https://orbilu.uni.lu/bitstream/10993/21149/1/Advantages%20and%20challenges%20of%20microfluidic%20cell%20culture%20in%20polydimethylsiloxane%20devices.pdf

Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.

This review provides an overview of major microengineering emulsification techniques for production of monodispersed droplets. The main emphasis has been put on membrane emulsification using Shirasu Porous Glass and microsieve membrane, microchannel emulsification using grooved-type and straight-through microchannel plates, microfluidic junctions and flow focusing microfluidic devices. Microfabrication methods for production of planar and 3D poly(dimethylsiloxane) devices, glass capillary microfluidic devices and single-crystal silicon microchannel array devices have been described including soft lithography, glass capillary pulling and microforging, hot embossing, anisotropic wet etching and deep reactive ion etching. In addition, fabrication methods for SPG and microseive membranes have been outlined, such as spinodal decomposition, reactive ion etching and ultraviolet LIGA (Lithography, Electroplating, and Moulding) process. The most widespread application of micromachined emulsification devices is in the synthesis of monodispersed particles and vesicles, such as polymeric particles, microgels, solid lipid particles, Janus particles, and functional vesicles (liposomes, polymersomes and colloidosomes). Glass capillary microfluidic devices are very suitable for production of core/shell drops of controllable shell thickness and multiple emulsions containing a controlled number of inner droplets and/or inner droplets of two or more distinct phases. Microchannel emulsification is a very promising technique for production of monodispersed droplets with droplet throughputs of up to 100 l h−1.

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Production of uniform droplets using membrane, microchannel and microfluidic emulsification devices

An attractive option for tissue engineering is to use of multicellular spheroids as microtissues, particularly with stem cell spheroids. Conventional approaches of fabricating spheroids suffer from low throughput and polydispersity in size, and fail to supplement cues from extracellular matrix (ECM) for enhanced differentiation. In this study, we report the application of microfluidics-generated water-in-oil-in-water (w/o/w) double-emulsion (DE) droplets as pico-liter sized bioreactor for rapid cell assembly and well-controlled microenvironment for spheroid culture. Cells aggregated to form size-controllable (30-80 μm) spheroids in DE droplets within 150 min and could be retrieved via a droplet-releasing agent. Moreover, precursor hydrogel solution can be adopted as the inner phase to produce spheroid-encapsulated microgels after spheroid formation. As an example, the encapsulation of human mesenchymal stem cells (hMSC) spheroids in alginate and alginate-arginine-glycine-aspartic acid (-RGD) microgel was demonstrated, with enhanced osteogenic differentiation further exhibited in the latter case.

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Rapid formation of multicellular spheroids in double-emulsion droplets with controllable microenvironment

Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies.

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Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency.

A modern flow cytometer can analyze and sort particles on a one by one basis at rates of 50 000 particles per second. Flow cytometers can also measure as many as 17 channels of fluorescence, several angles of scattered light, and other non-optical parameters such as particle impedance. More specialized flow cytometers can provide even greater analysis power, such as single molecule detection, imaging, and full spectral collection, at reduced rates. These capabilities have made flow cytometers an invaluable tool for numerous applications including cellular immunophenotyping, CD4+ T-cell counting, multiplex microsphere analysis, high-throughput screening, and rare cell analysis and sorting. Many bio-analytical techniques have been influenced by the advent of microfluidics as a component in analytical tools and flow cytometry is no exception. Here we detail the functions and uses of a modern flow cytometer, review the recent and historical contributions of microfluidics and microfabricated devices to field of flow cytometry, examine current application areas, and suggest opportunities for the synergistic application of microfabrication approaches to modern flow cytometry.

The intersection of flow cytometry with microfluidics and microfabrication.

Microfluidic techniques have been recently developed for cell-based assays. In microfluidic systems, the objective is for these microenvironments to mimic in vivo surroundings. With advantageous characteristics such as optical transparency and the capability for automating protocols, different types of cells can be cultured, screened, and monitored in real time to systematically investigate their morphology and functions under well-controlled microenvironments in response to various stimuli. Recently, the study of stem cells using microfluidic platforms has attracted considerable interest. Even though stem cells have been studied extensively using bench-top systems, an understanding of their behavior in in vivo-like microenvironments which stimulate cell proliferation and differentiation is still lacking. In this paper, recent cell studies using microfluidic systems are first introduced. The various miniature systems for cell culture, sorting and isolation, and stimulation are then systematically reviewed. The main focus of this review is on papers published in recent years studying stem cells by using microfluidic technology. This review aims to provide experts in microfluidics an overview of various microfluidic systems for stem cell research.

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Stem cells in microfluidics.

This study reports an integrated microfluidic system capable of isolation, counting, and sorting of hematopoietic stem cells (HSCs) from cord blood in an automatic format by utilizing a magnetic-bead-based immunoassay. Three functional modules, including cell isolation, cell counting, and cell sorting modules are integrated on a single chip by using microfluidic technology. The cell isolation module is comprised of a four-membrane-type micromixer for binding of target stem cells and magnetic beads, two pneumatic micropumps for sample transport, and an S-shaped channel for isolation of HSCs using a permanent magnet underneath. The counting and sorting of HSCs are performed by utilizing the cell counting and sorting modules. Experimental results show that a separation efficiency as high as 88% for HSCs from cord blood is achieved within 40 min for a sample volume of 100 μl. Therefore, the development of this integrated microfluidic system may be promising for various applications such as stem cell research and cell therapy.

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An integrated microfluidic system for isolation, counting, and sorting of hematopoietic stem cells

Recently, there has been considerable interest in stem cells which have the potential to differentiate into multiple lineages for cell therapy. Special attention has been paid, in particular, to the use of alternative sources of stem cells with fewer ethical issues. For example, mesenchymal stem cells (MSCs) from bone marrow have been proven to be multipotent for transplantation and tissue engineering. In this study, an integrated microfluidic chip capable of chemically and mechanically stimulating human mesenchymal stem cells (hMSCs) for adipogenic differentiation was presented. It was composed of a dilution module for controlling the insulin concentrations, and pneumatically-modulated membrane structures for precisely applying shear stresses on cells to perform chemical and mechanical stimulation, respectively. With this approach, a long-term culture and differentiation of MSCs were performed in an automatic manner. The accumulation of lipids that represented the results of insulin simulation was evaluated by Oil Red O staining. The measurements including lipid droplet numbers, optical density (OD) values, and expression of the PPARγ2 gene were used to assess the level of differentiation of the MSCs. The experimental result showed that the maximum oil droplets were induced under an optimal insulin concentration of 10 μg/ml. It was also revealed that, under mechanical stimulation, adipogenesis was inhibited under stronger levels of applied shear stresses and at higher pulsation frequencies. This proposed microfluidic chip has great potential as a powerful tool for MSC studies.

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A microfluidic device for chemical and mechanical stimulation of mesenchymal stem cells

In this study, a new microfluidic system is presented which can culture and differentiate MSCs in situ. It is composed of several micro-components, including seeding reservoirs, culture areas, micropumps, microgates, waste reservoirs and fluid microchannels. All of them are fabricated by using micro-electro-mechanical-systems (MEMS) technology. The automated system allows for the long-term culture and differentiation of MSCs. Two methods, including Oil Red O staining for adipogenic cells, and alkaline phosphatase staining for osteogenic cells are used to assess the differentiation of MSCs. Experimental results clearly demonstrate that the MSCs can be cultured for proliferation and different types of differentiation are possible in this microfluidic system.

Clulture and diferentiation of amniotic stem cells in a microfluidic system

This study presents a new microfluidic device for particle separation utilizing tilted eaves-like structures to separate small particles from a large volume of biosamples with various particle sizes without clogging in the microchannel. Experimental results showed that the microchip was capable of separating microparticles with diameters of 5, 20 and 60 μm using the proposed device. The developed microfluidic device also provides a simple way to perform a reasonable separation (86%).

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Huei-Wen Wu

Papers

Stem cells in microfluidics.

An integrated microfluidic system for isolation, counting, and sorting of hematopoietic stem cells

A microfluidic device for chemical and mechanical stimulation of mesenchymal stem cells

Clulture and diferentiation of amniotic stem cells in a microfluidic system

A microfluidic device for particle separation utilizing eaves structures