Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO₂ sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.     

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.     

Novel fabrication of morphology tailored nanostructures with Gelatin/Chitosan Co-polymeric bio-composited hydrogel system to accelerate bone fracture healing and hard tissue nursing care management

In this study, we are successfully fabricated on a hydrogel consisting of TiO₂ nanoparticles loaded onto a gelatin/chitosan matrix to control the acceleration of bone fracture healing and improved the nursing care applications. Each specimen (chitosan, gelatin and titanium dioxide) were characterized and confirmed by using different techniques, Fourier transforms infrared spectroscopy, X-ray diffraction analysis, Scanning Electron Microscopy with Elemental dispersive X-ray analysis, Thermo-gravimetric and Differential thermal analysis. In addition, the cell cytotoxicity results verified that the TiO₂/gelatin-chitosan hydrogel are nontoxic to osteoblasts. And cell fixation outcome after 5 days of incubation condition revels that the enhanced in vitro cell survival and cell spreading on the prepared TiO₂ incorporated hydrogel with respect to gelatin/chitosan hydrogel. Furthermore, TiO₂/gelatin-chitosan hydrogel nanostructures can modulate the bone fracture healing, indicating a potential application on nursing care.     

Synthesis and utilization of E. coli‐encapsulated PEG‐based microdroplet using a microfluidic chip for biological application

We report herein an effective strategy for encapsulating Escherichia coli in polyethylene glycol diacrylate (PEGDA) microdroplets using a microfluidic device and chemical polymerization. PEGDA was employed as a reactant due to the biocompatibility, high porosity, and hydrophilic property. The uniform size and shape of microdroplets are obtained in a single‐step process using microfluidic device. The size of microdroplets can be controlled through the changing continuous flow rate. The combination of microdroplet generation and chemical polymerization techniques provide unique environment to produce non‐toxic ways of fabricating microorganism‐encapsulated hydrogel microbeads. Due to these unique properties of micro‐sized hydrogel microbeads, the encapsulated E. coli can maintain viability inside of microbeads and green fluorescent protein (GFP) and red fluorescent protein (RFP) genes are efficiently expressed inside of microbeads after isopropyl‐β‐D ‐thiogalactopyranoside induction, suggesting that there is no low‐molecular weight substrate transfer limitation inside of microbeads. Furthermore, non‐toxic, gentle, and outstanding biocompatibility of microbeads, the encapsulated E. coli can be used in various applications including biotransformation, biosensing, bioremediation, and engineering of artificial cells. Biotechnol. Bioeng. 2010;107:747–751. © 2010 Wiley Periodicals, Inc.     

Cell Squeezing as a Robust,Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.     

Effect of nanoheat stimulation mediated by magnetic nanocomposite hydrogel on the osteogenic differentiation of mesenchymal stem cells

Hyperthermia has been considered as a promising healing treatment in bone regeneration. We designed a tissue engineering hydrogel based on magnetic nanoparticles to explore the characteristics of hyperthermia for osteogenic regeneration. This nanocomposite hydrogel was successfully fabricated by incorporating magnetic Fe₃O₄ nanoparticles into chitosan/polyethylene glycol (PEG) hydrogel, which showed excellent biocompatibility and were able to easily achieve increasing temperatures under an alternative magnetic field (AMF). With uniformly dispersed nanoparticles, the composite hydrogel resulted in high viability of mesenchymal stem cells (MSCs), and the elevated temperature contributed to the highest osteogenic differentiation ability compared with direct heat treatment applied under equal temperatures. Therefore, the nanoheat stimulation method using the magnetic nanocomposite hydrogel under an AMF may be considered as an alternative candidate in bone tissue engineering regenerative applications.     

Microfluidic bioassay system based on microarrays of hydrogel sensing elements entrapping quantum dot-enzyme conjugates

This paper presents a simple method to fabricate a microfluidic biosensor that is able to detect substrates for H(2)O(2)-generating oxidase. The biosensor consists of three components (quantum dot-enzyme conjugates, hydrogel microstructures, and a set of microchannels) that were hierarchically integrated into a microfluidic device. The quantum dot (QD)-enzyme conjugates were entrapped within the poly(ethylene glycol) (PEG)-based hydrogel microstructures that were fabricated within the microchannels by a photopatterning process. Glucose oxidase (GOX) and alcohol oxidase (AOX) were chosen as the model oxidase enzymes, conjugated to carboxyl-terminated CdSe/ZnS QDs, and entrapped within the hydrogel microstructures, which resulted in a fluorescent hydrogel microarray that was responsive to glucose or alcohol. The hydrogel-entrapped GOX and AOX were able to perform enzyme-catalyzed oxidation of glucose and alcohol, respectively, to produce H(2)O(2), which subsequently quenched the fluorescence of the conjugated QDs. The fluorescence intensity of the hydrogel microstructures decreased as the glucose and alcohol concentrations increased, and the detection limits of this system were found to be 50 μM of glucose and 70 μM of alcohol. Because each microchannel was able to carry out different assays independently, the simultaneous detection of glucose and alcohol was possible using our novel microfluidic device composed of multiple microchannels.     

Novel microbial photobioelectrochemical cell using an invasive ultramicroelectrode array and a microfluidic chamber

Objective

To fabricate a novel microbial photobioelectrochemical cell using silicon microfabrication techniques.

Results

High-density photosynthetic cells were immobilized in a microfluidic chamber, and ultra-microelectrodes in a microtip array were inserted into the cytosolic space of the cells to directly harvest photosynthetic electrons. In this way, the microbial photobioelectrochemical cell operated without the aid of electron mediators. Both short circuit current and open circuit voltage of the microbial photobioelectrochemical cell responded to light stimuli, and recorded as high as 250 pA and 45 mV, respectively.

Conclusion

A microbial photobioelectrochemical cell was fabricated with potential use in next-generation photosynthesis-based solar cells and sensors.

     

Phenotype of Hepatocyte Spheroids Behavior within Thermo-Sensitive Poly(NiPAAm-co-PEG-g-GRGDS) Hydrogel as a Cell Delivery Vehicle

Aggregates (spheroids) of specific cells are often regarded as a better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. Freshly harvested primary rat hepatocytes, cultivated as spheroids and entrapped in an adhesion molecules of Arg–Gly–Asp (RGD)-conjugated extracellular matrix, have been examined for differentiated morphology and enhanced liver-specific functions. A copolymer of RGD conjugated p(NiPAAm-co-PEG) hydrogel was used to entrap hepatocytes in the forms of spheroids. Over 28 days, entrapped the spheroids had a higher viability and produced albumin and urea at constant rates, while there was slight increase in cell numbers and reduction of albumin secretion in single cell culture in the hydrogel. Hepatocytes cultured in this way are a potentially useful three-dimensional cell system for application in a bioartificial liver device and bioreactor.The first two authors (Keun-Hong Park and Kun Na) are equally contributed to this work.     

用于药物筛选的微流控细胞阵列芯片

细胞区域分布培养以及如何有效地对微流体进行操控是微流控阵列芯片在细胞药物研究中的关键技术。本研究介绍了一种利用SU-8负性光刻胶模具和PDMS制作双层结构的微流控细胞阵列芯片的方法,该芯片通过C型的坝结构将进样细胞拦截在芯片的细胞培养的固定区域,键合双层PDMS构成阀控制层,阀网络的开关作用成功实现了芯片通道内微流体的操控,同时芯片设计了药物浓度梯度网络,产生6个不同浓度的药物刺激细胞。通过对芯片3种共培养细胞活性的检测和药物伊立替康(CTP-11)对肝癌细胞的浓度梯度刺激等实验结果验证该芯片在细胞研究和药物筛选等方面的可行性。     

Simple and Versatile 3D Printed Microfluidics Using Fused Filament Fabrication

The uptake of microfluidics by the wider scientific community has been limited by the fabrication barrier created by the skills and equipment required for the production of traditional microfluidic devices. Here we present simple 3D printed microfluidic devices using an inexpensive and readily accessible printer with commercially available printer materials. We demonstrate that previously reported limitations of transparency and fidelity have been overcome, whilst devices capable of operating at pressures in excess of 2000 kPa illustrate that leakage issues have also been resolved. The utility of the 3D printed microfluidic devices is illustrated by encapsulating dental pulp stem cells within alginate droplets; cell viability assays show the vast majority of cells remain live, and device transparency is sufficient for single cell imaging. The accessibility of these devices is further enhanced through fabrication of integrated ports and by the introduction of a Lego^®-like modular system facilitating rapid prototyping whilst offering the potential for novices to build microfluidic systems from a database of microfluidic components.     

Implantable microfluidic device for the formation of three-dimensional vasculature by human endothelial progenitor cells

Vasculogenesis is an important morphogenetic event for vascular tissue engineering and ischemic disease treatment. Stem and progenitor cells can contribute to vasculogenesis via endothelial differentiation and direct participation in blood vessel formation. In this study, we developed an implantable microfluidic device to facilitate formation of three-dimensional (3D) vascular structures by human endothelial progenitor cells (hEPCs). The microfluidic device was made of biodegradable poly(lactic-co-glycolic acid) (PLGA) using a microchannel patterned silicon wafer made by soft lithography. A collagen type I (Col I) hydrogel containing hEPCs filled the microfluidic channels to reconstitute a 3D microenvironment for facilitating vascular structure formation by hEPCs. The device seeded with hEPCs was implanted into the subcutaneous space of athymic mice and retrieved one and four weeks after implantation. Histology and immunohistochemistry revealed that hEPCs formed a 3D capillary network expressing endothelial cell-specific proteins in the channel of the PLGA microfluidic device. This result indicates that a 3D microscale extracellular matrix reconstituted in the microchannel can promote the endothelial differentiation of hEPCs and in turn hEPC-mediated vasculogenesis. The PLGA microfluidic device reported herein may be useful as an implantable tissue-engineering scaffold for vascularized tissue reconstruction and therapeutic angiogenesis.     

Low density cell culture of locust neurons in closed-channel microfluidic devices

Microfluidic channel systems were fabricated out of polydimethylsiloxane (PDMS) and used as culture vessels for primary culture of neurons from locust thoracic ganglia. In a biocompatibility study it was shown that cell adhesion and neuronal cell growth of locust neurons on uncoated PDMS was restricted. Coating with concanavalin A improved cell adhesion. In closed-channel microfluidic devices neurons were grown in static-bath culture conditions for more than 15 days. Cell densities of up to 20 cells/channel were not exceeded in low-density cultures but we also found optimal cell growth of single neurons inside individual channels. The first successful cultivation of insect neurons in closed-channel microfluidic devices provides a prerequisite for the development of low density neuronal networks on multi electrode arrays combined with microfluidic devices.     

A Novel Wick-Like Paper-Based Microfluidic Device for 3D Cell Culture and Anti-Cancer Drugs Screening

Paper is increasingly recognized as a portable substrate for cell culture, due to its low-cost, flexible, and special porous property, which provides a native cellular 3D microenvironment. Therefore, paper-based microfluidics has been developed for cell culture and biomedical analysis. However, the inability of continuous medium supply limits the wide application of paper devices for cell culture. Herein, a paper-based microfluidic device is developed with novel folded paper strips as wick-like structure, which is used for medium self-driven perfusion. The paper with patterns of hydrophilic channel, culture areas, and hydrophobic barrier could be easily fabricated through wax-printing. After printing, the hydrophilic paper strip at the periphery of the lower layer is then folded at 90° and extended into the medium container for continuous automatic supply of medium to the cell culture area. Tumor cells cultured in the paper device are tested for anti-cancer drug screening. Visualized cell viability and chemical sensitivity testing can be achieved by colorimetry combined with simple smartphone imaging, effectively reducing precision instrument dependence. The wick paper-based microfluidic device for cell culture endows the method the advantages of lower cost, ease-of-operation, miniaturization, and shows a great potential for large-scale cell culture, antibody drug production, and efficient screening.     

Microfluidic formation of single cell array for parallel analysis of Ca release-activated Ca (CRAC) channel activation and inhibition

High-throughput single cell analysis is required for understanding and predicting the complex stochastic responses of individual cells in changing environments. We have designed a microfluidic device consisting of parallel, independent channels with cell-docking structures for the formation of an array of individual cells. The microfluidic cell array was used to quantify single cell responses and the distribution of response patterns of calcium channels among a population of individual cells. In this device, 15 cell-docking units in each channel were fabricated with each unit containing 5 sandbag structures, such that an array of individual cells was formed in 8 independent channels. Single cell responses to different treatments in different channels were monitored in parallel to study the effects of the specific activator and inhibitor of the Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Multichannel detection was performed to obtain the response patterns of the population of cells within this single cell array. The results demonstrate that it is possible to acquire single cell features in multichannels simultaneously with passive structural control, which provides an opportunity for high-throughput single cell response analysis in a microfluidic chip.     

Separation of apoptotic cells using a microfluidic device

A highly sensitive microfluidic device has been developed to separate apoptotic cells. Apoptotic Jurkat cells were selectively labeled with magnetic beads (0.8 μm diam) using the C2A protein which recognizes phosphatidylserine. The cell mixture was flowed through a microfluidic channel and apoptotic cells were separated by a 0.3 T permanent magnet. Separations using our device showed 96% agreement with those of a commercial flow cytometer, indicating our device can be used to sort apoptotic cells in a miniaturized system.     

Preparation and Characterization of Electrospun PLCL/Poloxamer Nanofibers and Dextran/Gelatin Hydrogels for Skin Tissue Engineering

In this study, two different biomaterials were fabricated and their potential use as a bilayer scaffold for skin tissue engineering applications was assessed. The upper layer biomaterial was a Poly(ε-caprolactone-co-lactide)/Poloxamer (PLCL/Poloxamer) nanofiber membrane fabricated using electrospinning technology. The PLCL/Poloxamer nanofibers (PLCL/Poloxamer, 9/1) exhibited strong mechanical properties (stress/strain values of 9.37±0.38 MPa/187.43±10.66%) and good biocompatibility to support adipose-derived stem cells proliferation. The lower layer biomaterial was a hydrogel composed of 10% dextran and 20% gelatin without the addition of a chemical crosslinking agent. The 5/5 dextran/gelatin hydrogel displayed high swelling property, good compressive strength, capacity to present more than 3 weeks and was able to support cells proliferation. A bilayer scaffold was fabricated using these two materials by underlaying the nanofibers and casting hydrogel to mimic the structure and biological function of native skin tissue. The upper layer membrane provided mechanical support in the scaffold and the lower layer hydrogel provided adequate space to allow cells to proliferate and generate extracellular matrix. The biocompatibility of bilayer scaffold was preliminarily investigated to assess the potential cytotoxicity. The results show that cell viability had not been affected when cocultured with bilayer scaffold. As a consequence, the bilayer scaffold composed of PLCL/Poloxamer nanofibers and dextran/gelatin hydrogels is biocompatible and possesses its potentially high application prospect in the field of skin tissue engineering.     

Development of a Microfluidic Culture Paradigm for Ex Vivo Maintenance of Human Glioblastoma Tissue: A New Glioblastoma Model?

BACKGROUND: One way to overcome the genetic and molecular variations within glioblastoma is to treat each tumour on an individual basis. To facilitate this, we have developed a microfluidic culture paradigm that maintains human glioblastoma tissue ex vivo. METHODS: The assembled device, fabricated using a photolithographic process, is composed of two layers of glass bonded together to contain a tissue chamber and a network of microchannels that allow continued tissue perfusion. RESULTS: A total of 128 tissue biopsies (from 33 patients) were maintained in microfluidic devices for an average of 72 hours. Tissue viability (measured with Annexin V and propidium iodide) was 61.1% in tissue maintained on chip compared with 68.9% for fresh tissue analysed at commencement of the experiments. Other biomarkers, including lactate dehydrogenase absorbance and trypan blue exclusion, supported the viability of the tissue maintained on chip. Histological appearances remained unchanged during the tissue maintenance period, and immunohistochemical analysis of Ki67 and caspase 3 showed no significant differences when compared with fresh tissues. A trend showed that tumours associated with poorer outcomes (recurrent tumours and Isocitrate Dehydrogenase - IDH wildtype) displayed higher viability on chip than tumours linked with improved outcomes (low-grade gliomas, IDH mutants and primary tumours). conclusions: This work has demonstrated for the first time that human glioblastoma tissue can be successfully maintained within a microfluidic device and has the potential to be developed as a new platform for studying the biology of brain tumours, with the long-term aim of replacing current preclinical GBM models and facilitating personalised treatments.     

Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds as a cell delivery vehicle: Characterization of PC12 cell response

The central nervous system (CNS) has a low intrinsic potential for regeneration following injury and disease, yet neural stem/progenitor cell (NPC) transplants show promise to provide a dynamic therapeutic in this complex tissue environment. Moreover, biomaterial scaffolds may improve the success of NPC‐based therapeutics by promoting cell viability and guiding cell response. We hypothesized that a hydrogel scaffold could provide a temporary neurogenic environment that supports cell survival during encapsulation, and degrades completely in a temporally controlled manner to allow progression of dynamic cellular processes such as neurite extension. We utilized PC12 cells as a model cell line with an inducible neuronal phenotype to define key properties of hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds that impact cell viability and differentiation following release from the degraded hydrogel. Adhesive peptide ligands (RGDS, IKVAV, or YIGSR), were required to maintain cell viability during encapsulation; as compared to YIGSR, the RGDS, and IKVAV ligands were associated with a higher percentage of PC12 cells that differentiated to the neuronal phenotype following release from the hydrogel. Moreover, among the hydrogel properties examined (e.g., ligand type, concentration), total polymer density within the hydrogel had the most prominent effect on cell viability, with densities above 15% w/v leading to decreased cell viability likely due to a higher shear modulus. Thus, by identifying key properties of degradable hydrogels that affect cell viability and differentiation following release from the hydrogel, we lay the foundation for application of this system towards future applications of the scaffold as a neural cell delivery vehicle. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1255–1264, 2013     

Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology.