Nebulization of siRNA for inhalation therapy based on a microfluidic surface acoustic wave platform.

The local delivery of therapeutic small interfering RNA or siRNA to the lungs has the potential to improve the prognosis for patients suffering debilitating lung diseases. Recent advances in materials science have been aimed at addressing delivery challenges including biodistribution, bioavailability and cell internalization, but an equally important challenge to overcome is the development of an inhalation device that can deliver the siRNA effectively to the lung, without degrading the therapeutic itself. Here, we report the nebulization of siRNA, either naked siRNA or complexed with polyethyleneimine (PEI) or a commercial transfection agent, using a miniaturizable acoustomicrofluidic nebulization device. The siRNA solution could be nebulised without significant degradation into an aerosol mist with tunable mean aerodynamic diameters of approximately 3 µm, which is appropriate for deep lung deposition via inhalation. The nebulized siRNA was tested for its stability, as well as its toxicity and gene silencing properties using the mammalian lung carcinoma cell line A549, which demonstrated that the gene silencing capability of siRNA is retained after nebulization. This highlights the potential application of the acoustomicrofluidic device for the delivery of efficacious siRNA via inhalation, either for systemic delivery via the alveolar epithelium or local therapeutic delivery to the lung.

Enhanced Neural Differentiation Using Simultaneous Application of 3D Scaffold Culture, Fluid Flow, and Electrical Stimulation in Bioreactors.

Neural differentiation is studied using a simultaneous application of 3D scaffold culture and hydrodynamic and electrical stimuli in purpose-designed recirculation bioreactors operated with continuous fluid flow. Pheochromocytoma (PC12) cells are seeded into nonwoven microfibrous viscose-rayon scaffolds functionalized with poly-l-lysine and laminin. Compared with the results from static control cultures with and without electrical stimulation and bioreactor cultures with the fluid flow without electrical stimulation, expression levels of the differentiation markers ß3-tubulin, shootin1, and ephrin type-A receptor 2 are greatest when cells are cultured in bioreactors with fluid flow combined with in-situ electrical stimulus. Immunocytochemical assessment of neurite development and morphology within the scaffolds confirm the beneficial effects of exposing the cells to concurrent hydrodynamic and electrical treatments. Under the conditions tested, electrical stimulation by itself produces more pronounced levels of cell differentiation than fluid flow alone; however, significant additional improvements in differentiation are achieved by combining these treatments. Fluid flow and electrical stimuli exert independent and noninteractive effects on cellular differentiation, suggesting that interference between the mechanisms of differentiation enhancement by these two treatments is minimal during their simultaneous application. This work demonstrates the beneficial effects of combining several different potent physical environmental stimuli in cell culture systems to promote neurogenesis.

Label-Free Isolation and Single Cell Biophysical Phenotyping Analysis of Primary Cardiomyocytes Using Inertial Microfluidics.

To advance the understanding of cardiomyocyte (CM) identity and function, appropriate tools to isolate pure primary CMs are needed. A label-free method to purify viable CMs from mouse neonatal hearts is developed using a simple particle size-based inertial microfluidics biochip achieving purities of over 90%. Purified CMs are viable and retained their identity and function as depicted by the expression of cardiac-specific markers and contractility. The physico-mechanical properties of sorted cells are evaluated using downstream real-time deformability cytometry. CMs exhibited different physico-mechanical properties when compared with non-CMs. Taken together, this CM isolation and phenotyping method could serve as a valuable tool to progress the understanding of CM identity and function, and ultimately benefit cell therapy and diagnostic applications.

Fast three-dimensional micropatterning of PC12 cells in rapidly crosslinked hydrogel scaffolds using ultrasonic standing waves.

The ability to spatially organise the microenvironment of tissue scaffolds unlocks the potential of many scaffold-based tissue engineering applications. An example application is to aid the regeneration process of peripheral nerve injuries. Herein, we present a promising approach for three-dimensional (3D) micropatterning of nerve cells in tissue scaffolds for peripheral nerve repair. In particular, we demonstrate the 3D micropatterning of PC12 cells in a gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogel using ultrasound standing waves (USWs). PC12 cells were first aligned in 3D along nodal planes by the USWs in Gtn-HPA hydrogel precursor solution. The precursor was then crosslinked using horseradish peroxidase (HRP) and diluted hydrogen peroxide (H2O2), thus immobilising the aligned cells within 90-120 s. This micropatterning process is cost effective and can be replicated easily without the need for complex and expensive specialised equipment. USW-aligned PC12 cells showed no adverse effect in terms of viability or ability to proliferate. To our best knowledge, this is the first report on the effect of USW alignment on neural cell differentiation. Differentiated and USW-aligned PC12 cells showed directional uniformity after 20 d, making this technique a promising alternative approach to guide the nerve regeneration process.

Electrical stimulation of cell growth and neurogenesis using conductive and nonconductive microfibrous scaffolds.

The effect of exogenous electrical stimulation on cell viability, attachment, growth, and neurogenesis was examined using PC12 cells in microfibrous viscose-rayon scaffolds immersed in culture medium. The scaffolds were applied either in their nonconductive state or after coating the fibres with 200 nm of gold to give a scaffold sheet resistivity of (13 ± 1.3) Ω square-1. The cells were treated for 12 days using direct current electrical stimulation of 2 h per day. No cytotoxic effects were observed when up to 500 mV (8.3 mV mm-1) was applied to the scaffolds without gold, or when up to 100 mV (1.7 mV mm-1) was applied to the scaffolds with gold. Compared with unstimulated cells, whereas electrical stimulation significantly enhanced cell growth and attachment in the nonconductive scaffolds without gold, similar effects were not found for the conductive scaffolds with gold. Neural differentiation in the presence of nerve growth factor was improved by electrical stimulation in both scaffolds; however, neurite development and the expression of key differentiation markers were greater in the nonconductive scaffolds without gold than in the scaffolds with gold. Application of the same current to scaffolds with and without gold led to much higher levels of neurogenesis in the scaffolds without gold. This work demonstrates that substantial benefits in terms of cell growth and neural differentiation can be obtained using electric fields exerted across nonconductive microfibrous scaffolds, and that this approach to electrical stimulation can be more effective than when the stimulus is applied to cells on conductive scaffolds.

Printing in situ tissue sealant with visible-light-crosslinked porous hydrogel.

To reflect the rapidly growing interest in producing tissue sealants using various chemical/physical processes, we report an approach using visible light to control the crosslinking of 3D printable hydrogels as in situ tissue sealant. Gelatin-hydroxyphenylpropionic acid conjugate (Gtn-HPA) is shown to crosslink effectively within 30 s under visible light in the presence of [RuII(bpy)3]2+ and sodium persulphate, which is sufficiently rapid for surgery use. Porous structure can be also introduced by including carboxylmethyl cellulose-tyramine (CMC-Tyr) as a precursor. The detailed parameters involved in the hydrogel formation, including irradiation time and distance, are investigated in this study. The results suggested that a longer exposure time would result in a hydrogel with higher crosslinking density, while sufficient photocrosslinking can be achieved using a routine visible light source at a distance of 100 mm. Surface morphology of the photocrosslinked hydrogels are studied using scanning electron microscopy (SEM) and environmental SEM with results confirming the expected porosity. The tensile strength of the photocrosslinked hydrogels has been tested for both non-porous and porous samples. Notably, the adhesive strengths (adhesion) of the photocrosslinked hydrogels was demonstrated to be significantly higher compared to that of commercial fibrin glue. Finally, a prototype of hand-held applicator has been developed and demonstrated to print out Gtn-HPA/CMC-Tyr hydrogel of designed properties with controlled spatial resolution. The development of both material and applicator in this study provides a promising tissue sealant solution for wound closure in future surgical procedures.

Microparticle Delivery of Protein Markers for Single-Cell Western Blotting from Microwells.

Immunoblotting confers protein identification specificity beyond that of immunoassays by prepending protein electrophoresis (sizing) to immunoprobing. To accurately size protein targets, sample analysis includes concurrent analysis of protein markers with known molecular masses. To incorporate protein markers in single-cell western blotting, microwells are used to isolate individual cells and protein marker-coated microparticles. A magnetic field directs protein-coated microparticles to >75% of microwells, so as to 1) deliver a quantum of protein marker to each cell-laden microwell and 2) synchronize protein marker solubilization with cell lysis. Nickel-coated microparticles are designed, fabricated, and characterized, each conjugated with a mixture of histidine-tagged proteins (42.3-100 kDa). Imidazole in the cell lysis buffer solubilizes protein markers during a 30 s cell lysis step, with an observed protein marker release half-life of 4.46 s. Across hundreds of individual microwells and different microdevices, robust log-linear regression fits (R2 > 0.97) of protein molecular mass and electrophoretic mobility are observed. The protein marker and microparticle system is applied to determine the molecular masses of five endogenous proteins in breast cancer cells (GAPDH, ß-TUB, CK8, STAT3, ER-α), with <20% mass error. Microparticle-delivered protein standards underpin robust, reproducible electrophoretic cytometry that complements single-cell genomics and transcriptomics.

Large-scale production of stem cells utilizing microcarriers: A biomaterials engineering perspective from academic research to commercialized products.

Human stem cells, including pluripotent, embryonic and mesenchymal, stem cells play pivotal roles in cell-based therapies. Over the past decades, various methods for expansion and differentiation of stem cells have been developed to satisfy the burgeoning clinical demands. One of the most widely endorsed technologies for producing large cell quantities is using microcarriers (MCs) in bioreactor culture systems. In this review, we focus on microcarriers properties that can manipulate the expansion and fate of stem cells. Here, we provide an overview of commercially available MCs and focus on novel stimulus responsive MCs controlled by temperature, pH and field changes. Different features of MCs including composition, surface coating, morphology, geometry/size, surface functionalization, charge and mechanical properties, and their cellular effects are also highlighted. We then conclude with current challenges and outlook on this promising technology.

Rapid Capture and Release of Nucleic Acids through a Reversible Photo-Cycloaddition Reaction in a Psoralen-Functionalized Hydrogel.

Reversible immobilization of DNA and RNA is of great interest to researchers who seek to manipulate DNA or RNA in applications such as microarrays, DNA hydrogels, and gene therapeutics. However, there is no existing system that can rapidly capture and release intact nucleic acids. To meet this unmet need, we developed a functional hydrogel for rapid DNA/RNA capture and release based on the reversible photo-cycloaddition of psoralen and pyrimidines. The functional hydrogel can be easily fabricated through copolymerization of acrylamide with the synthesized allylated psoralen. The psoralen-functionalized hydrogel exhibits effective capture and release of nucleic acids spanning a wide range of lengths in a rapid fashion; over 90 % of the capture process is completed within 1 min, and circa 100 % of the release process is completed within 2 min. We observe no deleterious effects on the hybridization to the captured targets.

Acoustically-driven thread-based tuneable gradient generators.

Thread-based microfluidics offer a simple, easy to use, low-cost, disposable and biodegradable alternative to conventional microfluidic systems. While it has recently been shown that such thread networks facilitate manipulation of fluid samples including mixing, flow splitting and the formation of concentration gradients, the passive capillary transport of fluid through the thread does not allow for precise control due to the random orientation of cellulose fibres that make up the thread, nor does it permit dynamic manipulation of the flow. Here, we demonstrate the use of high frequency sound waves driven from a chip-scale device that drives rapid, precise and uniform convective transport through the thread network. In particular, we show that it is not only possible to generate a stable and continuous concentration gradient in a serial dilution and recombination network, but also one that can be dynamically tuned, which cannot be achieved solely with passive capillary transport. Additionally, we show a proof-of-concept in which such spatiotemporal gradient generation can be achieved with the entire thread network embedded in a three-dimensional hydrogel construct to more closely mimic the in vivo tissue microenvironment in microfluidic chemotaxis studies and cell culture systems, which is then employed to demonstrate the effect of such gradients on the proliferation of cells within the hydrogel.

Assessment of the potential of a high frequency acoustomicrofluidic nebulisation platform for inhaled stem cell therapy.

Despite the promise of stem cell therapy for lung therapeutics and repair, there are few viable means for directly delivering stem cells to locally target the respiratory airways via inhalation. This is not surprising given the significant challenges in aerosolising stem cells, particularly given their susceptibility to damage under the large stresses involved in the nebulisation process. Here, we present promising results using a microfluidic acoustic nebulisation platform that is not only low cost and portable, but also its high MHz order frequencies are effective for preserving the structural and functional integrity of mesenchymal stem cells (MSCs) during the nebulisation process. This is verified through an assessment of the viability, structure, metabolic activity, proliferation ability and genetic makeup of the nebulised MSCs using a variety of assays, including cell viability staining, flow cytometry, reverse transcription and quantitative polymerase chain reaction, and immunophenotyping, thus demonstrating the platform as a promising method for efficient pulmonary stem cell delivery.

Rapid Enhancement of Cellular Spheroid Assembly by Acoustically Driven Microcentrifugation.

Intense acoustically driven microcentrifugation flows are employed to enhance the assembly of cellular spheroids in the microwell of a tissue culture well plate. This ability to interface microfluidics with commonly used tissue culture plasticware is a significant advantage as it can potentially be parallelized for high throughput operation and allows existing analytical equipment designed to fit current laboratory formats to be retained. The microcentrifugation flow, induced in the microwell coated with a low adhesive hydrogel, is shown to rapidly enhance the concentration of cells into tight aggregates within a minute-considerably faster than the conventional hanging drop and liquid overlay methods, which typically require days-while maintaining their viability. The proposed method also affords better control of the compaction force and hence the spheroid dimension simply by tuning the input power, which is a significant improvement over other microfluidic methods that require the fabrication of different geometries and microstructures to generate spheroids of different sizes. The spheroids produced are observed to exhibit the concentric heterogeneous cell populations and tight cell-cell interfaces typical of in vivo tumors, and are potentially useful in a broad spectrum of cancer biology and drug screening studies.

Hydrogels with Tunable Properties.

This chapter describes the preparation of tissue engineered constructs by immobilizing chondrocytes in hydrogel with independently tunable porosity and mechanical properties. This chapter also presents the methods to characterize these tissue engineered constructs. The resulting tissue engineered constructs can be useful for the generation of cartilage tissue both in vitro and in vivo.

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.

Effects of GDNF-loaded injectable gelatin-based hydrogels on endogenous neural progenitor cell migration.

Brain repair following disease and injury is very limited due to difficulties in recruiting and mobilizing stem cells towards the lesion. More importantly, there is a lack of structural and trophic support to maintain viability of the limited stem/progenitor cells present. This study investigates the effectiveness of an injectable gelatin-based hydrogel in attracting neural progenitor cells (NPCs) from the subventricular zone (SVZ) towards the implant. Glial cell-line-derived neurotrophic factor (GDNF) encapsulated within the hydrogel and porosity within the hydrogel prevents glial scar formation. By directly targeting the hydrogel implant towards the SVZ, neuroblasts can actively migrate towards and along the implant tract. Significantly more doublecortin (DCX)-positive neuroblasts surround implants at 7 d post-implantation (dpi) compared with lesion alone controls, an effect that is enhanced when GDNF is incorporated into the hydrogels. Neuroblasts are not observed at the implant boundary at 21 dpi, indicating that neuroblast migration has halted, and neuroblasts have either matured or have not survived. The development of an injectable gelatin-based hydrogel has significant implications for the treatment of some neurodegenerative diseases and brain injuries. The ability of GDNF and porosity to effectively prevent glial scar formation will allow better integration and interaction between the implant and surrounding neural tissue.

Hydroxypropyl cellulose methacrylate as a photo-patternable and biodegradable hybrid paper substrate for cell culture and other bioapplications.

In addition to the choice of appropriate material properties of the tissue construct to be used, such as its biocompatibility, biodegradability, cytocompatibility, and mechanical rigidity, the ability to incorporate microarchitectural patterns in the construct to mimic that found in the cellular microenvironment is an important consideration in tissue engineering and regenerative medicine. Both these issues are addressed by demonstrating a method for preparing biodegradable and photo-patternable constructs, where modified cellulose is cross-linked to form an insoluble structure in an aqueous environment. Specifically, hydroxypropyl cellulose (HPC) is rendered photocrosslinkable by grafting with methylacrylic anhydride, whose linkages also render the cross-linked construct hydrolytically degradable. The HPC is then cross-linked via a photolithography-based fabrication process. The feasibility of functionalizing these HPC structures with biochemical cues is verified post-fabrication, and shown to facilitate the adhesion of mesenchymal progenitor cells. The HPC constructs are shown to be biocompatible and hydrolytically degradable, thus enabling cell proliferation and cell migration, and therefore constituting an ideal candidate for long-term cell culture and implantable tissue scaffold applications. In addition, the potential of the HPC structure is demonstrated as an alternative substrate to paper microfluidic diagnostic devices for protein and cell assays.

Injectable 3D hydrogel scaffold with tailorable porosity post-implantation.

Since rates of tissue growth vary significantly between tissue types, and also between individuals due to differences in age, dietary intake, and lifestyle-related factors, engineering a scaffold system that is appropriate for personalized tissue engineering remains a significant challenge. In this study, a gelatin-hydroxyphenylpropionic acid/carboxylmethylcellulose-tyramine (Gtn-HPA/CMC-Tyr) porous hydrogel system that allows the pore structure of scaffolds to be altered in vivo after implantation is developed. Cross-linking of Gtn-HPA/CMC-Tyr hydrogels via horseradish peroxidase oxidative coupling is examined both in vitro and in vivo. Post-implantation, further alteration of the hydrogel structure is achieved by injecting cellulase enzyme to digest the CMC component of the scaffold; this treatment yields a structure with larger pores and higher porosity than hydrogels without cellulase injection. Using this approach, the pore sizes of scaffolds are altered in vivo from 32-87 µm to 74-181 µm in a user-controled manner. The hydrogel is biocompatible to COS-7 cells and has mechanical properties similar to those of soft tissues. The new hydrogel system developed in this work provides clinicians with the ability to tailor the structure of scaffolds post-implantation depending on the growth rate of a tissue or an individual's recovery rate, and could thus be ideal for personalized tissue engineering.

Thermoresponsive cellulosic hydrogels with cell-releasing behavior.

Here we report the preparation and characterization of thermoresponsive cellulosic hydrogels with cell-releasing behavior. Hydroxypropyl cellulose (HPC) was modified with methacrylic anhydride (MA). The resultant macromonomer, HPC-MA, retains the characteristic thermoresponsive phase behavior of HPC, with an onset temperature of 36 °C and a lower critical solution temperature (LCST) of 37-38 °C, as determined by turbidity measurement. Homogenous HPC-MA hydrogels were prepared by UV-cross-linking the aqueous solutions of the macromonomer at room temperature, and characterized by water contact angle and swelling ratio measurements, and dynamic mechanical analysis. These hydrogels exhibit temperature-dependent surface hydrophilicity and hydrophobicity, equilibrium water content as well as mechanical properties. Cell-releasing characteristics were demonstrated using African green monkey kidney cell line (COS-7 cells) and murine-derived embryonic stem cell line (Oct4b2). By reducing temperature to 4 °C, the cultivated cells spontaneously detached from the hydrogels without the need of trypsin treatment. These unique properties make our HPC-MA hydrogels potential substrates for cell sheet engineering.

Three-dimensional nanocharacterization of porous hydrogel with ion and electron beams.

Porous hydrogels provide an excellent environment for cell growth and tissue regeneration, with high permeability for oxygen, nutrients, and other water-soluble metabolites through their high water-content matrix. The ability to image three-dimensional (3D) cell growth is crucial for understanding and studying various cellular activities in 3D context, particularly for designing new tissue engineering scaffold, but it is still challenging to study cell-biomaterial interfaces with high resolution imaging. We demonstrate using focused ion beam (FIB) milling, electron imaging, and associated microanalysis techniques that novel 3D characterizations can be performed effectively on cells growing inside 3D hydrogel scaffold. With FIB-tomography, the porous microstructures were revealed at nanometer resolution, and the cells grown inside. The results provide a unique 3D measurement of hydrogel porosity, as compared with those from porosimetry, and offer crucial insights into material factors affecting cell proliferation at specific regions within the scaffold. We also proved that high throughput correlative imaging of cell growth is viable through a silicon membrane based environment. The proposed approaches, together with the protocols developed, provide a unique platform for analysis of the microstructures of novel biomaterials, and for exploration of their interactions with the cells as well.

Preparation of a soft and interconnected macroporous hydroxypropyl cellulose methacrylate scaffold for adipose tissue engineering.

This study describes the preparation and characterization of a biodegradable 3D hydrogel constructed from hydroxypropyl cellulose (HPC), modified with bifunctional methacrylic anhydride (MA) to form hydroxypropyl cellulose methacrylate (HPC-MA), for adipose tissue engineering applications. The hydrogels were prepared from three different concentrations (10 wt%, 15 wt% and 20 wt%) of HPC-MA with 0.35 degree of substitution. HPC-MA hydrogel scaffolds with open biphasic features were prepared by exploiting the thermal responsive phase behavior of HPC and temperature mediated phase separation of HPC-MA. The resulting scaffolds exhibited pore sizes ranging from 30 to 300 µm and an interconnected porosity of ∼90%. The swelling ratio (SR) and storage modulus of HPC-MA scaffolds were in the range of 12.94 to 35.83 and 0.75 to 4.28 kPa, respectively. The swelling ratio and storage modulus suggested that the scaffold exhibits high water retention, allowing medium exchange during cell culturing and that it is suitable for adipose tissue regeneration. The HPC-MA scaffolds were found to be biocompatible to human adipose-derived stem cells (ASCs). ASCs were successfully differentiated into the adipocytes inside the scaffolds, and therefore demonstrated the potential application of these HPC-MA scaffolds for adipose tissue engineering.