Avoiding the side effects of electric current pulse application to electroporated cells in disposable small volume cuvettes assures good cell survival.

BACKGROUND: The harmful side effects of electroporation to cells due to local changes in pH, the appearance of toxic electrode products, temperature increase, and the heterogeneity of the electric field acting on cells in the cuvettes used for electroporation were observed and discussed in several laboratories. If cells are subjected to weak electric fields for prolonged periods, for example in experiments on cell electrophoresis or galvanotaxis the same effects are seen. In these experiments investigators managed to reduce or eliminate the harmful side effects of electric current application. METHODS: For the experiments, disposable 20 µl cuvettes with two walls made of dialysis membranes were constructed and placed in a locally focused electric field at a considerable distance from the electrodes. Cuvettes were mounted into an apparatus for horizontal electrophoresis and the cells were subjected to direct current electric field (dcEF) pulses from a commercial pulse generator of exponentially declining pulses and from a custom-made generator of double and single rectangular pulses. RESULTS: More than 80% of the electroporated cells survived the dcEF pulses in both systems. Side effects related to electrodes were eliminated in both the flow through the dcEF and in the disposable cuvettes placed in the focused dcEFs. With a disposable cuvette system, we also confirmed the sensitization of cells to a dcEF using procaine by observing the loading of AT2 cells with calceine and using a square pulse generator, applying 50 ms single rectangular pulses. CONCLUSIONS: We suggest that the same methods of avoiding the side effects of electric current pulse application as in cell electrophoresis and galvanotaxis should also be used for electroporation. This conclusion was confirmed in our electroporation experiments performed in conditions assuring survival of over 80% of the electroporated cells. If the amplitude, duration, and shape of the dcEF pulse are known, then electroporation does not depend on the type of pulse generator. This knowledge of the characteristics of the pulse assures reproducibility of electroporation experiments using different equipment.

Efficacy of Local Anesthetics in Detachment of Normal 3T3 Mouse Fibroblasts and Prostate Cancer AT-2 Cells from Substrata, in Maintenance of Viable Cells in a Non-Adherent State, and in Preservation of Cell Surface Markers Detected with FlowSight Image Cytometry.

The local anesthetics procaine, lidocaine and tetracaine permit the reversible detachment of viable cells and their passaging or preservation in a non-adherent state in the absence of proteolytic enzymes. The effects of these anesthetics, dissolved in various media, on cell viability, cell detachment from substrata and preservation of cells in a non-adherent state, were compared using the AT-2 line of rat prostate carcinoma cells of moderate malignancy and the 3T3 mouse fibroblast cell line. It was found that all three local anesthetics can induce cell rounding followed by detachment of over 95% of viable cells in both lines in Ca2+/Mg(2+)-free PBS. Tetracaine in 1 mM concentration was the most effective in induction of fast cell detachment. However, procaine and lidocaine in 16 mM concentrations were found to be optimal for preservation of cells in a non-adherent state and for the maintenance of cell viability for at least 2 h. The tested anesthetics also cause cell rounding and detachment when present in various cell culture media but these processes occurred much more slowly and less efficiently than in Ca2+/Mg(2+)-free PBS. Normal 3T3 mouse fibroblasts after detachment and passaging undertake growth reaching the same saturation density in cultures after detachment with procaine or lidocaine as after passaging using trypsin solution. The results suggest that the application of local anesthetics can be a very simple and effective technique for cell passaging in tissue cultures. This technique might decrease side-effects and cell injury caused by trypsinization or cell scraping. The preservation of cells in suspension in a non-adherent state may facilitate analysis of cell surface properties and fractionation of cell mixtures. Avoiding the use of trypsin allows for the preservation of cell surface proteins ICAM, CXCR4, and HCAM analyzed with FlowSight image flow cytometry.

Functional links between Snail-1 and Cx43 account for the recruitment of Cx43-positive cells into the invasive front of prostate cancer.

Suppressive function of connexin(Cx)43 in carcinogenesis was recently contested by reports that showed a multifaceted function of Cx43 in cancer progression. These studies did not attempt to model the dynamics of intratumoral heterogeneity involved in the metastatic cascade. An unorthodox look at the phenotypic heterogeneity of prostate cancer cells in vitro enabled us to identify links between Cx43 functions and Snail-1-regulated functional speciation of invasive cells. Incomplete Snail-1-dependent phenotypic shifts accounted for the formation of phenotypically stable subclones of AT-2 cells. These subclones showed diverse predilection for invasive behavior. High Snail-1 and Cx43 levels accompanied high motility and nanomechanical elasticity of the fibroblastoid AT-2_Fi2 subclone, which determined its considerable invasiveness. Transforming growth factor-ß and ectopic Snail-1 overexpression induced invasiveness and Cx43 expression in epithelioid AT-2 subclones and DU-145 cells. Functional links between Snail-1 function and Cx43 expression were confirmed by Cx43 downregulation and phenotypic shifts in AT-2_Fi2, DU-145 and MAT-LyLu cells upon Snail-1 silencing. Corresponding morphological changes and Snail-1 downregulation were seen upon Cx43 silencing in AT-2_Fi2 cells. This indicates that feedback loops between both proteins regulate cell invasive behavior. We demonstrate that Cx43 may differentially predispose prostate cancer cells for invasion in a coupling-dependent and coupling-independent manner. When extrapolated to in vivo conditions, these data show the complexity of Cx43 functions during the metastatic cascade of prostate cancer. They may explain how Cx43 confers a selective advantage during cooperative invasion of clonally evolving, invasive prostate cancer cell subpopulations.

Decreasing the thresholds for electroporation by sensitizing cells with local cationic anesthetics and substances that decrease the surface negative electric charge.

The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissues.

Reversible and irreversible electroporation of cell suspensions flowing through a localized DC electric field.

Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0-1000 V/cm for a selected duration in the range 10-1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permits the elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports on cell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation.

Solute-dependent activation of cell motility in strongly hypertonic solutions in Dictyostelium discoideum, human melanoma HTB-140 cells and walker 256 carcinosarcoma cells.

Published data concerning the effects of hypertonicity on cell motility have often been controversial. The interpretation of results often rests on the premise that cell responses result from cell dehydration, i.e. osmotic effects. The results of induced hypertonicity on cell movement of Dictyostelium discoideum amoebae and human melanoma HTB-140 cells reported here show that: i) hypertonic solutions of identical osmolarity will either inhibit or stimulate cell movement depending on specific solutes (Na(+) or K(+), sorbitol or saccharose); ii) inhibition of cell motility by hypertonic solutions containing Na(+) ions or carbohydrates can be reversed by the addition of calcium ions; iii) various cell types react differently to the same solutions, and iv) cells can adapt to hypertonic solutions. Various hypertonic solutions are now broadly used in medicine and to study modulation of gene expression. The observations reported suggest the need to examine whether the other responses of cells to hypertonicity can also be based on the solute-dependent cell responses besides cell dehydration due to the osmotic effects.

Involvement of cytoskeleton in orientation of cell division in contact guided cells.

Single human skin fibroblasts and the skin keratinocyte cell line HaCaT show contact guidance and elongate along narrow (1-2 microm) scratches in glass substratum. During cell division these cells orientate their mitotic spindles along the long axis of the cell. Immunofluorescence staining of actin, tubulin, chromatin, and the nuclear NuMA protein complex demonstrated that cell elongation along scratches is accompanied by a corresponding rearrangement in the cytoskeleton. The results and literature suggest the following steps in the interplay between outside-in and inside-out signalling in the regulation of cell division orientation by extracellular factors. The interaction of cell surface with an anisotropy in the local environment causes changes in F-actin organization, cell elongation and alignment of stress fibres along the cell axis. This is accompanied by a corresponding reorientation of microtubules. Microtubules mediate between cell shape changes dependent upon cell interaction with substratum or other cells, the cortical actin and the position of centrosomes. Centrosomes determine the position and orientation of the mitotic spindle. The astral and central microtubules of the mitotic spindle control the localization of contraction-relaxation in the cell cortex and the position of the constriction ring and cell division plane.

Cell electrophoresis--a method for cell separation and research into cell surface properties.

In this paper, we discuss the application of various methods of cell electrophoresis in research into cell surface properties (analytical methods), and the separation of uniform cell subpopulations from cell mixtures (preparative methods). The emphasis is on the prospects of the development of simplified and versatile methodologies, i.e. microcapillary cell electrophoresis and horizontal cell electrophoresis under near-isopycnic conditions. New perspectives are considered on the use of analytical and preparative cell electrophoresis in research on cell differentiation, neoplastic transformation, cell-cell interactions and the biology of stem cells.

Cell separation with horizontal cell electrophoresis under near-isopycnic conditions on a "density cushion".

This report describes an improvement made to the horizontal cell electrophoresis methodology. It involves using two liquid layers differing in density to produce an interface described as a "density cushion". The electrophoretic system that employed an anti-convective porous matrix to separate red blood cells (RBC) and charged dyes effectively was found to be unsuitable for some other mammalian cells. The "density cushion" method was found to be more versatile and applicable to studies on the separation of a variety of cell types. The experiments described show the differences between the electrophoretic mobilities of a human eosinophilic leukaemia cell line (Eol-1) and RBC, both with and without the modification of the cell surface properties.

Surface anisotropy orients cell divisions in contact guided cells.

The orientation of cell elongation and the plane of cell division were determined in cells growing under isotropic conditions on a plain glass surface or under anisotropic conditions on a scratched glass surface. Four cell lines were analysed, each showing various degrees of contact guidance. Human skin fibroblasts and the skin keratinocyte cell line HaCaT oriented randomly on the smooth isotropic surface of glass, grew and divided randomly. By contrast, on an anisotropic scratched surface these cells showed contact guidance, elongated along scratches, and their planes of division were perpendicular to the long axis of the cell. In these two cell types there was a high degree of correlation between the cell alignment and the plane of cell division, which shows that extracellular factors can influence or even determine the latter. In cell lines in which contact guidance under anisotropic conditions was less evident, viz. the lung endothelial cell line HLMEC and the skin endothelial cell line HSkMEC, the alignment of cell division planes was less ordered. This report concentrates on quantitative phenomenological descriptions of the orientation of cell division as determined by contact guidance.

Reversible inhibition of movement in the amoebae Dictyostelium discoideum and its effect on chemoattractant recognition.

The cell fixatives formaldehyde and KMnO4 at low concentrations reversibly inhibit the movement of D. discoideum amoebae without directly interfering with cell viability. This inhibition of cell movement is accompanied by the decreased attachment of cells to substratum. When the tenacity and attachment of immobilized cells are artificially increased by compressing cells between two glass surfaces, the amoebae begin to move even in the presence of the fixatives. Amoebae starved for 24 hours, subjected to fixatives and a mineral salt solution in which they remained motionless, maintained chemotactic responses to folic acid and only after a few hours of active locomotion became reactive to cAMP, in contrast to amoebae that reacted to cAMP after starvation in the absence of fixatives.

Electrotaxis: Cell Directional Movement in Electric Fields.

Electrotaxis plays an important role during embryogenesis, inflammation, wound healing, and tumour metastasis. However, the mechanisms at play during electrotaxis are still poorly understood. Therefore intensive studies on signaling pathways involved in this phenomenon should be carried out. In this chapter, we described an experimental system for studying electrotaxis of Amoeba proteus, mouse embryonic fibroblasts (MEF), Walker carcinosarcoma cells WC256, and bone marrow adherent cells (BMAC).

Motile activities of Dictyostelium discoideum differ from those in Protista or vertebrate animal cells.

Cell movement in the amoebae Dictyostelium discoideum has been examined in media differing in monovalent cation concentration (i.e. Na+ and K+). Under isotonic or even slightly hypertonic conditions, the cells move equally well in solutions in which either potassium or sodium ions dominate. However, in strongly hypertonic solutions the amoebae showed motility in a 2% potassium chloride solution, but remained motionless in a hypertonic 2% sodium chloride solution. This inhibition of D. discoideum amoebae movement in a hypertonic sodium chloride solution was fully reversible. Such behaviour corresponds to that of plant, fungi, and some invertebrate animal cells rather than protozoan or vertebrate cells. These observations suggest that studies using D. discoideum as a model for cell motility in vertebrate animal tissue cells should be considered with caution, and would seem to confirm the classification of cellular slime moulds as related rather to Fungi than to Protista. This also shows that the cell membrane models should consider the asymmetry in sodium/potassium ion concentrations found in vertebrate animal cells as one of various possibilities.

Some difficulties in research into cell motile activity under isotropic conditions.

Movement of Dictyostelium discoideum amoebae under isotropic and anisotropic conditions was recorded and analysed with computer-aided methods and the results are presented in various manners as described in the subject literature. Cell movement under isotropic conditions showed great diversity. Some cells moved almost in a straight path whereas others in close proximity turned around with little net translocation. When cell movement under isotropic conditions was observed, no direct correlation was found between the total length of cell trajectories and the length of final displacements of the cells. It was necessary to present the results in the form of histograms, circular diagrams of cell trajectories or in scatter correlation diagrams showing the motile behaviour of many individual cells. These methods of presentation are more informative than methods which present only average values, the "representative" behaviour of single cells, or start and end points of cell tracks. The latter methods can only illustrate but do not document the results of experiments. The use of statistical methods appears necessary in cases when it is difficult to monitor the same cells before and during experimental treatment. However, when cell movement under anisotropic conditions becomes oriented and ordered as during tactic cell movements, then the diversity in cell behaviour decreases and methods based on estimation of starting and end points of cell positions appear more credible.

A new method for the preperative and analytical electrophoresis of cells.

In this paper, a new method is described for the horizontal electrophoresis of cells on a density cushion under near-isopycnic conditions. When cell sedimentation is minimized, the electrophoresis of red blood cells (RBC) used as model cells within an anti-convective porous matrix (with pores over 300 microm in diameter) was capable of separating a mixture of human and chicken RBC according to their electrophoretic mobilities. Samples taken from the separated RBC bands show over 90% purity for each species. The simultaneous electrophoresis of several RBC samples carried out under identical conditions permitted the use of comparative data based on the electrophoretic mobility of cells which differ in their surface properties. We believe that this relatively simple system, in which cell sedimentation and convection are minimized, has the potential to be modified and adapted for the separation of other cell types/organelles.

9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts.

The present study found that, similarly to 5-fluorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.

Comparison of proliferation and motile activity between human keratinocytes isolated from skin and oral mucosa.

OBJECTIVE: Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. For healing of skin wounds the skin keratinocytes can be replaced by oral mucosa epithelial cells grown in vitro. The presented experiments were carried out in order to compare the proliferation, morphology, and migration between human keratinocytes isolated from human skin and oral mucosa. MATERIALS AND METHODS: Human epidermal and oral mucosa keratinocytes from primary culture were used in all experiments. Cell motility and shape were determined using computer-aided methods. RESULTS AND CONCLUSIONS: It was demonstrated that although both cell types exhibit the same typical epithelial morphology, oral mucosa keratinocytes locomote significantly faster than skin keratinocytes. They also differ in proliferation activity. Oral mucosa keratinocytes exhibited faster growth and different actin cytoskeleton organisation than skin keratinocytes under in vitro conditions. Autologous oral mucosa keratinocytes may be expanded in vitro and used for skin wound healing in vivo.

Procaine-induced enhancement of fluid-phase endocytosis and inhibition of exocytosis in human skin fibroblasts.

Local anaesthetics are often applied directly onto the skin, and for this reason the effect of some local anaesthetics upon morphology and cytoskeleton organisation in human skin fibroblasts was investigated. In this paper the authors report that procaine (p-aminobenzoic acid diethylamino-etyl ester hydrochloride) induced vacuolisation of cytoplasm and great enhancement of neutral red accumulation in human skin fibroblasts cultured in vitro. Procaine-induced vacuolisation of cell's cytoplasm was observed to be associated with the enhanced uptake and inhibited release of fluid taken by endocytosis. All these effects appeared fully reversible. The cell vacuolisation cannot be prevented by 3-methyadenine, brefeldine A, and cytochalasine D. On the other hand, nocodazole and caffeine prevent cytoplasm vacuolisation induced by procaine. These observations suggest that procaine-induced formation of great vacuoles is due to an impairment of membrane traffic between endosomes. The authors' results also demonstrate that neutral red uptake assay, if used as a cell viability test, must be interpreted with great caution.

Three-dimensional visualization of connexin 43 on the human cardiomyocytes.

Gap junctions created by a family of connexin proteins play an important role in the development of human heart. It has been previously shown that the abnormalities of right ventricular outflow tract are related to an altered level of expression of connexin 43. The right ventricular outflow tract narrowing, stenosis, or atresia of the main pulmonary artery and hypertrophy of the right ventricle are observed in tetralogy of Fallot. The aim of the current study was to determine the distribution of connexin 43 on the surface of human cardiomyocytes obtained during reparative surgery for tetralogy of Fallot. Connexin 43 distribution in these cells was compared with distribution of connexin 43 in cardiomyocytes obtained from patients without right ventricular outflow tract pathology. Cardiomyocytes isolated from tissue biopsy were cultured on collagen substratum, fixed with paraformaldehyde, and incubated with goat antihuman connexin 43 antibodies and secondary donkey antigoat antibodies conjugated with fluorescent indocarbocyanine. Z-series of optical sections were recorded using a laser scanning confocal microscope. Three-dimensional data stacks were visualized using volume-rendering techniques. Images of connexin 43 fluorescence revealed a pattern of three-dimensional distribution of connexin on the surface of an individual cardiomyocyte. Cardiomyocytes from tetralogy of Fallot and hearts with normal right ventricular outflow tract differ in the organization of connexin 43. Cardiomyocytes from tetralogy of Fallot hearts revealed disturbed distribution of connexin 43. The protein is located irregularly on the entire surface of the cell. In the controls, connexin 43 can be visualized within the intercalated disks only. These disturbances may influence heart maturation, cause hypertrophy of the right ventricle, and induce severe arrhythmias in children with tetralogy of Fallot.

Folic acid, ascorbic acid and sodium selenite restore the motility of Dictyostelium discoideum inhibited by triethyllead.

The effect of triethyllead (TriEL) on motile activity, structure of cytoskeleton and chemotaxis of Dictyostelium discoideum amoebae in developing concentration gradients of folic acid (FA) and cAMP has been studied. It was observed that 3 microM TriEL had little or no effect on locomotion and chemotactic response of cells, whereas 5 microM TriEL strongly reduced the motile activity of Dictyostelium discoideum amoebae and inhibited their chemotaxis towards cAMP, but not towards FA. FA was found to restore the motile activity of Dictyostelium discoideum, inhibited by TriEL. A similar effect was observed in the presence of other antioxidants, i.e. ascorbic acid and sodium selenite, suggesting that oxidative stress may be involved in the action of TriEL. Moreover, the treatment of Dictyostelium amoebae with 5 microM TriEL caused disruption of microtubules while 3 microM TriEL had little effect on their structure. FA caused restoration of microtubules only in some cells within 1 h of incubation, i.e. when the directional movement of cells towards this chemoattractant was already observed. However, their organization was significantly different from that observed in the untreated cells, suggesting that microtubule undisturbed organisation may be not necessary for Dictyostelium discoideum amoebae locomotion and chemotaxis