Biomaterial-based microfluidics for cell culture and analysis

Accepted Manuscript Title: Biomaterial-based microfluidics for cell culture and analysis Author: Ruizhi Ning, Feng Wang, Ling Lin PII: DOI: Reference:

S0165-9936(15)30016-9 http://dx.doi.org/doi: 10.1016/j.trac.2015.08.017 TRAC 14585

To appear in:

Trends in Analytical Chemistry

Please cite this article as: Ruizhi Ning, Feng Wang, Ling Lin, Biomaterial-based microfluidics for cell culture and analysis, Trends in Analytical Chemistry (2015), http://dx.doi.org/doi: 10.1016/j.trac.2015.08.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Biomaterial-based microfluidics for cell culture and analysis

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Ruizhi Ning a, Feng Wang a, Ling Lina,b,* a.

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State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, P. R. China b. Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

Highlights

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● We reviewed materials for microfluidic fabrication and summarized three tendencies.

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● Biomaterials play key role in 2D and 3D cell culture on-chip.

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● Organ-on-chip was introduced with promising potential.

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● The ways biomaterials participating in cell analysis were elaborated.

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ABSTRACT

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To make the microfluidics more functional and sensitive in biological applications,

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biomaterials for chip organization and function turn to be the key factor for leading

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microfluidics to a new area. Biomaterials used in microfluidics turned to be more

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various, complicated, and integrated, and polymers gradually take the chief position in

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bio microfluidics. The previous stage for microfluidics is microanalyzing in chemical

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and biology, including biomolecular analyzing. We believe that the biomaterial-based

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micro platform will take main responsibility for cell culture and analyzing in vitro in

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future, and it will bring a revolution to biology and medicine research and applications.

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In this review, we first conclude commonly used biomaterials in microfluidic

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construction. Then, biomaterials for cell culture on chip in 2D and 3D, as well as

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organ-on-chip mimicking are elaborated. Finally, cell observation and analysis also *

Corresponding author: Tel/Fax: +86-10- 64433585, E-mail: [email protected] (L. Lin) 1 Page 1 of 44

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adopt some biomaterials for higher sensitivity.

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Keywords：Biomaterials; Microfluidic; Cell culture; Organ-on-chip; Cell observation;

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Cell analysis

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1. Introduction

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Microfluidics, with controllable chambers and channels in micrometer (10~1000

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μm), have been widely investigated and applied in chemical microanalysis, medical

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evaluation, environmental monitoring, and biological fields. Among these, microfluidic

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devices show promising applications in cell manipulating due to their dimensional

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consistency with cells [1]. Microfluidics are also proved to be a powerful tool in dealing

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with cell analysis, because they have substantial superiority in microanalysis and

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dynamic monitoring, with small reagent volume and low detection limit, which is

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profitable for analyzing slight cellular and extracellular secretions, even for single cell

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analysis. The cell sorting and culture, cell imaging, cell analysis can be integrated in a

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single device with abundant parallel function channels on it [2], which makes cell

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experiments convenient and integrated.

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Microfluidic chips have emerged for a longtime since 1970s, and aside from

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structure design, materials for microfluidic fabrication and realizing specific functions

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act increasingly significant role in microfluidics development. The development of

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materials for microfluidics and functional blocks in microchip is the basic and key

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factor for solving current problems appeared in cell research. Materials for microfluidics

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has experienced three stages, the initial glass and silicon based inorganic system for

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analyte detection [3], the polymeric substitution for cell manipulation and analysis [4],

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and the recent integrated microfluidics with hydrogel or smart biomaterials that can

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assist in realizing cell-cell communication and cell-surface interaction with better

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biocompatibility [5].

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Microfluidic-based cell analysis is an interdiscipline contains various techniques,

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such as fluidic machinery, cell culture, materials engineering, and analytic chemistry.

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However, the chief target of microfluidic-based cell analysis is to settle issues

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concerned with cells in vitro, thus cell culture on-chip comes to the prior position in this

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field. Cell culture on microfluidic platform originally was performed in the ways as in

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commercial culture plates, that is, 2D culture. To improve cell adhesion and

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proliferation, surface modification and coating with biomaterials was carried out in

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microchannels. However, simple 2D culture can hardly make cells exert their instinct

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functions as in vivo, which induced great problems for in vitro cell research [6].

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Consequently, the on-chip cell culture varied from 2D culture to 2D/3D culture, and

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then finally to complex 3D culture that can mimic in vivo environment. Though great

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efforts are still needed in constructing a more accurate microenvironment for cells, the

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development of biomaterials in microfluidics has got plenty results for in vitro

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stimulation, and organ-on-chip has emerged and processed to some extent.

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As another significant portion in bio-microfluidics, cell detection and analysis

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on-chip also keep attracting attentions in both biology and chemistry. Transparency of

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microdevices gives them superiority in non-invasive and high sensitive cell observation

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[7], such as fluorescent imaging. Meanwhile, the dynamic microfluidic platform

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provides advantages in better investigating the process of cell metabolism, and

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combined with highly sensitive detection methods, such as mass spectrometry (MS),

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fluorescence with low detection limit could be accomplished.

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In this review, some issues related with key functional components of

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bio-microfluidics, microfluidic fabrication, on-chip cell culture, cell observation and

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analysis, are surveyed from the view of biomaterials. For further and better making use

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of microfluidics in cell research, biomaterial exploitation and flexible applications is of

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great significance, hence, materials for microfluidic fabrication and materials assisting

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in forming 2D and 3D cell culture in microfluidics with good biocompatibility were

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introduced. As the ultimate purpose for culturing cells on-chip, even building up

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organ-on-chip system, cell observation and analysis are also involved in this paper.

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2. Materials for microfluidics

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The initial microfluidic device was reported by Terry in 1979 [8]. A miniature gas

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analysis system based on gas chromatography (GC) was fabricated on silicon wafer

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using photolithography and chemical etching techniques. Though the GC system did not

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need to a further development, this platform opened a door for microscale analysis and

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manipulation for chemical micromolecule and biological macromolecule. Due to the

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mature technology of micro-electromechanical system (MEMS), inorganic materials,

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silicon and glass, were selected for constructing early microfluidic devices. These

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materials came to a limitation in biological applications due to their physical and

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mechanical properties. With the development of polymer science and engineering, as

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well as novel chip fabrication method, natural and synthesized polymer materials

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gradually replace traditional inorganic materials. Both thermalplastic and thermalsetting

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polymers are widely employed in various circumstances. Since polymers being widely

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applied in biology, soft materials with unique functions are applied to meet specific

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biological requirements. For demands of cell culture, cell capture, tissue engineering,

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and microenvironment construction in vitro, cross-linking structure hydrogels are

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recently emerging as a new generation of materials for microfluidics.

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2.1 "Hard" inorganic materials

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Early microfluidics emerged on the basic of MEMS development, accordingly, just

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like semiconductor manufacturing technology, silicon and glass were selected as the

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microfluidic materials. The main techniques for "hard" MEMS are as follows: (1)

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thin-film deposition, (2) lithography, (3) etching, and (4) packaging. After these

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processes, microfluidics with designed pattern channels can be achieved.

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For first step, thin-film deposition, various deposition methods can be applied in

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this stage to form a layer on inorganic substrate surface [9, 10]. Chemical vapor

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deposition (CVD), physical vapor deposition (PVD), oxidation and electrodeposition

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are four mainly used deposition methods. After thin-film deposition, a lithography

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process is employed to etch the thin-film layer with designed patterns [11], and then the

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patterns can be transferred from photomask to the inorganic substrates. In the third

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etching process, the techniques can be divided into two main classes: wet etching [12]

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and dry etching [13]. The wet etching is always isotropic, which makes this method

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difficult to provide a vertical side wall on amorphous substrates. However, commonly

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used anisotropic silicon etchants prefer crystallization direction, thus single crystal

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silicon can be utilized to perform anisotropic etching. Compared with wet method, dry

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etching method has smaller etching limitation and higher anisotropicity, which fits

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fabricating microchannels on different substrates. There are three chief dry etching

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techniques, high-pressure plasma etching, reactive ion etching, and ion milling. Through

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dry etching process, microchannels with better vertical side walls can reach to a relative

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high aspect ratio [14]. Finally, the packaging step, also called bonding process, always

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employs an adhesive layer or thermal fusion treatment to encapsulate the microfluidic

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system [15].

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Recently, quartz-based microfluidics show more excellent performances in cell

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identification and protein separation than microchips which are made of glass.

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According to Dochow's work [16], in Raman-activated cell sorting (RACS), compared

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with the glass, the quartz substrate shows much lower Raman background in fingerprint

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region at near-infrared laser excitation, which could settle the limits of RACS

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constructed by glasses in cell identification. Quartz also assisted in protein separation in

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a hybrid chip [17] that applied a bottom quartz substrate to increase heat dissipation due

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to superior thermal conductivity of the quartz, which allows larger electric fields to be

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used in isoelectric focusing (IEF) of protein.

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2.2 "Soft" polymer materials

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Though traditional inorganic materials were widely applied in microfluidics for

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chemical analysis in early studies, when employed to biological applications, the system

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emerged various problems. Opaque silicon material made difficulty for cell observation

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by microscope, and cells could not survive long on these surfaces due to gas

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impermeability. Under this situation, polymers, normally have smaller Young's modulus,

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turning into the main materials for microfluidics for biological applications. In addition,

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the cheap organic materials could easily be processed with diverse methods, and there

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are plenty categories of polymers that surface modifications can be made more facile.

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The polymer materials for microfluidics can mainly be divided into two groups: plastics

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and elastomers.

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2.2.1 Plastics

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Plastic is a kind of polymer that has low elastic deformation ability. According to

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classification of plastics, the biomaterial for microfluidics in this category can also be

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set into two classes, thermoplastics and thermosetting plastics.

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Thermoplastic polymer chains are always linear, which introduces a low glass

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transition temperature (Tg) for them to melt, and consequently makes the materials easy

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processing with thermal treatment (hot embossing, thermal molding, thermal bonding).

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Some popular plastics, such as polystyrene (PS, a widely used material in commercial

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culture dishes), polytetrafluoroethylene (PTFE, also be called Teflon), polymethyl

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methacrylate (PMMA, commonly known as organic glass), and polyethylene

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terephthalate (PET), are some ideal candidates for dealing with cells in a microfluidic

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system, because these materials are extensively utilized in agriculture, food industry,

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medicine, and clinic treatment, proved to be biocompatible [18-20]. However, they can

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easily diffuse low molecular compounds from the bulk and can be dissolved by organic

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solvents, and their poor gas-permeable property may be a dominant reason which makes

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them not suitable for culture cells, which limits the thermalplastics being applied for

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microfluidics. So far, some platforms based on thermalplastics have been established.

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For example, Battle et al. built up a PMMA microfluidic solid-phase extraction (μSPE)

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device by hot embossing, the micropillars in main channel can effectively enrich and

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purify membrane protein from whole cell lysates [21]. Midwoud et al. compared

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adsorption properties and biocompatibility of device in several different thermalplastics,

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such as PMMA, PS, polycarbnate (PC), and cyclic olefin copolymer (COC) [22].

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For thermosetting plastics, the polymer chains can crosslink into a network

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structure, making the bulk resistant to heating. Once the network formed, it is difficult

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to reshape the material again. At the same time, the cross-linking network also gives a

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strong mechanical property to this material, which makes thermosetting polymer high

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stiffness. The thermosetting is always applied as photolithographic mask layer for

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microfluidic fabrication, such as SU-8 negative photoresist, and it can also be employed

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to establish a free-standing microstructure [23]. Due to hard processing, rigid, and

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expensive, very few system applied thermosetting polymers for microfluidic fabrication.

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2.2.2 Elastomers

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Elastomers show significant advantage in biological applications, because this kind

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of material has a strong elastic deformation ability, resulting in soft and flexible

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properties. Loaded on external force, the material can stretch more than half than the

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original length, and the deformation will recover after removing the force. Compared

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with stiff surface, cells prefer to proliferate on soft substrates [24]. Actually, the

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elastomer, especially polydimethylsiloxane (PDMS), is the most popular material for

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microfluidic fabrication. PDMS is a transparent, gas permeable, biocompatible, and

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flexible material that completely fit for cell-related research. Recently, the fabrication of

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PDMS-based microfluidic devices have turned to be a routine method, that is, soft

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lithography technique. In this way, hybrid microfluidics like glass/PDMS that commonly

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used for cell culture are constructed. The soft lithography method applied replica

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molding and rapid prototyping, which makes PDMS has been sufficiently presented in

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microfluidics. Though widely investigated and applied in biology researches, PDMS

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comes to a limitation in microfluidics due to the hydrophobic surface can lead to

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unspecific adhesion of proteins or analytes. To settle this problem and integrate

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functional biomoleculars, various surface modification methods [25] have been

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developed, such as plasma treatment [26], silanization [27], and surface coating [28].

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After surface modification, the PDMS-based microfluidics have been successfully

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applied in biomolecular separation, protein/cell capture and release, and cell culture.

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Aside from PDMS, few synthesized elastomers also find a position in constructing

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microfluidics. For instance, Roy et al. claimed that PDMS is inadequate for industrial

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fabrication and applications. They used styrenic thermoplastic elastomers (TPE) for

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fabricating multilayer microfluidic devices [29]. They successfully employed DNA and

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protein solution for microfluidic spotting, and cultured human cell on surface of

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isothermal TPE microstructure. Perfluoropolyethers (PFPEs) is also a kind of elastomer

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that can be used for microfluidic fabrication. Rolland et al. reported the fabrication of

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microfluidic device by photocurable "Liquid Teflon" material that can resist to organic

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solvents, which may extend microfluidics applications to novel fields [30].

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2.3 Hydrogel and Paper

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In recent years, some untraditionally microfluidic materials, such as hydrogel and

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papers, have been employed to take the responsibility of microfluidic construction to

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realize better cell viability and some functions.

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2.3.1 Hydrogel

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Hydrogel is a kind of hydrophilic polymer that has linked networks, which leads to

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high water content, easy mass transportation, better cell viability and proliferation, thus

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can be used as an ideal candidate for mimicking native extracellular matrix. Both nature

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hydrogel and synthesized hydrogel have been used in microfluidic systems. Poly lactic

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acid (PLA) and polyethylene glycol (PEG) are two mainly used synthesized polymer in

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microfluidic devices. Compared with the artificial materials, the natural polymers, such

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as collagen, calcium alginate, cellulose, gelatin, and chitosan are widely applied for

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being as biomaterials applied in microfluidics [31, 32], because the natural substances

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have better biocompatibility and bioactivity. Though the hydrogels have several

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advantages in dealing with cells, there are also some challenges of these materials, such

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as insolubility, incontrollable pore distribution, low mechanical durability and

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microstructure reproducibility, to be further solved. Recently, composite hydrogels

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prepared with natural and synthesized materials take the chief position in hydrogels

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applied in biology-related researches, because properties of the composites can be tuned

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to meet with biological requirements. Blending with synthesized polymer, such as PEG

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or polyvinyl acetate (PVA), or changing the solution concentration, structures and 11 Page 11 of 44

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properties of cellulose hydrogels could be controlled, thus the cellulose hydrogel can

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performed as bulk materials for microfluidic devices. Pei et al. fabricated a

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cellulose-based hydrogel as bulk materials for microchips [33]. They successfully

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fabricated cross-linked cellulose (RCC) hydrogel and cellulose-collagen (RCC/C)

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hybrid hydrogel-based integrated microfluidics, which with well-controlled pore size,

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good mechanical durability, and good biocompatibility in both 2D and 3D cell culture.

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However, compared with acting as bulk materials for microfluidic fabrication,

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hydrogels are more frequently employed as scaffolds in microchips for culturing cells in

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3D, which will be detailed illustrated in chapter "3.2 3D culture".

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2.3.2 Paper

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Apart from above natural and synthesized hydrogels, paper is also a significant

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material for constructing microfluidic systems, because paper is easy to access, process,

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modify, and dispose. Paper is a sheet deposited by fibers, which permits the paper could

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integrate several functions, such as flow, filtering, and separation. Furthermore, the

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capillary action of paper makes paper-based microfluidics widely applied in rapid

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diagnostic tests [34]. For paper-based microfluidics, choosing a proper kind of paper

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that can meet with the requirements of assay is very important. Surface chemical and

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physical properties, capillary flow rate, porosity and pore size are some key factors in

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controlling the performance of papers. Commonly, the most frequently used paper in

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microfluidics is cellulosic materials [35, 36], such as filter paper and chromatography

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paper, and nitrocellulose paper [37] is also a big family in this category. Designing and

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constructing hydrophobic barriers in paper substrates acts as a significant role in

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fabricating 2D and 3D paper-based microfluidic devices. By constructing hydrophobic

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barriers in horizontal and vertical directions, the regents can be divided in several

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separated channels and multichannel detection can be realized at the same time. Wax

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patterning, alkyl ketene dimer printing, PDMS plotting, paper shaping and cutting are

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some common ways for fabricating paper-based microfluidics. Thuo et al. [38] applied

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omniphobic paper being as channel the substrate, the open-channel 2D and 3D

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microfluidic devices were generated by using embossing and "cut-and-stack" method

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respectively. The laminar flow and droplet generation could be realized on these two

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paper-based platforms, and the gas-permeable devices can be applied in fields of

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analysis, environmental monitoring, and droplet-based synthesis.

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Microfluidics fabricated based on different materials all have advantages in some

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views, and no one material can cover all requirements of diverse applications. Though

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in biology related fields, soft polymers and natural sources fabricated papers are more

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frequently adopted, normally, a microfluidic chip integrated with various materials tends

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to be more effective in cell culture, cell imaging and analysis.

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3. Cell culture

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Cell culture makes great significance to many disciplines, such as pharmacology,

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medicine, biology, and tissue engineering. Vast mysterious issues in human body are

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still need to be explored. Before human trial, preliminary experiments should be carried

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out on animal. However, animal experiments are expensive, troublesome, and restricted,

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as well as sometimes getting suspectable results due to genetic differences. In addition,

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in some disease, such as cancer, rare tumor cells isolated from blood or tumor tissues

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need to be proliferated to a large amount for further individual treatment and process

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evaluation. Thus, the in vitro cell culture is of great important and demand for settle

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these problems. Commercial culture plates and bottles can be applied to proliferating

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cells statically, however, dynamic system can be more benefit for culture in vitro,

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because controllable culture medium supply, drug stimulation, and metabolite extraction

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and analysis can be realized and integrated on microfluidic chips.

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3.1 Flat surface culture

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Cell culture is primarily carried out on flat substrates in microfluidics for some

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experiments those do not require a strict spatial culture, just as the routine culture on

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dish or in bottle. Majority of the recent microfluidics are based on polymer materials,

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such as PS, polycarbonate (PC), TPE, and PDMS. Though these materials find huge

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potentials for cell manipulation and analysis, the hydrophobic property of them can

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irreversibly adhere abundant albumin that can block cell adhesion. Thus, the

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hydrophobicity brings some issues for cell culture. Accordingly, numerous efforts have

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been done to change the hydrophobicity [39-41], such as physical protein adsorption,

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covalent surface chemical modification, and surface biomaterial modification.

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Physical protein adsorption provides a matrix protein layer on the surface of

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polymer substrates. Normally, the hydrophobic surface of polymer materials can

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nonspecifically adsorb serum proteins (albumin), and reduce the affinity of cells to

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adhere. Due to the good affinity between extracellular matrix (ECM) protein and cells,

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an ECM protein solution can be introduced in channels to modify the substrate surface

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through physical adhesion. Fibronectin (FN), laminin and collagen are some

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conventional physical modification proteins for enhance cell adhesion and proliferation.

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The physical adsorption mainly depend on some weak interaction between protein and

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material surface, so the adsorbed protein can easily detached from interaction surface,

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which leads to a short maintenance of cell adhesion.

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Covalent modification can induce strong interaction between protein and the

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materials. Convenient physical plasma treatment can bring hydrophilicity to material

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surface, but this method also has hydrophobic recovery after a short time [42]. Chemical

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modification is a process that is able to form steady covalent bond among protein and

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the surface of substrates, and shows efficient immobilization of matrix protein on the

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material surface. Kuddannaya et al. [43] reported that (3-aminopropyl)triethoxy silane

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(APTES) and cross-linker glutaraldehyde (GA) chemistry could be utilized to modify

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either FN or collagen type 1 (C1) on PDMS. They analyzed the cell adhesion and

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viability of mesenchymal stem cells (MSCs) on these surfaces, and the results showed

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the surface modification could effectively reduce hydrophobicity, and MSCs preferred

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to adhere on PDMS modified with APTES, GA and protein.

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Aside from natural biomaterials, such as ECM proteins, peptides and

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carbohydrates, synthesized biomaterials also assist in cell culture. Similar with above

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surface modification, the biomaterials modification also aim at increasing hydrophilicity.

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Smart materials is now a popular topic in this field, because the wettability of the smart

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surface can be controllably switched under some stimulating factors, such as

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temperature, pH value, light, and solvent. Among these researches, the most promising

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and widely investigated material is the thermal responsive surface.

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responsive systems, poly(N-isopropylacrylamide) (PNIPAAm) is the most frequently

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applied biomaterial [44, 45]. The PNIPAAm chain can have a reversible conformational

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transition when the temperature altered around its lower critical solution temperature

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(LCST) [46]. As shown in Fig. 1a, when temperature is lower than LCST, abundant

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complex H-bond can range along the polymer chain, whereas alter to be blocked when

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exposed in an atmosphere above LCST, and the surface wettability changes accordingly.

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Many works employed this kind of material in 2D cell culture. Fig. 1b displays how

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Schmidt switched cell adhesion by using PNIPAAm microgel films [47]. In their work,

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microgels’ physico-chemical parameters in the adsorbed state and their changes within

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temperature variation were illustrated. They showed fibroblast adhesion strongly

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depends on temperature, and the microgels slightly above LCST favor cell adhesion and

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proliferation. Figure 1

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In many thermal

3.2 3D culture

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The purpose for people to conduct cell culture in vitro is to mimic

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microenvironment for drug evaluation, cell research, and biological process

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investigation. Consequently, whether the cells can behave as they do in vivo is the key

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issue for in vitro experiments. The 2D cell culture could be easily performed on various

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substrates, however, plenty works have proved that cells need very special spatial

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structures to maintain their functions [48-50], which leads to the development of 3D cell

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culture. To realize controlling cell pattern and arrangement in a space, multilayer

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microfluidic structure and filling the channels with scaffold materials, especially

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hydrogels, have been adopted, as well as combining the two techniques.

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By employing the multi soft lithography technique, microchannels with different

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height, width, and shape could be integrated in a single chip for 3D cell culture. The

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spatial cell culture in chip can be realized in various ways, such as by microwells,

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height-gradient chambers, and intercellular porous films. The microwells and

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height-gradient chambers are fabricated by several times UV exposure with a group of

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masks. Kang developed a multilayer microfluidic platform with concave microwells and

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flat chambers to culture embryonic stem (ES) cells and regulate uniform-sized embryoid

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body formation [51]. Lin’s group have done many researches in this field. For example,

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Wu applied a 3D microfluidic chip with height-gradient chambers to imitate the

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diffusion process between blood vessels and tissues, and studied the quantum dot (QD)

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cytotoxicity to HepG2 cells on this platform [52]. In particular, sandwich structure

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systems with porous polymer films being as intercellular films were widely researched

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and successfully applied in cell co-culture. Fig. 2 gives a typical example of how the

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"sandwich" co-culture platform works [53]. As illustrated in Fig. 2a, normal mouse

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embryonic fibroblasts (mEFs) (without inactivation) and mouse embryonic stem (mES)

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cells were co-cultured on two sides of a PDMS porous membrane layer. Fig. 2b

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demonstrates significant advantages of efficiency and simplicity of the established

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platform. Figure 2 The above methods have achieved the 3D cell culture in chip, however, cells

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cultured in these chips still adhere on channels walls just as they performed on flat plate,

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which is not a totally real spatial cell culture. To fill up the channels with cells, a

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scaffold material is needed to support cell proliferation and migration, as well as mass

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transportation. Generally, some biocompatible hydrogel materials are ideal candidates

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for being as the cell scaffold [54, 55], which allows the encapsulation of cells in gel.

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The hydrogel comes both natural biomaterials and synthesized biopolymers. The ECM

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proteins, such as collagen, fibrin, matrigel, FN, PEG, and the mixtures of these proteins

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have been initially considered as the hydrogel scaffold basis for natural hydrogels for

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embedding disperse cells in 3D. Kamm group did several excellent works by employing

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hydrogel, such as collage, in researches that requires cells performed in 3D structure.

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For example, they adopted a 3D microfluidic assay filled with collagen gel to define the

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effect of endothelial KLF2 expression on smooth muscle cell (MSC) migration [56] and

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the results proved KLF2-expressing endothelial cells (EC) cultured with SMC could

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significantly reduce SMC migration. Besides, they also used collagen as 3D scaffolds in

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studying neurite turning under a growth factor gradient [57], and more remarkably, they

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successfully induced physiologically relevant 3D capillary morphogenesis on hydrogel

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microfluidic platforms stimulated by growth factors [58] and cells [59]. Meanwhile,

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calcium alginate and gelatin are also some kinds of natural substances that hold good

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biocompatibility for being as cell culture scaffolds. According to Choong Kim's work,

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fibroblast cell beads could be fabricated by alginate beads, and the cell-encapsulating

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beads could make an influence on EC monolayer in a collagen scaffold and formed

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circular lumen-like structures [60]. However, the hydrogel-based cell culture meets with

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some issues when applied hydrogel scaffolds for 3D culture. Hydrogels, especially

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natural hydrogels, are always unsteady in properties, and transportation of nutrients and

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oxygen is gradient along the thickness of hydrogels. Furthermore, experimental model

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that requires dense cell concentration, such as multicellular tumor ball, is hard to be

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constructed in hydrogel scaffolds. Therefore, 3D cell culture without using gel is

382

developed to settle this problem. The gel-free 3D culture adopts intercellular polymeric

383

linker that can cause cell aggregation [61]. Microwells on chip [62] and microbubbles

384

fabricated in chip [63] can also assist in forming 3D cell culture structure. For example,

385

Liu et al. [64] fabricated quasi-spherical microwells on PDMS substrates by an ice

386

lithography-based bench-top method. The concave microwells were capable of forming

387

dense and homogenous multicellular tumor spheroids. The microwells could eliminate

388

cell lose in manipulation and long-term culture could be facilitated on-chip.

389

4. Organ-on-chip system

390

Cells are the basic units for undertaking life functions, however, cells cultured on

391

flat substrates in vitro can hardly perform normal functions as they do in vivo. To

392

veritably realize cell functions, mimicking the microenvironment is of great importance

393

for cell analysis on chip. Building up an organ system is a very tough work in tissue

394

engineering, because manipulating different cells and keeping them maintain a 3D

19 Page 19 of 44

395

structure with steady supplements is not easy. Microfluidics have extraordinary

396

superiority in creating blood supply system with tunable channel shape, making

397

organ-on-chip systems promising for drug delivery, tissue engineering, and other

398

biological applications. Organ-on-chip system comes from the 3D cell culture on chip,

399

combined with mass transportation controlling.

400

Organ-on-chip system is a 3D cell culture platform that can mimic the

401

physiological activity, function, and mechanism of organoids in vitro. As proved in

402

many researches, the simple bulk 3D culture of one specific cell type can hardly realize

403

its functions. Intercellular communication is a very essential factor for supporting

404

normal behavior of cells [65, 66], thus multicellular culture system with controllable

405

cell arrangement is the main task for constructing organs on chip. Dongeun Huh built a

406

famous lung-on-a-chip microsystem [67], and the alveolar-capillary interface of the

407

human lung was biomimicked and showed complex integrated organ-level responses to

408

bacteria and inflammatory cytokines that were brought to the alveolar space. Besides,

409

"heat on a chip" with higher throughput was also successfully prepared by using soft

410

elastomers to process sub millimeter sized thin film cantilevers, and muscular thin films

411

(MTFs) were then obtained through engineering anisotropic cardiac microtissues on the

412

cantilevers [68]. According to the microfluidic technology of constructing organs, the

413

organ-on-chip system could be divided into two categories. One is bottom-up tissue

414

engineering, and the other is 3D hydrodynamic flow focusing by using a microdevice,

415

some representative examples are given in Fig. 3. The bottom-up tissue engineering

416

could be applied in simulating tissue interface in similar way of making sandwich

20 Page 20 of 44

417

structure, that is, from bottom to top, accumulating layer by layer. In this tissue

418

engineering platform, two or more cell types are diversely cultured in different

419

microchambers that are separated by porous membrane. By using this structure, fluidic

420

shear stress could be applied, thus lung alveolar-capillary interface, as well as

421

blood-brain barrier (BBB) could be better simulated. The BBB is an unique

422

phenomenon existed in central nervous system, and the tight junction in BBB structure

423

can prevent exogenetic substances from entering brain tissues. Fig. 3a exhibits a

424

microfluidic-built BBB structure. Two exogenetic flow channels are applied in one

425

microchip, and endothelial cells and astrocytes were respectively cultured on the two

426

sides of the membrane [69]. As a result, endothelial cell showed excellent viability even

427

after a relatively long time, with high tight junction expression. C8-D1A (astrocyte)

428

cells on PC membrane exhibited typical astrocytic morphology. They detected

429

trans-endothelial electrical resistance (TEER) of co-culture system of the artificial

430

microchip and transwell, and results proved TEER value of dynamic system (exceeded

431

250 Ω cm2) was significantly larger than static system (only 25 Ω cm2), which may due

432

to fluidic shear stress induced mechano-transductive effect on the endothelial molecular

433

pathways.

434

In another technique, 3D hydrodynamic flow focusing, spherical and fibrous cell

435

assembly with simple or more complicated cell distribution could be realized. Spherical

436

and fibrous structure possess great significance in organ construction in vitro, because

437

these two geometries widely existing in tissues and organs. Spherical structure finds

438

important application in simulating tumor tissues and investigating cell-cell

21 Page 21 of 44

439

communication between tumor cells and neighboring cells. As shown in Fig. 3b,

440

Alessanbri et al. developed a coextrusion microfluidic device that can produce 3D

441

cell-based assays [70]. They applied 3D hydrodynamic flow focusing to form alginate

442

microcapsules, and CT26 spheroids were successfully synthesized by gel-free and gel

443

encapsulating methods respectively. Fibrous structure fabrication is same as spheroids

444

from view of fabricating mechanism. They both apply coextrusion to from a coaxial

445

geometry. The injection flow rate determines how the final geometry will be, and to

446

form spheroids, a side flow channel that provides shear force to cut the flow into droplet

447

is always needed. Hydrogel fibers embedded with cells have potential applications in

448

mimicking fibrous structures in vivo, such as vessels, nerve fibres, and muscle fibres.

449

Takeuchi's group fabricated several metre-long biological fibres with core-shell

450

structures by adopting hydrodynamic focusing technique [71], which is exhibited in Fig.

451

3c. In that case, they used a double-coaxial microchip to continuously extrude a coaxial

452

structure fibre, with ECM protein with cells being as the core and Ca-alginate hydrogel

453

as shell. Using this platform, Cardiomyocyte-Fib, HUVEC-ACol, and Cortical

454

cell-PCol fibres were successfully fabricated. Furthermore, these functional biological

455

microfibres can be assembled by waving and reeling, which may finds applications in

456

reconstructing fibre-shaped tissues and organs.

457

Figure 3

458

Aside from forming multicellular structures, designing microfluidic devices with

459

different functional blocks is also a way for on-chip tissue and organ construction. For

460

example, Lee et al. developed a 3D liver-on-chip system for investigating the paracrine

22 Page 22 of 44

461

effect of hepatic stellate cells (HECs) on hepatocytes [72]. The formation of hepatocyte

462

spheroids was using PDMS concave microwells. The two cell types did not contact

463

directly, they communicated by culture medium flow. By comparing spheroids structure,

464

as well as the level of albumin and cytochrome P450 reductase of mono-cultured

465

hepatocyte spheroids and co-cultured spheroids, they proved paracrine actions of HSCs

466

made functional and structural benefit to hepatocyte spheroids.

467

5. On-chip cell observation

468

Microfluidic devices find significant positions in cell researches and applications

469

due to their accordance with cells on size, minimal demands for regents, and high

470

sensitivity in detection. Different from traditional cell experiments performed on culture

471

dishes, cell experiments carried out on dynamic flowing microfluidic platform is of

472

great importance for real-time monitoring on specific conditions. Accordingly, cell

473

observation on chip is quite another thing than normal ones and huge issues emerged

474

consequently. Due to on-chip experiments are processed in a flowing system, cells are

475

introduced into the microdevices in the way of cell suspension, so it is impossible to do

476

any observation before cells are immobilized. After successfully sorting cells at a

477

certain area on chip, culturing cells for further use, and observing cell morphology and

478

behavior by some cell imaging techniques could be realized.

479

5.1 Cell immobilization on chip

480

For on-chip cell observation, one major challenge at the primary stage is to

23 Page 23 of 44

481

efficiently immobilizing and sorting cells to a specific position on chip. To isolate cells

482

from suspensions, there are three main ways: 1) Cell adhesion on channel surface; 2)

483

Isolation by porous membranes; 3) Cells encapsulation by porous polymer. These three

484

methods are always performed in different systems, and people should choose the

485

proper one according to experimental requirements.

486

Cell adhesion on channel surface mainly proceeds in 2D system. By altering

487

chemical and physical properties of the channel surface, cell adhesion could be

488

enhanced. As widely acknowledged, surface modification is an efficient way for

489

improving adhesion. The charge property, wettability, and functional groups of the

490

channel surface make great influence on adhesive capacity. Cells always possess

491

negative charge on cytomembrane, thus the cell adhesion could be improved by coating

492

some cationic species on surface, such as ECM proteins, Poly-L-lysine, and APTES.

493

Hydrophobicity is a major drawback of PDMS microfluidics and induces poor affinity

494

between cells and matrix surface. Thus, numerous attempts have been made to adjust

495

wettability from hydrophobic to hydrophilic. There are several approaches for

496

improving surface hydrophilicity, such as physical protein adsorption, covalent surface

497

chemical modification. Changing surface morphology can also act as an effective way

498

for cell isolation. In Chen's work, microvoids immobilized of aptamer could specifically

499

isolate target Ramos cells and realized single cell capture with an occupancy of 88.2 %

500

in average [73]. Wu also subtly applied microwell arrays being as cell density generator

501

to controllably store cell suspension by altering microwell number, and the stored cells

502

could further be cultured with high viability after flushing and inverting in turn [74].

24 Page 24 of 44

503

Based on the size distinction between different cells, filtering cells by porous

504

membrane can achieve cell immobilization in a simple way. This method is mainly

505

applied in a 2D/3D system, that is, the "sandwich" structure platform. The filtering

506

membrane is in 2D to isolate target cells, and the whole microchip is in 3D structure

507

with more than two zones located on two sides of the membrane. Circulating tumor cell

508

(CTC) enrichment based on porous membrane sets a typical example in this part. Lin et

509

al. used a parylene membrane-based microdevice to capture CTC from human

510

peripheral blood, and achieved > 90 % recovery when only five tumor cells were seeded

511

in 7.5 mL blood [75].

512

Cell encapsulation for cell immobilization is always conducted by porous polymer,

513

especially hydrogels. Hydrogel-based cells trapping approach mainly was utilized in 3D

514

systems to provide a 3D microenvironment for cells. Gao applied photolithography

515

approach to fabricate encapsulated cell arrays in PEG hydrogel, and HepG2 cells and

516

A549 cells were simultaneously immobilized for evaluating anticancer drug effect on

517

cell viability and intracellular redox parameters [76].

518

5.2 Cell imaging

519

Cells are transparent under lamps, and cytomembrane behavior, intracellular

520

structures and substances can hardly be directly or clearly observed, thus proper cell

521

imaging means are very necessary to provide real-time monitoring. Numerous probes

522

combined with diverse detecting instruments have been widely studied for cell imaging

523

[77]. Techniques used in cell imaging are prone to be non-invasive and high sensitive,

25 Page 25 of 44

524

such as photoluminescence (PL), magnetic resonance imaging (MRI), and surface

525

enhanced Raman scattering (SERS). However, fluorescent imaging was purposely

526

selected to demonstrate biomaterials applied in cell imaging because fluorescence is a

527

most commonly employed method in cell observation.

528

In fluorescent spectroscopic imaging system, though cells can emit fluorescence by

529

some fluorescent proteins, such as green fluorescent protein (GFP), biomaterials are still

530

needed to assist in cell imaging in some cases. Countless efforts in investigating metal,

531

polymer, and composite biomaterials, nano-biomaterial in particular, have been done for

532

cell imaging aimed at locating cytomembrane, cytoplasm, and cell nucleus. The

533

bio-nanomaterials employing in cell imaging can be inorganic, organic, and

534

inorganic-organic composite nanomaterials. Inorganic nanomaterials, especially

535

quantum dots (QDs), are widely researched and applied due to their intrinsic

536

fluorescence emission performance and the emission wavelength could be tuned by

537

altering QD diameter. Transition metal QDs, carbon dots, graphene QDs, and metal

538

QDs are some commonly used fluorescent emitting materials, which has been detailed

539

reviewed by Li et al. [78]. However, these inorganic nanomaterials are restricted by

540

photoblinking and poor hydrophility. This problem could be settled by polymer coating

541

[79] or aptamer modification [80]. Recently, a kind of inorganic nanomaterial with

542

excellent

543

nanoparticles (UCNPs), has been synthesized and applied in biological imaging with

544

low cytotoxicity [81]. In this system, Yang prepared hydrophilic hollow NaREF4 (RE=Y,

545

Yb, and Lu) NPs through a facile liquid-liquid two-phase method. The NPs in in vitro

energy converting

properties,

rare

earth

(RE)-based

upconversion

26 Page 26 of 44

546

system showed bright-red emission without noise background, which proved UCNPs is

547

suitable for cell imaging. Polymer and inorganic-organic composite nanomaterials are

548

extremely attractive imaging regents for optical detection because the soft organics are

549

transparent, biocompatible, and easy biofunctionalized. The instinctively fluorescent

550

emission NPs, fluorescent conjugated NPs, and degradable polymer that encapsulating

551

fluorophores are some regularly adopted pathways for achieving cell imaging. For

552

example, Zheng et al. [82] employed a single-step assemble and nanoprecipitation

553

approach to construct folate receptor-targeted (FA) indocyanine green (ICG) dye-doped

554

poly(D,L-lactide-co-glycolide) (PLGA) liquid NPs for cell imaging with good

555

biocompatibility and excellent stability against photobleaching. With the assistance of

556

functional biomaterials shell, poor aqueous stability and target specificity and rapid

557

elimination of ICG was overcame. Compared with folate receptor negative A549 cells,

558

endocytosis of FA-ICG-PLGA-liquid NPs in folate receptor over-expressed MCF-7 cell

559

was much more efficient.

560

6. On-chip cell analysis

561

Microfluidic chip is an integrated platform that combines cell capture, cell culture,

562

metabolite enrichment and analysis. Biomaterials could participate in the whole process

563

for on-chip biological experiments. The ultimate goal of lab-on-chip applied in biology

564

is for cell analysis which could provide theoretical foundations for cytotoxicity,

565

metabolic pathway, drug evaluation, and intercellular interactions.

566

Microfluidic platform only provide a limited space for cell manipulation, and the

27 Page 27 of 44

567

extremely inadequate quantity and complicated construction of metabolite generated

568

from cells for analysis is a challenging issue in biomicrofluidics. Accumulating and

569

purifying the target metabolites with high efficiency in flowing system is of great

570

demand. Solid-phase extraction (SPE) technique is highly suitable for on-chip cell

571

analysis because the microchip is a dynamic system with high throughput. Gao [83]

572

developed

573

spectrometer (ESI-Q-TOF MS) to characterize drug absorption and cytotoxicity. Two

574

functional parts, cell culture chambers combined with drug gradient generator and

575

on-chip SPE column, were connected by PE tubes. In cell culture section, 0.1 %

576

poly-L-lysine (PLL) was utilized to enhance cell adhesion. After cells were stimulated

577

by gradient drug concentrations, an on-chip SPE sample purification procedure was

578

applied to evaluate drug absorption. Similarly, Gao et al. also used the SPE columns for

579

investigating drug permeability [84]. In their study, as shown in Fig. 4a, a

580

semipermeable PC membrane was adopted to build up sandwich-structure microdevice.

581

The model drug was released in one side of the channel to let the drug permeate into the

582

other side of the channel. ESI-Q-TOF MS was applied to determine the concentration of

583

curcumin permeation. Results showed the established platform only need around 30 min

584

to complete the analysis, and only 6 μL drug solution was required, which proved the

585

microdevice an efficient platform for drug discovery and development. Moreover, we

586

employed a porous polymer monolithic column for SPE and chemiluminescence (CL)

587

detection [85]. Porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) column

588

modified with ethylenediamine was applied to concentrate catechins and let it react with

an

online

electrospray ionization

quadrupole

time-of-flight

mass

28 Page 28 of 44

589

potassium permanganate to produce CL. No elution step was needed in this method, and

590

the limit of detection (LOD) was 1.0×10-9 M with the recovery ranging from 90 % to

591

110 %. We also fabricated a serious of poly(ethylene glycol) diacrylate (PEG-DA)

592

microcolumns arrays in microchip channels for being as the probe detecting proteins

593

and glucose [86].

594

Cell analysis is always performed on large amount of cells exposed in specific

595

conditions and the analytic data is an average exhibition of the whole cells. However,

596

cell has a property of unique, and single cell is not completely identical with the block.

597

For clearly understand cell-cell communication and cell-surface interaction, single-cell

598

analysis is significantly important. To isolate single cell in microchip and for further

599

analysis, our group made a lot of efforts. One efficient way for single cell isolation and

600

analysis is as Fig. 4b exhibits, Liu fabricated a single-cell trapped microwell arrays in

601

PDMS by virtue of PS NPs assembly on glass substrate [87]. Then, real-time single cell

602

enzyme activity analysis was conducted, and through analyzing fluorescence intensity

603

of calcein AM stained HeLa cells, dissimilarity of viability and conditions among the

604

trapped cells was confirmed. Liu developed a modified microscope projection

605

photolithography that can drive photopolymerization of PEG-DA in the microchannels,

606

and single cell encapsulation was achieved with an efficiency of 80 % [88].

607

Different from traditional static analysis, one crucial factor for bringing

608

microfluidics to cell analysis is real-time monitoring, which has significant sense for

609

understanding the process of cell metabolism. MS detection is an effective tool for

610

realizing on-line monitoring with low LOD, Fig. 4c-d are two methods for real-time cell

29 Page 29 of 44

611

analysis

combined

MS

detection.

612

microdialysis-paper spray ionization MS system to on-line monitor the glucose

613

concentration in cell culture medium, the device construction and working principle are

614

shown in Fig. 4c. A microdialysis hollow fiber was utilized to generate microdroplet at

615

the outlet of the capillary, and silicon-coated paper with triangular shape was applied to

616

accomplish a more sensitive MS detection. The established platform allowed high

617

temporal resolution and could reflect dynamic variation of analyte concentration, which

618

made the "MS sensor" a powerful tool in studying cellular metabolism. Zhang [90] used

619

matrix-assisted laser desorption mass spectrometry (MALDI-MS) to in situ analyze

620

lipids in cells. As Fig. 4d describes, mammalian cells were cultured on ITO-coated glass

621

substrate directly, and then a matrix layer on samples was applied by electrospray

622

coating. The platform could generate a profile of abundant membrane lipid, which could

623

be a characteristic of cell type.

example,

Liu

[89]

established

a

Figure 4

624

625

For

7. Conclusions

626

Microfluidics, a powerful tool for cell research, could be further improved by

627

applying more functional biomaterials. Biomaterials could participate in each portion of

628

microfluidic-based cell analysis:

629 630 631

(1) Materials for microfluidic fabrication have three tendencies: from inorganic to polymer, from supporting to functional and smart, from simple to integrated. (2) Cell culture on-chip on longer focuses on flat surface, achieving 3D culture that

30 Page 30 of 44

632

can better imitate microenvironment with dynamic flowing is the superiority of on-chip

633

cell culture. Particularly, organ-on-chip system makes microfluidic devices promising

634

in biology, and biomaterials that can support spatial cell growth, such as hydrogels and

635

bio-membranes will play a key role in the development of this field.

636

(3) Biomaterials assisting in cell immobilization and cell imaging on chip could

637

make non-invasive cell observations more facile and sensitive. Traditional inorganic

638

QDs have found vast application for cell imaging. Inorganic/organic composites could

639

perform better biocompatibility and imaging for on-chip cell observation.

640

(4) Microfluidic system could exhibit dynamic stimulation on cells, and combined

641

with mass spectrometry, on-line analysis with real-time monitoring could be

642

accomplished. Biomaterials could participate in cell analysis in ways of improving

643

metabolites accumulation, realizing single-cell analysis, and enhancing sensitivity. With

644

the employment of biomaterials, microfluidic chips can be more productive in cell

645

analysis.

646

Biomaterials take important positions in cell researches on-chip. Though plenty

647

achievements have obtained, there are still lots of problems remained to be solved. Due

648

to materials participating each step in cell researches on-chip, development of

649

biomaterials will bring a revolution to biology and medicine research and applications.

650

Acknowledgements

651 652

This work was supported by National Natural Science Foundation of China (No. 51125007).

653

31 Page 31 of 44

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References

655

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Figure Captions

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Fig. 1. Thermal responsive smart material PNIPAAm applied in bioresearch. (a)

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PNIPAAm chain of reversible conformational transition around LCST leads to

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wettability change (Reprinted with permission from [46]); (b) Tunable cell adhesion on

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PNIPAAm microgel films (Reprinted with permission from [47]).

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Fig. 2. A typical "sandwich" co-culture platform (Reprinted with permission from [53]).

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(a) Schematic of PDMS porous membrane-assembled microfluidic co-culture platform.

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(b) Proliferation and viabilities of mEFs and mES cells on the co-culture microdevice.

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Fig. 3. Two ways for constructing organ-on-chip system based on biomaterials. (a) Using

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bottom-up tissue engineering built BBB structure on chip (Reprinted with permission

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from [69]); 3D hydrodynamic flow focusing devices for fabricating (b) cell spheroids

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(Reprinted with permission from [70]) and (c) cell fibres with core-shell structure

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(Reprinted with permission from [71]).

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Fig. 4. On-chip cell analysis. (a) Drug permeation analysis based on semipermeable PC

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membrane combined SPE technique (Reprinted with permission from [84]); (b)

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Microwell arrays for single cell isolating and analyzing platform (Reprinted with

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permission from [87]); (c) Real-time cell analysis combined MS detection by using

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microdialysis hollow fiber to generate microdroplet; (d) Real-time cell analysis

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detection on a rapid MALDI-MS platform (Reprinted with permission from [90]).

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