Hydrogel microfluidic co-culture device for photothermal therapy and cancer migration.

We developed the photo-crosslinkable hydrogel microfluidic co-culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi-permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co-culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)-treated GelMA hydrogel-embedding microchannels. The main advantage of this hydrogel microfluidic co-culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod-mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co-culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.

Uniform-sized neurosphere-mediated motoneuron differentiation in microwell arrays.

We developed the photocrosslinkable hydrogel microwell arrays for uniform-sized neurosphere-mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform-sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform-sized neurospheres harvested from microwell arrays were replated into laminin-coated substrate. In parallel, uniform-sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three-dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150 µm in diameter) on motoneuron differentiation, showing that the largest uniform-sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform-sized neurosphere-mediated motoneuron differentiation.

Concave microwell array-mediated three-dimensional tumor model for screening anticancer drug-loaded nanoparticles.

We investigated the effect of anticancer drug-loaded functional polymeric nanoparticles on drug resistance of three-dimensional (3D) breast tumor spheroids. 3D tumor models were built using concave microwells with different diameters (300-700µm) and nanoparticles were prepared using thermo-responsive poly(N-isopropylacrylamide) (PNIPAM)-co-acrylic acid (AA). Upon culturing with doxorubicin-loaded PNIPAM-co-AA nanoparticles for 96hours, the smallest tumor spheroids were extensively disrupted, resulting in a reduction in spheroid diameter. In contrast, the sizes of the largest tumor spheroids were not changed. Scanning electron microscopy revealed that the circular shape of 3D spheroids treated with doxorubicin-loaded PNIPAM-co-AA nanoparticles had collapsed severely. Cell viability assays also demonstrated that the largest tumor spheroids cultured with doxorubicin-loaded PNIPAM-co-AA nanoparticles were highly resistant to the anticancer drug. We confirmed that tight cell-cell contacts within largest tumor spheroids significantly improved the anticancer drug resistance. Therefore, this uniform-sized 3D breast tumor model could be a potentially powerful tool for anticancer drug screening applications. FROM THE CLINICAL EDITOR: The battle against cancer is a big challenge. With new anti-cancer drugs being developed under the nanotechnology platform, there is a need to have a consistent and reliable testing system that mimics the in-vivo tumor scenario. The authors successfully designed a 3D tumor model using concave microwells to produce different tumor diameters. This will be of value for future drug screening.

Thermo-responsive polymeric nanoparticles for enhancing neuronal differentiation of human induced pluripotent stem cells.

We report thermo-responsive retinoic acid (RA)-loaded poly(N-isopropylacrylamide)-co-acrylamide (PNIPAM-co-Am) nanoparticles for directing human induced pluripotent stem cell (hiPSC) fate. Fourier transform infrared spectroscopy and (1)H nuclear magnetic resonance analysis confirmed that RA was efficiently incorporated into PNIAPM-co-Am nanoparticles (PCANs). The size of PCANs dropped with increasing temperatures (300-400 nm at room temperature, 80-90 nm at 37°C) due to its phase transition from hydrophilic to hydrophobic. Due to particle shrinkage caused by this thermo-responsive property of PCANs, RA could be released from nanoparticles in the cells upon cellular uptake. Immunocytochemistry and quantitative real-time polymerase chain reaction analysis demonstrated that neuronal differentiation of hiPSC-derived neuronal precursors was enhanced after treatment with 1-2 µg/ml RA-loaded PCANs. Therefore, we propose that this PCAN could be a potentially powerful carrier for effective RA delivery to direct hiPSC fate to neuronal lineage. FROM THE CLINICAL EDITOR: The use of induced pluripotent stem cells (iPSCs) has been at the forefront of research in the field of regenerative medicine, as these cells have the potential to differentiate into various terminal cell types. In this article, the authors utilized a thermo-responsive polymer, Poly(N-isopropylacrylamide) (PNIPAM), as a delivery platform for retinoic acid. It was shown that neuronal differentiation could be enhanced in hiPSC-derived neuronal precursor cells. This method may pave a way for future treatment of neuronal diseases.

Retinoic acid-polyethyleneimine complex nanoparticles for embryonic stem cell-derived neuronal differentiation.

We synthesized functional retinoic acid (RA)-polyethyleneimine (PEI) complex nanoparticles. NH groups of branched PEI chains were electrostatically interacted with carboxyl groups of RA surfaces to form cationic RA-PEI complex nanoparticles. We observed that the average diameter of RA-PEI complex nanoparticles was approximately 70 nm and the morphology of complex nanoparticles was homogeneous circular shape. To confirm the synthesis of RA-PEI complex nanoparticles, we characterized complex nanoparticles using (1)H nuclear magnetic resonance (NMR), indicating that hydrophilic branched PEI chains were covered on hydrophobic RA surfaces. Furthermore, we demonstrated that pH enabled the control of amounts of RA released from RA-PEI complex nanoparticles, showing that RA exposed to acidic pH 5 was steadily released (∼76%) from complex nanoparticles, whereas RA was rapidly released (∼97%) at pH 7.4 on day 11. We also observed that RA-PEI complex nanoparticles induced embryonic stem (ES) cell-derived neuronal differentiation. Therefore, this RA-PEI complex nanoparticle is a potentially powerful tool for directing murine ES cell fate.

Highly porous core-shell polymeric fiber network.

Core-shell nanofibers are of great interest in the field of tissue engineering and cell biology. We fabricated porous core-shell fiber networks using an electrospinning system with a water-immersed collector. We hypothesized that the phase separation and solvent evaporation process would enable the control of the pore formation on the core-shell fiber networks. To synthesize porous core-shell fiber networks, we used polycaprolactone (PCL) and gelatin. Quantitative analysis showed that the sizes of gelatin-PCL core-shell nanofibers increased with PCL concentrations. We also observed that the shapes of the pores created on the PCL fiber networks were elongated, whereas the gelatin-PCL core-shell fiber networks had circular pores. The surface areas of porous nanofibers were larger than those of the nonporous nanofibers due to the highly volatile solvent and phase separation process. The porous core-shell fiber network was also used as a matrix to culture various cell types, such as embryonic stem cells, breast cancer cells, and fibroblast cells. Therefore, this porous core-shell polymeric fiber network could be a potentially powerful tool for tissue engineering and biological applications.

Microfluidic synthesis of pure chitosan microfibers for bio-artificial liver chip.

We developed microfluidic-based pure chitosan microfibers (approximately 1 meter long, 70-150 microm diameter) for liver tissue engineering applications. Despite the potential of the chitosan for creating bio-artificial liver chips, its major limitation is the inability to fabricate pure chitosan-based microstructures with controlled shapes because of the mechanical weakness of the pure chitosan. Previous studies have shown that chitosan micro/nanofibers can be fabricated by using chemicals and electrospinning techniques. However, there is no paper regarding pure chitosan-based microfibers in a microfluidic device. This paper suggests a unique method to fabricate pure chitosan microfibers without any chemical additive. We also analyzed the chemical, mechanical, and diffusion properties of pure chitosan microfibers. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrometry and electron spectroscopy for chemical analysis (ESCA) were used to analyze the chemical composition of the synthesized chitosan microfibers. We measured the mechanical axial-force and diffusion coefficient in pure chitosan-based microfibers using fluorescence recovery after photobleaching (FRAP) techniques. Furthermore, to evaluate the capability of the microfibers for liver tissue formation, hepatoma HepG2 cells were seeded onto the chitosan microfibers. The functionality of these hepatic cells cultured on chitosan microfibers was analyzed by measuring albumin secretion and urea synthesis. Therefore, this pure chitosan-based microfiber chip could be a potentially useful method for liver tissue engineering applications.

A computational and experimental study inside microfluidic systems: the role of shear stress and flow recirculation in cell docking.

In this paper, microfluidic devices containing microwells that enabled cell docking were investigated. We theoretically assessed the effect of geometry on recirculation areas and wall shear stress patterns within microwells and studied the relationship between the computational predictions and experimental cell docking. We used microchannels with 150 microm diameter microwells that had either 20 or 80 microm thickness. Flow within 80 microm deep microwells was subject to extensive recirculation areas and low shear stresses (<0.5 mPa) near the well base; whilst these were only presented within a 10 microm peripheral ring in 20 microm thick microwells. We also experimentally demonstrated that cell docking was significantly higher (p < 0.01) in 80 microm thick microwells as compared to 20 microm thick microwells. Finally, a computational tool which correlated physical and geometrical parameters of microwells with their fluid dynamic environment was developed and was also experimentally confirmed.

Plasmonic heating-based portable digital PCR system.

A miniaturized polymerase chain reaction (PCR) system is not only important for medical applications in remote areas of developing countries, but also important for testing at ports of entry during global epidemics, such as the current outbreak of the coronavirus. Although there is a large number of PCR sensor systems available for this purpose, there is still a lack of portable digital PCR (dPCR) heating systems. Here, we first demonstrated a portable plasmonic heating-based dPCR system. The device has total dimensions of 9.7 × 5.6 × 4.1 cm and a total power consumption of 4.5 W, allowing for up to 25 dPCR experiments to be conducted on a single charge of a 20 000 mAh external battery. The dPCR system has a maximum heating rate of 10.7 °C s-1 and maximum cooling rate of 8 °C s-1. Target DNA concentrations in the range from 101 ± 1.4 copies per µL to 260 000 ± 20 000 copies per µL could be detected using a poly(dimethylsiloxane) (PDMS) microwell membrane with 22 080 well arrays (20 µm diameter). Furthermore, the heating system was demonstrated using a mass producible poly(methyl methacrylate) PMMA microwell array with 8100 microwell arrays (80 µm diameter). The PMMA microwell array could detect a concentration from 12 ± 0.7 copies per µL to 25 889 ± 737 copies per µL.

Stop-flow lithography to generate cell-laden microgel particles.

Encapsulating cells within hydrogels is important for generating three-dimensional (3D) tissue constructs for drug delivery and tissue engineering. This paper describes, for the first time, the fabrication of large numbers of cell-laden microgel particles using a continuous microfluidic process called stop-flow lithography (SFL). Prepolymer solution containing cells was flowed through a microfluidic device and arrays of individual particles were repeatedly defined using pulses of UV light through a transparency mask. Unlike photolithography, SFL can be used to synthesize microgel particles continuously while maintaining control over particle size, shape and anisotropy. Therefore, SFL may become a useful tool for generating cell-laden microgels for various biomedical applications.

High-throughput screening of cell responses to biomaterials.

Biomaterials have emerged as powerful regulators of the cellular microenvironment for drug discovery, tissue engineering research and chemical testing. Although biomaterial-based matrices control the cellular behavior, these matrices are still far from being optimal. In principle, efficacy of biomaterial development for the cell cultures can be improved by using high-throughput techniques that allow screening of a large number of materials and manipulate microenvironments in a controlled manner. Several cell responses such as toxicity, proliferation, and differentiation have been used to evaluate the biomaterials thus providing basis for further selection of the lead biomimetic materials or microenvironments. Although high-throughput techniques provide an initial screening of the desired properties, more detailed follow-up studies of the selected materials are required to understand the true value of a 'positive hit'. High-throughput methods may become important tools in the future development of biomaterials-based cell cultures that will enable more realistic pre-clinical prediction of pharmacokinetics, pharmacodynamics, and toxicity. This is highly important, because predictive pre-clinical methods are needed to improve the high attrition rate of drug candidates during clinical testing.

Generation of uniform-sized multicellular tumor spheroids using hydrogel microwells for advanced drug screening.

Even though in vitro co-culture tumor spheroid model plays an important role in screening drug candidates, its wide applications are currently limited due to the lack of reliable and high throughput methods for generating well-defined and 3D complex co-culture structures. Herein, we report the development of a hydrogel microwell array to generate uniform-sized multicellular tumor spheroids. Our developed multicellular tumor spheroids are structurally well-defined, robust and can be easily transferred into the widely used 2D culture substrates while maintaining our designed multicellular 3D-sphere structures. Moreover, to develop effective anti-cancer therapeutics we integrated our recently developed gold-graphene hybrid nanomaterial (Au@GO)-based photothermal cancer therapy into a series of multicellular tumor spheroid co-culture system. The multicellular tumor spheroids were harvested onto a two-dimensional (2D) substrate, under preservation of their three-dimensional (3D) structure, to evaluate the photothermal therapy effectiveness of graphene oxide (GO)-wrapped gold nanoparticles (Au@GO). From the model of co-culture spheroids of HeLa/Ovarian cancer and HeLa/human umbilical vein endothelial cell (HUVEC), we observed that Au@GO nanoparticles displayed selectivity towards the fast-dividing HeLa cells, which could not be observed to this extent in 2D cultures. Overall, our developed uniform-sized 3D multicellular tumor spheroid could be a powerful tool for anticancer drug screening applications.

A microfluidic multi-injector for gradient generation.

This paper describes a microfluidic multi-injector (MMI) that can generate temporal and spatial concentration gradients of soluble molecules. Compared to conventional glass micropipette-based methods that generate a single gradient, the MMI exploits microfluidic integration and actuation of multiple pulsatile injectors to generate arbitrary overlapping gradients that have not previously been possible. The MMI device is fabricated in poly(dimethylsiloxane) (PDMS) using multi-layer soft lithography and consists of fluidic channels and control channels with pneumatically actuated on-chip barrier valves. Repetitive actuation of on-chip valves control pulsatile release of solution that establishes microscopic chemical gradients around the orifice. The volume of solution released per actuation cycle ranged from 30 picolitres to several hundred picolitres and increased linearly with the duration of valve opening. The shape of the measured gradient profile agreed closely with the simulated diffusion profile from a point source. Steady state gradient profiles could be attained within 10 minutes, or less with an optimized pulse sequence. Overlapping gradients from 2 injectors were generated and characterized to highlight the advantages of MMI over conventional micropipette assays. The MMI platform should be useful for a wide range of basic and applied studies on chemotaxis and axon guidance.

Microfluidic fabrication of microengineered hydrogels and their application in tissue engineering.

Microfluidic technologies are emerging as an enabling tool for various applications in tissue engineering and cell biology. One emerging use of microfluidic systems is the generation of shape-controlled hydrogels (i.e., microfibers, microparticles, and hydrogel building blocks) for various biological applications. Furthermore, the microfluidic fabrication of cell-laden hydrogels is of great benefit for creating artificial scaffolds. In this paper, we review the current development of microfluidic-based fabrication techniques for the creation of fibers, particles, and cell-laden hydrogels. We also highlight their emerging applications in tissue engineering and regenerative medicine.

Two-phase bioreactor system for cell-laden hydrogel assembly.

Bottom-up approach is a potentially useful tool for hydrogel assembly of cell-laden individual building blocks. In this article, we assembled individual building blocks of photocrosslinkable microgels in a rapid and controlled manner. Individual building blocks of poly(ethylene glycol) (PEG) microgels with square and hexagonal shapes were fabricated by using a photolithography technique. Individual building blocks of PEG microgels were assembled on a hydrophobic mineral oil phase in a bioreactor with a magnetic stirrer. The hydrophobic mineral oil minimized the surface free energy to assemble hydrophilic PEG microgels on a two-phase oil-aqueous solution interface. We used the hydrophobic effect as a driving force for the hydrogel assembly. Various types of the hydrogel assembly were generated by controlling the stirring rate. As stirring speed increased, the percentage of linear, branched, and closely packed hydrogel assembly was increased. However, the percentage of random assembly was reduced by increasing stirring rate. The stirring time also played an important role in controlling the types of hydrogel assembly. The percentage of linear, branched, and closely packed hydrogel assembly was improved by increasing stirring time. Therefore, we performed directed cell-laden hydrogel assembly using a two-phase bioreactor system and optimized the stirring rate and time to regulate the desired types of hydrogel assembly. Furthermore, we analyzed cell viability of hydrogel linear assembly with square shapes, showing highly viable even after secondary photocrosslinking reaction. This bioreactor system-based hydrogel assembly could be a potentially powerful approach for creating tissue microarchitectures in a three-dimensional manner.

Microfabricated multilayer parylene-C stencils for the generation of patterned dynamic co-cultures.

Co-culturing different cell types can be useful to engineer a more in vivo-like microenvironment for cells in culture. Recent approaches to generating cellular co-cultures have used microfabrication technologies to regulate the degree of cell-cell contact between different cell types. However, these approaches are often limited to the co-culture of only two cell types in static cultures. The dynamic aspect of cell-cell interaction, however, is a key regulator of many biological processes such as early development, stem cell differentiation, and tissue regeneration. In this study, we describe a micropatterning technique based on microfabricated multilayer parylene-C stencils and demonstrate the potential of parylene-C technology for co-patterning of proteins and cells with the ability to generate a series of at least five temporally controlled patterned co-cultures. We generated dynamic co-cultures of murine embryonic stem cells in culture with various secondary cell types that could be sequentially introduced and removed from the co-cultures. Our studies suggested that dynamic co-cultures generated by using parylene-C stencils may be applicable in studies investigating cellular interactions in controlled microenvironments such as studies of ES cell differentiation, wound healing and development.