Multiorgan microfluidic platform with breathable lung chamber for inhalation or intravenous drug screening and development.

Efficient and economical delivery of pharmaceuticals to patients is critical for effective therapy. Here we describe a multiorgan (lung, liver, and breast cancer) microphysiological system ("Body-on-a-Chip") designed to mimic both inhalation therapy and/or intravenous therapy using curcumin as a model drug. This system is "pumpless" and self-contained using a rocker platform for fluid (blood surrogate) bidirectional recirculation. Our lung chamber is constructed to maintain an air-liquid interface and contained a "breathable" component that was designed to mimic breathing by simulating gas exchange, contraction and expansion of the "lung" using a reciprocating pump. Three cell lines were used: A549 for the lung, HepG2 C3A for the liver, and MDA MB231 for breast cancer. All cell lines were maintained with high viability (>85%) in the device for at least 48 hr. Curcumin is used to treat breast cancer and this allowed us to compare inhalation delivery versus intravenous delivery of the drug in terms of effectiveness and potentially toxicity. Inhalation therapy could be potentially applied at home by the patient while intravenous therapy would need to be applied in a clinical setting. Inhalation therapy would be more economical and allow more frequent dosing with a potentially lower level of drug. For 24 hr exposure to 2.5 and 25 µM curcumin in the flow device the effect on lung and liver viability was small to insignificant, while there was a significant decrease in viability of the breast cancer (to 69% at 2.5 µM and 51% at 25 µM). Intravenous delivery also selectively decreased breast cancer viability (to 88% at 2.5 µM and 79% at 25 µM) but was less effective than inhalation therapy. The response in the static device controls was significantly reduced from that with recirculation demonstrating the effect of flow. These results demonstrate for the first time the feasibility of constructing a multiorgan microphysiological system with recirculating flow that incorporates a "breathable" lung module that maintains an air-liquid interface.

Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening.

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm2 on day 3 on chip and were sustained above 2000 Ω · cm2 up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184-194. © 2016 Wiley Periodicals, Inc.

Design and demonstration of a pumpless 14 compartment microphysiological system.

We describe a human "Body-on-a-chip" device (or microphysiological system) that could be used to emulate drug distribution, metabolism, and action in the body. It is based upon a physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) model, where multiple chambers representing different organs are connected with fluidic channels to mimic multi-organ interactions within the body. Here we describe a pumpless 14 chamber (13 organs) microfluidic cell culture device that provides a separation between barrier and nonbarrier types of cell cultures. Our barrier chamber layer (skin, GI tract, and lung) allows for direct access and/or exposures to chemical or biological reagents forcing these reagents to pass through a barrier of cells established on a microfabricated membrane before exposing the nonbarrier tissue chambers (fat, kidney, heart, adrenal glands, liver, spleen, pancreas, bone marrow, brain, muscle) or entering the microfluidic circulation within the device. Our nonbarrier tissue chambers were created as three-dimensional configurations by resuspending cells in hydrogel (PGMatrix). We used cell lines to represent five of these organs (barrier lines-A549 [lung] and Caco2 [GI]) (nonbarrier lines-HepG2 C3A [liver], Meg01 [bone marrow], and HK2 [kidney]). The dimensions of our straight duct-like channels to each organ chamber were designed to provide the appropriate flow of a culture medium. The organ volumes and organ flow rates that have been reported for an average human male were used to estimate the desired fluid retention times in each organ chamber. The flow through the channels was induced by gravity on a custom programmed rocker platform which enabled pumpless operation and minimized bubble entrapment. The purpose of this paper is to describe the design and operation of a 14 chamber multi-organ system representing 13 tissues/organs with both barrier and nonbarrier tissue chambers and to study the interactive responses among the various cell lines. We demonstrate that five different cell lines survived with high viability (above 85%) for 7 days. We compared the individual observed flow rates to the compartments to the desired or estimated flow rates. This work demonstrates the feasibility of constructing, operating and maintaining a simple, gravity-driven, multi-organ microphysiological system with the capability of measuring cellular functions such as CYP1A1 and CYP3A4 activities, albumin release, urea, maintenance of tight junctions, and presence of surfactant for a sustained period. Biotechnol. Bioeng. 2016;113: 2213-2227. © 2016 Wiley Periodicals, Inc.

Body-on-a-chip systems for animal-free toxicity testing.

Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a prodrug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing.

Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1.

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.

Relationship between near infrared spectroscopy and intra-compartmental pressures.

BACKGROUND: Near infrared spectroscopy (NIRS) has been suggested as a possible means for detecting perfusion deficits in patients with acute compartment syndrome (ACS). STUDY OBJECTIVES: To longitudinally examine NIRS in an ACS model to determine its responsiveness to decreasing perfusion pressure. METHODS: A NIRS sensor pad was placed under a tourniquet over the anterior compartment in the mid-tibia region on 20 volunteers. Initial perfusion pressures and NIRS values were recorded. The tourniquet pressure was sequentially raised by 10 mm Hg in 10-min intervals until systolic pressure was surpassed. NIRS values and perfusion pressure were determined at the end of each 10-min interval. RESULTS: There was no change in mean NIRS values from the initial baseline until 30 mm Hg of perfusion pressure was reached. Additionally, a statistically significant drop in mean NIRS values was observed as perfusion pressures dropped from 10 mm Hg to 0 mm Hg, and again with subsequent decreases of 10 mm Hg perfusion pressure until systolic pressure was surpassed. CONCLUSIONS: These results coincide with previously published studies using alternative methods of measuring blood flow or perfusion. NIRS values were responsive to decreasing perfusion pressures over a longitudinal period of time in an ACS model. These results suggest that NIRS may be useful for continuous, non-invasive monitoring of patients for whom ACS is a concern. Additional studies on traumatized patients are required.

Identification of optimal control compartments for serial near-infrared spectroscopy assessment of lower extremity compartmental perfusion.

Near-infrared spectroscopy (NIRS) has shown promise in detecting ischemic changes in acute compartment syndrome. The objectives of this study were to 1) assess the correlation in NIRS values between upper and lower extremity control sites for bilateral lower extremity trauma and 2) investigate the effect of skin pigmentation on NIRS values. Forty-four volunteers (14 male, 30 female) were monitored over separate 1-hour sessions. NIRS leads were placed over leg and upper extremity compartments. Colorimeters were used to document skin pigmentation. NIRS values between corresponding contralateral compartments were extremely well correlated (r = 0.76-0.90). Upper extremity NIRS values were correlated to leg values in the following order: volar (r = 0.65-0.71), dorsal (r = 0.36-0.60), and deltoid (r = 0.42-0.51). A negative correlation was observed between melanin and NIRS values. Analogous leg compartments are the optimal site of control for each other. The volar forearm may be the best upper extremity control. Skin pigmentation may affect absolute NIRS values.

Development of disposable PDMS micro cell culture analog devices with photopolymerizable hydrogel encapsulating living cells.

Microscale cell culture devices with two or more cell types, such as the micro cell culture analog (microCCA), are promising devices to predict mammalian response to toxic drug and chemical exposure. A polydimethylsiloxane (PDMS) version of such microfluidic devices has been challenging to construct due to the difficulty of patterning multi cell types directly into designated individual cell culture chambers in an oxygen plasma bonded PDMS device. Approaches with micro-valves for flow control are complex, expensive and inconvenient to use. In this study, an alternative approach using polyethylene glycol diacrylate (PEG-DA) for spatially controlled multi-cell type patterning inside a bonded microCCA device is described. We constructed a three-cell type PDMS microCCA following a human physiologically based pharmacokinetic (PBPK) modeling, and applied continuous cell culture medium recirculation within the device as a blood surrogate. A fluorescence microscope based direct pattern writing method was used to form cell/hydrogel microstructures with higher cell viability than the traditional UV lamp based method. The positive effect of mixed molecular weight PDG-DA on hydrogel-encapsulated cell membrane integrity was also studied. This prototype PDMS microCCA device was then tested with Triton X-100 as a model toxicant. The combination of hydrogel photo-patterning and the microfluidic cell culture platform enables the fabrication of simple and low cost multi-cell type biosensors for drug development, toxicity study and clinical diagnosis.

On chip porous polymer membranes for integration of gastrointestinal tract epithelium with microfluidic 'body-on-a-chip' devices.

We describe a novel fabrication method that creates microporous, polymeric membranes that are either flat or contain controllable 3-dimensional shapes that, when populated with Caco-2 cells, mimic key aspects of the intestinal epithelium such as intestinal villi and tight junctions. The developed membranes can be integrated with microfluidic, multi-organ cell culture systems, providing access to both sides, apical and basolateral, of the 3D epithelial cell culture. Partial exposure of photoresist (SU-8) spun on silicon substrates creates flat membranes with micrometer-sized pores (0.5-4.0 µm) that--supported by posts--span across 50 µm deep microfluidic chambers that are 8 mm wide and 10 long. To create three-dimensional shapes the membranes were air dried over silicon pillars with aspect ratios of up to 4:1. Space that provides access to the underside of the shaped membranes can be created by isotropically etching the sacrificial silicon pillars with xenon difluoride. Depending on the size of the supporting posts and the pore sizes the overall porosity of the membranes ranged from 4.4 % to 25.3 %. The microfabricated membranes can be used for integrating barrier tissues such as the gastrointestinal tract epithelium, the lung epithelium, or other barrier tissues with multi-organ "body-on-a-chip" devices.

Batch, fed-batch, and microcarrier cultures with CHO cell lines in a pressure-cycle driven miniaturized bioreactor.

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage-dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed-batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 10(7) /mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed-batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale-up.

Metacridamides A and B, macrocycles from conidia of the entomopathogenic fungus Metarhizium acridum.

Metarhizium acridum, an entomopathogenic fungus, has been commercialized and used successfully for biocontrol of grasshopper pests in Africa and Australia. Its conidia produce two novel 17-membered macrocycles, metacridamides A and B, which consist of a Phe unit condensed with a nonaketide. Planar structures were elucidated by a combination of mass spectrometric and NMR techniques. Following hydrolysis of 1, chiral amino acid analysis assigned the L-configuration to the Phe unit. A crystal structure established the absolute configuration of the eight remaining stereogenic centers in 1. Metacridamide A showed cytotoxicity to three cancer lines with IC50's of 6.2, 11.0, and 10.8 µM against Caco-2 (epithelial colorectal adenocarcinoma), MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell lines, respectively. In addition, metacridamide B had an IC50 of 18.2 µM against HepG2/C3A, although it was inactive at 100 µM against Caco-2 and MCF-7. Neither analogue showed antimicrobial, phytotoxic, or insecticidal activity.

Ability of near infrared spectroscopy to measure oxygenation in isolated upper extremity muscle compartments.

PURPOSE: Near infrared spectroscopy (NIRS), a noninvasive means for monitoring muscle oxygenation, may be useful in the diagnosis of acute compartment syndrome, a condition characterized by poor tissue perfusion. This study used the decrease in muscle oxygenation caused by exercise to investigate the ability of anatomic placement of NIRS sensor pads over compartments of the forearm to isolate perfusion values of a specific compartment. METHODS: We recruited 63 uninjured volunteers from a private clinic-based setting and placed NIRS sensor pads over the dorsal, volar, and mobile wad compartments of 1 forearm. A total of 49 participants also had the contralateral forearm monitored, which served as an internal control. Participants performed a series of 3 exercises designed to sequentially activate the muscles of each compartment. A washout period separated each exercise to allow perfusion to return to baseline. We compared NIRS values of each compartment recorded during muscle contraction with baseline values. RESULTS: Mean NIRS values decreased significantly from baseline during muscle contraction for all compartments, whereas mean NIRS values of muscle compartments that remained at rest showed little or no change. We observed no changes in NIRS values of the contralateral arm, which remained at rest during the entire data collection period. CONCLUSIONS: Although lack of an existing method for quantifying muscle perfusion precludes validation of this technique against a reference standard, this study suggests that NIRS can provide oxygenation values that are both sensitive and specific to muscle compartments of the forearm. Future studies should investigate NIRS among patients with upper extremity injuries. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic III.

An uncertain role for Cu(II) in stimulating Mn(II) oxidation by Leptothrix discophora SS-1.

In an effort to improve understanding of the role of Cu(II) in bacterial Mn(II) oxidation, a model Mn(II)-oxidizing bacterium, Leptothrix discophora SS-1, was grown in presence of toxic and non-toxic concentrations of Cu(II), Cd(II) and Mn(II). Mn(II)-oxidizing activity increased by 40% when cells were grown in the presence of 0.05 microM of Cu(II) and increased twofold at 0.18 microM Cu(II). Toxic levels of Cd(II) did not stimulate Mn(II) oxidizing activity, indicating that Mn(II) oxidation is not a response to metal toxicity. Stimulation by Cu(II) confirms the specific role of Cu(II) in Mn(II) oxidation. Comparison of transcript levels of the multicopper oxidase mofA gene in the presence and absence of added Cu(II) do not indicate a statistically significant change in mofA transcript levels in cultures supplemented with Cu(II). Thus, the exact role of Cu(II) in Mn(II) oxidation and its affect on mofA gene expression remain uncertain.

Pumpless, unidirectional microphysiological system for testing metabolism-dependent chemotherapeutic toxicity.

Drug development is often hindered by the failure of preclinical models to accurately assess and predict the efficacy and safety of drug candidates. Body-on-a-chip (BOC) microfluidic devices, a subset of microphysiological systems (MPS), are being created to better predict human responses to drugs. Each BOC is designed with separate organ chambers interconnected with microfluidic channels mimicking blood recirculation. Here, we describe the design of the first pumpless, unidirectional, multiorgan system and apply this design concept for testing anticancer drug treatments. HCT-116 colon cancer spheroids, HepG2/C3A hepatocytes, and HL-60 promyeloblasts were embedded in collagen hydrogels and cultured within compartments representing "colon tumor", "liver," and "bone marrow" tissue, respectively. Operating on a pumpless platform, the microfluidic channel design provides unidirectional perfusion at physiologically realistic ratios to multiple channels simultaneously. The metabolism-dependent toxic effect of Tegafur, an oral prodrug of 5-fluorouracil, combined with uracil was examined in each cell type. Tegafur-uracil treatment induced substantial cell death in HCT-116 cells and this cytotoxic response was reduced for multicellular spheroids compared to single cells, likely due to diffusion-limited drug penetration. Additionally, off-target toxicity was detected by HL-60 cells, which demonstrate that such systems can provide useful information on dose-limiting side effects. Collectively, this microscale cell culture analog is a valuable physiologically-based pharmacokinetic drug screening platform that may be used to support cancer drug development.

Retrospective analysis of trends in surgery volumes between 2016 and 2019 and impact of the insurance deductible: Cross-sectional study.

BACKGROUND: Understanding trends in surgical volumes can help Ambulatory Surgery Centers (ASCs) prevent clinician burnout and provide adequate staffing while maintaining the quality of patient care throughout the year. Health insurance deductibles reset in January each year and may contribute to an annual rhythm where the levee of year-end deductibles is breached in the last few months of every year, resulting in a flood of cases and several accompanying challenges. This study aims to identify and analyze monthly and yearly surgical volume patterns in ASCs and explore a relationship with the deductible reset. METHODS: De-identified, aggregate visit data for 2016-2019 were obtained retrospectively from 14 ambulatory surgery centers within the same benchmarking consortium in the Southeast. The ASCs subspecialty types consisted of orthopedics, urology, otolaryngology, and multispecialty. Kaiser Family Foundation survey data from 2016 to 2019 was used to inform deductible trends. Augmented Dickey-Fuller tests, linear regressions, and two-sample T-tests were conducted to explore and establish patterns in surgical volume between 2016 and 2019. RESULTS: Overall, average orthopedic surgical volume increased 38.04% from January to December in 2016-2019 with an average difference of 64 cases (95% CI: 47-80), while that of all ASCs combined increased 19.24% within the same timeframe with an average difference of 37 cases (95% CI: 21-52). Average health insurance deductibles rose 12% from $1476 to $1655 within the same timeframe. Regression analysis showed a stronger association between year and volume for orthopedic ASCs (R (Claxton et al., 2019) [2] = 0.796) than for all ASCs combined (R (Claxton et al., 2019) [2] = 0.645). Regression analysis also showed a stronger association between month and volume for orthopedic ASCs (R (Claxton et al., 2019) [2] = 0.488-0.805) than for all ASCs combined (R (Claxton et al., 2019) [2] = 0.115-0.493). CONCLUSION: This study is first to identify regular and predictable yearly and monthly increases in orthopedic ASCs surgical volume. The study also identifies yearly increases in surgical volume for all ASCs. The combination of increasing yearly demand for orthopedic surgery and growing association between month and volume leads to an unnecessary year-end rush. The study aims to inform future policy decisions as well as help ASCs better manage resources throughout the year.

A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics.

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01-20 µg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).

Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

A microfluidic device for a pharmacokinetic-pharmacodynamic (PK-PD) model on a chip.

Drug discovery is often impeded by the poor predictability of in vitro assays for drug toxicity. One primary reason for this observation is the inability to reproduce the pharmacokinetics (PK) of drugs in vitro. Mathematical models to predict the pharmacokinetics-pharmacodynamics (PK-PD) of drugs are available, but have several limitations, preventing broader application. A microscale cell culture analog (microCCA) is a microfluidic device based on a PK-PD model, where multiple cell culture chambers are connected with fluidic channels to mimic multi-organ interactions and test drug toxicity in a pharmacokinetic-based manner. One critical issue with microfluidics, including the microCCA, is that specialized techniques are required for assembly and operation, limiting its usability to non-experts. Here, we describe a novel design, with enhanced usability while allowing hydrogel-cell cultures of multiple types. Gravity-induced flow enables pumpless operation and prevents bubble formation. Three cell lines representing the liver, tumor and marrow were cultured in the three-chamber microCCA to test the toxicity of an anticancer drug, 5-fluorouracil. The result was analyzed with a PK-PD model of the device, and compared with the result in static conditions. Each cell type exhibited differential responses to 5-FU, and the responses in the microfluidic environment were different from those in static environment. Combination of a mathematical modeling approach (PK-PD modeling) and an in vitro experimental approach (microCCA) provides a novel platform with improved predictability for testing drug toxicity and can help researchers gain a better insight into the drug's mechanism of action.