Selective production of hybridoma cells: antigenic-based pre-selection of B lymphocytes for electrofusion with myeloma cells

Methods for pre-selecting B lymphocytes were studied and investigated. First, biotinylated antigen was used for selecting B lymphocytes. These pre-selected B lymphocytes were then combined with biotinylated myeloma cells by adding streptavidin. The final formula of the selected B cell-myeloma cell was as follows: B cell-(antigen-biotin-strept-avidin-biotin)-myeloma cell. Then, this B cell-myeloma cell conjugate was fused by the pulsed electric field (PEF) method, which fused only those conjugated cells. The fusion efficiency obtained by this method was 3-15-times higher than that obtained by the non-specific poly(ethylene glycol) (PEG) fusion method. Second, avidin-antigen conjugate was used to select B lymphocytes. For this purpose, bifunctional cross-linkers such as N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and m-maleimidobenzoyl N-hydroxysuccinimide (MBS) were chosen. Each reagent contains two heterofunctional groups which can make covalent bond with both Lys and Cys residues. Typical avidin-antigen conjugate is expressed as avidin-SPDP (or MBS)-antigen. Thus, final B cell-myeloma cell conjugate was B cell-antigen-SPDP (or MBS)-avidin-biotin-myeloma cell. The yield of this procedure was of the order of 10(-2). Here, we suggest that the pre-selection of B lymphocytes by biotinylated antigen or avidin-antigen conjugate is a new method of obtaining selected hybridoma cells which produce specific monoclonal antibodies against the antigen used for selecting B lymphocytes.     

Mechanically facilitated cell-cell electrofusion.

Apparatus and methods were developed to enable mechanically facilitated cell-cell electrofusion to be performed. The apparatus and methods mechanically place cells in contact before fusion. The key component of this fusion system was a newly developed fusion chamber. The chamber was composed of two functionally identical electrodes that were housed in a multi-layer structure. The layers functioned as support for the electrodes. They also allowed adjustment of the distance between opposing electrode faces. The electrodes were constructed in a manner that allowed cells to be deposited, by vacuum, onto each face. Electrode faces were positioned at a predetermined distance from each other to mechanically force cell-cell contact between the deposited cells. Fusion was induced by delivering direct current pulses to the juxtaposed cells. Fusion products were detected and quantitated by flow cytometry. Details of the chamber design and a protocol for using the fusion chamber are given. Mechanically facilitated cell-cell electrofusion was demonstrated by using the chamber to produce fusion products from like fusion partners. The practical applicability of the chamber was demonstrated by fusing unlike cell types. Mechanically facilitated cell-cell electrofusion is not specific to the cells used in this study; the chamber can be adapted for use with other cell types.     

Hybridoma cells produced by electrofusion in a homogeneous electric field

In the customary technique will be used a fusion chamber with an inhomogeneous electric field. The disadvantage is that the fusion conditions are not constant in the chamber. Yet for di-electrophoretic collection of the cells their own field-inhomogenizing influence is sufficient that is, the electrofusion can proceed also in very simple chambers with homogeneous fields.     

An improved electrofusion technique for production of mouse hybridoma cells

An experimental procedure is described for the reproducible production of hybridoma cells using the electrofusion technique. High yields can be obtained when fusion is performed in isotonic inositol solutions containing Ca2+ and Mg2+ in a ratio of 1:5 in the millimolar range. The hybridoma cells are transferred 10 min after the field pulse application into a balanced salt solution for 30 min at 37 degrees C.     

Selective staining of immature hemopoietic cells with merocyanine 540 in flow cytometry

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.     

Homokaryons from animal and plant cells generated by electrofusion

A new apparatus was constructed which enables the use of the electrofusion method to obtain polynuclear cells of various mammalian cell lines, erythrocytes and plant protoplasts. This technique was applied to both suspensions and monolayers. Electrical and other physical parameters were monitored to find optimal conditions for mutual contact of cells (dielectrophoresis) and subsequent fusion. In the suspension technique, dielectrophoresis of mouse erythrocytes occurred at a field frequency of 20 kHz and a strength of 500 V.cm-1, whereas cultured mammalian cells and plant protoplasts required a frequency of 1-1.4 MHz and a strength of 250-800 V.cm-1. Fusion of cells was induced after the application of 1 to 10 high-voltage pulses of 1-5 kV.cm-1, 10-36 microseconds duration. After these high-voltage pulses were to the monolayer of mouse L cells, about 12% viable homokaryons were obtained.     

Selective gene mutation in MEL cells.

MEL cells, undergoing erythroid differentiation and parasynchronized by dimethyl sulfoxide (DMSO) induction, were irradiated with a 3-s pulse of UV light at sublethal dose. A large number of clones deficient in different gene functions are found in the progeny of the treated cells, if the pulse irradiation is performed 18-24 h from the start of DMSO induction. Kinetics of thymidine incorporation into DNA show that the period of sensitivity corresponds to the S phase. The results show that the activities of the tested genes are differently affected depending on the exact time of cell irradiation. Maximum percent inhibition of cells not expressing glucose-6-phosphate dehydrogenase (G-6-PD) (70%) is produced by irradiating at 20 h from the start of DMSO induction; 6-phosphogluconate dehydrogenase (6-PGD) (55%), and hypoxanthine (guanine) phosphoribosyltransferase (HPRT) (33%), at 21 h; hemoglobin (50%), at 22 h. The time difference in the sensitivity to UV light is highly reproducible and has been exploited to isolate, with high efficiency, cellular clones deficient in any one of the tested functions. Determinations of enzymatic activities on cell lysates show that the expression of tested genes is actually altered in cells that, on the basis of cytochemical tests, appear unaffected by UV irradiation. While the production of mutant clones is observed only during the S phase of the cell cycle, immediate statistical damage of the cellular DNA is produced at all times of irradiation. This finding excludes that the two types of phenotypic alterations, blocked or altered gene expression, both propagated in the progeny of the cells as clonal properties, may derive from a preferential alteration of those functions during the S phase.     

Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts

Mesophyll derived protoplasts ofCatharanthus roseus cv. Little pinkie were fused with protoplasts derived from an habituated cell line ofC. roseus. Polyethylene glycol was used as agglutinating agent while fusions were induced by square pulses. Best results were obtained by fusing protoplasts from primary leaves with those from three-day-old cell cultures. Adding calcium ions considerably enhanced heterofusion rate. Good cell viabilities indicated that this fusion process was not cytotoxic. The heterofusion frequency was up to 10% or more. Most of the heterokaryons were able to regenerate their cell walls and underwent division. Communicated by J. TUPY     

Characterization of PEG-mediated electrofusion of human erythrocytes.

Polyethylene glycol (PEG) and electrofusion were applied together in a simple and highly efficient cell fusion method. PEG (8000 M(r)) was used to bring human erythrocytes into contact, and a single 4.4 kV/cm, 80 microseconds duration pulse was applied to cell suspensions. The fusion yield (FY) is PEG concentration-dependent. A maximum FY (50%) was found at about 10% PEG. Higher PEG concentrations (> 10%) suppressed FY caused by colloid osmotic shrinkage. Morphological changes, such as colloidal osmotic swelling and shrinking, and the expanding and contraction of fusion lumen, when suspension media were changed from PBS to isotonic 15% dextran solutions, was examined by microscopy. FY was found to depend on both simple osmotic and colloidal-osmotic swelling. From the swelling behavior, we propose two types of electropores: the pre-fusion sites between cell pairs, and electropores on each individual cell connecting intracellular and extracellular space. The latter type is responsible for the colloidal osmotic swelling and shrinking of cell which, together with simple osmotic swelling, is responsible for expanding the pre-fusion sites into fusion lumens. Resealing of electropores resulted in reducing FY, but the FY can be restored by simple osmotic shock. Apparently, PEG plays two opposite roles in this fusion method; one is to promote pre-pulse and post-pulse cell-cell contact, protecting pre-fusion sites, and the other suppresses FY by colloid osmotic shrinkage of cells after pulsing, especially when high PEG concentration is used. 10% PEG 8000 represents the optimal combination of these properties.     

Selective cytotoxicity of antibody-ricin-A-chain conjugate for human tumor B cells. II. Comparison of activity of immunotoxins conjugated with poly- and monoclonal antibodies

The A-chain of a plant toxin ricin has been coupled to poly- and monoclonal antibodies specific to the L-chains of human IgG. The inhibitory effect of the conjugates has been compared with the ability of the antibodies to bind to target cells. Cytotoxicity of the conjugates has been monitored following incorporation of 14C-leucine radioactivity into Burkitt lymphoma cells with surface Ig. The 50% inhibition of protein synthesis is observed 18 h after treatment of cells with immunotoxins, when the concentration of the conjugates with poly- and monoclonal antibodies is 1.2.10(-9) M and 0.7.10(-9) M, respectively. The data take into account that only part of the polyclonal antibodies molecules is able to react with target cells. The control conjugates containing either monoclonal antibodies that do not react with the lymphoma cells surface L-chains or nonimmune serum IgG proved to have no effect on target cells even at the level of 10(-7) M. The immunotoxins with poly- and monoclonal antibodies produce almost the same kinetics of protein synthesis inhibition, when incubated with lymphoma cells for 60 min. However, a 30 min treatment reveals a considerably higher cytotoxicity of the conjugate with monoclonal antibodies.     

Kinetics and mechanism of cell membrane electrofusion.

A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts.     

Spectrofluorometric assay for the quantitation of cell-tissue electrofusion.

Fluorometric assay for quantitating electrofusion, FAQE, was developed to measure the number of cells fused to intact tissue. The fluorescent vital dye hydroethidine is used in this method. The fluorescent intensity detected in the cell tissue hybrids is proportional to the number of individual cells fused. The number of cells fused was determined after fusion by lysing the epithelial layer with 0.2% sodium dodecyl sulfate and the supernatant fluids were measured in a spectrofluorometer and compared to established standard curves. The mean number of cells fused, in five separate experiments, was determined to be approximately 5000. All the experimental corneas had approximately the same number of fused cells with less than 10% variation. In addition, the technique was used to demonstrate an increase in the number of cells fused when multiple fusions were applied to the cell-tissue electrofusion system. These results demonstrate that FAQE can be utilized to quantitatively analyze the fusion yields.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

Exposure to the electrofusion process can increase the immunogenicity of human cells

The cellular products obtained following electrofusion (EF) of dendritic cells (DC) and tumour cells have shown promise as cancer vaccines. The immunogenicity of these preparations has been attributed to the presence of small numbers of DC-tumour hybrids and the contribution of the non-hybrid tumour cells present has received little attention. In this report, we investigated the effect of the EF process on the immunogenicity of allogeneic human cells, in particular the colorectal cell line, SW620. EF conditions were optimised to yield the maximum number of DC-SW620 hybrids co-expressing tumour associated antigen (TAA) and DC associated antigens. Exposure of SW620 to EF induced significant increases (P<0.05) in apoptosis and necrosis. Pre-exposure of SW620 to the EF buffer alone [0.3 M glucose, 0.1 mM Ca(CH₃COO)₂ and 0.5 mM Mg(CH₃COO)₂] resulted in significant increases in TAA uptake by DC during co-culture (P<0.05). DC phenotype was, however, not altered by exposure to EF treated tumour cells. In co-cultures of PBMC responders with SW620, the levels of IFN release and cytotoxic activity were significantly increased (P<0.05) by pre-exposure of the SW620 to EF. Pre-exposure of allogeneic non-T cells, the colorectal cell line Lovo and a breast cancer cell line (MCF7) to EF also significantly (P<0.05) increased the levels of IFN release by responding PBMC. These results demonstrate that the EF process itself can increase the immunogenicity of at least some human cell types independently of hybrid formation. These findings suggest that EF protocols should be evaluated with regard to the possibility that DC-tumour hybrids may not contribute all, or even most, of the immunostimulatory capacity present in preparations of EF treated cells.BH and GR contributed equally as senior authors     

Dynamic changes of microtubule and actin structures in CV-1 cells during electrofusion

To study the involvement of the cytoskeletal system in the fusion of animal cells, we examined the dynamic changes of cytoskeletal proteins during the various stages of cell fusion. CV-1 cells were fused by applying a radio-frequency electrical pulse. Structural changes of microtubules (MTs) and F-actin were monitored simultaneously by double-label fluorescence microscopy. It was observed that in a few minutes after the initiation of cell fusion, MT bundles began to extend into the cytoplasmic bridges which were formed by fusing the membranes of neighboring cells. Later, a network of parallel MT bundles appeared between the adjacent nuclei of the fusing cells; such MT bundles may provide the mechanical links that are responsible for nuclear aggregation. The structural changes of F-actin during cell fusion were more complicated. We observed many different patterns of actin distribution in the fusing cells, including some giant, ring-shaped structures. Reorganization of actin is unlikely to be involved in the nuclear aggregation process. Instead, actin bundles condensed at the cell edges may help to widen the cytoplasmic bridges to allow merging of cellular contents between the fusing cells.     

Facilitation of electrofusion of mouse lymphoma cells by the proteolytic action of proteases

Cell fusion of mouse lymphoma (L5178Y) was achieved by applying electrical pulses under dielectrophoresis. The presence of dispase, pronase or trypsin facilitated the electric pulse-induced cell fusion. Heat-inactivated pronase was no longer effective. Protease inhibitors (aprotinin and p-tosyl-L-lysine chloromethylketone) suppressed the effect of trypsin. Even in the absence of proteases, the cells pretreated with dispase or pronase underwent fusion with high probabilities, as far as free calcium ions were present in the external solution. It is concluded that facilitation of electrofusion by proteases is due to their proteolytic activities.     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

Selective membrane protein trafficking: vectorial flow and filter.

Membrane proteins trafficking along cellular pathways encounter molecular filters. These filters can introduce them to new pathways and thus direct them towards new destinations. Many proteins carry molecular signals within their cytoplasmic domains that allow them to be selected by the filters. The detailed characterization of these signals is providing new insights into the pathways themselves and indicating the locations of other, as yet unidentified filters.     

Factors affecting the electrofusion of mouse and ferret oocytes with ferret somatic cells

The domestic ferret, Mustela putorius furos, holds great promise as a genetic model for human lung disease, provided that key technologies for somatic cell nuclear transfer (SCNT) are developed. In this report, we extend our understanding of SCNT in this species by defining conditions for efficient cell fusion by electrical pulse. Two experimental systems were employed in this study. First, in vivo-matured mouse oocytes and ferret somatic cells were used to establish general parameters for fusion. One fibroblast, or cumulus cell, was agglutinated to nucleate, zona pellucida-free, mouse oocytes, and subjected to an electrical pulse. Similar electrical pulse conditions were also tested with 1 or 2 somatic cells inserted into the perivitelline space (PVS) of intact mouse oocytes. The fusion rate for a single fibroblast with a zona-free oocyte was 80.2%, significantly higher (P < 0.05) than that observed for 1, or 2, fibroblasts placed in the PVS (52.0% and 63.8%, respectively). The fusion rate (44.1%) following insertion of two cumulus cells was significantly higher (P < 0.05) than that following insertion of one cumulus cell (25.1%). Second, in vitro-matured ferret oocytes were enucleated, and one to three fibroblasts or cumulus cells were inserted into the PVS. Zona pellucida-free ferret oocytes were fragile and excluded from the study. The fusion rates with two or three fibroblasts were 71.4% and 76.8%, respectively; significantly higher (P < 0.05) than that for one fibroblast (48.6%). This cell number-dependent difference in fusion efficiency was also observed with cumulus cells. Fusion-derived (ferret-ferret) NT embryos cleaved, formed blastocysts in vitro, and underwent early-stage fetal development following embryo transfer. The rate of development was cell type-independent, in contrast to the cell type-dependent differences observed in fusion efficiency. In conclusion, fibroblasts fused more efficiently than cumulus cells and the efficiency of single cell fusions was improved when two or more cells were inserted into the PVS. These studies define conditions for efficient cell fusion with ferret oocytes and should facilitate SCNT and the development of genetically defined animal models in this species.