Hanry Yu

A novel 3D mammalian cell perfusion-culture system in microfluidic channels

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.

A practical guide to microfluidic perfusion culture of adherent mammalian cells

Culturing cells at microscales allows control over microenvironmental cues, such as cell-cell and cell-matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.

Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits.

Prolonged delivery of neurotrophic proteins to the target tissue is valuable in the treatment of various disorders of the nervous system. We have tested in this study whether sustained release of nerve growth factor (NGF) within nerve guide conduits (NGCs), a device used to repair injured nerves, would augment peripheral nerve regeneration. NGF-containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site-specific delivery of NGF. The conduits were used to bridge a 10 mm gap in a rat sciatic nerve model. Three months after implantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower G-ratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, as compared with those from control conduits loaded with either saline alone, BSA microspheres, or NGF protein without microencapsulation. Beneficial effects on fiber diameter, G-ratio and fiber density were also observed in the permeable PPE NGCs. Thus, the results confirm a long-term promoting effect of exogenous NGF on morphological regeneration of peripheral nerves. The tissue-engineering approach reported in this study of incorporation of a microsphere protein release system into NGCs holds potential for improved functional recovery in patients whose injured nerves are reconstructed by entubulation.

A gel-free 3D microfluidic cell culture system

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

A new nerve guide conduit material composed of a biodegradable poly(phosphoester)

There is a resurgence of interest in the development of degradable and biocompatible polymers for fabrication of nerve guide conduits (NGCs) in recent years. Poly(phosphoester) (PPE) polymers are among the attractive candidates in this context, in view of their high biocompatibility, adjustable biodegradability, flexibility in coupling fragile biomolecules under physiological conditions and a wide variety of physicochemical properties. The feasibility of using a biodegradable PPE, P(BHET-EOP/TC), as a novel NGC material was investigated. Two types of conduits were fabricated by using two batches of P(BHET-EOP/TC) with different weight-average molecular weights (Mw) and polydispersity indexes (PI). The polymers as well as conduits were non-toxic to all six types of cells tested, including primary neurones and neuronally differentiated PC12 cells. After in situ implantation in the sciatic nerve of the rat, two types of conduits triggered a similar tissue response, inducing the formation of a thin tissue capsule composed of approximately eight layers of fibroblasts surrounding the conduits at 3 months. Biological performances of the conduits were examined in the rat sciatic nerve model with a 10 mm gap. Although tube fragmentation, even tube breakage, was observed within less than 5 days post-implantation, successful regeneration through the gap occurred in both types of conduits, with four out of 10 in the Type I conduits (Mw 14,900 and PI 2.57) and 11 out of 12 in the Type II conduits (Mw 18,900 and PI 1.72). The degradation of conduits was further evidenced by increased roughness on the tube surface in vivo under scanning electron microscope and a mass decrease in a time-dependent manner in vitro. The Mw of the polymers dropped 33 and 24% in the Type I and II conduits, respectively, in vitro within 3 months. Among their advantages over other biodegradable NGCs, the PPE conduits showed negligible swelling and no crystallisation after implantation. Thus, these PPE conduits can be effective aids for nerve regeneration with potential to be further developed into more sophisticated NGCs that have better control of the conduit micro-environment for improved nerve regeneration.

Multi-layered microcapsules for cell encapsulation.

Mechanical stability, complete encapsulation, selective permeability, and suitable extra-cellular microenvironment, are the major considerations in designing microcapsules for cell encapsulation. We have developed four types of multi-layered microcapsules that allow selective optimization of these parameters. Primary hepatocytes were used as model cells to test these different microcapsule configurations. Type-1 microcapsules with an average diameter of 400 microm were formed by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 microm. Cells in these microcapsules exhibited improved cellular functions over those cultured on collagen monolayers. Type-II microcapsules were formed by encapsulating the Type-I microcapsules in another 2-5 microm ter-polymer shell and a approximately 5 microm collagen layer between the two ter-polymer shells to ensure complete cell encapsulation. Type-II microcapsules comprised of a macro-porous exoskeleton with materials such as alumina sol-gel coated on the Type-I microcapsules. Nano-indendation assay indicated an improved mechanical stability over the Type-I microcapsules. Type-IV microcapsules were created by encapsulating Type-III microcapsules in another 2-5 microm ter-polymer shell, with the aim of imparting a negatively charged smooth surface to minimize plasma protein absorption and ensure complete cell encapsulation. The permeability for nutrient exchange, cellular functions in terms of urea production and mechanical stability of the microcapsules were characterized. The advantages and limitations of these microcapsules for tissue engineering are discussed.

Mechanotransduction in vivo by repeated talin stretch-relaxation events depends upon vinculin.

The focal adhesion protein talin undergoes cycles of stretching and relaxation in living cells, suggesting a role in the transduction of mechanical into biochemical signals.

Enhanced activation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML

To further investigate potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R. Gene profiling reveals up-regulation of FLT3LG (FLT3 ligand) and BIRC5 (survivin), but down-regulation of SOCS1, SOCS2, and SOCS3 in MV4-11-R cells. Hypermethylation of these SOCS genes leads to their transcriptional silencing. Survivin is directly regulated by STAT3. Stimulation of the parental MV4-11 cells with FLT3 ligand increases the expression of survivin and phosphorylated protein STAT1, STAT3, STAT5. Targeting survivin by short-hairpin RNA (shRNA) in MV4-11-R cells induces apoptosis and augments ABT-869-mediated cytotoxicity. Overexpression of survivin protects MV4-11 from apoptosis. Subtoxic dose of indirubin derivative (IDR) E804 resensitizes MV4-11-R to ABT-869 treatment by inhibiting STAT signaling activity and abolishing survivin expression. Combining IDR E804 with ABT-869 shows potent in vivo efficacy in the MV4-11-R xenograft model. Taken together, these results demonstrate that enhanced activation of STAT pathways and overexpression of survivin are important mechanisms of resistance to ABT-869, suggesting that the STAT pathways and survivin could be potential targets for reducing resistance developed in patients receiving FLT3 inhibitors.

Polyphosphoester microspheres for sustained release of biologically active nerve growth factor

Controlled delivery of neurotrophic proteins to a target tissue by biodegradable polymer microspheres has been explored widely for its potential applications in the treatment of various disorders in the nervous system. We investigated in this study the potential of polyphosphoester microspheres as carriers for the sustained release of nerve growth factor (NGF), a water-soluble neurotrophic protein. Two polyphosphoesters (PPEs), P(BHET-EOP/TC) and P(DAPG-EOP), as well as poly(lactide/glycolic acid) (PLGA), were used to fabricate microspheres by a W/O/W emulsion and solvent evaporation/extraction method. With bovine serum albumin as a model protein to optimize processing parameters. P(DAPG-EOP) microspheres exhibited a lower burst effect but similar protein entrapment levels and efficiencies when compared with those made of PLGA. Bioactive NGF could be released for at least 10 weeks from the P(DAPG-EOP) microspheres, as confirmed by a neurite outgrowth assay of the PC12 cells. These NGF containing microspheres were incorporated into the nerve guide conduits that were implanted to bridge a 10 mm gap in a rat sciatic nerve model. Two weeks after implantation, immunostaining with an antibody against the neurofilament protein confirmed the presence of axons at the distal end of regenerated cables within the NGF microsphere-loaded conduits. These results demonstrated the feasibility of using biodegradable PPEs for microencapsulation of NGF and provided a basis for future therapeutic application of the microspheres.

Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies

Three-dimensional (3D) in vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in vivo conditions. In cancer research, the multi-cellular tumor spheroid (MCTS) model is an established 3D cancer model that exhibits microenvironmental heterogeneity close to that of tumors in vivo. However, the established process of MCTS formation is time-consuming and often uncontrolled. Here, we report a method for engineering MCTS using a transient inter-cellular linker which facilitates cell-cell interaction. Using C3A cells (a hepatocellular carcinoma cell line) as a model, we formed linker-engineered spheroids which grew to a diameter of 250 microm in 7 days, as compared to 16 days using conventional non-adherent culture. Seven-day old linker-engineered spheroids exhibited characteristics of mature MCTS, including spheroid morphology, gene expression profile, cell-cell interaction, extracellular matrix secretion, proliferation and oxygen concentration gradients, and cellular functions. Linker-engineered spheroids also displayed a resistance to drug penetration similar to mature MCTS, with dose-dependent extracellular accumulation of the drug. The linker-engineered spheroids thus provide a reliable accelerated 3D in vitro tumor model for drug penetration studies.