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Growth of human × human hybridomas in protein-free medium supplemented with ethanolamine
Journal of Immunological Methods, 97 (1987) 29-35 Elsevier
29
JIM 04224
Growth of human × human hybridomas in protein-free medium supplemented with ethanolamine S.P.C. Cole 1,., E.H. Vreeken, S.E.L. Mirski a n d B.G. C a m p l i n g a,** Departments of I.-, Oncologv, I Microbiology and Immunology and 2 Medicine, Queen's Universi(v, Kingston, Ontario K7L 3N6, and Ontario Cancer Treatment and Research Foundation, Kingston Regional Cancer Centre, King Street West, Kingston, Ontario K7L 2 V7, Canada (Received 27 August 1986, accepted 17 October 1986)
A protein-free medium was developed which supports the growth of human x human hybridomas derived from the fusion partner KR-4. This medium consists of a 1:1 mixture of Iscove's modified Dulbecco's medium and Ham's F-12 medium supplemented with 300 #M ethanolamine. The addition of ethanolamine was essential to maintain cell viability and proliferation. Phosphoethanolamine could not substitute for ethanolamine. Although the maximal cell density achieved in protein-free medium was lower than that in medium supplemented with albumin and transferrin, immunoglobulin production was still supported. This protein-free medium may be useful for large-scale cultivation and for metabolic and immunologic studies of human hybridomas. Key words: Protein-free medium; Ethanolamine; Monoclonal antibody, human
Introduction
Human monoclonal antibodies (HuMAbs) are expected to have many advantages over their murine counterparts in terms of therapeutic potential. A host immune response in patients treated with murine MAbs may well limit the
Correspondence to: S.P.C. Cole, Kingston Regional Cancer Centre, King Street West, Kingston, Ontario K7L 2V7, Canada. * Recipient of grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. ** Recipient of grants from the National Cancer Institute of Canada and the Clare Nelson Bequest of Kingston General Hospital. Abbreviations: FBS, fetal bovine serum; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; Tf, transferrin; IMDM, Iscove's modified Dulbecco's medium; T3, 3,3',5-triiodothyronine; HuMAbs, human monoclonal antibodies; EBV, Epstein-Barr virus; E2, estradiol; H, hydrocortisone; TE, trace element; BM, basal medium.
effectiveness of these agents (Sears et al., 1982; Goodman et al., Shawler et al., 1985). This antiimmunoglobulin response is expected to be less with HuMAbs. Furthermore, it is reasonable to assume that HuMAbs will interact more effectively than murine MAbs with human effector cells a n d / o r complement. For these reasons, considerable effort has been devoted to optimizing the production of HuMAbs (Engleman et al., 1985). Three cellular approaches to the production of HuMAbs may be described as follows: (1) immortalization of antibody-producing B cells with Epstein-Barr virus (EBV), (2) generation of heterohybrids using murine myeloma fusion partners, and (3) generation of human x human hybrids. Our efforts have been directed towards the last approach, since human x human hybridomas are expected to yield the most stable antibody production (Cole et al., 1984a; Campling et al., 1986).
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
30 To be useful agents for therapeutic purposes, it will be necessary to have the means to grow human × human hybrids in large quantities. For a large scale tissue culture system to be economically feasible, serum-free culture is essential and protein-free culture would be desirable. We have previously shown that human × human hybridomas may be grown in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin ( B S A / T f medium) (Cole et al., 1985). In the present study, we describe the growth of human × human hybridomas in a protein-free medium. The key component in this medium is ethanolamine, a precursor of membrane phospholipid.
Human × human hybridomas The human hybridomas AIH, B5C, BC-MCE3-A2 and B5C2A are subclones of a hybridoma obtained from the fusion of EBV-transformed peripheral blood B lymphocytes of a patient with small cell lung cancer and KR-4, a thioguanineand ouabain-resistant human B lymphoblastoid cell line (Kozbor et al., 1982; Cole et al., 1984b, 1985). Cells were frozen in RPMI 1640/20% fetal bovine serum (FBS)/10% DMSO at - 7 0 ° C or in liquid nitrogen. Cells were routinely cultured in the absence of antibiotics in 25 cm 2 flasks in a 5 % CO 2 atmosphere at 37°C. All cells were checked for mycoplasma contamination using the DAPI (4',6-diamidino-2-phenylindole) DNA-binding assay (Russell et al., 1975) and found to be negative.
Materials and methods
Cell growth experiments Cells were thawed into RPMI 1640/20% FBS medium and then adapted to growth in serum-free B S A / T f medium (RPMI 1640 with 0.5% BSA and 1 0 / x g / m l Tf) as previously described (Cole et al., 1985) for at least 2 weeks. For the protein-free medium experiments cells were harvested from B S A / T f medium by low-speed centrifugation and washed with the medium to be used. In most experiments, cells were seeded in 25 cm 2 flasks at 2 × 105 cells/ml in triplicate flasks and were counted by trypan blue exclusion using a hemocytometer 5 days later. In some preliminary experiments, cells from single flasks were counted.
Source of materials Ham's nutrient mixture F-12 (N-6760) and Iscove's modified Dulbecco's medium (IMDM) (I-7633) were obtained from Sigma Chemical Co. (St. Louis, MO). Basal medium (BM) consisted of F 1 2 / I M D M ( 1 : 1 ) supplemented with progesterone (20 n g / m l ) and a-thioglycerol (6.3/~1/1) as described by Cleveland et al. (1983). Dulbecco's modified Eagle medium (DMEM), RPMI 1640 and minimum essential medium alpha (a-MEM) were from Gibco. Bovine serum albumin (BSA) (A-7888), progesterone (P-0130), transferrin (Tf) (T-2252), 3,3',5-triiodo-L-thyronine (T3) (T-5516), phosphoethanolamine (P-0503), fl-estradiol (E-8875) and hydrocortisone (H-4001) were obtained from Sigma. Ethanolamine (M-251) was obtained from Fisher Scientific Co. and a-thioglycerol(3-mercapto-l,2-propanediol) from Aldrich Chemical Co. Trace elements were obtained from the following manufacturers: Fisher Scientific (MnSO 4 H20; NazSiO 2 • 5H20; ( N H 4 ) 6 M 0 7 0 2 4 . 4 H 2 0 ; NaF; AgNO3) , BDH (SnC12-2H20; A1C13 • 6H/O; KBr; CrE(SO4)3.15H20; KI), Aldrich Chemical Co. (V205; CdC12 • 2 1 / 2 H 2 0 ; ZrOC12 • H20), Allied Chemical Co. (NiSO 4 • 6H20; Ba(C 2 H 3 0 2 ) 2 ) , Sigma Chemical Co. (RbCI), Alfa Producers (GeO2) and McArthur Chemicals (CoC12 • 6 H 2 0 ). Trace element mixtures TEI and TEII were made up as described by Cleveland et al. (1983).
Human immunoglobulin assay Human immunoglobulin IgM was quantitated using an enzyme-linked immunosorbent assay (ELISA) as previously described (Cole et al., 1985). An IgM standard curve was computer generated using software written for this purpose (Immunosoft, Dynatech).
Results
Human × human hybridomas were thawed into RPMI 1640 medium containing 10% FBS. Once growth was well established (usually less than 1 week), cells were directly transferred to serum-free B S A / T f medium or gradually adapted to B S A / T f medium over several weeks (Cole et al., 1985). The
31 cells thrived e q u a l l y well with b o t h m e t h o d s . T r a n s f e r to p r o t e i n - f r e e m e d i u m was m o s t successful if the h y b r i d o m a s were allowed to g r o w in B S A / T f m e d i u m for at least 1 week ( p r e f e r a b l y 2). H y b r i d s have b e e n m a i n t a i n e d in the B S A / T f m e d i u m for over 10 months. I n a first set of experiments, h u m a n h y b r i d s t h a t h a d b e e n g r o w n in R P M I 1 6 4 0 / 1 0 % F B S or in B S A / T f m e d i u m were transferred d i r e c t l y to B M s u p p l e m e n t e d with 1 / 2 T E I , T E I or T E I I at several cell densities. U n l i k e the m u r i n e hyb r i d o m a s d e s c r i b e d b y C l e v e l a n d et al. (1983), o u r h u m a n h y b r i d s failed to grow in these m e d i a a n d cell viability was r e d u c e d to less t h a n 10% within a w e e k (results n o t shown). I n a s e c o n d set of e x p e r i m e n t s , a variety of c o m m o n l y used n o n - p r o t e i n s u p p l e m e n t s were tested for their a b i l i t y to s u p p o r t cell g r o w t h in B M with T E I or 1 / 2 T E I m e d i u m . These supplem e n t s i n c l u d e d 0.1 n M t r i i o d o t h y r o n i n e (T3), 20 /~M e t h a n o l a m i n e , 10 n M h y d r o c o r t i s o n e , 10 n M fl-estradiol a n d a c o m b i n a t i o n of these four c o m p o u n d s ( T E E E H m e d i u m ) . T h e results are shown in T a b l e I. W h i l e n o n e of these m e d i a s u p p o r t e d cell g r o w t h as m e a s u r e d at 7 days, cell v i a b i l i t y was m a r k e d l y e n h a n c e d in e t h a n o l a m i n e - c o n t a i n ing a n d T E E z H - c o n t a i n i n g media. These results suggested that e t h a n o l a m i n e was a k e y g r o w t h factor. O n the basis of this a n d other p r e l i m i n a r y e x p e r i m e n t s , it was d e c i d e d that b e t t e r results
w o u l d be o b t a i n e d if the p r o t e i n - f r e e e x p e r i m e n t s were set u p at a lower cell d e n s i t y (i.e., 2 × 105 c e l l s / m l ) a n d cell counts d e t e r m i n e d on d a y 5. T E I was c o m p a r e d to T E I I in B M supplem e n t e d with T E E 2 H to see if one trace element m i x t u r e was s u p e r i o r to the other. W h i l e results v a r i e d to s o m e degree within four s e p a r a t e experiments, there was no consistent significant adv a n t a g e to using TEI, T E I I , or 1 / 2 ( T E I + T E I I ) c o m p a r e d to B M a l o n e (results n o t shown). H o w ever, since the T E mixtures h a d no i n h i b i t o r y effect a n d in some e x p e r i m e n t s h a d a m a r g i n a l g r o w t h e n h a n c i n g effect, the B M was r o u t i n e l y s u p p l e m e n t e d with 1 / 2 ( T E I + T E I I ) in all subseq u e n t experiments. T h e next objective was to d e t e r m i n e the optim a l c o n c e n t r a t i o n of e t h a n o l a m i n e . The g r o w t h r e s p o n s e to various doses of e t h a n o l a m i n e was tested in B M with 1 / 2 ( T E I + T E I I ) a n d T E 2 H a n d the results are shown in Fig. 1. E t h a n o l a m i n e e n h a n c e d g r o w t h at c o n c e n t r a t i o n s as low as 5 /zM. M a x i m a l e n h a n c e m e n t was seen at 300 /zM e t h a n o l a m i n e . In the e x p e r i m e n t shown, the viab i l i t y was similar (range 7 2 - 7 5 % ) for all doses of e t h a n o l a m i n e tested. I n a second e x p e r i m e n t (results n o t shown), e t h a n o l a m i n e was tested at higher c o n c e n t r a t i o n s (1 a n d 3 m M ) ; however, a slight decrease in viability was o b s e r v e d at these doses a l t h o u g h the cell d e n s i t y was similar to that obt a i n e d with 3 0 0 / x M . These results suggested that
TABLE I GROWTH OF HUMAN × HUMAN HYBRIDOMAS IN PROTEIN-FREE MEDIUM Medium b
T = T3 (0.1 nM) E = Ethanolamine (20/xM) E 2 = estradiol (10 nM) H = hydrocortisone (10 nM) TEE2H No additions
Clone AIH a
Clone B5C a
TEI cells/ml ~ (×10 5)
1/2 TEI cells/ml ~ (xlO-5)
TEI cells/ml ~ (×10-5)
1/2 TEI cells/ml ~ (×10 5)
1.5 (20) 6.0 (37) 0.9 (12) 1.2 (20) 7.8 (43) 0.8 (11)
0.3 (8) 1.4 (18) 0.4 (5) 0.4 (8) 6.9 (44) 0.8 (9)
0.6 (10) 2.2 (22) 0.7 (8) 1.3 (13) 6,2 (37) 2.2 (24)
1.7 (18) 3.7 (30) 1.2 (13) 1.3 (16) 6.2 (36) 1.0 (13)
a Human hybridomas AIH and B5C are two subclones of a hybridoma obtained from the fusion of EBV-transformed B lymphocytes of a small cell lung cancer patient and KR-4. b The media compared are BM (see materials and methods section) with TEI or 1/2 TEI and supplements added at the indicated concentrations. c The density of viable cells was determined by trypan blue exclusion 7 days after seeding flasks at 5 × 105 cells/ml. Viability as a percentage is given in parentheses. The results shown are counts from a single flask.
32 m
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5
20
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ETHANOLAMINE,
4
-
2
300 H.M
Fig. 1. Dose response to ethanolamine. Clone A I H cells, maintained in B S A / T f medium, were resuspended at 2 × 1 0 5 cells/ml in BM supplemented with 1 / 2 ( T E I + T E I I ) , TE2H (see Table I for details) and ethanolamine at the concentrations indicated. Cells were counted and viability determined on day 5. The results shown are the mean + SD of counts from three flasks. The experiment was repeated twice with similar results.
300 ffM ethanolamine was optimal and this concentration was used in all subsequent experiments. To determine whether ethanolamine alone was sufficient to support protein-free growth or the other components of the T E E 2 H medium were also important, media with various combinations of ethanolamine, T3, fl-estradiol and hydrocortisone were tested for their ability to support cell growth. No synergistic effects were observed and hydrocortisone and fl-estradiol had no effect on cell growth or viability. The effect of T3 was somewhat variable; in some experiments it had no effect while in others, it enhanced cell growth. For this reason, various concentrations of T3 were tested for growth-enhancing activity to determine if there was a dose-effect relationship. The results of one experiment are shown in Fig. 2. At 10 nM T3, the cell density was greater at 8.0 + 0.1 × 105 cells/ml compared to 6.9 _+ 0.2 × 105 cells/ml in medium without T3. While this difference is statistically significant, it is not dramatic. In a second experiment, using the same batch of T3, the difference was not significant (results not shown). The effect of phosphoethanolamine on cell growth was determined and the results are shown in Fig. 3. Phosphoethanolamine stimulated growth in a dose-related manner although it was far less potent than ethanolamine. Thus, the density of viable cells reached 4.2 + 0.2 x 105 cells/ml with 300 ffM phosphoethanolamine compared to
I I
0 0
OI
T3,
I
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Fig. 2. Dose response to T3. Clone A I H cells, maintained in B S A / T f medium, were resuspended at 2 × 1 0 5 / m l in BM supplemented with 1 / 2 ( T E I + TEII), ethanolamine (300 # M ) and T3 at the concentrations indicated. Cells were counted and viability determined on day 5. The results shown are the mean _+SD of counts from three flasks.
8.6 +__0.3 × 105 cells/ml with 300 ~M ethanolamine. The cell density without phosphoethanolamine or ethanolamine was 2.3 _+ 0.3 × 105 cells/ml. In addition, cell viability was significantly lower in phosphoethanolamine-supplemented cultures compared to ethanolamine-supplemented cultures. The growth of clone AIH in serum-free B S A / T f medium and protein-free BM with 1 / 2 (TEl +
~
8
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oU
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5
20
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300
PI4OSPHOETHA NOL AMI N [ , /~M Fig. 3. Dose response to phosphoethanolamine. Clone B5C 2A
cells, maintained in B S A / T f medium, were resuspended at 2 × 1 0 5 cells/ml in BM with 1 / 2 ( T E I + T E I I ) and phosphoethanolamine at the concentrations indicated (open bars) and ethanolamine at 300 ~ M (hatched bar). Cells were counted and viability determined on Day 5. The results shown are the mean + SD of counts from three flasks.
33
TEll) and ethanolamine (300/~M) was compared and the results are shown in Fig. 4. In BSA/Tf medium, the hybrid grew to a density of 8.1 4- 0.3 × 105 cells/ml while in protein-free medium the maximal cell density was lower at 5.9 _+0.2 × 105 cells/ml. In addition, the decrease in cell viability once maximal cell density was achieved was more pronounced in protein-free medium than in serum-free medium (Fig. 4). Various basal media were tested for their ability to support protein-free cell growth. Media tested included RPMI 1640, IMDM, F12, DMEM and a-MEM which were compared to BM (F12/IMDM 1 : 1). The results are shown in Table II. Clone B5C2A grew best in F12/IMDM (BM), moderately well in DMEM and IMDM but poorly in RPMI 1640, a-MEM and F12 media. Similarly, clone AIH grew well in F12/IMDM but poorly in RPMI 1640. Concurrent experiments indicated that the a-thioglycerol and progesterone supplements to the BM described by Cleveland et al. (1983) were unnecessary (results not shown). To determine how long cells could be mainrained in protein-free medium, cultures were initiated and then refed with fresh protein-free medium and the cell density readjusted to 2 × 105/ml every 3 days when cell viability starts to diminish. It was found that cell growth could be
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E F F E C T OF D I F F E R E N T BASAL M E D I A ON PROTEINFREE GROWTH OF HUMAN x HUMAN HYBRIDOMAS Medium a
Cells/ml (xl0 -s) B5C2A
AIH
F12/IMDM(BM) a-MEM RPMI1640 DMEM F12 IMDM
7.8+0.2 2.7±0.2 3.4±0.2 5.9±0.4 3.3±0.1 6.7±0.2
8.5±0.2 n,d. ¢ 2.9±0.3 n.d. n,d. n.d.
b
Media were supplemented with 1 / 2 ( T E l + T E l l ) and 300 # M ethanolamine. b Cells were seeded at 2 x 105/ml on day 0. Cells were counted and viability determined on day 5. The results shown are the mean_+ SD of counts from three flasks, ¢ Not determined.
supported for at least 14 days following this schedule (results not shown). Production of human IgM by three different human hybrid clones in protein-free medium was quantitated by ELISA. The concentration of IgM was determined in culture supernatants harvested 5 days after initiating protein-free cultures. The supernatants were stored at - 2 0 ° C and then tested in an ELISA. The results are presented in Table III. As indicated, IgM production of all three clones was supported in protein-free medium.
b) p r o t e i n - f r e e medium
o
tJ
T A B L E II
I
I
I
I
L
l
l
~
0 1 2 3 4 5 6 7 DAYS
Fig. 4. Growth curves of A I H h u m a n x h u m a n hybridOmas in serum-free and protein-free medium. Cells were seeded on day 0 at 2 x 105 cells/ml in ( a ) serum-free B S A / T f medium consisting of R P M I 1640 medium supplemented with 0.5% BSA and 10 # g / m l transferrin, or (b) protein-free medium consisting of BM with 1 / 2 ( T E I + T E I I ) and 300 # M ethanolamine. The open symbols represent total cell counts while the solid symbols represent total viable cells. Each point represents the m e a n + SD of cell counts from three replicate flasks.
T A B L E III H U M A N I M M U N O G L O B U L I N P R O D U C T I O N BY H U M A N × H U M A N H Y B R I D O M A S IN P R O T E I N - F R E E MEDIUM Clone a
IgM b (~g/ml)
Cells/ml c ( X 10 -6)
IgM ( # g / m l per 106 cells)
B5C B5C2A BC-MC-E3-A2
4.3 7.7 3.2
0.46 0.52 0,52
9.4 14.8 6.2
a Cells were seeded at 2 × 105 cells/ml in BM with 1 / 2 ( T E I + TEII) and 300/zM ethanolamine. b H y b f i d o m a s u p e m a t a n t s were collected 5 days after plating, diluted 1 : 5, 1 : 25 and 1 : 125 and tested in an ELISA for IgM content by comparison to affinity purified standards. The values shown are the mean of duplicate determinations. c The density of viable cells was determined by trypan blue exclusion. The values shown are the mean of triplicate cultures.
34
Discussion In the present study, we have shown that human x human hybridomas may be successfully grown in a protein-free chemically defined medium. Furthermore, the hybridomas retained their capacity to synthesize human immunoglobulin in such medium. The hybrids do not achieve as high a cell density in protein-free medium as compared to serum-free B S A / T f medium. However, if higher cell densities are required, a more frequent feeding schedule may circumvent this problem. We have found that the protein-free medium which best supports growth consists of F 1 2 / I M D M ( 1 : 1 ) supplemented with ethanolamine (300/zM). Addition of trace elements, while not essential, marginally improved growth in some cases. The same was true of T3. However, since neither of these supplements were ever detrimental to cell growth, we recommend that they be included. Ethanolamine has been reported to be an essential nutrient for a number of tissue culture systems. Tsao et al. (1982) found that either ethanolamine or phosphoethanolamine was an absolute requirement for proliferation of human epidermal keratinocytes. Murakami et al. (1982) found ethanolamine to be an essential growth factor for their murine hybridomas. In both studies, as in the present study, phosphoethanolamine was considerably less potent than ethanolamine in its ability to stimulate growth. The reason for this is unknown although Murakami et al. (1982) have suggested that either phosphoethanolamine is converted to ethanolamine before being taken up by the cells, or phosphoethanolamine is taken up much less efficiently by the cells. The mechanism by which ethanolamine supports cell growth has not been elucidated. In a study using rat m a m m a r y carcinoma cells, KanoSueoka and Errick (1981) found that the majority of ethanolamine taken up by the cells was efficiently converted to phosphatidylethanolamine which was then incorporated into membrane phospholipid. These authors suggested that the synthesis of phosphatidylethanolamine may be rate limiting and that by supplying exogenous ethanolamine, cells can readily synthesize the critical amount of phosphatidylethanolamine neces-
sary for proliferation. Such may be the case with our human x h u m a n hybrids. In contrast, several investigators have reported growth of human cells in protein-free media which do not contain ethanolamine (Okabe and Takaku, 1984; Alderman et al., 1985; Yamaguchi et al., 1985; Shive et al., 1986). These cells may be able to synthesize sufficient phosphatidylethanolamine and thus not require exogenous ethanolamine for cell growth. Further studies are required to fully elucidate the role of ethanolamine in stimulating cell growth. It has been shown that murine MAbs derived from various myeloma fusion partners differ in their dependence on individual components of serum-free medium for growth (Kovar and Franek, 1984). In the present study we have found that four out of four human hybrids derived from K R - 4 grew in protein-free medium; further studies are required to determine if this medium will support the growth of hybrids derived from human fusion partners other than KR-4. The protein-flee medium described in this study will be useful for both immunologic and metabolic studies of human x human hybridomas. In addition, it should facilitate purification of H u M A b s from spent medium. Whether or not this medium will be adaptable to large-scale cultivation methods for mass production of H u M A b s remains to be determined (Arathoon and Birch, 1986).
Acknowledgement We thank C. Jackson for her help in the preparation of the manuscript.
References Alderman, E.M., Lobb, R.R. and Fett, J.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5771. Arathoon, W.R. and Birch, J.R. (1986) Science 232, 1390. Campling, B.G., Cole, S.P.C., Atlaw, T., Kozbor, D. and Roder, J.C. (1986) In: A.J. Strelkauskas (Ed.), Human Monoclonal Antibodies (Marcel Dekker, New York) p. 3. Cleveland, W.L., Wood, I. and Erlanger, B.F. (1983) J. Immunol. Methods 56, 221. Cole, S.P.C., Campling, B.G., Atlaw, T., Kozbor, D. and Roder, J.C. (1984a) Mol. Cell. Biochem. 62, 109. Cole, S.P.C., Campling, B.G., Louwman, I.H., Kozbor, D. and Roder, J.C. (1984b) Cancer Res. 44, 2750.
35 Cole, S.P.C., Vreeken, E.H. and Roder, J.C. (1985) J. Immunol. Methods 78, 271. Engleman, E.G., Foung, S.K.H., Larrick, J. and Raubitschek, A. (Eds.) (1985) Human Hybridomas and Monoclonal Antibodies (Plenum Press, New York). Goodman, G.E.,, Beaumier, P., Hellstrom, I., Fernyhough, B. and Hellstrom, K.-E. (1985) J. Clin. Oncol. 3, 340. Kano-Sueoka, T. and Errick, J.E. (1981) Exp. Cell Res. 136, 137. Kovar, J. and Franek, F. (1984) Immunol. Lett. 7, 339. Kozbor, D., Lagarde, A.E. and Roder, J.C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6651. Murakami, H., Masui, H., Sato, G.S., Sueoko, N., Chow, T.P. and Kano-Sueoka, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1158.
Okabe, T. and Takaku, F. (1984) Cancer Res: 44, 4503. Russell, W.C., Newman, C. and Williamson, D.H. (1975) Nature 253, 461. Sears, H.F., Mattis, J., Herlyn, D., Ayry, P.H., Atkinson, B., Ernst, C., Steplewski, Z. and Kroprowski, H. (1982) Lancet i, 762. Shawler, D.L., Bartholomew, R.M., Smith, L.M. and Dillman, R.O. (1985) J. Immunol. 135, 1530. Shive, W., Pinkerton, F., Humphreys, J., Johnson, M.M., Hamilton, W.G. and Matthews, K.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9. Tsao, M.C., Walthall, B.J. and Ham, R.G. (1982) J. Cell. Physiol. 110, 219. Yamaguchi, N., Okabe, T. and Kawai, K. (1985) J. Cancer Res. Clin. Oncol. 110, 42.
29
JIM 04224
Growth of human × human hybridomas in protein-free medium supplemented with ethanolamine S.P.C. Cole 1,., E.H. Vreeken, S.E.L. Mirski a n d B.G. C a m p l i n g a,** Departments of I.-, Oncologv, I Microbiology and Immunology and 2 Medicine, Queen's Universi(v, Kingston, Ontario K7L 3N6, and Ontario Cancer Treatment and Research Foundation, Kingston Regional Cancer Centre, King Street West, Kingston, Ontario K7L 2 V7, Canada (Received 27 August 1986, accepted 17 October 1986)
A protein-free medium was developed which supports the growth of human x human hybridomas derived from the fusion partner KR-4. This medium consists of a 1:1 mixture of Iscove's modified Dulbecco's medium and Ham's F-12 medium supplemented with 300 #M ethanolamine. The addition of ethanolamine was essential to maintain cell viability and proliferation. Phosphoethanolamine could not substitute for ethanolamine. Although the maximal cell density achieved in protein-free medium was lower than that in medium supplemented with albumin and transferrin, immunoglobulin production was still supported. This protein-free medium may be useful for large-scale cultivation and for metabolic and immunologic studies of human hybridomas. Key words: Protein-free medium; Ethanolamine; Monoclonal antibody, human
Introduction
Human monoclonal antibodies (HuMAbs) are expected to have many advantages over their murine counterparts in terms of therapeutic potential. A host immune response in patients treated with murine MAbs may well limit the
Correspondence to: S.P.C. Cole, Kingston Regional Cancer Centre, King Street West, Kingston, Ontario K7L 2V7, Canada. * Recipient of grants from the National Cancer Institute of Canada and the Medical Research Council of Canada. ** Recipient of grants from the National Cancer Institute of Canada and the Clare Nelson Bequest of Kingston General Hospital. Abbreviations: FBS, fetal bovine serum; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; Tf, transferrin; IMDM, Iscove's modified Dulbecco's medium; T3, 3,3',5-triiodothyronine; HuMAbs, human monoclonal antibodies; EBV, Epstein-Barr virus; E2, estradiol; H, hydrocortisone; TE, trace element; BM, basal medium.
effectiveness of these agents (Sears et al., 1982; Goodman et al., Shawler et al., 1985). This antiimmunoglobulin response is expected to be less with HuMAbs. Furthermore, it is reasonable to assume that HuMAbs will interact more effectively than murine MAbs with human effector cells a n d / o r complement. For these reasons, considerable effort has been devoted to optimizing the production of HuMAbs (Engleman et al., 1985). Three cellular approaches to the production of HuMAbs may be described as follows: (1) immortalization of antibody-producing B cells with Epstein-Barr virus (EBV), (2) generation of heterohybrids using murine myeloma fusion partners, and (3) generation of human x human hybrids. Our efforts have been directed towards the last approach, since human x human hybridomas are expected to yield the most stable antibody production (Cole et al., 1984a; Campling et al., 1986).
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
30 To be useful agents for therapeutic purposes, it will be necessary to have the means to grow human × human hybrids in large quantities. For a large scale tissue culture system to be economically feasible, serum-free culture is essential and protein-free culture would be desirable. We have previously shown that human × human hybridomas may be grown in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin ( B S A / T f medium) (Cole et al., 1985). In the present study, we describe the growth of human × human hybridomas in a protein-free medium. The key component in this medium is ethanolamine, a precursor of membrane phospholipid.
Human × human hybridomas The human hybridomas AIH, B5C, BC-MCE3-A2 and B5C2A are subclones of a hybridoma obtained from the fusion of EBV-transformed peripheral blood B lymphocytes of a patient with small cell lung cancer and KR-4, a thioguanineand ouabain-resistant human B lymphoblastoid cell line (Kozbor et al., 1982; Cole et al., 1984b, 1985). Cells were frozen in RPMI 1640/20% fetal bovine serum (FBS)/10% DMSO at - 7 0 ° C or in liquid nitrogen. Cells were routinely cultured in the absence of antibiotics in 25 cm 2 flasks in a 5 % CO 2 atmosphere at 37°C. All cells were checked for mycoplasma contamination using the DAPI (4',6-diamidino-2-phenylindole) DNA-binding assay (Russell et al., 1975) and found to be negative.
Materials and methods
Cell growth experiments Cells were thawed into RPMI 1640/20% FBS medium and then adapted to growth in serum-free B S A / T f medium (RPMI 1640 with 0.5% BSA and 1 0 / x g / m l Tf) as previously described (Cole et al., 1985) for at least 2 weeks. For the protein-free medium experiments cells were harvested from B S A / T f medium by low-speed centrifugation and washed with the medium to be used. In most experiments, cells were seeded in 25 cm 2 flasks at 2 × 105 cells/ml in triplicate flasks and were counted by trypan blue exclusion using a hemocytometer 5 days later. In some preliminary experiments, cells from single flasks were counted.
Source of materials Ham's nutrient mixture F-12 (N-6760) and Iscove's modified Dulbecco's medium (IMDM) (I-7633) were obtained from Sigma Chemical Co. (St. Louis, MO). Basal medium (BM) consisted of F 1 2 / I M D M ( 1 : 1 ) supplemented with progesterone (20 n g / m l ) and a-thioglycerol (6.3/~1/1) as described by Cleveland et al. (1983). Dulbecco's modified Eagle medium (DMEM), RPMI 1640 and minimum essential medium alpha (a-MEM) were from Gibco. Bovine serum albumin (BSA) (A-7888), progesterone (P-0130), transferrin (Tf) (T-2252), 3,3',5-triiodo-L-thyronine (T3) (T-5516), phosphoethanolamine (P-0503), fl-estradiol (E-8875) and hydrocortisone (H-4001) were obtained from Sigma. Ethanolamine (M-251) was obtained from Fisher Scientific Co. and a-thioglycerol(3-mercapto-l,2-propanediol) from Aldrich Chemical Co. Trace elements were obtained from the following manufacturers: Fisher Scientific (MnSO 4 H20; NazSiO 2 • 5H20; ( N H 4 ) 6 M 0 7 0 2 4 . 4 H 2 0 ; NaF; AgNO3) , BDH (SnC12-2H20; A1C13 • 6H/O; KBr; CrE(SO4)3.15H20; KI), Aldrich Chemical Co. (V205; CdC12 • 2 1 / 2 H 2 0 ; ZrOC12 • H20), Allied Chemical Co. (NiSO 4 • 6H20; Ba(C 2 H 3 0 2 ) 2 ) , Sigma Chemical Co. (RbCI), Alfa Producers (GeO2) and McArthur Chemicals (CoC12 • 6 H 2 0 ). Trace element mixtures TEI and TEII were made up as described by Cleveland et al. (1983).
Human immunoglobulin assay Human immunoglobulin IgM was quantitated using an enzyme-linked immunosorbent assay (ELISA) as previously described (Cole et al., 1985). An IgM standard curve was computer generated using software written for this purpose (Immunosoft, Dynatech).
Results
Human × human hybridomas were thawed into RPMI 1640 medium containing 10% FBS. Once growth was well established (usually less than 1 week), cells were directly transferred to serum-free B S A / T f medium or gradually adapted to B S A / T f medium over several weeks (Cole et al., 1985). The
31 cells thrived e q u a l l y well with b o t h m e t h o d s . T r a n s f e r to p r o t e i n - f r e e m e d i u m was m o s t successful if the h y b r i d o m a s were allowed to g r o w in B S A / T f m e d i u m for at least 1 week ( p r e f e r a b l y 2). H y b r i d s have b e e n m a i n t a i n e d in the B S A / T f m e d i u m for over 10 months. I n a first set of experiments, h u m a n h y b r i d s t h a t h a d b e e n g r o w n in R P M I 1 6 4 0 / 1 0 % F B S or in B S A / T f m e d i u m were transferred d i r e c t l y to B M s u p p l e m e n t e d with 1 / 2 T E I , T E I or T E I I at several cell densities. U n l i k e the m u r i n e hyb r i d o m a s d e s c r i b e d b y C l e v e l a n d et al. (1983), o u r h u m a n h y b r i d s failed to grow in these m e d i a a n d cell viability was r e d u c e d to less t h a n 10% within a w e e k (results n o t shown). I n a s e c o n d set of e x p e r i m e n t s , a variety of c o m m o n l y used n o n - p r o t e i n s u p p l e m e n t s were tested for their a b i l i t y to s u p p o r t cell g r o w t h in B M with T E I or 1 / 2 T E I m e d i u m . These supplem e n t s i n c l u d e d 0.1 n M t r i i o d o t h y r o n i n e (T3), 20 /~M e t h a n o l a m i n e , 10 n M h y d r o c o r t i s o n e , 10 n M fl-estradiol a n d a c o m b i n a t i o n of these four c o m p o u n d s ( T E E E H m e d i u m ) . T h e results are shown in T a b l e I. W h i l e n o n e of these m e d i a s u p p o r t e d cell g r o w t h as m e a s u r e d at 7 days, cell v i a b i l i t y was m a r k e d l y e n h a n c e d in e t h a n o l a m i n e - c o n t a i n ing a n d T E E z H - c o n t a i n i n g media. These results suggested that e t h a n o l a m i n e was a k e y g r o w t h factor. O n the basis of this a n d other p r e l i m i n a r y e x p e r i m e n t s , it was d e c i d e d that b e t t e r results
w o u l d be o b t a i n e d if the p r o t e i n - f r e e e x p e r i m e n t s were set u p at a lower cell d e n s i t y (i.e., 2 × 105 c e l l s / m l ) a n d cell counts d e t e r m i n e d on d a y 5. T E I was c o m p a r e d to T E I I in B M supplem e n t e d with T E E 2 H to see if one trace element m i x t u r e was s u p e r i o r to the other. W h i l e results v a r i e d to s o m e degree within four s e p a r a t e experiments, there was no consistent significant adv a n t a g e to using TEI, T E I I , or 1 / 2 ( T E I + T E I I ) c o m p a r e d to B M a l o n e (results n o t shown). H o w ever, since the T E mixtures h a d no i n h i b i t o r y effect a n d in some e x p e r i m e n t s h a d a m a r g i n a l g r o w t h e n h a n c i n g effect, the B M was r o u t i n e l y s u p p l e m e n t e d with 1 / 2 ( T E I + T E I I ) in all subseq u e n t experiments. T h e next objective was to d e t e r m i n e the optim a l c o n c e n t r a t i o n of e t h a n o l a m i n e . The g r o w t h r e s p o n s e to various doses of e t h a n o l a m i n e was tested in B M with 1 / 2 ( T E I + T E I I ) a n d T E 2 H a n d the results are shown in Fig. 1. E t h a n o l a m i n e e n h a n c e d g r o w t h at c o n c e n t r a t i o n s as low as 5 /zM. M a x i m a l e n h a n c e m e n t was seen at 300 /zM e t h a n o l a m i n e . In the e x p e r i m e n t shown, the viab i l i t y was similar (range 7 2 - 7 5 % ) for all doses of e t h a n o l a m i n e tested. I n a second e x p e r i m e n t (results n o t shown), e t h a n o l a m i n e was tested at higher c o n c e n t r a t i o n s (1 a n d 3 m M ) ; however, a slight decrease in viability was o b s e r v e d at these doses a l t h o u g h the cell d e n s i t y was similar to that obt a i n e d with 3 0 0 / x M . These results suggested that
TABLE I GROWTH OF HUMAN × HUMAN HYBRIDOMAS IN PROTEIN-FREE MEDIUM Medium b
T = T3 (0.1 nM) E = Ethanolamine (20/xM) E 2 = estradiol (10 nM) H = hydrocortisone (10 nM) TEE2H No additions
Clone AIH a
Clone B5C a
TEI cells/ml ~ (×10 5)
1/2 TEI cells/ml ~ (xlO-5)
TEI cells/ml ~ (×10-5)
1/2 TEI cells/ml ~ (×10 5)
1.5 (20) 6.0 (37) 0.9 (12) 1.2 (20) 7.8 (43) 0.8 (11)
0.3 (8) 1.4 (18) 0.4 (5) 0.4 (8) 6.9 (44) 0.8 (9)
0.6 (10) 2.2 (22) 0.7 (8) 1.3 (13) 6,2 (37) 2.2 (24)
1.7 (18) 3.7 (30) 1.2 (13) 1.3 (16) 6.2 (36) 1.0 (13)
a Human hybridomas AIH and B5C are two subclones of a hybridoma obtained from the fusion of EBV-transformed B lymphocytes of a small cell lung cancer patient and KR-4. b The media compared are BM (see materials and methods section) with TEI or 1/2 TEI and supplements added at the indicated concentrations. c The density of viable cells was determined by trypan blue exclusion 7 days after seeding flasks at 5 × 105 cells/ml. Viability as a percentage is given in parentheses. The results shown are counts from a single flask.
32 m
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.J .J bJ (D
03
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bJ 0
5
20
I00
ETHANOLAMINE,
4
-
2
300 H.M
Fig. 1. Dose response to ethanolamine. Clone A I H cells, maintained in B S A / T f medium, were resuspended at 2 × 1 0 5 cells/ml in BM supplemented with 1 / 2 ( T E I + T E I I ) , TE2H (see Table I for details) and ethanolamine at the concentrations indicated. Cells were counted and viability determined on day 5. The results shown are the mean + SD of counts from three flasks. The experiment was repeated twice with similar results.
300 ffM ethanolamine was optimal and this concentration was used in all subsequent experiments. To determine whether ethanolamine alone was sufficient to support protein-free growth or the other components of the T E E 2 H medium were also important, media with various combinations of ethanolamine, T3, fl-estradiol and hydrocortisone were tested for their ability to support cell growth. No synergistic effects were observed and hydrocortisone and fl-estradiol had no effect on cell growth or viability. The effect of T3 was somewhat variable; in some experiments it had no effect while in others, it enhanced cell growth. For this reason, various concentrations of T3 were tested for growth-enhancing activity to determine if there was a dose-effect relationship. The results of one experiment are shown in Fig. 2. At 10 nM T3, the cell density was greater at 8.0 + 0.1 × 105 cells/ml compared to 6.9 _+ 0.2 × 105 cells/ml in medium without T3. While this difference is statistically significant, it is not dramatic. In a second experiment, using the same batch of T3, the difference was not significant (results not shown). The effect of phosphoethanolamine on cell growth was determined and the results are shown in Fig. 3. Phosphoethanolamine stimulated growth in a dose-related manner although it was far less potent than ethanolamine. Thus, the density of viable cells reached 4.2 + 0.2 x 105 cells/ml with 300 ffM phosphoethanolamine compared to
I I
0 0
OI
T3,
I
I0
nM
Fig. 2. Dose response to T3. Clone A I H cells, maintained in B S A / T f medium, were resuspended at 2 × 1 0 5 / m l in BM supplemented with 1 / 2 ( T E I + TEII), ethanolamine (300 # M ) and T3 at the concentrations indicated. Cells were counted and viability determined on day 5. The results shown are the mean _+SD of counts from three flasks.
8.6 +__0.3 × 105 cells/ml with 300 ~M ethanolamine. The cell density without phosphoethanolamine or ethanolamine was 2.3 _+ 0.3 × 105 cells/ml. In addition, cell viability was significantly lower in phosphoethanolamine-supplemented cultures compared to ethanolamine-supplemented cultures. The growth of clone AIH in serum-free B S A / T f medium and protein-free BM with 1 / 2 (TEl +
~
8
"jq
21t
- -
" ..
oU
i;~l O
5
20
I00
300
PI4OSPHOETHA NOL AMI N [ , /~M Fig. 3. Dose response to phosphoethanolamine. Clone B5C 2A
cells, maintained in B S A / T f medium, were resuspended at 2 × 1 0 5 cells/ml in BM with 1 / 2 ( T E I + T E I I ) and phosphoethanolamine at the concentrations indicated (open bars) and ethanolamine at 300 ~ M (hatched bar). Cells were counted and viability determined on Day 5. The results shown are the mean + SD of counts from three flasks.
33
TEll) and ethanolamine (300/~M) was compared and the results are shown in Fig. 4. In BSA/Tf medium, the hybrid grew to a density of 8.1 4- 0.3 × 105 cells/ml while in protein-free medium the maximal cell density was lower at 5.9 _+0.2 × 105 cells/ml. In addition, the decrease in cell viability once maximal cell density was achieved was more pronounced in protein-free medium than in serum-free medium (Fig. 4). Various basal media were tested for their ability to support protein-free cell growth. Media tested included RPMI 1640, IMDM, F12, DMEM and a-MEM which were compared to BM (F12/IMDM 1 : 1). The results are shown in Table II. Clone B5C2A grew best in F12/IMDM (BM), moderately well in DMEM and IMDM but poorly in RPMI 1640, a-MEM and F12 media. Similarly, clone AIH grew well in F12/IMDM but poorly in RPMI 1640. Concurrent experiments indicated that the a-thioglycerol and progesterone supplements to the BM described by Cleveland et al. (1983) were unnecessary (results not shown). To determine how long cells could be mainrained in protein-free medium, cultures were initiated and then refed with fresh protein-free medium and the cell density readjusted to 2 × 105/ml every 3 days when cell viability starts to diminish. It was found that cell growth could be
°%0..
0) B S A I T f m e d i u m
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E F F E C T OF D I F F E R E N T BASAL M E D I A ON PROTEINFREE GROWTH OF HUMAN x HUMAN HYBRIDOMAS Medium a
Cells/ml (xl0 -s) B5C2A
AIH
F12/IMDM(BM) a-MEM RPMI1640 DMEM F12 IMDM
7.8+0.2 2.7±0.2 3.4±0.2 5.9±0.4 3.3±0.1 6.7±0.2
8.5±0.2 n,d. ¢ 2.9±0.3 n.d. n,d. n.d.
b
Media were supplemented with 1 / 2 ( T E l + T E l l ) and 300 # M ethanolamine. b Cells were seeded at 2 x 105/ml on day 0. Cells were counted and viability determined on day 5. The results shown are the mean_+ SD of counts from three flasks, ¢ Not determined.
supported for at least 14 days following this schedule (results not shown). Production of human IgM by three different human hybrid clones in protein-free medium was quantitated by ELISA. The concentration of IgM was determined in culture supernatants harvested 5 days after initiating protein-free cultures. The supernatants were stored at - 2 0 ° C and then tested in an ELISA. The results are presented in Table III. As indicated, IgM production of all three clones was supported in protein-free medium.
b) p r o t e i n - f r e e medium
o
tJ
T A B L E II
I
I
I
I
L
l
l
~
0 1 2 3 4 5 6 7 DAYS
Fig. 4. Growth curves of A I H h u m a n x h u m a n hybridOmas in serum-free and protein-free medium. Cells were seeded on day 0 at 2 x 105 cells/ml in ( a ) serum-free B S A / T f medium consisting of R P M I 1640 medium supplemented with 0.5% BSA and 10 # g / m l transferrin, or (b) protein-free medium consisting of BM with 1 / 2 ( T E I + T E I I ) and 300 # M ethanolamine. The open symbols represent total cell counts while the solid symbols represent total viable cells. Each point represents the m e a n + SD of cell counts from three replicate flasks.
T A B L E III H U M A N I M M U N O G L O B U L I N P R O D U C T I O N BY H U M A N × H U M A N H Y B R I D O M A S IN P R O T E I N - F R E E MEDIUM Clone a
IgM b (~g/ml)
Cells/ml c ( X 10 -6)
IgM ( # g / m l per 106 cells)
B5C B5C2A BC-MC-E3-A2
4.3 7.7 3.2
0.46 0.52 0,52
9.4 14.8 6.2
a Cells were seeded at 2 × 105 cells/ml in BM with 1 / 2 ( T E I + TEII) and 300/zM ethanolamine. b H y b f i d o m a s u p e m a t a n t s were collected 5 days after plating, diluted 1 : 5, 1 : 25 and 1 : 125 and tested in an ELISA for IgM content by comparison to affinity purified standards. The values shown are the mean of duplicate determinations. c The density of viable cells was determined by trypan blue exclusion. The values shown are the mean of triplicate cultures.
34
Discussion In the present study, we have shown that human x human hybridomas may be successfully grown in a protein-free chemically defined medium. Furthermore, the hybridomas retained their capacity to synthesize human immunoglobulin in such medium. The hybrids do not achieve as high a cell density in protein-free medium as compared to serum-free B S A / T f medium. However, if higher cell densities are required, a more frequent feeding schedule may circumvent this problem. We have found that the protein-free medium which best supports growth consists of F 1 2 / I M D M ( 1 : 1 ) supplemented with ethanolamine (300/zM). Addition of trace elements, while not essential, marginally improved growth in some cases. The same was true of T3. However, since neither of these supplements were ever detrimental to cell growth, we recommend that they be included. Ethanolamine has been reported to be an essential nutrient for a number of tissue culture systems. Tsao et al. (1982) found that either ethanolamine or phosphoethanolamine was an absolute requirement for proliferation of human epidermal keratinocytes. Murakami et al. (1982) found ethanolamine to be an essential growth factor for their murine hybridomas. In both studies, as in the present study, phosphoethanolamine was considerably less potent than ethanolamine in its ability to stimulate growth. The reason for this is unknown although Murakami et al. (1982) have suggested that either phosphoethanolamine is converted to ethanolamine before being taken up by the cells, or phosphoethanolamine is taken up much less efficiently by the cells. The mechanism by which ethanolamine supports cell growth has not been elucidated. In a study using rat m a m m a r y carcinoma cells, KanoSueoka and Errick (1981) found that the majority of ethanolamine taken up by the cells was efficiently converted to phosphatidylethanolamine which was then incorporated into membrane phospholipid. These authors suggested that the synthesis of phosphatidylethanolamine may be rate limiting and that by supplying exogenous ethanolamine, cells can readily synthesize the critical amount of phosphatidylethanolamine neces-
sary for proliferation. Such may be the case with our human x h u m a n hybrids. In contrast, several investigators have reported growth of human cells in protein-free media which do not contain ethanolamine (Okabe and Takaku, 1984; Alderman et al., 1985; Yamaguchi et al., 1985; Shive et al., 1986). These cells may be able to synthesize sufficient phosphatidylethanolamine and thus not require exogenous ethanolamine for cell growth. Further studies are required to fully elucidate the role of ethanolamine in stimulating cell growth. It has been shown that murine MAbs derived from various myeloma fusion partners differ in their dependence on individual components of serum-free medium for growth (Kovar and Franek, 1984). In the present study we have found that four out of four human hybrids derived from K R - 4 grew in protein-free medium; further studies are required to determine if this medium will support the growth of hybrids derived from human fusion partners other than KR-4. The protein-flee medium described in this study will be useful for both immunologic and metabolic studies of human x human hybridomas. In addition, it should facilitate purification of H u M A b s from spent medium. Whether or not this medium will be adaptable to large-scale cultivation methods for mass production of H u M A b s remains to be determined (Arathoon and Birch, 1986).
Acknowledgement We thank C. Jackson for her help in the preparation of the manuscript.
References Alderman, E.M., Lobb, R.R. and Fett, J.W. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5771. Arathoon, W.R. and Birch, J.R. (1986) Science 232, 1390. Campling, B.G., Cole, S.P.C., Atlaw, T., Kozbor, D. and Roder, J.C. (1986) In: A.J. Strelkauskas (Ed.), Human Monoclonal Antibodies (Marcel Dekker, New York) p. 3. Cleveland, W.L., Wood, I. and Erlanger, B.F. (1983) J. Immunol. Methods 56, 221. Cole, S.P.C., Campling, B.G., Atlaw, T., Kozbor, D. and Roder, J.C. (1984a) Mol. Cell. Biochem. 62, 109. Cole, S.P.C., Campling, B.G., Louwman, I.H., Kozbor, D. and Roder, J.C. (1984b) Cancer Res. 44, 2750.
35 Cole, S.P.C., Vreeken, E.H. and Roder, J.C. (1985) J. Immunol. Methods 78, 271. Engleman, E.G., Foung, S.K.H., Larrick, J. and Raubitschek, A. (Eds.) (1985) Human Hybridomas and Monoclonal Antibodies (Plenum Press, New York). Goodman, G.E.,, Beaumier, P., Hellstrom, I., Fernyhough, B. and Hellstrom, K.-E. (1985) J. Clin. Oncol. 3, 340. Kano-Sueoka, T. and Errick, J.E. (1981) Exp. Cell Res. 136, 137. Kovar, J. and Franek, F. (1984) Immunol. Lett. 7, 339. Kozbor, D., Lagarde, A.E. and Roder, J.C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6651. Murakami, H., Masui, H., Sato, G.S., Sueoko, N., Chow, T.P. and Kano-Sueoka, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1158.
Okabe, T. and Takaku, F. (1984) Cancer Res: 44, 4503. Russell, W.C., Newman, C. and Williamson, D.H. (1975) Nature 253, 461. Sears, H.F., Mattis, J., Herlyn, D., Ayry, P.H., Atkinson, B., Ernst, C., Steplewski, Z. and Kroprowski, H. (1982) Lancet i, 762. Shawler, D.L., Bartholomew, R.M., Smith, L.M. and Dillman, R.O. (1985) J. Immunol. 135, 1530. Shive, W., Pinkerton, F., Humphreys, J., Johnson, M.M., Hamilton, W.G. and Matthews, K.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9. Tsao, M.C., Walthall, B.J. and Ham, R.G. (1982) J. Cell. Physiol. 110, 219. Yamaguchi, N., Okabe, T. and Kawai, K. (1985) J. Cancer Res. Clin. Oncol. 110, 42.