43
L-63
18or63t
3
53
71
6
L-704
18
2
FEC§
82
81
FEC§
MB13-705
18
40
FEC§
11
FEC
MB13-705
20
4
0
19
36
5
RM3-56
56
4J
FEC
46
64
61
RMS-F17
F17
2
50
76
72
RM3-73
79
3
20
45
41
RSI-56
56
2
31
55
55
RS1-F17
F17
3
FEC
30
49
59
RT6-56
56
4
FEC
28
52
54
U12-705
18 or 705f
3
57
60
20
U12-63
18or63t
3
71
76
13
U12-79
79 or 18f
4
0
45
16
FEC
U12-80
79 or 80f
3
0
51
6
0
U12-82
79 or 18t
3
35
10
0
LÃŽ12-87
79
3
0
39
8
0
U12-89
79 or 18f
5
09
FEC
0
* Basal medium is solution to which glycerol is added for storage of cells.
t Results were the same with each medium indicated.
ÕIn one or more of the experiments, the cells were refrigerated at 4°C. for 18 hours prior to storage
at —70°
C. In all other experiments indicated in Table 3, the cells were frozen by placing the sealed
tubes directly into the dry-ice chest.
§
FEC = so few extended cells that counting not feasible; few cultures survived on continued incu
bation at 37°C. Numerical values in columns 4-7 indicate per cent survival of cells.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1958 American Association for Cancer Research.
714
Vol. 18, July, 1958
Cancer Research
0.5 ml. of cell suspensions were placed directly
into the dry-ice chest, all the cell strains survived
satisfactorily, provided the proper medium was
used (Table 3). The initial survival of many
of the strains indicated in Table 2 was comparable
when the tubes were placed in the deep freeze
at —20°
C. This method was discontinued, how
ever, when it was observed that none of the strains
survived for more than 6 weeks under these con
ditions. Several strains do not withstand freezing
when they are refrigerated at 4°C. for 18 hours
prior to storage in the dry-ice chest. On the
other hand, refrigeration for 1-6 hours prior to
storage at —70°C. did not appear to alter the
results obtained with any of the stains studied.
The failure of strains CM-56, U12-79, U12-80,
and U12-82 to withstand freezing after 18 hours
at 4°C. may be related to the fact that these
strains deteriorate within 1 or 2 weeks when
intact cultures are stored at 4°C. under conditions
(26) which permit the remaining strains indicated
in Table 2 to survive for 6-10 weeks. In contrast
with the results obtained when freezing is per
formed by simply placing the sealed tubes in the
dry-ice chest, only strain HeLa and, to a lesser
extent, L and MB13 withstand the more rapid
freezing accomplished by immersing the tubes
in a mixture of dry-ice and ethanol prior to storage
at -70°C.
The rate at which the cells were thawed after
storage at —
70°C. appeared to be equally as
important as the method of freezing. The data
in Table 2 were obtained when the cells were
thawed in a water bath at 37°C. immediately
TABLE 4
SURVIVAL
OFCELLSASAFUNCTIONOFTIMEANDCOMPOSITION
OFSTORAGE
MEDIUM
CELL
STRAIN
CM-56
BASAL
MEDIUM*
56
No.
EXPERIMENTS
TIME
(MONTHS)
(MONTHS)
1- 2
12-14
20-24
GLYCEKOLCONCENTRATION,
PEB CENTV/V
5
10
20
29
6
FEC
«0
24
9
76
19
2
21
10
40
19
8
FS-FIS
18
1
56
30
12
21
5
24
FEC
11
1- 3
FS4-705
18
21
73
12
6
39
15
24
0
1- 8
HeLa-19
18
81
St
77
18-20
77
67
26-34
02
S
07
1- 3
L-705
18
43
87
80
11-14
15
32
61
20-24
6
50
8
30-36
FEC
FEC
46
MB13-705
1
24
FEC
16
14
20
8
0
RM3-F17
F17
59
1
66
73
12
5
15
41
20
FEC
14
6
RSI-56
50
1
66
55
31
12-14
10
30
26
24-26
5
20
25
RT6-56
1
54
54
28
15
6
23
IS
24-26
FEC
14
17
1- 3
U12-705
18
57
20
60
20-24
40
14
FEC
30-34
FEC
0
29
1- 3
U12-63
18
71
76
13
16-18
FEC
49
40
U12-79
2
79
17
1
fil
12
FEC
18
* Basal medium is solution to which glycerol is added for storage of cells (see Table 1).
t In one or more of the experiments the cells were refrigerated at 4°C. for 18 hours prior
to storage in dry-ice chest. In all other experiments indicated in Table 4, the cells were frozen
by placing the sealed tubes directly into the dry-ice chest.
Numerical values in columns 4-7 indicate per cent survival of cells.
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1958 American Association for Cancer Research.
SWIM et al.—Storage of Mammalian
upon removal from the dry-ice chest (see "Meth
ods") . On the other hand, relatively few viable cells
were recovered when they were thawed by allowing
the tubes to stand at room temperature
(air).
Survival of cells as a function of the composition
of the medium.—The results summarized in Table
3 point up the importance of supplementing vari
ous tissue culture media with glycerol for the
successful storage of cells at —70°
C. Media con
taining 30 per cent or more of glycerol were
unsuitable for all strains examined. Of particular
significance is the fact that the concentration
of glycerol permitting
maximal survival varied
with the strain of cells employed. The results
of a limited number of experiments indicate that
media containing either serum or dialyzed serum
are somewhat superior to those devoid of protein.
The importance of serum in the storage of MB 13
is indicated by the fact that more cells survived
in medium 20 supplemented with glycerol than was
the case in the corresponding solutions prepared
with medium 18. None of the rabbit fibroblasts
survived as well (particularly
on long-term stor
age) in solutions prepared with medium 18 as
in the media indicated in Table 3. This may be
related to the fact that rabbit fibroblasts cannot
be propagated
serially in media prepared with
solution 703 (14). Although solution 18 is an ex
cellent basal medium, with the exceptions already
noted, it does not offer any advantages over the
usual growth media. On the basis of these con
siderations, the method in current use consists
of adding the appropriate
concentration
of glyc
erol to the medium used to propagate the par
ticular strain of cells.
Survival of cells as a function of time and con
centration of glycerol in the storage medium.—The
results of a number of experiments in which the
cells were examined after periods of storage rang
ing from 1-36 months are summarized in Table
4. It is apparent that the cells gradually deterio
rated at —70°
C. and that the rate of decline
in the number of cells was a function both of
the strain of cells and the concentration of glycerol
in the storage medium. For example, media con
taining 5 or 10 per cent glycerol appeared to
be equally satisfactory for strains L-705, U12-705,
FS4-705, or RM3-F17, as judged by microscopic
appearance as well as number of cells recovered,
when tests were performed within 1-3 months
On prolonged storage at —70°C , however, the
number of cells decreased much more rapidly
in media containing
5 per cent glycerol than
in solutions containing 10 per cent glycerol. On
the other hand, strains FS-F15 and U12-63 were
equally resistant to storage at —70°
C. in media
Cells ai —70°C.
715
containing either 5 or 10 per cent glycerol. Further
variations between strains were indicated by the
fact that U12-79 was preserved satisfactorily only
in media containing 5 per cent glycerol, whereas
HeLa and the rabbit fibroblasts survived well
when 20 per cent glycerol was employed.
DISCUSSION
The foregoing data demonstrate that a variety
of mammalian
cells can be stored at —70°
C.
for periods of at least 3 years. The results with
strain L and HeLa confirm and extend the studies
of Scherer and Hoogasian (23) on the storage
of these strains for 6 months. It is clear that the
success of the procedure is dependent
on the
method of freezing, concentration
of glycerol in
the medium, and the strain of cells employed.
Although many investigators
have studied the
effects of rapid and slow freezing and thawing
and the effect of glycerol on the survival of normal
tissues (1, 15, 18, 24), cells (3, 22), and tumor
tissues (4) from various sources, it is difficult
to compare the results because of differences in
materials and technics employed (see 3, 4, 16,
and 17 for reviews which include biophysics of
freezing and thawing). The terms "rapid" and
"slow" as applied to freezing and thawing have
usually been used in a relative sense, and the
present studies are no exception. The data in
Table 2 indicate that rapid freezing is of limited
value when applied to cultured mammalian cells.
All the strains tested survive, however, if the
cells are permitted to freeze at a slower rate by
simply placing the sealed tubes in the dry-ice
chest. Storage of cells in media containing glycerol
at 4°C. for 18 hours prior to freezing is satisfactory
for some strains but results in the destruction
of others. It is emphasized that the procedure
for slow freezing of cells has been employed only
under the conditions described under "Methods,"
and its applicability
to storage of cells en masse
remains to be determined experimentally.
The data presented in Tables 3 and 4 demon
strate not only that glycerol is essential for satis
factory storage of cells but that the concentration
is a critical factor. Of particular significance is
the fact that under certain circumstances neither
the microscopic appearance of the cells nor their
ability to attach to the glass after 1-3 months
at —70°
C. may be adequate criteria in predicting
the outcome of continued storage. For example,
a number of strains which survived well for a
few months in media containing 5 per cent glyc
erol, as judged by the number of cells and their
appearance,
deteriorated
rapidly on more porlonged storage. This points up the importance
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716
Cancer Research
Vol. 18, July, 1958
of checking strains periodically and of employing and newly isolated lines (one to five passages
more than one concentration of glycerol when in vitro) derived from several species and with
dealing with a strain whose behavior at —70°
C. diverse histories have been stored successfully
C. (dry-ice chest) for periods of at least
has not been examined experimentally. The crucial at —70°
effect of glycerol concentration on cell survival 3 years. The conditions for storage were found
to be dependent on the method of freezing and
is further illustrated by the fact that nutritional
variants isolated from a given culture may require thawing and on the concentration of glycerol
in the medium over the range of 5-20 per cent.
concentrations of glycerol for optimal survival
at —70°
C. different from those of the parent
Conditions for optimal survival were observed
to vary not only among strains from different
strain. For example, all strains of U12 indicated
in Tables 2, 3, and 4 were derived from strain sources, but also among nutritional variants and
U12-705 by a process of selection from a hetero
the stock cultures from which these were isolated.
Cells deteriorated at —70°C. at a rate which
geneous population. Heretofore, these variants
have been indistinguishable from U12-705 except was a function of the conditions of storage and
the strain of cells. Under conditions for optimal
on the basis of nutrition (28, 29). Similar varia
tions are observed with strains L and RM3 and survival, one group of cell strains had a half-life
C. of about 3 years, whereas a second
between strains derived from different sources. at —70°
group
had
a half-life of 1-1.5 years.
Likewise, variations in the ability of cells to with
stand freezing by a number of procedures are
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Some Practical Aspects of Storing Mammalian Cells in the
Dry-Ice Chest
H. E. Swim, R. F. Haff and R. F. Parker
Cancer Res 1958;18:711-717.
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