Orwik

Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues.

Bio-electrospraying (BES) has demonstrated great promise as a rapidly evolving strategy for tissue engineering and regenerative biology/medicine. Since its discovery in 2005, many studies have confirmed that cells (immortalized, primary and stem cell... expand abstracts) and whole organisms (Danio rerio, Xenopus tropicalis, Caenorhabditis elegans to Drosophila) remain viable post-bio-electrospraying. Although this bio-protocol has achieved much, it suffers from one crucial problem, namely the ability to precisely control the number of cells within droplets and or encapsulations. If overcome, BES has the potential to become a high-efficiency biotechnique for controlled cell encapsulation, a technique most useful for a wide range of applications in biology and medicine ranging from the forming of three-dimensional cultures to an approach for treating diseases such as type I diabetes. In this communication, we address this issue by demonstrating the coupling of BES with droplet-based microfluidics for controlling live cell numbers within droplets and residues. collapse abstract

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning dem... expand abstractonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine. collapse abstract

Development and fertility studies on post-bio-electrosprayed Drosophila melanogaster embryos.

Bio-electrosprays (BESs) provide a means of precisely manipulating cells and thus have the potential for many clinical uses such as the generation of artificial tissuesorgans. Previously we tested the biological safety of this technology with a varie... expand abstractty of living cells and also embryos from the vertebrate model organisms Danio rerio (zebrafish) and Xenopus tropicalis (frog). However, the viability and fertility of the treated embryos could not be fully assessed due to animal licensing laws. Here we assay the viability and fertility of Drosophila melanogaster (fruit fly) embryos in conjunction with the bio-electrospray procedure. Bio-electrosprayed Drosophila embryos developed into fully fertile adult flies that were indistinguishable from wild-type. Thus, we demonstrate that the bio-electrospray procedure does not induce genetic or physical damage that significantly affects the development or fertility of a multicellular organism. This study along with our previous investigations demonstrates the potential of this approach to be developed for the precise manipulation of sensitive biological materials. collapse abstract

The differentiation and engraftment potential of mouse hematopoietic stem cells is maintained after bio-electrospray.

The bio-electrospray technique has been recently pioneered to manipulate living, immortalised and primary cells, including a wide range of stem cells. Studies have demonstrated that the creation of viable, fully functional in vitro microenvironments ... expand abstractis possible using this technique. By modifying the bio-electrospray procedure (referred to as cell electrospinning), a variety of microenvironment morphologies have been fabricated. Because bio-electrospraying of biological material is a relatively new technique, it is important to determine if there are any unwanted consequences to the manipulated cells as a result of the procedure. Here, we establish the validity of the process using a heterogeneous, living population of hematopoietic stem/progenitor cells, using a functional in vitro assay and in vivo mouse model to investigate for side-effects that previous in vitro assays may not have detected. Our studies demonstrate that these bio-protocols have no obvious negative effects, thus indicating significant promise for utility in biological sciences and for a plethora of healthcare applications. collapse abstract

Bio-electrospraying whole human blood: analysing cellular viability at a molecular level.

Bio-electrosprays, pioneered in 2005, have undergone several developmental studies which have seen this technique evolve as a novel direct in vivo tissue engineering and regenerative medicinal strategy. Those studies have been a hallmark for electros... expand abstractprays; however, in this communication we report our on-going developmental investigations for exploring bio-electrosprays as a potential medical device and diagnostic protocol. The studies reported here demonstrate the ability to directly jet whole human blood without affecting the genetic make-up, which has been interrogated by way of reverse transcription-polymerase chain reaction (RT-PCR) in comparison to controls (p = 0.7337). These studies demonstrate bio-electrosprays as a possible diagnostic protocol. collapse abstract

Bio-electrospraying the nematode Caenorhabditis elegans: studying whole-genome transcriptional responses and key life cycle parameters.

Bio-electrospray, the direct jet-based cell handling approach, is able to handle a wide range of cells (spanning immortalized, primary to stem cells). Studies at the genomic, genetic and the physiological levels have shown that, post-treatment, cellu... expand abstractlar integrity is unperturbed and a high percentage (more than 70%, compared with control) of cells remain viable. Although, these results are impressive, it may be argued that cell-based systems are oversimplistic. Therefore, it is important to evaluate the bio-electrospray technology using sensitive and dynamically developing multi-cellular organisms that share, at least some, similarities with multi-cell microenvironments encountered with tissues and organs. This study addressed this issue by using a well-characterized model organism, the non-parasitic nematode Caenorhabditis elegans. Nematode cultures were subjected to bio-electrospraying and compared with positive (heat shock) and negative controls (appropriate laboratory culture controls). Overall, bio-electrospraying did not modulate the reproductive output or induce significant changes in in vivo stress-responsive biomarkers (heat shock proteins). Likewise, whole-genome transcriptomics could not identify any biological processes, cellular components or molecular functions (gene ontology terms) that were significantly enriched in response to bio-electrospraying. This demonstrates that bio-electrosprays can be safely applied directly to nematodes and underlines its potential future use in the creation of multi-cellular environments within clinical applications. collapse abstract

The role of surface wettability and surface charge of electrosprayed nanoapatites on the behaviour of osteoblasts.

A new deposition method is presented, based on electrospraying, that can build bioceramic structures with desirable surface properties. This technology allows nanoapatite crystals, including hydroxyapatite (nHA), carbonate-substituted HA (nCHA) and s... expand abstractilicon-substituted HA (nSiHA), to be electrosprayed on glass substrates. Human osteoblast cells cultured on nSiHA showed enhanced cell attachment, proliferation and protein expression, namely alkaline phosphatase, type 1 collagen and osteocalcin, as compared to nHA and nCHA. The modification of nanoapatite by the addition of silicon into the HA lattice structure renders the electrosprayed surface more hydrophilic and electronegatively charged. collapse abstract

Direct jetting approaches for handling stem cells.

Cell handling by means of jets has recently been highlighted as having significant implications for tissue engineering and regenerative medicine. Bio-electrosprays and aerodynamically assisted bio-jetting, two recently discovered direct cell jetting ... expand abstractmethods, have undergone extensive developmental studies which have seen these techniques have many implications for the life sciences. In our previous investigations both these techniques have only been explored for the direct handling of primary living cells, which have demonstrated great promise. However, stem cells play a critical role in tissue engineering and regenerative medicine and hence these jetting protocols must be applied to stem cells if these approaches are to be employed for wider applications in both biology and medicine. Thus, the investigations reported herein, which are the first of their kind, elucidate the ability to explore these jetting methodologies for safe handling of stem cells. Our studies report cellular viability on several controls in comparison to those post-jetted stem cells over a 72 h time frame. In addition, we have explored flow cytometry and apoptosis assays, further providing evidence that those stem cells handled by means of either bio-electrosprays or aerodynamically assisted bio-jetting have not incurred any gross cellular damage. These pilot studies provide the much needed proof-of-principle for these techniques to progress for their exploration as an advanced strategy in tissue engineering and regenerative medicine. collapse abstract

Bio-electrospraying living Xenopus tropicalis embryos: investigating the structural, functional and biological integrity of a model organism.

Bio-electrosprays, a recently pioneered direct cell engineering approach, have been demonstrated to handle living cells including stem cells for the development of active specialized and unspecialized microenvironments. This electric field driven tec... expand abstracthnique is currently undergoing vigorous development where the technique is racing towards possible clinical utility. Although this direct cell engineering approach has been elucidated to have no significant effects on the processed cells from a molecular level upwards, the technique needs to demonstrate its potential for use with whole organisms (multi-cellular systems). We believe this is mandatory for whole organisms such as model embryos; developing multi-cellular biological structures are sensitive systems and could possibly be prone to a wide range of embryological disruptions during their dynamic development, post-treatment. Therefore our studies presented herein have investigated the effects on embryos in terms of their structure, function and biological integrity post-bio-electrospraying in comparison to several controls. Our investigations demonstrate the absence of any detectable gross effects on the embryos from a genetic level upwards on post-treated embryos. In fact, these studies clearly elucidate no significant disruptions on the dynamic development of these treated embryos in comparison to those respective controls, thus validating the utility of bio-electrosprays for the careful handling of dynamically developing multi-cellular organisms. collapse abstract

Bio-electrospraying embryonic stem cells: interrogating cellular viability and pluripotency.

Bio-electrospraying, a recently discovered, direct electric field driven cell engineering process, has been demonstrated to have no harmful effects on treated cells at a molecular level. Although several cell types from both immortalized and primary ... expand abstractcultures have been assessed post-treatment as a function of time in comparison to controls, the protocol has yet to be applied on embryonic stem cells. This is most important if bio-electrosprays are to further their applicability, in particular with regard to tissue engineering and regenerative medicine, where embryonic stem cells play a fundamental role. In the study presented herein the chosen stem cells are mouse embryonic stem (ES) cells. Hence, these first examples where embryonic stem cells have been jetted by way of bio-electrosprays, demonstrate the cellular viability and the cell's pluripotency indistinguishable when comparing those post-treated cells with their respective controls. collapse abstract

Aerodynamically assisted jetting and threading for processing concentrated suspensions containing advanced structural, functional and biological materials.

In recent years material sciences have been interpreted right across the physical and the life sciences. Essentially this discipline broadly addresses the materials, processing, and/or fabrication right up to the structure. The materials and structur... expand abstractes areas can range from the micro- to the nanometre scale and, in a materials sense, span from the structural, functional to the most complex, namely biological (living cells). It is generally recognised that the processing or fabrication is fundamental in bridging the materials with their structures. In a global perspective, processing has not only contributed to the materials sciences but its very nature has bridged the physical with the life sciences. In this review we discuss one such swiftly emerging fabrication approach having a plethora of applications spanning the physical and life sciences. collapse abstract

Genomic, genetic and physiological effects of bio-electrospraying on live cells of the model yeast Saccharomyces cerevisiae.

The ability to directly engineer living cells is rapidly becoming a hot field of research for a wide range of applications within the life sciences. 'Bio-electrospraying' cells, a recently developed technique, has great potential in this area. In thi... expand abstracts paper, we quantify genetic, genomic and physiological effects of bio-electrospraying cells of a model eukaryote, the yeast Saccharomyces cerevisiae. Our results demonstrate that yeast cells bio-electrosprayed at 30 kV have not incurred any detectable damage at a genomic or genetic level, and that the detectable physiological stress of the procedure is negligible. These results support our proposal to use yeast as a model system to develop bio-electrospray devices and protocols. collapse abstract

Advanced processing routes for tissue engineering and regenerative medicine.

Cell engineering: spearheading the next generation in healthcare.

Manipulating living mammalian cells present fascinating possibilities for a plethora of applications within our healthcare. These imply several possibilities in tissue engineering and regenerative medicine, to those of a therapeutic nature. The physi... expand abstractcal sciences are increasingly playing a pivotal role in this endeavour by both advancing existing cell engineering technology and pioneering new protocols for the creation of biologically viable structures. In this paper, the author introduces several direct needle/channel/orifice-based cell engineering protocols, currently undergoing intense investigation for a whole host of bio-applications. Hence, each protocol's advantages and disadvantages are clearly identified, whilst recognizing their future biological and engineering challenges. In conclusion, a few selected biotechnological applications present possibilities where these protocols could undergo focused exploration. Successful development of these bio-protocols sees the emergence of unique future strategies within a clinical environment having far-reaching consequences for our healthcare. collapse abstract

Genetic, genomic and physiological state studies on single-needle bio-electrosprayed human cells.

Bio-electrospraying, a non-contact jet-based direct cell engineering approach, was recently pioneered and demonstrated for handling a wide range of primary living cells. In those studies, post-treated cells were biologically assessed in comparison to... expand abstract several controls by way of flow cytometry. Although flow cytometry accurately assesses those viable populations of cells, subtle effects at a sub-cellular level could have been missed. Therefore, in the present study we demonstrate metaphase chromosome breakage studies carried out on single-needle bio-electrosprayed human T-lymphocytes, which are compared with several controls. The results indicate that post-treated T-lymphocytes do not demonstrate any increase in chromosome damage in comparison to control cells. These studies further validate bio-electrospraying as a technique with potential for clinical utility. collapse abstract

Pilot study to investigate the possibility of cytogenetic and physiological changes in bio-electrosprayed human lymphocyte cells.

BACKGROUND: We recently pioneered the ability to directly electrospray and electrospin living cells without compromising their viability. These protocols, now referred to as 'bio-electrosprays' and 'cell electrospinning', are rapidly emerging bio-tec... expand abstracthniques with a plethora of promising applications within the life sciences, in particular to regenerative and therapeutic medicine. Our studies to date, with both bio-electrosprays and cell electrospinning, have demonstrated that a large population of viable cells exist post-treatment, in comparison to controls over both short and long periods as assessed by flow cytometry. METHODS: Post-treated mammalian cells are investigated in comparison to controls (culture and needle controls) at a cytogenetic and physiological level. In particular, the study addresses chromosome integrity following these protocols to assess any protocol-inflicted aberrations. RESULTS: The procedures explored failed to inflict any process-driven gross chromosomal aberrations post-treatment. CONCLUSIONS: Our preliminary investigations demonstrate no significant compromising affects on the cell's structure at a cytogenetic or physiological level, post-treatment. Thus, further establishing these protocols as unique direct cell-engineering approaches with a host of biological and medical applications, from the development of tissues to perhaps even organs in the future. collapse abstract

A hybrid bio-jetting approach for directly engineering living cells.

This paper reports developments on a hybrid cell-engineering protocol coupling both bio-electrosprays and aerodynamically assisted bio-jets for process-handling living cells. The current work demonstrates the ability to couple these two cell-jetting ... expand abstractprotocols for handling a wide range of cells for deposition. The post-treated cells are assessed for their viability by way of flow cytometry, which illustrates a significant population of viable cells post-treatment in comparison to those controls. This work is the first example of coupling these two protocols for the process handling of living cells. The hybrid protocol demonstrates the achievement of stable cone jetting of a cellular suspension in the single-needle configuration which was previously unachieved with single-needle bio-electrosprays. Furthermore the living cells explored in these investigations expressed GFP, thus demonstrating the ability to couple gene therapy with this hybrid protocol. Hence, this approach could one day be explored for building biologically viable tissues incorporating a therapeutic payload for combating a range of cellular/tissue-based pathologies. collapse abstract

Deposition of nano-hydroxyapatite particles utilising direct and transitional electrohydrodynamic processes.

Electrohydrodynamic spraying is a well established process used to deposit, coat, analyse and synthesise materials within the biomedical remit. Recently, electrohydrodynamic printing has been developed to afford structures for potential applications ... expand abstractin the biomedical and medical engineering fields. Both of these processes rely on the formation of an electrically-induced jet, however the resulting products can be made strikingly different and offer potential in broader applications. Here we show how spraying and printing are linked by elucidating the ease of transition between the processes. Changes in the deposition distance can result in either spray (>10 mm) or print formation (<3 mm), with an overlap of the two in between this range. For the optimal printing distance of 0.5 mm, gradual changes in the applied voltage (0-4.5 kV) encounters transitional printing modes (dripping, micro-dripping, rapid micro-dripping, unstable and stable jetting) which can be utilised for patterning. The results indicate the robustness of the electrohydrodynamic route in the nano-materials processing arena, with emphasis on biomedical materials. collapse abstract

Influence of nanohydroxyapatite patterns deposited by electrohydrodynamic spraying on osteoblast response.

Electrohydrodynamic spraying has been used to produce patterns of line width up to 100 microm in size on glass discs, using nanohydroxyapatite (nHA). A human osteoblast (HOB)-like cell model was then used to study the interaction between the HOB cell... expand abstracts and nHA patterns in vitro. Growth of the cells was significantly increased (p < 0.05) on the nHA surfaces. In addition, HOBs attached and spread well, secreting extracellular matrix. It was found that a confluent, aligned cell layer was achieved on nHA patterns by day 9. Immunofluorescent staining indicated that these cells showed elongated nuclei, enhanced adhesion (vinculin adhesion plaques) and a well-aligned cytoskeleton (actin stress fibres). This work suggests that this type of spraying may provide a route for the production of nanoscale features on implants for biomedical applications. collapse abstract

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 enginee... expand abstractring 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. collapse abstract

Living scaffolds (specialized and unspecialized) for regenerative and therapeutic medicine.

The physical sciences have increasingly demonstrated a significant influence on the life sciences. Engineering in particular has shown its input through the development of novel medical devices and processes having significance to the biomedical fiel... expand abstractd. This review introduces and discusses several fiber generation protocols, which have recently undergone development and exploration for directly handling living cells from which continuous cell-bearing or living threads to scaffolds and membranes have been fabricated. In doing so these protocols have not only demonstrated their versatility but also opened several unique possibilities for the use of these scaffolds in a plethora of biological and medical applications. In particular, these living fibrous structural units could be explored for regeneration purposes, e.g., from accelerated wound healing to combating a wide range of pathologies when coupled with gene therapy. Thus, "living entities" such as these scaffolds could be most useful in surgery/medicine, including its exploration with stem cells for the preparation of unspecialized living scaffolds and membranes. collapse abstract

The role of electrosprayed apatite nanocrystals in guiding osteoblast behaviour.

Apatite nanocrystals, which mimic the dimensions of natural bone mineral, were electrosprayed on glass substrates, as a suitable synthetic biomedical material for osteoblast outgrowth was explored. A variety of topographic patterns were deposited and... expand abstract the influence of these designs on osteoblast alignment and cell differentiation was investigated. Patterned cell growth and enhanced cell differentiation were seen. Osteoblasts were also cultured on apatite nanocrystals chemically modified with either carbonate or silicon ions. Enhanced cell proliferation and early formation of mineral nodules were observed on apatite nanocrystals with silicon addition. This work highlights the importance of the combined effects of surface topography and surface chemistry in the guidance of cell behaviour. collapse abstract

Bio-electrosprayed multicellular zebrafish embryos are viable and develop normally.

Bio-electrosprays are rapidly emerging as a viable protocol for directly engineering living cells. This communication reports the bio-electrospraying of multicellular organisms, namely zebrafish embryos. The results demonstrate that the bio-electrosp... expand abstractray protocol fails to induce any embryological perturbations. In addition to analysing overall embryo morphology, we use transgenic embryos that express green fluorescent protein in specific brain neurons to determine that neuronal numbers and organization are completely normal. These results demonstrate that the bio-electrospraying protocol does not interfere with the complex gene regulation and cell movements required for the development of a multicellular organism. collapse abstract

A unique physical-chemistry approach for fabricating cell friendly surfaces.

Recent interests in the fabrication of bio/cell-friendly surfaces are consistently gaining much scientific coverage as these methods could be explored as novel regenerative and therapeutic medicinal protocols. Essentially two main components govern t... expand abstracthis aspect, the processing methodology possessing the required robustness to fabricate a wide range of materials and, not least, the synthesised materials that need to be cell-compatible both in the short and long term after processing. In the study reported here we have combined one such robust jetting approach with a specially formulated siloxane sol. This has several unique properties in itself, and these have been demonstrated here to have a positive effect on the seeded cells. The current work demonstrates that this approach has great promise as a novel methodology for surface engineering for a wide range of applications spanning the physical to the life science areas of research. collapse abstract

Advanced jet protocols for directly engineering living cells: a genesis to alternative biohandling approaches for the life sciences.

Processing methodologies possessing the ability to directly handle living cells imply tremendous possibilities for a whole host of applications in the regenerative and therapeutic medicinal themes of R&D. Most cell-handling techniques have, in the pa... expand abstractst, been unearthed in the physical sciences, which have subsequently undergone rapid development for a plethora of applications within the life sciences. In this review, the author wishes to introduce current and swiftly emerging direct cell-handling jet protocols whilst identifying their advantages and disadvantages in comparison to each approach. The article extends to elucidating their applicability for a few life science-based research themes, where these protocols are currently undergoing intense investigation. It is the opinion of this author that these protocols generate a range of opportunities for the life sciences, which have previously not been explored and hence could have an overwhelming affect in a biological and clinical standpoint. These methods and protocols have evidently bridged the physical with the life sciences during this endeavor. collapse abstract