Bio-electrosprays: the next generation of electrified jets

Biological electrosprays are rapidly becoming a robust means by which to engineer living organisms for applications ranging from tissue repair to developmental biology. We previously reported the ability to electrospray living organisms without compromising their viability, but found it challenging to achieve stability in the jetting of these organisms as a result of the chemical properties of the living cellular suspensions. Jet stability is required for the generation of a near-mono distribution of droplets, which is necessary for the development of electrospray technology as a "drop and place" biotechnique. Recently, we determined the conditions needed to achieve jet stability and were able to generate droplets with a near-mono distribution (<50 microm). In this communication, we elucidate the relationship between jet behaviour and droplet size under stable jetting conditions, with a view to further reducing the droplet size to deposit a single living cell within a droplet. We believe that this level of resolution will make electrospray jetting superior amongst the jet-based biotechnologies presently being developed for the engineering of biological architectures comprised of living cells.     

Coaxial Aerodynamically Assisted Bio‐jets: A Versatile Paradigm for Directly Engineering Living Primary Organisms

In this paper, a coaxial jetting methodology is demonstrated as a first example (non‐electric field driven) completely run by aerodynamic forces which are brought about by the application of a differential pressure for the safe handling of primary living organisms by means of jets as encapsulated droplets. Previously this jetting technique in this configuration has only been investigated for processing combinations of liquid‐liquid and liquid‐gas systems. These developmental studies into aerodynamically assisted jets (AAJ) have unearthed a versatile bio‐jetting approach referred to here as coaxial aerodynamically assisted bio‐jetting (CAABJ). In the current work, this flexible approach is demonstrated to handle two primary cell types for drop‐and‐placing onto several different substrates. Furthermore, the study assesses cellular viability of the post‐treated cells in comparison to controls by way of flow cytometry. These first steps demonstrate the promise this protocol has in exploring the creation of biologically viable structures to form encapsulations of cells which would be useful as a direct tissue engineering to the immuno‐hinding methodology in bio‐repair and therapeutics. Therefore, these investigations place CAABJ into the cell jetting pursuit together with bio‐electrosprays, which will undergo an explosive developmental research.     

Single-Molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.     

Bacterial extracellular electron transfer in bioelectrochemical systems

Bioelectrochemical systems (BES), typically microbial fuel cells (MFCs), have attracted increasing attention in the past decade due to their promising applications in many fields, such as bioremediation, energy generation and biosynthesis. Current-generating microorganisms play a key role in BES. The process of transferring electrons to electrode has been considered as a novel anaerobic bacteria respiration, and more and more bacteria capable of exchanging electrons with electrodes have been isolated. Among those bacteria, Shewanella and Geobacter genera are the most frequently used model organisms in the studies of BES, as well as the bacteria-electrode electron transfer mechanisms. Many significant new findings in the field of the bacterial extracellular electron transfer in BES have been reported recently. A better understanding of the mechanisms of bacterial extracellular electron transfer would provide more efficient strategies to enhance the applicability of BES. This review summarizes the recent advances of extracellular electron transfer mechanisms with foci on Shewanella and Geobacter species in BES.     

Electric field driven jetting: an emerging approach for processing living cells

This paper reports for the first time the ability to process living cellular materials by means of electrified jets at electric field strengths of up to 2 kV/mm. Bio-suspensions containing living human Jurkat cells at different concentrations were processed via this jetting approach. The jetting process was carried out at an electric field strength between 0.67 kV/mm and 2 kV/mm, corresponding to an applied voltage of 10-30 kV between two electrodes approximately 15 mm apart. The Jurkat cells were jetted under sterile conditions, collected in petri dishes and incubated for 24 and 48 hours. During and after incubation, cells were assessed for survival and structural damage; cells were found to be unharmed and to retain their integrity under all electric field strengths examined. At all field strengths jetting took place in the unstable mode. Good correlation was observed between droplet distribution plots generated by way of laser spectroscopy and estimated values from measurements of droplet relics.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Bronchial epithelial spheroids: an alternative culture model to investigate epithelium inflammation-mediated COPD

Background

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal lung inflammation that exceeds the protective response. Various culture models using epithelial cell lines or primary cells have been used to investigate the contribution of bronchial epithelium in the exaggerated inflammation of COPD. However, these models do not mimic in vivo situations for several reasons (e.g, transformed epithelial cells, protease-mediated dissociation of primary cells, etc.). To circumvent these concerns, we developed a new epithelial cell culture model.

Methods

Using non transformed non dissociated bronchial epithelium obtained by bronchial brushings from COPD and non-COPD smokers, we developed a 3-dimensional culture model, bronchial epithelial spheroids (BES). BES were analyzed by videomicroscopy, light microscopy, immunofluorescence, and transmission electron microscopy. We also compared the inflammatory responses of COPD and non-COPD BES. In our study, we chose to stimulate BES with lipopolycaccharide (LPS) and measured the release of the pro-inflammatory mediators interleukin-8 (IL-8) and leukotriene B4 (LTB4) and the anti-inflammatory mediator prostaglandin E2 (PGE2).

Results

BES obtained from both COPD and non-COPD patients were characterized by a polarized bronchial epithelium with tight junctions and ciliary beating, composed of basal cells, secretory cells and ciliated cells. The ciliary beat frequency of ciliated cells was not significantly different between the two groups. Of interest, BES retained their characteristic features in culture up to 8 days. BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS. Interestingly, LPS induced a higher release of IL-8, but not PGE2 and LTB4 in COPD BES (p < 0.001) which correlated with lung function changes.

Conclusion

This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium. More importantly, BES represent an attractive culture model to investigate the mechanisms of injuring agents that mediate epithelial cell inflammation and its contribution to COPD pathogenesis.     

Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition

Background

The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron.

Methods

These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays.

Results

This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized.

Conclusions

Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis.

General significance

Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer.     

Information storing by biomagnetites

Since the discovery of the presence of biogenic magnetites in living organisms, there have been speculations on the role that these biomagnetites play in cellular processes. It seems that the formation of biomagnetite crystals is a universal phenomenon and not an exception in living cells. Many experimental facts show that features of organic and inorganic processes could be indistinguishable at nanoscale levels. Living cells are quantum “devices” rather than simple electronic devices utilizing only the charge of conduction electrons. In our opinion, due to their unusual biophysical properties, special biomagnetites must have a biological function in living cells in general and in the brain in particular. In this paper, we advance a hypothesis that while biomagnetites are developed jointly with organic molecules and cellular electromagnetic fields in cells, they can record information about the Earth’s magnetic vector potential of the entire flight in migratory birds.     

An in vitro approach to the study of target organ toxicity of drugs and chemicals

Summary A major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.     

Cell electrospinning: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds

Jet-based technologies are increasingly being explored as potential high-throughput and high-resolution methods for the manipulation of biological materials. Previously shown to be of use in generating scaffolds from biocompatible materials, we were interested to explore the possibility of using electrospinning technology for the generation of scaffolds comprised of living cells. For this, it was necessary to identify appropriate parameters under which viable threads containing living cells could be produced. Here, we describe a method of electrospinning that can be used to deposit active biological threads and scaffolds. This has been achieved by use of a coaxial needle arrangement where a concentrated living biosuspension flows through the inner needle and a medical-grade poly(dimethylsiloxane) (PDMS) medium with high viscosity (12,500 mPa s) and low electrical conductivity (10-15 S m-1) flows through the outer needle. Using this technique, we have identified the operational conditions under which the finest cell-bearing composite microthreads are formed. Collected cells that have been cultured, postelectrospinning, have been viable and show no evidence of having incurred any cellular damage during the bionanofabrication process. This study demonstrates the feasibility of using coaxial electrospinning technology for biological and biomedical applications requiring the deposition of living cells as composite microthreads for forming active biological scaffolds.     

Cellular MR imaging

Cellular MR imaging is a young field that aims to visualize targeted cells in living organisms. In order to provide a different signal intensity of the targeted cell, they are either labeled with MR contrast agents in vivo or prelabeled in vitro. Either (ultrasmall) superparamagnetic iron oxide [(U)SPIO] particles or (polymeric) paramagnetic chelates can be used for this purpose. For in vivo cellular labeling, Gd3+- and Mn2+-chelates have mainly been used for targeted hepatobiliary imaging, and (U)SPIO-based cellular imaging has been focused on imaging of macrophage activity. Several of these magnetopharmaceuticals have been FDA-approved or are in late-phase clinical trials. As for prelabeling of cells in vitro, a challenge has been to induce a sufficient uptake of contrast agents into nonphagocytic cells, without affecting normal cellular function. It appears that this issue has now largely been resolved, leading to an active research on monitoring the cellular biodistribution in vivo following transplantation or transfusion of these cells, including cell migration and trafficking. New applications of cellular MR imaging will be directed, for instance, towards our understanding of hematopoietic (immune) cell trafficking and of novel guided (stem) cell-based therapies aimed to be translated to the clinic in the future.     

Biology of size and gravity]

Gravity is a force that acts on mass. Biological effects of gravity and their magnitude depend on scale of mass and difference in density. One significant contribution of space biology is confirmation of direct action of gravity even at the cellular level. Since cell is the elementary unit of life, existence of primary effects of gravity on cells leads to establish the firm basis of gravitational biology. However, gravity is not limited to produce its biological effects on molecules and their reaction networks that compose living cells. Biological system has hierarchical structure with layers of organism, group, and ecological system, which emerge from the system one layer down. Influence of gravity is higher at larger mass. In addition to this, actions of gravity in each layer are caused by process and mechanism that is subjected and different in each layer of the hierarchy. Because of this feature, summing up gravitational action on cells does not explain gravity for biological system at upper layers. Gravity at ecological system or organismal level can not reduced to cellular mechanism. Size of cells and organisms is one of fundamental characters of them and a determinant in their design of form and function. Size closely relates to other physical quantities, such as mass, volume, and surface area. Gravity produces weight of mass. Organisms are required to equip components to support weight and to resist against force that arise at movement of body or a part of it. Volume and surface area associate with mass and heat transport process at body. Gravity dominates those processes by inducing natural convection around organisms. This review covers various elements and process, with which gravity make influence on living systems, chosen on the basis of biology of size. Cells and biochemical networks are under the control of organism to integrate a consolidated form. How cells adjust metabolic rate to meet to the size of the composed organism, whether is gravity responsible for this feature, are subject we discuss in this article. Three major topics in gravitational and space biology are; how living systems have been adapted to terrestrial gravity and evolved, how living systems respond to exotic gravitational environment, and whether living systems could respond and adapt to microgravity. Biology of size can contribute to find a way to answer these question, and answer why gravity is important in biology, at explaining why gravity has been a dominant factor through the evolutional history on the earth.     

Modulation of channel activity and gadolinium block of MscL by static magnetic fields

The magnetic field of the Earth has for long been known to influence the behaviour and orientation of a variety of living organisms. Experimental studies of the magnetic sense have, however, been impaired by the lack of a plausible cellular and/or molecular mechanism providing meaningful explanation for detection of magnetic fields by these organisms. Recently, mechanosensitive (MS) ion channels have been implied to play a role in magnetoreception. In this study we have investigated the effect of static magnetic fields (SMFs) of moderate intensity on the activity and gadolinium block of MscL, the bacterial MS channel of large conductance, which has served as a model channel to study the basic physical principles of mechanosensory transduction in living cells. In addition to showing that direct application of the magnetic field decreased the activity of the MscL channel, our study demonstrates for the first time that SMFs can reverse the effect of gadolinium, a well-known blocker of MS channels. The results of our study are consistent with a notion that (1) the effects of SMFs on the MscL channels may result from changes in physical properties of the lipid bilayer due to diamagnetic anisotropy of phospholipid molecules and consequently (2) cooperative superdiamagnetism of phospholipid molecules under influence of SMFs could cause displacement of Gd³⁺ ions from the membrane bilayer and thus remove the MscL channel block.     

In vitro distinction between primary and secondary cellular response to alloantigens by means of a subcellular antigenic preparation]

Living spleen cells and a subcellular product of spleen cells have been compared, in mice, for their ability to elicit, for a long term culture (4-5 days), a cellular cytotoxic response against alloantigens. Contrary to living spleen cells which could induce the same high level of cytotoxic activity in a primary or in a secondary response, the antigenic preparation was only able to mount a very low primary response while it could render highly cytotoxic a lymphoid population proved to be able to mount in vitro an anamnestic response.     

Defining Life: The Virus Viewpoint

Are viruses alive? Until very recently, answering this question was often negative and viruses were not considered in discussions on the origin and definition of life. This situation is rapidly changing, following several discoveries that have modified our vision of viruses. It has been recognized that viruses have played (and still play) a major innovative role in the evolution of cellular organisms. New definitions of viruses have been proposed and their position in the universal tree of life is actively discussed. Viruses are no more confused with their virions, but can be viewed as complex living entities that transform the infected cell into a novel organism—the virus—producing virions. I suggest here to define life (an historical process) as the mode of existence of ribosome encoding organisms (cells) and capsid encoding organisms (viruses) and their ancestors. I propose to define an organism as an ensemble of integrated organs (molecular or cellular) producing individuals evolving through natural selection. The origin of life on our planet would correspond to the establishment of the first organism corresponding to this definition.     

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50–100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.     

Gene expression studies on bio-electrosprayed primary cardiac myocytes

Cardiovascular pathology accounts for the greatest number of mortalities in the western world and thus the development of ex vivo cardiac tissue has vast potential in cardiac therapy. Bio-electrosprays (BES), a recently discovered direct cell engineering protocol, has demonstrated tremendous applicability for regenerative and therapeutic medicine. For bio-electrospraying to be carried forward as a novel method of cardiac tissue engineering, it is important that the process does not adversely affect cellular physiology. Our previous work has shown that bio-electrospraying does not induce cell death, activate intracellular stress pathways or induce DNA damage in primary cardiac myocytes. Here we show for the first time using genome-wide microarray analysis, that bio-electrospraying has no negative effects on global gene expression in cardiac myocytes. Moreover, we show that bio-electrospraying does not lead to endothelial cell activation. These data suggest that BES has minimal effect upon the physiology of cardiac myocytes and endothelial cells and thus paves the way for the development of BES in cardiac tissue engineering.