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  • 1.
    Kandušer, Maša
    et al.
    University of Ljubljana, Slovenia.
    Ušaj, Marko
    University of Ljubljana, Slovenia.
    Cell electrofusion: past and future perspectives for antibody production and cancer cell vaccines2014In: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 11, no 12, p. 1885-1898Article in journal (Refereed)
    Abstract [en]

    Introduction: In the past few decades, new methods for drug and gene delivery have been developed, among which electroporation and electrofusion have gained noticeable attention. Lately, advances in the field of immunotherapy have enabled new cancer therapies based on immune response, including monoclonal antibodies and cell vaccines. Efficient cell fusion is needed for both hybridoma production and cell vaccine preparation, and electrofusion is a promising method to achieve this goal.Areas covered: In the present review, we cover new strategies of cancer treatment related to antibody production and cell vaccines. In more detail, cell electroporation and electrofusion are addressed. We briefly describe principles of cell electroporation and focus on electrofusion and its influential factors, with special attention on the fusogenic state of the cell membrane, contact formation, the effect of electrofusion media and cell viability. We end the review with an overview of the very promising field of microfluidic devices for electrofusion.Expert opinion: In our opinion, electrofusion can be a very efficient method for hybridoma and cell vaccine production. Advances in the development of microfluidic devices and a better understanding of the underlying (biological) mechanisms will overcome the current limitations.

  • 2.
    Klavins, Maris
    et al.
    University of Latvia, Latvia.
    Burlakovs, Juris
    University of Latvia, Latvia.
    Ozola, Ruta
    University of Latvia, Latvia.
    Muter, Olga
    University of Latvia, Latvia.
    Composite clay sorbents for immobilisation of biomolecules and cells2015In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 208, no supplement, p. S56-Article in journal (Refereed)
  • 3.
    Kroon, Martin
    et al.
    Royal Institute of Technology.
    Holzapfel, Gerhard
    Graz University of Technology, Austria.
    A model for saccular cerebral aneurysm growth by collagen fibre remodelling2007In: Journal of Theoretical Biology, ISSN 0022-5193, E-ISSN 1095-8541, Vol. 247, p. 775-787Article in journal (Refereed)
    Abstract [en]

    The first structural model for saccular cerebral aneurysm growth is proposed. It is assumed that the development of the aneurysm isaccompanied by a loss of the media, and that only collagen fibres provide load-bearing capacity to the aneurysm wall. The aneurysm ismodelled as an axisymmetric multi-layered membrane, exposed to an inflation pressure. Each layer is characterized by an orientationangle, which changes between different layers. The collagen fibres and fibroblasts within a specific layer are perfectly aligned. The growthand the morphological changes of the aneurysm are accomplished by the turnover of collagen. Fibroblasts are responsible for collagenproduction, and the related deformations are assumed to govern the collagen production rate. There are four key parameters in themodel: a normalized pressure, the number of layers in the wall, an exponent in the collagen mass production rate law, and the pre-stretchunder which the collagen is deposited. The influence of the model parameters on the aneurysmal response is investigated, and a stabilityanalysis is performed. The model is able to predict clinical observations and mechanical test results, for example, in terms of predictedaneurysm size, shape, wall stress and wall thickness.

  • 4. Ohlson, Sten
    et al.
    Zopf, David
    Weak affinity chromatography1993In: Handbook of affinity chromatography / [ed] Toni Kline, New York: Marcel Dekker, 1993, 1, p. 299-314Chapter in book (Other academic)
  • 5.
    Rems, Lea
    et al.
    University of Ljubljana, Slovenia.
    Ušaj, Marko
    University of Ljubljana, Slovenia.
    Kandušer, Maša
    University of Ljubljana, Slovenia.
    Reberšek, Matej
    University of Ljubljana, Slovenia.
    Miklavčič, Damijan
    University of Ljubljana, Slovenia.
    Pucihar, Gorazd
    University of Ljubljana, Slovenia.
    Cell electrofusion using nanosecond electric pulses2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, article id 3382Article in journal (Refereed)
    Abstract [en]

    Electrofusion is an efficient method for fusing cells using short-duration high-voltage electric pulses. However, electrofusion yields are very low when fusion partner cells differ considerably in their size, since the extent of electroporation (consequently membrane fusogenic state) with conventionally used microsecond pulses depends proportionally on the cell radius. We here propose a new and innovative approach to fuse cells with shorter, nanosecond (ns) pulses. Using numerical calculations we demonstrate that ns pulses can induce selective electroporation of the contact areas between cells (i.e. the target areas), regardless of the cell size. We then confirm experimentally on B16-F1 and CHO cell lines that electrofusion of cells with either equal or different size by using ns pulses is indeed feasible. Based on our results we expect that ns pulses can improve fusion yields in electrofusion of cells with different size, such as myeloma cells and B lymphocytes in hybridoma technology.

  • 6.
    Ušaj, Marko
    et al.
    University of Ljubljana, Slovenia.
    Flisar, Karel
    University of Ljubljana, Slovenia.
    Miklavcic, Damijan
    University of Ljubljana, Slovenia.
    Kanduser, Masa
    University of Ljubljana, Slovenia.
    Electrofusion of B16-F1 and CHO cells: the comparison of the pulse first and contact first protocols2013In: Bioelectrochemistry, ISSN 1567-5394, E-ISSN 1878-562X, Vol. 89, p. 34-41Article in journal (Refereed)
    Abstract [en]

    High voltage electric pulses induce permeabilisation (i.e. electroporation) of cell membranes. Electric pulses also induce fusion of cells which are in contact. Contacts between cells can be established before electroporation, in so-called contact first or after electroporation in pulse first protocol. The lowest fusion yield was obtained by pulse first protocol (0.8%±0.3%) and it was only detected by phase contrast microscopy. Higher fusion yield detected by fluorescence microscopy was obtained by contact first protocol. The highest fusion yield (15%) was obtained by modified adherence method whereas fusion yield obtained by dielectrophoresis was lower (4%). The results are in agreement with current understanding of electrofusion process and with existing electrochemical models. Our data indicate that probability of stalk formation leading to fusion pores and cytoplasmic mixing is higher in contact first protocol where cells in contact are exposed to electric pulses. Another contribution of present study is the comparison of two detection methods. Although fusion yield can be more precisely determined with fluorescence microscopy we should note that by using this detection method single coloured fused cells cannot be detected. Therefore low fusion yields are more reliably detected by phase contrast microscopy.

  • 7.
    Ušaj, Marko
    et al.
    University of Ljubljana, Slovenia.
    Kandušer, Maša
    University of Ljubljana, Slovenia.
    Modified Adherence Method (MAM) for Electrofusion of Anchorage-Dependent Cells2015In: Cell Fusion: Overviews and Methods, New York, NY: Humana Press, 2015, p. 203-216Chapter in book (Refereed)
    Abstract [en]

    The artificially induced cell fusion is a useful experimental tool in biology, biotechnology and medicine. The electrofusion is a physical method for cell fusion that applies high-voltage electric pulses. The use of electric pulses causes cell membrane structural changes which bring the cell membrane in the so-called fusogenic state. When such fusogenic membranes are in close contact cell fusion takes place. Physical contact between fusion partners can be achieved by various methods and one of them is modified adherence method (MAM) described in detail here on B16-F1 cell line. The method is based on the fact that living cells form contacts in confluent culture. However, instead of using confluent cell culture, in modified adherence method cells are plated in suitable concentration and allowed to form contacts for only short predetermined period of time. During that time the cells are only slightly attached to the dish surface maintaining the spherical shape. Observed high fusion yields up to 50 % obtained by MAM in situ by dual-color fluorescence microscopy are among the highest in field of electrofusion. The method can be readily adapted to other anchorage-dependent cell lines.

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