Micropatterning of a nanoporous alumina membrane with poly(ethylene glycol) hydrogel to create cellular micropatterns on nanotopographic substrates

In this paper, we describe a simple method for fabricating micropatterned nanoporous substrates that are capable of controlling the spatial positioning of mammalian cells. Micropatterned substrates were prepared by fabricating poly(ethylene glycol) (PEG) hydrogel microstructures on alumina membranes with 200 nm nanopores using photolithography. Because hydrogel precursor solution could infiltrate and become crosslinked within the nanopores, the resultant hydrogel micropatterns were firmly anchored on the substrate without the use of adhesion-promoting monolayers, thereby allow tailoring of the surface properties of unpatterned nanoporous areas. For mammalian cell patterning, arrays of microwells of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding nanoporous bottoms that were modified with cell-adhesive Arg-Gly-Asp (RGD) peptides. Because the PEG hydrogel was non-adhesive towards proteins and cells, cells adhered selectively and remained viable within the RGD-modified nanoporous regions, thereby creating cellular micropatterns. Although the morphology of cell clusters and the number of cells inside one microwell were dependent on the lateral dimension of the microwells, adhered cells that were in direct contact with nanopores were able to penetrate into the nanopores by small extensions (filopodia) for all the different sizes of microwells evaluated.     

The use of glass substrates with bi-functional silanes for designing micropatterned cell-secreted cytokine immunoassays

It is often desirable to sequester cells in specific locations on the surface and to integrate sensing elements next to the cells. In the present study, surfaces were fabricated so as to position cytokine sensing domains inside non-fouling poly(ethylene glycol) (PEG) hydrogel microwells. Our aim was to increase sensitivity of micropatterned cytokine immunoassays through covalent attachment of biorecognition molecules. To achieve this, glass substrates were functionalized with a binary mixture of acrylate- and thiol-terminated methoxysilanes. During subsequent hydrogel photopatterning steps, acrylate moieties served to anchor hydrogel microwells to glass substrates. Importantly, glass attachment sites within the microwells contained thiol groups that could be activated with a hetero-bifunctional cross-linker for covalent immobilization of proteins. After incubation with fluorescently-labeled avidin, microwells fabricated on a mixed acryl/thiol silane layer emitted ～ 6 times more fluorescence compared to microwells fabricated on an acryl silane alone. This result highlighted the advantages of covalent attachment of avidin inside the microwells. To create cytokine immunoassays, micropatterned surfaces were incubated with biotinylated IFN-γ or TNF-α antibodies (Abs). Micropatterned immunoassays prepared in this manner were sensitive down to 1?ng/ml or 60?pM IFN-γ. To further prove utility of this biointerface design, macrophages were seeded into 30?μm diameter microwells fabricated on either bi-functional (acryl/thiol) or mono-functional silane layers. Both types of microwells were coated with avidin and biotin-anti-TNF-α prior to cell seeding. Short mitogenic activation followed by immunostaining for TNF-α revealed that microwells created on bi-functional silane layer had 3 times higher signal due to macrophage-secreted TNF-α compared to microwells fabricated on mono-functional silane. The rational design of cytokine-sensing surfaces described here, will be leveraged in the future for rapid detection of multiple cytokines secreted by individual immune cells.     

A microwell cell culture platform for the aggregation of pancreatic β-cells

Cell-cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D β-cell aggregates of defined sizes from 25 to 210 μm in diameter. Using this platform, mouse insulinoma 6 (MIN6) β-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability (>90%) over 7 days in culture, while the β-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single β-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional β-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered.     

Efficient Dielectrophoretic Patterning of Embryonic Stem Cells in Energy Landscapes Defined by Hydrogel Geometries

In this study, we have developed an integrated microfluidic platform for actively patterning mammalian cells, where poly(ethylene glycol) (PEG) hydrogels play two important roles as a non-fouling layer and a dielectric structure. The developed system has an embedded array of PEG microwells fabricated on a planar indium tin oxide (ITO) electrode. Due to its dielectric properties, the PEG microwells define electrical energy landscapes, effectively forming positive dielectrophoresis (DEP) traps in a low-conductivity environment. Distribution of DEP forces on a model cell was first estimated by computationally solving quasi-electrostatic Maxwell’s equations, followed by an experimental demonstration of cell and particle patterning without an external flow. Furthermore, efficient patterning of mouse embryonic stem (mES) cells was successfully achieved in combination with an external flow. With a seeding density of 10⁷ cells/mL and a flow rate of 3 μL/min, trapping of cells in the microwells was completed in tens of seconds after initiation of the DEP operation. Captured cells subsequently formed viable and homogeneous monolayer patterns. This simple approach could provide an efficient strategy for fabricating various cell microarrays for applications such as cell-based biosensors, drug discovery, and cell microenvironment studies.     

三维微小凹图式的制备及其与C17.2神经干细胞的复合

以紫外光光刻及氢氟酸湿法蚀刻加工硅阳模,采用基于聚二甲基硅氧烷(PDMS)的软光刻技术制备9种不同结构尺寸的聚乳酸-羟基乙酸共聚物(PLGA)和PMDS三维微小凹图式。PLGA及PDMS三维微小凹图式经等离子氧蚀刻和多聚赖氨酸裱衬处理后进行C17.2神经干细胞培养。随着在图式上培养时细胞的增殖,C17.2神经干细胞逐渐在微小凹中聚集,表现出明显的三维生长行为;通过羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA-SE)染色后进行激光共聚焦显微扫描与三维重构,显示大部分细胞生长于微小凹中离底面30～90μm的区间内;免疫荧光结果显示C17.2神经干细胞在三维微结构中复合培养2d后呈现均一的巢蛋白(Nestin)阳性。结论:本文设计的微小凹图式适用于C17.2神经干细胞的三维培养及后续的分化研究,细胞于微小凹图式培养过程中可以保持均一的干细胞特性。     

Therapeutic Micro and Nanotechnology Section: Microwell Array Photoimprint Lithography: Micropost Sensors and Guidance of PC12 Neurite Processes

Microwell array photoimprint lithography (PIL) is demonstrated by fabricating a patterned chemical sensing layer that is suitable for use with cultured biological cells and inverted epifluorescence microscopes. A microwell array is a 50,000-count, coherent fiber-optic bundle whose distal face was etched chemically. Individual microwells had 3 m depths and 8 m widths. The microwell array PIL process patterned an array of 50,000 individual polymeric micropost sensors on a glass coverslip. Individual microposts were 3 m tall and 8 m wide. O₂-sensitive micropost array sensors (MPASs) were fabricated using a ruthenium complex encapsulated in a gas permeable photopolymerizable siloxane. pH-sensitive MPASs were fabricated using a fluorescein conjugate encapsulated in a photocrosslinkable poly(vinyl alcohol)-based polymer. PO₂ and pH were quantitated by acquiring MPAS luminescence images with an epifluorescence microscope/charge coupled device imaging system. The advantage of a patterned MPAS layer relative to a planar sensing layer is the ability to direct the growth of biological cells. Preliminary data are presented whereby nerve growth factor-differentiated rat pheochromocytoma (PC12) cells grew neurite-like processes that extended along paths defined by the micropost architecture.     

Two-photon fabrication of hydrogel microstructures for excitation and immobilization of cells

We investigate in vitro fabrication of hydrogel microstructures by two photon laser lithography for single cell immobilization and excitation. Fluorescent yeast cells are embedded in water containing the hydrogel precursor mixtures and cross-linking is used to selectively immobilize a particular cell. Cell viability within the hydrogel precursor is estimated using a life/dead assay and elastic and stiff hydrogel structures are fabricated, immobilizing cells in a microfluidic environment. Additionally, we demonstrate the illumination of cells by on-the-fly fabricated hydrogel waveguide networks connected to an external light source, thereby exciting a fluorescence signal in a single immobilized cell.     

Suspension arrays of hydrogel microparticles prepared by photopatterning for multiplexed protein-based bioassays

Suspension arrays for protein-based assays have been developed using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles to overcome the problems with current systems which use color-coded rigid microparticles as protein supports. Various shapes of hydrogel microparticles were fabricated by a two-step process consisting of photopatterning and flushing using a poly(dimethylsiloxane) (PDMS) channel as a molding insert. Hydrogel microparticles with lateral dimensions ranging from 50 to 300 μm were fabricated using different molecular weights of PEG (700, 3,400, and 8,000 Da), by which the water content and swelling behavior of the hydrogel microparticles could be controlled. Protein-entrapped hydrogel microparticles were prepared in a suspension array format, and PEG hydrogel could encapsulate proteins without deactivation for a week due to its high water content and soft nature. The sequential bienzymatic reaction of hydrogel-entrapped glucose oxidase (GOX) and peroxidase (POD) was successfully investigated using fluorescence detection, demonstrating one possible application of suspension arrays. Furthermore, a mixture of two different shapes of hydrogel microparticles containing GOX/POD and alkaline phosphatase (AP), respectively, was prepared and the shape-coded suspension array was used for simultaneous characterization of two different enzyme-catalyzed reactions.     

Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells

Embryonic stem (ES) cells have the ability to self-replicate and differentiate into cells from all three germ layers, holding great promise for tissue regeneration applications. However, controlling the differentiation of ES cells and obtaining homogenous cell populations still remains a challenge. We hypothesize that a supportive three-dimensional (3D) environment provides ES cell-derived cells an environment that more closely mimics chondrogenesis in vivo. In the present study, the chondrogenic differentiation capability of ES cell-derived embryoid bodies (EBs) encapsulated in poly(ethylene glycol)-based (PEG) hydrogels was examined and compared with the chondrogenic potential of EBs in conventional monolayer culture. PEG hydrogel-encapsulated EBs and EBs in monolayer were cultured in vitro for up to 17 days in chondrogenic differentiation medium in the presence of transforming growth factor (TGF)-beta1 or bone morphogenic protein-2. Gene expression and protein analyses indicated that EB-PEG hydrogel culture upregulated cartilage-relevant markers compared with a monolayer environment and induction of chondrocytic phenotype was stimulated with TGF-beta1. Histology of EBs in PEG hydrogel culture with TGF-beta1 demonstrated basophilic extracellular matrix deposition characteristic of neocartilage. These findings suggest that EB-PEG hydrogel culture, with an appropriate growth factor, may provide a suitable environment for chondrogenic differentiation of intact ES cell-derived EBs.     

A microwell array system for stem cell culture

Directed embryonic stem (ES) cell differentiation is a potentially powerful approach for generating a renewable source of cells for regenerative medicine. Typical in vitro ES cell differentiation protocols involve the formation of ES cell aggregate intermediates called embryoid bodies (EBs). Recently, we demonstrated the use of poly(ethylene glycol) (PEG) microwells as templates for directing the formation of these aggregates, offering control over parameters such as size, shape, and homogeneity. Despite these promising results, the previously developed technology was limited as it was difficult to reproducibly obtain cultures of homogeneous EBs with high efficiency and retrievability. In this study, we improve the platform by optimizing a number of features: material composition of the microwells, cell seeding procedures, and aggregate retrieval methods. Adopting these modifications, we demonstrate an improved degree of homogeneity of the resulting aggregate populations and establish a robust protocol for eliciting high EB formation efficiencies. The optimized microwell array system is a potentially versatile tool for ES cell differentiation studies and high-throughput stem cell experimentation.     

A new dimension in suspension fusion techniques with polyethylene glycol

Polyethylene glycol (PEG) induces the hybridization of mammalian cells at a much higher frequency when the cells are attached to a substrate during treatment than when the cells are treated in suspension. Since many cell types, e.g., lymphocytes, cannot attach to a substrate, a new technique for the PEG-induced fusion of cells in suspension was developed. This technique, referred to as pancake fusion, is based on the centrifugation of suspended cells onto a coverslip and the PEG treatment of the cells on the coverslip as if they were attached to a substrate. With this technique, the frequency of hybridization of human white blood cells, which are incapable of attaching to a substrate, can be greatly increased.     

Biospecific anchoring and spatially confined germination of bacterial spores in non-biofouling microwells

In this paper, we report a simple method for spatially confining Bacillus subtilis (BS) spores into semi-three dimensional, non-biofouling microwells by using biospecific (such as biotin–streptavidin) interactions. Non-biofouling poly(ethylene glycol) (PEG)-based microwells were fabricated by employing a process of capillary molding on a glass slide. The biospecific interactions between biotinylated BS spores and streptavidin led to the selective deposition of BS spores onto the bottom of the microwells of which presented streptavidin. The viability of the patterned spores was confirmed by the induction of germination. Bacterial spores were found to maintain extreme robustness until they were exposed to favorable conditions. This work suggests that the use of bacterial spore-based sensors would increase the shelf-life (such as long-term storage and stability) of cell-based sensors.     

DNA delivery from matrix metalloproteinase degradable poly(ethylene glycol) hydrogels to mouse cloned mesenchymal stem cells

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation and to study molecular pathways in three dimensions than protein delivery. In this report, we explored gene transfer to infiltrating MSCs into matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP-degradable peptides via Michael addition chemistry. A large fraction of encapsulated polyplexes remained active after encapsulation (65%) and the mechanical properties of the hydrogels were unchanged by the encapsulation of the polyplexes. Cells were seeded inside the hydrogel scaffolds using two different approaches: clustered and homogeneous. The viability of MSCs was similar in hydrogels with and without polyplexes. Transgene expression was characterized with time using a secreted reporter gene and showed different profiles for clustered and homogeneously seeded cells. Clustered cells resulted in cumulative transgene expression that increased through the 21-day incubation, while homogeneously seeded cells resulted in cumulative transgene expression that plateaued after 7 days of culture. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long lasting signals in vivo, which are essential for the regeneration of functional tissues.     

Poly(acrylamide‐co‐itaconic acid) and Semi‐IPNS with Poly(ethylene glycol): Preparation and Characterization

Summary: A pH‐responsive poly(acrylamide‐co‐itaconic acid) (PAAm/IA) hydrogel and semi‐interpenetrating networks (semi‐IPNs) with 5, 10 and 15 wt.‐% of poly(ethylene glycol) (PAAm/IA/PEG), were synthesized. Their swelling behavior was studied in the pH range from 1.76 to 7.81, as well as their oscillatory swelling behavior at pH = 7.81 and pH = 1.7. Throughout these studies, the gels maintained their mechanical strengths and shape. The shear storage (G′) and loss (G″) moduli, obtained as a function of frequency, for the gels as formed and at equilibrium swelling were higher for the semi‐IPNs than for the copolymer hydrogel. The shear storage moduli of copolymer hydrogel and semi‐IPNs as formed were independent of frequency over the whole experimental range, whereas the values for the gels at equilibrium swelling decreased with increasing degree of swelling, i.e., the PAAm/IA hydrogel which exhibited the largest swelling had the lowest G′ value. The G′ and G″ values also depended on the content of PEG.

Diffusion exponent vs. pH for PAAm, copolymer hydrogel PAAm/IA and semi‐IPN with PEG.     

Control of neural cell composition in poly(ethylene glycol) hydrogel culture with soluble factors

Poly(ethylene glycol) (PEG) hydrogels are being developed as cell delivery vehicles that have great potential to improve neuronal replacement therapies. Current research priorities include (1) characterizing neural cell growth within PEG hydrogels relative to standard culture systems and (2) generating neuronal-enriched populations within the PEG hydrogel environment. This study compares the percentage of neural precursor cells (NPCs), neurons, and glia present when dissociated neural cells are seeded within PEG hydrogels relative to standard monolayer culture. Results demonstrate that PEG hydrogels enriched the initial cell population for NPCs, which subsequently gave rise to neurons, then to glia. Relative to monolayer culture, PEG hydrogels maintained an increased percentage of NPCs and a decreased percentage of glia. This neurogenic advantage of PEG hydrogels is accentuated in the presence of basic fibroblast growth factor and epidermal growth factor, which more potently increase NPC and neuronal expression markers when applied to cells cultured within PEG hydrogels. Finally, this work demonstrates that glial differentiation can be selectively eliminated upon supplementation with a γ-secretase inhibitor. Together, this study furthers our understanding of how the PEG hydrogel environment influences neural cell composition and also describes select soluble factors that are useful in generating neuronal-enriched populations within the PEG hydrogel environment.     

An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells

With the goal to investigate the relation of shape and function of single cells or clusters of cells in a 3-dimensional (3-D) microenvironment, we present a novel platform technology to create arrays of microwells on polystyrene (PS) chips for hosting cells in a local microenvironment characterized by controlled shape and surface chemistry. The micro-3-D cell culturing combines 2-dimensional chemical patterning with topographical microstructuring presenting to the cells a local 3-D host structure. Microwells of controlled dimensions were produced by a two-step replication process, based on standard microfabrication of Si, replica molding into poly(dimethylsiloxane), and hot embossing of PS. This allowed the production of large numbers of microstructured surfaces with high reproducibility and fidelity of replication. Using inverted micro contact printing, the plateau surface between the microwells was successfully passivated to block adsorption of proteins and prevent cell attachment by transfer of a graft-copolymer, poly(l-lysine)-g-poly(ethylene glycol). The surface inside the microwells was subsequently modified by spontaneous adsorption of proteins or functionalized PLL-g-PEG/PEG-X (X=biotin or specific, cell-interactive peptide) to elicit specific responses inside the wells. Preliminary cell experiments demonstrated the functionality of such a device to host single epithelial cells (MDCK II) inside the functionalized microwells and thus to control their 3-D shape. This novel platform is useful for fundamental cell-biological studies and applications in the area of cell-based sensing.     

Investigation of translational motion of poly(ethylene glycol) macromolecules in poly(methacrylic acid) hydrogels

The translational mobility of linear macromolecules of poly(ethylene glycol) (PEG) within a weakly cross-linked poly(methacrylic acid) (PMAA) hydrogel was investigated by means of the pulse field gradient (PFG) NMR method in order to reveal the effect of PMAA/PEG complex formation. It was found that inside the collapsed gel a fraction of the PEG molecules has self-diffusion characteristics like those of the network chains. This suggests the formation of an interpolymer complex, as a result of which some linear molecules acquired the dynamic properties of the network chains. Another fraction of the PEG macromolecules inside the collapsed gel enjoyed free diffusion, for they were not included in the complex with PMAA. In contrast, within the swollen gel (at concentrations of PEG higher than 5 wt.-%) the self-diffusion coefficient of all PEG molecules was independent of the diffusion time, which indicates an absence of the interpolymer complex (or at least that its lifetime is negligibly short).     

Agarose microwell assay for cell-mediated cytotoxicity

A colony-inhibition micromethod for tumour cells is described, which conbines the high cloning efficiency of the dilute agar test (Heppner, 1973) with the quantitation advantages of actylamide microwells (Haskill, 1973), simply by moulding microwells in agarose over a feeder layer. It uses 150 tumour cells per 30 mm dish of 200 wells, with cloning efficiency of 50–80%. At an effector: target cell ratio of 10 : 1 the agarose microwell assay method gives a colony-inhibition of 25–85%.     

Poly(ethylene glycol) hydrogels conjugated with a collagenase-sensitive fluorogenic substrate to visualize collagenase activity during three-dimensional cell migration

We have developed collagenase-sensitive hydrogels by incorporating a collagenase-sensitive fluorogenic substrate (CS-FS) within the backbone of a polyethylene glycol (PEG) copolymer to visualize collagenase activity during three-dimensional cell migration. CS-FS was synthesized by conjugating Bodipy dyes to a peptide with collagenase-sensitive sequence, Leu-Gly-Pro-Ala (LGPA), and the products were grafted into the collagenase-sensitive PEG hydrogels. CS-FS both in solution and hydrogels had an increase in the fluorescence intensity after proteolytic degradation by collagenase, but not by non-targeted proteases nor in the absence of an enzyme. Fibroblasts inside the hydrogels conjugated with CS-FS spread and extended lamellipodia in three dimensions over several days, and their pericellular collagenase-mediated proteolysis of the hydrogel was visualized via confocal microscopy. A matrix metalloproteinase inhibitor, served as a negative control, significantly reduced the degradation rate of CS-FS by collagenase and prevented cell migration and cell-mediated collagenase activity inside these hydrogels. In summary, we have fabricated collagenase-sensitive hydrogels incorporated with CS-FS and successfully visualized the collagenase activity during three-dimensional cell migration.