Marko Ušaj

Cell electrofusion using nanosecond electric pulses

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.

Cell counting tool parameters optimization approach for electroporation efficiency determination of attached cells in phase contrast images

In this paper a novel parameter optimization approach for cell detection tool and counting cells procedure in phase contrast images are presented. Manual counting of the attached cells in phase contrast images is time‐consuming and subjective. For evaluation of electroporation efficiency of attached cells, we often perform manual counting of the cells which is needed to determine the percentage of electroporated cells under different experimental conditions. Here we present an automated cell counting procedure based on novel artificial neural network optimization of Image‐based Tool for Counting Nuclei algorithm parameters to fit the training image set based on counts from an expert. Comparing the results of automated cell counting to user manual counting a 90,31% average agreement was achieved which is reasonably good especially taking into account inter‐person error which can be up to 10%. Even more, our procedure can also be used for fluorescent cell images with similar counting accuracy (>90%) enabling us to determine electroporation efficiency. In our experiments, the electroporation efficiency determined by manual cell counting was virtually the same as the one obtained by the automated procedure.

Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization

Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization Background. Various electrofusion parameters have to be adjusted to obtain the optimal electrofusion efficiency. Based on published data, good electrofusion conditions can be achieved with the hypotonic treatment. However, the duration of the hypotonic treatment before electroporation and buffer hypoosmolarity have to be adjusted in order to cause cell swelling, to avoid regulatory volume decrease and to preserve cell viability. The aims of our study were to determine cell size dynamics and viability of four different cell lines in hypotonic buffer and to study the influence of the electroporation on the selected cell line in hypotonic buffer. Materials and methods. Cell size dynamics of different cell lines exposed to hypotonic buffer and electroporation were analyzed by time-resolved cell size measurements. The viability of hypotonically treated or/and electroporated cells was determined 24 h after the experiment by a modified crystal violet (CV) viability assay. Results. In our experimental conditions the hypotonic treatment at 100 mOsm was efficient for CHO, V79 and B16-F1 cell lines. The optimal duration of the treatment was between two and five minutes. On the other hand the same hypotonic treatment did not cause cell swelling of NS1 cells. Cell swelling was also observed after electroporation of B16-F1 in isotonic buffer and it was amplified when hypotonic buffer was used. In addition, the regulatory volume decrease was successfully inhibited with electroporation. Conclusions. Cell size dynamics in hypotonic conditions should be studied for each cell line since they differ in their sensitivity to the hypotonic treatment. The inhibition of cell regulatory volume decrease by electroporation may be beneficial in achieving higher electrofusion efficiency. The hypotonic treatment in itself did not significantly affect the cell viability; however, electric field parameters for electroporation should be carefully selected taking into account the hypotonically induced volume increase of cells.

Cell electrofusion: past and future perspectives for antibody production and cancer cell vaccines.

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.

Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia

ABSTRACT Mitochondria respond to environmental cues and stress conditions. Additionally, the disruption of the mitochondrial network dynamics and its distribution is implicated in a variety of neurodegenerative diseases. Here, we reveal a new function for Myo19 in mitochondrial dynamics and localization during the cellular response to glucose starvation. Ectopically expressed Myo19 localized with mitochondria to the tips of starvation-induced filopodia. Corollary to this, RNA interference (RNAi)-mediated knockdown of Myo19 diminished filopodia formation without evident effects on the mitochondrial network. We analyzed the Myo19–mitochondria interaction, and demonstrated that Myo19 is uniquely anchored to the outer mitochondrial membrane (OMM) through a 30–45-residue motif, indicating that Myo19 is a stably attached OMM molecular motor. Our work reveals a new function for Myo19 in mitochondrial positioning under stress. Summary: Myo19 is an effector of starvation-induced filopodia formation and is directly anchored to the outer mitochondrial membrane.

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.

Electrofusion of B16-F1 and CHO cells: The comparison of the pulse first and contact first protocols

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.

Automatic cell detection in phase-contrast images for evaluation of electroporation efficiency in vitro

In the research of electroporation, we often need to know the percent of electroporated cells under different experimental conditions. Manual counting of the cells in digital images is time-consuming and subjective, especially on phase contrast images. In this paper, we present an automatic cell counting method based on optimization of ITCN (Image-based Tool for Counting Nuclei) algorithm’s parameters to fit training data that is based on counts from user or expert. In comparing the results of automatic cell counting and user manual counting 94,21 % average agreement was achieved what is good.

Cell Electrofusion Visualized with Fluorescence Microscopy

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. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

ROS induced distribution of mitochondria to filopodia by Myo19 depends on a class specific tryptophan in the motor domain

The role of the actin cytoskeleton in relation to mitochondria function and dynamics is only recently beginning to be recognized. Myo19 is an actin-based motor that is bound to the outer mitochondrial membrane and promotes the localization of mitochondria to filopodia in response to glucose starvation. However, how glucose starvation induces mitochondria localization to filopodia, what are the dynamics of this process and which enzymatic adaptation allows the translocation of mitochondria to filopodia are not known. Here we show that reactive oxygen species (ROS) mimic and mediate the glucose starvation induced phenotype. In addition, time-lapse fluorescent microscopy reveals that ROS-induced Myo19 motility is a highly dynamic process which is coupled to filopodia elongation and retraction. Interestingly, Myo19 motility is inhibited by back-to-consensus-mutation of a unique residue of class XIX myosins in the motor domain. Kinetic analysis of the purified mutant Myo19 motor domain reveals that the duty ratio (time spent strongly bound to actin) is highly compromised in comparison to that of the WT motor domain, indicating that Myo19 unique motor properties are necessary to propel mitochondria to filopodia tips. In summary, our study demonstrates the contribution of actin-based motility to the mitochondrial localization to filopodia by specific cellular cues.