Microfluidic Cell Culture Research Articles

Fabrication of Microfluidic Cell Culture Platform for Real-time Monitoring of Lidocaine Concentration

We describe a microfluidic cell culture platform for real-time measurement of drug concentrations during cell cultivation. This is the first attempt to quantify drug concentrations during cell cultivation in real-time combining ultraviolet–visible (UV–Vis) spectroscopy with a microfluidic cell culture system in a single microfluidic chip. The microfluidic chip has two specific micro-chambers, one for spectroscopic measurements and the other for cell culture. NIH/3T3 fibroblast cells were seeded and cultured in the microfluidic chip, and then the local anesthetic lidocaine was applied and its concentration was monitored in real time. The test results showed that lidocaine negatively affected the cell growth. The lidocaine concentration was decreased due to its intake by cells for the first 3 h, after which an equilibrium concentration was reached. This is a simple but innovative method that can observe and analyze the correlation between cells and drugs in real time. The microfluidic system presented here can be a useful tool in various fields such as in cytotoxicity and drug screening studies.

Real-time physiological sensor-based liver-on-chip device for monitoring drug toxicity

Organ-on-chip models, known as microphysiological systems, are created to mimic the anatomy and physiology of a human organ at the micro-level. Besides being pivotal in the reverse engineering of human organs and pathogenesis studies, they serve as an alternative to animal testing and the development of pharmaceutics. Monitoring the extracellular stromal environment is the basis for gaining in-depth knowledge of the pathophysiology of cell culture. Hence, it is extensively employed as an essential tool in the fields of organ-on-chip and in vitro toxicology. In this study, we explore the vitality of a microfluidic system for the automated, online detection of drug-induced physical changes in cellular viability by continual monitoring of a microfluidic 2D monolayer cell culture. Trans-epithelial electrical resistance (TEER) values and pH changes of the immortal HepG2 cell line were measured continuously using microfluidic-based electrical and photoelectric sensors. A chip-embedded transparent, flat, non-toxic sensor and in-house 3D manufactured portable digital microscope supersedes the conventional manual, expensive confocal microscopic assays, and off-line operated isolated sensor systems. The cytotoxicity was induced by various concentrations of doxorubicin, epirubicin and lapatinib, and the acute metabolic and physical response of cells was examined by detecting the variations in TEER, pH and other biological markers. Thus, our liver-on-chip device provides real-time online data on drug-induced liver injury in vitro.

Fabrication of a 3D microfluidic cell culture device for bone marrow-on-a-chip

Abstract Conventional biomedical research models are confined to static cell culture models and animal testing, both of them representing suboptimal preclinical models. Organs-on-chips (OOCs) are novel 3D microfluidic cell culture devices lined with living cells that allow for faithful mimicry of the physiology and function of a vital human organ unit. Bone marrow-on-a-chip (BMoC) systems are currently limited to in vitro maintenance of the hematopoietic potential, supported with 3D scaffold matrices. Herein, we introduce a purely in vitro and scaffold-free bone marrow-on-a-chip device, intended for both the generation and sustainment of the perivascular hematopoietic niche, to serve as a study platform for the chronic autoimmune disease of systemic lupus erythematosus (SLE). The device consists of three layers of poly(dimethylsiloxane) (PDMS), including two cylindrical microchambers separated by an intervening porous membrane, altogether bonded to a glass slide. In this communication, the fabrication steps for a reliable and reproducible BMoC device are presented, from its design to its final assembly. As a first step towards the full recapitulation of the perivascular niche on-chip, the bone marrow stromal niche was replicated inside sealed type I collagen-coated microchambers hosting the in vitro 3D microfluidic culture of mesenchymal stem cells (MSCs) for 8 days. Culture of the MSCs revealed significant cell proliferation and progressive formation of 3D stromal tissue, a promising outcome for the subsequent 3D stromal matrix growth on the bone marrow-on-a-chip platform.

Metabolic profiling of material-limited cell samples by dimethylaminophenacyl bromide derivatization with UPLC-MS/MS analysis

The ability to dissect the intracellular metabolome is vital in the study of diverse biological systems and models. However, limited cell availability is a challenge in metabolic profiling due to the low concentrations affecting the sensitivity. This is further exacerbated by modern technologies such as 3D microfluidic cell culture devices that provide a physiologically realistic environment, compared to traditional techniques such as cell culture in 2D well-plates. Attempts to address sensitivity issues have been made via advances in microscale separation such as CE and micro/nano-LC coupled to mass spectrometers with low-diameter ionization emitter sources. An alternative approach is sample derivatization, which improves the chromatographic separation, enhances the MS ionization, and promotes favourable fragmentation in terms of sensitivity and specificity. Although chemical derivatization is widely used for various applications, few derivatization methods allow sensitive analysis below 1 × 104 cells. Here, we conduct RPLC-MS/MS analysis of HepG2 cells ranging from 250 cells to 1 × 105 cells, after fast and accessible derivatization by dimethylaminophenacyl bromide (DmPABr), which labels the primary amine, secondary amine, thiol and carboxyl submetabolome, and also utilizes the isotope-coded derivatization (ICD). The analysis of 1 × 104 HepG2 cells accomplished quantification of 37 metabolites within 7-minute elution, and included amino acids, N-acetylated amino acids, acylcarntines, fatty acids and TCA cycle metabolites. The metabolic coverage includes commonly studied metabolites involved in the central carbon and energy-related metabolism, showing applicability in various applications and fields. The limit of detection of the method was below 20 nM for most amino acids, and sub 5 nM for the majority of N-acetylated amino acids and acylcarnitines. Good linearity was recorded for derivatized standards in a wide biological range representing expected metabolite levels in 2–10,000 cells. Intraday variability in 5 × 103 HepG2 cells was below 20% RSD for concentrations measured of all but two metabolites. The method sensitivity at the highest dilution of cell extract, 250 HepG2 cells, enabled the quantification of twelve metabolites and the detection of three additional metabolites below LLOQ. Where possible, performance parameters were compared to published methodologies that measure cell extract samples. The presented work shows a proof of concept for harnessing a derivatization method for sensitive analysis of material-limited biological samples. It offers an attractive tool with further potential for enhanced performance when coupled to low-material suitable technologies such as CE-MS and micro/nano LC-MS.

A modular brain-on-a-chip for modelling epileptic seizures with functionally connected human neuronal networks

Epilepsies are a group of neurological disorders characterised by recurrent epileptic seizures. Seizures, defined as abnormal transient discharges of neuronal activity, can affect the entire brain circuitry or remain more focal in the specific brain regions and neuronal networks. Human pluripotent stem cell (hPSC)-derived neurons are a promising option for modelling epilepsies, but as such, they do not model groups of connected neuronal networks or focal seizures. Our solution is a Modular Platform for Epilepsy Modelling In Vitro (MEMO), a lab-on-chip device, in which three hPSC-derived networks are separated by a novel microfluidic cell culture device that allows controlled network-to-network axonal connections through microtunnels. In this study, we show that the neuronal networks formed a functional circuitry that was successfully cultured in MEMO for up to 98 days. The spontaneous neuronal network activities were monitored with an integrated custom-made microelectrode array (MEA). The networks developed spontaneous burst activity that was synchronous both within and between the axonally connected networks, i.e. mimicking both local and circuitry functionality of the brain. A convulsant, kainic acid, increased bursts only in the specifically treated networks. The activity reduction by an anticonvulsant, phenytoin, was also localised to treated networks. Therefore, modelling focal seizures in human neuronal networks is now possible with the developed chip.

Microfluidic Device Setting by Coculturing Endothelial Cells and Mesenchymal Stem Cells.

The construction of vascular networks is essential for developing functional organ/tissue constructs in terms of oxygen and nutrient supply. Although recent advances in microfluidic techniques have allowed for the construction of microvascular networks using microfluidic devices, their structures cannot be maintained for extended periods of time due to a lack of perivascular cells. To construct long-lasting microvascular networks, it is important that perivascular cells are present to provide structural support to vessels, because in vivo microvessels are covered by perivascular cells and stabilized. Here, we describe a microfluidic cell culture platform for the construction of microvascular networks with supportive perivascular cells. Our results showed that microvascular networks covered by pericyte-like perivascular cells formed in a microfluidic device and their structures were maintained for at least 3weeks in vitro.

Rapid Fabrication of Membrane-Integrated Thermoplastic Elastomer Microfluidic Devices.

Leveraging the advantageous material properties of recently developed soft thermoplastic elastomer materials, this work presents the facile and rapid fabrication of composite membrane-integrated microfluidic devices consisting of FlexdymTM polymer and commercially available porous polycarbonate membranes. The three-layer devices can be fabricated in under 2.5 h, consisting of a 2-min hot embossing cycle, conformal contact between device layers and a low-temperature baking step. The strength of the FlexdymTM-polycarbonate seal was characterized using a specialized microfluidic delamination device and an automated pressure controller configuration, offering a standardized and high-throughput method of microfluidic burst testing. Given a minimum bonding distance of 200 μm, the materials showed bonding that reliably withstood pressures of 500 mbar and above, which is sufficient for most microfluidic cell culture applications. Bonding was also stable when subjected to long term pressurization (10 h) and repeated use (10,000 pressure cycles). Cell culture trials confirmed good cell adhesion and sustained culture of human dermal fibroblasts on a polycarbonate membrane inside the device channels over the course of one week. In comparison to existing porous membrane-based microfluidic platforms of this configuration, most often made of polydimethylsiloxane (PDMS), these devices offer a streamlined fabrication methodology with materials having favourable properties for cell culture applications and the potential for implementation in barrier model organ-on-chips.

Quantitative Image-Based Cell Viability (QuantICV) Assay for Microfluidic 3D Tissue Culture Applications.

Microfluidic 3D tissue culture systems are attractive for in vitro drug testing applications due to the ability of these platforms to generate 3D tissue models and perform drug testing at a very small scale. However, the minute cell number and liquid volume impose significant technical challenges to perform quantitative cell viability measurements using conventional colorimetric or fluorometric assays, such as MTS or Alamar Blue. Similarly, live-dead staining approaches often utilize metabolic dyes that typically label the cytoplasm of live cells, which makes it difficult to segment and count individual cells in compact 3D tissue cultures. In this paper, we present a quantitative image-based cell viability (QuantICV) assay technique that circumvents current challenges of performing the quantitative cell viability assay in microfluidic 3D tissue cultures. A pair of cell-impermeant nuclear dyes (EthD-1 and DAPI) were used to sequentially label the nuclei of necrotic and total cell populations, respectively. Confocal microscopy and image processing algorithms were employed to visualize and quantify the cell nuclei in the 3D tissue volume. The QuantICV assay was validated and showed good concordance with the conventional bulk MTS assay in static 2D and 3D tumor cell cultures. Finally, the QuantICV assay was employed as an on-chip readout to determine the differential dose responses of parental and metastatic 3D oral squamous cell carcinoma (OSCC) to Gefitinib in a microfluidic 3D culture device. This proposed technique can be useful in microfluidic cell cultures as well as in a situation where conventional cell viability assays are not available.

Single-cell analysis of autoreactive B cells in patients with systemic sclerosis revealed that responses to therapy are determined by effector and regulatory B cell balance

Abstract Objective Systemic sclerosis (SSc) is a connective tissue disease with poor prognosis. Although the standard treatment for skin and lung fibrosis is cyclophosphamide (CYC), there are not a few non-responders to the treatment. Recent studies showed that autoreactive B cells play a critical role in SSc. We hypothesized that these B cells determine the effectiveness of CYC treatment. We focused on cytokine profiles of single autoreactive B cells in patients with SSc. Methods We simulated the interaction of B cells and endothelial cells in microfluidic cell culture systems. We cultured endothelial cells in a microchannel. Then, we loaded B cells of patients with SSc. We collected single B cells which adhere to endothelial cells by trypsin treatment. We stimulated these B cells, with PMA and ionomycin, and measured the levels of produced interleukin (IL)-10 and IL-6 to detect the percentages of regulatory and effector B cells, by our original micro fluidic-ELISA system. Results In SSc patients, the number of B cells adhering to endothelial cells significantly increased compared with healthy controls. Most of these B cells produced anti-endothelial cells antibodies. The frequencies of IL-10-producing regulatory B cells after the CYC treatment increased in responders and decresed in non-responders. Those of IL-6-producing effector B cells after the treatment decreased in responders and increased in non-responders. Conclusions The effectiveness of CYC treatment to SSc patients is associated with cytokine profiles of B cells, which interact with endothelial cells. Checking the profiles of these B cells, after the first few months on from initiation of CYC therapy, may help to decide whether or not we should continue the treatment.

Polylactic is a Sustainable, Low Absorption, Low Autofluorescence Alternative to Other Plastics for Microfluidic and Organ-on-Chip Applications.

Organ-on-chip (OOC) devices are miniaturized devices replacing animal models in drug discovery and toxicology studies. The majority of OOC devices are made from polydimethylsiloxane (PDMS), an elastomer widely used in microfluidic prototyping, but posing a number of challenges to experimentalists, including leaching of uncured oligomers and uncontrolled absorption of small compounds. Here we assess the suitability of polylactic acid (PLA) as a replacement material to PDMS for microfluidic cell culture and OOC applications. We changed the wettability of PLA substrates and demonstrated the functionalization method to be stable over a time period of at least 9 months. We successfully cultured human cells on PLA substrates and devices, without coating. We demonstrated that PLA does not absorb small molecules, is transparent (92% transparency), and has low autofluorescence. As a proof of concept of its manufacturability, biocompatibility, and transparency, we performed a cell tracking experiment of prostate cancer cells in a PLA device for advanced cell culture.

A Microfluidic Perfusion Platform for In Vitro Analysis of Drug Pharmacokinetic-Pharmacodynamic (PK-PD) Relationships.

Static in vitro cell culture studies cannot capture the dynamic concentration profiles of drugs, nutrients, and other factors that cells experience in physiological systems. This limits the confidence in the translational relevance of in vitro experiments and increases the reliance on empirical testing of exposure-response relationships and dose optimization in animal models during preclinical drug development, introducing additional challenges owing to species-specific differences in drug pharmacokinetics (PK) and pharmacodynamics (PD). Here, we describe the development of a microfluidic cell culture device that enables perfusion of cells under 2D or 3D culture conditions with temporally programmable concentration profiles. Proof-of-concept studies using doxorubicin and gemcitabine demonstrated the ability of the microfluidic PK-PD device to examine dose- and time-dependent effects of doxorubicin as well as schedule-dependent effects of doxorubicin and gemcitabine combination therapy on cell viability using both step-wise drug concentration profiles and species-specific (i.e., mouse, human) drug PK profiles. The results demonstrate the importance of including physiologically relevant dynamic drug exposure profiles during in vitro drug testing to more accurately mimic in vivo drug effects, thereby improving translatability across nonclinical studies and reducing the reliance on animal models during drug development.

A Non-Cytotoxic Resin for Micro-Stereolithography for Cell Cultures of HUVECs.

Three-dimensional (3D) printing of microfluidic devices continuously replaces conventional fabrication methods. A versatile tool for achieving microscopic feature sizes and short process times is micro-stereolithography (µSL). However, common resins for µSL lack biocompatibility and are cytotoxic. This work focuses on developing new photo-curable resins as a basis for µSL fabrication of polymer materials and surfaces for cell culture. Different acrylate- and methacrylate-based compositions are screened for material characteristics including wettability, surface roughness, and swelling behavior. For further understanding, the impact of photo-absorber and photo-initiator on the cytotoxicity of 3D-printed substrates is studied. Cell culture experiments with human umbilical vein endothelial cells (HUVECs) in standard polystyrene vessels are compared to 3D-printed parts made from our library of homemade resins. Among these, after optimizing material composition and post-processing, we identify selected mixtures of poly(ethylene glycol) diacrylate (PEGDA) and poly(ethylene glycol) methyl ethyl methacrylate (PEGMEMA) as most suitable to allow for fabricating cell culture platforms that retain both the viability and proliferation of HUVECs. Next, our PEGDA/PEGMEMA resins will be further optimized regarding minimal feature size and cell adhesion to fabricate microscopic (microfluidic) cell culture platforms, e.g., for studying vascularization of HUVECs in vitro.

Targeting tumor-associated macrophage (TAM) mediated inhibition of T-cell migration in prostate cancer using epigenetic modifying agents.

166 Background: Cytotoxic T lymphocytes (CTLs) perform vital anti-tumor functions and are critical to the efficacy of many anticancer therapies. In prostate cancer, the characteristic paucity of activated CTLs within the tumor microenvironment (TME) may be a key factor in disease progression and likely underlies the limited role for immune checkpoint inhibitors (ICIs) in prostate cancer treatment. In this study, we utilized novel microfluidic technologies to evaluate whether TAMs may be driving the exclusion of T cells from the prostate TME and whether the immunosuppressive functions of TAMs could be modified by epigenetic modifying agents. Methods: Primary macrophages and autologous T cells were derived from peripheral blood samples of prostate cancer patients at the University of Wisconsin. Mono-, co-, and tri-culture systems of macrophages, T cells, and 22RV1 cells (androgen-dependent prostate cancer cell line) were cultured in 2D and 3D in microfluidic cell culture platforms. Culture systems were treated with the EZH2 inhibitors (EZH2i) DZNep or EPZ-6438 or left untreated. Macrophages were also treated with M1 (IFN-g) and M2 (IL-4) polarizing cytokines. Systems were analyzed for T cell migration as well as mRNA and protein expression in each cell population. Results: Autologous macrophages inhibited activated T cell migration towards tumor cells in a multi-cellular microscale TME. T cell migration was restored through treatment with EZH2i. Gene expression analysis identified that EZH2i altered macrophage gene expression in the unpolarized and M1/M2 polarized states. In particular, there was increased expression of genes involved in T cell recruitment/chemotaxis, including CXCL10, CXCL11, CXCL12, following EZH2i treatment. Conclusions: We used novel microfluidic technologies to model and analyze multicellular TMEs using primary, patient-derived cells. We demonstrate that TAM-mediated suppression of T cell migration is mediated, in part, through epigenetic pathways, which can be targeted with EZH2i. Treatment with EZH2i, alone or in combination other therapies such as ICIs, may enhance cytotoxic T cell migration and activity in primary prostate cancer.

Hydrogels as artificial matrices for cell seeding in microfluidic devices.

Hydrogel-based artificial scaffolds play a vital role in shifting in vitro models from two-dimensional (2D) cell culture to three-dimensional (3D) cell culture. Microfluidic 3D cell culture systems with a hydrogel matrix encourage biomedical researchers to replace in vivo models with 3D in vitro models with a cellular microenvironment that resembles physiological conditions with greater fidelity. Hydrogels can be designed as an artificial extracellular matrix scaffold for providing spatial orientation and promoting cellular interactions with surroundings. Selecting the appropriate hydrogels and their fabrication techniques are the key to mimic the in vivo mechanical environment. Moreover, combining a microfluidic technique with a hydrogel-based 3D cell culture system can create a complex and controlled microenvironment for the cells by placing small biosamples inside the microchannel. This paper provides an overview of the structural similarities of the hydrogels as an extracellular matrix (ECM), their classification and fabrication techniques as an ECM, and their use in microfluidic 3D cell culture systems. Finally, the paper presents the current challenges and future perspectives of using hydrogel scaffolds in microfluidic 3D cell culture systems.

Chitosan-polydopamine hydrogel complex: a novel green adhesion agent for reversibly bonding thermoplastic microdevice and its application for cell-friendly microfluidic 3D cell culture.

Owing to biocompatible characteristics and supporting cell growth capability, hydrogels have been widely used for scaffold fabrication and surface coating for cell culture. To employ the advantages of hydrogels, in the present study, we introduce a biocompatible chitosan (CS)-polydopamine (pDA) hydrogel complex as a green adhesion agent for the reversible bonding of thermoplastics assisted by UV irradiation. Poly(methyl methacrylate) (PMMA) substrates were bonded due to the covalent bond network formed between the amine groups of either CS or pDA in the hydrogel complex and the aldehyde groups of the oxidized PMMA surface via the Schiff-base reaction during the UV irradiation. Furthermore, the introduced method allowed for reversible bonding, which is highly appropriate for the fabrication of microdevices for cell-related applications. Surface characterizations such as water contact angle measurement, scanning electron microscopy analysis (SEM), atomic force microscopy analysis (AFM), and Fourier-transform infrared microscopy analysis (FTIR) were performed to confirm the successful coating of the hydrogel complex on the PMMA surface. Moreover, the bonding between two PMMAs or PMMA with other thermoplastics was successfully investigated with high bond strengths ranging from 0.4 to 0.7 MPa. The potential for reversible bonding of this method was verified by repeating the bonding/debonding cycle of the bonded PMMAs for three times, which maintained the bond strength at approximately 0.5 MPa. The compatibility of the bonding method in biological applications was examined by culturing mesenchymal stem cells (MSCs) inside a microchannel where multiple uniform-sized MSC spheroids were successfully formed. Then, spheroids were harvested for off-chip experiments enabled by the reversibility of the introduced bonding strategy. The bonding strategy employing a green hydrogel complex as a cell-friendly and eco-friendly adhesion agent could have a high impact on the fabrication of microdevices suitable for advanced organ-on-a-chip studies.

Controlled pharmacokinetic anti-cancer drug concentration profiles lead to growth inhibition of colorectal cancer cells in a microfluidic device.

We present a microfluidic device to expose cancer cells to a dynamic, in vivo-like concentration profile of a drug, and quantify efficacy on-chip. About 30% of cancer patients receive drug therapy. In conventional cell culture experiments drug efficacy is tested under static concentrations, e.g. 1 μM for 48 hours, whereas in vivo, drug concentration follows a pharmacokinetic profile with an initial peak and a decline over time. With the rise of microfluidic cell culture models, including organs-on-chips, there are opportunities to more realistically mimic in vivo-like concentrations. Our microfluidic device contains a cell culture chamber and a drug-dosing channel separated by a transparent membrane, to allow for shear stress-free drug exposure and label-free growth quantification. Dynamic drug concentration profiles in the cell culture chamber were controlled by continuously flowing controlled concentrations of drug in the dosing channel. The control over drug concentrations in the cell culture chambers was validated with fluorescence experiments and numerical simulations. Exposure of HCT116 colorectal cancer cells to static concentrations of the clinically used drug oxaliplatin resulted in a sensible dose-effect curve. Dynamic, in vivo-like drug exposure also led to statistically significant lower growth compared to untreated control. Continuous exposure to the average concentration of the in vivo-like exposure seems more effective than exposure to the peak concentration (Cmax) only. We expect that our microfluidic system will improve efficacy prediction of in vitro models, including organs-on-chips, and may lead to future clinical optimization of drug administration schedules.

Development of microfluidic cell culture system in preparation for human organ on a chip

Development of microfluidic cell culture system in preparation for human organ on a chip

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.

A rapidly prototyped lung-on-a-chip model using 3D-printed molds

Organ-on-a-chip is a microfluidic cell culture model that replicates key organ-specific microarchitecture and pathophysiology in vitro . The current methods to fabricate these devices rely on softlithography, which is usually tedious, laborious, and requires adroit users as well as cleanroom facilities. Recently, the use of 3D-printing technologies for the rapid fabrication of molds for polydimethylsiloxane (PDMS) casting is on the rise. However, most of the 3D-printed materials are unsuitable for PDMS casting. To address this issue, we have improved the existing techniques and introduced a modified protocol for the surface treatment of 3D-printed molds, making them ideal for repeated long-term PDMS casting. Using this protocol, we have fabricated a simple open well lung-on-a-chip model to simulate the in vivo environment of airway at air-liquid interface under dynamic condition. To validate the functionality of the developed chip, Calu-3 cells were cultured in the chip and maintained at an air-liquid interface. The model demonstrated that the cultured cells replicated the 3D culture-specific-morphology, maintained excellent barrier integrity, secreted mucus, and expressed cell surface functional P-glycoprotein; all indicative of a promising in vitro model for permeability assays, toxicological tests, and pulmonary drug delivery studies. To validate the suitability of this lung-on-a-chip in vitro model, the effects of cigarette smoke extract (CSE) on Interleukin-6 (IL-6) and Interleukin-8 (IL-8) release from cultured Calu-3 cells were examined. CSE treated cells showed significantly higher secretion of IL-6 and IL-8 over 24 h compared to the cells treated with both CSE and Budesonide, an anti-inflammatory drug. Moreover, our results illustrated that CSE reduced the expression of E-cadherin as an adherent junctional protein. In conclusion, the proposed protocol demonstrated an easy and low-cost fabrication technique which will allow a biologist with minimal technical skills to rapidly prototype molds for different/versatile organ-on-a-chip models.

Tetrafluoroethylene-Propylene Elastomer for Fabrication of Microfluidic Organs-on-Chips Resistant to Drug Absorption.

Organs-on-chips are microfluidic devices typically fabricated from polydimethylsiloxane (PDMS). Since PDMS has many attractive properties including high optical clarity and compliance, PDMS is very useful for cell culture applications; however, PDMS possesses a significant drawback in that small hydrophobic molecules are strongly absorbed. This drawback hinders widespread use of PDMS-based devices for drug discovery and development. Here, we describe a microfluidic cell culture system made of a tetrafluoroethylene-propylene (FEPM) elastomer. We demonstrated that FEPM does not absorb small hydrophobic compounds including rhodamine B and three types of drugs, nifedipine, coumarin, and Bay K8644, whereas PDMS absorbs them strongly. The device consists of two FEPM layers of microchannels separated by a thin collagen vitrigel membrane. Since FEPM is flexible and biocompatible, this microfluidic device can be used to culture cells while applying mechanical strain. When human umbilical vein endothelial cells (HUVECs) were subjected to cyclic strain (~10%) for 4 h in this device, HUVECs reoriented and aligned perpendicularly in response to the cyclic stretch. Moreover, we demonstrated that this device can be used to replicate the epithelial–endothelial interface as well as to provide physiological mechanical strain and fluid flow. This method offers a robust platform to produce organs-on-chips for drug discovery and development.