Journal of Immunological Methods, 134 (1990) 35-42
35
Elsevier JIM05726
Development of microfusion techniques to generate human hybridomas S. F o u n g 1, S. Perkins 1, K. K a f a d a r 2, p. G e s s n e r 3 a n d U. Z i m m e r m a n n 3 1 Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A., 2 Statistics and Surveillance Branch, Epidemiology Program Office, Centers for Disease Control, Atlanta, GA 30333, U.S.A., and 3 Institute of Biotechnology, University of Wiirzburg, Ve'iirzburg, F.R.G.
(Received8 May 1990, accepted 3 July 1990) The rarity of antigen-specific B cells in peripheral blood and lymphoid tissues is a major limitation in the production of human monoclonal antibodies. This has led to a requirement for techniques capable of fusing small numbers of cells and achieving a higher hybridoma formation efficiency than currently is possible. The approach used in these studies to generate human hybridomas is based on the observation that under hypo-osmolar conditions electric field induced cell fusion or electrofusion is facilitated. Electrofusion parameters have been defined in strongly hypo-osmolar solutions which have resulted in a hybridoma formation efficiency greater than 5 x 10 -3 under optimal conditions. Furthermore, this has been accomplished with total input B cells of 1-2 x 105. This is a ten-fold reduction in the required number of input B cells and is associated with a hybridoma formation efficiency at least equal to that achieved with a higher input B cell number. An important factor in the development of this microfusion technique appears to be the duration of exposure to the hypo-osmolar solution by B cells to be immortalized. Other parameters which may affect hybridoma yield include the electrical field strength used for cell alignment and membrane breakdown, ratio of human B cells to fusion partner, washing procedure, post-fusion incubation time, and the elimination of toxic molecules. Key words: Electrofusion;Hybridoma, human
Introduction
A major obstacle in the generation of human monoclonal antibodies is the rarity of antigenspecific B calls in peripheral blood and lymphoid tissues. While the problem is overcome partly by expanding the B cell pool with secondary in vitro activation (e.g., by Epstein-Barr virus), the proportion of antigen-specific B cells remains low (James Abbreviations." EBV, Epstein-Barr virus; IMDM, Iscove's modified Dulbecco's medium; FCS, fetal calf serum. Correspondence to: S.K.H. Foung, Stanford University Blood Bank, 800 Welch Road, Palo Alto, CA 94304, U.S.A.
and Bell, 1987). Furthermore, the total cell number available for immortalization by somatic cell hybridization is often less than 106 in a variety of in vitro activation systems used to expand the antigen-specific B cell pool. Because of this limitation, there is a requirement to develop approaches to immortalize small numbers of cells with a high fusion or hybridoma formation efficiency. The development of electric field-induced cell fusion or electrofusion has led to techniques capable of achieving a higher fusion efficiency with a corresponding higher hybridoma formation efficiency in comparison to polyethylene glycol-induced cell fusion (Vienken and Zimmermann, 1985; Ohnishi et al., 1987; Glassy, 1988; Foung and Perkins,
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
36 1989). One approach to further enhance fusion efficiency by electrofusion is based on the observation that electrofusion is facilitated under hypo-osmolar conditions. The feasibility of this approach has been demonstrated recently in a murine model system for hybridoma formation (Schmitt and Zimmermann, 1989; Schmitt et al., 1989; Zimmermann et al., 1989). In this report, we describe the effects of hypo-osmolar conditions on the generation of human hybridomas by electrofusion. This has led to the development of microfusion techniques with a high efficiency of hybridoma formation.
Materials and methods
B cell isolation and activation Peripheral blood lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation (B~Syum, 1968), and B cells were separated from T cells by rosetting with 2-aminoethylisothiouronium bromide hydrobromide treated sheep red blood cells (Saxon et al., 1976). B cells were activated with Epstein-Barr virus (EBV) using supernatant from the B95-8 marmoset line as a source of virus (Foung et al., 1989; Perkins et al., 1989). Composition of fusion media Iso-osmolar fusion medium (300 L3) consisted of 280 mM sorbitol (E.M. Science, Cherry Hill, NJ, U.S.A.), 0.1 mM Ca 2+ acetate (E.M. Science), 0.5 mM Mg 2+ acetate (E.M. Science), and 1 m g / m l pure bovine serum albumin (Serva Biochemicals, Westbury, NY, U.S.A.) with a final osmolarity of 300 mosM. Hypo-osmolar fusion medium (75 L3) consisted of 70 mM sorbitol, 0.1 mM Ca z+ acetate, 0.5 m M Mg 2+ acetate, and 1 m g / m l pure bovine serum albumin with a final osmolarity of 75 mosM. Fusion protocols Open chamber fusions were performed to establish the approximate electrical parameters necessary for eventual hybridoma formation. The open chamber consists of two parallel electrodes (200/~m in diameter and 200/~m apart) attached to a glass slide. This configuration permits direct microscopic observation of the fusion process. The
cell populations, EBV activated B cells and the mouse-human heteromyeloma K 6 H 6 / B 5 (generously provided by Dr. Levy, Stanford University) (Carroll et al., 1986) were fused at room temperature, first separately and then in combination, to establish the appropriate electrical range. Based on these studies, helical chamber fusions with EBV activated B cells and K 6 H 6 / B 5 were performed with similar electrical parameters using different cell ratios, wash conditions and osmolar solutions. The cells were pooled, washed and fused in the medium indicated. After washing, cells were resuspended in 170 /~1 of fusion medium and transferred to a helical fusion chamber (GCA, Chicago, IL, U.S.A.) consisting of a receptacle to hold the cells and an electrical assembly of two electrodes of 200 /~m diameter wound in a helix 200/~m apart that fits inside the receptacle. Electrical current was applied with the Model Z1000 Zimmermann Cell Fusion System (GCA). Cells were aligned in a non-uniform alternating field (AC) of I MHz frequency, and fusion was induced by three pulses of direct current (DC) of high intensity with 15 t~s duration, 1 s between pulses, and 10 ms of alignment off time. Alignment was tapered off gradually over 30 s after fusion. After incubating in the chamber, cells were washed out and plated in 96 well microtiter plates in pre-selection medium containing complete Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY, U.S.A.) with 15% fetal calf serum (FCS), 100 ~M hypoxanthine (Sigma, St. Louis, MO, U.S.A.), and 15/~M thymidine (Sigma), and grown in a 6-6.5% CO 2 incubator at 37°C. Unfused cells were also plated in this medium and served as controls. After 24 h, and for a period of 2 weeks, the cells were maintained in selection medium consisting of pre-selection medium with the addition of 0.8 /~M aminopterin (Sigma) and 0.1 /~M ouabain (Sigma). After 2 weeks, the hybridomas were grown in the preselection medium. Hybrid yield was determined by counting the macroscopic growth in wells after no new colonies appeared in new wells (on the order of 6 - 7 weeks with these cells). Because of the difficulty in counting a large number of wells and colonies and the potential for counting errors, it seems reasonable to characterize the efficiency of hybridoma formation as a range of likely values rather than
37
as a single number. The concept of range in hybridoma formation efficiency has been defined and illustrated previously where it was referred to as the fusion efficiency (Foung and Perkins, 1989). It is the number of wells with growth multiplied by two values corresponding to the extremes in the number of colonies in over half of the wells which were counted, divided by 105 input EBV activated B cells. This definition attempts to incorporate some of the uncertainty and variation inherent in counting large numbers of wells with large numbers of colonies which are not always clearly delineated. To summarize a series of such ranges (in Tables III, IV and VI), data are presented as an overall range of the lowest of the low endpoints and the highest of the high endpoints to indicate the extremes in efficiency. A median range is also presented and is computed as the median of the low end points and the median of the high end points for each series of ranges. The median range is used along with the overall range to compare the hybridoma formation efficiency with different sets of parameters.
Fusions in iso-osmolar medium (300 L3) 1-3.5 X 105 EBV activated B cells were fused to K 6 H 6 / B 5 at a ratio of I EBV : 1-10 K 6 H 6 / B 5 . The cells were washed two times in 300 L3 fusion medium. The parameters for fusions in 300 L3 were a 20-30 s alignment time at 250-300 AC V / c m and three D C fusion pulses of 1.5-2.0 k V / c m . Cells were washed out of the chamber 30
rain after fusion and plated in I M D M pre-selection medium at 5 x 103-9 X 104 cells/well.
Fusions in hypo-osmolar medium (75 L3) 1-2.5 X 105 EBV activated B ceils were fused to K 6 H 6 / B 5 at a ratio of 1 EBV : 0.5-2 K 6 H 6 / B 5 . The cells were washed one or two times in 300 L3 medium and fused in 75 L3 fusion medium. The electrical parameters were a 30 s alignment time at 300 AC V / c m and three D C fusion pulses of 1.0 k V / c m . The fusion process was performed between 5 and 15 min after the cells were suspended in hypo-osmolar medium. Cells were washed out of the chamber 10-15 min after fusion and plated at 5 x 103-104 cells/well in I M D M preselection medium containing no p H (phenol) indicator. After 24 h they were maintained in selection medium containing p H indicator.
Results
EBV activated B cells when fused to a mousehuman heteromyeloma in iso-osmolar medium by electrofusion have produced a hybridoma formation efficiency of 32-229 hybridomas per 105 input B cells (Foung and Perkins, 1989). This enhanced range in fusion efficiency of hybridoma formation over polyethylene glycol-induced fusion methods was achieved with as few as 106 input B cells. In a similar series of fusions in iso-osmolar fusion medium using only 1-3.5 X 105 input EBV,
TABLE I H U M A N H Y B R I D O M A F O R M A T I O N IN ISO-OSMOLAR S O L U T I O N BY E L E C T R O F U S I O N Fusion
Input EBV (105)
Fusion ratio a
DC V (kV/cm)
Percent wells with hybrids
Hybridoma formation efficiency
1
1.0
1 : 10
2 3 4 5 6 7 8 9
1.0 2.0 2.0 2.0 2.3 3.3 2.4 3.5
1 : 10 1:3 1:1 1 :1 1 :5 1:3 1 :5 1:3
2.0 2.0 2.0 1.5 2.0 2.0 1.5 1.5 2.0
75 25 45 44 18 47 66 44 58
14 5 3-24 11 4 6- 9 5-15 7-13 5- 6
a Ratio of EBV activated B cells fused to K6H6//B5.
38
however, a substantial drop in the range in efficiency of hybridoma formation was observed (Table I). The decrease in efficiency to an overall range of 3-24 hybridomas per 105 input B cells was present with a wide range of fusion ratios between EBV activated B cells and the mouse-human heteromyeloma cell line, K6H6/B5. To ascertain the effects of hypo-osmolar solutions on hybridoma formation by electrofusion, multiple series of fusions were performed with EBV activated B cells and K6H6//B5. The approximate electrical parameters necessary for hybridoma formation were first determined in open chamber fusions. In iso-osmolar fusion medium (300 L3), a small number of K 6 H 6 / B 5 cells could be observed fusing to themselves over a 20 min period at a wide range of DC voltages (1.5-3.5 kV/cm). At low cell density, more cells were fused with three DC pulses of 1.5 k V / c m , although the number was still small (5-10% of the total). A marked increase in fusion efficiency occurred in hypo-osmolar medium (75 L3). 50-90% of KrH6//B5 fused within five rain using a lower DC voltage (1.0 k V / c m with three pulses) than in iso-osmolar solution. EBV-activated B cells in 300 L3 were observed to fuse in a similar manner to K r H 6 / B 5 . A small number of cells were fused over a wide range in DC voltage (1.5-3.5 kV/cm). More cell fusions occurred at lower voltages when the cell density was low. To determine the optimal conditions in 75 L3, over 100 experiments were performed in
the open chamber with different alignment field strength (1-2 MHz, 250-300 V / c m AC), fusion voltage (0.5-1.5 k V / c m DC), pulse number (3-9), cell density (5x105-107 cells/ml), wash medium osmolarity (38-300 mosM), fusion medium osmolarity (38-150 mosM), and time exposure to the hypo-osmolar medium (2 min-1 h). The most critical factor appeared to be the duration of cell exposure to the hypo-osmolar medium prior to fusion. Higher fusion efficiency was obtained in 75 L3 only when the cells' exposure time to the medium was limited. Poor fusion efficiency in 75 L3 resulted when time exposures were less than 5 rain or greater than 15 min. Based on these observations, a series of hypoosmolar electrofusions were performed in helical chambers with EBV-activated B cells and K6H6/B5 a t a fixed ratio with different durations of exposure to 75 L3 before fusion (Table II). The electrical parameters chosen for all hypo-osmolar fusions were based on the results obtained with K 6 H 6 / B 5 fusions with the open chamber. The alignment time was 30 s at 300 AC V / c m and the fusion event initiated with three DC pulses of 1.0 k V / c m . Highest hybrid yield was observed after 10 rain in the medium, and the lowest after 30-40 min. Hybrid yield and exposure time to the hypoosmolar solution appeared to be inversely related. The number of times cells were washed in 300 L3 prior to the electrofusion procedure was also noted to affect hybrid yield with a slightly higher efficiency after one wash (Table II). This may be
TABLE II D U R A T I O N OF E X P O S U R E TO H Y P O - O S M O L A R S O L U T I O N P R I O R TO E L E C T R O F U S 1 O N a Fusion
Exposure duration b
Number of washes
Percent wells with hybrids
Hybridoma formation efficiency
1 2 3 4
10 20 30 40
1 1 1 1
100 92 72 80
80-200 36-144 16- 80 16- 96
5 6 7 8
10 20 30 40
2 2 2 2
90 80 45 25
28-193 16-133 10- 30 4 - 24
a EBV activated B cells fused to K 6 H 6 / B 5 at 1 : 1 ratio in all experiments. b Minutes cells exposed to 75 L3 prior to electrofusion.
39 TABLE III D U R A T I O N OF POST-FUSION INCUBATION USED FOR H Y B R I D O M A F O R M A T I O N a Duration (min)
N u m b e r of fusions
Percent wells with hybrids
Hybridoma formation efficiency
Range
Mean
Overall range
Median range
10-15 25-30
4 4
50-94 22-94
76 62
10-180 6-118
20-120 14- 77
Minutes of incubation after fusion in the helical chambers. All fusions performed in 75 L3 at 1 : 1 ratio of EBV activated B cells to K6H6/B5.
a
due to factors other than cell loss from washing since the relative drop in hybridoma formation between 10 min and 40 min exposure time in 75 L3 is less in the one wash series of electrofusions. Other parameters that appear to affect hybrid yield include post-fusion incubation time and the ratio between input B cell and fusion partner (Tables III and IV). A shorter post-fusion incubation of 10-15 min in the chamber resulted in an overall hybridoma formation efficiency range of 10-180 hybridomas per 105 input B cells, which was greater than the 6-118 hybridomas per 105 input B cells obtained with the longer post-fusion incubation duration of 25-30 min (Table III). The fusion ratio has an even greater effect on the efficiency of hybridoma formation (Table IV). Although only two fusions were performed with a fusion ratio of one EBV activated B cell to two K6H6/B5, the hybrid yield was substantially greater using this ratio than other fusion ratios, producing a median efficiency range of 38-464 hybridomas per 105 input B cells. Previous studies have suggested that molecules not ordinarily toxic to intact cells such as pH
indicator can have a toxic effect shortly after electrofusion. A series of fusions was performed and maintained for 24 h in I M D M pre-selection medium without pH indicator (Table V). In this series, hybridoma formation efficiency of up to 600 hybridomas per 105 input B cells was obtained. A marked increase in the percent wells with hybrid growth was noted with fusions performed in 75 L3 compared to those performed in 300 L3 (Table VI). When the cells were fused under hypo-osmolar conditions and grown for 24 h in medium without pH indicator, a 6-20-fold increase in the median range of hybridoma formation efficiency was achieved compared to isoosmolar fusions.
Discussion
A major strategy used to generate antigenspecific human monoclonal antibodies involves initial expansion of the antigen-specific B cell pool by in vitro activation with EBV and subsequent immortalization by somatic cell hybridization with
TABLE IV E F F E C T OF F U S I O N RATIO ON H Y B R I D O M A YIELD a d Cell ratio EBV : K r H 6 / B 5
Number of fusions
Percent wells with hybrids
Hybridoma formation efficiency
Range
Mean
Overall range
Median range
2:1 1:1 1:2
7 10 2
20- 75 58-100 89- 94
54 84 92
3-209 20-525 29-635
11- 82 32-196 38-464
Fusions performed in 75 L3.
TABLE V HUMAN HYBRIDOMA YIELD IN MEDIUM WITHOUT pH INDICATOR a Fusion
Wash number
Input EBV (105)
Fusion b ratio
Percent w e l l s with hybrids
Hybridoma formation Efficiency
1 2 3 4 5 6 7 8 9
1 1 1 1 2 2 2 2 2
1.0 1.0 1.0 2.0 2.0 2.0 2.0 2.0 2.0
1:1 1:2 2:1 1:1 1:1 1:1 1:1 1:2 2:1
92 94 70 100 94 90 82 89 75
35-525 29-635 15- 82 80-200 36-180 28-193 24-193 47-293 12-209
a Fusions performed in 75 L3 and plated in IMDM without pH indicator. b Ratio of EBV activated B cells fused to K6H6//B5.
an a p p r o p r i a t e fusion partner. I n this a n d other in vitro activation systems used to e x p a n d the relev a n t B cell p o p u l a t i o n , the total cell n u m b e r available for s u b s e q u e n t cell fusion is often less t h a n ] 0 6 . Therefore, it is desirable to have techniques c a p a b l e of fusing small n u m b e r s of cells ( 1 - 2 x 10 5) with an efficiency of h y b r i d o m a f o r m a t i o n a p p r o a c h i n g 10 -2, or one h y b r i d o m a per 100 input B cells. T h e d e v e l o p m e n t of these m i c r o f u s i o n techniques will lead to a b r o a d e r application of h y b r i d o m a technology to s t u d y the h u m a n h u m o r a l i m m u n e response in disease states. D e v e l o p m e n t of electric field-induced cell fusion or electrofusion already has led to techniques capable of achieving a h u m a n h y b r i d o m a formation efficiency a p p r o a c h i n g 1 0 - 3 with a n i n p u t n u m b e r of 10 6 or more EBV activated B cells ( F o u n g a n d Perkins, 1989). T o m a i n t a i n this high fusion or h y b r i d o m a f o r m a t i o n efficiency with a lower n u m b e r of i n p u t B cells (105), the effects of
h y p o - o s m o l a r c o n d i t i o n s were explored. This approach is based on the o b s e r v a t i o n that the surface area of m e m b r a n e c o n t a c t b e t w e e n two cells established in an a l t e r n a t i n g electric field is markedly increased u n d e r strongly h y p o - o s m o l a r c o n d i t i o n s p r o v i d i n g more p o t e n t i a l area for cell fusion. A n o t h e r factor is the n u c l e a r swelling occurring u n d e r c o n d i t i o n s of h y p o - o s m o l a r i t y which m a y facilitate nuclear fusion in the fused cells (Bertsche et al., 1988). Both are necessary steps in h y b r i d o m a formation. As shown in these studies, the fusion process initiated b y a p p l i c a t i o n of direct current is achieved with less field strength (1.0 k V / c m ) c o m p a r e d to those iso-osmolar fusions (1.5-2.0 k V / c m ) . F u r t h e r m o r e , the fusion process in h y p o - o s m o l a r solution occurs m o r e rapidly t h a n in iso-osmolar solution a n d with more cells b e i n g fused. H y p o - o s m o l a r electrofusions have resulted in a h y b r i d o m a f o r m a t i o n efficiency greater t h a n 5 × 1 0 3 with total i n p u t B cells of 1 - 2 x 1 0 5
TABLE VI HUMAN HYBRIDOMA FORMATION EFFICIENCY IN ISO-OSMOLAR AND HYPO-OSMOLAR SOLUTIONS Fusion medium
pH indicator
Number of fusions
Percent wells with hybrids
Hybridoma formation efficiency
Range
Mean
Overall Range
Median Range
300 L3 75 L3 75 L3
+ + a -
9 10 9
18- 75 20- 98 70-100
45 58 80
3- 24 3-347 12-635
5- 11 12-108 29-200
a Fused cells harvested from helical chamber in RPMI without pH indicator, diluted and plated in IMDM with indicator.
41
under optimal conditions. This is a ten-fold decrease in the required number of input B cells and is associated with a fusion efficiency equal to or greater than that achieved using a higher input B cell number. A most critical factor in the development of this microfusion technique appears to be the duration of exposure to the hypo-osmolar solution by EBV activated B cells. A range of 5-15 min was optimal in these studies. It is likely that a slightly higher osmolar solution or modified fusion voltage parameters will lead to a greater tolerance of EBV activated B cell exposure to hypo-osmolar conditions. Other parameters that appear to affect hybridoma yield include the ratio of human B cells to fusion partner, washing procedure, post-fusion incubation time, and the elimination of molecules not ordinarily toxic to intact cells (e.g., phenol) from the growth medium used to support human hybridomas within the first 24 h post-fusion. The development of appropriate electrical parameters to achieve maximum fusion efficiency is best accomplished with open chamber fusions. The open chamber allows direct visualization of the effects of different electrical parameters on the fusion process. The defined electrical parameters can then be applied to helical chamber electrofusions with slight modifications. By elucidating the effects of these parameters, it has been possible to develop microfusion techniques with increased fusion or hybridoma formation efficiency using other mouse-human heteromyelomas (Zimmerman et al., 1990). The improvements in hybridoma technology achieved in these studies should enable researchers to use a combination of human B cell selection and in vitro activation to isolate virtually any antibody represented within the human B cell repertoire. It is possible that the recently described recombinant DNA technique used to generate and select the immunoglobulins of a desirefl specificity may represent a more efficient appro~tch (Huse et al., 1989). This technique based on random recombination of heavy and light chain genes may potentially generate an even greater diversity of antibodies than are represented by the repertoire of B cells. However, to define antibodies which are involved in human disease, the hybridoma approach will yield immunoglobulins which are en-
coded by B cells under the constraints of the disease process in vivo. Therefore, the improvements in hybridoma technology described in these studies should provide a better tool for the evaluation of the role of antibodies in the pathogenesis or resolution of human disease.
Acknowledgements The authors wish to thank Judy Campbell and Norma Secord who typed this manuscript. This study was supported in part by Grants HL33811, AI22557, AI26031 and DA06596 from the National Institutes of Health to S.K.H.F., and grants of the Deutsche Forschungsgemeinschaft (SFB 176) and of the Federal Ministry of Research and Technology, F . R . G . ( D F V L R 01QV354) to U.Z.
References Bertsche, U., Mader, A. and Zimmermann, U. (1988) Nuclear membrane fusion in electrofused mammalian cells. Biochim. Biophys. Acta 939, 509. BSyum, A. (1968) A one-stage procedure for isolation of granulocytes and lymphocytes from human blood. Scand. J. Clin. Lab. Invest. 2197 (Suppl.), 51. Carroll, W.L., Thielemans, K., Dilley, J. and Levy, R. (1986) Mouse x human heterohybridomas as fusion partners with human B cell tumors. J. Immunol. Methods 89, 61. Foung, S.K.H. and Perkins, S. (1989) Electric field-induced cell fusion and human monoclonal antibodies. J. Immunol. Methods 116, 117. Foung, S.K.H., Perkins, S., Bradshaw, P., Rowe, J., Rabin, L.B., Reyes, G.R. and Lennette, E.T. (1989) Human monoclonal antibodies to human cytomegalovirus. J. Infect. Dis. 159, 436. Glassy, M.C. (1988) Creating hybridomas by electrofusion. Nature 333, 579. Huse, W.D., Sastry, L., Iverson, S.A., Kang, A.S., Alting-Mees, M., Burton, D.R., Benkovic, S.J. and Lerner, R.A. (1989) Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275. James, K. and Bell, G.T. (1987) Human monoclonal antibody production, current status and future prospects. J. Immunol. Methods 100, 5. Ohnishi, K., Chiba, J., Goto, Y. and Tokumaga, T. (1987) Improvement in the basic technology of electrofusion for generation of antibody-producing hybridomas. J. Immunol. Methods 100, 181. Perkins, S., Zimmermann, U., Gessner, P. and Foung, S.K.H.
42 (1989) Formation of hybridomas secreting human monoclonal antibodies with mouse-human fusion partners. In: C. Borrebaeck and I. Hagen, (Eds.) Electromanipulation in Hybridoma Technology. Stockton Press, New York, p. 47. Saxon, A., Feldhaus, J. and Robbins, R.A. (1976) Single step separation of human T and B cells using AET treated SRBC rosettes. J. Immunol. Methods 12, 285. Schmitt, J.J. and Zimmermann, U. (1989) Enhanced hybridoma production by electrofusion in strongly hypoosmolar solutions. Biochim. Biophys. Acta 983, 42. Schmitt, J.J., Zimmermann, U. and Gessner, P. (1989) Electrofusion of osmotically treated cells. High and reproductible yields of hybridoma cells. Naturwissenschaften 76, 122.
Vienken, J. and Zimmermann, U. (1985) An improved electrofusion technique for production of mouse hybridoma cells. FEBS Lett. 182, 278. Zimmermann, U., Gessner, P., Wander, M. and Foung, S.K.H. (1989) Electroinjection and electrofusion in hypo-osmolar solution. In: C. Borrebaeck and I. Hagen, (Eds.), Electromanipulation in Hybridoma Technology. Stockton Press, New York, p. 1. Zimmermann, U., Gessner, P., Schnettler, R., Perkins, S. and Foung, S.K.H. (1990) Efficient hybridization of mouse-human cell lines by means of hypo-osmolar electrofusion. J. Immunol. Methods 134, 43.
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Elsevier JIM05726
Development of microfusion techniques to generate human hybridomas S. F o u n g 1, S. Perkins 1, K. K a f a d a r 2, p. G e s s n e r 3 a n d U. Z i m m e r m a n n 3 1 Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, U.S.A., 2 Statistics and Surveillance Branch, Epidemiology Program Office, Centers for Disease Control, Atlanta, GA 30333, U.S.A., and 3 Institute of Biotechnology, University of Wiirzburg, Ve'iirzburg, F.R.G.
(Received8 May 1990, accepted 3 July 1990) The rarity of antigen-specific B cells in peripheral blood and lymphoid tissues is a major limitation in the production of human monoclonal antibodies. This has led to a requirement for techniques capable of fusing small numbers of cells and achieving a higher hybridoma formation efficiency than currently is possible. The approach used in these studies to generate human hybridomas is based on the observation that under hypo-osmolar conditions electric field induced cell fusion or electrofusion is facilitated. Electrofusion parameters have been defined in strongly hypo-osmolar solutions which have resulted in a hybridoma formation efficiency greater than 5 x 10 -3 under optimal conditions. Furthermore, this has been accomplished with total input B cells of 1-2 x 105. This is a ten-fold reduction in the required number of input B cells and is associated with a hybridoma formation efficiency at least equal to that achieved with a higher input B cell number. An important factor in the development of this microfusion technique appears to be the duration of exposure to the hypo-osmolar solution by B cells to be immortalized. Other parameters which may affect hybridoma yield include the electrical field strength used for cell alignment and membrane breakdown, ratio of human B cells to fusion partner, washing procedure, post-fusion incubation time, and the elimination of toxic molecules. Key words: Electrofusion;Hybridoma, human
Introduction
A major obstacle in the generation of human monoclonal antibodies is the rarity of antigenspecific B calls in peripheral blood and lymphoid tissues. While the problem is overcome partly by expanding the B cell pool with secondary in vitro activation (e.g., by Epstein-Barr virus), the proportion of antigen-specific B cells remains low (James Abbreviations." EBV, Epstein-Barr virus; IMDM, Iscove's modified Dulbecco's medium; FCS, fetal calf serum. Correspondence to: S.K.H. Foung, Stanford University Blood Bank, 800 Welch Road, Palo Alto, CA 94304, U.S.A.
and Bell, 1987). Furthermore, the total cell number available for immortalization by somatic cell hybridization is often less than 106 in a variety of in vitro activation systems used to expand the antigen-specific B cell pool. Because of this limitation, there is a requirement to develop approaches to immortalize small numbers of cells with a high fusion or hybridoma formation efficiency. The development of electric field-induced cell fusion or electrofusion has led to techniques capable of achieving a higher fusion efficiency with a corresponding higher hybridoma formation efficiency in comparison to polyethylene glycol-induced cell fusion (Vienken and Zimmermann, 1985; Ohnishi et al., 1987; Glassy, 1988; Foung and Perkins,
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
36 1989). One approach to further enhance fusion efficiency by electrofusion is based on the observation that electrofusion is facilitated under hypo-osmolar conditions. The feasibility of this approach has been demonstrated recently in a murine model system for hybridoma formation (Schmitt and Zimmermann, 1989; Schmitt et al., 1989; Zimmermann et al., 1989). In this report, we describe the effects of hypo-osmolar conditions on the generation of human hybridomas by electrofusion. This has led to the development of microfusion techniques with a high efficiency of hybridoma formation.
Materials and methods
B cell isolation and activation Peripheral blood lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation (B~Syum, 1968), and B cells were separated from T cells by rosetting with 2-aminoethylisothiouronium bromide hydrobromide treated sheep red blood cells (Saxon et al., 1976). B cells were activated with Epstein-Barr virus (EBV) using supernatant from the B95-8 marmoset line as a source of virus (Foung et al., 1989; Perkins et al., 1989). Composition of fusion media Iso-osmolar fusion medium (300 L3) consisted of 280 mM sorbitol (E.M. Science, Cherry Hill, NJ, U.S.A.), 0.1 mM Ca 2+ acetate (E.M. Science), 0.5 mM Mg 2+ acetate (E.M. Science), and 1 m g / m l pure bovine serum albumin (Serva Biochemicals, Westbury, NY, U.S.A.) with a final osmolarity of 300 mosM. Hypo-osmolar fusion medium (75 L3) consisted of 70 mM sorbitol, 0.1 mM Ca z+ acetate, 0.5 m M Mg 2+ acetate, and 1 m g / m l pure bovine serum albumin with a final osmolarity of 75 mosM. Fusion protocols Open chamber fusions were performed to establish the approximate electrical parameters necessary for eventual hybridoma formation. The open chamber consists of two parallel electrodes (200/~m in diameter and 200/~m apart) attached to a glass slide. This configuration permits direct microscopic observation of the fusion process. The
cell populations, EBV activated B cells and the mouse-human heteromyeloma K 6 H 6 / B 5 (generously provided by Dr. Levy, Stanford University) (Carroll et al., 1986) were fused at room temperature, first separately and then in combination, to establish the appropriate electrical range. Based on these studies, helical chamber fusions with EBV activated B cells and K 6 H 6 / B 5 were performed with similar electrical parameters using different cell ratios, wash conditions and osmolar solutions. The cells were pooled, washed and fused in the medium indicated. After washing, cells were resuspended in 170 /~1 of fusion medium and transferred to a helical fusion chamber (GCA, Chicago, IL, U.S.A.) consisting of a receptacle to hold the cells and an electrical assembly of two electrodes of 200 /~m diameter wound in a helix 200/~m apart that fits inside the receptacle. Electrical current was applied with the Model Z1000 Zimmermann Cell Fusion System (GCA). Cells were aligned in a non-uniform alternating field (AC) of I MHz frequency, and fusion was induced by three pulses of direct current (DC) of high intensity with 15 t~s duration, 1 s between pulses, and 10 ms of alignment off time. Alignment was tapered off gradually over 30 s after fusion. After incubating in the chamber, cells were washed out and plated in 96 well microtiter plates in pre-selection medium containing complete Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY, U.S.A.) with 15% fetal calf serum (FCS), 100 ~M hypoxanthine (Sigma, St. Louis, MO, U.S.A.), and 15/~M thymidine (Sigma), and grown in a 6-6.5% CO 2 incubator at 37°C. Unfused cells were also plated in this medium and served as controls. After 24 h, and for a period of 2 weeks, the cells were maintained in selection medium consisting of pre-selection medium with the addition of 0.8 /~M aminopterin (Sigma) and 0.1 /~M ouabain (Sigma). After 2 weeks, the hybridomas were grown in the preselection medium. Hybrid yield was determined by counting the macroscopic growth in wells after no new colonies appeared in new wells (on the order of 6 - 7 weeks with these cells). Because of the difficulty in counting a large number of wells and colonies and the potential for counting errors, it seems reasonable to characterize the efficiency of hybridoma formation as a range of likely values rather than
37
as a single number. The concept of range in hybridoma formation efficiency has been defined and illustrated previously where it was referred to as the fusion efficiency (Foung and Perkins, 1989). It is the number of wells with growth multiplied by two values corresponding to the extremes in the number of colonies in over half of the wells which were counted, divided by 105 input EBV activated B cells. This definition attempts to incorporate some of the uncertainty and variation inherent in counting large numbers of wells with large numbers of colonies which are not always clearly delineated. To summarize a series of such ranges (in Tables III, IV and VI), data are presented as an overall range of the lowest of the low endpoints and the highest of the high endpoints to indicate the extremes in efficiency. A median range is also presented and is computed as the median of the low end points and the median of the high end points for each series of ranges. The median range is used along with the overall range to compare the hybridoma formation efficiency with different sets of parameters.
Fusions in iso-osmolar medium (300 L3) 1-3.5 X 105 EBV activated B cells were fused to K 6 H 6 / B 5 at a ratio of I EBV : 1-10 K 6 H 6 / B 5 . The cells were washed two times in 300 L3 fusion medium. The parameters for fusions in 300 L3 were a 20-30 s alignment time at 250-300 AC V / c m and three D C fusion pulses of 1.5-2.0 k V / c m . Cells were washed out of the chamber 30
rain after fusion and plated in I M D M pre-selection medium at 5 x 103-9 X 104 cells/well.
Fusions in hypo-osmolar medium (75 L3) 1-2.5 X 105 EBV activated B ceils were fused to K 6 H 6 / B 5 at a ratio of 1 EBV : 0.5-2 K 6 H 6 / B 5 . The cells were washed one or two times in 300 L3 medium and fused in 75 L3 fusion medium. The electrical parameters were a 30 s alignment time at 300 AC V / c m and three D C fusion pulses of 1.0 k V / c m . The fusion process was performed between 5 and 15 min after the cells were suspended in hypo-osmolar medium. Cells were washed out of the chamber 10-15 min after fusion and plated at 5 x 103-104 cells/well in I M D M preselection medium containing no p H (phenol) indicator. After 24 h they were maintained in selection medium containing p H indicator.
Results
EBV activated B cells when fused to a mousehuman heteromyeloma in iso-osmolar medium by electrofusion have produced a hybridoma formation efficiency of 32-229 hybridomas per 105 input B cells (Foung and Perkins, 1989). This enhanced range in fusion efficiency of hybridoma formation over polyethylene glycol-induced fusion methods was achieved with as few as 106 input B cells. In a similar series of fusions in iso-osmolar fusion medium using only 1-3.5 X 105 input EBV,
TABLE I H U M A N H Y B R I D O M A F O R M A T I O N IN ISO-OSMOLAR S O L U T I O N BY E L E C T R O F U S I O N Fusion
Input EBV (105)
Fusion ratio a
DC V (kV/cm)
Percent wells with hybrids
Hybridoma formation efficiency
1
1.0
1 : 10
2 3 4 5 6 7 8 9
1.0 2.0 2.0 2.0 2.3 3.3 2.4 3.5
1 : 10 1:3 1:1 1 :1 1 :5 1:3 1 :5 1:3
2.0 2.0 2.0 1.5 2.0 2.0 1.5 1.5 2.0
75 25 45 44 18 47 66 44 58
14 5 3-24 11 4 6- 9 5-15 7-13 5- 6
a Ratio of EBV activated B cells fused to K6H6//B5.
38
however, a substantial drop in the range in efficiency of hybridoma formation was observed (Table I). The decrease in efficiency to an overall range of 3-24 hybridomas per 105 input B cells was present with a wide range of fusion ratios between EBV activated B cells and the mouse-human heteromyeloma cell line, K6H6/B5. To ascertain the effects of hypo-osmolar solutions on hybridoma formation by electrofusion, multiple series of fusions were performed with EBV activated B cells and K6H6//B5. The approximate electrical parameters necessary for hybridoma formation were first determined in open chamber fusions. In iso-osmolar fusion medium (300 L3), a small number of K 6 H 6 / B 5 cells could be observed fusing to themselves over a 20 min period at a wide range of DC voltages (1.5-3.5 kV/cm). At low cell density, more cells were fused with three DC pulses of 1.5 k V / c m , although the number was still small (5-10% of the total). A marked increase in fusion efficiency occurred in hypo-osmolar medium (75 L3). 50-90% of KrH6//B5 fused within five rain using a lower DC voltage (1.0 k V / c m with three pulses) than in iso-osmolar solution. EBV-activated B cells in 300 L3 were observed to fuse in a similar manner to K r H 6 / B 5 . A small number of cells were fused over a wide range in DC voltage (1.5-3.5 kV/cm). More cell fusions occurred at lower voltages when the cell density was low. To determine the optimal conditions in 75 L3, over 100 experiments were performed in
the open chamber with different alignment field strength (1-2 MHz, 250-300 V / c m AC), fusion voltage (0.5-1.5 k V / c m DC), pulse number (3-9), cell density (5x105-107 cells/ml), wash medium osmolarity (38-300 mosM), fusion medium osmolarity (38-150 mosM), and time exposure to the hypo-osmolar medium (2 min-1 h). The most critical factor appeared to be the duration of cell exposure to the hypo-osmolar medium prior to fusion. Higher fusion efficiency was obtained in 75 L3 only when the cells' exposure time to the medium was limited. Poor fusion efficiency in 75 L3 resulted when time exposures were less than 5 rain or greater than 15 min. Based on these observations, a series of hypoosmolar electrofusions were performed in helical chambers with EBV-activated B cells and K6H6/B5 a t a fixed ratio with different durations of exposure to 75 L3 before fusion (Table II). The electrical parameters chosen for all hypo-osmolar fusions were based on the results obtained with K 6 H 6 / B 5 fusions with the open chamber. The alignment time was 30 s at 300 AC V / c m and the fusion event initiated with three DC pulses of 1.0 k V / c m . Highest hybrid yield was observed after 10 rain in the medium, and the lowest after 30-40 min. Hybrid yield and exposure time to the hypoosmolar solution appeared to be inversely related. The number of times cells were washed in 300 L3 prior to the electrofusion procedure was also noted to affect hybrid yield with a slightly higher efficiency after one wash (Table II). This may be
TABLE II D U R A T I O N OF E X P O S U R E TO H Y P O - O S M O L A R S O L U T I O N P R I O R TO E L E C T R O F U S 1 O N a Fusion
Exposure duration b
Number of washes
Percent wells with hybrids
Hybridoma formation efficiency
1 2 3 4
10 20 30 40
1 1 1 1
100 92 72 80
80-200 36-144 16- 80 16- 96
5 6 7 8
10 20 30 40
2 2 2 2
90 80 45 25
28-193 16-133 10- 30 4 - 24
a EBV activated B cells fused to K 6 H 6 / B 5 at 1 : 1 ratio in all experiments. b Minutes cells exposed to 75 L3 prior to electrofusion.
39 TABLE III D U R A T I O N OF POST-FUSION INCUBATION USED FOR H Y B R I D O M A F O R M A T I O N a Duration (min)
N u m b e r of fusions
Percent wells with hybrids
Hybridoma formation efficiency
Range
Mean
Overall range
Median range
10-15 25-30
4 4
50-94 22-94
76 62
10-180 6-118
20-120 14- 77
Minutes of incubation after fusion in the helical chambers. All fusions performed in 75 L3 at 1 : 1 ratio of EBV activated B cells to K6H6/B5.
a
due to factors other than cell loss from washing since the relative drop in hybridoma formation between 10 min and 40 min exposure time in 75 L3 is less in the one wash series of electrofusions. Other parameters that appear to affect hybrid yield include post-fusion incubation time and the ratio between input B cell and fusion partner (Tables III and IV). A shorter post-fusion incubation of 10-15 min in the chamber resulted in an overall hybridoma formation efficiency range of 10-180 hybridomas per 105 input B cells, which was greater than the 6-118 hybridomas per 105 input B cells obtained with the longer post-fusion incubation duration of 25-30 min (Table III). The fusion ratio has an even greater effect on the efficiency of hybridoma formation (Table IV). Although only two fusions were performed with a fusion ratio of one EBV activated B cell to two K6H6/B5, the hybrid yield was substantially greater using this ratio than other fusion ratios, producing a median efficiency range of 38-464 hybridomas per 105 input B cells. Previous studies have suggested that molecules not ordinarily toxic to intact cells such as pH
indicator can have a toxic effect shortly after electrofusion. A series of fusions was performed and maintained for 24 h in I M D M pre-selection medium without pH indicator (Table V). In this series, hybridoma formation efficiency of up to 600 hybridomas per 105 input B cells was obtained. A marked increase in the percent wells with hybrid growth was noted with fusions performed in 75 L3 compared to those performed in 300 L3 (Table VI). When the cells were fused under hypo-osmolar conditions and grown for 24 h in medium without pH indicator, a 6-20-fold increase in the median range of hybridoma formation efficiency was achieved compared to isoosmolar fusions.
Discussion
A major strategy used to generate antigenspecific human monoclonal antibodies involves initial expansion of the antigen-specific B cell pool by in vitro activation with EBV and subsequent immortalization by somatic cell hybridization with
TABLE IV E F F E C T OF F U S I O N RATIO ON H Y B R I D O M A YIELD a d Cell ratio EBV : K r H 6 / B 5
Number of fusions
Percent wells with hybrids
Hybridoma formation efficiency
Range
Mean
Overall range
Median range
2:1 1:1 1:2
7 10 2
20- 75 58-100 89- 94
54 84 92
3-209 20-525 29-635
11- 82 32-196 38-464
Fusions performed in 75 L3.
TABLE V HUMAN HYBRIDOMA YIELD IN MEDIUM WITHOUT pH INDICATOR a Fusion
Wash number
Input EBV (105)
Fusion b ratio
Percent w e l l s with hybrids
Hybridoma formation Efficiency
1 2 3 4 5 6 7 8 9
1 1 1 1 2 2 2 2 2
1.0 1.0 1.0 2.0 2.0 2.0 2.0 2.0 2.0
1:1 1:2 2:1 1:1 1:1 1:1 1:1 1:2 2:1
92 94 70 100 94 90 82 89 75
35-525 29-635 15- 82 80-200 36-180 28-193 24-193 47-293 12-209
a Fusions performed in 75 L3 and plated in IMDM without pH indicator. b Ratio of EBV activated B cells fused to K6H6//B5.
an a p p r o p r i a t e fusion partner. I n this a n d other in vitro activation systems used to e x p a n d the relev a n t B cell p o p u l a t i o n , the total cell n u m b e r available for s u b s e q u e n t cell fusion is often less t h a n ] 0 6 . Therefore, it is desirable to have techniques c a p a b l e of fusing small n u m b e r s of cells ( 1 - 2 x 10 5) with an efficiency of h y b r i d o m a f o r m a t i o n a p p r o a c h i n g 10 -2, or one h y b r i d o m a per 100 input B cells. T h e d e v e l o p m e n t of these m i c r o f u s i o n techniques will lead to a b r o a d e r application of h y b r i d o m a technology to s t u d y the h u m a n h u m o r a l i m m u n e response in disease states. D e v e l o p m e n t of electric field-induced cell fusion or electrofusion already has led to techniques capable of achieving a h u m a n h y b r i d o m a formation efficiency a p p r o a c h i n g 1 0 - 3 with a n i n p u t n u m b e r of 10 6 or more EBV activated B cells ( F o u n g a n d Perkins, 1989). T o m a i n t a i n this high fusion or h y b r i d o m a f o r m a t i o n efficiency with a lower n u m b e r of i n p u t B cells (105), the effects of
h y p o - o s m o l a r c o n d i t i o n s were explored. This approach is based on the o b s e r v a t i o n that the surface area of m e m b r a n e c o n t a c t b e t w e e n two cells established in an a l t e r n a t i n g electric field is markedly increased u n d e r strongly h y p o - o s m o l a r c o n d i t i o n s p r o v i d i n g more p o t e n t i a l area for cell fusion. A n o t h e r factor is the n u c l e a r swelling occurring u n d e r c o n d i t i o n s of h y p o - o s m o l a r i t y which m a y facilitate nuclear fusion in the fused cells (Bertsche et al., 1988). Both are necessary steps in h y b r i d o m a formation. As shown in these studies, the fusion process initiated b y a p p l i c a t i o n of direct current is achieved with less field strength (1.0 k V / c m ) c o m p a r e d to those iso-osmolar fusions (1.5-2.0 k V / c m ) . F u r t h e r m o r e , the fusion process in h y p o - o s m o l a r solution occurs m o r e rapidly t h a n in iso-osmolar solution a n d with more cells b e i n g fused. H y p o - o s m o l a r electrofusions have resulted in a h y b r i d o m a f o r m a t i o n efficiency greater t h a n 5 × 1 0 3 with total i n p u t B cells of 1 - 2 x 1 0 5
TABLE VI HUMAN HYBRIDOMA FORMATION EFFICIENCY IN ISO-OSMOLAR AND HYPO-OSMOLAR SOLUTIONS Fusion medium
pH indicator
Number of fusions
Percent wells with hybrids
Hybridoma formation efficiency
Range
Mean
Overall Range
Median Range
300 L3 75 L3 75 L3
+ + a -
9 10 9
18- 75 20- 98 70-100
45 58 80
3- 24 3-347 12-635
5- 11 12-108 29-200
a Fused cells harvested from helical chamber in RPMI without pH indicator, diluted and plated in IMDM with indicator.
41
under optimal conditions. This is a ten-fold decrease in the required number of input B cells and is associated with a fusion efficiency equal to or greater than that achieved using a higher input B cell number. A most critical factor in the development of this microfusion technique appears to be the duration of exposure to the hypo-osmolar solution by EBV activated B cells. A range of 5-15 min was optimal in these studies. It is likely that a slightly higher osmolar solution or modified fusion voltage parameters will lead to a greater tolerance of EBV activated B cell exposure to hypo-osmolar conditions. Other parameters that appear to affect hybridoma yield include the ratio of human B cells to fusion partner, washing procedure, post-fusion incubation time, and the elimination of molecules not ordinarily toxic to intact cells (e.g., phenol) from the growth medium used to support human hybridomas within the first 24 h post-fusion. The development of appropriate electrical parameters to achieve maximum fusion efficiency is best accomplished with open chamber fusions. The open chamber allows direct visualization of the effects of different electrical parameters on the fusion process. The defined electrical parameters can then be applied to helical chamber electrofusions with slight modifications. By elucidating the effects of these parameters, it has been possible to develop microfusion techniques with increased fusion or hybridoma formation efficiency using other mouse-human heteromyelomas (Zimmerman et al., 1990). The improvements in hybridoma technology achieved in these studies should enable researchers to use a combination of human B cell selection and in vitro activation to isolate virtually any antibody represented within the human B cell repertoire. It is possible that the recently described recombinant DNA technique used to generate and select the immunoglobulins of a desirefl specificity may represent a more efficient appro~tch (Huse et al., 1989). This technique based on random recombination of heavy and light chain genes may potentially generate an even greater diversity of antibodies than are represented by the repertoire of B cells. However, to define antibodies which are involved in human disease, the hybridoma approach will yield immunoglobulins which are en-
coded by B cells under the constraints of the disease process in vivo. Therefore, the improvements in hybridoma technology described in these studies should provide a better tool for the evaluation of the role of antibodies in the pathogenesis or resolution of human disease.
Acknowledgements The authors wish to thank Judy Campbell and Norma Secord who typed this manuscript. This study was supported in part by Grants HL33811, AI22557, AI26031 and DA06596 from the National Institutes of Health to S.K.H.F., and grants of the Deutsche Forschungsgemeinschaft (SFB 176) and of the Federal Ministry of Research and Technology, F . R . G . ( D F V L R 01QV354) to U.Z.
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42 (1989) Formation of hybridomas secreting human monoclonal antibodies with mouse-human fusion partners. In: C. Borrebaeck and I. Hagen, (Eds.) Electromanipulation in Hybridoma Technology. Stockton Press, New York, p. 47. Saxon, A., Feldhaus, J. and Robbins, R.A. (1976) Single step separation of human T and B cells using AET treated SRBC rosettes. J. Immunol. Methods 12, 285. Schmitt, J.J. and Zimmermann, U. (1989) Enhanced hybridoma production by electrofusion in strongly hypoosmolar solutions. Biochim. Biophys. Acta 983, 42. Schmitt, J.J., Zimmermann, U. and Gessner, P. (1989) Electrofusion of osmotically treated cells. High and reproductible yields of hybridoma cells. Naturwissenschaften 76, 122.
Vienken, J. and Zimmermann, U. (1985) An improved electrofusion technique for production of mouse hybridoma cells. FEBS Lett. 182, 278. Zimmermann, U., Gessner, P., Wander, M. and Foung, S.K.H. (1989) Electroinjection and electrofusion in hypo-osmolar solution. In: C. Borrebaeck and I. Hagen, (Eds.), Electromanipulation in Hybridoma Technology. Stockton Press, New York, p. 1. Zimmermann, U., Gessner, P., Schnettler, R., Perkins, S. and Foung, S.K.H. (1990) Efficient hybridization of mouse-human cell lines by means of hypo-osmolar electrofusion. J. Immunol. Methods 134, 43.
