Dynamic microarray system with gentle retrieval mechanism for cell-encapsulating hydrogel beads

This paper describes a selective retrieval method for arrayed monodisperse hydrogel beads containing cells. We implemented modifications such as: (i) the incorporation of cavities as nucleation sites, (ii) indirect retrieval using bubble powered jets and (iii) the use of low boiling point fluid in our device to realize a gentle optical-based retrieval method. Parametric studies confirmed that these modifications dramatically reduced both the intensity and duration of applied laser for bubble formation. We also demonstrated for the first time the formation of a bead-based dynamic cell microarray by introducing cell-encapsulating alginate beads into our dynamic microfluidic system, and successfully retrieved an alginate bead from a fluidic trap. Tests with trypan blue revealed that membrane integrity of the encapsulated cells was not compromised by the retrieval process.     

Preparation,Mass Diffusion,and Biocompatibility Analysis of Porous-Channel Controlled Calcium-Alginate-Gelatin Hybrid Microbeads for In Vitro Culture of NSCs

The Ca-alginate/gelatin (CAG) microbeads were prepared and evaluated through assays for their mechanical strength, permeability, and the feasibility as a cell carrier for in vitro culture of neural stem cells. The effects of different concentrations of sodium alginate, gelatin, and calcium chloride on the mechanical strength of CAG microbeads were determined using a self-made puncture force tester. Following this, the microbeads were immersed in DMEM media for a specified period to test its decay resistance. A diffusion model including a calculation formula of diffusion coefficient was built to investigate the diffusion of glucose and bovine serum albumin (BSA) through the wall of the microbeads. Furthermore, the feasibility of the microbeads for in vitro culture was identified using neural stem cells from Kunming mouse. Through a systematic approach and comprehensive analysis, the optimal gelatin conditions for microbead preparation were determined; the final combination of parameters of 1.5 % (wt%) sodium alginate (SA), 0.5 % (wt%) gelatin, and 4 % (wt%) CaCl₂ were the best conditions for NSC cultures. This experiment demonstrated that CAG microbeads had good cytocompatibility that made it suitable for the culture and successfully maintained stemness of neural stem cells.     

In vitro angiogenesis assay for the study of cell-encapsulation therapy

Cell encapsulation within alginate beads has potential as a sustained release system for delivering therapeutic agents in vivo while protecting encapsulated cells from the immune system. There is, however, no in vitro model for cell-encapsulation therapy that provides a suitable platform for quantitative assessment of physiological responses to secreted factors. Here we introduce a new microfluidic system specifically designed to evaluate and quantify the pro-angiogenic potential of factors secreted from human fetal lung fibroblasts encapsulated in beads on an intact endothelial cell monolayer. We confirmed that cell-encapsulating beads induced an angiogenic response in vitro, demonstrated by a strong correlation between the encapsulated cell density in the beads and the length of the vascular lumen formed in vitro. Conditions established by in vitro tests were then further shown to exert a pro-angiogenic response in vivo using a subcutaneous mouse model, forming an extensive network of functional luminal structures perfused with red blood cells.     

Microfluidic biofunctionalisation protocols to form multi‐valent interactions for cell rolling and phenotype modification investigations

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro‐fluidic protocols. Many available processes either require expensive and time‐consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi‐valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin–biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC‐I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC‐I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC‐I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC‐I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.     

Development of an integrated microsystem for injection, transport and manipulation of encoded microbeads

An integrated microsystem for injection, transport and manipulation of encoded microbeads on a single microchip is presented. The device also incorporates a customized reaction chamber to process individual, optically encoded microbeads. This research illustrates how microfabrication technologies enable convenient integration of multiple capabilities of microbeads, controlled microfluidic injection, integration of heater elements and temperature sensors and detection of microbeads in a single microfluidic chip. A practical application for the integrated microsystem is confirmed by the ability to select a specific DNA sequence of interest from a 4 x 4 cDNA library. This application emphasizes the advantages of component integration for rapid bio-assay development in a complete microsystem.     

Development of 3D Biofabricated Cell Laden Hydrogel Vessels and a Low‐Cost Desktop Printed Perfusion Chamber for In Vitro Vessel Maturation

The vascular system represents the key supply chain for nutrients and oxygen inside the human body. Engineered solutions to produce sophisticated alternatives for autologous or artificial vascular implants to sustainably replace diseased vascular tissue still remain a key challenge in tissue engineering. In this paper, cell‐laden 3D bioplotted hydrogel vessel‐like constructs made from alginate di‐aldehyde (ADA) and gelatin (GEL) are presented. The aim is to increase the mechanical stability of fibroblast‐laden ADA‐GEL vessels, tailoring them for maturation under dynamic cell culture conditions. BaCl₂ is investigated as a crosslinker for the oxidized alginate‐gelatin system. Normal human dermal fibroblast (NHDF)‐laden vessel constructs are optimized successfully in terms of higher stiffness by increasing ADA concentration and using BaCl₂, with no toxic effects observed on NHDF. Contrarily, BaCl₂ crosslinking of ADA‐GEL accelerates cell attachment, viability, and growth from 7d to 24h compared to CaCl₂. Moreover, alignment of cells in the longitudinal direction of the hydrogel vessels when extruding the cell‐laden hydrogel crosslinked with Ba²⁺ is observed. It is possible to tune the stiffness of ADA‐GEL by utilizing Ba²⁺ as crosslinker. In addition, a customized, low‐cost 3D printed polycarbonate (PC) perfusion chamber for perfusion of vessel‐like constructs is introduced.     

Cell sheet engineering: intelligent polymer patterned surfaces for tissue engineered liver

We describe a new culture system utilizing the temperature-responsive polymer grafted surface for designing of cell position and layered tissue reconstruction. Organizing of the hepatic tissue structure by controlling the culture system, that is patterned co-culture and layered cell sheet co-culture achieved by moving the cultured cells from the culture surface, resulted in regulation of the hepatocyte function. The technique for cell sheet manipulation would promote the liver tissue engineering in quality.     

Progress in Articular Cartilage Tissue Engineering: A Review on Therapeutic Cells and Macromolecular Scaffolds

Repair and regeneration of articular cartilage lesions have always been a major challenge in the medical field due to its peculiar structure (e.g., sparsely distributed chondrocytes, no blood supply, no nerves). Articular cartilage tissue engineering is considered as one promising strategy to achieve reconstruction of cartilage. With this perspective, the articular cartilage tissue engineering has been widely studied. Here, the recent progress of articular cartilage tissue engineering is reviewed. The ad hoc therapeutic cells and growth factors for cartilage regeneration are summarized and discussed. Various types of bio/macromolecular scaffolds together with their pros and cons are also reviewed and elaborated.     

Responsive microgrooves for the formation of harvestable tissue constructs

Given its biocompatibility, elasticity, and gas permeability, poly(dimethylsiloxane) (PDMS) is widely used to fabricate microgrooves and microfluidic devices for three-dimensional (3D) cell culture studies. However, conformal coating of complex PDMS devices prepared by standard microfabrication techniques with desired chemical functionality is challenging. This study describes the conformal coating of PDMS microgrooves with poly(N-isopropylacrylamide) (PNIPAAm) by using initiated chemical vapor deposition (iCVD). These microgrooves guided the formation of tissue constructs from NIH-3T3 fibroblasts that could be retrieved by the temperature-dependent swelling property and hydrophilicity change of the PNIPAAm. The thickness of swollen PNIPAAm films at 24 °C was approximately 3 times greater than at 37 °C. Furthermore, PNIPAAm-coated microgroove surfaces exhibit increased hydrophilicity at 24 °C (contact angle θ = 30° ± 2) compared to 37 °C (θ = 50° ± 1). Thus PNIPAAm film on the microgrooves exhibits responsive swelling with higher hydrophilicity at room temperature, which could be used to retrieve tissue constructs. The resulting tissue constructs were the same size as the grooves and could be used as modules in tissue fabrication. Given its ability to form and retrieve cell aggregates and its integration with standard microfabrication, PNIPAAm-coated PDMS templates may become useful for 3D cell culture applications in tissue engineering and drug discovery.     

Effect of the gelation process on the production of alginate microbeads by microfluidic chip technology

The present paper reports the production of Ba-alginate microspheres by microfluidic chip technology. The general production strategy is based on the formation of an alginate multiphase flow by a 'Y' junction squeezing mechanism. Special emphasis is given to the relationship existing between the gelation process and the final morphological characteristics of the produced microbeads. A series of different gelation strategies, namely: 'external gelation', 'internal gelation' and 'partial gelation' were compared in terms of size, size distribution and morphology of the produced microbeads.     

Applications of Gelatin Methacryloyl (GelMA) Hydrogels in Microfluidic Technique-Assisted Tissue Engineering

In recent years, the microfluidic technique has been widely used in the field of tissue engineering. Possessing the advantages of large-scale integration and flexible manipulation, microfluidic devices may serve as the production line of building blocks and the microenvironment simulator in tissue engineering. Additionally, in microfluidic technique-assisted tissue engineering, various biomaterials are desired to fabricate the tissue mimicking or repairing structures (i.e., particles, fibers, and scaffolds). Among the materials, gelatin methacrylate (GelMA)-based hydrogels have shown great potential due to their biocompatibility and mechanical tenability. In this work, applications of GelMA hydrogels in microfluidic technique-assisted tissue engineering are reviewed mainly from two viewpoints: Serving as raw materials for microfluidic fabrication of building blocks in tissue engineering and the simulation units in microfluidic chip-based microenvironment-mimicking devices. In addition, challenges and outlooks of the exploration of GelMA hydrogels in tissue engineering applications are proposed.     

Cell‐Friendly Inverse Opal‐Like Hydrogels for a Spatially Separated Co‐Culture System

Three‐dimensional macroporous scaffolds have extensively been studied for cell‐based tissue engineering but their use is mostly limited to mechanical support for cell adhesion and growth on the surface of macropores. Here, a templated fabrication method is described to prepare cell‐friendly inverse opal‐like hydrogels (IOHs) allowing both cell encapsulation within the hydrogel matrix and cell seeding on the surface of macropores. Ionically crosslinked alginate microbeads and photocrosslinkable biocompatible polymers are used as a sacrificial template and as a matrix, respectively. The alginate microbeads are easily removed by a chelating agent, with minimal toxicity for the encapsulated cells during template removal. The outer surface of macropores in IOHs can also provide a space for cell adherence. The cells encapsulated or attached in IOHs are able to remain viable and to proliferate over time. The elastic modulus and cell‐adhesion properties of IOHs can be easily controlled and tuned. Finally, it is demonstrated that IOH can be used to co‐culture two distinct cell populations in different spatial positions. This cell‐friendly IOH system provides a 3D scaffold for organizing different cell types in a controllable microenvironment to investigate biological processes such as stem cell niches or tumor microenvironments.

     

DNA-templated assembly of droplet-derived PEG microtissues

Patterning multiple cell types is a critical step for engineering functional tissues, but few methods provide three-dimensional positioning at the cellular length scale. Here, we present a "bottom-up" approach for fabricating multicellular tissue constructs that utilizes DNA-templated assembly of 3D cell-laden hydrogel microtissues. A flow focusing-generated emulsion of photopolymerizable prepolymer is used to produce 100 μm monodisperse microtissues at a rate of 100 Hz (10(5) h(-1)). Multiple cell types, including suspension and adherently cultured cells, can be encapsulated into the microtissues with high viability (~97%). We then use a DNA coding scheme to self-assemble microtissues "bottom-up" from a template that is defined using "top-down" techniques. The microtissues are derivatized with single-stranded DNA using a biotin-streptavidin linkage to the polymer network, and are assembled by sequence-specific hybridization onto spotted DNA microarrays. Using orthogonal DNA codes, we achieve multiplexed patterning of multiple microtissue types with high binding efficiency and >90% patterning specificity. Finally, we demonstrate the ability to organize multicomponent constructs composed of epithelial and mesenchymal microtissues while preserving each cell type in a 3D microenvironment. The combination of high throughput microtissue generation with scalable surface-templated assembly offers the potential to dissect mechanisms of cell-cell interaction in three dimensions in healthy and diseased states, as well as provides a framework for templated assembly of larger structures for implantation.     

Microfluidic chip-based fabrication of PLGA microfiber scaffolds for tissue engineering

In this paper, we have developed a method to produce poly(lactic- co-glycolic acid) (PLGA) microfibers within a microfluidic chip for the generation of 3D tissue engineering scaffolds. The synthesis of PLGA fibers was achieved by using a polydimethylsiloxane (PDMS)-based microfluidic spinning device in which linear streams of PLGA dissolved in dimethyl sulfoxide (DMSO) were precipitated in a glycerol-containing water solution. By changing the flow rate of PLGA solution from 1 to 50 microL/min with a sheath flow rate of 250 or 1000 microL/min, fibers were formed with diameters that ranged from 20 to 230 microm. The PLGA fibers were comprised of a dense outer surface and a highly porous interior. To evaluate the applicability of PLGA microfibers generated in this process as a cell culture scaffold, L929 fibroblasts were seeded on the PLGA fibers either as-fabricated or coated with fibronectin. L929 fibroblasts showed no significant difference in proliferation on both PLGA microfibers after 5 days of culture. As a test for application as nerve guide, neural progenitor cells were cultured and the neural axons elongated along the PLGA microfibers. Thus our experiments suggest that microfluidic chip-based PLGA microfiber fabrication may be useful for 3D cell culture tissue engineering applications.     

Generation of core-shell microcapsules with three-dimensional focusing device for efficient formation of cell spheroid

We present a microfluidic device generating three-dimensional (3D) coaxial flow by the addition of a simple hillock to produce an alginate core-shell microcapsule for the efficient formation of a cell spheroid. A hillock tapered at downstream of the two-dimensional focusing channel enables outside flow to enclose the core flow. The aqueous solution in the core flow was focused and surrounded by 1.8% alginate solution to be solidified as a shell. The double-layered coaxial flow (aqueous phase) was broken up into a droplet by the shear flow of oleic acid (oil phase) containing calcium chloride for the polymerization of the alginate shell. The droplet generated from the laminar coaxial flow maintained a double-layer structure and gelation of the alginate solution made a core-shell microcapsule. The shell-thickness of the microcapsule was adjusted from 8-21 μm by the variation of two aqueous flow rates. The inner shape of the shell was almost spherical when the ratio of the water-glycol mixture in the core flow exceeded 20%. The microcapsule was used to form a spheroid of embryonic carcinoma cells (embryoid body; EB) by injecting a cell suspension into the core flow. The cells inside the microcapsule aggregated into an EB within 2 days and the EB formation rate was more than 80% with strong compaction. The microcapsule formed single spherical EBs without small satellite clusters or a bumpy shape as observed in solid microbeads. The microfluidic chip for encapsulation of cells could generate a number of EBs with high rate of EB formation when compared with the conventional hanging drop method. The core-shell microcapsule generated by 3D focusing in the microchannel was effective in forming large number of spherical cell clusters and the encapsulation of cells in the microcapsule is expected to be useful in the transplantation of islet cells or cancer stem cell enrichment.     

Multi-Material Scaffolds for Tissue Engineering

A trend in developing biocompatible scaffolds for tissue engineering has been to seek an ideal single material for which a given cell type will exhibit favorable behavior. While an ideal single material has proven elusive, scaffold manufacture using combinations of specialist materials can produce more versatile structures. By controlling the percentage and architecture of material components, mechanical properties, cell attachment, and proliferation may be optimized for a given function. Three specialist materials, poly-ϵ-caprolactone (PCL), fibrin, and alginate, were incorporated into multi-component scaffolds for a series of experiments testing each component with culture of fibroblasts. The rigid and formable PCL provided structure, the fibrin pore-filler allowed for cell attachment, and alginate thread provided a nutrient transfer pathway in lieu of a vascular system. The efficacy of these scaffolds was judged on fibroblast distribution and population after 7-12 days of culture.     

结冷胶与聚乙二醇丙烯酸酯双网络凝胶的制备及生物相容性评价

针对结冷胶脆性较大的问题,将聚乙二醇丙烯酸酯(PEGDA)引入结冷胶,通过紫外交联制备了结冷胶/PEGDA双网络凝胶,并对单组分凝胶和双网络凝胶的溶胀性能、微观形貌、拉伸力学性能、动态压缩性能和流变性能等进行比较.结果表明,双网络凝胶在类生理环境中具有较小的溶胀率和较好的尺寸稳定性,PEGDA的引入能够大幅度提高结冷胶的韧性,双网络凝胶的拉断伸长率可达340%,断裂能达1.01×103J/m2,与天然关节软骨相当.将成纤维细胞种植在凝胶内部进行体外三维立体培养,结果显示,细胞在凝胶内部生存状态良好,双网络凝胶的细胞负载率高于单网络结冷胶,说明该体系在生物医用领域具有良好的应用前景.     

Microfluidics technology for manipulation and analysis of biological cells

Analysis of the profiles and dynamics of molecular components and sub-cellular structures in living cells using microfluidic devices has become a major branch of bioanalytical chemistry during the past decades. Microfluidic systems have shown unique advantages in performing analytical functions such as controlled transportation, immobilization, and manipulation of biological molecules and cells, as well as separation, mixing, and dilution of chemical reagents, which enables the analysis of intracellular parameters and detection of cell metabolites, even on a single-cell level. This article provides an in-depth review on the applications of microfluidic devices for cell-based assays in recent years (2002–2005). Various cell manipulation methods for microfluidic applications, based on magnetic, optical, mechanical, and electrical principles, are described with selected examples of microfluidic devices for cell-based analysis. Microfluidic devices for cell treatment, including cell lysis, cell culture, and cell electroporation, are surveyed and their unique features are introduced. Special attention is devoted to a number of microfluidic devices for cell-based assays, including micro cytometer, microfluidic chemical cytometry, biochemical sensing chip, and whole cell sensing chip.