Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Synthesizing Living Tissues with Microfluidics Wenfu Zheng† and Xingyu Jiang*,†,§,‡ †
Acc. Chem. Res. Downloaded from pubs.acs.org by UNIV OF NORTH DAKOTA on 11/20/18. For personal use only.
Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, Beijing 100190, P. R. China § Department of Biomedical Engineering, Southern University of Science and Technology, No. 1088 Xueyuan Rd, Nanshan District, Shenzhen, Guangdong 518055, P. R. China ‡ The University of Chinese Academy of Sciences, Beijing 100049, P. R. China CONSPECTUS: In native tissues, various cell types organize and spatiotemporally function and communicate with neighboring or remote cells in a highly regulated way. How can we replicate these amazing functional structures in vitro? From the view of a chemist, the heterogeneous cells and extracellular matrix (ECM) could be regarded as various chemical substrate materials for “synthetic” reactions during tissue engineering. But how can we accelerate these reactions? Microfluidics provides ideal solutions. Microfluidics could be metaphorically regarded as a miniature “biofactory”, whereas the on-chip critical chemical cues such as biomolecule gradients and physical cues such as geometrical confinement, topological guidance, and mechanical stimulations, along with the external stimulations such as light, electricity, acoustics, and magnetics, could be regarded as “catalytic cues” which can accelerate the “synthetic reactions” by precisely and effectively manipulating a series of cell behaviors including cell adhesion, migration, growth, proliferation, differentiation, cell−cell interaction, and cell−matrix interaction to reduce activation energy of the “synthetic reactions”. Thus, on the microfluidics platform, the “biofactory”, various “synthetic” reactions take place to change the substrate materials (cells and ECM) into products (tissues) in a nonlinear way, which is a typical feature of a biological process. By precisely organizing the substrate materials and spatiotemporally controlling the activity of the products, as a “biofactory”, the microfluidics system can not only “synthesize” living tissues but also recreate physiological or pathophysiological processes such as immune responses, angiogenesis, wound healing, and tumor metastasis in vitro to bring insights into the mechanisms underlying these processes taking place in vivo. In this Account, we borrow the concept of chemical “synthesis” to describe how to “synthesize” artificial tissues using microfluidics from a chemist’s view. Accelerated by the built-in physiochemical cues on microfluidics and external stimulations, various tissues could be “synthesized” on a microfluidics platform. We summarize that there are “step-by-step synthesis” and “one-step synthesis” on microfluidics for creating desired tissues with unprecedented precision, accuracy, and speed. In recent years, researchers developed various microfluidic techniques including creating adhesive domains for mediating reverse and precise adhesion, chemical gradients for directing cell growth, geometrical confinements and topological cues for manipulating cell migration, and mechanics for stimulating cell differentiation. By employing and orchestrating these on-chip tissue “synthetic” conditions, “step-by-step synthesis” could be realized on chips to develop multilayered tissues such as blood vessels. “One-step synthesis” on chips could develop functional three-dimensional tissue structures such as neural networks or nephronlike structures. Based on these on-chip studies, many critical physiological and pathophysiological processes such as wound healing, tumor metastasis, and atherosclerosis could be deeply investigated, and the drugs or therapeutic approaches could also be evaluated or screened conveniently. The “synthetic tissues on microfluidics” system would pave an avenue for precise creation of artificial tissues for not only fundamental research but also biomedical applications such as tissue engineering.
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INTRODUCTION “Organ-on-a-chip” is an explosive field with exciting applications ranging from drug discovery to regenerative medicine. A microfluidics chip could be metaphorically regarded as a “biofactory” to “synthesize” various tissues in vitro in a highly efficient and precise way. The cells and scaffold materials could be metaphorically regarded as substrate molecules, whereas the built-in chemical cues (e.g., biomolecule gradient) or physical cues (e.g., geometrical confinement, topographical guidance, and mechanical stimulations) in the microfluidics system could © XXXX American Chemical Society
combine external stimulations such as light, electricity, acoustics, and magnetics to serve as “catalytic cues” to decrease the activation energy and accelerate the “synthetic reactions” for producing desired tissues that mimic the native tissues. Also, the built-in cues and external stimulations could be individually or collectively introduced in the microfluidics environment to manipulate cell behaviors including cell adhesion, migration, Received: August 17, 2018
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DOI: 10.1021/acs.accounts.8b00417 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research
cells. Moreover, these cues on microfluidic chips could accelerate the “synthesis” of artificial tissues. Gradients of biomolecules play key roles in various cellular behaviors in human body. Microfluidic devices can generate serially diluted analytes on a surface7 or serve as a modular system to produce chemical gradients of any profiles at will.8 Microfluidics can also simulate combinational or individual influences of chemical gradient and geometrical confinement on cell polarity, which is crucial for directing cell migration and tissue development.9 Chemical gradients generated on microfluidics platforms are central for studying neurobiology. For example, a sharp change of the laminin concentration can guide neurite growth,10 whereas a gradient of Slit protein can induce axon fasciculation.11 Geometry is a major factor influencing the spatial distribution of chemical cues or ligands at the cell−scaffold interface. By utilizing microcontact printing techniques, we patterned thiols to form SAMs and confined cells into rectangular patterns. By electrochemical desorption of the SAMs, we released the patterned cells from the geometrical constraints (Figure 2a).12 Furthermore, we constrain individual cells to tear-drop shape, which polarized the morphology of the cell. After electrochemical desorption of the SAMs, the cells were released from the constraints and tended to move toward their blunt ends (Figure 2b).13 By fabricating laminin patterns of different densities and geometry widths,14 we found that both a sharp change of laminin density and substrate geometry can stimulate axon branching (Figure 2c). Using a microfluidic chip, we can electrochemically desorb thiols to active the inert surface and release the confined cells to migrate freely (Figure 2d)15 and mimic several in vivo cell migration modes (Figure 2e).16 By patterning Matrigel on geometric constraints, we established a 2D neural circuit and studied collective neuronal migration (Figure 2f).17 To achieve different chemical patterns in a single microchannel, we combined colloidal lithography with microfluidics to generate sharp gradients on a substrate to form bifunctional chemical nanopatterns to enable site-specific adsorption of different proteins on the nanopatterned surface (Figure 2g),18 on which we revealed that about 14 or more integrin molecules on the cell membrane are needed for mediating adhesion of one cell.19 Extracellular matrix (ECM), providing physical supporting for living cells, is a 3D architecture composed of various nano- to microscale geometrically featured structures. Various topological features including grooves, fibers, pores, ridges and pillars play roles in influencing cell behaviors.20,21 For example, we studied the behaviors of different cell groups on same interfaces with microgrooves and observed that the cells with different cell−cell intercellular interactions showed distinct migration behaviors on the microgroove patterned surface.22 Micropatterned substrates of varying sizes can determine cell fates: alive or apoptotic.23 For example, a honeycomb microframe can trigger differentiation of human induced pluripotent stem cells (hiPSCs) toward a uniform cardiac layer.24 Cells reside in a microenvironment with biomechanics, which is a key physical cue determining cell fates. To address the influence of biomechanics on cell behavior, we designed an elastic membrane-based device to study the arrangement of F-actin of cells responding to stretch.25,26 We also observed the immediate response of mesenchymal stem cells (MSCs) to fluid shear stress on microfluidic chips.27 Besides the built-in chemical or physical cues, external measures such as light, electricity, acoustics, and magnetics
and differentiation, which are helpful for investigation of basic physiological or pathophysiological processes such as immune responses, angiogenesis, and tumor metastasis. To summarize recent progress in “synthesizing” artificial tissues on microfluidics platforms, the “synthetic” strategies could be classified into two categories: (1) “Step-by-step synthesis” is composed of multistep procedures. For instance, to synthesize a multilayered tubular structure (e.g., blood vessel), we prepare a stress-induced rolling membrane (SIRM) system and subsequently pattern multiple cell types including endothelial cells, smooth muscle cells, and fibroblasts on the SIRM by microfluidic chips; the resulting flat multi-cell-type-laden matrix film finally forms a multilayered cell-laden tubular structure by the releasing of the SIRM.1 (2) “One-step synthesis” can be finished in one step. For instance, to synthesize a three-dimensional (3D) neuronal network, we put neurons, astrocytes, and scaffolds (borosilicate microspheres) into microchambers interconnected by multilayered microchannels to form neuronal networks in one step.2 In this Account, we summarize recent developments in how to “synthesize” various functional tissues using microfluidics (Figure 1). We highlight the strategy to “synthesize” tubular
Figure 1. Schematic diagram shows the concept of “synthesizing” tissues on a microfluidics platform. The microfluidics platform could serve as a “biofactory” for tissue “synthetic” reactions. Various types of cells plus scaffolds of different materials could be metaphorically regarded as substrate materials. The built-in chemical cues such as biomolecule gradients and physical cues such as geometrical confinement, topological guidance, and mechanical stimulations could accelerate the “synthetic” reactions. External stimulations such as light, electricity, acoustics, and magnetics can modulate the tissue “synthetic” reactions on the chip. Various functional tissues including flat tissues, tubular tissues, and solid tissues could be “synthesized” on the chip.
tissues by microfluidics,1,3−6 which shows great potential in creating highly sophisticated tissues on microfluidics in a straightforward way.
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CREATING “CATALYTIC CUES” ON A MICROFLUIDICS PLATFORM FOR MANIPULATING CELLS Microfluidics includes versatile measures to create chemical cues and physical cues, which are capable of simulating a variety of physiological and pathophysiological conditions of the native tissues or organs in vitro. By orchestrating these chemical and physical cues, along with external controlling methods such as light, electricity, acoustics, and magnetics, microfluidics provides a variety of approaches for manipulating B
DOI: 10.1021/acs.accounts.8b00417 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research
Figure 3. Light or electricity-triggered cell adhesion manipulation. (a) Working mechanism of a photoresponsive surface. Reproduced with permission from ref 28. Copyright 2009 Wiley. (b) A cartoon illustrates a method to coat surface dynamically. Glass slides are coated with APP, which can be activated by UV irradiation and patterned with fibronectin to create specific cell-repellent areas. Cell adhesion is switched on by addition of BCN-RGD to the cell culture medium to trigger migration. Reproduced with permission from ref 29. Copyright 2013 Wiley. (c) Schematic representation of a switchable surface for controlling cell adhesion. Reproduced with permission from ref 30. Copyright 2015 Wiley.
Figure 2. Geometrical confinement and dynamic control of cell adhesion and migration. (a) Cells are confined to microislands, and a voltage pulse releases the cells from the micropatterns. Reproduced with permission from ref 12. Copyright 2003 American Chemical Society. (b) A tear-drop shaped pattern can polarize a NIH 3T3 cell on a gold surface. Reproduced with permission from ref 13. Copyright 2005 National Academy of Sciences. (c) Axon branching control on microfluidics by differently sized micropatterns. Reproduced with permission from ref 14. Copyright 2013 Royal Society of Chemistry. (d) Time-lapse micrographs show the potential-triggered release of cells on the surface. Reproduced with permission from ref 15. Copyright 2007 Wiley. (e) A strategy for patterning different types of cells that can simulate cell−cell interactions taking place in vivo. Reproduced with permission from ref 16. Copyright 2009 Wiley. (f) Geometric regulation of clustering location. Scale bar, 200 μm. Reproduced with permission from ref 17. Copyright 2011 Public Library of Science. (g) The cartoon shows patterning of different proteins inside a “Y”-shaped channel. Reproduced with permission from ref 18. Copyright 2015 Royal Society of Chemistry.
glass. The cells confined in the specific areas could be released (Figure 3b).29 Electricity-triggered electrochemical reactions on an adhesive or inert surface could be used to control cell adhesion and desorption. Cyclic RGD peptides usually have a higher binding affinity to cells than linear RGD peptides. We designed a quaternary ammonium-terminated RGD-containing peptide (RGD-NMe3), which can undergo reversible switch between cyclic and linear conformations under different electrochemical potentials to realize reversible control of cell adhesion (Figure 3c).30 Acoustics is another external source for cell control on chip.31 For example, a droplet-based surface acoustic wave (SAW) system realized detachment of different types of cells from a surface and label-free sorting of the cell types based on differences in adhesion strength.32 Magnetics can facilitate not only on-chip diagnosis but also cell manipulations. For example, a microfluidic cell culture device was designed to fit commercially available nuclear magnetic resonance (NMR) equipment to realize real-time monitoring of cultured neurospheres. Necrotic areas in the interior of the neurospheres could be detected during the dynamic culturing.33 Collectively, the external cues could serve together with the built-in chemical or physical cues to accelerate the assembly of the cells, scaffolds, and biomolecules for on-chip tissue engineering.
could also manipulate cell behaviors on chip. Light is a facile tool for mediating adhesion of cells on adhesive or inert surfaces. We tethered RGD peptide on azobenzene unit and formed SAMs on a gold surface. UV light irradiation can trigger the conversion of the azobenzene isoform from E to Z, which can present RGD to promote cell adhesion, while visible light can reverse the conversion to conceal RGD to desorb the adhered cells (Figure 3a).28 A recent work demonstrated that azido-(PLL-g-PEG) (APP) modification on glass can produce an inert surface that could be activated by photomaskerdirected, UV irradiation-induced destruction of the APP and the adhered cells at specific areas. The addition of a strained cyclooctyne bicyclo[6.1.0]- nonyne (BCN)−RGD peptide to the cell culture medium allows the strain-promoted azide− alkyne cycloaddition (SPAAC) reaction between the APP and BCN−RGD and the activation of the residual areas on the C
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Figure 4. Flat tissues on microfluidics. (a, left) Schematic diagram of a cross-section through the small airway-on-a-chip. (a, right) 3D reconstruction image shows pseudostratified airway epithelium formed on-chip (green, F-actin) with human pulmonary microvascular endothelial cells (red, F-actin) on the opposite side of the membrane. Blue denotes DAPI-stained nuclei; scale bar, 30 μm. Reproduced with permission from ref 35. Copyright 2016 Nature. (b) Human−microbial crosstalk on microfluidic chips. Left, conceptual diagram of the human−microbial crosstalk model for the representative coculture of human epithelial cells with gastrointestinal microbiota; right, photograph of the assembled human− microbial crosstalk device. Scale bar, 1 cm. Reproduced with permission from ref 37. Copyright 2016 Nature.
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“SYNTHESIZING” TISSUES BY MICROFLUIDICS Conventional methods for tissue engineering, usually lacking chemical or physical cues for guiding hierarchical growth of cells and directional evolution of tissues, are time and labor consuming and make it difficult to construct desirable artificial tissues in vitro. Microfluidics has the potential to address these issues. By integrating cells, matrices, chemical and physical cues, and appropriate external stimulations (temperature, compartment, light, electricity, acoustics, and magnetics), microfluidics provides a platform for “synthesizing” various tissues. According to the shapes and structures, the on-chip tissues could be classified into three categories: (1) flat tissues, which contain most of the basic functional units of tissues concerning material exchanges, such as the liquid/air interface in alveoli; (2) tubular tissues, such as the blood vessel; (3) solid tissues, such as the lobule in liver. Flat tissues refer to thin and flat cell−ECM layers such as liquid/air interface in alveoli, blood−urine interface in renal tubules, and blood−gastrointestinal tract interfaces in the digestive system. The common feature of these flat tissues is a sandwich shaped structure with a thin layer of ECM in the middle and two heterogeneous cell layers on both sides. Microfluidics can model the sandwich structure via step-by-step synthesis of two cell layers on one piece of porous membrane. Researchers built an alveolar−capillary system on a microdevice34 and simulated pathological conditions of the lung such as chronic obstructive pulmonary disease (COPD) (Figure 4a).35 Microfluidics can also construct the blood−urine interface in renal tubules to simulate structure and function of proximal tubule and collecting duct.36 Recently, researchers studied human− microbe interactions on a microfluidic device. On the chip, the coculture of human epithelial cells with the obligate anaerobe Bacteroides caccae and Lactobacillus rhamnosus GG (LGG) resulted in a transcriptional response, which is distinct from that of a coculture solely comprising LGG (Figure 4b).37 Tubular tissues are ubiquitous structures in human body. Blood vessels, lymph vessels, guts, and windpipes are typical tubular tissues. Tubular tissues are composed of multilayered
structures with different cells and matrices. For example, blood vessels have a three-layer structure: intima (inner layer), media (medium layer), and adventitia (outer layer) from inner to outer side. Conventional methods to prepare artificial blood vessels usually need months to years for integration of cells and materials, which greatly hinder the clinical translation of these methods. An ideal solution is to rapidly transit well-defined 2D culture to a 3D culture.1 We developed a strategy to synthesize cell-laden, multilayered tubular structures mimicking native blood vessels using a stress-induced rolling membrane (SIRM) technique. We performed step-by-step synthesis: first, we used microchannels to deliver and pattern different types of cells on a chemically modified 2D SIRM; second, we released the SIRM, which can roll up automatically to form a 3D tube. The resulting tube has multiple types of cells located specifically, mimicking the structural and functional features of a real blood vessel. This strategy may have wide applications in 3D tissue fabrication by transforming a 2D patterned structure to a 3D structure (Figure 5a).1 To mimic 3D cell−cell interactions in blood vessel, we “synthesized” 2D multilayered cell constructions by biotin−streptavidin (SA) interaction and further formed 3D, multilayered artificial blood vessels by applying the SIRM technique (Figure 5b).3 The SIRM technique could be further extended to “synthesize” various tubular structures such as wrinkled tubes, multitube structures, and spirals that were difficult to fabricate with ordinary methods. Thus, the SIRM technique is a useful strategy for rapid “synthesis” of 3D tubular tissues, which may have a variety of applications (Figure 5c).4 Solid tissues such as liver, heart, kidney, and brain can hardly be recapitulated by conventional 2D cultures, whereas microfluidics provides precisely and chemically defined synthetic methods for 3D tissue construction and mimicking the complex 3D cell−cell and cell−matrix interactions in these organs. For these tissues, one-step synthesis is a facile and effective method. For example, we presented a method to produce 3D microchannel networks in hydrogel utilizing a microfluidic liquid mold and collagen. The model composed a nephron-like structure for mimicking passive diffusion of soluble organic D
DOI: 10.1021/acs.accounts.8b00417 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 6. Tissues on microfluidic chips by “one-step synthesis”. (a) A microfluidic nephron model. Left panel shows schematic illustration of structure and function of a nephron. Right panel displays images of the artificial nephron structure. Reproduced with permission from ref 38. Copyright 2013 Royal Society of Chemistry. (b) A microfluidic model of the brain. Top panel shows schematics of the “synthesis” processes of a 3D neural network on a multilayered microchip. Bottom panel shows neurite-connected networks between different chambers. Reproduced with permission from ref 2. Copyright 2014 Wiley.
Figure 5. Stress-induced rolling membrane (SIRM) for “synthesizing” tubular structures. (a, left) Working mechanism of a SIRM and the resulting tube containing multiple types of cells. (a, right) Fluorescent images show the morphologies of different types of cells on a flat SIRM and a rolled SIRM. Reproduced with permission from ref 1. Copyright 2012 Wiley. (b) Combination of cell layer assembly and SIRM to construct multilayer tubes shown in schematics and confocal images. Scale bar: 200 μm. Reproduced with permission from ref 3. Copyright 2013 Wiley. (c) The production process of tube, multitube, and spiral by SIRM and the sample images. Reproduced with permission from ref 4. Copyright 2013 Wiley.
device, pluripotent stem cell-derived cardiomyocytes formed junction complexes and showed superior cardiac differentiation and electrical and mechanical coupling.43
molecules between capillary vessels and kidney tubules (Figure 6a).38 We also “synthesized” a structurally and functionally biomimetic 3D neuronal network on a microchip by onestep synthesis. We used gravity-driven, microchamber-assisted assembly of borosilicate glass microspheres to fabricate scaffolds and introduced neurons and astrocytes onto the scaffolds. The neurons grew and protruded neurites into multilayered microchannels interconnecting the microchambers to construct neuronal networks in one step. The product, 3D neural networks, showed spontaneous activities like those found in the brain (Figure 6b).2 The sinusoid is a basic functional unit of the liver. To recapitulate this structure in vitro, researchers developed a microfluidic chip that can maintain long-term (28 days) normal functions of the liver such as albumin synthesis and urea excretion.39 Besides mimicking the normal functions of the liver, researchers developed microfluidic devices that can recapitulate nonalcoholic fatty liver disease40 and hepatitis B virus infection.41 The heart is a special organ that has contraction/relaxation phases to function in the body. Thus, the realization of controlled physiological uniaxial cyclic strains in vitro is highly desirable for drug toxicity evaluation. Researchers developed a fluidic heart on a chip device for analyzing the behavior of muscular thin film in response to isoproterenol.42 Another work used an array of hanging posts to confine cell-laden gels, which was subjected to homogeneous uniaxial cyclic strains generated by a pneumatic actuation system. On this microfluidic
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BIOMEDICAL APPLICATION OF “SYNTHETIC” TISSUES ON MICROFLUIDICS PLATFORMS Tissues on microfluidics provide convenient platforms for in vitro investigation of key physiological or pathological processes, fabrication of tissue-engineered implants, and screening of drugs. By simulating physiological or pathological processes on microfluidics, researchers can study mechanisms of various disorders in vitro, under similar conditions as those in vivo. Thus, the on-chip disease models can partially or even totally replace experimental animals for the related studies. Wound healing is an important process involving cell migration and complex cell−cell interactions. We constructed a three-channel microfluidic chip for simulating the in vivo multicellular environment and studied the influence of damaged cells on collective migration of epithelial cells (Figure 7a).44 We also designed a microfluidic wound model to simulate skin wounds and screened the performance of wound dressings made from electrospun mats in promoting wound healing. The model provides a new way for screening candidates of wound dressings conveniently (Figure 7b).45 Based on this model, we further evaluated the performance of a biosynthesized material, bacterial cellulose (BC), as wound dressing to treat cutaneous wounds (Figure 7c).46 To promote the treatment of bacteriainfected wounds, we developed small molecule modified nanoparticles (Au-DAPT NPs) and decorated BC with the E
DOI: 10.1021/acs.accounts.8b00417 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 7. Wound healing models or wound dressing screening on microfluidics. (a) A microfluidic wound model for coculture of cells and selective producing of wounds. Scale bar: 500 μm. Reproduced with permission from ref 44. Copyright 2011 Royal Society of Chemistry. (b) Schematic illustration of a wound model on a microfluidic device. Reproduced with permission from ref 45. Copyright 2012 Wiley. (c) A schematic illustrates the difference between two sides of a BC film as wound dressings evaluated by onchip wound healing model. Reproduced with permission from ref 46. Copyright 2015 American Chemical Society. (d) A schematic illustration of the preparation process of the BC-Au-DAPT nanocomposites. Reproduced with permission from ref 47. Copyright 2017 Wiley. (e) The “synthesis” process of antibacterial AuNPs and their applications for promoting wound healing. Reproduced with permission from ref 48. Copyright 2017 America Chemical Society.
Figure 8. Drug screening on model tissues on microfluidics platform. (a) GNC-siRNA inhibits pancreatic cancer development. Top panel shows scheme of a microfluidic chip for neuron−pancreatic cancer cell coculture. Bottom panel shows on-chip migration assay of Panc-1 cells. Scale bar: 100 μm. Reproduced with permission from ref 50. Copyright 2017 Nature Publishing Group. (b, left) Schematic of a flow-stretch microchip. (b, right) Fluorescence images show the reorganization of stress fibers of MSCs under different mechanical stimulations. Reproduced with permission from ref 51. Copyright 2012 Royal Society of Chemistry. (c) Schematic of an early stage atherosclerosis model. In the left panel, the cartoon shows the different responses of cells to drugs between petri dish and chip. Right panel shows the morphological features of endothelial cells on the Petri dish and the chip under Probucol treatment. Reproduced with permission from ref 52. Copyright 2016 Wiley.
nanoparticles to prepare antibacterial wound dressings, which showed excellent performance to treat multidrug resistant (MDR) bacteria-infected wounds (Figure 7d).47 We also doped 6-aminopenicillanic acid (APA)-coated Au nanoparticles into PCL/gelatin electrospun nanofibers to fabricate wound dressing that was capable of remedying MDR bacteria wound infection (Figure 7e).48 We also developed a TGF-β1 inhibitor-doped PCL/gelatin electrospun scaffold for treating hypertrophic scarring.49 The synthetic tissues on microfluidics devices provide a platform for efficient drug evaluation and screening. On a simple nerve−tumor interactions model on a microfluidics platform, we simulated in vivo nerve−tumor crosstalk, such as peripheral nerve invasion (PNI), which is proposed to be another route for tumor metastasis. We assessed the efficiency of gold nanocluster-assisted delivery of siRNA of nerve growth factor (NGF) (GNC-siRNA) to pancreatic tumor cells and the resultant antitumor effects on the chip (Figure 8a).50 The on-chip assay showed that GNC-siRNA induced about 60% inhibition of tumor cell migration, which was excellent accordance with the reduction (∼60%) of tumor metastasis of orthotopic pancreatic patient-derived xenograft tumor. This means that our on-chip model can accurately predict the drug efficiency in vivo, and it
could be used to partially or even completely replace the animal models for drug screening. The on-chip tissues, which recapitulate the mechanical microenvironment of the human body, may help find some new pharmaceutical effects of the tested drugs. For example, we developed a flow-stretch microchip on which a monolayer of endothelial cells was constructed and stimulated by integrated fluid shear stress and cyclic stretch to simulate the hemodynamic environment of the artery (Figure 8b).51 Based on this chip, we built a microchip reconstituting early stage pathology of atherosclerosis. The cells on the microfluidic chip were more sensitive to chemical cues than the cells on a Petri dish. On the chip, we found cellular level evidence of the clinical side-effects of probucol (an antiatherosclerotic drug), which causes arrhythmia in patients (Figure 8c).52
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FUTURE PERSPECTIVES In this Account, we highlighted the concept of synthesis of tissues on microfluidics platforms and summarized recent developments of microfluidic approaches to synthesize desirable F
DOI: 10.1021/acs.accounts.8b00417 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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cellular products at various levels to mimic advanced structural and functional features of native tissues or organs and simulate the physiological or pathophysiological conditions in the human body. Among the important synthetic products summarized in this Account, the tubular structures are the most representative ones due to their special structure and crucial role in living tissues. For example, vascularization is among the central concepts of tissue engineering because the artificial tissues built in bulk scaffolds are difficult to keep alive or proliferate without full supply of nutrients and drainage of waste products. That is to say, like native tissues or organs, the compartmentalization and vascularization of the artificial tissues are prerequisites for the tissue development and maintenance. Microfluidics provides facile and excellent measures for the precise construction and manipulation of 3D artificial tissues with built-in vasculature mimicking the microenvironment of the native tissues. Here, we highlight novel strategies such as the stress-induced rolling membrane (SIRM) technique in synthesizing tubular structures that could be extended to many areas that deserve exploration such as tissue or organ evolution or complex tissue regeneration. For example, embryonic stem cell-laden multilayered tubes could be synthesized on a microfluidics platform to mimic the three primary germ layers (endoderm, mesoderm, ectoderm) of the very early embryo. By tuning the proliferation and differentiation of cells in each layer using gene regulating approaches (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) system, a gene editing toolbox), researchers may build desirable tissue development models or disease models in vitro, which are promising for biomedical research and drug development industry. Another example is to synthesize functional units of tissues or organs based on vasculature made by the SIRM technique. By integrating human-derived nerve cells, stromal cells, and functioning cells (e.g., myocytes, neuron, parenchymal cells) with the vasculature, most of the tissues in human body could be simulated. The on-chip tissue models with human-derived cells have the most similar responses to drug treatment as in human body and have the potential to replace parts or all animal experiments in the future to serve as in vitro platforms for drug screening.
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Wenfu Zheng received his Ph.D. in Biophysics from Peking University (China) in 2008, he is currently a Professor at National Center for NanoScience and Technology of China (NCNST). Xingyu Jiang received his PhD (2004) from Harvard University (Chemistry), He is currently a Professor of chemistry at National Center for NanoScience and Technology of China (NCNST).
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AUTHOR INFORMATION
Corresponding Author
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[email protected]. ORCID
Xingyu Jiang: 0000-0002-5008-4703 Author Contributions
The manuscript was written through contributions of both authors. All authors have given approval to the final version of the manuscript. Funding
This work was supported by Minister of Science and Technology of China (Grant 2017YFA0205901), National Nature Science Foundation of China (Grants 81361140345, 21535001, 81730051, 21761142006, 81671784, 21505027, 31470911, and 81673039), the Chinese Academy of Sciences (Grant 121D11KYSB20170026), and the CAS/SAFEA International Partnership Program for Creative Research Teams. Notes
The authors declare no competing financial interest. G
DOI: 10.1021/acs.accounts.8b00417 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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