Trends in Droplet Microfluidics: From Droplet Generation to Biomedical Applications.

Over the past decade, droplet microfluidics has attracted growing interest in biology, medicine, and engineering. In this feature article, we review the advances in droplet microfluidics, primarily focusing on the research conducted by our group. Starting from the introduction to the mechanisms of microfluidic droplet formation and the strategies for cell encapsulation in droplets, we then focus on droplet transformation into microgels. Furthermore, we review three biomedical applications of droplet microfluidics, that is, 3D cell culture, single-cell analysis, and in vitro organ and disease modeling. We conclude with our perspective on future directions in the development of droplet microfluidics for biomedical applications.

Multiple Myeloma Cell Drug Responses Differ in Thermoplastic vs PDMS Microfluidic Devices.

Poly(dimethylsiloxane) (PDMS) is a commonly used elastomer for fabricating microfluidic devices, but it has previously been shown to absorb hydrophobic molecules. Although this has been demonstrated for molecules such as estrogen and Nile Red, the absorption of small hydrophobic molecules in PDMS specifically used to treat cancer and its subsequent impact on cytotoxicity measurements and assays have not been investigated. This is critical for the development of microfluidic chemosensitivity and resistance assay (CSRA) platforms that have shown potential to help guide clinical therapy selection and which rely on the accuracy of the readout involving interactions between patient-derived cells and cancer drugs. It is thus important to address the issue of drug absorption into device material. We investigated drug absorption into microfluidic devices by treating multiple myeloma (MM) tumor cells with two MM drugs (bortezomib (BTZ) and carfilzomib (CFZ)) in devices fabricated using three different materials (polystyrene (PS), cyclo-olefin polymer (COP), and PDMS). Half-maximal inhibitory concentrations (IC50) were obtained for each drug-material combination, and an increase in IC50 of ∼4.3× was observed in PDMS devices compared to both thermoplastic devices. Additionally, each MM drug was exposed to polymer samples, and samples were analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS) to characterize adsorption and absorption of the drugs into each material. ToF-SIMS data showed the bias observed in IC50 values found in PDMS devices was directly related to the absorption of drug during dose-response experiments. Specifically, BTZ and CFZ absorption in both PS and COP were all in the range of ∼100-300 nm, whereas BTZ and CFZ absorption in PDMS was ∼5.0 and ∼3.5 µm, respectively. These results highlight the biases that exist in PDMS devices and the importance of material selection in microfluidic device design, especially in applications involving drug cytotoxicity and hydrophobic molecules.

IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation.

Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and ß family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin ß family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response.

Microfluidic multiculture assay to analyze biomolecular signaling in angiogenesis.

Angiogenesis (the formation of blood vessels from existing blood vessels) plays a critical role in many diseases such as cancer, benign tumors, and macular degeneration. There is a need for cell culture methods capable of dissecting the intricate regulation of angiogenesis within the microenvironment of the vasculature. We have developed a microscale cell-based assay that responds to complex pro- and antiangiogenic soluble factors with an in vitro readout for vessel formation. The power of this system over traditional techniques is that we can incorporate the whole milieu of soluble factors produced by cells in situ into one biological readout (vessel formation), even if the identity of the factors is unknown. We have currently incorporated macrophages, endothelial cells, and fibroblasts into the assay, with the potential to include additional cell types in the future. Importantly, the microfluidic platform is simple to operate and multiplex to test drugs targeting angiogenesis in a more physiologically relevant context. As a proof of concept, we tested the effect of an enzyme inhibitor (targeting matrix metalloproteinase 12) on vessel formation; the triculture microfluidic assay enabled us to capture a dose-dependent effect entirely missed in a simplified coculture assay (p < 0.0001). This result underscores the importance of cell-based assays that capture chemical cross-talk occurring between cell types. The microscale dimensions significantly reduce cell consumption compared to conventional well plate platforms, enabling the use of limited primary cells from patients in future investigations and offering the potential to screen therapeutic approaches for individual patients in vitro.

Fluorescence-based assessment of plasma-induced hydrophilicity in microfluidic devices via Nile Red adsorption and depletion.

We present a simple method, called fluorescence-based assessment of plasma-induced hydrophilicity (FAPH), that enables spatial mapping of the local hydrophilicity of surfaces normally inaccessible by traditional contact angle measurement techniques. The method leverages the change in fluorescence of a dye, Nile Red, which is adsorbed on an oxygen plasma-treated surface, and its correlation with the contact angle of water. Using FAPH, we explored the effect of microchannel geometries on the penetration distance of oxygen plasma into a microchannel and found that entrance effects prevent uniform treatment. We showed that these variations have a significant impact on cell culture, and thus the design of cell-based microfluidic assays must consider this phenomenon to obtain repeatable and homogeneous results.

Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays.

Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays.

Microscale functional cytomics for studying hematologic cancers.

An important problem in translational cancer research is our limited ability to functionally characterize behaviors of primary patient cancer cells and associated stromal cell types, and relate mechanistic understanding to therapy selection. Functional analyses of primary samples face at least 3 major challenges: limited availability of primary samples for testing, paucity of functional information extracted from samples, and lack of functional methods accessible to many researchers. We developed a microscale cell culture platform that overcomes these limitations, especially for hematologic cancers. A key feature of the platform is the ability to compartmentalize small populations of adherent and nonadherent cells in controlled microenvironments that can better reflect physiological conditions and enable cell-cell interaction studies. Custom image analysis was developed to measure cell viability and protein subcellular localizations in single cells to provide insights into heterogeneity of cellular responses. We validated our platform by assessing viability and nuclear translocations of NF-κB and STAT3 in multiple myeloma cells exposed to different conditions, including cocultured bone marrow stromal cells. We further assessed its utility by analyzing NF-κB activation in a primary chronic lymphocytic leukemia patient sample. Our platform can be applied to myriad biological questions, enabling high-content functional cytomics of primary hematologic malignancies.

Interrogating Matrix Stiffness and Metabolomics in Pancreatic Ductal Carcinoma Using an Openable Microfluidic Tumor-on-a-Chip.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma that contributes to aggressive tumor biology and therapeutic resistance. Current in vitro PDAC models lack sufficient optical and physical access for fibrous network visualization, in situ mechanical stiffness measurement, and metabolomic profiling. Here, we describe an openable multilayer microfluidic PDAC-on-a-chip platform that consists of pancreatic tumor cells (PTCs) and pancreatic stellate cells (PSCs) embedded in a 3D collagen matrix that mimics the stroma. Our system allows fibrous network visualization via reflected light confocal (RLC) microscopy, in situ mechanical stiffness testing using atomic force microscopy (AFM), and compartmentalized hydrogel extraction for PSC metabolomic profiling via mass spectrometry (MS) analysis. In comparing cocultures of gel-embedded PSCs and PTCs with PSC-only monocultures, RLC microscopy identified a significant decrease in pore size and corresponding increase in fiber density. In situ AFM indicated significant increases in stiffness, and hallmark characteristics of PSC activation were observed using fluorescence microscopy. PSCs in coculture also demonstrated localized fiber alignment and densification as well as increased collagen production. Finally, an untargeted MS study putatively identified metabolic contributions consistent with in vivo PDAC studies. Taken together, this platform can potentially advance our understanding of tumor-stromal interactions toward the discovery of novel therapies.

Tunable In Situ Synthesis of Ultrathin Extracellular Matrix-Derived Membranes in Organ-on-a-Chip Devices.

Thin cell culture membranes in organ-on-a-chip (OOC) devices are used to model a wide range of thin tissues. While early and most current platforms use microporous or fibrous elastomeric or thermoplastic membranes, there is an emerging class of devices using extra-cellular matrix (ECM) protein-based membranes to improve their biological relevance. These ECM-based membranes present physiologically relevant properties, but they are difficult to integrate into OOC devices due to their relative fragility. Additionally, the specialized fabrication methods developed to date make comparison between methods difficult. This work presents the development and characterization of a method to produce ultrathin matrix-derived membranes (UMM) in OOC devices that requires only a preassembled thermoplastic device and a micropipette, decoupling the device and UMM fabrication processes. Control over the thickness and permeability of the UMM is demonstrated, along with integration of the UMM in a device enabling high-resolution on-chip microscopy. The reliability of the UMM fabrication method is leveraged to develop a medium-throughput well-plate format device with 32 independent UMM-integrated samples. Finally, proof-of-concept cell culture experiments are demonstrated. Due to its simplicity and controllability, the presented method has the potential to overcome technical barriers preventing wider adoption of physiologically relevant ECM-based membranes in OOC devices.

A Microfluidic Platform for Evaluating the Internalization of Liposome Drug Carriers in Tumor Spheroids.

The development of in vitro models recapitulating nanoparticle transport under physiological flow conditions is of great importance for predicting the efficacy of nanoparticle drug carriers. Liposomes are extensively used for drug delivery owing to their biocompatibility and biodegradability and the ability to carry both hydrophilic and hydrophobic compounds. Here, we used a library of liposomes with various dimensions and a microfluidic platform comprising a large array of uniformly sized breast cancer spheroids to explore size-dependent liposome internalization and retention in the spheroids under close-to-physiological interstitial conditions. Such a platform showed promising applications in the preclinical screening of small molecule drugs; however, the capability to deliver nanoparticles in the spheroid interior under close-to-physiological flow conditions was not explored. For the liposomes with diameters in the range of 45-200 nm, we show experimentally and by simulations that in comparison with liposome delivery solely by diffusion, flow significantly enhances liposome internalization in the microgels and mitigates the size-dependent spheroid penetration by the liposomes. The utility of the microfluidic platform was validated by evaluating the efficacy of clinically approved doxorubicin-loaded liposomes (Doxil), which exhibited superior retention in the spheroids under flow conditions, in comparison with free doxorubicin. This MF platform can serve as an in vitro model for screening the efficacy of drugs encapsulated in liposomes and find applications for screening other types of nanoparticle carriers for vaccine delivery, diagnostics, and skincare.

Assessment of enhanced autofluorescence and impact on cell microscopy for microfabricated thermoplastic devices.

Thermoplastics such as polystyrene (PS) and cyclo-olefin polymer (COP) have become common materials for fabrication of microfluidic cell-based systems because of a number of attractive properties. However, thermoplastics are also known to exhibit autofluorescence levels that may hinder their utility for cell-based and imaging applications. Here, we identify and characterize a phenomenon causing an increase in the autofluorescence of polystyrene after thermal treatment. This effect is of particular importance for plastic microfluidic device fabrication because the ranges of pressures and temperatures causing this effect match the same range as those used for polystyrene bonding. Further, we find that the enhanced autofluorescence has significant impact on the image quality, accuracy, and ability to identify and quantify fluorescently labeled cells. We tested two alternative strategies, solvent bonding of PS or thermal bonding of COP, to alleviate the adverse effects of heterogeneous and enhanced autofluorescence on cell image analysis, and demonstrate that both strategies are viable options to thermal bonding of PS for specific applications where cellular imaging is of primary interest.

Bidirectional airflow in lung airway-on-a-chip with matrix-derived membrane elicits epithelial glycocalyx formation.

Organ-on-a-chip systems are rapidly advancing as a viable alternative to existing experimental models in respiratory research. To date, however, epithelial cell cultures within lung airway-on-a-chip devices have yet to demonstrate the presence of an epithelial glycocalyx, a thin layer of proteoglycans, glycoproteins, and glycolipids known to play an important role in regulating epithelial function. Here, we demonstrate that an airway-on-a-chip device that incorporates bidirectional flow mimicking breathing cycles in combination with an ultra-thin matrix-derived membrane (UMM) layer can generate a glycocalyx layer comprised of heparan sulfate. Results with this device and airflow system showed dramatic differences of airway epithelial cell viability and expression of tight junctions, cilia, and mucus over a wide range of flow rates when cultured under oscillatory flow. More importantly, for the first time in a microfluidic organ-on-a-chip setting, we achieved the visualization of an airflow-induced epithelial glycocalyx layer. Our experiments highlight the importance of physiological mimicry in developing in vitro models, as bidirectional airflow showed more representative mucociliary differentiation compared to continuous unidirectional airflow. Thus, the lung airway-on-a-chip platform demonstrated in this study holds great potential as a lung epithelial barrier model for studying the mechanisms of various respiratory diseases and for testing the efficacy of therapeutic candidates in the presence of bidirectional airflow and the glycocalyx.

Integrating spheroid-on-a-chip with tubeless rocker platform: A high-throughput biological screening platform.

Spheroid-on-a-chip platforms are emerging as promising in vitro models that enable screening of the efficacy of biologically active ingredients. Generally, the supply of liquids to spheroids occurs in the steady flow mode with the use of syringe pumps; however, the utilization of tubing and connections, especially for multiplexing and high-throughput screening applications, makes spheroid-on-a-chip platforms labor- and cost-intensive. Gravity-induced flow using rocker platforms overcomes these challenges. Here, a robust gravity-driven technique was developed to culture arrays of cancer cell spheroids and dermal fibroblast spheroids in a high-throughput manner using a rocker platform. The efficiency of the developed rocker-based platform was benchmarked to syringe pumps for generating multicellular spheroids and their use for screening biologically active ingredients. Cell viability, internal spheroid structure as well as the effect of vitamin C on spheroids' protein synthesis was studied. The rocker-based platform not only offers comparable or enhanced performance in terms of cell viability, spheroids formation, and protein production by dermal fibroblast spheroids but also, from a practical perspective, offers a smaller footprint, requires a lower cost, and offers an easier method for handling. These results support the application of rocker-based microfluidic spheroid-on-a-chip platforms for in vitro screening in a high-throughput manner with industrial scaling-up opportunities.

Laser interstitial thermal therapy of lung lesions near large vessels: a numerical study.

Objective.Laser interstitial thermal therapy (LITT) is an evolving hyperthermia-based technology that may offer a minimally invasive alternative to inoperable lung cancer. LITT of perivascular targets is challenged by higher risk of disease recurrence due to vascular heat sinks, as well as risk of damage to these vascular structures. The objective of this work is to examine the impact of multiple vessel parameters on the efficacy of the treatment and the integrity of the vessel wall in perivascular LITT.Approach.A finite element model is used to examine the role of vessel proximity, flow rate, and wall thickness on the outcome of the treatment. Main result. The simulated work indicates that vessel proximity is the major factor in driving the magnitude of the heat sink effect. Vessels situated near the target volume may act as a protective measure for reducing healthy tissue damage. Vessels with thicker walls are more at risk of damage during treatment. Interventions to reduce the flow rate may reduce the vessel's heat sink effect but may also result in increased risk of vascular wall damage. Lastly, even at reduced blood flow rates, the volume of blood reaching the threshold of irreversible damage (>43 °C) is negligible compared to the volume of blood flow throughout the treatment duration.Significance.This investigative simulation yields results that may help guide clinicians on treatment planning near large vessels.

Microfluidic Arrays of Breast Tumor Spheroids for Drug Screening and Personalized Cancer Therapies.

One of the obstacles limiting progress in the development of effective cancer therapies is the shortage of preclinical models that capture the dynamic nature of tumor microenvironments. Interstitial flow strongly impacts tumor response to chemotherapy; however, conventional in vitro cancer models largely disregard this key feature. Here, a proof of principle microfluidic platform for the generation of large arrays of breast tumor spheroids that are grown under close-to-physiological flow in a biomimetic hydrogel is reported. This cancer spheroids-on-a-chip model is used for time- and labor-efficient studies of the effects of drug dose and supply rate on the chemosensitivity of breast tumor spheroids. The capability to grow large arrays of tumor spheroids from patient-derived cells of different breast cancer subtypes is shown, and the correlation between in vivo drug efficacy and on-chip spheroid drug response is demonstrated. The proposed platform can serve as an in vitro preclinical model for the development of personalized cancer therapies and effective screening of new anticancer drugs.

Rapid prototyping of arrayed microfluidic systems in polystyrene for cell-based assays.

Microfluidic cell-based systems have enabled the study of cellular phenomena with improved spatiotemporal control of the microenvironment and at increased throughput. While poly(dimethylsiloxane) (PDMS) has emerged as the most popular material in microfluidics research, it has specific limitations that prevent microfluidic platforms from achieving their full potential. We present here a complete process, ranging from mold design to embossing and bonding, that describes the fabrication of polystyrene (PS) microfluidic devices with similar cost and time expenditures as PDMS-based devices. Emphasis was placed on creating methods that can compete with PDMS fabrication methods in terms of robustness, complexity, and time requirements. To achieve this goal, several improvements were made to remove critical bottlenecks in existing PS embossing methods. First, traditional lithographic techniques were adapted to fabricate bulk epoxy molds capable of resisting high temperatures and pressures. Second, a method was developed to emboss through-holes in a PS layer, enabling creation of large arrays of independent microfluidic systems on a single device without need to manually create access ports. Third, thermal bonding of PS layers was optimized in order to achieve quality bonding over large arrays of microsystems. The choice of materials and methods was validated for biological function in two different cell-based applications to demonstrate the versatility of our streamlined fabrication process.

Fundamentals of microfluidic cell culture in controlled microenvironments.

Microfluidics has the potential to revolutionize the way we approach cell biology research. The dimensions of microfluidic channels are well suited to the physical scale of biological cells, and the many advantages of microfluidics make it an attractive platform for new techniques in biology. One of the key benefits of microfluidics for basic biology is the ability to control parameters of the cell microenvironment at relevant length and time scales. Considerable progress has been made in the design and use of novel microfluidic devices for culturing cells and for subsequent treatment and analysis. With the recent pace of scientific discovery, it is becoming increasingly important to evaluate existing tools and techniques, and to synthesize fundamental concepts that would further improve the efficiency of biological research at the microscale. This tutorial review integrates fundamental principles from cell biology and local microenvironments with cell culture techniques and concepts in microfluidics. Culturing cells in microscale environments requires knowledge of multiple disciplines including physics, biochemistry, and engineering. We discuss basic concepts related to the physical and biochemical microenvironments of the cell, physicochemical properties of that microenvironment, cell culture techniques, and practical knowledge of microfluidic device design and operation. We also discuss the most recent advances in microfluidic cell culture and their implications on the future of the field. The goal is to guide new and interested researchers to the important areas and challenges facing the scientific community as we strive toward full integration of microfluidics with biology.

TANDEM: biomicrofluidic systems with transverse and normal diffusional environments for multidirectional signaling.

Biomicrofluidic systems that can recapitulate complex biological processes with precisely controlled 3D geometries are a significant advancement from traditional 2D cultures. To this point, these systems have largely been limited to either laterally adjacent channels in a single plane or vertically stacked single-channel arrangements. As a result, lateral (or transverse) and vertical (or normal) diffusion have been isolated to their respective designs only, thus limiting potential access to nutrients and 3D communication that typifies in vivo microenvironments. Here we report a novel device architecture called "TANDEM", an acronym for "T̲ransverse A̲nd N̲ormal D̲iffusional E̲nvironments for M̲ultidirectional Signaling", which enables multiplanar arrangements of aligned channels where normal and transverse diffusion occur in tandem to facilitate multidirectional communication. We developed a computational transport model in COMSOL and tested diffusion and culture viability in one specific TANDEM configuration, and found that TANDEM systems demonstrated enhanced diffusion in comparison to single-plane counterparts. This resulted in improved viability of hydrogel-embedded cells, which typically suffer from a lack of sufficient nutrient access during long-term culture. Finally, we showed that TANDEM designs can be expanded to more complex alternative configurations depending on the needs of the end-user. Based on these findings, TANDEM designs can utilize multidirectional enhanced diffusion to improve long-term viability and ultimately facilitate more robust and more biomimetic microfluidic systems with increasingly more complex geometric layouts.

Angiogenic Sprouting Dynamics Mediated by Endothelial-Fibroblast Interactions in Microfluidic Systems.

Angiogenesis, the development of new blood vessels from existing vasculature, is a key process in normal development and pathophysiology. In vitro models are necessary for investigating mechanisms of angiogenesis and developing antiangiogenic therapies. Microfluidic cell culture models of angiogenesis are favored for their ability to recapitulate 3D tissue structures and control spatiotemporal aspects of the microenvironments. To capture the angiogenesis process, microfluidic models often include endothelial cells and a fibroblast component. However, the influence of fibroblast organization on resulting angiogenic behavior remains unclear. Here a comparative study of angiogenic sprouting on a microfluidic chip induced by fibroblasts in 2D monolayer, 3D dispersed, and 3D spheroid culture formats, is conducted. Vessel morphology and sprout distribution for each configuration are measured, and these observations are correlated with measurements of secreted factors and numerical simulations of diffusion gradients. The results demonstrate that angiogenic sprouting varies in response to fibroblast organization with correlating variations in secretory profile and secreted factor gradients across the microfluidic device. This study is anticipated to shed light on how sprouting dynamics are mediated by fibroblast configuration such that the microfluidic cell culture design process includes the selection of a fibroblast component where the effects are known and leveraged.

Computational Modelling and Big Data Analysis of Flow and Drug Transport in Microfluidic Systems: A Spheroid-on-a-Chip Study.

Microfluidic tumour spheroid-on-a-chip platforms enable control of spheroid size and their microenvironment and offer the capability of high-throughput drug screening, but drug supply to spheroids is a complex process that depends on a combination of mechanical, biochemical, and biophysical factors. To account for these coupled effects, many microfluidic device designs and operating conditions must be considered and optimized in a time- and labour-intensive trial-and-error process. Computational modelling facilitates a systematic exploration of a large design parameter space via in silico simulations, but the majority of in silico models apply only a small set of conditions or parametric levels. Novel approaches to computational modelling are needed to explore large parameter spaces and accelerate the optimization of spheroid-on-a-chip and other organ-on-a-chip designs. Here, we report an efficient computational approach for simulating fluid flow and transport of drugs in a high-throughput arrayed cancer spheroid-on-a-chip platform. Our strategy combines four key factors: i) governing physical equations; ii) parametric sweeping; iii) parallel computing; and iv) extensive dataset analysis, thereby enabling a complete "full-factorial" exploration of the design parameter space in combinatorial fashion. The simulations were conducted in a time-efficient manner without requiring massive computational time. As a case study, we simulated >15,000 microfluidic device designs and flow conditions for a representative multicellular spheroids-on-a-chip arrayed device, thus acquiring a single dataset consisting of ∼10 billion datapoints in ∼95 GBs. To validate our computational model, we performed physical experiments in a representative spheroid-on-a-chip device that showed excellent agreement between experimental and simulated data. This study offers a computational strategy to accelerate the optimization of microfluidic device designs and provide insight on the flow and drug transport in spheroid-on-a-chip and other biomicrofluidic platforms.