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 is 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.
Responses of valve endothelial cells (VECs) to shear stresses are important for the regulation of valve durability. However, the effect of flow patterns subjected to VECs on the opposite surfaces of the valves on the production of extracellular matrix (ECM) has not yet been investigated. This study aims to investigate the response of side-specific flow patterns, in terms of ECM synthesis and/or degradation in porcine aortic valves. Aortic and ventricular sides of aortic valve leaflets were exposed to oscillatory and laminar flow generated by a Cone-and-Plate machine for 48 h. The amount of collagen, GAGs and elastin was quantified and compared to samples collected from the same leaflets without exposing to flow. The results demonstrated that flow is important to maintain the amount of GAGs and elastin in the valve, as compared to the effect of static conditions. Particularly, the laminar waveform plays a crucial role on the modulation of elastin in side-independent manner. Furthermore, the ability of oscillatory flow on the aortic surface to increase the amount of collagen and GAGs cannot be replicated by exposure of an identical flow pattern on the ventricular side of the valve. Side-specific responses to the particular patterns of flow are important to the regulation of ECM components. Such understanding is imperative to the creation of tissue-engineered heart valves that must be created from the “appropriate” cells that can replicate the functions of the native VECs to regulate the different constituents of ECM.
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 methods, 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.
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, cellular 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 microenviorments 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.
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