RESUMEN
Cancer cell-immune cell hybrids and cancer immunotherapy have attracted much attention in recent years. The design of efficient cell pairing and fusion chips for hybridoma generation has been, subsequently, a subject of great interest. Here, we report a three-layered integrated Microfluidic Flip-Chip (MFC) consisting of a thin through-hole membrane sandwiched between a mirrored array of microfluidic channels and saw-tooth shaped titanium electrodes on the glass. We discuss the design and operation of MFC and show its applicability for cell fusion. The proposed device combines passive hydrodynamic phenomenon and gravitational sedimentation, which allows the transportation and trapping of homotypic and heterotypic cells in large numbers with pairing efficiencies of 75~78% and fusion efficiencies of 73%. Additionally, we also report properties of fused cells from cell biology perspectives, including combined fluorescence-labeled intracellular materials from THP1 and A549, mixed cell morphology, and cell viability. The MFC can be tuned for pairing and fusion of cells with a similar protocol for different cell types. The MFC can be easily disconnected from the test setup for further analysis.
Asunto(s)
Fusión Celular , Hidrodinámica , Microfluídica , Células A549 , Fusión Celular/instrumentación , Supervivencia Celular , Electricidad , Humanos , Imagenología Tridimensional , Microfluídica/instrumentación , Células THP-1RESUMEN
Once a good immune response has developed in an animal and an appropriate screening procedure has been developed, the construction of hybridomas is ready to begin. The electro cell fusion (electrofusion) method uses an electrical field in the form of short, intense pulses to increase the permeability of the membrane. The resulting local perforation of the cell membrane induces the cells to fuse, forming hybridomas. Electrofusion is accomplished in three steps: Prealignment of the cells (convergence and cell contact), membrane fusion, and postalignment (rounding off the fused cells). This method has been applied successfully to hybridoma production with higher efficiency than routine polyethylene glycol fusion, allowing production of more hybrid cells.
Asunto(s)
Fusión Celular/métodos , Técnicas Electroquímicas/métodos , Hibridomas , Animales , Linfocitos B/citología , Fusión Celular/instrumentación , Línea Celular Tumoral , Separación Celular/métodos , Técnicas de Cocultivo , Células Híbridas/citología , Ratones , Mieloma Múltiple/patologíaRESUMEN
Based on a previous finding that fusion of a somatic cell with an embryonic stem (ES) cell reprogrammed the somatic cell, genes for reprogramming transcription factors were selected and induced pluripotent stem (iPS) cell technology was developed. The cell fusion itself produced a tetraploid cell. To avoid nuclear fusion, a method for cytoplasmic fusion using a microtunnel device was developed. However, the ES cell was too small for cell pairing at the device. Therefore, in the present study, ES cell enlargement was carried out with the colchicine derivative demecolcine (DC). DC induced enlargement of ES cells without loss of their stemness. When an enlarged ES cell was paired with a somatic cell in the microtunnel device, cytoplasmic fusion was observed. The present method may be useful for further development of reprogramming techniques for iPS cell preparation without gene transfection.
Asunto(s)
Fusión Celular/instrumentación , Citoplasma , Células Madre Embrionarias/citología , Animales , Fusión Celular/métodos , Tamaño de la Célula , Células Cultivadas , Demecolcina/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Diseño de Equipo , Regulación de la Expresión Génica/efectos de los fármacos , Dispositivos Laboratorio en un Chip , Ratones , Células Madre Pluripotentes/fisiologíaRESUMEN
The excessive cell death rate caused by electrofusion with unipolar pulses (UPs) has been a bottleneck to increasing cell fusion efficiency in monoclonal antibody technology. Several studies have confirmed that compared with UPs, bipolar pulses (BPs) with microsecond pulse widths can increase electropermeabilization while reducing cell death. Given these characteristics, BPs were used to increase cell fusion efficiency in this study. Cell staining and hybridoma culture experiments were performed using SP2/0 mouse myeloma cells and lymphocytes. Based on the equal energy principle, UPs and BPs were delivered to electrodes at a distance of 3.81â¯mm, with electric field intensities ranging from 2â¯kV/cm to 3â¯kV/cm and pulse duration of 40⯵s for the UPs and 20-20⯵s for the BPs. The results of cell staining experiments showed that cell fusion efficiency was 3-fold greater with BPs than with UPs. Similarly, the results of the hybridoma culture experiments showed that the hybridoma yields were 0.26 and 0.23 (2.5â¯kV/cm and 3â¯kV/cm, respectively) in the UP groups and increased to 0.46 and 0.35 in the BP groups. Taken together, the results show that the efficiency of heterologous cell fusion can be greatly increased if BPs are used instead of the commonly applied UPs. This study may provide a promising method for monoclonal antibody technology.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Fusión Celular/métodos , Hibridomas/citología , Linfocitos/citología , Animales , Fusión Celular/instrumentación , Células Cultivadas , Electricidad , Electroporación/instrumentación , Electroporación/métodos , Diseño de Equipo , Hibridomas/metabolismo , Linfocitos/metabolismo , Masculino , RatonesRESUMEN
Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.
Asunto(s)
Fusión Celular/métodos , Vidrio/química , Macrófagos/citología , Fusión Celular/instrumentación , Células Gigantes/citologíaRESUMEN
Dendritic cells/tumor fusions have shown to elicit anti-cancer immunity in different cancer types. However, the application of these vaccines for human cancer immunotherapy are limited by the instable quality and insufficient quanity of fusion cells. We present a cell electrofusion chip fabricated using soft lithography technique, which combines the rapid and precise cell pairing microstructures and the high yield electrofusion micro-electrodes to improve the cell fusion. The design uses hydrodynamic trapping in combination with positive dielectrophoretic force (pDEP) to achieve cell fusion. The chip consists of total 960 pairs of trapping channels, which are capable of pairing and fusing both homogeneous and heterogeneous types of cells. The fused cells can be easily taken out of the chip that makes this device a distinguishable from other designs. We observe pairing efficiency of 68% with fusion efficiency of 64%.
Asunto(s)
Fusión Celular/métodos , Hibridomas/citología , Inmunoterapia/métodos , Dispositivos Laboratorio en un Chip , Microfluídica/métodos , Fusión Celular/instrumentación , Línea Celular Tumoral , Humanos , Hibridomas/inmunología , Leucemia Monocítica Aguda/patología , Neoplasias Pulmonares/patología , Microelectrodos , Microfluídica/instrumentaciónRESUMEN
In this study, we analyzed the electrofusion of two cells in a biochip that has been developed to perform the capture by dielectrophoresis and the electrofusion of pairs of cells. The good transparency of the microsystem allowed analyzing the details of the fusion events. By staining one of the cells, the mixing of the two cytosols could be observed during the electrofusion experiment. We show for the first time the rapidity of the mixing of the two cytosols: less than 5 s under our experimental conditions. By comparing these experimental results to a numerical simulation, we found that the rate of this phenomenon is compatible with a diffusion-only mechanism, showing that during the fusion, the two cell membranes in contact are affected by very rapid structural changes and do not limit the exchange of the cytosols between the two cells. A point of interest is the use of dielectric structures to concentrate the electric field and of positive dielectrophoresis to capture cells in the area where the electric field is more intense. This technique allows the increase of the cell-to-cell contact and limits cell cytosol leakages during the fusion process.
Asunto(s)
Fusión Celular/instrumentación , Fusión Celular/métodos , Técnicas Electroquímicas/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Animales , Línea Celular Tumoral , Simulación por Computador , Difusión , Diseño de Equipo , Ratones , Espectrometría de FluorescenciaRESUMEN
The prospect of novel therapeutic approaches has renewed the current interest in the fusion of rare cells, like stem cells or primary immune cells. While conventional techniques are only capable of mass fusion, lab-on-a-chip systems often still lack an acceptable method for making the cells available after processing. Here, we present a microfluidic approach for electrofusion on the single-cell level that offers high control over the cells both before and after fusion. For cell pairing and fusion, we employed dielectrophoresis and AC voltage pulses, respectively. Each cell has been characterized and selected before they were paired, fused and released from the fluidic system for subsequent analysis and cultivation. The successful experimental evaluation of our system was further corroborated by numerical simulations. We obtained fusion efficiencies of more than 30% for individual pairs of mouse myeloma and B cell blasts and showed the proliferating ability of the hybrid cells 3 d after fusion. Since aggregates of more than two cells can be fused, the technique could also be developed further for generating giant cells for low-noise electrophysiology in the context of semi-automated pharmaceutical screening procedures.
Asunto(s)
Linfocitos B/citología , Electroforesis por Microchip/instrumentación , Electroforesis por Microchip/métodos , Mieloma Múltiple/patología , Animales , Fusión Celular/instrumentación , Fusión Celular/métodos , Línea Celular , Proliferación Celular , Supervivencia Celular , Humanos , Ratones , Células Madre/citología , Células Madre/patología , Células U937RESUMEN
This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 µs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.
Asunto(s)
Linfocitos B/citología , Fusión Celular/instrumentación , Técnicas Electroquímicas/instrumentación , Hibridomas/citología , Técnicas Analíticas Microfluídicas/instrumentación , Mieloma Múltiple/patología , Animales , Fusión Celular/métodos , Separación Celular/métodos , Humanos , Hibridomas/fisiología , Ratones , Técnicas Analíticas Microfluídicas/métodosRESUMEN
Cell fusion is a fundamental biological process that can be artificially induced by different methods. Although femtosecond (fs) lasers have been successfully employed for cell fusion over the past few years, the underlying mechanisms are still unknown. In our experimental study, we investigated the correlation between fs laser-induced cell fusion and membrane perforation, and the influence of laser parameters on the fusion efficiency of nonadherent HL-60 cells. We found that shorter exposure times resulted in higher fusion efficiencies with a maximum of 21% at 10 ms and 100 mJ/cm(2) (190 mW). Successful cell fusion was indicated by the formation of a long-lasting vapor bubble in the irradiated area with an average diameter much larger than in cell perforation experiments. With this knowledge, we demonstrated, for the first time, the fusion of very large parthenogenetic two-cell porcine embryos with high efficiencies of 55% at 20 ms and 360 mJ/cm(2) (670 mW). Long-term viability of fused embryos was proven by successful development up to the blastocyst stage in 70% of cases with no significant difference to controls. In contrast to previous studies, our results indicate that fs laser-induced cell fusion occurs when the membrane pore size exceeds a critical value, preventing immediate membrane resealing.
Asunto(s)
Fusión Celular/instrumentación , Fusión Celular/métodos , Rayos Láser , Animales , Blastocisto/citología , Blastocisto/efectos de la radiación , Membrana Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Diseño de Equipo , Células HL-60 , Humanos , Partenogénesis/efectos de la radiación , Tamaño de la Partícula , Porosidad , PorcinosRESUMEN
In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two-dimensional micro-orifice array. The sheet is sandwiched by a pair of micro-chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size-independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two-dimensional orifice arrangement at the pitch of 50 µm realizes simultaneous fusion of 6 × 10³ cells on a 4 mm diameter chip, and the fusion yield of 78-90% is achieved for various sizes and types of cells.
Asunto(s)
Fusión Celular/instrumentación , Fusión Celular/métodos , Electroforesis/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Análisis de Matrices Tisulares/instrumentación , Animales , Línea Celular Tumoral , Electrodos , Diseño de Equipo , Fluoresceínas/química , Humanos , Ratones , Tamaño de la PartículaRESUMEN
A high-throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon-on-insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40-µm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2-µm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150-nm thickness SiO2 film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42-68% cells were aligned to form cell-cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (â¼12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.
Asunto(s)
Fusión Celular/instrumentación , Fusión Celular/métodos , Electroforesis/instrumentación , Electroporación/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Aluminio/química , Diseño de Equipo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Microelectrodos , Protoplastos/citología , Nicotiana/citologíaRESUMEN
Nuclear transfer (NT) cloning involves manual positioning of individual donor-recipient cell couplets for electrofusion. This is time-consuming and introduces operator-dependent variation as a confounding parameter in cloning trials. In order to automate the NT procedure, we developed a micro-fluidic device that integrates automated cell positioning and electrofusion of isolated cell couplets. A simple two layer micro-fluidic device was fabricated. Thin film interdigitated titanium electrodes (300 nm thick, 250 microm wide and 250 microm apart) were deposited on a solid borosilicate glass substrate. They were coated with a film of electrically insulating photosensitive epoxy polymer (SU-8) of either 4 or 22 microm thickness. Circular holes ("micropits") measuring 10, 20, 30, 40 or 80 microm in diameter were fabricated above the electrodes. The device was immersed in hypo-osmolar fusion buffer and manually loaded with somatic donor cells and recipient oocytes. Dielectrophoresis (DEP) was used to attract cells towards the micropit and form couplets on the same side of the insulating film. Fusion pulses between 80 V and 120 V were applied to each couplet and fusion scored under a stereomicroscope. Automated couplet formation between oocytes and somatic cells was achieved using DEP. Bovine oocyte-oocyte, oocyte-follicular cells and oocyte-fibroblast couplets fused with up to 69% (n = 13), 50% (n = 30) and 78% (n = 9) efficiency, respectively. Fusion rates were comparable to parallel plate or film electrodes that are conventionally used for bovine NT. This demonstrates proof-of-principle that a micropit device is capable of both rapid cell positioning and fusion.
Asunto(s)
Fusión Celular/instrumentación , Electroforesis/instrumentación , Técnicas Analíticas Microfluídicas , Técnicas de Transferencia Nuclear/instrumentación , Animales , Automatización , Bovinos , Electrodos , Femenino , Modelos Teóricos , Oocistos/citología , Integración de SistemasRESUMEN
Microorifice-based fusion makes use of electric field constriction to assure high-yield one-to-one fusion of selected cell pairs. The aim of this paper is to verify feasibility of high-yield cell fusion on a microfluidic chip. This paper also examines viability of the fusant created on the chip. We fabricated a microfluidic chip to fuse selected cell pairs and to study postfusion behavior. We used a self-forming meniscus-based fabrication process to create microorifice with a diameter of 2-10 microm on the vertical walls in a microfluidic channel. When 1 MHz was applied to electrodes located on both sides of the microorifice, dielectrophoretic force attracted the cells toward microorifice to form a cell pair. Once the cells get into contact, fusion pulse was applied. Real time imaging of cells during fusion and cytoplasmic dye transfer between cells indicated success of cell fusion. We found that when high frequency voltage for dielectrophoresis was swept from 1 MHz to 10 kHz in 100 micros, cell fusion was initiated. The effective electric field strength was 0.1-0.2 kV/cm. We analyzed viability by imaging fusant going into cell division phase after 48 h of incubation. We conclude that fabricated microfluidic chip is suitable for high-yield one-to-one fusion and creation of viable fusants. This technology should be a useful tool to study fusion phenomena and viability of fusants, as it allows imaging of the cells during and after the fusion.
Asunto(s)
Fusión Celular/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Animales , Ingeniería Biomédica , Fusión Celular/métodos , Línea Celular , Supervivencia Celular , Electricidad , Diseño de Equipo , Humanos , Células Jurkat , Ratones , Técnicas Analíticas Microfluídicas/métodos , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Microscopía de Contraste de FaseRESUMEN
Cell fusion is a powerful tool for understanding the molecular mechanisms of epigenetic reprogramming. In hybrid cells of somatic cells and pluripotential stem cells, including embryonic stem (ES) and embryonic germ cells, somatic nuclei acquire pluripotential competence. ES and embryonic germ cells retain intrinsic trans activity to induce epigenetic reprogramming. For generating hybrid cells, we have used the technique of electrofusion. Electrofusion is a highly effective, reproducible, and biomedically safe in vitro system. For successful cell fusion, two sequential steps of electric pulse stimulation are required for the alignment (pearl chain formation) of two different types of cells between electrodes in response to alternating current stimulation and for the fusion of cytoplasmic membranes by direct current stimulation. Optimal conditions for electrofusion with a pulse generator are introduced for ES and somatic cell fusion. Topics in the field of stem cell research include the successful production of cloned animals via the epigenetic reprogramming of somatic cells and contribution of spontaneous cell fusion to generating intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of epigenetic reprogramming and regenerative medicine.
Asunto(s)
Fusión Celular/métodos , Núcleo Celular/genética , Embrión de Mamíferos/citología , Epigénesis Genética , Células Híbridas/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Fusión Celular/instrumentación , Células Clonales , Electricidad , Células Híbridas/citología , Ratones , Células Madre Pluripotentes/citologíaRESUMEN
OBJECTIVE: The lack of meaningful axon regeneration after central nervous system damage and poor functional recovery after serious peripheral nervous system nerve injuries have been long-standing problems of substantial interest to both neurosurgeons and neurobiologists. As an alternative to strategies that seek to promote the regeneration of adult axons, our research group has taken advantage of advances in microtechnology to develop a paradigm of direct axon repair involving the substitution of damaged axon regions with healthy segments from donor axons. METHODS: This repair methodology uses a novel combination of microtechnology, electrokinetic axon manipulation, and the well-established biological principle of cell fusion. These three fields of research have been integrated in a multidisciplinary approach to develop a solution for a significant clinical problem that currently has no specific treatment. RESULTS: The findings reported here provide some initial proof of principle for the core technologies we intend to use for axon repair. Functional recovery from nerve damage of course is clinically challenging, and many obstacles would need to be overcome before such axon repair procedures can be contemplated for therapeutic use. We identify some of the clinical issues that must be addressed for microtechnology-assisted axon repair to transition from the realm of research into actual surgical settings. CONCLUSION: It is hoped that each advance in axon repair technology will spur additional research to provide us with a comprehensive understanding on how best to pursue neurosurgical intervention at the microscale.
Asunto(s)
Axones/ultraestructura , Microcirugia/instrumentación , Microcirugia/métodos , Animales , Axones/fisiología , Fusión Celular/instrumentación , Fusión Celular/métodos , Humanos , Regeneración Nerviosa/fisiologíaRESUMEN
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.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Fusión Celular/métodos , Células Dendríticas/inmunología , Células Híbridas/inmunología , Factores Inmunológicos/uso terapéutico , Inmunoterapia/métodos , Interleucina-12/uso terapéutico , Neoplasias Pulmonares/secundario , Melanoma Experimental/terapia , Células Madre Neoplásicas/inmunología , Vacunación , Adyuvantes Inmunológicos , Animales , Presentación de Antígeno , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Fusión Celular/instrumentación , Terapia Combinada , Células Dendríticas/citología , Estimulación Eléctrica , Femenino , Células Híbridas/trasplante , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/terapia , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/citología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Organismos Libres de Patógenos Específicos , beta-Galactosidasa/genética , beta-Galactosidasa/inmunologíaRESUMEN
The recently emerging technique of laser microsurgery (optical tweezers, optical scissors, etc.) is providing a new precise, sterile method for the cell engineering practices such as introduction of external gene into an object cell, cell-fusion, and trapping or transportation of microscopic objects (cells or chromosomes etc.). The thermal effects thus induced usually proved to be critical factors for successful operation of this method. In order to meet the requirement for the rapid development in this territory, some important bio-thermal physical problems and the corresponding research subjects in this area were comprehensively summarized. Difficulties and critical issues were discussed. The latest advancement of the laser cell engineering was also described. This review is attempted to bridge up the gap between bioengineering and thermal science fields and then to enhance the rapid progress of laser microsurgery.
Asunto(s)
Fusión Celular/métodos , Calor , Terapia por Láser/instrumentación , Rayos Láser , Micromanipulación/métodos , Biotecnología/instrumentación , Biotecnología/métodos , Fusión Celular/instrumentación , Células Cultivadas , Transferencia de Energía , Ingeniería Genética/instrumentación , Ingeniería Genética/métodos , Terapia por Láser/métodos , Micromanipulación/instrumentaciónRESUMEN
A fusion chamber and an appropriate procedure are described which allow to fuse a sample of 15 to 25 microliters of cell suspension every two minutes. The cells can be observed throughout the process. They are not exposed to mechanical stress after the fusion pulse. Electrofusion between the heteromyeloma line CB-F7 and human mononuclear cells from peripheral blood of immunized donors is shown to provide stable hybridomas producing IgG against tetanustoxin. Pronase treatment, calmodulin, PEG, lanthanum and a number of variations in the fusion conditions were investigated as to whether they influence physical fusion of the cells, hybridoma yield, and immunoglobulin production.