A N E W ASSAY S Y S T E M F O R Q U A L I T Y C O N T R O L T E S T I N G

OF A CHEMICALLY DEFINED MEDIUM

Jindrich Cinatl, Jr. 1, Jaroslav Cinatl, Holger Rabenau,

and H a n s W. Doerr

Centre of Hygiene, Department of Virology (J. C. Jr., H. R., H. IV. D.) and Centre of Pediatry,
Department of Hematology and Oncology (J. C.), Clinics of the J. IV. Goethe University
Frankfurt a.M., Federal Republic of Germany

SUMMARY: Mouse NCTC clone 929 L (L-929) cells were propagated continuously over 2 yr in protein-free
Eagles's minimal essential medium (EMEM). The cells designated L-929-WS were used for quality-control
testing of protein-free EMEM as a model for more general medium quality tests. The parameter for the
suggested quality control assay was the growth-promoting activity of the medium for L-929-WS cells. As an
example of quality tests we assayed the growth promoting activity of EMEM exposed to various conditions of
storage time and temperature. We established the sensitivity of the new assay by comparing it to the original ceils
L-929 grown in E M E M supplemented with 10% fetal bovine serum (FBS). The assay system using L-929-WS
cells propagated continuously in protein-free medium proved to be much more sensitive. The sensitivity,
however, was abolished by the addition of FBS in a concentration as low as 1% to a culture medium.

Key words: protein-free medium; quality control testing; growth-promoting activity.

I. INTRODUCTION II. MATERIALS

Quality control testing of media and cell culture A. Equipment

medium constituents depends on highly sensitive assay Incubator, model B 5061 EK/CO2, Heraeus 1
procedures. In this context, tests for growth-promoting Laminar-flow hood, model BSB 4, Flow Lab2
activity of a chemically defined medium deserve special B. Chemicals and reagents
attention. To detect significant deviations from the Powdered formulation of Eagle's minimal essential
designated formula, the presence of chemical con- medium (EMEM) without b-glutamine, no. 072-
taminants, or a negative influence of storage conditions 01700, Gibco3
the use of sensitive cell lines grown in serum-free media L-Glutamine, no. 066-010513
has been suggested 19,10). To achieve sustained cell Penicillin G potasium, no. 066-18303
growth, however, such media usually have to be sup- Streptomycin sulfate, no. 066-186(P
plemented with various protein growth factors and Fetal bovine serum (FBS) sterile, mycoplasma- and
hormones (1,2) that possibly prevent the effect of virus-free, no. S-0115, Biochrom4
contaminants. Phosphate buffered saline (PBS) without Ca ÷÷ and
Several cell lines, including mouse NCTC clone 929 L Mg ÷÷,no. 182-1(P
{L-929) cells, have been established in chemically Trypsin 1:250, no. 0152-13-1, Difcos
defined media without any protein supplement Trypan blue solution, 0.5%, no. 47285, Serva6
(3,6,7,12-14,20,22,23). The present study shows that L- Crystal violet, no. 273356
929 cells grown continuously in such a medium can be Sodium bicarbonate, no. 6329, Merck 7
used as a highly sensitive and simple cell culture system C. Supplies
for quality control testing. Petri dishes, disposable, sterile, plastic (35 X 10 mm)
no. 153066, Nunc s
Tissue culture flasks, polystyrene, sterile, 80 cm2, no.
147589s
Disposable plastic pipettes, 1 ml, no. 604181; 5 ml, no.
606180; 10 ml, no. 607180, Greiner9
i T o w h o m correspondence should be addressed at Centre of Silicone rubber policeman, manufactured in our
Hygiene, D e p a r t m e n t of Virology, Clinics of the J. W. Goethe laboratory or disposable cell scraper, no. 3010,
University, Paul-Ehrlieh-Str. 40, D-6000 Frankfurt/Main, FRG. Costar 'o

Journal of Tissue Cukure Methods Vol. 12, No. 4, 1989 67 @)1989 Tissue Culture Association, Inc.
 CINATL ET AL.

Hemacytometer (Fuehs-Rosenthal), no. 191832421,

Kalensee 11
Sterivex filter units, no. SVGVB 1010 {0.22 gmL (::3
MiUipore '2 t- i00
D. Cells O
N C T C clone 929 L ( L-929; cells, murine {5,15 }2
L-929-WS ceils were established in protein-free E M E M o 80
by "adaptation" of L-929 cells. The adaptation was
accomplished by reducing the concentration of FBS
stepwise in a culture medium as described
previously (4L L-929-WS cells used in the ex-
periments were propagated continuously for more GJ
than 120 passages in protein-free E M E M .
o
o
-
40

III. PROCEDURE
re 20
A. Preparation of solutions O3
1. E M E M (1000 ml)
a. Dissolve E M E M with or without L-glutamine in
900 ml double-glass distilled water (DGDW) t 1 I 1 I I I 1 I I

b. Add 2.2 g sodium bicarbonate 0123456789

Number of passages
i00 FIG. 2. Effect on E M E M stored at 4 ° C for 160 d on saturation
density of L-929-WS cells grown in protein-free E M E M I~4 a n d of L-
929 cells grown in E M E M supplemented with 10% FBS {-4 over
80 several passages.

60 c. Adjust to pH 7.4 if necessary by adding 1 N

NaOH or HCI
d. Bring the volume with D G D W to 1000 ml
o 40
e. Sterilize by filtration using a 0.22-#m filter and
c-"
store in the dark at - - 2 0 ° and 4 ° C.
20 Note: Medium used for propagation of stock
C3 cultures {dissolved with L-glutamine) is stored at
4 ° C u~ to 3 wk.
- - 100 2. Glutamine
. - -
a. Dissolve L-glutamine in D G D W to achieve
r--- concentration of 200 m M
80 b. Add fresh L-glutamine directly before use to assay
c- EMEM
O 3. Trypsin ( 1000 ml), 0.2 and 0.02% solution
:~ 60
rD a. Dissolve 2 and 0.2 g trypsin in PBS, pH 7.4
b. Store at --70 ° C in 100 ml aliquots
CO 4. Antibiotics (100 X )
r.~ 40
a. Dissolve 5000 U / m l penicillin and 5000 /~g/ml
streptomycin in D G D W
20 b. Store at - - 70 ° C
B. Propagation of stock cell cultures
1. L-929 cells
a. Remove spent medium by pipetting from a
20 40 80 t60 confluent cell layer in the culture flask
Days of storage of FMEH b. Wash cells 3 times with PBS (20 ml for each
washing)
FIG. 1. Effect of E M E M stored at various temperatures for various c. Pour 10 ml of 0.2% trypsin in the culture flask
time intervals on saturation density of L-929-WS cells grown in
d. Discard the trypsin by pipetting after 20 s.
protein-free E M E M {A) and of L-929 cells grown in E M E M sup-
p|emented with 10% FBS (BL E M E M was stored at - - 2 0 ° C (13) and e. Incubate the culture flask with the residual
4 ° C (11) for 20, 40, 80, a n d 160d. trypsin for 5 min at 37 ° C

68 Journal of Tissue Culture Methods VoL 12, No. 4, 1989

 CINATL ET AL.

f. Resuspend the cells in 6 ml EMEM supplemented io

with 10% FBS, penicillin 150 U/mlL strep-
tomycin (50 gg/ml}
g. Dilute 2 ml of cell suspension in 48 ml EMEM
supplemented with 10% FBS, penicillin I50
U/mlL streptomycin {50 #g/ml}
h. Incubate the cells in new culture flasks {25 ml
medium per flask} at 37° C with 5% CO2 in air
Note: L-929 cells are subcultured in 6-d in-
tervals.
2. L-929-WS ceils
a. Remove spent medium by pipetting
b. Wash cells 3 times with PBS
c. Scrape the cells gently from the plastic surface
with the silicone-rubber policeman in 10 mi
EMEM supplemented with penicillin i50 U/mb ?0 ,10 80 160 21} 40 80 ISO
and streptomycin i50 eg/ml} Daysof storageof EME~q
d. Dilute the cell suspension in 40 ml EMEM FIG. 4. Effect of E M E M stored at various temperatures for various
supplemented with antibiotics time intervals and supplemented with various concentrations of FBS
e. Incubate the cells in new culture flasks {25 ml on saturation density of L-929-WS cells. E M E M stored at --20 ° C {O1
and 4° C {11) for 20, 40, 80, and 160 d was supplemented with 0.1%
medium per flask} at 37° C with 5% CO2 in air
FBS {AL 0.2% FBS (BI, 0.5% FBS tCt, and 1% FBS {DL
Note: L-929-WS cells were subcultured at
weekly intervals.
C. Viability of cells only show a slight blue coloration are not con-
1. Detach cells from the culture surface by tryp- sidered viable.
sinization {L-929) or by scraping {L-929-WS} D. Assay for growth-promoting activity
2. Resuspend the cells in the appropriate volume of In the experiments, the growth characteristic used is
culture medium to obtain approximately 1@ saturation density. EMEM is stored in the dark at
cells/ml
3. Dilute 0.1 ml of the cell suspension with 3.6 ml PBS
4. Add 3.6 ml of trypan blue solution
5. Incubate 2 to 5 min at 37° C
6. Mix suspension well, but not vigorously, with a
pipette C3
7. Count the viable cells with a hemacytometer
t" 100
t--
Note: All cells whose nucleus or cytoplasm show C3
blue staining are defective. Even those cells that (.-3

C3 80
cz)
r.__

cn
u 100 O3
60
¢--
(D 03
~ 8O "E3
c'-"
C3
40
~O'3 60
c-
20
c-
O CO
co
ft._
20

co
0 I I I I I I I I 'j

~ 0 0 1 2 3 4 5 6 7 8 9
2O 40 8O t60
Days of storage of EMEM Number of subcultures
FIG. 5. Effect of E M E M stored at 4° C for 160 d after sup-
FIG. 3. Effect of E ~ , ~ M with fresh glutamine on saturation ptementation with various concentrations of serum on saturation
density of I~929-WS cells grown in protein-free EMEM. Medium density of L-929-WS cells over several passages. E M E M was sup-
prepared without glutamlne was stored at --20 ° C {D} and 4° C { i } plemented with 0.1% FBS ~:~, 0.2% FBS fO~, 0.5% FBS (1~}, and
for 20, 40, 80, and 160 d. 1% FBS ~j.

Journal of Tissue Culture Methods Vol. 12, No. 4, 1989 69

 CINATL ET AL.
--20 ° and 4° C for 20, 40, 80, and 160 d and then used
for the examination of growth-promoting activity with
and without the addition of FBS. Saturation density and
plating efficiency of the cells in the assay media are
compared in all cases to that of the cells in the control
medium. Freshly prepared E M E M with and without
FBS is used as a control medium.
1. Prepare a suspension of L-929-cells in EMEM
supplemented with 10% FBS (step BlJ, and of L-
929-WS cells in protein-free E M E M or in EMEM
supplemented with 0.1, 0.2, 0.5 and 1% FBS (step
B2~
2. Count the number of viable cells {step C}
3. Dilute the cells using culture medium to obtain a
final concentration of 10s cells/ml
4. Plate 2 ml of the cell suspension in 21 dishes (2 ml
per dish)
5. Incubate at 37 ° C with 5% COs in air for 7 d
6. Detach L-929 and L-929-WS cells from the plastic
surface at 24-h intervals during the 7 d incubation
with 0.2 or 0.02% trypsin solution, respectively
7. Count the number of viable cells in three dishes for
each cell line (step C}
Note: Saturation density of the cell population is
estimated as the maximal cell number attained
under specified culture conditions in a culture
vessel (17 ~.
FIG. 6. L-929-WS cells grown in protein-free E M E M which was stored 160 d at 4 ° C. Cells do not form a confluent layer,
b u t instead form piles around n u m e r o u s " e m p t y " areas; 7 d of culture. P h a s e contrast microscopy. X350.
70
IV. DISCUSSION
This procedure demonstrates that L-929-WS cells
grown continuously in a protein-free medium may be
used as a highly sensitive and simple cell culture system
for testing the growth-promoting activity of a chemically
defined medium, of different medium constituents, or
the influence of storage conditions. We showed
previously that L-929 cells may be easily adapted to
protein-free growth using commercially available cell
culture media and supplies (4). In addition, L-929-WS
cells are available in our laboratory for common use.
Experimental results are shown in Figs. 1-5. L-929-
WS cells grown in protein-free EMEM indicate very
precisely any decrease in the quality of the medium, in
contrast to L-929 cells grown in EMEM supplemented
with 10% FBS {Figs. 1 A, B and 2k A prolonged
storage of liquid medium at 4 ° C, as recommended by
many suppliers (GIBCO BRL, Produktkatalog fiir die
Zellbiologie und Bioproze/~technik, 1988:19-28;
Biochrom KG, Biologisch-Medizinische Pr'~iparate,
Poduktirdormation, 19871, may negatively influence the
quality of a medium, even if fresh glutamine is added
directly before use (Fig. 3~. The addition of 0.5% FBS
to E M E M decreases the sensitivity of the assay
significantly {Figs. 4 C and 5). FBS added at a con-
centration of 1% to EMEM completely abolishes the
sensitivity (Figs. 4 D and 5}. FBS added at con-
Journal of Tissue Culture Methods Vol. 12, No. 4, 1989
 CINATL ET AL.
FIG. 7. L-929°WScells grown in protein-free EMEM which was stored for 160 d at 4° C and supplemented with 0.5% FBS.
Cells do not show any morphologic changes; 7 d of culture. Phase contrast microscopy. X540.
centrations of 0.1 and 0.2% has little influence on the
sensitivity (Figs. 4 A, B and 5}. The decreased quality of
a chemically defined medium can easily be demon-
strated by microscopic examination of the cells for
typical changes in cell morphology (Fig. 6L We
routinely use this aspect of the method for rapid
orientation when the quality of a culture medium is
being tested. The deleterious effect of a medium on cell
morphology may, however, be masked by the addition
of FBS in a concentration as low as 0.5% (Fig. 7t.
The assays for growth-promoting activity of a
chemically defined medium in general were insensitive
when serum was added to a culture medium; this might
be due to the ability of serum to react as a detoxifying
agent with chemical contaminants of a medium
(8,18,21~. In addition, deviations from the designated
formula were often concealed because of the presence of
complementary nutrients in serum ~11,16,191.
Similarly, any deterioration in the activity of labile
components caused by inappropriate storage conditions
were masked. For these reasons, the use of sensitive cell
lines propagated in sermn-free medium has been
recommended (9,10}. Growth medium for these cells
usually has to be supplemented with protein growth
factors and hormones to obtain sustained cell growth
tl,2~.
Journal of Tissue Culture Methods Vol. 12, No. 4, 1989
Various cell lines, including mouse L-929 cells, were
"adapted" to continuous growth in a chemically defined
medium without any protein supplement (3,6,7, 12-14,
20,22,23}. It has been suggested that the successful
growth of these cells in protein-free medium can be
attributed to several factors, including the commercial
availability of high-quality chemicals, instrumentation
for obtaining pure water, and equipment and
procedures to ensure chemically clean glassware t14t.
Cells in protein-free medium may be superior to any
other culture system to test medium for the presence of
various chemical contaminants. It has been suggested
that some proteins added in microgram concentrations
to a defined medium l e.g., transferrin~ bind heavy metal
ions which otherwise would have inhibitory effects on
cell growth tl,81. This fact may contribute to the
decreased sensitivity of assay systems using cells
cultured in serum-free medium supplemented with such
proteins. On the other hand, cells grown continuously in
protein-free medium may be insensitive to deviations
from the designated formula of distinct nutrients in a
medium to be tested. Therefore, metabolic studies of
various cell lines adapted to continuous growth in
protein-free medium are strongly encouraged. These
cells should also be compared with cells grown in serum-
free medium supplemented with defined proteins in
71
 CINATL ET AL.
terms of their sensitivity to medium deterioration. The
availability of cells cultured in protein-free medium and
knowledge of their metabolic characteristics may
contribute a great deal to more general quality control
testing of medium and other products used for cell
culture.
V. REFERENCES
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2 Flow Laboratories, Meckenheim, West Germany
GIBCO, Eggenstein, West Germany
Bioehrom KG, Berlin, West Germany
Ditco, Detroit, MI
Serva, Heidelberg, West Germany
We are grateful to Alena Cinatlova for photomicrographs, We thank Iris Fairtey for help with preparation of
the manuscript and Barbara Steinthal for reviewing the manuscript.
72
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7 Merck, Darmstadt, West Germany
* Nunc, Wiesbaden, West Germany
9 Greiner, Ni/rtingen, West Germany
~oCostar Corporation, Cambridge, MA
" Kalensee, Giessen, West Germany
12 Millipore, Eschborn, West Germany
Journal of Tissue Culture Methods Vol. 12, No. 4, 1989