Bio-electrosprays: a novel electrified jetting methodology for the safe handling and deployment of primary living organisms

Electrohydrodynamic jetting (EHDJ) which is also known as electrosprays (ES) has recently been elucidated as a unique electrified biotechnique for the safe handling and deployment of living organisms. This high intensity electric field driven jetting methodology is now referred to as "bioelectrosprays" (BES). Previously these charged jets have only been shown to jet-process immortalized cells which have undergone expected cellular behavior when compared with control cells. In this paper we demonstrate the ability to jet process primary living organisms in the stable conejetting mode. Finally the viability of the bio-electrosprayed living organisms has been assessed employing a flow cytometry approach which forms the discussion in this paper. Our findings further establish BES as a competing biotechnique, which could be employed for the deposition of primary living organisms according to a predetermined active cellular architecture. One day this could be used for the fabrication of viable tissues and organs for repair or replacement. These advanced studies carried out on BES have direct widespread applications ranging from developmental biology to regenerative and therapeutic medicine, which are a few amongst several other areas of study within the life sciences.     

A mutant alphaII-spectrin designed to resist calpain and caspase cleavage questions the functional importance of this process in vivo

alpha- and beta-spectrins are components of molecular scaffolds located under the lipid bilayer and named membrane skeletons. Disruption of these scaffolds through mutations in spectrins demonstrated that they are involved in the membrane localization or the maintenance of proteins associated with them. The ubiquitous alphaII-spectrin chain bears in its central region a unique domain that is sensitive to several proteases such as calpains or caspases. The conservation of this region in vertebrates suggests that the proteolysis of alphaII-spectrin by these enzymes could be involved in important functions. To assess the role of alphaII-spectrin cleavage in vivo, we generated a murine model in which the exons encoding the region defining this cleavage sensitivity were disrupted by gene targeting. Surprisingly, homozygous mice expressing this mutant alphaII-spectrin appeared healthy, bred normally, and had no histological anomaly. Remarkably, the mutant alphaII-spectrin assembles correctly into the membrane skeleton, thus challenging the notion that this region is required for the stable biogenesis of the membrane skeleton in nonerythroid cells. Our finding also argues against a critical role of this particular alphaII-spectrin cleavage in either major cellular functions or in normal development.     

Non-immunoglobulin based protein scaffolds

Non-immunoglobulin based protein scaffolds have been reported as promising alternatives to traditional monoclonal antibodies for over a decade and are often mentioned as part of the next-generation immunotherapeutics. Today, this class of biologics is beginning to demonstrate its potential for therapeutic applications and several are currently in preclinical or clinical development. A common denominator for most of these new scaffolds is the attractive properties that differentiate them from monoclonal antibodies including small size, cysteine-free sequence, flexible pharmacokinetic properties, and ease of generating multispecific molecules. In addition to therapeutic applications, substantial evidence point to superior performance of several of these scaffolds in molecular imaging compared to full-length antibodies. Here we review the most recent progress using alternative protein scaffolds for therapy and medical imaging.     

Synthesis,Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.     

Progress in the use of dental pulp stem cells in regenerative medicine

The field of tissue engineering is emerging as a multidisciplinary area with promising potential for regenerating new tissues and organs. This approach requires the involvement of three essential components: stem cells, scaffolds and growth factors. To date, dental pulp stem cells have received special attention because they represent a readily accessible source of stem cells. Their high plasticity and multipotential capacity to differentiate into a large array of tissues can be explained by its neural crest origin, which supports applications beyond the scope of oral tissues. Many isolation, culture and cryopreservation protocols have been proposed that are known to affect cell phenotype, proliferation rate and differentiation capacity. The clinical applications of therapies based on dental pulp stem cells demand the development of new biomaterials suitable for regenerative purposes that can act as scaffolds to handle, carry and implant stem cells into patients. Currently, the development of xeno-free culture media is emerging as a means of standardization to improve safe and reproducibility. The present review aims to describe the current knowledge of dental pulp stem cells, considering in depth the key aspects related to the characterization, establishment, maintenance and cryopreservation of primary cultures and their involvement in the multilineage differentiation potential. The main clinical applications for these stem cells and their combination with several biomaterials is also covered.     

Vascular biomaterials: from biomedical engineering to tissue engineering

Biomaterials are already widely used in medical sciences. The field of biomaterials began to shift to produce materials able to stimulate specific cellular responses at the molecular level. The combined efforts of cell biologists, engineers, materials scientists, mathematicians, geneticists, and clinicians are now used in tissue engineering to restore, maintain, or improve tissue functions or organs. This rapidly expanding approach combines the fields of material sciences and cell biology for the molecular design of polymeric scaffolds with appropriate 3D configuration and biological responses. Future developments for new blood vessels will require improvements in technology of materials and biotechnology together with the increased knowledge of the interactions between materials, blood, and living tissues. Biomaterials represent a crucial mainstay for all these studies.     

Aqueous polymer two-phase systems: effective tools for plasma membrane proteomics

Plasma membranes (PMs) are of particular importance for all living cells. They form a selectively permeable barrier to the environment. Many essential tasks of PMs are carried out by their proteinaceous components, including molecular transport, cell-cell interactions, and signal transduction. Due to the key role of these proteins for cellular function, they take center-stage in basic and applied research. A major problem towards in-depth identification and characterization of PM proteins by modern proteomic approaches is their low abundance and immense heterogeneity in different cells. Highly selective and efficient purification protocols are hence essential to any PM proteome analysis. An effective tool for preparative isolation of PMs is partitioning in aqueous polymer two-phase systems. In two-phase systems, membranes are separated according to differences in surface properties rather than size and density. Despite their rare application to the fractionation of animal tissues and cells, they represent an attractive alternative to conventional fractionation protocols. Here, we review the principles of partitioning using aqueous polymer two-phase systems and compare aqueous polymer two-phase systems with other methods currently used for the isolation of PMs.     

A brief review of extrusion‐based tissue scaffold bio‐printing

Extrusion‐based bio‐printing has great potential as a technique for manipulating biomaterials and living cells to create three‐dimensional (3D) scaffolds for damaged tissue repair and function restoration. Over the last two decades, advances in both engineering techniques and life sciences have evolved extrusion‐based bio‐printing from a simple technique to one able to create diverse tissue scaffolds from a wide range of biomaterials and cell types. However, the complexities associated with synthesis of materials for bio‐printing and manipulation of multiple materials and cells in bio‐printing pose many challenges for scaffold fabrication. This paper presents an overview of extrusion‐based bio‐printing for scaffold fabrication, focusing on the prior‐printing considerations (such as scaffold design and materials/cell synthesis), working principles, comparison to other techniques, and to‐date achievements. This paper also briefly reviews the recent development of strategies with regard to hydrogel synthesis, multi‐materials/cells manipulation, and process‐induced cell damage in extrusion‐based bio‐printing. The key issue and challenges for extrusion‐based bio‐printing are also identified and discussed along with recommendations for future, aimed at developing novel biomaterials and bio‐printing systems, creating patterned vascular networks within scaffolds, and preserving the cell viability and functions in scaffold bio‐printing. The address of these challenges will significantly enhance the capability of extrusion‐based bio‐printing.     

Next generation organoids for biomedical research and applications

Organoids are in vitro cultures of miniature fetal or adult organ-like structures. Their potentials for use in tissue and organ replacement, disease modeling, toxicology studies, and drug discovery are tremendous. Currently, major challenges facing human organoid technology include (i) improving the range of cellular heterogeneity for a particular organoid system, (ii) mimicking the native micro- and matrix-environment encountered by cells within organoids, and (iii) developing robust protocols for the in vitro maturation of organoids that remain mostly fetal-like in cultures. To tackle these challenges, we advocate the principle of reverse engineering that replicates the inner workings of in vivo systems with the goal of achieving functionality and maturation of the resulting organoid structures with the input of minimal intrinsic (cellular) and environmental (matrix and niche) constituents. Here, we present an overview of organoid technology development in several systems that employ cell materials derived from fetal and adult tissues and pluripotent stem cell cultures. We focus on key studies that exploit the self-organizing property of embryonic progenitors and the role of designer matrices and cell-free scaffolds in assisting organoid formation. We further explore the relationship between adult stem cells, niche factors, and other current developments that aim to enhance robust organoid maturation. From these works, we propose a standardized pipeline for the development of future protocols that would help generate more physiologically relevant human organoids for various biomedical applications.     

Can we predict biological activity of antimicrobial peptides from their interactions with model phospholipid membranes?

Cationic antibacterial peptides are produced in all living organisms and possess either selective activity toward a certain type of cell or microorganism, or a broad spectrum of activity toward several types of cells including prokaryotic and mammalian cells. In order to exert their activity, peptides first interact with and traverse an outer barrier, e.g., mainly LPS and peptidoglycan in bacteria or a glycocalix layer and matrix proteins in mammalian cells. Only then, can the peptides bind and insert into the cytoplasmic membrane. The mode of action of many antibacterial peptides is believed to be the disruption of the lipidic plasma membrane. Therefore, model phospholipid membranes have been used to study the mode of action of antimicrobial peptides. These studies have demonstrated that peptides that act preferentially on bacteria are also able to interact with and permeate efficiently anionic phospholipids, whereas peptides that lyse mammalian cells bind and permeate efficiently both acidic and zwitterionic phospholipids membranes, mimicking the plasma membranes of these cells. It is now becoming increasingly clear that selective activity of these peptides against different cells depends also on other parameters that characterize both the peptide and the target cell. With respect to the peptide's properties, these include the volume of the molecule, its structure, and its oligomeric state in solution and in membranes. Regarding the target membrane, these include the structure, length, and complexity of the hydrophilic polysaccharide found in its outer layer. These parameters affect the ability of the peptides to diffuse through the cell's outer barrier and to reach its cytoplasmic plasma membrane.     

Recombinant viruses as tools to induce protective cellular immunity against infectious diseases.

Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.     

Piezoelectric poly(vinylidene fluoride) microstructure and poling state in active tissue engineering

Tissue engineering strategies rely on suitable membranes and scaffolds, providing the necessary physicochemical stimuli to specific cells. This review summarizes the main results on piezoelectric polymers, in particular poly(vinylidene fluoride), for muscle and bone cell culture. Further, the relevance of polymer microstructure and surface charge on cell response is demonstrated. Together with the necessary biochemical cues, the proper design of piezoelectric polymers can open the way to novel and more reliable tissue engineering strategies for cells in which electromechanical stimuli are present in their environment.     

Modeling Huntington disease in yeast: Perspectives and future directions

Yeast have been extensively used to model aspects of protein folding diseases, yielding novel mechanistic insights and identifying promising candidate therapeutic targets. In particular, the neurodegenerative disorder Huntington disease (HD), which is caused by the abnormal expansion of a polyglutamine tract in the huntingtin (htt) protein, has been widely studied in yeast. This work has led to the identification of several promising therapeutic targets and compounds that have been validated in mammalian cells, Drosophila and rodent models of HD. Here we discuss the development of yeast models of mutant htt toxicity and misfolding, as well as the mechanistic insights gleaned from this simple model. The role of yeast prions in the toxicity/misfolding of mutant htt is also highlighted. Furthermore, we provide an overview of the application of HD yeast models in both genetic and chemical screens, and the fruitful results obtained from these approaches. Finally, we discuss the future of yeast in neurodegenerative research, in the context of HD and other diseases.Key words: Huntington disease, yeast, neurodegeneration, genetic modifiers, prionsThe single-celled eukaryote Saccharomyces cerevisiae has long been involved with the technological advancement of mankind. Commonly known as baker''s yeast, for millennia this organism has been employed for the requisite fermentation in the production of bread, wine, beer and other food products.¹ Louis Pasteur first described the critical role of yeast in fermentation in 1860, and conclusively showed that living yeast cells are required for this process.² Since this time, yeast have been used extensively in biological sciences to explore the fundamental properties of the cell, and have become a vital genetic weapon in the arsenal of modern day medical scientists. This review provides an overview of the development, characterization and utilization of yeast models of Huntington disease (HD). These simple models have provided striking insights into the mechanisms underlying cellular toxicity in this disease, and have also uncovered many promising candidate drug targets for HD, several of which have been validated in animal models and hold great therapeutic promise.     

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.     

Coupling spatial segregation with synthetic circuits to control bacterial survival

Engineered bacteria have great potential for medical and environmental applications. Fulfilling this potential requires controllability over engineered behaviors and scalability of the engineered systems. Here, we present a platform technology, microbial swarmbot, which employs spatial arrangement to control the growth dynamics of engineered bacteria. As a proof of principle, we demonstrated a safeguard strategy to prevent unintended bacterial proliferation. In particular, we adopted several synthetic gene circuits to program collective survival in Escherichia coli: the engineered bacteria could only survive when present at sufficiently high population densities. When encapsulated by permeable membranes, these bacteria can sense the local environment and respond accordingly. The cells inside the microbial swarmbot capsules will survive due to their high densities. Those escaping from a capsule, however, will be killed due to a decrease in their densities. We demonstrate that this design concept is modular and readily generalizable. Our work lays the foundation for engineering integrated and programmable control of hybrid biological–material systems for diverse applications.     

Toward minimal bacterial cells: evolution vs. design

Recent technical and conceptual advances in the biological sciences opened the possibility of the construction of newly designed cells. In this paper we review the state of the art of cell engineering in the context of genome research, paying particular attention to what we can learn on naturally reduced genomes from either symbiotic or free living bacteria. Different minimal hypothetically viable cells can be defined on the basis of several computational and experimental approaches. Projects aiming at simplifying living cells converge with efforts to make synthetic genomes for minimal cells. The panorama of this particular view of synthetic biology lead us to consider the use of defined minimal cells to be applied in biomedical, bioremediation, or bioenergy application by taking advantage of existing naturally minimized cells.     

Single-molecule recognition force spectroscopy of transmembrane transporters on living cells

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.     

Novel physical chemistry approaches in biophysical researches with advanced application of lasers: Detection and manipulation

Novel methodologies utilizing pulsed or intense CW irradiation obtained from lasers have a major impact on biological sciences. In this article, recent development in biophysical researches fully utilizing the laser irradiation is described for three topics, time-resolved fluorescence spectroscopy, time-resolved thermodynamics, and manipulation of the biological assemblies by intense laser irradiation. First, experimental techniques for time-resolved fluorescence spectroscopy are concisely explained in Section 2. As an example of the recent application of time-resolved fluorescence spectroscopy to biological systems, evaluation of the viscosity of lipid bilayer membranes is described. The results of the spectroscopic experiments strongly suggest the presence of heterogeneous membrane structure with two different viscosity values in liposomes formed by a single phospholipid. Section 3 covers the time-resolved thermodynamics. Thermodynamical properties are important to characterize biomolecules. However, measurement of these quantities for short-lived intermediate species has been impossible by traditional thermodynamical techniques. Recently, development of a spectroscopic method based on the transient grating method enables us to measure these quantities and also to elucidate reaction kinetics which cannot be detected by other spectroscopic methods. The principle of the measurements and applications to some protein reactions are reviewed. Manipulation and fabrication of supramolecues, amino acids, proteins, and living cells by intense laser irradiation are described in Section 4. Unconventional assembly, crystallization and growth, amyloid fibril formation, and living cell manipulation are achieved by CW laser trapping and femtosecond laser-induced cavitation bubbling. Their spatio-temporal controllability is opening a new avenue in the relevant molecular and bioscience research fields. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato.     

From inflammation to pain: experimental gene therapy

Chronic pain is frequently associated with profound alterations of neuronal systems involved in pain processing and should be considered as a real disease state of the nervous system. Unfortunately, some forms of chronic pain remain difficult to be satisfactorily treated. In the search for new therapeutic strategies, the gene-based approaches are of potential interest as they offer the possibility to introduce a therapeutic protein into some relevant structures and to drive its continuous production in the near vicinity of targeted cells. Recently, these techniques have been experimented in several animal models of chronic pain, showing that transfer at the spinal level of some genes, in particular those of opioid precursors proopiomelanocortin or proenkephalin A, leading to the overproduction of products that they encode, attenuated persistent pain of both inflammatory and neuropathic origin. Thus, in polyarthritic rat, a model of chronic inflammatory pain, we demonstrated that herpes simplex virus vector mediated overexpression of proenkephalin A in primary sensory neurons at the lumbar level elicited both antihyperalgesic and anti-inflammatory activities. Apart from opioids, numerous other molecules involved in pain processing are of potential therapeutic interest for gene-based protocols. For instance, targeting some molecules involved in pain induction and perpetuation, such as proinflammatory cytokines, raises an interesting possibility to block the "development" of pain. The clinical application of these approaches remains to be established, and, presently, one of the main problems to be solved is the innocuity of virus-derived vectors. However, the experimental use of gene-based techniques might be particularly useful for the evaluation of the therapeutic interest of some recently identified molecules involved in pain processing and might finally lead to the development of new "classical" pharmacological tools.     

The collagen type XVIII endostatin domain is co-localized with perlecan in basement membranes in vivo.

The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo.