Indocyanine-Green-Loaded Liposomes for Photodynamic and Photothermal Therapies: Inducing Apoptosis and Ferroptosis in Cancer Cells with Implications beyond Oral Cancer.

Oral cancer represents a global health burden, necessitating novel therapeutic strategies. Photodynamic and photothermal therapies using indocyanine green (ICG) have shown promise due to their distinctive near-infrared (NIR) light absorption characteristics and FDA-approved safety profiles. This study develops ICG-loaded liposomes (Lipo-ICGs) to further explore their potential in oral cancer treatments. We synthesized and characterized the Lipo-ICGs, conducted in vitro cell culture experiments to assess cellular uptake and photodynamic/photothermal effects, and performed in vivo animal studies to evaluate their therapeutic efficacy. Quantitative cell apoptosis and gene expression variation were further characterized using flow cytometry and RNA sequencing, respectively. Lipo-ICGs demonstrated a uniform molecular weight distribution among particles. The in vitro studies showed a successful internalization of Lipo-ICGs into the cells and a significant photodynamic treatment effect. The in vivo studies confirmed the efficient delivery of Lipo-ICGs to tumor sites and successful tumor growth inhibition following photodynamic therapy. Moreover, light exposure induced a time-sensitive photothermal effect, facilitating the further release of ICG, and enhancing the treatment efficacy. RNA sequencing data showed significant changes in gene expression patterns upon Lipo-ICG treatment, suggesting the activation of apoptosis and ferroptosis pathways. The findings demonstrate the potential of Lipo-ICGs as a therapeutic tool for oral cancer management, potentially extending to other cancer types.

Rapid time-lapse 3D oxygen tension measurements within hydrogels using widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) and image segmentation.

Understanding the influence of oxygen tension on cellular functions and behaviors is crucial for investigating various physiological and pathological conditions. In vitro cell culture models, particularly those based on hydrogel extracellular matrices, have been developed to study cellular responses in specific oxygen microenvironments. However, accurately characterizing oxygen tension variations with great spatiotemporal resolutions, especially in three dimensions, remains challenging. This paper presents an approach for rapid time-lapse 3D oxygen tension measurements in hydrogels using a widely available inverted fluorescence microscope. Oxygen-sensitive fluorescent microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) are utilized for oxygen tension estimation. To incorporate the third dimension, a motorized sample stage is implanted that enables automated image acquisition in the vertical direction. A machine learning algorithm based on K-means clustering is employed for microbead position identification. Using an upside-down microfluidic device, 3D oxygen gradients are generated within a hydrogel sample, and z-stack images are acquired using the FD-FLIM system. Analyses of the acquired images, involving microbead position identification, lifetime calculation, and oxygen tension conversion, are then performed offline. The results demonstrate the functionality of the developed approach for rapid time-lapse 3D oxygen tension measurements in hydrogels. Furthermore, the 3D oxygen tension adjacent to a tumor spheroid within a hydrogel during media exchange is characterized. The results further confirm that the 3D spatiotemporal oxygen tension profiles can be successfully measured quantitatively using the established setup and analysis process and that the approach may have great potential for investigating cellular activities within oxygen microenvironments.

Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.

Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

Efficient single-cell oxygen consumption rate characterization based on frequency domain fluorescence lifetime imaging microscopy measurement and microfluidic platform.

Cell metabolism is critical in regulating normal cell functions to maintain energy homeostasis. In order to monitor cell metabolism, the oxygen consumption rate (OCR) of cells has been characterized as an important factor. In conventional cell analysis, the cells are characterized in bulk due to technical limitations. However, the heterogeneity between the cells cannot be identified. Therefore, single-cell analysis has been proposed to reveal cellular functions and their heterogeneity. In this research, an approach integrating a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell OCR characterization in an efficient manner is developed. The microfluidic device provides an efficient platform to trap and isolate single cells in microwells with the buffer saline containing an oxygen-sensitive phosphorescent dye. The oxygen tension variation within the microwells can be efficiently estimated by measuring the fluorescence lifetime change using the FD-FLIM, and the OCR values of the single cells can then be calculated. In the experiments, breast cancer (MCF-7) cells are exploited for the OCR measurement. The results demonstrate the functionality of the developed approach and show the heterogeneity among the cells. The developed approach possesses great potential to advance cellular metabolism studies with single-cell resolution.

Studying sprouting angiogenesis under combination of oxygen gradients and co-culture of fibroblasts using microfluidic cell culture model.

Sprouting angiogenesis is an essential process for expanding vascular systems under various physiological and pathological conditions. In this paper, a microfluidic device capable of integrating a hydrogel matrix for cell culture and generating stable oxygen gradients is developed to study the sprouting angiogenesis of endothelial cells under combinations of oxygen gradients and co-culture of fibroblast cells. The endothelial cells can be cultured as a monolayer endothelium inside the device to mimic an existing blood vessel, and the hydrogel without or with fibroblast cells cultured in it provides a matrix next to the formed endothelium for three-dimensional sprouting of the endothelial cells. Oxygen gradients can be stably established inside the device for cell culture using the spatially-confined chemical reaction method. Using the device, the sprouting angiogenesis under combinations of oxygen gradients and co-culture of fibroblast cells is systematically studied. The results show that the oxygen gradient and the co-culture of fibroblast cells in the hydrogel can promote sprouting of the endothelial cells into the hydrogel matrix by altering cytokines in the culture medium and the physical properties of the hydrogel. The developed device provides a powerful in vitro model to investigate sprouting angiogenesis under various in vivo-like microenvironments.

A 3D culture system for evaluating the combined effects of cisplatin and anti-fibrotic drugs on the growth and invasion of lung cancer cells co-cultured with fibroblasts.

Fibrosis and fibroblast activation usually occur in the tissues surrounding a malignant tumor; therefore, anti-fibrotic drugs are used in addition to chemotherapy. A reliable technique for evaluating the combined effects of anti-fibrotic drugs and anticancer drugs would be beneficial for the development of an appropriate treatment strategy. In this study, we manufactured a three-dimensional (3D) co-culture system of fibroblasts and lung cancer cell spheroids in Matrigel supplemented with fibrin (fibrin/Matrigel) that simulated the tissue microenvironment around a solid tumor. We compared the efficacy of an anticancer drug (cisplatin) with or without pretreatments of two anti-fibrotic drugs, nintedanib and pirfenidone, on the growth and invasion of cancer cells co-cultured with fibroblasts. The results showed that the addition of nintedanib improved cisplatin's effects on suppressing the growth of cancer cell spheroids and the invasion of cancer cells. In contrast, pirfenidone did not enhance the anticancer activity of cisplatin. Nintedanib also showed higher efficacy than pirfenidone in reducing the expression of four genes in fibroblasts associated with cell adhesion, invasion, and extracellular matrix degradation. This study demonstrated that the 3D co-cultures in fibrin/Matrigel would be useful for assessing the effects of drug combinations on tumor growth and invasion.

Extracellular Matrix-Derived Biophysical Cues Mediate Interstitial Flow-Induced Sprouting Angiogenesis.

Sprouting angiogenesis is orchestrated by an intricate balance of biochemical and mechanical cues in the local tissue microenvironment. Interstitial flow has been established as a potent regulator of angiogenesis. Similarly, extracellular matrix (ECM) physical properties, such as stiffness and microarchitecture, have also emerged as important mediators of angiogenesis. However, the interplay between interstitial flow and ECM physical properties in the initiation and control of angiogenesis is poorly understood. Using a three-dimensional (3D) microfluidic tissue analogue of angiogenic sprouting with defined interstitial flow superimposed over ECM with well-characterized physical properties, we found that the addition of hyaluronan (HA) to collagen-based matrices significantly enhances sprouting induced by interstitial flow compared to responses in collagen-only hydrogels. We confirmed that both the stiffness and matrix pore size of collagen-only hydrogels were increased by the addition of HA. Interestingly, interstitial flow-potentiated sprouting responses in collagen/HA matrices were not affected when functionally blocking the HA receptor CD44. In contrast, enzymatic depletion of HA in collagen/HA matrices with hyaluronidase (HAdase) resulted in decreased stiffness, pore size, and interstitial flow-mediated sprouting to the levels observed in collagen-only matrices. Taken together, these results suggest that HA enhances interstitial flow-mediated angiogenic sprouting through its alterations to collagen ECM stiffness and pore size.

Study Hypoxic Response under Cyclic Oxygen Gradients Generated in Microfluidic Devices Using Real-Time Fluorescence Imaging.

Oxygen plays important roles in regulating various biological activities under physiological and pathological conditions. However, the response of cells facing temporal variation in oxygen microenvironments has seldom been studied due to technical limitations. In this paper, an integrated approach to studying hypoxic response under cyclic oxygen gradients is developed. In the experiments, a cell culture system based on a microfluidic device is constructed to generate cyclic oxygen gradients with desired periods by alternately introducing gases with specific compositions into the microfluidic channels next to the cell culture channel separated by thin channel walls. Observation of the hypoxic responses is performed using real-time fluorescence imaging of dyes sensitive to extra- and intracellular oxygen tensions as well as intracellular calcium concentrations. Cellular hypoxic responses of human aortic smooth muscle cells (AoSMCs) and lung carcinoma epithelium (A549) cells, including intracellular oxygen and calcium levels, are measured. The results show that the two types of cells have different hypoxic responses to the applied cyclic oxygen gradients. With the capability of real-time cellular response monitoring under cyclic oxygen gradients, the developed approach provides a useful scheme to investigate hypoxic responses in vitro under microenvironments mimicking various in vivo physiological and pathological conditions.

Cav 3.2 T-type calcium channel regulates mouse platelet activation and arterial thrombosis.

BACKGROUND: Cav 3.2 is a T-type calcium channel that causes low-threshold exocytosis. T-type calcium channel blockers reduce platelet granule exocytosis and aggregation. However, studies of the T-type calcium channel in platelets are lacking. OBJECTIVE: To examine the expression and role of Cav 3.2 in platelet function. METHODS: Global Cav 3.2-/- and platelet-specific Cav 3.2-/- mice and littermate controls were used for this study. Western blot analysis was used to detect the presence of Cav 3.2 and activation of the calcium-responsive protein extracellular signal-regulated kinase (ERK). Fura-2 dye was used to assess platelet calcium. Flow cytometry and light transmission aggregometry were used to evaluate platelet activation markers and aggregation, respectively. FeCl3 -induced thrombosis and a microfluidic flow device were used to assess in vivo and ex vivo thrombosis, respectively. RESULTS: Cav 3.2 was expressed in mouse platelets. As compared with wild-type controls, Cav 3.2-/- mouse platelets showed reduced calcium influx. Similarly, treatment with the T-type calcium channel inhibitor Ni2+ decreased the calcium influx in wild-type platelets. As compared with controls, both Cav 3.2-/- and Ni2+ -treated wild-type platelets showed reduced activation of ERK. ATP release, P-selectin exposure, and αIIb ß3 activation were reduced in Cav 3.2-/- and Ni2+ -treated wild-type platelets, as was platelet aggregation. On in vivo and ex vivo thrombosis assay, Cav3.2 deletion caused delayed thrombus formation. However, tail bleeding assay showed intact hemostasis. CONCLUSION: These results suggest that Cav 3.2 is required for the optimal activation of platelets.

Comparison of Hydrogen Peroxide Secretion From Living Cells Cultured in Different Formats Using Hydrogel-Based LSPR Substrates.

Reactive oxygen species (ROS), a number of reactive molecules and free radicals derived from molecular oxygen, are generated as by-products during mitochondrial electron transport within cells. Physiologically, cells are capable of metabolizing the ROS exploiting specific mechanisms. However, if excessive ROS accumulate inside the cells, it will cause the cells apoptosis or necrosis. Hydrogen peroxide (H2O2) is one of the essential ROS often participating in chemical reactions in organisms and regulating homeostasis in the body. Therefore, rapid and sensitive detection of H2O2 is a significant task in cell biology research. Furthermore, it has been found that cells cultured in different formats can result in different cellular responses and biological activities. In order to investigate the H2O2 secretion from the cells cultured in different formats, a hydrogel-based substrate is exploited to separate relatively large molecular (e.g., proteins) for direct measurement of H2O2 secreted from living cells in complete cell culture medium containing serum. The substrate takes advantage of the localized surface plasmon resonance (LSPR) method based on enzyme immunoprecipitation. In addition, the H2O2 secreted from the cells cultured in different dimensions (suspension of single cells and three-dimensional cell spheroids) treated with identical drugs is measured and compared. The spheroid samples can be prepared with ample amount using a designed microfluidic device with precise control of size. The results show that the H2O2 secretion from the cells are great affected by their culture formats.

Revealing anisotropic elasticity of endothelium under fluid shear stress.

Endothelium lining interior surface of blood vessels experiences various physical stimulations in vivo. Its physical properties, especially elasticity, play important roles in regulating the physiological functions of vascular systems. In this paper, an integrated approach is developed to characterize the anisotropic elasticity of the endothelium under physiological-level fluid shear stress. A pressure sensor-embedded microfluidic device is developed to provide fluid shear stress on the perfusion-cultured endothelium and to measure transverse in-plane elasticities in the directions parallel and perpendicular to the flow direction. Biological atomic force microscopy (Bio-AFM) is further exploited to measure the vertical elasticity of the endothelium in its out-of-plane direction. The results show that the transverse elasticity of the endothelium in the direction parallel to the perfusion culture flow direction is about 70% higher than that in the direction perpendicular to the flow direction. Moreover, the transverse elasticities of the endothelium are estimated to be approximately 120 times larger than the vertical one. The results indicate the effects of fluid shear stress on the transverse elasticity anisotropy of the endothelium, and the difference between the elasticities in transverse and vertical directions. The quantitative measurement of the endothelium anisotropic elasticity in different directions at the tissue level under the fluid shear stress provides biologists insightful information for the advanced vascular system studies from biophysical and biomaterial viewpoints. STATEMENT OF SIGNIFICANCE: In this paper, we take advantage an integrated approach combining microfluidic devices and biological atomic force microscopy (Bio-AFM) to characterize anisotropic elasticities of endothelia with and without fluidic shear stress application. The microfluidic devices are exploited to conduct perfusion cell culture of the endothelial cells, and to estimate the in-plane elasticities of the endothelium in the direction parallel and perpendicular to the shear stress. In addition, the Bio-AFM is utilized for characterization of the endothelium morphology and vertical elasticity. The measurement results demonstrate the very first anisotropic elasticity quantification of the endothelia. Furthermore, the study provides insightful information bridging the microscopic sing cell and macroscopic organ level studies, which can greatly help to advance vascular system research from material perspective.

Identifying distinct oxygen diffusivity through type I pneumocyte-like cell layers using microfluidic device.

Oxygen is necessary for cellular respiration in aerobic organisms. In animals, such as human, inhaled oxygen moves from the alveoli to the blood through alveolar epithelium into pulmonary capillaries. Up to now, different studies have been reported to examine experimental oxygen diffusivity for simple membrane or single-celled organisms; however, devices capable of precisely characterizing oxygen transportation through cell layers with dimensions similar to their physiological ones have not been developed. In this study, we establish an integrated approach exploiting a multi-layer microfluidic device and relative fluorescence lifetime detection apparatus to reliably measure oxygen diffusivity through a cell layer. In the experiments, different types of cells, including A549 and 3T3 cell lines, lung stem/progenitor cells, and the differentiated type I pneumocyte-like cells, are used to form cell layers within the devices for their oxygen diffusivity evaluation. A distinct facilitated oxygen transportation behavior of the differentiated type I pneumocyte-like cells that has never been discussed before is identified using the approach. The study offered a new in vitro approach to evaluate the oxygen diffusivity across cell layers in a microfluidic device and open a door to construct more physiologically meaningful in vitro model system to study respiratory systems.

Transwell Insert-Embedded Microfluidic Devices for Time-Lapse Monitoring of Alveolar Epithelium Barrier Function under Various Stimulations.

This paper reports a transwell insert-embedded microfluidic device capable of culturing cells at an air-liquid interface (ALI), mimicking the in vivo alveolar epithelium microenvironment. Integration of a commercially available transwell insert makes the device fabrication straightforward and eliminates the tedious device assembly processes. The transwell insert can later be detached from the device for high-resolution imaging of the cells. In the experiments, the cells showing type-I pneumocyte markers are exploited to construct an in vitro alveolar epithelium model, and four culture conditions including conventional liquid/liquid culture (LLC) and air-liquid interface (ALI) cell culture in normal growth medium, and ALI cell culture with inflammatory cytokine (TNF-α) stimulation and ethanol vapor exposure are applied to investigate their effects on the alveolar epithelium barrier function. The barrier permeability is time-lapse monitored using trans-epithelial electrical resistance (TEER) measurement and immunofluorescence staining of the tight junction protein (ZO-1). The results demonstrate the functionalities of the device, and further show the applications and advantages of the constructed in vitro cell models for the lung studies.

Study 3D Endothelial Cell Network Formation under Various Oxygen Microenvironment and Hydrogel Composition Combinations Using Upside-Down Microfluidic Devices.

Formation of 3D networks is a crucial process for endothelial cells during development of primary blood vessels under both normal and pathological conditions. In order to investigate effects of oxygen microenvironment and matrix composition on the 3D network formation, an upside-down microfluidic cell culture device capable of generating oxygen gradients is developed in this paper. In cell experiments, network formation of human umbilical vein endothelial cells (HUVECs) within fibrinogen-based hydrogels with different concentrations of hyaluronic acid (HA) is systematically studied. In addition, five different oxygen microenvironments (uniform normoxia, 5%, and 1% O2 ; oxygen gradients under normoxia and 5% O2 ) are also applied for the cell culture. The generated oxygen gradients are characterized based on fluorescence lifetime measurements. The experimental results show increased 3D cell network length when the cells are cultured under the oxygen gradients within the hydrogels with the HA addition suggesting their roles in promoting network formation. Furthermore, the formed networks tend to align along the direction of the oxygen gradients indicating the presence of gradient-driven cellular response. The results demonstrate that the developed upside-down microfluidic device can provide an advanced platform to investigate 3D cell culture under the controlled oxygen microenvironments for various biomedical studies in vitro.

Evaluation of Nanoparticle Penetration in the Tumor Spheroid Using Two-Photon Microscopy.

Mesoporous silica nanoparticles (MSNs) have emerged as a prominent nanomedicine platform, especially for tumor-related nanocarrier systems. However, there is increasing concern about the ability of nanoparticles (NPs) to penetrate solid tumors, resulting in compromised antitumor efficacy. Because the physicochemical properties of NPs play a significant role in their penetration and accumulation in solid tumors, it is essential to systematically study their relationship in a model system. Here, we report a multihierarchical assessment of the accumulation and penetration of fluorescence-labeled MSNs with nine different physicochemical properties in tumor spheroids using two-photon microscopy. Our results indicated that individual physicochemical parameters separately could not define the MSNs' ability to accumulate in a deeper tumor region; their features are entangled. We observed that the MSNs' stability determined their success in reaching the hypoxia region. Moreover, the change in the MSNs' penetration behavior postprotein crowning was associated with both the original properties of NPs and proteins on their surfaces.

Comparison of VEGF-A secretion from tumor cells under cellular stresses in conventional monolayer culture and microfluidic three-dimensional spheroid models.

Vascular endothelial growth factor (VEGF) is a major cytokine in tumor biology affecting tumor survival, aggressiveness and pro-angiogenetic activities. In addition, cellular stresses often result in aggressive pro-angiogenetic behavior in tumors. For in vitro study, conventional monolayer cell culture has been broadly exploited; however, it often provides limited information due to its different microenvironment from that in vivo. Recently, three-dimensional (3D) cell spheroid culture provides in vivo-like microenvironments to study tumor biology and their survival mechanisms with better predictive power. In this work, vascular endothelial growth factor of type A (VEGF-A) secretion from osteosarcoma (MG-63) cells cultured using monolayer and 3D spheroid models under two stress conditions: nutrient deficiency (reduced serum culture) and hypoxia-inducible factor (HIF) inhibition (HIF inhibitor, YC-1) are characterized and systematically compared. In order to obtain ample sample size for consistent characterization of cellular responses from cancer spheroids under the stresses and compare the responses to those from the conventional monolayer model, a microfluidic spheroid formation and culture device is utilized in the experiments. In the analysis, cell viability is estimated from captured images, and quantification of VEGF-A secreted from the cells is achieved using enzyme-linked immunosorbent assay (ELISA). The experimental results show that the viabilities decrease when the cells face higher stress levels in both monolayer and 3D spheroid culture models; however, the VEGF-A secretion profiles between the cell culture models are different. The VEGF-A secretion decreases when the cells face higher stress conditions in the monolayer cell culture. In contrast, for the 3D spheroid culture, the VEGF-A concentration decreases for low stress levels but increases while the stress level is high. The VEGF-A regulation in the 3D models mimics in vivo cases of tumor survival and can provide insightful information to investigate tumor angiogenesis in vitro. The approach developed in this paper provides an efficient method to quantitatively and statistically study tumor growth kinetics and stress responses from highly uniform samples and it can also be applied to compare the underlying biomolecular mechanisms in monolayer and 3D spheroid culture models to elucidate the effects of microenvironments on cellular response in cancer research.

Editorial for the Special Issue on Organs-on-Chips.

Recent advances in microsystems technology and cell culture techniques have led to the development of organ-on-chip microdevices to model functional units of organs [...].

Epidermal growth factor-like repeats of SCUBE1 derived from platelets are critical for thrombus formation.

AIMS: SCUBE1 [signal peptide-CUB-epidermal growth factor (EGF) domain-containing protein 1], expressed in endothelial cells (ECs) and platelets, exists in soluble or membrane forms. We previously showed that soluble SCUBE1 is a biomarker for platelet activation and also an active participant of thrombosis. However, whether the adhesive module of its EGF-like repeats is essential and the specific contribution of SCUBE1 synthesized in ECs or platelets to thrombosis in vivo remain unclear. METHODS AND RESULTS: We generated new mutant (Δ2) mice lacking the entire EGF-like repeats to evaluate the module's functional importance during thrombogenesis in vivo. The Δ2 platelet-rich plasma showed markedly impaired platelet aggregation induced by agonists including adenosine diphosphate, collagen, the thrombin agonist PAR-4 peptide and the thromboxane A2 analogue U46619. Consistently, genetic ablation of the EGF-like repeats diminished arterial thrombosis and protected Δ2 mice against lethal thromboembolism. On flow chamber assay, whole blood isolated from Δ2 or wild-type (WT) mice pre-treated with blocking antibodies against the EGF-like repeats showed a significant decrease in platelet deposition and thrombus formation on collagen-coated surfaces under arterial shear rates. Moreover, we created animals expressing SCUBE1 only in ECs (S1-EC) or platelets (S1-PLT) by reciprocal bone-marrow transplantation between WT and Δ2 mice. The time of carotid arterial thrombosis induced by ferric chloride was normal in S1-PLT chimeric mice but much prolonged in S1-EC animals. CONCLUSIONS: We demonstrate that platelet-derived SCUBE1 plays a critical role in arterial thrombosis via its adhesive EGF-like repeats in vivo and suggest targeting these adhesive motifs of SCUBE1 for potential anti-thrombotic strategy.

A Low-Power CMOS Microfluidic Pump Based on Travelling-Wave Electroosmosis for Diluted Serum Pumping.

Microfluidic pump is an essential component in lab-on-chip applications. It is of importance to develop an active microfluidic pump with low-power and low-cost characteristics for portable and miniaturized diagnostic systems. Taking advantages of CMOS technologies, in this work, we report a low-power microfluidic pump based on travelling-wave electroosmosis (TWEO). Utilizing an integrated driving circuit, this monolithic CMOS microfluidic pump can be operated at 1.5 V driving voltage with a power consumption of 1.74 mW. The integrated driving circuit consist of a resistor-capacitor (RC) oscillator, a 90-degrees phase-shift square wave generator, and buffer amplifiers. Moreover, capabilities of the developed CMOS TWEO pump to drive diluted human serum are characterized. The flow rate of diluted human serum with dilution ratio of 1:1000 can achieve 51 µm/s. This is the first time demonstrating an in-situ CMOS-based microfluidic pump to drive the clinical diluted serum sample. As a consequence, this work demonstrates an essential component of CMOS biotechnologies for potential applications of portable in vitro diagnosis (IVD) systems.