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An improved nutrient solution for diploid Chinese hamster and human cell lines
Experimental Cell Research 29, .515-526 (1963)
AN IMPROVED NUTRIENT
515 S O L U T I O N FOR D I P L O I D
C H I N E S E H A M S T E R A N D H U M A N CELL L I N E S 1 R. G. H A M
Department qf Biophysics, Florence R. Sabin Laboratories, University of Colorado Medical Center, Denver, Colorado, U.S.A.
lReeeived June 1, 1962
IN a previous article [21, a synthetic nutrient mixture was described, which, w h e n s u p p l e m e n t e d with two purified s e r u m protein fractions, w o u l d s u p p o r t elonal growth of a diploid Chinese h a m s t e r o v a r y cell line, CHBOC1 D 1. This nutrient mixture, designated F7, was d e v e l o p e d f r o m earlier nutrient mixtures w h i c h s u p p o r t e d elonal g r o w t h of the $3 H e L a strain u n d e r similar conditions [1]. I n the present study, n e a r l y all of the c o m p o n e n t s of mixture F7 were omitted one at a time a n d a d d e d b a c k over a wide r a n g e of c o n c e n t r a tions in o r d e r to d e t e r m i n e for each the m i n i m u m c o n c e n t r a t i o n w h i c h will p e r m i t growth, the o p t i m u m concentration, a n d the m a x i m u m permissible non-toxic concentration. T h e results of these titrations indicated that nutrient mixture F7 was not o p t i m a l for the h a m s t e r cell line. A n e w mixture, F10, ( T a b l e I) was p r e p a r e d , containing e a c h nutrient at the o p t i m u m level suggested b y the titrations. This mixture was f o u n d to be superior to F7, both for s e r u m - f r e e growth of the h a m s t e r o v a r y line, a n d for growth of a n u m b e r of other cell lines in s e r u m c o n t a i n i n g media. MATERIALS
AND
METHODS
Cell lines.--STI~AIN CHD-3 is a subclone of diploid Chinese hamster ovary strain CHBOC1D1, which was used in the development of nutrient mixture F7 [2]. It appears to be identical in all particulars, including chromosome number and tendency to display non-disjunction to strain CHBOC1D1. Strain CHD-3 was used for all of the growth factor titrations. During those studies it was maintained in medium F9FC. Currently, medium F10HIFC is used. Strain CHD-3 and its parental strains have been in continuous culture for 4} years. STRAIN CHL-1 was derived from the lung of a newborn Chinese hamster. This strain, which has been in continuous culture for over two years, exhibits a predominantly diploid chromosome pattern similar to that previously described for
1 Contribution No. 195. This work was aided by a grant from the National Foundation, Inc., and U.S.P.H.S. Graduate Training Grant No. 2G-781. Experimental Cell Research 29
R. G. Ham
516
TABLE I. Composition of nutrient mixture FIO.
Stock solution
Amount used per l i t e r of F I O
A1
10 ml (100 x )
As
Aa
A4
10 m I ( 1 0 0 ×
10 m l ( 1 0 0 ×
10 m l ( 1 0 0 x
Component
L-Arginine HC1
I0 ml (100 x
21.1
Final concentration in nutrient m i x t u r e ]710 (moles/l)
1.0 × 1 0 -8
L-Histidine HC1
2.10
1.0 x 1 0 - 4
L-Lysine HC1
2.93
1.0 x 10 -~
L-Tryptophane
0.060
3.0 x 1 0 - 6
L-Methionine
0.448
3.0 x 10-5
L-Phenylalanine
0.496
3.0 x 10-~
L-Tyrosine
0.181
1.0 x 10 _5
L-Alanine
0.891
1.0 x 10 -4
Glycine
0.751
1.0 x 10 -4
L-Serine
1.05
1.0 x 10 -4
L-Tbreonine
0.357
3 . 0 x 10 - s
L-Aspartic acid
1.33
1,0 x 10 -4
L-Glutamic
1.47
1.0 × 10 -4
acid
L-Asparagine • H~O
Aa
Concentration in stock solution (g/l)
1.50
1.0 × 10 - a
L-Glutamine
14.62
1.0 x 10 - a
L-Isoleucine
0.26
2 . 0 x 10 -5
L-Leucine
1.31
1.0 × 1 0 -4
L-Proline
1.15
1.0 x 10 -4
L-Valine
0.35
3.0 × 10 -5
3.15
2.0
A6
10 n i l ( 1 0 0 x )
L-cysteine HC1
B
10 m l ( l O O x )
Biotin
0.0024
1.0 x 10 -7
Calcium pantothenate
0.0715
3.0 x 10 -6
Choline chloride
0.0698
5.0 x 10 -6
myo-Inositol
0.0541
3 . 0 x 10 -6
Niacinamide
0,0615
5 . 0 x 10 -6
0.0206
1.0 x 10 -6
Pyridoxine
HC1
Riboflavin
D
10 m l ( 1 0 0 × )
1.0 m l ( 1 0 0 0 x )
ExperimeNal Cell Research 2 9
10 -4
0.0376
1.0 x 1 0 -~
0.1012
3.0 x 10 -6
Hypoxanthine
0.408
3.0 x 10 -5
Folic acid
0.132
3.0 x 10 -6
Thymidine
0.0727
3 . 0 × 10 -6
Vitamin
0.136
1.0
x
10 -6
FeSO~ a
0.834
3.0
x
10-6
CuSO4 a
0.0025
1.0
x
10-s
ZnSO4 a
0.0288
1.0 x 10 -~
Thiamine C
x
HC1
Bi~
Ceil culture nutrient solution
517
Table I - - continued
Concentration in stock solution (g/l)
Final concentration in nutrient mixture (moles/l)
4.41
3.0 x 10 -4
15.28
6.2 x 10 _4
Stock solution
Amount used per liter of FIO
E
10 ml (100 x )
CaC12 • 2H20
F
10 ml (100 x )
MgSO~ • 7H20
G
50 ml (20 ×)
NaCI ~ KC1 b NaeHPO 4 • 7H20 KH2PO 4
L
1.0 ml (1000 x )
Lipoie acid
0.200
il.0 × 10 -6
P
1.0 ml
Phenol red
1.2
3.3 x 10 .6
None s None s None s
1.10 g 0.110 g 1.20 g
Glucose Sodium pyruvate NaHCO3 b
----
6.1 x 10 -3 1.0 × 10 -s 1.43 x 10-2
Component
148. 5.7 5.8 1.66
1.2 3.8 1.1 4.8
x 10 -1 x 10 -3 x 10 .8 x 10 .4
"Specpure" Grade, Johnson, Matthey and Co., London. "Biological" Grade, Baker and Addamson, New York. c No stock solutions. Dry reagents are added directly to the final mixture.
C h i n e s e h a m s t e r o v a r y line C H B O C 1 [9] a n d its s u b c l o n e s , C H B O C 1 D 1 [2] a n d C H D - 3 . S t o c k s of C H L - 1 are c u r r e n t l y m a i n t a i n e d in m e d i u m F 1 0 H I F C . STRAIN S 3 - 9 - I V is a s u b c l o n a l s t r a i n d e r i v e d f r o m H e L a c l o n e $3, a n d h a s b e e n d e s c r i b e d e l s e w h e r e [6]. S t o c k c u l t u r e s h a v e g e n e r a l l y b e e n m a i n t a i n e d in m e d i u m N 1 6 H H F [3]. STRAIN H F 3 6 5 is a d i p l o i d h u m a n line w i t h a n o r m a l f e m a l e k a r y o t y p e i s o l a t e d in F e b r u a r y 1959 f r o m a p a t i e n t w i t h f e m a l e p s e u d o h e r m a p h r o d i t i s m [10]. T h e p r e s e n t c u l t u r e w a s d e r i v e d f r o m cells f r o z e n in J u l y 1959 a n d t h a w e d f i v e m o n t h s b e f o r e t h e e x p e r i m e n t s d e s c r i b e d in t h i s p a p e r . T h u s , t h e cells u s e d h a d b e e n a t o t a l of t e n m o n t h s in a c t i v e c u l t u r e . S t o c k c u l t u r e s are c u r r e n t l y m a i n t a i n e d in m e d i u m F10FC~HC15. Synthetic nutrient solution.--Nutrient M i x t u r e F 1 0 is n o r m a l l y p r e p a r e d f r o m c o n c e n t r a t e d s t o c k s o l u t i o n s , as i n d i c a t e d in T a b l e I. STOCKS A1, A2, As, As, B, E , F, a n d G are p r e p a r e d b y d i s s o l v i n g t h e c o m p o n e n t s in t r i p l e d i s t i l l e d w a t e r ( t . d . w . ) w i t h v i g o r o u s m e c h a n i c a l s t i r r i n g as n e e d e d . STOCK A 4 is p r e p a r e d as follows: T h e a s p a r t i c a n d g l u t a m i c a c i d s are a d d e d t o t h e f i n a l v o l u m e of t . d . w . , t o g e t h e r w i t h 1.2 rag~1 of p h e n o l r e d (1.0 ml/1 of s t o c k P). 1.0 N N a O H is t h e n a d d e d s l o w l y w i t h v i g o r o u s m e c h a n i c a l s t i r r i n g u n t i l a n o r a n g e color w h i c h d o e s n o t f a d e is o b t a i n e d a n d t h e solids are c o m p l e t e l y d i s s o l v e d . T h e g l u t a m i n e a n d a s p a r a g i n e are t h e n d i s s o l v e d in t h e s o l u t i o n a n d 1.0 N N a O H is a g a i n a d d e d u n t i l t h e i n d i c a t o r is o r a n g e . 33 - 631808
Experimental Cell Research 29
518
R. G. Ham
STOCK A 6 is readily prepared b y dissolving the cysteine HC1 in t.d.w. However, on standing, it gradually forms a highly insoluble precipitate (eystine) due to air oxidation. W h e n this occurs, the solution m u s t be discarded, since the precipitate does n o t readily redissolve on dilution into the final m e d i u m , and since the eysteine level is critical [2]. STOCK C is prepared as follows: The h y p o x a n t h i n e is completely dissolved in the final v o l u m e of t.d.w, with h e a t i n g a n d vigorous stirring. The solution is cooled and the folio acid is added. J u s t sufficient 1.0 N N a O H to dissolve the folie acid completely is t h e n added (not over 2-3 ml/1 of solution C). The t h y m i d i n e and v i t a m i n B12 are t h e n added a n d dissolved with additional stirring. STOCK D is prepared b y dissolving the "speepure" grade salts in t.d.w, a n d adding 0.5 ml/1 of 12 N HC1 to p r e v e n t gradual precipitation of the iron. STOCK L is prepared b y first dissolving the lipoie acid completely in a few drops of 1.0 N N a O H a n d t h e n diluting to the final volume with t.d.w. The lipoie acid dissolves slowly, b u t is actually quite soluble in 1.0 N NaOH. STOCK P is prepared b y dissolving 1.2 g phenol red a n d 0.4 g sodium b i c a r b o n a t e in 1 1 of t.d.w. Stocks A4, A6, B, a n d C should be stored frozen. Stocks A1, A2, A3, As, a n d L should be refrigerated or frozen. Stocks D, E, F, G, a n d P are stable at room t e m p e r a t u r e as long as t h e y are kept sterile a n d t i g h t l y stoppered. All solutions stored w i t h o u t freezing should be sterilized b y Millipore filtration. (Stocks E, F, a n d G m a y be autoclaved if it is more convenient.) The biochemieals used in n u t r i e n t m i x t u r e F10 are the best commercially available grades. The inorganie salts are " a n a l y t i c a l reagent" grade, except for the following: the sodium chloride, p o t a s s i u m chloride, a n d sodium b i c a r b o n a t e are Baker a n d A d d a m s o n "Biological" grade; a n d the ferrous sulphate, zinc sulphate, a n d copper sulphate are J o h n s o n a n d M a t t h e y "Specpure" grade. The water used is distilled once in a m e t a l still a n d twiee more in a quartz Hereaus "Bidistill" apparatus. The amino acid stock solutions as described are designed to p e r m i t easy omission of related groups for n u t r i t i o n a l studies. If desired, stocks A1, A~, A3, A4, and A 5 m a y be combined into a single 20 x stock, which should be stored frozen. PREPARATION OF F10 FROM STOCK SOLUTtONS: N u t r i e n t m i x t u r e F10 is prepared as follows: All the c o m p o n e n t s except the sodium b i c a r b o n a t e are added to t.d.w. a n d diluted to final volume. 1.0 N N a O H is t h e n added with stirring u n t i l the indicator becomes orange (pH 7.2-7.4). Finally, the sodium b i c a r b o n a t e is added a n d the solution is sterilized b y Millipore filtration a n d dispensed aseptically into sterile storage bottles. Short-term storage is satisfactory at 4°C. Freezing below - 2 0 ° C is r e e o m m e n d e d for storage of more t h a n a m o n t h . Growth m e d i a . - - T a b l e I I describes the growth media prepared from n u t r i e n t m i x t u r e F10, a n d lists the uses of each. All of these media are sterilized b y Millipore filtration, a n d should be stored frozen if kept for more t h a n two weeks. N u t r i e n t m i x t u r e F10 a n d all media derived from it are designed for use with a humidified atmosphere of 5 per cent carbon dioxide in air. Culture techniques.--Proeedures for m a i n t e n a n c e of stock cultures a n d p l a t i n g of single cell suspensions are the same as those previously described [2, 3], except t h a t the media listed in Table I I are used. Measurement of colony s i z e . - - G r o w t h rate is often more sensitive to n u t r i e n t
Experimental Cell Research 29
Cell cullure nutrient solution
519
TABLE II. Growth media prepared from nutrient mixture FIO. ml/1
Medium FIOAF
F10 Fetuin (50 mg/ml) a Albumin (250 mg/ml)b
978 20 2
Use: Serum-free growth; nutritional studies: Chinese hamster strains CHD-3 and CHL-1 and HeLa strain S3-9-IV. ml/1
Medium FIOFlo o
F10 Fetuin (50 m g / m l ) a Use: Nutritional studies: Chinese hamster strain CHD-3
008 2
per 1
Medium FIOHIFC
F10 Baeto Heart Infusion Broth Tested Fetal Calf Serum
980 ml 0.50 g 20 ml
Use: Control medium for single cell plating experiments; maintenance of stock cultures: Chinese hamster strains CHD-3 and CHL-1 ml/1
Medium FIOFCsHCI5
F10 Tested Fetal Calf Serum Tested H u m a n Cord Serum
800 50 150
Use: Single cell plating and maintenance of stock cultures: H u m a n diploid strain HF365 ml/1
Medium F10FCIsHC ~
FIO Tested Fetal Calf Serum Tested H u m a n Cord Serum
800 150 50
Use: Short term culture of h u m a n white blood cells for chromosomal analysis a Cf. [3] for preparation of fetuin. H u m a n Serum Albumin, " A l b n m i s o l " , Merck Sharpe and Dohme. c o n c e n t r a t i o n t h a n p l a t i n g efficiency. U n f o r t u n a t e l y , t h e closely p a c k e d g r o w t h p a t t e r n of t h e f i b r o b l a s t - l i k e cells u s e d in t h e s e s t u d i e s g e n e r a l l y m a k e s i t i m p o s s i b l e t o c o u n t t h e n u m b e r of cells p e r colony. C o l o n y size c a n b e r o u g h l y q u a n t i t a t e d , however, by placing the stained colony over a round opening in a mask over a p h o t o c e l l , a n d d e t e r m i n i n g p h o t o m e t r i c a l l y h o w m u c h of t h e o p e n i n g is c o v e r e d b y t h e colony. A n a r b i t r a r y scale of p h o t o c e l l u n i t s is u s e d , in w h i c h 0 c o r r e s p o n d s t o n o l i g h t a b s o r p t i o n , a n d 50 t o c o m p l e t e o p a c i t y . F o r a n y g i v e n t e s t series, a n o p e n i n g Experimental Cell Besearch 29
R. G. Ham
520
is used which is approximately the same diameter as the largest colonies in the series. Generally the largest colony, or in some cases, the largest five colonies on each plate are measured. This technique is limited in that it can only be used to compare colonies whose density of growth is similar. A large, diffuse colony will give a higher reading than a small, densely packed colony containing exactly the same number of cells. However, colony morphology depends primarily on the protein or serum supplement, and tends to be constant for tests carried out entirely in one type of medium. In such tests, the photometrically determined "colony areas" closely parallel visual estimates of colony size. RESULTS
Growth factor titrations.--Optimum c o n c e n t r a t i o n s for i n d i v i d u a l g r o w t h factors d e t e r m i n e d b y titration in m e d i u m F 7 A F o n l y a p p r o x i m a t e the c o m p o s i t i o n of a fully b a l a n c e d m e d i u m , since e a c h o p t i m u m is d e t e r m i n e d with other factors not at o p t i m u m levels. Nevertheless, the analysis of s u c h d a t a r e s u l t e d in a n i m p r o v e d n u t r i e n t m i x t u r e , F10, w h i c h is s u p e r i o r to F7 for s e v e r a l cell lines. Selected titrations w h i c h w e r e p a r t i c u l a r l y significant in the d e v e l o p m e n t of F10 are p r e s e n t e d below. AR6ININE: T h e c o n c e n t r a t i o n of arginine h a s little effect on plating efficiency o v e r a r a t h e r w i d e r a n g e (Fig. 1). H o w e v e r , c o l o n y size is s h a r p l y d e p e n d e n t on the arginine level, w i t h o p t i m u m g r o w t h o c c u r r i n g n e a r 1 × 10 -3 m o l a r arginine, far a b o v e the level of 3.6 × 10-~ m o l a r f o u n d in F7. TRYPTOPHANE: S e r u m a l b u m i n w a s o m i t t e d f r o m the m e d i u m u s e d for t r y p t o p h a n e titrations, since the c o m m e r c i a l p r e p a r a t i o n contains s o d i u m a c e t y l t r y p t o p h a n a t e as a stabilizing agent. T h e d a t a ( T a b l e I I I ) indicate a b r o a d o p t i m u m r a n g e b e t w e e n 10-7 a n d 10 -5 m o l a r , a n d a n a b s o l u t e toxicity of t r y p t o p h a n e at c o n c e n t r a t i o n s a b o v e 1 × 10 4 M. T h e a d d i t i o n of a l b u m i n i m p r o v e s g r o w t h in the o p t i m u m range, a n d also p a r t i a l l y r e v e r s e s the toxicity of higher c o n c e n t r a t i o n s of t r y p t o p h a n e . 4O
40 u
30
30
20
20
o o
o 10u
Concentration of arginine (moles per [iter)
Experimental Cell Research 29
Fig. 1.--Response of strain CHD-3 to arginine. Basal medium: F7AF [2] minus arginine. [], plating efficiency; ©, colony area.
Cell culture nulrienl solulion
521
PROL1NE: Unlike most well studied mammalian cells, strain CHD-3 requires proline for colonial growth (Table IV). GLYCINE, SERINE: In medium F7AF from which glutamic acid, aspartic acid, serine, and glyeine are omitted, either serine or glycine is needed for colonial growth of strain CHD-3 (Fig. 2). These data demonstrate how narrow TABLE III. Response of strain CHD-3 to tr~tptophane and albumin, a
Tryptophane concentration
M/1
Plating efficiency
Colony
%
No Albumin
Plus Albuminb
area (photocell units) ~ No albumin Plus albumin
0
26
62
2
1 × 10 -s
31
--
2
--
3 × 10 -s
43
--
3
--
1 × 1 0 -7
47
--
10
--
1 x 10-~
43
60
lO
19
3 x 10 -6
46
--
10
--
2 × 10-~
43
--
11
--
5 x 10-5
30
--
8
--
1 × 10 -4
20
--
6
--
2 ×10 -~
0
--
0
--
3 × 10 - 4
0
33
0
• ~
8
9
a B a s a l M e d i u m : F 7 A F [2] m i n u s T r y p t o p h a n e and serum albumin. b Human serum albumin ("Albumisol", Merck, Sharpe and Dohme), 0.5 mg/ml. This preparat i o n a l s o i n t r o d u c e s 4 × 1 0 - 5 M/1 e a c h s o d i u m a c e t y l t r y p t o p h a n a t e and sodium caprylate.
the optimum concentration range is for some nutrients, and strongly emphasize the need for a precisely balanced medium. IRON: The iron requirement of strain CHD-3 has already been described [2]. In an attempt to determine whether the effect was due to iron itself or to trace impurities, spectrographically analyzed "Specpure" grade iron compounds were obtained. In the presence of copper, zinc, and lipoic acid (see below), the high purity iron compounds not only replaced the "analytical reagent" grade ferric chloride previously used, but frequently resulted in improved growth, particularly in media containing very low levels of proteins. Because of this benefit, apparently due to the absence of toxic impurities, the "Specpure" grade of ferrous sulphate is now routinely used in the preparation of nutrient mixture F10. COPPER, ZINC, LiPom ACID: Small amounts of copper, zinc, and lipoic acid added either singly or together frequently improve growth of strain Experimenlal Cell Research 29
522
R. G. Ham
CHD-3, particularly in media containing very low levels of protein. Although the present data are not adequate to claim definite stimulation by any one of these factors, all three have been included as routine components of F t 0 in order to avoid possible difficulties due to borderline deficiencies. OPTIMUM LEVELS: For each titration, the growth and plating responses of the cells were plotted on semi-logarithmic graph paper against the logaTABLE IV. Response of strain CHD-3 to proline. ~ Proline concentration
M/1
0 1 x 10-6
Plating efficiency
0 0
Appearance of colonies b
---
3 x 10-8
0
1 x 10-5
57
3 x 10-5
61
normal
1 x 10 - 4
56
normal
3 x 10 -~
57
normal
1 × 10 -3
50
normal
a B a s a l M e d i u m : F 7 A F [2] m i n u s p r o l i n e . b Visual estimate only. This experiment was performed for measuring colony density.
-small
prior to development
of a p p a r a t u s
rithms of the nutrient concentrations (as in Fig. 1). The optimum range within which both plating efficiency and colony size remained unchanged was then determined, and the middle of this range on the semi-log plot was chosen as the optimum level for use in F10. For completeness and for applicability to a wide variety of cell lines, all 20 amino acids were included in F10, although the data suggested that several of them could be eliminated without noticeable effect on the growth of strain CHD-3. Results obtained using FIO media.--STRAIN CHD-3: Replacing nutrient mixture F7 with F10 has increased the growth rate of strain CHD-3 in serumfree media and has resulted in more consistently high plating efficiencies. Single cell plating experiments in m e d i u m F10AF now yield satisfactory colonies in 7 to 9 days, whereas 10 to 12 days were required using m e d i u m F7AF. Use of F10 has also reduced the amount of protein needed for satisfactory plating. W h e n sufficient care is taken to exclude environmental toxicities, the Experimental Cell Research 2 9
Cell culturenutrielTlsolution
523
serum albumin can be completely eliminated, and the fetuin can be reduced to 0.1 mg/ml ( m e d i u m F10F~00) or even lower. Although the results are still rather variable at these low protein levels, well formed colonies can usually be obtained in 8 to 10 days. Thus, it is now possible to plate strain CHD-3 with fair reliability in a defined nutrient solution supplemented with only 100 micrograms of purified protein per ml. 80
.49.~
~
70 8 B 60
~- so
Fig. 2 . - - R e s p o n s e of strain CHD-3 to glyeine or serine. Basal medium: F 7 A F [2] minus glycine, serine, glutamie acid and aspartic acid [], glycine; O , serine.
///
i\\
3O m 20
10 ~ 10-s 10_4 10 3 Concentration of amino acid (moles per hter)
10-2
Growth of strain CHD-3 in serum-containing media has also been greatly improved. Use of F10 has reduced the fetal calf serum needed for consistently good results from 15 per cent to 2 per cent or less, and also has m a d e possible the use of m a n y lots of fetal calf serum which would not support growth of strain CHD-3 at all in F7 media. The major factor responsible for this difference appears to be the increased level of arginine in F10. Addition of arginine to F7 permits colony formation using serum lots which otherwise would not support growth in F7, but which would support growth in F10 media. Growth of strain CHD-3 in m e d i u m F10HIFC is very good. Because of its low serum content, F10HIFC is a very economical medium. Preliminary experiments (Table V) suggest that further econom y can p r o b a b l y be accomplished b y replacing the relatively scarce fetal calf serum with a wide variety of other sera. However, the long term effects of these other sera on diploid cell lines must first be determined. In m a n y cases, nutrient mixture F10 plus two per cent fetal calf serum is satisfactory as a m e d i u m without added heart infusion broth. However, in a n u m b e r of low-serum and serum-free tests with several cell lines, heart infusion broth has tended to produce a slight overall i m p r o v e m e n t in growth, and also to reduce the frequency of unexplained growth failures. Since it appears to improve both reliability and growth, Baeto heart infusion broth has Experimenlal Cell Research 29
R. G. H a m
524
b e e n r o u t i n e l y i n c o r p o r a t e d i n t o m e d i u m F 1 0 H I F C , w h i c h is u s e d p r i m a r i l y for maintaining stock cultures and as a control in plating experiments. STRAIN C H L - I : T h i s s t r a i n , w h i c h c o u l d n o t b e p l a t e d at a l l in m e d i u m F7AF, plates well in medium F10AF, although colonial growth cannot be o b t a i n e d i n F10F100 o r s i m i l a r l o w p r o t e i n m e d i a w h i c h w i l l s u p p o r t p l a t i n g of CHD-3. TABLE V. Response of strain CHD-3 to various seraf Serum concentration 1%
Type of serum b
Plating efficiency (%)
Fetal calf Normal calf Ox Pregnant cow Sheep Goat Pig Horse
85 66 61 80 81 81 74 88
2% Colony density Plating (photoeell efficiency units) (%) 11 7 8 8 9 10 14c 11
90 72 70 89 81 73 87 84
5%
Colony density (photocell units) 12 10 10 13 7 5 12c 12
Plating efficiency (%)
Colony density (photocell units)
86 74 86 94 74 77 89 93
11 11 ll 12 4 6 9e 10
a Basal Medium: F10 plus 0.5 mg/ml Bacto heart infusion broth. b All sera were obtained from Colorado Serum Co., Denver, Colo. c Colonies diffuse. Larger area is responsible for higher readings, rather than increase in cell number. T h e r a p i d g r o w t h r a t e o f s t r a i n C H L - 1 m a k e s it p o t e n t i a l l y v e r y u s e f u l i n plating experiments. In serum-free medium F10AF, useful colonies can n o r m a l l y b e o b t a i n e d i n 6--7 d a y s . I n s e r u m - c o n t a i n i n g m e d i u m F 1 0 H I F C , v e r y d e n s e c o l o n i e s , e s t i m a t e d to c o n t a i n 1000 o r m o r e cells, c a n r o u t i n e l y b e o b t a i n e d a f t e r five d a y s ' i n c u b a t i o n , c o r r e s p o n d i n g to a d o u b l i n g t i m e o f 12 h r o r less. STRArN $ 3 - 9 - I V H e L a : G r o w t h o f t h i s s t r a i n i n m e d i u m F 1 0 A F is a b o u t e q u i v a l e n t to its g r o w t h i n t h e s e r u m - f r e e m e d i u m p r e v i o u s l y r e p o r t e d b y F i s h e r , P u c k a n d S a t o [1]. G r o w t h o f t h e H e L a s t r a i n i n m e d i u m F 1 0 H I F C is g o o d , a l t h o u g h t h e g r o w t h r a t e is s l o w e r t h a n i n m e d i u m N 16 H H F I3]. T h i s r e d u c e d r a t e o f g r o w t h c a n b e o v e r c o m e b y i n c r e a s i n g t h e f e t a l c a l f s e r u m c o n c e n t r a t i o n to 10 o r 15 p e r cent.
Experimental Cell Research 29
Cell culture nutrient soluliou
525
STRAIN HF365: Use of F10 containing media for this h u m a n diploid cell line has resulted in better and more consistent growth than was obtained with previous media. Well formed colonies of 1000 to 4000 cells are now reliably obtained after 8 to 10 days' incubation with plating efficiencies of 40 per cent or better. All diploid h u m a n strains thus far tested have been resistant to attempts at culture in either low-serum or serum-free media. HUMAN W~I~TE BLOOD CELLS: Preliminary studies by Robinson [7] using a modification of the peripheral blood technique of Hungerford et aI. [4] indicate that excellent short term growth of h u m a n white blood cells for chromosome preparations can be obtained with a high degree of reliability when m e d i u m F10FC15HC ~ is employed. DISCUSSION
The use of single cell plating in a medium containing a m i n i m u m protein supplement has provided a powerful tool for studying the nutritional requirements of m a m m a l i a n cells in culture. Under these conditions, the small number of cells present cannot appreciably alter the composition of the surrounding medium, and the limited amount of protein offers them relatively little protection from it. Thus, their growth response provides a very sensitive index to the adequacy and freedom from toxicity of the medium being tested. The deliberate selection of nutrient concentrations m i d w a y between the lower and upper limits of the optimum range has resulted in a balanced nutrient mixture which is not only superior for the cell strain used in the initial study, but also for several others, including a diploid h u m a n line. An important difference between nutrient mixtures F7 and F10 is the increased level of arginine in the latter. Similar effects of arginine have been previously reported [81, and m a y be related to the presence of arginase in m a n y cultured cell lines [5]. SUMMARY
1. Single cell plating of diploid Chinese hamster ovary cells (Strain CHD-3) in a m e d i u m containing a m i n i m u m supplement of purified proteins has been used to determine optimum concentrations for the components of a previously described nutrient mixture, F7. 2. The more significant results of these titrations include: (a) a high arginine requirement; (b) a low toxic limit for tryptophane; (c) a requirement for proline; and (d) a narrow optimum range for serine and glycine. Experimental Cell Research 29
R. G. Ham
526
3. A new nutrient mixture, F10, has been p r e p a r e d in which each component has been a dde d at the experimentally determined o p t i m u m concentration. 4. Tests of the new nutrient mixture using strain CHD-3 revealed: (a) i m p r o v e d growth rate and reliability in a fetuin and albumin supplemented medium; (b) clonal growth at total protein concentrations as low as 100 #g/ ml.; (c) excellent growth at r e d u c e d serum concentrations; (d) satisfactory clonal growth using batches of fetal calf serum which would not support such growth in F7 media; (e) ability to use sera other than fetal calf serum, for clonal growth. 5. Tests of media p r e p a r e d from the new nutrient mixture on other cell strains revealed: (a) good growth with a fetuin and albumin supplement of strain CHL-1 Chinese hamster lung cells and strain S3-9-IV H e L a cells; (b) rapid growth of strain CHL-1 on a 2 per cent serum m e d i u m yielding dense colonies estimated to contain 1000 or more cells in five days' incubation time; (c) i m p r o v e d growth of a h u m a n diploid cell line; (d) excellent short term growth of h u m a n white blood cells for c h r o m o s o m e preparations. The author wishes to express his thanks to Dr. Theodore T. Puck for his interest in this work and for his many valuable suggestions; also to Mrs. Catherine Fogg for excellent technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8.
FISHER, H. W., PUCK, T. T. and SATO, G., J. Exptl. Med. 109, 649 (1959). HA~, R. G., Exptl. Celt Res. 28, 489 (1962). HAM, R. G. and PUCK, T. T., Methods in Enzgmologg 5, 90 (1962). HUNGEBFORD~ D. A., DONNELLY, A. J., NOWELL, P. G. and BECK, S., A m . J. H u m a n Genetics 11, 215 (1959). KLEIN, E., Exptl. Cell Res. 22, 226 (1961). MARCUS, P. I., Virologg 9, 546 (1959). I:~OBINSON, n., Personal communication. THOraAS, W. J., ZIE~LEB, D. W., SCttEPARTZ, S. A. and McLIMANS, W. F., Science 127, 591
(195s). 9. TJlo, J. H. and PUCK, T. T., J. Exptl. Med. 108,259 (1958). 10. TJio, J. H. and PUCK, T. T. and ROBINSON, A., PFOC. ~'~ratl. Acad. Sci. 45, 1008 (1959).
Experimental Cell Research 29
AN IMPROVED NUTRIENT
515 S O L U T I O N FOR D I P L O I D
C H I N E S E H A M S T E R A N D H U M A N CELL L I N E S 1 R. G. H A M
Department qf Biophysics, Florence R. Sabin Laboratories, University of Colorado Medical Center, Denver, Colorado, U.S.A.
lReeeived June 1, 1962
IN a previous article [21, a synthetic nutrient mixture was described, which, w h e n s u p p l e m e n t e d with two purified s e r u m protein fractions, w o u l d s u p p o r t elonal growth of a diploid Chinese h a m s t e r o v a r y cell line, CHBOC1 D 1. This nutrient mixture, designated F7, was d e v e l o p e d f r o m earlier nutrient mixtures w h i c h s u p p o r t e d elonal g r o w t h of the $3 H e L a strain u n d e r similar conditions [1]. I n the present study, n e a r l y all of the c o m p o n e n t s of mixture F7 were omitted one at a time a n d a d d e d b a c k over a wide r a n g e of c o n c e n t r a tions in o r d e r to d e t e r m i n e for each the m i n i m u m c o n c e n t r a t i o n w h i c h will p e r m i t growth, the o p t i m u m concentration, a n d the m a x i m u m permissible non-toxic concentration. T h e results of these titrations indicated that nutrient mixture F7 was not o p t i m a l for the h a m s t e r cell line. A n e w mixture, F10, ( T a b l e I) was p r e p a r e d , containing e a c h nutrient at the o p t i m u m level suggested b y the titrations. This mixture was f o u n d to be superior to F7, both for s e r u m - f r e e growth of the h a m s t e r o v a r y line, a n d for growth of a n u m b e r of other cell lines in s e r u m c o n t a i n i n g media. MATERIALS
AND
METHODS
Cell lines.--STI~AIN CHD-3 is a subclone of diploid Chinese hamster ovary strain CHBOC1D1, which was used in the development of nutrient mixture F7 [2]. It appears to be identical in all particulars, including chromosome number and tendency to display non-disjunction to strain CHBOC1D1. Strain CHD-3 was used for all of the growth factor titrations. During those studies it was maintained in medium F9FC. Currently, medium F10HIFC is used. Strain CHD-3 and its parental strains have been in continuous culture for 4} years. STRAIN CHL-1 was derived from the lung of a newborn Chinese hamster. This strain, which has been in continuous culture for over two years, exhibits a predominantly diploid chromosome pattern similar to that previously described for
1 Contribution No. 195. This work was aided by a grant from the National Foundation, Inc., and U.S.P.H.S. Graduate Training Grant No. 2G-781. Experimental Cell Research 29
R. G. Ham
516
TABLE I. Composition of nutrient mixture FIO.
Stock solution
Amount used per l i t e r of F I O
A1
10 ml (100 x )
As
Aa
A4
10 m I ( 1 0 0 ×
10 m l ( 1 0 0 ×
10 m l ( 1 0 0 x
Component
L-Arginine HC1
I0 ml (100 x
21.1
Final concentration in nutrient m i x t u r e ]710 (moles/l)
1.0 × 1 0 -8
L-Histidine HC1
2.10
1.0 x 1 0 - 4
L-Lysine HC1
2.93
1.0 x 10 -~
L-Tryptophane
0.060
3.0 x 1 0 - 6
L-Methionine
0.448
3.0 x 10-5
L-Phenylalanine
0.496
3.0 x 10-~
L-Tyrosine
0.181
1.0 x 10 _5
L-Alanine
0.891
1.0 x 10 -4
Glycine
0.751
1.0 x 10 -4
L-Serine
1.05
1.0 x 10 -4
L-Tbreonine
0.357
3 . 0 x 10 - s
L-Aspartic acid
1.33
1,0 x 10 -4
L-Glutamic
1.47
1.0 × 10 -4
acid
L-Asparagine • H~O
Aa
Concentration in stock solution (g/l)
1.50
1.0 × 10 - a
L-Glutamine
14.62
1.0 x 10 - a
L-Isoleucine
0.26
2 . 0 x 10 -5
L-Leucine
1.31
1.0 × 1 0 -4
L-Proline
1.15
1.0 x 10 -4
L-Valine
0.35
3.0 × 10 -5
3.15
2.0
A6
10 n i l ( 1 0 0 x )
L-cysteine HC1
B
10 m l ( l O O x )
Biotin
0.0024
1.0 x 10 -7
Calcium pantothenate
0.0715
3.0 x 10 -6
Choline chloride
0.0698
5.0 x 10 -6
myo-Inositol
0.0541
3 . 0 x 10 -6
Niacinamide
0,0615
5 . 0 x 10 -6
0.0206
1.0 x 10 -6
Pyridoxine
HC1
Riboflavin
D
10 m l ( 1 0 0 × )
1.0 m l ( 1 0 0 0 x )
ExperimeNal Cell Research 2 9
10 -4
0.0376
1.0 x 1 0 -~
0.1012
3.0 x 10 -6
Hypoxanthine
0.408
3.0 x 10 -5
Folic acid
0.132
3.0 x 10 -6
Thymidine
0.0727
3 . 0 × 10 -6
Vitamin
0.136
1.0
x
10 -6
FeSO~ a
0.834
3.0
x
10-6
CuSO4 a
0.0025
1.0
x
10-s
ZnSO4 a
0.0288
1.0 x 10 -~
Thiamine C
x
HC1
Bi~
Ceil culture nutrient solution
517
Table I - - continued
Concentration in stock solution (g/l)
Final concentration in nutrient mixture (moles/l)
4.41
3.0 x 10 -4
15.28
6.2 x 10 _4
Stock solution
Amount used per liter of FIO
E
10 ml (100 x )
CaC12 • 2H20
F
10 ml (100 x )
MgSO~ • 7H20
G
50 ml (20 ×)
NaCI ~ KC1 b NaeHPO 4 • 7H20 KH2PO 4
L
1.0 ml (1000 x )
Lipoie acid
0.200
il.0 × 10 -6
P
1.0 ml
Phenol red
1.2
3.3 x 10 .6
None s None s None s
1.10 g 0.110 g 1.20 g
Glucose Sodium pyruvate NaHCO3 b
----
6.1 x 10 -3 1.0 × 10 -s 1.43 x 10-2
Component
148. 5.7 5.8 1.66
1.2 3.8 1.1 4.8
x 10 -1 x 10 -3 x 10 .8 x 10 .4
"Specpure" Grade, Johnson, Matthey and Co., London. "Biological" Grade, Baker and Addamson, New York. c No stock solutions. Dry reagents are added directly to the final mixture.
C h i n e s e h a m s t e r o v a r y line C H B O C 1 [9] a n d its s u b c l o n e s , C H B O C 1 D 1 [2] a n d C H D - 3 . S t o c k s of C H L - 1 are c u r r e n t l y m a i n t a i n e d in m e d i u m F 1 0 H I F C . STRAIN S 3 - 9 - I V is a s u b c l o n a l s t r a i n d e r i v e d f r o m H e L a c l o n e $3, a n d h a s b e e n d e s c r i b e d e l s e w h e r e [6]. S t o c k c u l t u r e s h a v e g e n e r a l l y b e e n m a i n t a i n e d in m e d i u m N 1 6 H H F [3]. STRAIN H F 3 6 5 is a d i p l o i d h u m a n line w i t h a n o r m a l f e m a l e k a r y o t y p e i s o l a t e d in F e b r u a r y 1959 f r o m a p a t i e n t w i t h f e m a l e p s e u d o h e r m a p h r o d i t i s m [10]. T h e p r e s e n t c u l t u r e w a s d e r i v e d f r o m cells f r o z e n in J u l y 1959 a n d t h a w e d f i v e m o n t h s b e f o r e t h e e x p e r i m e n t s d e s c r i b e d in t h i s p a p e r . T h u s , t h e cells u s e d h a d b e e n a t o t a l of t e n m o n t h s in a c t i v e c u l t u r e . S t o c k c u l t u r e s are c u r r e n t l y m a i n t a i n e d in m e d i u m F10FC~HC15. Synthetic nutrient solution.--Nutrient M i x t u r e F 1 0 is n o r m a l l y p r e p a r e d f r o m c o n c e n t r a t e d s t o c k s o l u t i o n s , as i n d i c a t e d in T a b l e I. STOCKS A1, A2, As, As, B, E , F, a n d G are p r e p a r e d b y d i s s o l v i n g t h e c o m p o n e n t s in t r i p l e d i s t i l l e d w a t e r ( t . d . w . ) w i t h v i g o r o u s m e c h a n i c a l s t i r r i n g as n e e d e d . STOCK A 4 is p r e p a r e d as follows: T h e a s p a r t i c a n d g l u t a m i c a c i d s are a d d e d t o t h e f i n a l v o l u m e of t . d . w . , t o g e t h e r w i t h 1.2 rag~1 of p h e n o l r e d (1.0 ml/1 of s t o c k P). 1.0 N N a O H is t h e n a d d e d s l o w l y w i t h v i g o r o u s m e c h a n i c a l s t i r r i n g u n t i l a n o r a n g e color w h i c h d o e s n o t f a d e is o b t a i n e d a n d t h e solids are c o m p l e t e l y d i s s o l v e d . T h e g l u t a m i n e a n d a s p a r a g i n e are t h e n d i s s o l v e d in t h e s o l u t i o n a n d 1.0 N N a O H is a g a i n a d d e d u n t i l t h e i n d i c a t o r is o r a n g e . 33 - 631808
Experimental Cell Research 29
518
R. G. Ham
STOCK A 6 is readily prepared b y dissolving the cysteine HC1 in t.d.w. However, on standing, it gradually forms a highly insoluble precipitate (eystine) due to air oxidation. W h e n this occurs, the solution m u s t be discarded, since the precipitate does n o t readily redissolve on dilution into the final m e d i u m , and since the eysteine level is critical [2]. STOCK C is prepared as follows: The h y p o x a n t h i n e is completely dissolved in the final v o l u m e of t.d.w, with h e a t i n g a n d vigorous stirring. The solution is cooled and the folio acid is added. J u s t sufficient 1.0 N N a O H to dissolve the folie acid completely is t h e n added (not over 2-3 ml/1 of solution C). The t h y m i d i n e and v i t a m i n B12 are t h e n added a n d dissolved with additional stirring. STOCK D is prepared b y dissolving the "speepure" grade salts in t.d.w, a n d adding 0.5 ml/1 of 12 N HC1 to p r e v e n t gradual precipitation of the iron. STOCK L is prepared b y first dissolving the lipoie acid completely in a few drops of 1.0 N N a O H a n d t h e n diluting to the final volume with t.d.w. The lipoie acid dissolves slowly, b u t is actually quite soluble in 1.0 N NaOH. STOCK P is prepared b y dissolving 1.2 g phenol red a n d 0.4 g sodium b i c a r b o n a t e in 1 1 of t.d.w. Stocks A4, A6, B, a n d C should be stored frozen. Stocks A1, A2, A3, As, a n d L should be refrigerated or frozen. Stocks D, E, F, G, a n d P are stable at room t e m p e r a t u r e as long as t h e y are kept sterile a n d t i g h t l y stoppered. All solutions stored w i t h o u t freezing should be sterilized b y Millipore filtration. (Stocks E, F, a n d G m a y be autoclaved if it is more convenient.) The biochemieals used in n u t r i e n t m i x t u r e F10 are the best commercially available grades. The inorganie salts are " a n a l y t i c a l reagent" grade, except for the following: the sodium chloride, p o t a s s i u m chloride, a n d sodium b i c a r b o n a t e are Baker a n d A d d a m s o n "Biological" grade; a n d the ferrous sulphate, zinc sulphate, a n d copper sulphate are J o h n s o n a n d M a t t h e y "Specpure" grade. The water used is distilled once in a m e t a l still a n d twiee more in a quartz Hereaus "Bidistill" apparatus. The amino acid stock solutions as described are designed to p e r m i t easy omission of related groups for n u t r i t i o n a l studies. If desired, stocks A1, A~, A3, A4, and A 5 m a y be combined into a single 20 x stock, which should be stored frozen. PREPARATION OF F10 FROM STOCK SOLUTtONS: N u t r i e n t m i x t u r e F10 is prepared as follows: All the c o m p o n e n t s except the sodium b i c a r b o n a t e are added to t.d.w. a n d diluted to final volume. 1.0 N N a O H is t h e n added with stirring u n t i l the indicator becomes orange (pH 7.2-7.4). Finally, the sodium b i c a r b o n a t e is added a n d the solution is sterilized b y Millipore filtration a n d dispensed aseptically into sterile storage bottles. Short-term storage is satisfactory at 4°C. Freezing below - 2 0 ° C is r e e o m m e n d e d for storage of more t h a n a m o n t h . Growth m e d i a . - - T a b l e I I describes the growth media prepared from n u t r i e n t m i x t u r e F10, a n d lists the uses of each. All of these media are sterilized b y Millipore filtration, a n d should be stored frozen if kept for more t h a n two weeks. N u t r i e n t m i x t u r e F10 a n d all media derived from it are designed for use with a humidified atmosphere of 5 per cent carbon dioxide in air. Culture techniques.--Proeedures for m a i n t e n a n c e of stock cultures a n d p l a t i n g of single cell suspensions are the same as those previously described [2, 3], except t h a t the media listed in Table I I are used. Measurement of colony s i z e . - - G r o w t h rate is often more sensitive to n u t r i e n t
Experimental Cell Research 29
Cell cullure nutrient solution
519
TABLE II. Growth media prepared from nutrient mixture FIO. ml/1
Medium FIOAF
F10 Fetuin (50 mg/ml) a Albumin (250 mg/ml)b
978 20 2
Use: Serum-free growth; nutritional studies: Chinese hamster strains CHD-3 and CHL-1 and HeLa strain S3-9-IV. ml/1
Medium FIOFlo o
F10 Fetuin (50 m g / m l ) a Use: Nutritional studies: Chinese hamster strain CHD-3
008 2
per 1
Medium FIOHIFC
F10 Baeto Heart Infusion Broth Tested Fetal Calf Serum
980 ml 0.50 g 20 ml
Use: Control medium for single cell plating experiments; maintenance of stock cultures: Chinese hamster strains CHD-3 and CHL-1 ml/1
Medium FIOFCsHCI5
F10 Tested Fetal Calf Serum Tested H u m a n Cord Serum
800 50 150
Use: Single cell plating and maintenance of stock cultures: H u m a n diploid strain HF365 ml/1
Medium F10FCIsHC ~
FIO Tested Fetal Calf Serum Tested H u m a n Cord Serum
800 150 50
Use: Short term culture of h u m a n white blood cells for chromosomal analysis a Cf. [3] for preparation of fetuin. H u m a n Serum Albumin, " A l b n m i s o l " , Merck Sharpe and Dohme. c o n c e n t r a t i o n t h a n p l a t i n g efficiency. U n f o r t u n a t e l y , t h e closely p a c k e d g r o w t h p a t t e r n of t h e f i b r o b l a s t - l i k e cells u s e d in t h e s e s t u d i e s g e n e r a l l y m a k e s i t i m p o s s i b l e t o c o u n t t h e n u m b e r of cells p e r colony. C o l o n y size c a n b e r o u g h l y q u a n t i t a t e d , however, by placing the stained colony over a round opening in a mask over a p h o t o c e l l , a n d d e t e r m i n i n g p h o t o m e t r i c a l l y h o w m u c h of t h e o p e n i n g is c o v e r e d b y t h e colony. A n a r b i t r a r y scale of p h o t o c e l l u n i t s is u s e d , in w h i c h 0 c o r r e s p o n d s t o n o l i g h t a b s o r p t i o n , a n d 50 t o c o m p l e t e o p a c i t y . F o r a n y g i v e n t e s t series, a n o p e n i n g Experimental Cell Besearch 29
R. G. Ham
520
is used which is approximately the same diameter as the largest colonies in the series. Generally the largest colony, or in some cases, the largest five colonies on each plate are measured. This technique is limited in that it can only be used to compare colonies whose density of growth is similar. A large, diffuse colony will give a higher reading than a small, densely packed colony containing exactly the same number of cells. However, colony morphology depends primarily on the protein or serum supplement, and tends to be constant for tests carried out entirely in one type of medium. In such tests, the photometrically determined "colony areas" closely parallel visual estimates of colony size. RESULTS
Growth factor titrations.--Optimum c o n c e n t r a t i o n s for i n d i v i d u a l g r o w t h factors d e t e r m i n e d b y titration in m e d i u m F 7 A F o n l y a p p r o x i m a t e the c o m p o s i t i o n of a fully b a l a n c e d m e d i u m , since e a c h o p t i m u m is d e t e r m i n e d with other factors not at o p t i m u m levels. Nevertheless, the analysis of s u c h d a t a r e s u l t e d in a n i m p r o v e d n u t r i e n t m i x t u r e , F10, w h i c h is s u p e r i o r to F7 for s e v e r a l cell lines. Selected titrations w h i c h w e r e p a r t i c u l a r l y significant in the d e v e l o p m e n t of F10 are p r e s e n t e d below. AR6ININE: T h e c o n c e n t r a t i o n of arginine h a s little effect on plating efficiency o v e r a r a t h e r w i d e r a n g e (Fig. 1). H o w e v e r , c o l o n y size is s h a r p l y d e p e n d e n t on the arginine level, w i t h o p t i m u m g r o w t h o c c u r r i n g n e a r 1 × 10 -3 m o l a r arginine, far a b o v e the level of 3.6 × 10-~ m o l a r f o u n d in F7. TRYPTOPHANE: S e r u m a l b u m i n w a s o m i t t e d f r o m the m e d i u m u s e d for t r y p t o p h a n e titrations, since the c o m m e r c i a l p r e p a r a t i o n contains s o d i u m a c e t y l t r y p t o p h a n a t e as a stabilizing agent. T h e d a t a ( T a b l e I I I ) indicate a b r o a d o p t i m u m r a n g e b e t w e e n 10-7 a n d 10 -5 m o l a r , a n d a n a b s o l u t e toxicity of t r y p t o p h a n e at c o n c e n t r a t i o n s a b o v e 1 × 10 4 M. T h e a d d i t i o n of a l b u m i n i m p r o v e s g r o w t h in the o p t i m u m range, a n d also p a r t i a l l y r e v e r s e s the toxicity of higher c o n c e n t r a t i o n s of t r y p t o p h a n e . 4O
40 u
30
30
20
20
o o
o 10u
Concentration of arginine (moles per [iter)
Experimental Cell Research 29
Fig. 1.--Response of strain CHD-3 to arginine. Basal medium: F7AF [2] minus arginine. [], plating efficiency; ©, colony area.
Cell culture nulrienl solulion
521
PROL1NE: Unlike most well studied mammalian cells, strain CHD-3 requires proline for colonial growth (Table IV). GLYCINE, SERINE: In medium F7AF from which glutamic acid, aspartic acid, serine, and glyeine are omitted, either serine or glycine is needed for colonial growth of strain CHD-3 (Fig. 2). These data demonstrate how narrow TABLE III. Response of strain CHD-3 to tr~tptophane and albumin, a
Tryptophane concentration
M/1
Plating efficiency
Colony
%
No Albumin
Plus Albuminb
area (photocell units) ~ No albumin Plus albumin
0
26
62
2
1 × 10 -s
31
--
2
--
3 × 10 -s
43
--
3
--
1 × 1 0 -7
47
--
10
--
1 x 10-~
43
60
lO
19
3 x 10 -6
46
--
10
--
2 × 10-~
43
--
11
--
5 x 10-5
30
--
8
--
1 × 10 -4
20
--
6
--
2 ×10 -~
0
--
0
--
3 × 10 - 4
0
33
0
• ~
8
9
a B a s a l M e d i u m : F 7 A F [2] m i n u s T r y p t o p h a n e and serum albumin. b Human serum albumin ("Albumisol", Merck, Sharpe and Dohme), 0.5 mg/ml. This preparat i o n a l s o i n t r o d u c e s 4 × 1 0 - 5 M/1 e a c h s o d i u m a c e t y l t r y p t o p h a n a t e and sodium caprylate.
the optimum concentration range is for some nutrients, and strongly emphasize the need for a precisely balanced medium. IRON: The iron requirement of strain CHD-3 has already been described [2]. In an attempt to determine whether the effect was due to iron itself or to trace impurities, spectrographically analyzed "Specpure" grade iron compounds were obtained. In the presence of copper, zinc, and lipoic acid (see below), the high purity iron compounds not only replaced the "analytical reagent" grade ferric chloride previously used, but frequently resulted in improved growth, particularly in media containing very low levels of proteins. Because of this benefit, apparently due to the absence of toxic impurities, the "Specpure" grade of ferrous sulphate is now routinely used in the preparation of nutrient mixture F10. COPPER, ZINC, LiPom ACID: Small amounts of copper, zinc, and lipoic acid added either singly or together frequently improve growth of strain Experimenlal Cell Research 29
522
R. G. Ham
CHD-3, particularly in media containing very low levels of protein. Although the present data are not adequate to claim definite stimulation by any one of these factors, all three have been included as routine components of F t 0 in order to avoid possible difficulties due to borderline deficiencies. OPTIMUM LEVELS: For each titration, the growth and plating responses of the cells were plotted on semi-logarithmic graph paper against the logaTABLE IV. Response of strain CHD-3 to proline. ~ Proline concentration
M/1
0 1 x 10-6
Plating efficiency
0 0
Appearance of colonies b
---
3 x 10-8
0
1 x 10-5
57
3 x 10-5
61
normal
1 x 10 - 4
56
normal
3 x 10 -~
57
normal
1 × 10 -3
50
normal
a B a s a l M e d i u m : F 7 A F [2] m i n u s p r o l i n e . b Visual estimate only. This experiment was performed for measuring colony density.
-small
prior to development
of a p p a r a t u s
rithms of the nutrient concentrations (as in Fig. 1). The optimum range within which both plating efficiency and colony size remained unchanged was then determined, and the middle of this range on the semi-log plot was chosen as the optimum level for use in F10. For completeness and for applicability to a wide variety of cell lines, all 20 amino acids were included in F10, although the data suggested that several of them could be eliminated without noticeable effect on the growth of strain CHD-3. Results obtained using FIO media.--STRAIN CHD-3: Replacing nutrient mixture F7 with F10 has increased the growth rate of strain CHD-3 in serumfree media and has resulted in more consistently high plating efficiencies. Single cell plating experiments in m e d i u m F10AF now yield satisfactory colonies in 7 to 9 days, whereas 10 to 12 days were required using m e d i u m F7AF. Use of F10 has also reduced the amount of protein needed for satisfactory plating. W h e n sufficient care is taken to exclude environmental toxicities, the Experimental Cell Research 2 9
Cell culturenutrielTlsolution
523
serum albumin can be completely eliminated, and the fetuin can be reduced to 0.1 mg/ml ( m e d i u m F10F~00) or even lower. Although the results are still rather variable at these low protein levels, well formed colonies can usually be obtained in 8 to 10 days. Thus, it is now possible to plate strain CHD-3 with fair reliability in a defined nutrient solution supplemented with only 100 micrograms of purified protein per ml. 80
.49.~
~
70 8 B 60
~- so
Fig. 2 . - - R e s p o n s e of strain CHD-3 to glyeine or serine. Basal medium: F 7 A F [2] minus glycine, serine, glutamie acid and aspartic acid [], glycine; O , serine.
///
i\\
3O m 20
10 ~ 10-s 10_4 10 3 Concentration of amino acid (moles per hter)
10-2
Growth of strain CHD-3 in serum-containing media has also been greatly improved. Use of F10 has reduced the fetal calf serum needed for consistently good results from 15 per cent to 2 per cent or less, and also has m a d e possible the use of m a n y lots of fetal calf serum which would not support growth of strain CHD-3 at all in F7 media. The major factor responsible for this difference appears to be the increased level of arginine in F10. Addition of arginine to F7 permits colony formation using serum lots which otherwise would not support growth in F7, but which would support growth in F10 media. Growth of strain CHD-3 in m e d i u m F10HIFC is very good. Because of its low serum content, F10HIFC is a very economical medium. Preliminary experiments (Table V) suggest that further econom y can p r o b a b l y be accomplished b y replacing the relatively scarce fetal calf serum with a wide variety of other sera. However, the long term effects of these other sera on diploid cell lines must first be determined. In m a n y cases, nutrient mixture F10 plus two per cent fetal calf serum is satisfactory as a m e d i u m without added heart infusion broth. However, in a n u m b e r of low-serum and serum-free tests with several cell lines, heart infusion broth has tended to produce a slight overall i m p r o v e m e n t in growth, and also to reduce the frequency of unexplained growth failures. Since it appears to improve both reliability and growth, Baeto heart infusion broth has Experimenlal Cell Research 29
R. G. H a m
524
b e e n r o u t i n e l y i n c o r p o r a t e d i n t o m e d i u m F 1 0 H I F C , w h i c h is u s e d p r i m a r i l y for maintaining stock cultures and as a control in plating experiments. STRAIN C H L - I : T h i s s t r a i n , w h i c h c o u l d n o t b e p l a t e d at a l l in m e d i u m F7AF, plates well in medium F10AF, although colonial growth cannot be o b t a i n e d i n F10F100 o r s i m i l a r l o w p r o t e i n m e d i a w h i c h w i l l s u p p o r t p l a t i n g of CHD-3. TABLE V. Response of strain CHD-3 to various seraf Serum concentration 1%
Type of serum b
Plating efficiency (%)
Fetal calf Normal calf Ox Pregnant cow Sheep Goat Pig Horse
85 66 61 80 81 81 74 88
2% Colony density Plating (photoeell efficiency units) (%) 11 7 8 8 9 10 14c 11
90 72 70 89 81 73 87 84
5%
Colony density (photocell units) 12 10 10 13 7 5 12c 12
Plating efficiency (%)
Colony density (photocell units)
86 74 86 94 74 77 89 93
11 11 ll 12 4 6 9e 10
a Basal Medium: F10 plus 0.5 mg/ml Bacto heart infusion broth. b All sera were obtained from Colorado Serum Co., Denver, Colo. c Colonies diffuse. Larger area is responsible for higher readings, rather than increase in cell number. T h e r a p i d g r o w t h r a t e o f s t r a i n C H L - 1 m a k e s it p o t e n t i a l l y v e r y u s e f u l i n plating experiments. In serum-free medium F10AF, useful colonies can n o r m a l l y b e o b t a i n e d i n 6--7 d a y s . I n s e r u m - c o n t a i n i n g m e d i u m F 1 0 H I F C , v e r y d e n s e c o l o n i e s , e s t i m a t e d to c o n t a i n 1000 o r m o r e cells, c a n r o u t i n e l y b e o b t a i n e d a f t e r five d a y s ' i n c u b a t i o n , c o r r e s p o n d i n g to a d o u b l i n g t i m e o f 12 h r o r less. STRArN $ 3 - 9 - I V H e L a : G r o w t h o f t h i s s t r a i n i n m e d i u m F 1 0 A F is a b o u t e q u i v a l e n t to its g r o w t h i n t h e s e r u m - f r e e m e d i u m p r e v i o u s l y r e p o r t e d b y F i s h e r , P u c k a n d S a t o [1]. G r o w t h o f t h e H e L a s t r a i n i n m e d i u m F 1 0 H I F C is g o o d , a l t h o u g h t h e g r o w t h r a t e is s l o w e r t h a n i n m e d i u m N 16 H H F I3]. T h i s r e d u c e d r a t e o f g r o w t h c a n b e o v e r c o m e b y i n c r e a s i n g t h e f e t a l c a l f s e r u m c o n c e n t r a t i o n to 10 o r 15 p e r cent.
Experimental Cell Research 29
Cell culture nutrient soluliou
525
STRAIN HF365: Use of F10 containing media for this h u m a n diploid cell line has resulted in better and more consistent growth than was obtained with previous media. Well formed colonies of 1000 to 4000 cells are now reliably obtained after 8 to 10 days' incubation with plating efficiencies of 40 per cent or better. All diploid h u m a n strains thus far tested have been resistant to attempts at culture in either low-serum or serum-free media. HUMAN W~I~TE BLOOD CELLS: Preliminary studies by Robinson [7] using a modification of the peripheral blood technique of Hungerford et aI. [4] indicate that excellent short term growth of h u m a n white blood cells for chromosome preparations can be obtained with a high degree of reliability when m e d i u m F10FC15HC ~ is employed. DISCUSSION
The use of single cell plating in a medium containing a m i n i m u m protein supplement has provided a powerful tool for studying the nutritional requirements of m a m m a l i a n cells in culture. Under these conditions, the small number of cells present cannot appreciably alter the composition of the surrounding medium, and the limited amount of protein offers them relatively little protection from it. Thus, their growth response provides a very sensitive index to the adequacy and freedom from toxicity of the medium being tested. The deliberate selection of nutrient concentrations m i d w a y between the lower and upper limits of the optimum range has resulted in a balanced nutrient mixture which is not only superior for the cell strain used in the initial study, but also for several others, including a diploid h u m a n line. An important difference between nutrient mixtures F7 and F10 is the increased level of arginine in the latter. Similar effects of arginine have been previously reported [81, and m a y be related to the presence of arginase in m a n y cultured cell lines [5]. SUMMARY
1. Single cell plating of diploid Chinese hamster ovary cells (Strain CHD-3) in a m e d i u m containing a m i n i m u m supplement of purified proteins has been used to determine optimum concentrations for the components of a previously described nutrient mixture, F7. 2. The more significant results of these titrations include: (a) a high arginine requirement; (b) a low toxic limit for tryptophane; (c) a requirement for proline; and (d) a narrow optimum range for serine and glycine. Experimental Cell Research 29
R. G. Ham
526
3. A new nutrient mixture, F10, has been p r e p a r e d in which each component has been a dde d at the experimentally determined o p t i m u m concentration. 4. Tests of the new nutrient mixture using strain CHD-3 revealed: (a) i m p r o v e d growth rate and reliability in a fetuin and albumin supplemented medium; (b) clonal growth at total protein concentrations as low as 100 #g/ ml.; (c) excellent growth at r e d u c e d serum concentrations; (d) satisfactory clonal growth using batches of fetal calf serum which would not support such growth in F7 media; (e) ability to use sera other than fetal calf serum, for clonal growth. 5. Tests of media p r e p a r e d from the new nutrient mixture on other cell strains revealed: (a) good growth with a fetuin and albumin supplement of strain CHL-1 Chinese hamster lung cells and strain S3-9-IV H e L a cells; (b) rapid growth of strain CHL-1 on a 2 per cent serum m e d i u m yielding dense colonies estimated to contain 1000 or more cells in five days' incubation time; (c) i m p r o v e d growth of a h u m a n diploid cell line; (d) excellent short term growth of h u m a n white blood cells for c h r o m o s o m e preparations. The author wishes to express his thanks to Dr. Theodore T. Puck for his interest in this work and for his many valuable suggestions; also to Mrs. Catherine Fogg for excellent technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8.
FISHER, H. W., PUCK, T. T. and SATO, G., J. Exptl. Med. 109, 649 (1959). HA~, R. G., Exptl. Celt Res. 28, 489 (1962). HAM, R. G. and PUCK, T. T., Methods in Enzgmologg 5, 90 (1962). HUNGEBFORD~ D. A., DONNELLY, A. J., NOWELL, P. G. and BECK, S., A m . J. H u m a n Genetics 11, 215 (1959). KLEIN, E., Exptl. Cell Res. 22, 226 (1961). MARCUS, P. I., Virologg 9, 546 (1959). I:~OBINSON, n., Personal communication. THOraAS, W. J., ZIE~LEB, D. W., SCttEPARTZ, S. A. and McLIMANS, W. F., Science 127, 591
(195s). 9. TJlo, J. H. and PUCK, T. T., J. Exptl. Med. 108,259 (1958). 10. TJio, J. H. and PUCK, T. T. and ROBINSON, A., PFOC. ~'~ratl. Acad. Sci. 45, 1008 (1959).
Experimental Cell Research 29