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Carboxyethyl γ-aminobutyric acid, a polyamine derivative molecule with a growth effect on hybridomas
Journal o f l m m u n o l o g i c a l M e t h o d s ,
77 (1985) 229 235
229
Elsevier JIM03401
Carboxyethyl 3,-Aminobutyric Acid, a Polyamine Derivative Molecule with a Growth Effect on Hybridomas Antonella Cerino l, Mara D e Amici 2, F e r n a n d o F. Fussi 3 and Giulia C.B. Astaldi Ricotti 1., J lstituto di Genetica Biochimica ed Eooluzionistica del C.N.R., Pavia, " Chntca Pedtatrtca, Universith di Pavia, Pavia, Italy, a n d ~ H e p a r Chimie S.A., Givisiez, Switzerland
(Received 26 October 1984, accepted 12 November 1984
An increased proliferation of hybridomas is induced by a polyamine derivative molecule, CEGABA (carboxyethyl gamma-aminobutyric acid). The effects of CEGABA and CR-ECGS (endothelial cell growth supplement) on hybridoma growth are similar. However, both factors are less efficient than HECS (human endothelial culture supernatant) in inducing hybridoma growth through all the steps of hybridoma production, including: recovery after fusion, cloning, and proliferation. Key words:
C E G A B A - growth factors - hybridoma cultures - media supplements
Introduction It is k n o w n that efficiency in h y b r i d o m a recovery a n d cloning is i m p r o v e d b y the presence of feeder cells in the culture m e d i u m ( H a m m e r l i n g et al., 1978; L e n h a r d t et al., 1978; F a z e k a s de S t . G r o t h a n d Scheidegger, 1980). It has been r e p o r t e d that soluble factors o b t a i n e d from h u m a n e n d o t h e l i a l culture s u p e r n a t a n t ( H E C S ) ( A s t a l d i et al., 1980, 1981; Astaldi, 1983; W e s t e r w o u d t et al., 1983), h u m a n u m b i l i c a l cord serum ( H U C S ) ( W e s t e r w o u d t et al., 1983), a n d bovine neural tissue ( C R - E C G S ) (Pintus et al., 1983) can f a v o r a b l y substitute for feeder cells in i m p r o v i n g h y b r i d o m a growth. The a d v a n t a g e s of using growth factors i n s t e a d of feeder cells are: (1) sterile c o n d i t i o n s are assured; (2) the work required to o b t a i n feeder cells from a n i m a l s is e l i m i n a t e d ; (3) h y b r i d o m a cultures are n o t c o n t a m i n a t e d b y o t h e r cells; (4) m o r e r e p r o d u c i b l e results are o b t a i n e d .
* Address correspondence to: G.C.B. Astaldi Ricotti, Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R., Via Abbiategrasso 207, 27100 Pavia, Italy. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
230 In this report we tested the effect of a polyamine derivate molecule, carboxyethyl gamma-aminobutyric acid (CEGABA) on hybridoma growth and compared it with that induced by HECS and CR-ECGS. CEGABA is a synthetic dicarboxylated polyamine derivative and is structurally related to spermidine, putreanine and isoputreanine. CEGABA was reported originally to be a growth promoting agent for plants (Curti and Fussi, 1981). Preliminary observations show a growth promoting activity in vitro on some specialized mammalian cells. Among the latter are: murine and human B lymphocytes and macrophages (G. Forni, personal communication) and secretory cells from the rat seminal vesicle and testes (J. Guardiola, personal communication).
Materials and Methods
Culture medium Hybridomas were cultured in RPMI 1640 Dutch modification (Flow Laboratories) supplemented with 10% FCS (fetal calf serum, Flow Laboratories), 2 mM MEM sodium pyruvate (Flow Laboratories), 4 mM L-glutamine (Flow Laboratories), 100 /~g/ml streptomycin, 100 U / m l penicillin at 37°C in an atmosphere of 5 6% CO 2.
Growth factors Human endothelial culture supernatant (HECS) was prepared as previously described (Astaldi et al., 1980, 1981; Astaldi, 1983). Endothelial cell growth supplement (CR-ECGS) is a commercial product from Collaborative Research, Lexington, MA. CEGABA ( H O O C - ( C H 2 ) 2 N H - ( C H 2 ) 3 COOH) was synthetized by previously described methods (Curti and Fussi, 1981) and obtained from Hepar Chimie in Givisiez, Switzerland.
Antigen A partially purified preparation of human glutamic pyruvic transaminase (GPT) was used to immunize 6-12-week-old B A L B / c mice (50 /~g/mouse for each immunization) and as an antigen to test the hybridoma supernatants by an ELISA method (Aiji et al., 1980).
Hybridoma production Spleen cells from immunized B A L B / c mice were fused with the Sp 2/0-Ag 14 non-secretory plasmacytoma line (kindly provided by Dr. G. K6hler) as previously reported (Astaldi et al., 1980), according to the method described by Galfr6 et al., 1977. Fused cells were plated in microtiter plates (0.5 and 1 × 10 s cells/well) in wells containing culture medium pure or supplemented with 10% HECS (v/v) or 30 /~g/ml CEGABA or 50 # g / m l CR-ECGS.
Limiting dilution Hybridomas were cultured at the single cell level by limiting dilution in microtiter tissue culture plates as reported (Astaldi et al., 1980) in the presence of culture
231
medium pure or supplemented with 10% HECS (v/v) or 3 0 / , g / m l CEGABA or 50 /~g/ml CR-ECGS.
[¢H]thymidine (3H-TdR) incorporation Hybridoma cells were cultured in microtiter tissue culture plates at a concentration of 100 cells/well in the presence of different concentrations of the various growth factors; the cultures were labeled after 5 days with 0.5/,Ci 3H-TdR (specific activity 2000 mCi/mmol. Radiochemical Centre, Amersham U.K.), harvested 24 h later and processed as described (Astaldi et al., 1980).
Results
Optimal concentration of the growth factors Fig. 1 shows that a significant effect of CEGABA on the growth of hybridoma 3H-thymidine
Uptake by Hybridomas in P r e s e n c e Different Growth Factors
1101 1OO
nOx< E
of
t ......
.,
.
..... t
90
80
t
•
t
""
HECS X..... X CEGABAo o ECGS ~- - - 4,
.
'
704
/
5o-
40 -
.
J
10 I
/
\ '\\
<
// o.
o. ~ 1:1eo
~,.:~ l:eO
"\~O a.3
1012.s
so 100 2~ ECGS and CEGABA (:40
-
~1:2o
ao;)l'O0 o
CONCENTRATION(~9/ml) ~1o HECS DILUTION
(V :
V)
Fig. 1. [3H]thymidine uptake by hybridomas in the presence of different growth factors. One hundred hybridoma cells were added to each well at the start of the culture in the presence of different growth factors. Incorporation of 3H-TdR was measured at day 6. Results are expressed as mean cpm of 6 determinations + standard deviation and are representative of 3 separate experiments.
232
cells was reached at 3.2 /2g/ml and this remained constant up to 200 /2g/ml. CR-ECGS induced optimal growth in the range between 25-200/~g/ml. As already reported (Astaldi et al., 1981), the effect of HECS was dose-dependent, giving an optimal growth when used at 10% (v/v). Based on these results, we decided to routinely use the following concentrations of the growth factors: HECS 10%, CEGABA 30/~g/ml, and CR-ECGS 5 0 / x g / m l (this concentration of CR-ECGS is also recommended by Collaborative Research).
Comparison of the different growth factors on hybrid recovery' after fusion After fusion, the cells were plated at 5 × 104 and 1 x l0 s cells/well. Table I shows that C E G A B A and CR-ECGS improve hybridoma recovery mainly when used to grow a rather high number of cells. In any case, HECS was far more effective. The low effect on the recovery of hybridomas after fusion observed with CEGABA and CR-ECGS can be explained by the fact that, contrary to HECS, these factors promote a more general growth of cells. Foreign cells can eventually compete with hybridoma growth.
Effect of different growth factors" on hybridomas cloning Table II shows that C E G A B A and C R - E C G S have a similar effect in promoting the growth of hybridomas cultured at the single cell level. The results obtained using
TABLE 1 EFFECT OF D I F F E R E N T GROWTH FACTORS ON THE RECOVERY OF HYBRIDS AFTER FUSION Results are expressed as following: (a) total number of wells; (b) number of wells containing growing hybrids 12 days after the fusion, and (c) number of wells containing hybridomas producing anti-GPT antibodies. Antibody production was tested 12 days after the fusion. Growth factor
Number of cells plated after fusion 0.5 x l0 s cells/well
1 × 105 cells/well
Exp. 1
Exp. 2
Exp. 3
Exp. 1
Exp. 2
Exp. 3
None
(a) (b) (c)
96 2 0
96 5 0
96 0 0
96 17 0
96 13 1
96 12 0
HECS
(a) (b) (c)
96 72 4
96 95 13
96 54 4
96 93 20
96 96 22
96 90 15
CR-ECGS
(a) (b) (c)
96 14 0
96 8 0
96 2 0
96 45 5
96 26 1
96 36 1
CEGABA
(a) (b) (c)
96 14 0
96 6 0
96 1 0
96 48 7
96 26 2
96 41 5
233 TABLE II EFFECT OF G R O W T H FACTORS ON HYBRIDOMA C L O N I N G a: 5 hybridomas were added to each well (36 wells per experiment) at the start of the culture, b: 1 hybridoma was added to each well (36 wells per experiment) at the start of the culture, c: the equivalent of 0.5 hybridoma cells was added to each well (24 wells per experiment) at the start of the culture. After 12 days, clones of hybrids became evident. Results are expressed as the mean of the wells containing growing hybridomas ± standard deviation of 3 different experiments. Number of wells containing growing hybridomas
Growth factor
None HECS CR-ECGS CEGABA
a
b
c
10±3 35±1 17±7 19±7
3±3 23±2 8±3 11±4
0.5±0.5 7 ±1 3 +1 4 ±2
CR-ECGS are in agreement with those reported by Collaborative Research who obtained circa 35% cloning efficiency by using CR-ECGS as a growth factor (Pintus et al., 1983). As already reported, HECS gives a cloning efficiency close to 100% (Astaldi et al., 1980).
Comparison of different growth factors on the proliferation of hybridomas Table III shows that in all the experiments, CR-ECGS (50 ~ g / m l ) and CEGABA (30 # g / m l ) increased the proliferation of hybridomas cultured at 100 cells/well. However, their growth promoting activity was less efficient than HECS (10%, v/v). Their effects were more similar when the 3 growth factors are used on hybridomas which are already adapted to culture conditions (experiments 9 and 10). Using CEGABA associated with CR-ECGS we found no evidence of any synergistic effect on the growth of 2 different clones of hybridomas (data not shown). TABLE IIl INCREASED PROLIFERATION OF HYBRIDOMAS BY G R O W T H FACTORS Results are expressed as: Proliferation Index (P.I.): P.I. -
cpm obtained from hybridomas cultured with growth factors cpm obtained from hybridomas cultured without growth factors
Each experiment was performed using a different hybridoma clone; the first 8 were hybrids not yet adapted to culture conditions ( - 1 month after fusion) and the last 2 were hybrids adapted to culture conditions ( - 3 months after fusion). Each experiment was calculated as mean cpm of 8 determinations. Growth factor
HECS CR-ECGS CEGABA
Experiment no.: 1
2
3
4
5
6
7
8
9
10
36.3 13.8 21.8
39.5 nd 15.1
49.7 11.2 10.6
55.1 26.0 17.4
43.6 3.9 4.0
22.2 4.4 5.5
20.6 2.2 2.1
23.2 nd 5.5
6.4 nd 2.8
4.1 2.1 3.2
234
Discussion
We have previously reported that HECS is a very powerful growth factor for hybridomas since it enhances the recovery of hybridomas after fusion, gives 100% cloning efficiency and dramatically increases the growth of hybridomas as measured by their 3H-TdR incorporation (Astaldi et al., 1980, 1981; Astaldi, 1983). However, HECS preparation requires sophisticated facilities for the culturing of human endothelial cells. We therefore investigated whether commercially available media supplements have growth promoting activity as compared to HECS. For this purpose, we have used C R - E C G S (Collaborative Research) which has been demonstrated to be a growth factor, both for endothelial cells (Maciag et al.. 1979) and hybridomas (Pintus et al., 1983) and a polyamine derivative, CEGABA. In fact it has already been shown that some cultured cells require the addition of polyamines (putrescine, spermine and spermidine) to obtain maximum growth and long term survival (Roszell et al., 1977). Unfortunately, polyamines act only in mixtures and at high concentrations they support lower growth rates and show increased toxicity to the cultured cells. Therefore, we tested a polyamine derivative, CEGABA, which has been demonstrated to have lower toxicity (Fussi and Curti. 1984) and higher growth stimulation (Curti and Fussi, 1984) in comparison to polyamines. CEGABA has not been isolated from animal tissues but some CEGABA mediated interrelationships between polyamines and gamma-aminobutyric acid have been postulated (Fussi and Curti, 1984). Furthermore, CEGABA has been demonstrated to be a growth factor for a variety of cells, cultured in vitro, including murine and human B cells and macrophages (G. Forni, personal communication). In this article, we have shown that CEGABA has a growth promoting activity on hybridomas which is similar to CR-ECGS. However, at all concentrations tested both factors are less efficient than HECS in enhancing hybridoma growth through the 3 steps of the hybridoma technology where feeder cells or growth factors are required, including recovery of hybrids after fusion, cloning of hybridomas and proliferation of hybridomas. (CEGABA and CR-ECGS showed a slightly less efficient improvement when tested on the recovery of hybridomas after fusion.) An explanation is that both factors induce a general cell growth; at the first transfer, all types of spleen cell are present and foreign cells can compete with hybridoma growth. In fact, CEGABA and CR-ECGS are more efficient when used in cultures of pure hybridomas. On the contrary, HECS, which enhances mainly the growth of hybridomas, gives a very high recovery of hybridomas after fusion. The effect of CEGABA and CR-ECGS is similar in supporting the growth of hybridomas cultured at the single cell level although a cloning efficiency of 100% was not reached. In contrast HECS consistently gave a cloning efficiency near to 100%. Both CEGABA and CR-ECGS support hybridoma proliferation, as measured by ~H-TdR incorporation, to an extent which is circa 2 25 times the controls cultured in the absence of growth factors. However, their growth activity is circa 2 10 times less efficient than that provided by HECS (Table III). The difference between the growth effect shown either by CEGABA or CR-ECGS and HECS is less pronounced when the growth factors are tested on hybridomas which are already adapted to culture
235 conditions. No synergistic effect was obtained between CEGABA and CR-ECGS indicating that these factors may act through similar mechanisms. In conclusion, both CEGABA and CR-ECGS improve hybridoma growth espec i a l l y w h e n t h e r e is n o c o n t a m i n a t i o n b y o t h e r cells. T h e s i g n i f i c a n t a d v a n t a g e o f C E G A B A o v e r o t h e r g r o w t h f a c t o r s is t h a t , as a p u r e m o l e c u l e , it p e r m i t s t h e i n v e s t i g a t o r to s t u d y s e p a r a t e l y t h e f a c t o r s i n v o l v e d in o p t i m a l cell g r o w t h in d e f i n e d m e d i a .
References Aiji, C. and N. Winkel, 1980, in: Developments in Clinical Biochemistry, Vol. I, lmmunoenzymatic Assay Techniques, ed. R. Malvano (Martinus Nijhoff, The Hague) p. 184. Astaldi, G.C.B., 1983, in: Methods in Enzymology, Vol. 92, lmmunochemical Techniques, eds. H. Van Vunekis and J.J. Langone (Academic Press, New York) p. 39. Astaldi, G.C.B., M.C. Janssen, P.M. Lansdorp, C. Willems, W.P. Zeijlemaker and F. Oosterhof, 1980, J. Immunol. 125, 1411. Astaldi, G.C.B., M.C. Janssen, P.M. Lansdorp, W.P. Zeijlemaker and C. Willems, 1981, J. lmmunol. 126, 1170. Curti, M. and F. Fussi, 1981, Ital. Pat. Appl. 21q5qA/81 and 21q6A/81. Curti, M. and F. Fussi, 1984, Ital. J. Biochem. 33, 31. Fazekas de St.Groth, S. and D. Scheidegger, 1980, J. Immunol. Methods 35, 1. Fussi, F. and M. Curti, 1984, in: Advances in Polyamines in Biomedical Science, eds. C.M. Caldarera and U. Bachrach (CLUEB, Bologna) p. 53. Galfr6, G., S.C. Howe, C. Milstein, G.W. Butcher and J.C. Howard, 1977, Nature (London) 266, 550. H~mmerling, G.J., H. Lemke, U. Nauerling, C. Hohmann, R. Wallich and K. Rajewsky, 1978, Curr. Top. Microbiol. lmmunol. 8, 100. Lenhardt, W., J. Andersen, A. Coutinho and F. Melchers, 1978, Exp. Cell Res. 111, 309. Maciag, T., J. Cerundolo, S. Ilsley, P. Kelley and R. Forand, 1979, Fed. Proc. 38, 634a. Pintus, C., J. Ranson and C. Evans, 1983, J. Immunol. Methods 61, 195. Roszell, J.A., C.J. Douglas and C.C. Irving, 1977, Can. Res. 37, 239. Westerwoudt, R.J., G. Blom, A.M. Naipal and J.J. Van Rood, 1983, J. Immunol. Methods 62, 59.
77 (1985) 229 235
229
Elsevier JIM03401
Carboxyethyl 3,-Aminobutyric Acid, a Polyamine Derivative Molecule with a Growth Effect on Hybridomas Antonella Cerino l, Mara D e Amici 2, F e r n a n d o F. Fussi 3 and Giulia C.B. Astaldi Ricotti 1., J lstituto di Genetica Biochimica ed Eooluzionistica del C.N.R., Pavia, " Chntca Pedtatrtca, Universith di Pavia, Pavia, Italy, a n d ~ H e p a r Chimie S.A., Givisiez, Switzerland
(Received 26 October 1984, accepted 12 November 1984
An increased proliferation of hybridomas is induced by a polyamine derivative molecule, CEGABA (carboxyethyl gamma-aminobutyric acid). The effects of CEGABA and CR-ECGS (endothelial cell growth supplement) on hybridoma growth are similar. However, both factors are less efficient than HECS (human endothelial culture supernatant) in inducing hybridoma growth through all the steps of hybridoma production, including: recovery after fusion, cloning, and proliferation. Key words:
C E G A B A - growth factors - hybridoma cultures - media supplements
Introduction It is k n o w n that efficiency in h y b r i d o m a recovery a n d cloning is i m p r o v e d b y the presence of feeder cells in the culture m e d i u m ( H a m m e r l i n g et al., 1978; L e n h a r d t et al., 1978; F a z e k a s de S t . G r o t h a n d Scheidegger, 1980). It has been r e p o r t e d that soluble factors o b t a i n e d from h u m a n e n d o t h e l i a l culture s u p e r n a t a n t ( H E C S ) ( A s t a l d i et al., 1980, 1981; Astaldi, 1983; W e s t e r w o u d t et al., 1983), h u m a n u m b i l i c a l cord serum ( H U C S ) ( W e s t e r w o u d t et al., 1983), a n d bovine neural tissue ( C R - E C G S ) (Pintus et al., 1983) can f a v o r a b l y substitute for feeder cells in i m p r o v i n g h y b r i d o m a growth. The a d v a n t a g e s of using growth factors i n s t e a d of feeder cells are: (1) sterile c o n d i t i o n s are assured; (2) the work required to o b t a i n feeder cells from a n i m a l s is e l i m i n a t e d ; (3) h y b r i d o m a cultures are n o t c o n t a m i n a t e d b y o t h e r cells; (4) m o r e r e p r o d u c i b l e results are o b t a i n e d .
* Address correspondence to: G.C.B. Astaldi Ricotti, Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R., Via Abbiategrasso 207, 27100 Pavia, Italy. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
230 In this report we tested the effect of a polyamine derivate molecule, carboxyethyl gamma-aminobutyric acid (CEGABA) on hybridoma growth and compared it with that induced by HECS and CR-ECGS. CEGABA is a synthetic dicarboxylated polyamine derivative and is structurally related to spermidine, putreanine and isoputreanine. CEGABA was reported originally to be a growth promoting agent for plants (Curti and Fussi, 1981). Preliminary observations show a growth promoting activity in vitro on some specialized mammalian cells. Among the latter are: murine and human B lymphocytes and macrophages (G. Forni, personal communication) and secretory cells from the rat seminal vesicle and testes (J. Guardiola, personal communication).
Materials and Methods
Culture medium Hybridomas were cultured in RPMI 1640 Dutch modification (Flow Laboratories) supplemented with 10% FCS (fetal calf serum, Flow Laboratories), 2 mM MEM sodium pyruvate (Flow Laboratories), 4 mM L-glutamine (Flow Laboratories), 100 /~g/ml streptomycin, 100 U / m l penicillin at 37°C in an atmosphere of 5 6% CO 2.
Growth factors Human endothelial culture supernatant (HECS) was prepared as previously described (Astaldi et al., 1980, 1981; Astaldi, 1983). Endothelial cell growth supplement (CR-ECGS) is a commercial product from Collaborative Research, Lexington, MA. CEGABA ( H O O C - ( C H 2 ) 2 N H - ( C H 2 ) 3 COOH) was synthetized by previously described methods (Curti and Fussi, 1981) and obtained from Hepar Chimie in Givisiez, Switzerland.
Antigen A partially purified preparation of human glutamic pyruvic transaminase (GPT) was used to immunize 6-12-week-old B A L B / c mice (50 /~g/mouse for each immunization) and as an antigen to test the hybridoma supernatants by an ELISA method (Aiji et al., 1980).
Hybridoma production Spleen cells from immunized B A L B / c mice were fused with the Sp 2/0-Ag 14 non-secretory plasmacytoma line (kindly provided by Dr. G. K6hler) as previously reported (Astaldi et al., 1980), according to the method described by Galfr6 et al., 1977. Fused cells were plated in microtiter plates (0.5 and 1 × 10 s cells/well) in wells containing culture medium pure or supplemented with 10% HECS (v/v) or 30 /~g/ml CEGABA or 50 # g / m l CR-ECGS.
Limiting dilution Hybridomas were cultured at the single cell level by limiting dilution in microtiter tissue culture plates as reported (Astaldi et al., 1980) in the presence of culture
231
medium pure or supplemented with 10% HECS (v/v) or 3 0 / , g / m l CEGABA or 50 /~g/ml CR-ECGS.
[¢H]thymidine (3H-TdR) incorporation Hybridoma cells were cultured in microtiter tissue culture plates at a concentration of 100 cells/well in the presence of different concentrations of the various growth factors; the cultures were labeled after 5 days with 0.5/,Ci 3H-TdR (specific activity 2000 mCi/mmol. Radiochemical Centre, Amersham U.K.), harvested 24 h later and processed as described (Astaldi et al., 1980).
Results
Optimal concentration of the growth factors Fig. 1 shows that a significant effect of CEGABA on the growth of hybridoma 3H-thymidine
Uptake by Hybridomas in P r e s e n c e Different Growth Factors
1101 1OO
nOx< E
of
t ......
.,
.
..... t
90
80
t
•
t
""
HECS X..... X CEGABAo o ECGS ~- - - 4,
.
'
704
/
5o-
40 -
.
J
10 I
/
\ '\\
<
// o.
o. ~ 1:1eo
~,.:~ l:eO
"\~O a.3
1012.s
so 100 2~ ECGS and CEGABA (:40
-
~1:2o
ao;)l'O0 o
CONCENTRATION(~9/ml) ~1o HECS DILUTION
(V :
V)
Fig. 1. [3H]thymidine uptake by hybridomas in the presence of different growth factors. One hundred hybridoma cells were added to each well at the start of the culture in the presence of different growth factors. Incorporation of 3H-TdR was measured at day 6. Results are expressed as mean cpm of 6 determinations + standard deviation and are representative of 3 separate experiments.
232
cells was reached at 3.2 /2g/ml and this remained constant up to 200 /2g/ml. CR-ECGS induced optimal growth in the range between 25-200/~g/ml. As already reported (Astaldi et al., 1981), the effect of HECS was dose-dependent, giving an optimal growth when used at 10% (v/v). Based on these results, we decided to routinely use the following concentrations of the growth factors: HECS 10%, CEGABA 30/~g/ml, and CR-ECGS 5 0 / x g / m l (this concentration of CR-ECGS is also recommended by Collaborative Research).
Comparison of the different growth factors on hybrid recovery' after fusion After fusion, the cells were plated at 5 × 104 and 1 x l0 s cells/well. Table I shows that C E G A B A and CR-ECGS improve hybridoma recovery mainly when used to grow a rather high number of cells. In any case, HECS was far more effective. The low effect on the recovery of hybridomas after fusion observed with CEGABA and CR-ECGS can be explained by the fact that, contrary to HECS, these factors promote a more general growth of cells. Foreign cells can eventually compete with hybridoma growth.
Effect of different growth factors" on hybridomas cloning Table II shows that C E G A B A and C R - E C G S have a similar effect in promoting the growth of hybridomas cultured at the single cell level. The results obtained using
TABLE 1 EFFECT OF D I F F E R E N T GROWTH FACTORS ON THE RECOVERY OF HYBRIDS AFTER FUSION Results are expressed as following: (a) total number of wells; (b) number of wells containing growing hybrids 12 days after the fusion, and (c) number of wells containing hybridomas producing anti-GPT antibodies. Antibody production was tested 12 days after the fusion. Growth factor
Number of cells plated after fusion 0.5 x l0 s cells/well
1 × 105 cells/well
Exp. 1
Exp. 2
Exp. 3
Exp. 1
Exp. 2
Exp. 3
None
(a) (b) (c)
96 2 0
96 5 0
96 0 0
96 17 0
96 13 1
96 12 0
HECS
(a) (b) (c)
96 72 4
96 95 13
96 54 4
96 93 20
96 96 22
96 90 15
CR-ECGS
(a) (b) (c)
96 14 0
96 8 0
96 2 0
96 45 5
96 26 1
96 36 1
CEGABA
(a) (b) (c)
96 14 0
96 6 0
96 1 0
96 48 7
96 26 2
96 41 5
233 TABLE II EFFECT OF G R O W T H FACTORS ON HYBRIDOMA C L O N I N G a: 5 hybridomas were added to each well (36 wells per experiment) at the start of the culture, b: 1 hybridoma was added to each well (36 wells per experiment) at the start of the culture, c: the equivalent of 0.5 hybridoma cells was added to each well (24 wells per experiment) at the start of the culture. After 12 days, clones of hybrids became evident. Results are expressed as the mean of the wells containing growing hybridomas ± standard deviation of 3 different experiments. Number of wells containing growing hybridomas
Growth factor
None HECS CR-ECGS CEGABA
a
b
c
10±3 35±1 17±7 19±7
3±3 23±2 8±3 11±4
0.5±0.5 7 ±1 3 +1 4 ±2
CR-ECGS are in agreement with those reported by Collaborative Research who obtained circa 35% cloning efficiency by using CR-ECGS as a growth factor (Pintus et al., 1983). As already reported, HECS gives a cloning efficiency close to 100% (Astaldi et al., 1980).
Comparison of different growth factors on the proliferation of hybridomas Table III shows that in all the experiments, CR-ECGS (50 ~ g / m l ) and CEGABA (30 # g / m l ) increased the proliferation of hybridomas cultured at 100 cells/well. However, their growth promoting activity was less efficient than HECS (10%, v/v). Their effects were more similar when the 3 growth factors are used on hybridomas which are already adapted to culture conditions (experiments 9 and 10). Using CEGABA associated with CR-ECGS we found no evidence of any synergistic effect on the growth of 2 different clones of hybridomas (data not shown). TABLE IIl INCREASED PROLIFERATION OF HYBRIDOMAS BY G R O W T H FACTORS Results are expressed as: Proliferation Index (P.I.): P.I. -
cpm obtained from hybridomas cultured with growth factors cpm obtained from hybridomas cultured without growth factors
Each experiment was performed using a different hybridoma clone; the first 8 were hybrids not yet adapted to culture conditions ( - 1 month after fusion) and the last 2 were hybrids adapted to culture conditions ( - 3 months after fusion). Each experiment was calculated as mean cpm of 8 determinations. Growth factor
HECS CR-ECGS CEGABA
Experiment no.: 1
2
3
4
5
6
7
8
9
10
36.3 13.8 21.8
39.5 nd 15.1
49.7 11.2 10.6
55.1 26.0 17.4
43.6 3.9 4.0
22.2 4.4 5.5
20.6 2.2 2.1
23.2 nd 5.5
6.4 nd 2.8
4.1 2.1 3.2
234
Discussion
We have previously reported that HECS is a very powerful growth factor for hybridomas since it enhances the recovery of hybridomas after fusion, gives 100% cloning efficiency and dramatically increases the growth of hybridomas as measured by their 3H-TdR incorporation (Astaldi et al., 1980, 1981; Astaldi, 1983). However, HECS preparation requires sophisticated facilities for the culturing of human endothelial cells. We therefore investigated whether commercially available media supplements have growth promoting activity as compared to HECS. For this purpose, we have used C R - E C G S (Collaborative Research) which has been demonstrated to be a growth factor, both for endothelial cells (Maciag et al.. 1979) and hybridomas (Pintus et al., 1983) and a polyamine derivative, CEGABA. In fact it has already been shown that some cultured cells require the addition of polyamines (putrescine, spermine and spermidine) to obtain maximum growth and long term survival (Roszell et al., 1977). Unfortunately, polyamines act only in mixtures and at high concentrations they support lower growth rates and show increased toxicity to the cultured cells. Therefore, we tested a polyamine derivative, CEGABA, which has been demonstrated to have lower toxicity (Fussi and Curti. 1984) and higher growth stimulation (Curti and Fussi, 1984) in comparison to polyamines. CEGABA has not been isolated from animal tissues but some CEGABA mediated interrelationships between polyamines and gamma-aminobutyric acid have been postulated (Fussi and Curti, 1984). Furthermore, CEGABA has been demonstrated to be a growth factor for a variety of cells, cultured in vitro, including murine and human B cells and macrophages (G. Forni, personal communication). In this article, we have shown that CEGABA has a growth promoting activity on hybridomas which is similar to CR-ECGS. However, at all concentrations tested both factors are less efficient than HECS in enhancing hybridoma growth through the 3 steps of the hybridoma technology where feeder cells or growth factors are required, including recovery of hybrids after fusion, cloning of hybridomas and proliferation of hybridomas. (CEGABA and CR-ECGS showed a slightly less efficient improvement when tested on the recovery of hybridomas after fusion.) An explanation is that both factors induce a general cell growth; at the first transfer, all types of spleen cell are present and foreign cells can compete with hybridoma growth. In fact, CEGABA and CR-ECGS are more efficient when used in cultures of pure hybridomas. On the contrary, HECS, which enhances mainly the growth of hybridomas, gives a very high recovery of hybridomas after fusion. The effect of CEGABA and CR-ECGS is similar in supporting the growth of hybridomas cultured at the single cell level although a cloning efficiency of 100% was not reached. In contrast HECS consistently gave a cloning efficiency near to 100%. Both CEGABA and CR-ECGS support hybridoma proliferation, as measured by ~H-TdR incorporation, to an extent which is circa 2 25 times the controls cultured in the absence of growth factors. However, their growth activity is circa 2 10 times less efficient than that provided by HECS (Table III). The difference between the growth effect shown either by CEGABA or CR-ECGS and HECS is less pronounced when the growth factors are tested on hybridomas which are already adapted to culture
235 conditions. No synergistic effect was obtained between CEGABA and CR-ECGS indicating that these factors may act through similar mechanisms. In conclusion, both CEGABA and CR-ECGS improve hybridoma growth espec i a l l y w h e n t h e r e is n o c o n t a m i n a t i o n b y o t h e r cells. T h e s i g n i f i c a n t a d v a n t a g e o f C E G A B A o v e r o t h e r g r o w t h f a c t o r s is t h a t , as a p u r e m o l e c u l e , it p e r m i t s t h e i n v e s t i g a t o r to s t u d y s e p a r a t e l y t h e f a c t o r s i n v o l v e d in o p t i m a l cell g r o w t h in d e f i n e d m e d i a .
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