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Cloning of human hybridoma, myeloma and lymphoma cell lines using enriched human monocytes as feeder layer
Journal of Immunological Methods, 60 (1983) 1-7
1
Elsevier Biomedical Press
Cloning of Human Hybridoma, Myeloma and Lymphoma Cell Lines Using Enriched Human Monocytes as Feeder Layer * Thomas Brodin l,**, Lennart Olsson 2 and Hans-Olov Sj6gren 1 1 Department of Tumor Immunology, The Wallenberg Laboratory, University of Lund, Lund, Sweden and 2 Medical Department A, State University Hospital, Copenhagen, Denmark
(Received 30 June 1982, accepted 10 December 1982)
Human monocytes were prepared from peripheral blood by buoyant density centrifugation and subsequent absorption-elution on a column of gelatin beads. The eluted fraction containing 60-80% monocytes was used as feeder layer in cloning of the human lymphoma line RH-L4, the human myeloma line SKO-007, and a human hybridoma cell derived from the latter line. Cloning efficiencies were high in both liquid and semisolid media with all 3 cell lines tested. Feeder monocytes could also be successfully used after having been stored in liquid nitrogen. Key words: human hybridoma - - cloning - - monocyte feeder layer
Introduction S i n c e t h e first r e p o r t s of successful c u l t u r e o f h u m a n a n t i b o d y p r o d u c i n g hyb r i d o m a s ( O l s s o n a n d K a p l a n , 1980; C r o c e et al., 1980), m o s t t e c h n i c a l i m p r o v e m e n t s h a v e b e e n p r o m p t e d b y the search for n e w m a l i g n a n t h u m a n cell lines to b e u s e d as f u s i o n p a r t n e r s . T h e i n t r o d u c t i o n of faster g r o w i n g , n o n - I g - p r o d u c i n g h u m a n cell lines t h a t give a h i g h n u m b e r of h y b r i d s w i t h a stable Ig p r o d u c t i o n , a n d o p t i m a l a n t i g e n - p r i m i n g of h u m a n B - l y m p h o c y t e s are b o t h of p r i m e i m p o r t a n c e , in t h e m o u s e s y s t e m ( F a z e k a s de St. G r o t h a n d S c h e i d e g g e r , 1980; St~ihli et al., 1980). * This work was supported by Public Health Service Grant CA 14924 from the National Cancer Institute through the National Large Bowel Cancer Project, NIH Grant no. CA-29876 and by grants from the Swedish Cancer Society, John and Augusta Persson's Foundation, and the Medical Faculty, University of Lund. ** Correspondence to: Thomas Brodin, The Wallenberg Laboratory, University of Lund, Box 7031. S-220 07 LUND, Sweden. Abbrevations: DMSO, dimethylsulphoxide; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; Ig, immunoglobulin; PBS, phosphate-buffered saline; PMC, peripheral blood mononuclear cells. 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
The present report emphasizes the role of feeder cells both as growth stimulators of hybrids at a very early stage after fusion and as an absolute requirement in the cloning step in the human system, aspects of the technique which have not been described previously. We here report studies on the use of human phagocytic cells (peripheral blood monocytes) as feeder cells to support the growth of human hybridoma, myeloma, and lymphoma clones.
Materials and Methods
Cells The human myeloma cell line SKO-007 is an 8-azaguanine resistant variant (Olsson and Kaplan, 1980) of the originally described myeloma line U-266 (Nilsson et al., 1970). The lymphoma line RH-L4 was established from a patient with histiocytic lymphoma. It does not produce Ig, but carries significant amounts of 3,-heavy chains and x-light chains on its cell surface and is resistant to 8-azaguanine (Olsson and Kaplan, 1982). The cell lines are cultured in RPMI 1640 medium supplemented with 10-15% FCS, 40 # g / m l 8-azaguanine, 4 mM L-glutamine (in addition to the original content) and 1 mM sodium pyruvate and buffered with 15 mM NaHCO 3 and 10 mM HEPES. Cell fusion and cultivation of a human hybridoma A human hybridoma cell line was established by fusion of the myeloma line SKO-007 with peripheral blood mononuclear cells (PMC) obtained by Ficoll-Hypaque (Pharmacia) separation of heparinized blood. The blood was taken from a normal blood group A donor who had a low but persistent agglutinating titre against blood group B. The fusion and cultivation protocol was as follows. Ten million PMC were centrifuged together with 1.0 x 10 7 SKO-007 cells at 200 x g for 5 rain. One millilitre polyethylene glycol (MW 1000, Baker), 37% in RPMI was added for 1 min and gradually diluted with RPMI. After fusion, the cells were centrifuged and resuspended in culture medium supplemented with insulin (10 ng/ml) and distributed in 96-well plates (Falcon, Oxnard, CA, or Nunc, Denmark) at a total cell concentration of 2 X 105 per well. In some experiments, 5 x 10 4 human monocytes in 50 ffl medium were distributed in each well one day before fusion. One day after fusion, half the medium was replaced with HAT-medium containing 1.4 x 10 -8 aminopterin, 3 x 10 -4 M hypoxanthine and 1.6 X 10 - 6 M thymidine. The medium was replaced with this medium in this way every 2 or 3 days for 14 days. Cultures were fed with medium containing only hypoxanthine and thymidine (HT-medium) for another 1-2 weeks and finally without these additives. Dense hybrid cultures were tested 3-5 weeks after fusion for production of IgG and IgM by a sandwich ELISA. Ig-containing supernatants were also tested for anti-blood group B activity by an ELISA method. Supernatants were incubated in microtitre wells coated with 1 ~ g / m l blood group B active glycoprotein preparation (gift from Dr. W.M. Watkins, London). One supernatant showed significant reactiv-
ity against this preparation and this hybrid culture was used in the cloning experiments.
Preparation of human monocytes The separation of human monocytes from PMC has recently been described in more detail (SjOgren et al., 1983). Buffy coats from healthy donors were used as the source of human leukocytes. The buffy coats were mixed with an equal volume of phosphate-buffered saline (PBS), pH 7.3 containing 50 I E / m l heparin (Novo) and layered on top of a cushion of Ficoll 400-diatrizoate sodium (density 1.077 g/ml, Ficoll-Hypaque, Pharmacia Fine Chemicals, Uppsala) and centrifuged at 400 × g for 30 min. The interface with mononuclear cells was collected and washed twice in PBS-heparin and finally suspended in 3 ml of RPMI 1640 medium containing 10% human plasma and 50 I E / m l heparin (R10-hep). The cell suspension was loaded on a 10 ml column of gelatin beads (Nilsson and Mosbach, 1980) and the column washed with a total volume of 15 ml Rl0-hep at a flow rate of 1-1.5 ml/min. Loosely attached cells were washed out by adding another 20 ml R10-hep while the beads were stirred with a Pasteur pipette. Finally, cells adherent to the beads were eluted with 50 mM EDTA in PBS containing 10 IE heparin/ml. A total of 15 ml of elution buffer was passed through the column, and the beads were resuspended by pipetting. The eluted cells were washed thrice in culture medium and either used immediately or frozen in RPMI 1640 with 20% FCS and 10% DMSO and stored in liquid nitrogen.
Cloning in liquid medium Human hybridoma, myeloma and lymphoma cells were seeded into microtitre plates at concentrations of 1.0 cell/well and 10 cells/well in a volume of 0.2 ml culture medium. Plates were prepared with feeder cells (5 x 10a/well) the day before cloning. Freshly prepared or liquid nitrogen stored human monocytes, peritoneal cells, obtained by peritoneal lavage of 2-3 month-old B A L B / c mice and peritoneal cells of 2-3-month-old Wistar-Furth rats, respectively, were seeded into 50 btl of culture medium. Thymocytes were also used as feeder cells. These were freshly prepared from 4-week-old B A L B / c mice and were seeded together with the cell lines to be cloned. Eighteen different combinations of feeder cells and tumor cells, including a control without feeder cells, were used on 2 different occasions. The plates were fed once a week by replacing half the medium. Growing clones were visible on microscopy after 10-20 days. Wells with cultures covering at least half the bottom area were counted between 20 and 35 days after seeding.
Cloning in semisolid medium One hundred cells were suspended in 1.0 ml Of 0.3% agar (Nobel, Difco) in RPMI 1640 with 15% FCS and seeded in a well of a 24-well plate (Nunc, Denmark). Wells with feeder cells were prepared by seeding 105 monocytes per well into each well the day before seeding hybridoma, myeloma, or lymphoma cells. Clones ( > 50 cells) were scored 2 weeks after cloning.
A
B
C
Fig. 1. A: H u m a n monocytes seeded at appropriate density in a well of a 96-well plate. B: H u m a n hybridoma clone surrounded by monocyte feeder cells. C: H u m a n hybridoma cells in culture without monocyte feeder cells.
Results
The yield of monocytes by absorption-elution from the gelatin column was 60-80% and the purity 50-70% by analysis of size and staining for non-specific esterase activity (Sj6gren et al., 1983). About 65% of the cells from this preparation were reproducibly recovered after storage in liquid nitrogen. The viability after thawing was 80% as estimated by trypan blue exclusion, and the proportion of monocytes estimated by cell size analysis was approximately the same as in the original preparation. Monocytes adherent to the bottom of the wells could be easily identified by microscopy during the growth of the clones (Fig. 1). Some of these adherent monocytes altered in microscopic appearance from the small ovoid monocytes to enlarged, elongated macrophage-like cells. The results of cloning in liquid medium are summarized in Table I. When human monocytes or mouse thymocytes were used as feeder cells clones arose from all 3 cell lines seeded at a concentration as low as one cell per well. In the wells with mouse and rat peritoneal cells there was initial growth of clones of tumor cells, but after about 2 weeks large adherent cells appeared that microscopically gave the impression of intense phagocytic activity towards the tumor cells. These cultures were continued for as long as 6 weeks without any sign of clonal growth. No growth of clones was observed in medium without feeder cells unless a 100-1000-fold higher number of tumor cells were used. Conditioned media from monocyte or tumor cell cultures did not improve cloning efficiency without feeder cells. Human monocytes, mouse thymocytes and mouse peritoneal cells all improved cloning efficiency dramatically (Table II). In semisolid medium, no inhibitory effect of peritoneal cells on the growth of clones was observed. As described in Materials and Methods, cells were also mixed directly after fusion with human monocyte feeder cells and seeded into plates. The effect of the monocytes in clearing the culture from dead cells and debris and promoting early hybrid growth is illustrated by the micrographs in Fig. 1. TABLE I CLONING EFFICIENCY IN LIQUID MEDIUM T u m o r cell type H u m a n h y b r i d o m a SKO-007 ( N u m b e r of c e l l s / w e l l )
RH-L4
F e e d e r cell type
1
10
1
10
1
10
None Human monocytes BALB/c thymocytes BALB/c peritoneal macrophages W/F peritoneal macrophages
<1 6.50 a 2.37 < 1 < 1
<1 94.94 87.87 < 1 < 1
<1 6.69 13.37 < 1 < 1
<1 100.100 100.100 < 1 < 1
<1 44.87 21.63 < 1 < 1
<1 87.100 94.94 < 1 < 1
a Results are expressed as frequency (%) of wells with clones as a percentage of total n u m b e r of wells ( 1 6 - 4 8 ) seeded with 1 or 10 t u m o r c e l l s / w e l l . Results are from 2 experiments.
TABLE II C L O N I N G EFFICIENCY IN SEMISOLID M E D I U M The results are mean ± S.D. of 4 experiments. 102 tumor cells are seeded per well in 24-well plates and a minimum of 6 wells per feeder cell type were seeded. Monocytes/macrophages were seeded at 105/well, thymocytes at 106/well. Feeder cell type
None Human monocytes BALB/c thymocytes BALB/c peritoneal macrophages
Tumor cell type Human hybridoma
SKO-007
RH-L4
< 1% 14 ±_7% 9 ± 4% 15 + 3%
< 1% 9 + 2,% 5 + 3% 8 _+3 %
< 1% 16 ± 4% 10_+ 3% 14 +_4%
Discussion
The use of feeder cells as support of hybrid growth early after fusion and in cloning procedures is essential in the mouse and rat hybridoma systems. This study demonstrates that feeder cells facilitate cloning of human lymphoid cell lines, including hybridomas, and also the feasibility of using human feeder cells. Peripheral blood monocytes were chosen, because they are easily obtained in sufficient amounts. Furthermore, they have a phagocytic capacity, which is most likely important during the period of HAT selection for hybrids soon after fusion when a large number of cells are dying. The gelatin column method of separation is quick with a high yield and acceptable purity of monocytes. It also has a large separation capacity allowing separation of 3-4 × 108 mononuclear cells on a 10 ml column. The fact that monocytes can be used successfully after storage in liquid nitrogen is very important in practice, since it makes it possible to use monocytes prepared in large batches. The great increase in cloning efficiency when human monocytes are used as feeder cells means not only that a 100-1000-fold reduction is possible in the number of cells needed in the cloning of a primary hybridoma culture, but, more important, that selection against slower growing hybridoma cells is reduced. Tissue-culture factors and growth characteristics of the hybridoma cells probably have less influence in general on the success of cloning in the presence of feeder cells. Although mouse thymocytes showed a feeding effect in the cloning experiments at the same level as human monocytes, we believe that the presence of phagocytic cells just after fusion is of importance in providing optimal conditions for hybrid growth. The possibility of running a hybridoma system with all cells of human origin will avoid effects due to xenogeneic components and possibly xenotropic viruses when monoclonal antibodies are intended for therapeutic purposes.
7
Acknowledgements We wish to acknowledge the skilful technical assistance of Mrs. Barbro Sj6gren, Hanne Kronstr~Sm and Lone Malte.
References Croce, C.M., A. Linnenbach, A.W. Hall, Z. Steplewski and H. Koprowski, 1980, Nature (London) 288, 488. Fazekas de St. Groth, S. and D. Scheidegger, 1980, J. Immunol. Methods 35, 1. Nilsson, L. and K. Mosbach, 1980, Febs Lett. 118, 145. Nilsson, K., H. Bennich, S.G.O. Johansson and J. Ponten, 1970, Clin. Exp. Immunol. 7, 477. Olsson, L. and H.S. Kaplan, 1980. Proc. Natl. Acad. Sci. U.S.A. 77: 9, 5429. Olsson, L. and H.S. Kaplan, in: Methods in Enzymology, 1982, ed. J. Langone, (Academic Press, New York) in press. Sj6gren, H.O., K. Nilsson, P. Malmstr6m and B. Exelsson, 1983, J. Immunol. Methods, 56, 285. Stahli, C., T. Staehelin, V. Miggiano, J. Schmidt and P. H/iring, 1980, J. Immunol. Methods 32, 297.
1
Elsevier Biomedical Press
Cloning of Human Hybridoma, Myeloma and Lymphoma Cell Lines Using Enriched Human Monocytes as Feeder Layer * Thomas Brodin l,**, Lennart Olsson 2 and Hans-Olov Sj6gren 1 1 Department of Tumor Immunology, The Wallenberg Laboratory, University of Lund, Lund, Sweden and 2 Medical Department A, State University Hospital, Copenhagen, Denmark
(Received 30 June 1982, accepted 10 December 1982)
Human monocytes were prepared from peripheral blood by buoyant density centrifugation and subsequent absorption-elution on a column of gelatin beads. The eluted fraction containing 60-80% monocytes was used as feeder layer in cloning of the human lymphoma line RH-L4, the human myeloma line SKO-007, and a human hybridoma cell derived from the latter line. Cloning efficiencies were high in both liquid and semisolid media with all 3 cell lines tested. Feeder monocytes could also be successfully used after having been stored in liquid nitrogen. Key words: human hybridoma - - cloning - - monocyte feeder layer
Introduction S i n c e t h e first r e p o r t s of successful c u l t u r e o f h u m a n a n t i b o d y p r o d u c i n g hyb r i d o m a s ( O l s s o n a n d K a p l a n , 1980; C r o c e et al., 1980), m o s t t e c h n i c a l i m p r o v e m e n t s h a v e b e e n p r o m p t e d b y the search for n e w m a l i g n a n t h u m a n cell lines to b e u s e d as f u s i o n p a r t n e r s . T h e i n t r o d u c t i o n of faster g r o w i n g , n o n - I g - p r o d u c i n g h u m a n cell lines t h a t give a h i g h n u m b e r of h y b r i d s w i t h a stable Ig p r o d u c t i o n , a n d o p t i m a l a n t i g e n - p r i m i n g of h u m a n B - l y m p h o c y t e s are b o t h of p r i m e i m p o r t a n c e , in t h e m o u s e s y s t e m ( F a z e k a s de St. G r o t h a n d S c h e i d e g g e r , 1980; St~ihli et al., 1980). * This work was supported by Public Health Service Grant CA 14924 from the National Cancer Institute through the National Large Bowel Cancer Project, NIH Grant no. CA-29876 and by grants from the Swedish Cancer Society, John and Augusta Persson's Foundation, and the Medical Faculty, University of Lund. ** Correspondence to: Thomas Brodin, The Wallenberg Laboratory, University of Lund, Box 7031. S-220 07 LUND, Sweden. Abbrevations: DMSO, dimethylsulphoxide; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; Ig, immunoglobulin; PBS, phosphate-buffered saline; PMC, peripheral blood mononuclear cells. 0022-1759/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
The present report emphasizes the role of feeder cells both as growth stimulators of hybrids at a very early stage after fusion and as an absolute requirement in the cloning step in the human system, aspects of the technique which have not been described previously. We here report studies on the use of human phagocytic cells (peripheral blood monocytes) as feeder cells to support the growth of human hybridoma, myeloma, and lymphoma clones.
Materials and Methods
Cells The human myeloma cell line SKO-007 is an 8-azaguanine resistant variant (Olsson and Kaplan, 1980) of the originally described myeloma line U-266 (Nilsson et al., 1970). The lymphoma line RH-L4 was established from a patient with histiocytic lymphoma. It does not produce Ig, but carries significant amounts of 3,-heavy chains and x-light chains on its cell surface and is resistant to 8-azaguanine (Olsson and Kaplan, 1982). The cell lines are cultured in RPMI 1640 medium supplemented with 10-15% FCS, 40 # g / m l 8-azaguanine, 4 mM L-glutamine (in addition to the original content) and 1 mM sodium pyruvate and buffered with 15 mM NaHCO 3 and 10 mM HEPES. Cell fusion and cultivation of a human hybridoma A human hybridoma cell line was established by fusion of the myeloma line SKO-007 with peripheral blood mononuclear cells (PMC) obtained by Ficoll-Hypaque (Pharmacia) separation of heparinized blood. The blood was taken from a normal blood group A donor who had a low but persistent agglutinating titre against blood group B. The fusion and cultivation protocol was as follows. Ten million PMC were centrifuged together with 1.0 x 10 7 SKO-007 cells at 200 x g for 5 rain. One millilitre polyethylene glycol (MW 1000, Baker), 37% in RPMI was added for 1 min and gradually diluted with RPMI. After fusion, the cells were centrifuged and resuspended in culture medium supplemented with insulin (10 ng/ml) and distributed in 96-well plates (Falcon, Oxnard, CA, or Nunc, Denmark) at a total cell concentration of 2 X 105 per well. In some experiments, 5 x 10 4 human monocytes in 50 ffl medium were distributed in each well one day before fusion. One day after fusion, half the medium was replaced with HAT-medium containing 1.4 x 10 -8 aminopterin, 3 x 10 -4 M hypoxanthine and 1.6 X 10 - 6 M thymidine. The medium was replaced with this medium in this way every 2 or 3 days for 14 days. Cultures were fed with medium containing only hypoxanthine and thymidine (HT-medium) for another 1-2 weeks and finally without these additives. Dense hybrid cultures were tested 3-5 weeks after fusion for production of IgG and IgM by a sandwich ELISA. Ig-containing supernatants were also tested for anti-blood group B activity by an ELISA method. Supernatants were incubated in microtitre wells coated with 1 ~ g / m l blood group B active glycoprotein preparation (gift from Dr. W.M. Watkins, London). One supernatant showed significant reactiv-
ity against this preparation and this hybrid culture was used in the cloning experiments.
Preparation of human monocytes The separation of human monocytes from PMC has recently been described in more detail (SjOgren et al., 1983). Buffy coats from healthy donors were used as the source of human leukocytes. The buffy coats were mixed with an equal volume of phosphate-buffered saline (PBS), pH 7.3 containing 50 I E / m l heparin (Novo) and layered on top of a cushion of Ficoll 400-diatrizoate sodium (density 1.077 g/ml, Ficoll-Hypaque, Pharmacia Fine Chemicals, Uppsala) and centrifuged at 400 × g for 30 min. The interface with mononuclear cells was collected and washed twice in PBS-heparin and finally suspended in 3 ml of RPMI 1640 medium containing 10% human plasma and 50 I E / m l heparin (R10-hep). The cell suspension was loaded on a 10 ml column of gelatin beads (Nilsson and Mosbach, 1980) and the column washed with a total volume of 15 ml Rl0-hep at a flow rate of 1-1.5 ml/min. Loosely attached cells were washed out by adding another 20 ml R10-hep while the beads were stirred with a Pasteur pipette. Finally, cells adherent to the beads were eluted with 50 mM EDTA in PBS containing 10 IE heparin/ml. A total of 15 ml of elution buffer was passed through the column, and the beads were resuspended by pipetting. The eluted cells were washed thrice in culture medium and either used immediately or frozen in RPMI 1640 with 20% FCS and 10% DMSO and stored in liquid nitrogen.
Cloning in liquid medium Human hybridoma, myeloma and lymphoma cells were seeded into microtitre plates at concentrations of 1.0 cell/well and 10 cells/well in a volume of 0.2 ml culture medium. Plates were prepared with feeder cells (5 x 10a/well) the day before cloning. Freshly prepared or liquid nitrogen stored human monocytes, peritoneal cells, obtained by peritoneal lavage of 2-3 month-old B A L B / c mice and peritoneal cells of 2-3-month-old Wistar-Furth rats, respectively, were seeded into 50 btl of culture medium. Thymocytes were also used as feeder cells. These were freshly prepared from 4-week-old B A L B / c mice and were seeded together with the cell lines to be cloned. Eighteen different combinations of feeder cells and tumor cells, including a control without feeder cells, were used on 2 different occasions. The plates were fed once a week by replacing half the medium. Growing clones were visible on microscopy after 10-20 days. Wells with cultures covering at least half the bottom area were counted between 20 and 35 days after seeding.
Cloning in semisolid medium One hundred cells were suspended in 1.0 ml Of 0.3% agar (Nobel, Difco) in RPMI 1640 with 15% FCS and seeded in a well of a 24-well plate (Nunc, Denmark). Wells with feeder cells were prepared by seeding 105 monocytes per well into each well the day before seeding hybridoma, myeloma, or lymphoma cells. Clones ( > 50 cells) were scored 2 weeks after cloning.
A
B
C
Fig. 1. A: H u m a n monocytes seeded at appropriate density in a well of a 96-well plate. B: H u m a n hybridoma clone surrounded by monocyte feeder cells. C: H u m a n hybridoma cells in culture without monocyte feeder cells.
Results
The yield of monocytes by absorption-elution from the gelatin column was 60-80% and the purity 50-70% by analysis of size and staining for non-specific esterase activity (Sj6gren et al., 1983). About 65% of the cells from this preparation were reproducibly recovered after storage in liquid nitrogen. The viability after thawing was 80% as estimated by trypan blue exclusion, and the proportion of monocytes estimated by cell size analysis was approximately the same as in the original preparation. Monocytes adherent to the bottom of the wells could be easily identified by microscopy during the growth of the clones (Fig. 1). Some of these adherent monocytes altered in microscopic appearance from the small ovoid monocytes to enlarged, elongated macrophage-like cells. The results of cloning in liquid medium are summarized in Table I. When human monocytes or mouse thymocytes were used as feeder cells clones arose from all 3 cell lines seeded at a concentration as low as one cell per well. In the wells with mouse and rat peritoneal cells there was initial growth of clones of tumor cells, but after about 2 weeks large adherent cells appeared that microscopically gave the impression of intense phagocytic activity towards the tumor cells. These cultures were continued for as long as 6 weeks without any sign of clonal growth. No growth of clones was observed in medium without feeder cells unless a 100-1000-fold higher number of tumor cells were used. Conditioned media from monocyte or tumor cell cultures did not improve cloning efficiency without feeder cells. Human monocytes, mouse thymocytes and mouse peritoneal cells all improved cloning efficiency dramatically (Table II). In semisolid medium, no inhibitory effect of peritoneal cells on the growth of clones was observed. As described in Materials and Methods, cells were also mixed directly after fusion with human monocyte feeder cells and seeded into plates. The effect of the monocytes in clearing the culture from dead cells and debris and promoting early hybrid growth is illustrated by the micrographs in Fig. 1. TABLE I CLONING EFFICIENCY IN LIQUID MEDIUM T u m o r cell type H u m a n h y b r i d o m a SKO-007 ( N u m b e r of c e l l s / w e l l )
RH-L4
F e e d e r cell type
1
10
1
10
1
10
None Human monocytes BALB/c thymocytes BALB/c peritoneal macrophages W/F peritoneal macrophages
<1 6.50 a 2.37 < 1 < 1
<1 94.94 87.87 < 1 < 1
<1 6.69 13.37 < 1 < 1
<1 100.100 100.100 < 1 < 1
<1 44.87 21.63 < 1 < 1
<1 87.100 94.94 < 1 < 1
a Results are expressed as frequency (%) of wells with clones as a percentage of total n u m b e r of wells ( 1 6 - 4 8 ) seeded with 1 or 10 t u m o r c e l l s / w e l l . Results are from 2 experiments.
TABLE II C L O N I N G EFFICIENCY IN SEMISOLID M E D I U M The results are mean ± S.D. of 4 experiments. 102 tumor cells are seeded per well in 24-well plates and a minimum of 6 wells per feeder cell type were seeded. Monocytes/macrophages were seeded at 105/well, thymocytes at 106/well. Feeder cell type
None Human monocytes BALB/c thymocytes BALB/c peritoneal macrophages
Tumor cell type Human hybridoma
SKO-007
RH-L4
< 1% 14 ±_7% 9 ± 4% 15 + 3%
< 1% 9 + 2,% 5 + 3% 8 _+3 %
< 1% 16 ± 4% 10_+ 3% 14 +_4%
Discussion
The use of feeder cells as support of hybrid growth early after fusion and in cloning procedures is essential in the mouse and rat hybridoma systems. This study demonstrates that feeder cells facilitate cloning of human lymphoid cell lines, including hybridomas, and also the feasibility of using human feeder cells. Peripheral blood monocytes were chosen, because they are easily obtained in sufficient amounts. Furthermore, they have a phagocytic capacity, which is most likely important during the period of HAT selection for hybrids soon after fusion when a large number of cells are dying. The gelatin column method of separation is quick with a high yield and acceptable purity of monocytes. It also has a large separation capacity allowing separation of 3-4 × 108 mononuclear cells on a 10 ml column. The fact that monocytes can be used successfully after storage in liquid nitrogen is very important in practice, since it makes it possible to use monocytes prepared in large batches. The great increase in cloning efficiency when human monocytes are used as feeder cells means not only that a 100-1000-fold reduction is possible in the number of cells needed in the cloning of a primary hybridoma culture, but, more important, that selection against slower growing hybridoma cells is reduced. Tissue-culture factors and growth characteristics of the hybridoma cells probably have less influence in general on the success of cloning in the presence of feeder cells. Although mouse thymocytes showed a feeding effect in the cloning experiments at the same level as human monocytes, we believe that the presence of phagocytic cells just after fusion is of importance in providing optimal conditions for hybrid growth. The possibility of running a hybridoma system with all cells of human origin will avoid effects due to xenogeneic components and possibly xenotropic viruses when monoclonal antibodies are intended for therapeutic purposes.
7
Acknowledgements We wish to acknowledge the skilful technical assistance of Mrs. Barbro Sj6gren, Hanne Kronstr~Sm and Lone Malte.
References Croce, C.M., A. Linnenbach, A.W. Hall, Z. Steplewski and H. Koprowski, 1980, Nature (London) 288, 488. Fazekas de St. Groth, S. and D. Scheidegger, 1980, J. Immunol. Methods 35, 1. Nilsson, L. and K. Mosbach, 1980, Febs Lett. 118, 145. Nilsson, K., H. Bennich, S.G.O. Johansson and J. Ponten, 1970, Clin. Exp. Immunol. 7, 477. Olsson, L. and H.S. Kaplan, 1980. Proc. Natl. Acad. Sci. U.S.A. 77: 9, 5429. Olsson, L. and H.S. Kaplan, in: Methods in Enzymology, 1982, ed. J. Langone, (Academic Press, New York) in press. Sj6gren, H.O., K. Nilsson, P. Malmstr6m and B. Exelsson, 1983, J. Immunol. Methods, 56, 285. Stahli, C., T. Staehelin, V. Miggiano, J. Schmidt and P. H/iring, 1980, J. Immunol. Methods 32, 297.