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Resistance to targeted therapies is a major clinical challenge in cancer treatment. Despite technological advances, robust biomarkers or platforms predictive of treatment response are lacking owing to the inherent nature of complex genomic landscape of carcinoma. Nevertheless, recent efforts centred on performing direct drug screening on patient-derived cells through their ex vivo expansion and maintenance have enabled personalized stratification of treatment modalities. Microfluidics is one such technology that allows high-throughput drug screening through parallelization and automation using small-volume sample. In this review, we present recent microfluidic platforms that have been successfully applied for the maintenance and expansion of patient-derived tumor cells spanning diverse cancer types and sources (solid tumors or liquid biopsies (circulating tumor cells)) for personalized drug screening applications.

Mechanistic understanding of atherosclerosis is largely hampered by the lack of a suitable in vitro human arterial model that recapitulates the arterial wall structure, and the interplay between different cell types and the surrounding extracellular matrix (ECM). This work introduces a novel microfluidic endothelial cell (EC)-smooth muscle cell (SMC) 3D co-culture platform that replicates the structural and biological aspects of the human arterial wall for modeling early atherosclerosis. Using a modified surface tension-based ECM patterning method, we established a well-defined intima-media-like structure, and identified an ECM composition (collagen I and Matrigel mixture) that retains the SMCs in a quiescent and aligned state, characteristic of a healthy artery. Endothelial stimulation with cytokines (IL-1β and TNFα) and oxidized low-density lipoprotein (oxLDL) was performed on-chip to study various early atherogenic events including endothelial inflammation (ICAM-1 expression), EC/SMC oxLDL uptake, SMC migration, and monocyte-EC adhesion. As a proof-of-concept for drug screening applications, we demonstrated the atheroprotective effects of vitamin D (1,25(OH)2D3) and metformin in mitigating cytokine-induced monocyte-EC adhesion and SMC migration. Overall, the developed arterial wall model facilitates quantitative and multi-factorial studies of EC and SMC phenotype in an atherogenic environment, and can be readily used as a platform technology to reconstitute multi-layered ECM tissue biointerfaces. Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) We would like to acknowledge financial support from the Singapore Ministry of Education Academic Research Fund Tier 1 (RG53/18) awarded to H. W. H., and National Medical Research Council (NMRC) Clinician Scientist Award (CSAINV17nov009) awarded to R. D. C. S. is supported by the NTU Interdisciplinary Graduate Programme Scholarship. H. C. is supported by the NTU Research Scholarship.

Background The application of intra-operative blood salvage autotransfusion(IBSA) in liver transplantation(LT) for hepatocellular carcinoma(HCC) remains controversial due to the theoretical risk of tumour cell(TC) reintroduction. Current studies evaluating for presence of TC are limited by suboptimal detection techniques. This study aims to analyze the presence of TC in HCC LT autologous blood using microfluidics technology. Methods A prospective study of HCC patients who underwent LT from February 2018–April 2019 was conducted. Blood samples were collected peri-operatively. TCs were isolated using microfluidics technology and stained with antibody cocktails for confirmation. Results A total of 15 HCC LT patients were recruited. All recipients had tumour characteristics within the University of California, San Francisco(UCSF) criteria pre-operatively. TC was detected in all of the autologous blood samples collected from the surgical field. After IOCS wash, five patients had no detectable TC, while 10 patients had detectable TC; of these two remained positive for TC after Leukocyte Depletion Filter(LDF) filtration. Conclusion The risk of tumour cell reintroduction using IBSA in HCC LT patients can be reduced with a single LDF. Future studies should evaluate the proliferation capacity and tumorigenicity of HCC TC in IBSA samples, and the effects of TC reintroduction in patients with pre-existing HCC TCs.

Blood vessel narrowing and arterial occlusion are pathological hallmarks of atherosclerosis, which involves a complex interplay of perturbed hemodynamics, endothelial dysfunction and inflammatory cascade. Herein, we report a novel circular microfluidic stenosis model that recapitulates atherogenic flow-mediated endothelial dysfunction and blood-endothelial cell (EC) interactions in vitro. 2D and 3D stenosis microchannels with different constriction geometries were fabricated using 3D printing to study flow disturbances under varying severity of occlusion and wall shear stresses (100 to 2000 dynecm-2). Experimental and fluid simulation results confirmed the presence of pathological shear stresses in the stenosis region, and recirculation flow post stenosis. The resultant pathological flow profile induced pro-inflammatory and pro-thrombotic EC state as demonstrated by orthogonal EC alignment, enhanced platelet adhesion at the stenosis, and aberrant leukocyte-EC interactions post stenosis. Clinical utility of the vascular model was further investigated by testing anti-thrombotic and immunomodulatory efficacy of aspirin and metformin, respectively. Overall, the platform enables multi-factorial analysis of critical atherogenic events including endothelial dysfunction, platelets and leukocyte adhesion, and can be further developed into a liquid biopsy tool for cardiovascular risk stratification.

The nanotopography of the cellular environment in vivo is an important factor that affects cellular phenomena such as adhesion, proliferation and migration. The capability of tumor cells to collectively migrate is critical during the process of tumor metastasis, which is significantly regulated by the nanotopography of the microenvironment such as its roughness. Herein, a simple and effective approach is developed to generate a controlled roughness contrast on the same poly(dimethylsiloxane) substrate using chemical etching and rapid molding, and a quantitative study is presented on the influence of surface roughness on cell collective migration. Specifically, the HuH7 (a human hepatocarcinoma) cell monolayer exhibits a slower migration mode on a nanoroughened substrate compared to its behaviour on a smooth substrate. Subsequent gene analyses indicate that the cell-substrate and cell-cell adhesion proteins are downregulated on the roughened substrate. This study shows the impact of substrate roughness on cell biochemical functioning, and hence on collective migration, suggesting that an engineered nanotopography could be applied in the design of biomedical devices in order to manipulate tumor cell behaviour.

Microfluidics is a highly promising technology platform for cancer diagnosis. It will need a change of mindset among engineers, clinicians and regulators to fully embrace its potential. [Image: see text]

Control of structure and function, at the nanometer scale, remains a formidable challenge in the arena of self-assembled soft materials. Here, we report on the design of a small molecule-based two-component assembly system in which the assembly partners can recognize each other through host–guest interactions. One component is hydrophobic and carries a donor–acceptor type of electronic structure. This is realized by employing a bucky-bowl corannulene derivative. The other component is hydrophilic and hollow. This is achieved by using γ-cyclodextrin, the largest and least studied member of the cyclic oligosaccharide family. In a chemically polar aqueous environment, the two partners come together to form an amphiphilic structure that assembles further into nanosized, quasicrystalline, dot-like, non-toxic, all-organic structures showing two-photon activity and bright green luminescence in water upon excitation at 800 nm. The devised synthesis is achieved by a simple mixing process carried out under continuous-flow conditions. Therefore, in a scalable manner, a constant supply of the assembly components results in continuous fabrication of the nanostructures. Non-linear optical activity and biocompatibility aspects suggest utility of the prepared new class of soft organic nano-dots as contrast agents or labeling tags for visualizing biological specimens. This aspect is examined and demonstrated through two-photon fluorescence imaging of cancer cell lines.

Microfluidics is a platform that is being widely used for developing in-vitro models to study various in-vivo phenomena. Our aim was to develop a simple, easy to use microfluidic device, which can be used for different cell-based applications. By performing cell patterning on-a-chip we were able to develop a microfluidic in-vitro model that could be used to perform cell-cell interaction studies and cell-substrate interaction studies. The same chip was also extended to test drugs on human cells where the efficacy of a photosensitizer to perform targeted cancer cell imaging and targeted photodynamic therapy was tested. The advantage of such a system is in the fact that they can be used to perform different types of cell studies apart from facilitating drug testing and hypothesis testing. DOCTOR OF PHILOSOPHY (SCBE)

In membrane biofouling studies, quantification of biofouling is often conducted destructively and the results reflect only a snapshot of the biofouling processes. This limitation is mainly due to the lack of tools that allow us to monitor dynamics of biofouling without the need to disassemble the membrane testing systems. In this study, we developed a novel multichannel fluidic membrane biofilm flow cell that allows nondestructive, real-time monitoring of biofouling dynamics on forward osmosis (FO) membranes using confocal laser scanning microscopy. As a proof of concept, we used green fluorescent protein-tagged Shewanella oneidensis as a model organism and examined its biofilm development on membranes in FO mode. The temporal profiles of quantitative biofouling parameters such as surface coverage, biovolume, and biofilm thickness were obtained without disrupting the continuous operation of the membrane testing system. We also demonstrated the applicability of the microfluidic membrane flow cells, reveali...

Vascular smooth muscle cells (SMCs) are located in the middle of the tunica media and regulate the vasodilation and vasoconstriction of the blood vessels. SMCs also play a critical role during the development of atherosclerotic lesions, which are mainly found at sites of disturbed blood flow such as arterial branch points and bifurcations. Although the migratory and proliferative activities of SMCs and their phenotypic switch have been widely studied, the mechanotransduction of the SMC layer underlying atherosclerotic plaques remains unclear. In this study, bifurcate micropatterns with different angles were fabricated with polydimethylsiloxane and polyacrylamide gel for SMC culture and characterization of cell traction force. The cellular morphology, density and orientation-specific adaptation during branched cell layer formation on this platform were monitored until they became confluence. The results indicated that the characteristic cell traction forces and the von Mises stresses were dependent on bifurcation angles, which might provide important geometrical cues associated with the development of atherosclerosis. Immunofluorescence staining and gene analysis further revealed the proliferative and migratory states of SMCs in response to different bifurcation angles, which might elucidate the localization and progression of atherosclerotic lesions.

We have developed a planar optofluidic chip for the analysis of tumor cell apoptosis by exciting dual fluorescence through the integrated fibers. In contrast to conventional cell apop- tosis study that requires commercial flow cytometer with a very high price tag and bulky optics, our design was realized via a sim- ple single-layer soft lithography fabrication process. The device performance was tested by characterizing the HeLa cell apoptosis induced by hydrogen peroxide. The performance of the proposed optofluidic chip was concurrently confirmed by fluorescent mi- croscopy and benchmarked against a commercial flow cytometer. This study demonstrated the capability of the proposed optofluidic chip for cell apoptosis assay. The major advantages of this device include simple fabrication process, compact optical components, reliable performance, and low manufacturing cost, making it a promising platform for future mass-producible, inexpensive, and disposable on-chip investigation of biological samples.

Combined near-infrared (NIR) fluorescence and photoacoustic imaging techniques present promising capabilities for noninvasive visualization of biological structures. Development of bimodal noninvasive optical imaging approaches by combining NIR fluorescence and photoacoustic tomography demands suitable NIR-active exogenous contrast agents. If the aggregation and photobleaching are prevented, squaraine dyes are ideal candidates for fluorescence and photoacoustic imaging. Herein, we report rational selection, preparation, and micelle encapsulation of an NIR-absorbing squaraine dye (D1) for in vivo fluorescence and photoacoustic bimodal imaging. D1 was encapsulated inside micelles constructed from a biocompatible nonionic surfactant (Pluoronic F-127) to obtain D1-encapsulated micelles (D1micelle) in aqueous conditions. The micelle encapsulation retains both the photophysical features and chemical stability of D1. D1micelle exhibits high photostability and low cytotoxicity in biological conditions. Unique properties of D1micelle in the NIR window of 800–900 nm enable the development of a squaraine-based exogenous contrast agent for fluorescence and photoacoustic bimodal imaging above 820 nm. In vivo imaging using D1micelle, as demonstrated by fluorescence and photoacoustic tomography experiments in live mice, shows contrast-enhanced deep tissue imaging capability. The usage of D1micelle proven by preclinical experiments in rodents reveals its excellent applicability for NIR fluorescence and photoacoustic bimodal imaging. NRF (Natl Research Foundation, S’pore) Accepted version

Detection of oil micro-droplets in water is important to environmental oil spill monitoring agencies. We present a microfluidic device for high-throughput trapping of oil droplets in water by using an AC electric field in a continuous flow. The electric field is applied through optically transparent indium tin oxide (ITO) electrodes fabricated on a glass substrate to enable direct visualization of the trapped oil droplets in water. The continuously flowing oil droplets in water are trapped by the opposing AC-driven electroosmotic (ACEO) flow due to the formation of electric double layers because of the deposition of polyelectrolytes. The deposition of these polyelectrolytes is necessary for the hydrophilization of the polydimethylsiloxane (PDMS) reactor. An array of SU8 micro-pillars was fabricated within the oil droplet entrapment zone to reduce the hydrodynamic drag force of the incoming fluids. The entrapment efficiency of oil droplets in water was studied as a function of the applied frequency of the electric field. We also observed the presence of a size-dependent negative dielectrophoretic (DEP) force as the droplets exit the entrapment zone. This can improve the entrapment efficiency for larger sized droplets as the strength of the DEP force would be higher.

Monocytes represent a highly heterogeneous leukocyte population with the ability to differentiate into macrophages, a major cell type involved in the pathogenesis of atherosclerotic plaque in cardiovascular diseases [1,2]. Label-free analysis of their native cellular phenotypes and functions not only reduces assay cost and time, but is also essential for understanding disease progression and development of novel therapeutic strategies. Impedance cytometry is an emerging technology for high throughput cell phenotyping based on intrinsic electrical properties without the use of antibodies [3,4]. While parallel electrodes offer higher detection sensitivity as compared to co-planar electrodes [3,4], it is limited by laborious microfabrication. Herein, we present the development of an efficient microfluidics impedance cytometer using coplanar electrodes for rapid monocyte identification and phenotyping based on differentiation status. The microfluidics impedance cytometer consists of a two-inlet, two-outlet polydimethylsiloxane (PDMS) microchannel (30 μm (width) × 20 μm (height)) bonded on patterned coplanar electrodes (20 μm in width with 20 μm separation gap). Sample and sheath fluid are injected into the device to hydrodynamically focus the cells at the channel center prior electrical detection. As the cell moves through the detection region, it disrupts the electric field generated by coplanar electrodes, thereby causing a change in electrical impedance. Using our setup (Fig. 1), the change in electrical impedance was quantified based on current change, and various cellular information were extracted at different frequencies of the excitation signal. We measured two impedance parameters namely the 1) opacity (ratio of impedance magnitude at 0.3MHz (|ZLF|) to impedance magnitude at 1.7MHz (|ZHF|)) which reflects cell membrane capacitance, and 2) |ZLF| which characterize cell size. For identification of different blood cell types, human monocytes and lymphocytes purified using Dean Flow Fractionation (DFF) [5], and diluted red blood cells (RBCs) samples were separately injected into the microdevice. As shown in figure 2, different cell types were clearly differentiated based on |ZLF| due to distinct cell size differences (monocytes: 10 – 12 µm, lymphocytes: 7 – 8 µm, and red blood cells: 6 – 8 µm). Characterization of primary monocytes and leukemic THP-1 monocytic cell line also showed distinct differences in size and opacity (Fig. 3). Lastly, we compared the impedance profile of THP-1 and differentiated macrophages (PMA stimulated), and found that macrophages were more heterogeneous in size with significantly lower opacity than THP-1, demonstrating the feasibility of real-time assessment of monocyte differentiation using impedance-based sensing (Fig. 4). In conclusion, the developed microdevice enables continuous label-free monocyte phenotyping using coplanar electrodes configuration with sufficient sensitivity. With the design simplicity and low cost fabrication, we envision our method will greatly facilitate immunology research and point-of-care monocyte profiling in patients with cardiovascular diseases.

Atherosclerosis, the leading cause of cardiovascular diseases [1–3], is a chronic inflammatory disorder characterized by deposition of cholesterol-containing low-density lipoproteins (LDL) in arterial walls, resulting in blood vessel narrowing and impaired perfusion, and increased leukocyte recruitment [4,5]. Current atherosclerosis in vitro models are 2-dimensional (2D), and fail to reproduce important features of atherogenesis including blood flow-induced shear stress and leukocyte-endothelial interactions [6–7]. Herein, we report a novel biomimetic blood vessel model to study the hemodynamics and leukocyte-endothelial interactions using a tunable, 3-dimensional (3D) endothelial barrier to mimic stenotic plaque. We first characterized the effects of THP-1 monocyte adhesion on activated endothelial monolayer, followed by whole blood perfusion at different channel constrictions to study leukocyte rolling phenotype. A schematic of the polydimethylsiloxane (PDMS) microfluidic device is shown in Figure 1A. It consists of 3 layers, with a top cell culture chamber (800×100 µm (H×W)) for endothelial (HUVECs) cell culture, and a bottom pneumatic channel (1000×100 µm (H×W)), separated by a thin PDMS membrane (10 µm thick). By varying air flow into the pneumatic channel, the membrane is deflected upwards, thereby creating tunable constrictions in the cell culture chamber (Figure 1B). Endothelial inflammation is mimicked by growing HUVECs to confluency and treating them with tumor necrosis factor-α (TNF-α) to study leukocyte-endothelial interactions in cell culture media (RPMI) and whole blood flow. We first performed confocal imaging to visualize channel constriction using FITC dye and 3D endothelial “stenotic plaque” (Figure 1B, C). The laminar flow profile over the 3D stenosis was studied from the rolling velocities of 10 µm beads, which varied significantly in the stenosis region (Figure 1D). As observed from Figure 2A, HUVECs expressed higher ICAM-1 due to TNF-α-treatment which facilitated binding of THP-1 cells. Interestingly, distinct adherence patterns of THP-1 for 50% and 80% constrictions was observed under flow (1 dyne/cm2) with increased adherence at the top of the constriction (Figure 2B, C). Upon increasing flow rate to 10 dyne/cm2, THP-1 adhesion was reduced in 80% constriction while completely eliminated in 50% constriction. (Figure 2D, E). Finally, whole blood was perfused through the device to study blood flow under stenosis conditions (Figure 3). As expected, significant leukocyte (mostly neutrophils) rolling and adhesion were observed, and average leukocyte rolling velocity was lowest with an 80% constriction (Figure 3D), possibly due to low shear at the top of the stenotic “plaque”. On-going studies are performed to gain better insights into the stenosis-induced hemodynamics effects on leukocyte-endothelial interactions. In conclusion, the developed atherosclerosis-on-a-chip mimics a physiologically-relevant 3D stenotic blood vessel which enables long-term perfusion cell culture and on-chip visualization of leukocyte-endothelial interactions (rolling, adhesion). This model can also be further developed to study thrombus formation and other endothelial-related dysfunctions in cardiovascular diseases.

Vessel geometries in microengineered in vitro vascular models are important to recapitulate a pathophysiological microenvironment for the study of flow-induced endothelial dysfunction and inflammation in cardiovascular diseases. Herein, we present a simple and novel extracellular matrix (ECM) hydrogel patterning method to create perfusable vascularized microchannels of different geometries based on the concept of capillary burst valve (CBV). No surface modification is necessary and the method is suitable for different ECM types including collagen, matrigel and fibrin. We first created collagen-patterned, endothelialized microchannels to study barrier permeability and neutrophil transendothelial migration, followed by the development of a biomimetic 3D endothelial-smooth muscle cell (EC-SMC) vascular model. We observed a significant decrease in barrier permeability in the co-culture model during inflammation, which indicates the importance of perivascular cells in ECM remodeling. Finally, we engineered collagen-patterned constricted vascular microchannels to mimic stenosis in atherosclerosis. Whole blood was perfused (1-10 dyne cm-2) into the microdevices and distinct platelet and leukocyte adherence patterns were observed due to increased shear stresses at the constriction, and an additional convective flow through the collagen. Taken together, the developed hydrogel patterning technique enables the formation of unique pathophysiological architectures in organ-on-chip microsystems for real-time study of hemodynamics and cellular interactions in cardiovascular diseases. MOE (Min. of Education, S’pore) MOH (Min. of Health, S’pore) Accepted version

This paper reports an in situ photopolymerization method based on a microfluidic device to produce 5-fluorouracil loaded biocompatible poly(ethylene glycol) diacrylate (PEGDA) microspheres with monodisperse size distribution for sustained drug release. Multiphase flow of 5-fluorouracil and PEGDA are dispersed to generate droplets in continuous hexadecane stream based on a T-junction microfluidic channel. The size of emulsion can be controlled from 16.7 to 85.7 µm in diameter by altering the flow rate ratio of the continuous phase and the dispersed phase. The droplets are polymerized in a serpentine channel region induced by UV irradiation. Optical microscopy and scanning electron microscopy are performed to characterize the dispersity and morphology of the microbeads. A quantitative drug release study is conducted within the drug dosage range from 0.1 to 0.5 % w/w. It is shown that the PEGDA microbead-mediated release of 5-fluorouracil exhibits relatively fast elution in the first 12 h and sustained release over the next 156 h, under which the proliferation of Huh-7 tumor cells is effectively inhibited in vitro. These results demonstrate that monodisperse PEGDA microbeads could be utilized as promising carriers with long-term stability in a drug delivery and screening system for sustained release of 5-fluorouracil during chemotherapy.

Deep tissue bioimaging with three-photon (3P) excitation using near-infrared (NIR) light in the second IR window (1.0-1.4 μm) could provide high resolution images with an improved signal-to-noise ratio. Herein, we report a photostable and nontoxic 3P excitable donor-π-acceptor system (GMP) having 3P cross-section (σ3 ) of 1.78×10(-80) cm(6) s(2) photon(-2) and action cross-section (σ3 η3 ) of 2.31×10(-81) cm(6) s(2) photon(-2) , which provides ratiometric fluorescence response with divalent zinc ions in aqueous conditions. The probe signals the Zn(2+) binding at 530 and 600 nm, respectively, upon 1150 nm excitation with enhanced σ3 of 1.85×10(-80) cm(6) s(2) photon(-2) and σ3 η3 of 3.33×10(-81) cm(6) s(2) photon(-2) . The application of this probe is demonstrated for ratiometric 3P imaging of Zn(2+) in vitro using HuH-7 cell lines. Furthermore, the Zn(2+) concentration in rat hippocampal slices was imaged at 1150 nm excitation after incubation with GMP, illustrating its potential as a 3P ratiometric probe for deep tissue Zn(2+) ion imaging.

Real time monitoring of blood aminothiol level by a near-IR dye assisted fluorescence–photoacoustic bimodal imaging is reported., The development of molecular probes for the detection and imaging of biological thiols is a major step forward diagnosing various types of diseases. Previously reported thiol imaging strategies were mainly based on a single mode of imaging with a limited application in vivo. In this work, we introduced an unsymmetrical near-infrared (NIR) squaraine dye (USq) as an exogenous contrast agent for photoacoustic and fluorescence bimodal imaging of thiol variations in live animals. USq exhibits a narrow absorption band at 680 nm that generates a photoacoustic signal and a strong NIR emission at 700 nm (ΦF = 0.27), which is applicable for deep tissue optical imaging. Both photoacoustic and fluorescence signals could selectively disappear in the presence of different thiols. Through in vitro and in vivo imaging studies, unique imaging capability of USq was demonstrated, and the effect of food uptake on the increased level of aminothiols in blood was confirmed.

Atherosclerosis, a chronic inflammatory disorder characterized by endothelial dysfunction and blood vessel narrowing, is the leading cause of cardiovascular diseases including heart attack and stroke. Herein, we present a novel tunable microfluidic atherosclerosis model to study vascular inflammation and leukocyte-endothelial interactions in 3D vessel stenosis. Flow and shear stress profiles were characterized in pneumatic-controlled stenosis conditions (0%, 50% and 80% constriction) using fluid simulation and experimental beads perfusion. Due to non-uniform fluid flow at the 3D stenosis, distinct monocyte (THP-1) adhesion patterns on inflamed [tumor necrosis factor-α (TNF-α) treated] endothelium were observed, and there was a differential endothelial expression of intercellular adhesion molecule-1 (ICAM-1) at the constriction region. Whole blood perfusion studies also showed increased leukocyte interactions (cell rolling and adherence) at the stenosis of healthy and inflamed endothelium, clearly highlighting the importance of vascular inflammation, flow disturbance, and vessel geometry in recapitulating atherogenic microenvironment. To demonstrate inflammatory risk assessment using leukocytes as functional biomarkers, we perfused whole blood samples into the developed microdevices (80% constriction) and observed significant dose-dependent effects of leukocyte adhesion in healthy and inflamed (TNF-α treated) blood samples. Taken together, the 3D stenosis chip facilitates quantitative study of hemodynamics and leukocyte-endothelial interactions, and can be further developed into a point-of-care blood profiling device for atherosclerosis and other vascular diseases.

As an alternative to complex and costly in vivo models, microfluidic in vitro models are being widely used to study various physiological phenomena. It is of particular interest to study cell migration in a controlled microenvironment because of its vital role in a large number of physiological processes, such as wound healing, disease progression, and tissue regeneration. Cell migration has been shown to be affected by variations in the biochemical and physical properties of the extracellular matrix (ECM). To study the combinatorial impact of the ECM physical properties on cell migration, we have developed a microfluidic assay to induce migration of human bone marrow derived mesenchymal stem cells (hBMSCs) on polydimethylsiloxane (PDMS) substrates with varying combinatorial properties (hydrophobicity, stiffness, and roughness). The results show that although the initial cell adhesion and viability appear similar on all PDMS samples, the cell spreading and migration are enhanced on PDMS samples exhibiting intermediate levels of hydrophobicity, stiffness, and roughness. This study suggests that there is a particular range of substrate properties for optimal cell spreading and migration. The influence of substrate properties on hBMSC migration can help understand the physical cues that affect cell migration, which may facilitate the development of optimized engineered scaffolds with desired properties for tissue regeneration applications.

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

Cell sheet engineering has been exploited as an alternative approach in tissue regeneration and the use of stem cells to generate cell sheets has further showed its potential in stem cell-mediated tissue regeneration. There exist vast interests in developing strategies to enhance the formation of stem cell sheets for downstream applications. It has been proved that stem cells are sensitive to the biophysical cues of the microenvironment. Therefore we hypothesized that the combinatorial substratum properties could be tailored to modulate the development of cell sheet formation and further influence its multipotency. For validation, polydimethylsiloxane (PDMS) of different combinatorial substratum properties (including stiffness, roughness and wettability) were created, on which the human bone marrow derived mesenchymal stem cells (BMSCs) were cultured to form cell sheets with their multipotency evaluated after induced differentiation. The results showed that different combinatorial effects of these substratum properties were able to influence BMSC behavior such as adhesion, spreading and proliferation during cell sheet development. Collagen formation within the cell sheet was enhanced on substrates with lower stiffness, higher hydrophobicity and roughness, which further assisted the induced chondrogenesis and osteogenesis, respectively. These findings suggested that combinatorial substratum properties had profound effects on BMSC cell sheet integrity and multipotency, which had significant implications for future biomaterials and scaffold designs in the field of BMSC-mediated tissue regeneration.

The living cells are arranged in a complex natural environment wherein they interact with extracellular matrix and other neighboring cells. Cell-cell interactions, especially those between distinct phenotypes, have attracted particular interest due to the significant physiological relevance they can reveal for both fundamental and applied biomedical research. To study cell-cell interactions, it is necessary to develop co-culture systems, where different cell types can be cultured within the same confined space. Although the current advancement in lab-on-a-chip technology has allowed the creation of in vitro models to mimic the complexity of in vivo environment, it is still rather challenging to create such co-culture systems for easy control of different colonies of cells. In this paper, we have demonstrated a straightforward method for the development of an on-chip co-culture system. It involves a series of steps to selectively change the surface property for discriminative cell seeding and to induce cellular interaction in a co-culture region. Bone marrow stromal cells (HS5) and a liver tumor cell line (HuH7) have been used to demonstrate this co-culture model. The cell migration and cellular interaction have been analyzed using microscopy and biochemical assays. This co-culture system could be used as a disease model to obtain biological insight of pathological progression, as well as a tool to evaluate the efficacy of different drugs for pharmaceutical studies. Published version

Cell growing behavior is significantly dependent on the surface chemistry of materials. SU-8 as an epoxy-based negative photoresist is commonly used for fabricating patterned layers in lab-on-a-chip devices. As a hydrophobic material, SU-8 substrate is not favorable for cell culture, and cell attachment on native SU-8 is limited attributed to poor surface biocompatibility. Although physical adsorption of proteins could enhance the cell adhesion, the effect is not durable. In this work, SU-8 surface chemistry is modified by immobilizing fibronectin (FN) and collagen type I (COL I) covalently using (3-aminopropyl)triethoxysilane (APTES) and cross-linker glutaraldehyde (GA) to increase surface biofunctionality. The effectiveness of this surface treatment to improve the adhesion and viability of mesenchymal stem cells (MSCs) is investigated. It is found that the wettability of SU-8 surface can be significantly increased by this chemical modification. In addition, the spreading area of MSCs increases on the SU-8 surfaces with covalently conjugated matrix proteins, as compared to other unmodified SU-8 surface or those coated with proteins simply by physical adsorption. Furthermore, cell proliferation is dramatically enhanced on the SU-8 surfaces modified under the proposed scheme. Therefore, SU-8 surface modification with covalently bound matrix proteins assisted by APTES+GA provides a highly biocompatible interface for the enhanced adhesion, spreading, and proliferation of MSCs.

The application of nanoparticles as delivery platforms in PDT is a promising alternative for resolving some of the major challenges associated with classic photosensitizers (PSs) such as porphyrin molecules. Polysilsesquioxane nanoparticles (PSQ-NPs) are crosslinked homopolymers formed by the condensation of functionalized trialkoxysilanes. Recent reports have shown that PSQ-NPs can provide an interesting platform for developing PS nanocarriers. Several advantages can be foreseen by using this platform such as carrying a large payload of PSmolecules; their surface and composition can be tailored to develop multifunctional systems. In this work, two PSQ-NP systems with a high payload of photosensitizers were synthesized, characterized, and applied in vitro. The network of these nanomaterials is formed by two different types of porphyrin-based photosensitizers, protoporphyrin-IX and tetrakis(4-carboxyphenyl)porphyrin, chemically connectedvia a redox-responsive linker. Both platforms can be further functionalized with polyethylene glycol and targeting ligands such as folic acid to improve their biocompatibility and target specificity, respectively. The effectiveness of these porphyrin-based hybrid nanomaterials was successfully demonstrated in vitro using cervical human cancer (HeLa), lung (H-460) and colon (HT-29) cancer cells.

The surface chemistry of materials has an interactive influence on cell behavior. The optimal adhesion of mammalian cells is critical in determining the cell viability and proliferation on substrate surfaces. Because of the inherent high hydrophobicity of a poly(dimethylsiloxane) (PDMS) surface, cell culture on these surfaces is unfavorable, causing cells to eventually dislodge from the surface. Although physically adsorbed matrix proteins can promote initial cell adhesion, this effect is usually short-lived. Here, (3-aminopropyl)triethoxy silane (APTES) and cross-linker glutaraldehyde (GA) chemistry was employed to immobilize either fibronectin (FN) or collagen type 1 (C1) on PDMS. The efficiency of these surfaces to support the adhesion and viability of mesenchymal stem cells (MSCs) was analyzed. The hydrophobicity of the native PDMS decreased significantly with the mentioned surface functionalization. The adhesion of MSCs was mostly favorable on chemically modified PDMS surfaces with APTES + GA + protein. Additionally, the spreading area of MSCs was significantly higher on APTES + GA + C1 surfaces than on other unmodified/modified PDMS surfaces with C1 adsorption. However, there were no significant differences in the MSC spreading area on the unmodified/modified PDMS surfaces with FN adsorption. Furthermore, there was a significant increase in cell proliferation on the PDMS surface with APTES + GA + protein functionalization as compared to the PDMS surface with protein adsorption only. Therefore, the covalent surface chemical modification of PDMS with APTES + GA + protein could offer a more biocompatible platform for the enhanced adhesion and proliferation of MSCs. Similar strategies can be applied for other substrates and cell lines by appropriate combinations of self-assembly monolayers (SAMs) and extracellular matrix proteins.

A study of dye-loaded organosilica for photo-oxidation., Periodic mesoporous organosilica (PMO) has been widely used for the fabrication of a variety of catalytically active materials. We report the preparation of novel photo-responsive PMO with azobenzene-gated pores. Upon activation, the azobenzene gate undergoes trans-cis isomerization, which allows an unsymmetrical near-infrared squaraine dye (Sq) to enter into the pores. The gate closure by cis-trans isomerization of the azobenzene unit leads to the safe loading of the monomeric dye inside the pores. The dye-loaded and azobenzene-gated PMO (Sq-azo@PMO) exhibits excellent generation of reactive oxygen species upon excitation at 664 nm, which can be effectively used for the oxidation of phenol into benzoquinone in aqueous solution. Furthermore, Sq-azo@PMO as the catalyst was placed inside a custom-built, continuous-flow device to carry out the photo-oxidation of phenol to benzoquinone in the presence of 664-nm light. By using the device, about 23% production of benzoquinone with 100% selectivity was achieved. The current research presents a prototype of transforming heterogeneous catalysts toward practical use.