Optimization of bulk cell electrofusion in vitro for production of human–mouse heterohybridoma cells

Trontelj, Katja; Reberšek, Matej; Kandušer, Maša; Šerbec, Vladka Čurin; Šprohar, Marjana; Miklavčič, Damijan

doi:10.1016/j.bioelechem.2008.06.003

Cited by 38 publications

(30 citation statements)

References 32 publications

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“…The results show that applying multiple pulses resulted in a higher fusion efficiency (data not shown). This is in agreement with the results described in the literature [19,23,27]. However, pulses of 200 ms directly initiated cell death (Supporting Information video).…”

Section: Fusion Efficiency Using Different Electric Parameterssupporting

confidence: 93%

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On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

et al. 2011

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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.

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Section: Fusion Efficiency Using Different Electric Parameterssupporting

confidence: 93%

“…Moreover, for therapeutic applications, such as cancer immunotherapy, human antibodies, and hence human B cells, are a prerequisite [20,21]. A human IgM-producing hybridoma can be generated via the fusion of a human B-cell with a mouse myeloma cell [21][22][23]. Furthermore, readily accessible human peripheral blood CD19…”

Section: Introductionmentioning

confidence: 99%

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

et al. 2011

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“…Such nonspecific fusion enables production of highly valuable hybrid cells and their products, such as monoclonal antibodies, and provides information about fundamental mechanisms of fusion [2]. Electrofusion is a potentially very effective method since it can be properly adjusted to different types of cells.…”

Section: Discussionmentioning

confidence: 99%

“…Experiments were performed on previously prepared cells of mouse melanoma cell line (B16-F1). Cells are grown in two separated 25 cm 2 culture flasks (TPP, ZDA) to 70-80 % confluence in DMEM culture medium (Dulbecco's modified Eagle's medium) supplemented with 10 % fetal bovine serum, 0.15 mg/ml L-glutamine, 16 mg/ml gentamicin (all from Sigma-Aldrich, Germany), 200 units/ml crystacillin (Pliva, Croatia), and incubated in 5% CO 2 at 37°C. 2.…”

Section: Video Linkmentioning

confidence: 99%

Cell Electrofusion Visualized with Fluorescence Microscopy

Trontelj

Ušaj

Miklavčič

2010

JoVE

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Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Video LinkThe video component of this article can be found at

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“…The phenomenon termed as electropermeabilization has been studied for several decades and has already found numerous applications for intracellular drug and gene delivery in oncology (electrochemotherapy), gene therapy, and cell biology, for transdermal drug delivery, insertion of proteins into cells, fusion of cells, sterilization, and tissue ablation [1,12,13,33,37,41,42,49,53].…”

Section: Introductionmentioning

confidence: 99%

Electropermeabilization

Encyclopedia of Microfluidics and Nanofluidics

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It has been reported previously that electric pulses of sufficiently high voltage and short duration can permeabilize the membranes of various organelles inside living cells. In this article, we describe electropermeabilization of endocytotic vesicles in B16 F1 mouse melanoma cells. The cells were exposed to short, high-voltage electric pulses (from 1 to 20 pulses, 60 ns, 50 kV/cm, repetition frequency 1 kHz). We observed that 10 and 20 such pulses induced permeabilization of membranes of endocytotic vesicles, detected by release of lucifer yellow from the vesicles into the cytosol. Simultaneously, we detected uptake of propidium iodide through plasma membrane in the same cells. With higher number of pulses permeabilization of the membranes of endocytotic vesicles by pulses of given parameters is accompanied by permeabilization of plasma membrane. However, with lower number of pulses only permeabilization of the plasma membrane was detected.

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Optimization of bulk cell electrofusion in vitro for production of human–mouse heterohybridoma cells

Cited by 38 publications

References 32 publications

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

Cell Electrofusion Visualized with Fluorescence Microscopy

Electropermeabilization

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