Engineering imaging: using particle image velocimetry to see physiology in a new light.

1. Despite the array of sophisticated imaging techniques available for biological applications, none of the standard biomedical techniques adequately provides the capability to measure motion and flow. Those techniques currently in use are particularly lacking in spatial and temporal resolution. 2. Herein, we introduce the technique of particle image velocimetry. This technique is a well-established tool in engineering research and industry. Particle image velocimetry is continuing to develop and has an increasing number of variants. 3. Three case studies are presented: (i) the use of microparticle image velocimetry to study flow generated by high-frequency oscillatory ventilation in a human airway model; (ii) the use of stereoparticle image velocimetry to study stirred cell and tissue culture devices; and (iii) a three-dimensional X-ray particle image velocimetry technique used to measure flow in an in vitro vascular flow model. 4. The case studies highlight the vast potential of applying the engineering technique of particle image velocimetry and its many variants to current research problems in physiology.

The leading-edge vortex on a rotating wing changes markedly beyond a certain central body size.

Stable attachment of a leading-edge vortex (LEV) plays a key role in generating the high lift on rotating wings with a central body. The central body size can affect the LEV structure broadly in two ways. First, an overall change in the size changes the Reynolds number, which is known to have an influence on the LEV structure. Second, it may affect the Coriolis acceleration acting across the wing, depending on the wing-offset from the axis of rotation. To investigate this, the effects of Reynolds number and the wing-offset are independently studied for a rotating wing. The three-dimensional LEV structure is mapped using a scanning particle image velocimetry technique. The rapid acquisition of images and their correlation are carefully validated. The results presented in this paper show that the LEV structure changes mainly with the Reynolds number. The LEV-split is found to be only minimally affected by changing the central body radius in the range of small offsets, which interestingly includes the range for most insects. However, beyond this small offset range, the LEV-split is found to change dramatically.

Bioreducible PEI-functionalized glycol chitosan: A novel gene vector with reduced cytotoxicity and improved transfection efficiency.

Non-viral gene delivery has been well recognised as a potential way to address the main safety limitations of viral gene carriers. A new redox-responsive PEI derivative was designed, synthesized and evaluated for non-viral delivery applications of GFP DNA. Glycol chitosan was covalently attached to highly branched LMW PEI via bio-cleavable disulfide bonds to synthesize a new redox-responsive gene carrier (GCS-ss-PEI). Results showed the enhanced buffering capacity of GCS-ss-PEI, 43.1%, compared to the buffering capacities of both LMW PEI and HMW PEI, 23.2% and 31.5%, respectively, indicating more likely endosomal escape of the entrapped gene for GCS-ss-PEI. Moreover, electrophoretic gel retardation assay, performed to investigate the binding strength of GCS-ss-PEI to GFP DNA, showed stronger complexation with GFP DNA in GCS-ss-PEI at non-GSH condition. Employing GCS and incorporation of disulfide bonds in the structure of the PEI-based gene carrier resulted in improved redox-responsivity, reduced toxicity, enhanced endosomal escape and GFP DNA transfection. The facilitated intracellular gene release along with excellent redox-responsive characteristics and dropped cytotoxicity suggests the potential of GCS-ss-PEI as a candidate for developing highly efficient and safe gene vectors.

Dual effect of F-actin targeted carrier combined with antimitotic drug on aggressive colorectal cancer cytoskeleton: Allying dissimilar cell cytoskeleton disrupting mechanisms.

A recent approach to colon cancer therapy is to employ selective drugs with specific extra/intracellular sites of action. Alteration of cytoskeletal protein reorganization and, subsequently, to cellular biomechanical behaviour during cancer progression highly affects the cancer cell progress. Hence, cytoskeleton targeted drugs are an important class of cancer therapy agents. We have studied viscoelastic alteration of the human colon adenocarcinoma cell line, SW48, after treatment with a drug delivery system comprising chitosan as the carrier and albendazole as the microtubule-targeting agent (MTA). For the first time, we have evaluated the biomechanical characteristics of the cell line, using the micropipette aspiration (MA) method after treatment with drug delivery systems. Surprisingly, employing a chitosan-albendazole pair, in comparison with both neat materials, resulted in more significant change in the viscoelastic parameters of cells, including the elastic constants (K1 and K2) and the coefficient of viscosity (µ). This difference was more pronounced for cancer cells after 48h of the treatment. Microtubule and actin microfilament (F-actin) contents in the cell line were studied by immunofluorescent staining. Good agreement was observed between the mechanical characteristics results and microtubule/F-actin contents of the treated SW48 cell line, which declined after treatment. The results showed that chitosan affected F-actin more, while MTA was more effective for microtubules. Toxicity studies were performed against two cancer cell lines (SW48 and MCF10CA1h) and compared to normal cells, MCF10A. The results showed cancer selectiveness, safety of formulation, and enhanced anticancer efficacy of the CS/ABZ conjugate. This study suggests that employing such a suitable pair of drug-carriers with dissimilar sites of action, thus allying the different cell cytoskeleton disrupting mechanisms, may provide a more efficient cancer therapy approach.

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells.

In recent times, the study and use of induced pluripotent stem cells (iPSC) have become important in order to avoid the ethical issues surrounding the use of embryonic stem cells. Therapeutic, industrial and research based use of iPSC requires large quantities of cells generated in vitro. Mammalian cells, including pluripotent stem cells, have been expanded using 3D culture, however current limitations have not been overcome to allow a uniform, optimized platform for dynamic culture of pluripotent stem cells to be achieved. In the current work, we have expanded mouse iPSC in a spinner flask using Cytodex 3 microcarriers. We have looked at the effect of agitation on the microcarrier survival and optimized an agitation speed that supports bead suspension and iPS cell expansion without any bead breakage. Under the optimized conditions, the mouse iPSC were able to maintain their growth, pluripotency and differentiation capability. We demonstrate that microcarrier survival and iPS cell expansion in a spinner flask are reliant on a very narrow range of spin rates, highlighting the need for precise control of such set ups and the need for improved design of more robust systems.

Microfluidic Manipulation of Core/Shell Nanoparticles for Oral Delivery of Chemotherapeutics: A New Treatment Approach for Colorectal Cancer.

A microfluidics approach to synthesize core-shell nanocarriers with high pH tunability is described. The sacrificial shell protects the core layer with the drugs and prevents their release in the severe pH conditions of the gastrointestinal tract, while allowing for drug release in the proximity of a tumor. The proposed nanoparticulate drug-delivery system is designed for the oral administration of cancer therapeutics.

Myocardial infarction: stem cell transplantation for cardiac regeneration.

It is estimated that by 2030, almost 23.6 million people will perish from cardiovascular disease, according to the WHO. The review discusses advances in stem cell therapy for myocardial infarction, including cell sources, methods of differentiation, expansion selection and their route of delivery. Skeletal muscle cells, hematopoietic cells and mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs)-derived cardiomyocytes have advanced to the clinical stage, while induced pluripotent cells (iPSCs) are yet to be considered clinically. Delivery of cells to the sites of injury and their subsequent retention is a major issue. The development of supportive scaffold matrices to facilitate stem cell retention and differentiation are analyzed. The review outlines clinical translation of conjugate stem cell-based cellular therapeutics post-myocardial infarction.

Cardiogenesis of embryonic stem cells with liquid marble micro-bioreactor.

A liquid marble micro-bioreactor is prepared by placing a drop of murine embryonic stem cell (ESC) (Oct4B2-ESC) suspension onto a polytetrafluoroethylene (PTFE) particle bed. The Oct4B2-ESC aggregates to form embryoid bodies (EBs) with relatively uniform size and shape in a liquid marble within 3 d. For the first time, the feasibility of differentiating ESC into cardiac lineages within liquid marbles is being investigated. Without the addition of growth factors, suspended EBs from liquid marbles express various precardiac mesoderm markers including Flk-1, Gata4, and Nkx2.5. Some of the suspended EBs exhibit spontaneous contraction. These results indicate that the liquid marble provides a suitable microenvironment to induce EB formation and spontaneous cardiac mesoderm differentiation. Some of the EBs are subsequently plated onto gelatin-coated tissue culture dishes. Plated EBs express mature cardiac markers atrial myosin light chain 2a (MLC2a) and ventricular myosin light chain (MLC2v), and the cardiac structural marker α-actinin. More than 60% of the plated EBs exhibit spontaneous contraction and express mature cardiomyocyte marker cardiac troponin T (cTnT), indicating that these EBs have differentiated into functional cardiomyocytes. Together, these results demonstrate that the liquid-marble technique is an easily employed, cost effective, and efficient approach to generate EBs and facilitating their cardiogenesis.

Flow characterization of a spinner flask for induced pluripotent stem cell culture application.

We present detailed quantitative measurement analyses for flow in a spinner flask with spinning rates between 20 to 45 RPM, utilizing the optical velocimetry measurement technique of Particle Image Velocimetry (PIV). A partial section of the impeller was immersed in the working fluid to reduce the shear forces induced on the cells cultured on microcarriers. Higher rotational speeds improved the mixing effect in the medium at the expense of a higher shear environment. It was found that the mouse induced pluripotent stem (iPS) cells achieved the optimum number of cells over 7 days in 25 RPM suspension culture. This condition translates to 0.0984 Pa of maximum shear stress caused by the interaction of the fluid flow with the bottom surface. However, inverse cell growth was obtained at 28 RPM culture condition. Such a narrow margin demonstrated that mouse iPS cells cultured on microcarriers are very sensitive to mechanical forces. This study provides insight to biomechanical parameters, specifically the shear stress distribution, for a commercially available spinner flask over a wide range of Reynolds number.

Optimisation of a stirred bioreactor through the use of a novel holographic correlation velocimetry flow measurement technique.

We describe a method for measuring three dimensional (3D) velocity fields of a fluid at high speed, by combining a correlation-based approach with in-line holography. While this method utilizes tracer particles contained within the flow, our method does not require the holographic reconstruction of 3D images. The direct flow reconstruction approach developed here allows for measurements at seeding densities in excess of the allowable levels for techniques based on image or particle reconstruction, thus making it suited for biological flow measurement, such as the flow in bioreactor. We outline the theory behind our method, which we term Holographic Correlation Velocimetry (HCV), and subsequently apply it to both synthetic and laboratory data. Moreover, because the system is based on in-line holography, it is very efficient with regard to the use of light, as it does not rely on side scattering. This efficiency could be utilized to create a very high quality system at a modest cost. Alternatively, this efficiency makes the system appropriate for high-speed flows and low exposure times, which is essential for imaging dynamic systems.

Surface-functionalization of PDMS for potential micro-bioreactor and embryonic stem cell culture applications.

This study presents a novel and inexpensive method to prepare a disposable micro-bioreactor for stem cell expansion. The micro-bioreactor was fabricated in the form of a fixed bed bioreactor with a microchannel reactor bed. The micro-bioreactor was constructed from polydimethylsiloxane (PDMS), and the microchannel was functionalized to enable cell adhesion and resistance to bovine serum albumin protein adsorption. The PDMS reactor bed surface was activated by oxygen plasma, then aminized with trimethoxysilylpropyl(polyethyleneimine), followed by grafting with carboxylmethyl cellulose (CMC) and gelatin in sequence. The functionalized PDMS surface demonstrated improved hydrophilicity and antifouling properties. The grafting of gelatin promoted cell adhesion. The functionalized surface was found to be biocompatible with MDA-MB-231 and Oct4b2 cells and was demonstrated to facilitate cell proliferation. The expanded Oct4b2 cells retained their proliferation potential, undifferentiated phenotype and pluripotency.

Altered lung motion is a sensitive indicator of regional lung disease.

Since lung diseases adversely affect airflow during breathing, they must also alter normal lung motion, which can be exploited to detect these diseases. However, standard imaging techniques such as CT and MRI imaging during breath-holds provide little or no information on lung motion and cannot detect diseases that cause subtle changes in lung structure. Phase-contrast X-ray imaging provides images of high contrast and spatial resolution with temporal resolutions that allow multiple images to be acquired throughout the respiratory cycle. Using X-ray phase-contrast imaging, coupled with velocimetry, we have measured lung tissue movement and determined velocity fields that define speed and direction of regional lung motion throughout a breath in normal Balb/c nude male mice and mice exposed to bleomycin. Regional maps of lung tissue motion reveal both the heterogeneity of normal lung motion, as well as abnormal motion induced by bleomycin treatment. Analysed histologically, bleomycin treatment caused pathological changes in lung structure that were heterogenous, occupying less than 12% of the lung at 6 days after treatment. Moreover, plethysmography failed to detect significant changes in compliance at either 36 h or 6 days after treatment. Detailed analysis of the vector fields demonstrated major differences (p < 0.001) in regional lung motion between control and bleomycin-treated mice at both 36 h and 6 days after treatment. The results of this study demonstrate that X-ray phase-contrast imaging, coupled with velocimetry, can detect early stage, subtle and non-uniform lung disease.

X-ray velocimetry and haemodynamic forces within a stenosed femoral model at physiological flow rates.

High resolution in vivo velocity measurements within the cardiovascular system are essential for accurate calculation of vessel wall shear stress, a highly influential factor for the progression of arterial disease. Unfortunately, currently available techniques for in vivo imaging are unable to provide the temporal resolution required for velocity measurement at physiological flow rates. Advances in technology and improvements in imaging systems are allowing a relatively new technique, X-ray velocimetry, to become a viable tool for such measurements. This study investigates the haemodynamics of pulsatile blood flow in an optically opaque in vitro model at physiological flow rates using X-ray velocimetry. The in vitro model, an asymmetric stenosis, is designed as a 3:1 femoral artery with the diameter and flow rate replicating vasculature of a mouse. Velocity measurements are obtained over multiple cycles of the periodic flow at high temporal and spatial resolution (1 ms and 29 µm, respectively) allowing accurate measurement of the velocity gradients and calculation of the wall shear stress. This study clearly illustrates the capability of in vitro X-ray velocimetry, suggesting it as a possible measurement technique for future in vivo vascular wall shear stress measurement.

A bioreactor model of mouse tumor progression.

The present study represents an investigation of a novel stirred bioreactor for culture of a transformed cell line under defined hydrodynamic conditions in vitro. Cell colonies of the EL-4 mouse lymphoma cell line grown for the first time in a rotating disc bioreactor (RDB), were observed to undergo changes in phenotype in comparison to standard, static flask cultures. RDB cultures, with or without agitation, promoted the formation of adherent EL-4 cell plaques that merged to form contiguous tumor-like masses in longer-term cultures, unlike the unattached spheroid aggregates of flask cultures. Plaques grown under agitated conditions were further altered in morphology and distribution in direct response to fluid mechanical stimuli. Plaque colonies growth in RDBs with or without agitation also exhibited significant increases in production of interleukin-4 (IL-4) and lactate, suggesting an inducible "Warburg effect." Increases in cell biomass in RDB cultures were no different to flask cultures, though a trend toward a marginal increase was observed at specific rotational speeds. The RDB may therefore be a suitable alternative method to study mechanisms of tumor progression and invasiveness in vitro, under more complex physicochemical conditions that may approximate natural tissue environments.

A fluid dynamics approach to bioreactor design for cell and tissue culture.

The problem of controlling cylindrical tank bioreactor conditions for cell and tissue culture purposes has been considered from a flow dynamics perspective. Simple laminar flows in the vortex breakdown region are proposed as being a suitable alternative to turbulent spinner flask flows and horizontally oriented rotational flows. Vortex breakdown flows have been measured using three-dimensional Stereoscopic particle image velocimetry, and non-dimensionalized velocity and stress distributions are presented. Regions of locally high principal stress occur in the vicinity of the impeller and the lower sidewall. Topological changes in the vortex breakdown region caused by an increase in Reynolds number are reflected in a redistribution of the peak stress regions. The inclusion of submerged scaffold models adds complexity to the flow, although vortex breakdown may still occur. Relatively large stresses occur along the edge of disks jutting into the boundary of the vortex breakdown region.