Electrofusion Of Cells Research Articles

Production of cloned goats by nuclear transfer of cumulus cells and long‐term cultured fetal fibroblast cells into abattoir‐derived oocytes

Dairy goats are ideal for the transgenic production of therapeutic recombinant proteins. The use of recombinant somatic cell lines for nuclear transfer (NT) allows the introduction of genes by transfection, increases the efficiency of transgenic animal production to 100%, and overcomes the problem of founder mosaicism. Although viable animals have been cloned via NT from somatic cells of 11 species, the efficiency has been extremely low. Both blastomere and somatic cell NT increased fetal loss and perinatal morbidity/mortality in cattle and sheep, but fetal loss and perinatal mortality appear to be relatively low in goats. In this study, we produced cloned goats by NT from cumulus cells and long-term cultured fetal fibroblast cells (FFCs) to abattoir-derived oocytes. NT embryos were constructed from electrofusion of cumulus cells (CCs), FFCs, or skin fibroblast cells (SFCs) with cytoplasts prepared from abattoir-derived ovaries. The NT embryos were activated with an optimized activating protocol (1 min exposure to 2.5 microM ionomycin followed by 2 hr incubation in 2mM 6-DMAP). Two viable cloned kids from CCs and one from long-term cultured FFCs (at passage 20-25) were born. Microsatellite analysis of 10 markers confirmed that all cloned offspring were derived from corresponding donor cells. To our knowledge, the production of cloned goat offspring using abattoir-derived oocytes receiving nuclei from CCs and long-term cultured FFCs has not been reported. The production of viable cloned animals after activation with reduced intensity of ionomycin and 6-DMAP treatment has also not been reported. Loss of cloned embryos was obvious after 45 and 90 days of pregnancy, and a lack of cotyledons, heart defects, and improperly closed abdominal wall were observed in the aborted fetuses and one cloned kid. The fusibility and in vitro developmental potential of embryos reconstructed from FFCs at passage 20-25 were significantly lower than those of embryos reconstructed from FFCs at passage 3-5, and the cloning efficiency of the long-term cultured cells was low (0.5%).

Active immunotherapy for advanced intracranial murine tumors by using dendritic cell-tumor cell fusion vaccines

Immunotherapy for malignant brain tumors by active immunization or adoptive transfer of tumor antigen-specific T lymphocytes has the potential to make up for some of the limitations of current clinical therapy. In this study, the authors tested whether active immunotherapy is curative in mice bearing advanced, rapidly progressive intracranial tumors. Tumor vaccines were created through electrofusion of dendritic cells (DCs) and irradiated tumor cells to form multinucleated heterokaryons that retained the potent antigen processing and costimulatory function of DCs as well as the entire complement of tumor antigens. Murine hosts bearing intracranial GL261 glioma or MCA 205 fibrosarcoma were treated with a combination of local cranial radiotherapy, intrasplenic vaccination with DC/tumor fusion cells, and anti-OX40R (CD134) monoclonal antibody (mAb) 7 days after tumor inoculation. Whereas control mice had a median survival of approximately 20 days, the treated mice underwent complete tumor regression that was immunologically specific. Seven days after vaccination treated mice demonstrated robust infiltration of CD4+ and CD8+ T cells, which was exclusively confined to the tumor without apparent neurological toxicity. Cured mice survived longer than 120 days with no evidence of tumor recurrence and resisted intracranial tumor challenge. These data indicate a strategy to achieve an antitumor response against tumors in the central nervous system that is highly focused from both immunological and anatomical perspectives.

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 IFNgamma 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 IFNgamma 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.

Clinical evolution of diabetic rats after transplant of electrofused pancreatic islet cells and dermic cells

This work was designed to study an alternative treatment of diabetes mellitus by using a transplant of hybrid cells obtained by the electrofusion of pancreatic islet cells from a healthy donor with dermic cells obtained from a recipient. The hybrid cells kept the capacity of insulin production, its regulation, and the natural control of glycemia, as well as the factors of histocompatibility to avoid the rejection. Four groups of four rats each were established: Group 1. Healthy animals (healthy control), Group 2. Diabetized non-treated animals (diabetic control), Group 3. Transplant recipient rats with extraction of dermic cells which were mixed with pancreatic insular cells from a healthy donor (transplant without fusion), and Group 4. Transplant recipient rats, with extraction of dermic cells which were electrofused with pancreatic insular cells from a healthy donor (transplant with fusion). For the Group 4, the cells were combined and they were submitted to dielectrophoresis conditions with an alternating current pulse of 15 s of 10 V RMS of 0.5 MHz. The fusion was made with a direct current pulse of 1 ms of 300 V. Clinical signs were registered (weight, diuresis, food and water intake), and several biochemical parameters in blood which included basal glycemia, uric acid, cholesterol, triglycerides, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, urea, creatinine, insulin, glycated hemoglobin were registered. Additionally, ketone bodies and glucose were also measured in urine. All determinations were made at 30, 60, and 90 days. Animals of Group 1 maintained its parameters within the normal ranges. Rats of Group 2 presented alterations corresponding to a diabetic state in almost all the parameters measured, none of the animals showed a tendency to improve spontaneously, two of the rats died at 66 and 72 days. The Group 3 showed a clinical profile similar to the diabetic control group without improvement, only one rat died at day 33, while in the rats transplanted with fusion (Group 4) an improvement was observed on some parameters including body weight, water intake and glycemia. Although insulin concentration was under the normal range, it was higher than in the Group 3. None rat died. These results indicate that it is possible to improve the diabetic profile by the transplant of dermic cells from a diabetic animal fused with insular cells from a healthy donor in the recipient animal.

Tumor-dendritic cell fusion as a basis for cancer immunotherapy

To establish the basis for use of allogeneic dendritic-tumor fusion cells. Fusion cells were created by electrofusion. We used 2 allogeneic murine tumor lines (D5 and 4T1) that were virally transduced to express the antigen (beta-galactosidase) as a surrogate tumor marker. Cross-immunization was achieved with irradiated allogenic tumor cells. Successful electrofusion of dendritic cells and tumor cells was confirmed by using fluorescence-activated cell sorting and cytospin. Significant responses were shown in immunized mice against tumor challenge and established 3-day pulmonary metastasis with fusion cells. Allogeneic tumor sharing a common tumor antigen can immunize against syngeneic tumor challenge. Fusion cells showed successful immunization against tumor challenge and showed regression of 3-day established pulmonary metastasis. These preclinical studies provide evidence that an allogenic tumor-dendritic cell fusion vaccine is a valid approach for head and neck cancer immunotherapy.

Microfluidics and Micropatterned Immobilization as a Tool for Improved Electrofusion of Dendritic Cells with Tumor Cells

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Dendritic Cells and Vaccines

Tumor–dendritic cell fusion as a basis for cancer immunotherapy

Problem: Patients with advanced head and neck cancer are at risk for metastasis - local and regional recurrence. One approach to addressing these issues is immunotherapy. Recent studies have identified the dendritic cell (DC) as a critical component in eliciting a T-cell response against neoplastic cells. One immunotherapy approach is fusion of DCs with irradiated tumor cells by electrofusion. Methods: Fusion cells were created using electrofusion technique. We used 2 allogeneic murine tumor lines (D5 and 4T1) virally transduced to express the antigen B-galactosidase. Using beta-galactosidase as a surrogate tumor marker, we crossed immunized mice with irradiated tumor or fusion cells with subsequent tumor challenge intradermally. Fusion cells were also used in the treatment of three-day established pulmonary metastasis. Results: Cross immunization was achieved with irradiated allogenic tumor cells sharing a common antigen given subcutaneously and intranodally. Successful electrofusion of DC and tumor cells was confirmed using fluorescent activated cell sorting (FACS) and cytospin. Significant responses were demonstrated in immunized mice against tumor challenge and established 3-day pulmonary metastasis with syngeneic-fusion cells. Conclusion: Allogeneic tumors sharing a common tumor antigen can immunize against syngeneic tumor challenge. The use of syngeneic tumor-DC fusion cells demonstrated successful immunization against tumor challenge. Fusion cells also resulted in regression of 3-day established pulmonary metastasis. Significance: Difficulties are encountered in successfully and reliably generating tumor cell lines from individual patients, especially in head and neck cancer patients. Findings over recent years suggest that many tumors of the same histological origin but from different individuals may express common antigens. Therefore, a tumor from a different individual (allogeneic) may be feasible for use as a source of antigens. These preclinical studies provide evidence that an allogeneic tumor-dendritic cell fusion vaccine may be a valid approach for head and neck cancer immunotherapy. Support: American Academy of Otolaryngology-Head and Neck Surgery Resident Research Award

A Microfluidic Device for Electrofusion of Biological Membranes

This paper reports a microfabricated device with high aspect-ratio electrodes and low power consumption for the electrofusion of liposomes and cells. The applications may range from gene transfection or cell tracking to biophysical studies of membrane proteins. The device consists of 250 /spl mu/m thick silicon electrodes bonded to a glass substrate and covered by a PDMS-coated glass slide. Liposomes were first aligned by AC voltage at 300 kHz and then fused with short DC pulses. The fusion yield can reach up 75% and is globally better for liposome diameters larger than 10 /spl mu/m. The encapsulation of microbeads inside liposomes has been also demonstrated and opens up the route towards fusion-based delivery of artificial microstructures into cells.

In vitro development of reconstructed goat oocytes after somatic cell nuclear transfer with fetal fibroblast cells

In the present study the developmental potential of fetal fibroblasts was evaluated using nuclear transfer. Studies were undertaken to workout suitable dc pulse length on electrofusion of fetal goat skin fibroblast cells with enucleated in vitro matured goat oocytes and their capability of forming embryos. Skin fibroblast cells from fetal skin were isolated and cultured in monolayer using RPMI-1640 media with 10% FCS in 5% CO 2 incubator at 38.5±1 °C with 95% humidity. Recipient oocytes recovered from slaughterhouse ovaries were matured in vitro for 22–24 h and enucleated using a Leitz micromanipulator. Serum starved cells, cytochalasin B blocked synchronized cells and growing cells were used for insertion into the perivitelline space of the enucleated oocyte and electrofused with constant ac pulse at 7–10 V, while dc pulses at 250, 300 and 350 V were applied for 5–20 μs followed by culture in a CO 2 incubator to observe proper electrofusion and cleavage. After fusion the embryos were activated with cytochalasin B for 2–4 h, and then were transferred to IVC media (without any supporting cells and growth factors). During the study, it was observed that fetal fibroblast became confluence within 2 days. Proportions of oocytes undergoing zona dissolution did not differ significantly among the three dc pulses during electrofusion after reconstruction of the embryos, and also there was no significant difference for the fusion rates among serum starved cells, cytochalasin B blocked cells and growing cells. It was concluded that around 300 V resulted in better electrofusion, and cytochalasin B blocked synchronized cells and fast growing skin fibroblast cells of goats could be used for nuclear cloning.

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 β-galactosidase (β-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 β-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-γ 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.

Cell Hybridization by Electrofusion on Filters

Electric field pulses induce permeabilization and associated fusogenicity in cell membranes. Electrofusion of cells is usually performed in two steps: the first is the creation of close intercellular contacts; the second is an application of electric pulses that induces membrane fusion. Very large cell contacts can be obtained by a filter aspiration method. A cell monolayer is created by controlled suction on biocompatible filter. No spontaneous fusion results. Just after filtration, electrofusion is obtained by field pulses applied parallel to the filter. Cell viability is not strongly affected and cells recover their spherical shape in the minute time range after filtration. The electrical parameters, the cell density, and the flow rate control fusion. Fusion is obtained with cells of different origins with very different adhesion properties. Hybrid cells are easily formed. This approach appears to be a very efficient method for cell hybridization with an easy-to-use protocol.

Regenerated bovine fetal fibroblasts support high blastocyst development following nuclear transfer.

Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.

In vivo cell electrofusion

In vitro electrofusion of cells brought into contact and exposed to electric pulses is an established procedure. Here we report for the first time the occurrence of fusion of cells within a tissue exposed in vivo to permeabilizing electric pulses. The dependence of electrofusion on the ratio of applied voltage to distance between the electrodes, and thus on the achievement of in vivo cell electropermeabilization (electroporation) is demonstrated in the metastasizing B16 melanoma tumor model. The kinetics of the morphological changes induced by cell electrofusion (appearance of syncytial areas or formation of giant cells) are also described, as well as the kinetics of mitosis and cell death occurrence. Finally, tissue dependence of in vivo cell electrofusion is reported and discussed, since electrofusion has been observed neither in liver nor in another tumor type. Particular microenvironmental conditions, such as the existence of reduced extracellular matrices, could be necessary for electrofusion achievement. Since biomedical applications of in vivo cell electropermeabilization are rapidly developing, we also discuss the influence of cell electrofusion on the efficacy of DNA electrotransfer for gene therapy and of antitumor electrochemotherapy, in which electrofusion could be an interesting advantage to treat metastasizing tumors.

INFLUENCE OF POLYPEPTIDES AND PROTEINS ON ELECTROFUSION OF HUMAN CANCER CELLS

The electrofusion of human cancer U937 and K562 cells was studied under the influence of a selected group of polypeptides and proteins adsorbed at their membranes. For poly-L-lysine, we found that the higher the molecular mass (Mm) the higher the relative fusion yield (Fr), whereas poly-L-arginine and poly-L-ornithine showed toxicity. As recently measured for barley protoplast fusion, the same rule of dependence on the isoelectric point (pI) could be verified:which means 0.5 < Fr < 4 in 0.3 M mannitol solution (pH 6). If the pH of the 0.3 M mannitol solution is the same as the pI of the protein, its influence on the membrane nearly disappears. Bridging by Ca ions increases Fr from 0.5 (bovine serum albumin) to Fr ∼ 1 by charge compensation. The value of Fr is commensurate with the pore resealing time τ—determined by trypan blue staining—in such a way that for pI > 8, τ is longer and for pI < 6, τ is shorter than the control without protein content. Qualitatively, plant and animal membranes studied show equal behavior. According to our technique, the adsorption of biopolymers and other substances can be detected by this bioelectrochemical method, which is also a tool for analysis of electric stress mechanisms.

ELECTROPORATION AND FUSION OF HUMAN CANCER CELLS MODIFIED BY AMINO ACIDS AND POLYPEPTIDES

The influence of adsorbed polypeptides on electrofusion of histiocytic U937 and myeloidic K562 cells was extended by the corresponding monomeric amino acids in 0.3 M mannitol solution at pH 6.6 and other pH values. In all cases the monomers showed a similar behavior to the polymers, namely, an increase in the relative fusion yield Fr, or, in the case of sulfur-containing compounds, a decrease in Fr at higher concentrations. These electrostatic effects—dominated by the isoelectric points pI and the pH of the medium—are modified by mechanical interventions (e.g., bridging), because the Fr value also depends on the molecular mass (Mm) of the polypeptides. In fact, it rises with increasing Mm. These results demonstrate the fundamental interactions among cell membranes and proteins, polypeptides, and amino acids, which are responsible for essential life processes.

Multiple-pulse-mediated electrofusion of intact erythrocyte onto human term placental amnion

The creation of surface modified human term placental amnion by electrofusing human cells onto its surface has been thought of. A multiple-pulse electrofusion protocol with 10 square pulses of 10-μs pulse length, and electric field of 0.2 kV cm −1, can make erythrocyte–amnion tissue electrofusion possible. The protocol devised merge the cell-tissue-adherence steps with fusogenic pulse. The finding opens up a new avenue of cell electrofusion onto human tissue with minimal procedural complexities.

Electropermeabilization and electrofusion of human cells modified by anaesthetic agents

Results on the interaction between anaesthetics and human cell membranes are presented in order to show their target behavior including changes in electropermeabilization and resealing. Using electroporation and electrofusion techniques, it is possible to characterize the effects of anaesthetic agents which act as modifiers of cell membrane permeability depending on their types, concentrations and contact times. The field-induced uptake of trypan blue, used as a reporter of membrane electropermeabilization, and electrofusion of human lymphoma cells U 937 and human breast cancer cells MCF 7 were investigated. The cell electropermeabilization decreased with increasing external medium conductivity and osmolarity. The resealing kinetics of both types of cells studied could be fitted by exponentially decaying curves, however the first order rate constant for lymphoma cells was about two orders of magnitude higher. Moreover, the resealing step was slowed down in the presence of anaesthetics. It is suggested that the impact of anaesthetics on cell membranes may be explained by the combined action of different interaction mechanisms, electrical and/or mechanical in nature, as well as by specific bindings to integral membrane molecules, depending on the anaesthetic agent applied. It is concluded that the influence of anaesthetics on in vitro cell electropermeabilization might be used as an additional indicator for clinical purposes.

Electropermeabilization and electrofusion of human lymphoma cells modified by proteolytic enzymes

Former results on protein-modified electrofusion of barley protoplasts have been extended by human cell membrane electropermeation and electrofusion after preincubation of cells with proteolytic enzymes. After pulsation, the amount of cells electroporated or broken down increased eight times and also the resealing time was prolonged. That means the lipid bilayer (protein content diminished by lysis) has a lower resistance against permeation. However, the rule of the relative electrofusion yield determination by the protein isoelectric point is still valid. This study of biopolymer interaction with human cell membranes is a prerequisite for drug incorporation (delivery) and electrotransformation by nucleic acids.

Transfer of foreign receptors to living cell surfaces: the bioelectrochemical approach

Studies of the structure function relationship in membrane receptors need to obtain a back insertion of purified molecules in recipient cells. Taking advantage of the induction of a localized out of equilibrium state of cell membrane, electropulsation offers several successful approaches for such transfers. Plasmids carrying the gene coding for specific receptors can be transfered in recipient cells and a high level of expression can be obtained at the membrane level. Purified soluble receptors can be spontaneously inserted in the membrane of pulsed cells giving them the properties associated to the grafted molecules. Fusion of enriched vesicles with target cells can be easily obtained by using the pulse first electrofusion approach. In vivo electrofusion of human cells to animal tissues confers to the naturally-resistant animal sensitivity to the human pathogen.

Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.

Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.