Microfluidic Cell Culture Model Research Articles

Prostaglandin E2 depolarises sensory axons in vitro in an ANO1 and Nav1.8 dependent manner

Prostaglandin E2 (PGE2) is a major contributor to inflammatory pain hyperalgesia, however, the extent to which it modulates the activity of nociceptive axons is incompletely understood. We developed and characterized a microfluidic cell culture model to investigate sensitisation of the axons of dorsal root ganglia neurons. We show that application of PGE2 to fluidically isolated axons leads to sensitisation of their responses to depolarising stimuli. Interestingly the application of PGE2 to the DRG axons elicited a direct and persistent spiking activity propagated to the soma. Both the persistent activity and the membrane depolarisation in the axons are abolished by the EP4 receptor inhibitor and a blocker of cAMP synthesis. Further investigated into the mechanisms of the spiking activity showed that the PGE2 evoked depolarisation was inhibited by Nav1.8 sodium channel blockers but was refractory to the application of TTX or zatebradine. Interestingly, the depolarisation of axons was blocked by blocking ANO1 channels with T16Ainh-A01. We further show that PGE2-elicited axonal responses are altered by the changes in chloride gradient within the axons following treatment with bumetanide a Na-K-2Cl cotransporter NKCC1 inhibitor, but not by VU01240551 an inhibitor of potassium-chloride transporter KCC2. Our data demonstrate a novel role for PGE2/EP4/cAMP pathway which culminates in a sustained depolarisation of sensory axons mediated by a chloride current through ANO1 channels. Therefore, using a microfluidic culture model, we provide evidence for a potential dual function of PGE2 in inflammatory pain: it sensitises depolarisation-evoked responses in nociceptive axons and directly triggers action potentials by activating ANO1 and Nav1.8 channels.

Abstract A048: Development and validation of a microfluidic cell culture model for examining cerebral microvascular barrier responses to the tumor microenvironment

Abstract Introduction: The blood-brain barrier (BBB) is composed of specialized endothelial cells that limit the passage of solutes from the blood to the brain. Microfluidic technology that allows formation of 3D tubular microvessels has been applied to in vitro BBB models, and having advantages of fluid flow, cell-cell contact, provide a more robust and predictive model for assessing vascular permeability. Use of the microfluidic model to examine the effects of the tumor microenvironment on brain endothelial cell permeability would provide mechanistic fundamental knowledge that could be used to more effectively deliver therapeutic agents to the treatment site. Methods: Human brain microvessel endothelial cells (hCMEC/d3) were seeded (10,000 cells/µL) into the vascular channel and following vascular capillary tube formation, various human brain tumor cell lines (SF8628, U87, and U251) were seeded in the adjacent brain channel of the Mimeta 3-channel microfluidic unit. Tumor-induced alterations in barrier function were examined over a 72-hour period using paracellular (fluorescein-labeled detran-FDX70kD; IRdye 800; sodium fluorescein - NaF) and transcellularly transported dyes (rhodamine 800-R800). Tumor induced changes in permeability of various marker molecules were correlated to changes in the expression of selected BBB specific genes and the tumor secretome. Results: The microfluidic BBB culture model showed reduced permeability to all the permeability markers examined compared to TranswellTM inserts with values similar to those previously reported in vivo. Compared to monoculture, no significant changes in permeability were observed with U251 co-culture model. However, SF8628 enhanced barrier properties, while U87 increased paracellular leakiness. Examination of the barrier enhancing microenvironment observed with the SF8628 co-culture model indicated a Sonic Hedgehog dependent process that stimulates the release of Angiopoietin 1 (Ang1) and Platelet derived growth factor (PDGF-BB) release from the SF8628 tumor cells as the likely factors driving the permeability effects observed. With regards to the increased permeability observed with the U87 co-culture, increased cytokines (ie. IL-6 and IL-8) and altered fatty acid secretion appear to play a significant role in the reduced barrier properties of brain endothelial cells. Conclusion: The brain tumor microenvironment can have either barrier enhancing (SF8628 co-culture) or barrier reducing (U87-co culture) effects based on tumor cell induced changes in secreted factors. The dynamic microfluidic blood-brain barrier - brain tumor co-culture model provides a robust system with which to mechanistically identify tumor-driven changes in brain endothelial cell permeability. Citation Format: Stacey Line, Magimairajan Issai Vanan, Donald W. Miller. Development and validation of a microfluidic cell culture model for examining cerebral microvascular barrier responses to the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A048.

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

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

Transient fluid flow improves photoimmunoconjugate delivery and photoimmunotherapy efficacy

Circulating drugs in the peritoneal cavity is an effective strategy for advanced ovarian cancer treatment. Photoimmunotherapy, an emerging modality with potential for the treatment of ovarian cancer, involves near-infrared light activation of antibody-photosensitizer conjugates (photoimmunoconjugates) to generate cytotoxic reactive oxygen species. Here, a microfluidic cell culture model is used to study how fluid flow-induced shear stress affects photoimmunoconjugate delivery to ovarian cancer cells. Photoimmunoconjugates are composed of the antibody, cetuximab, conjugated to the photosensitizer, and benzoporphyrin derivative. Longitudinal tracking of photoimmunoconjugate treatment under flow conditions reveals enhancements in subcellular photosensitizer accumulation. Compared to static conditions, fluid flow-induced shear stress at 0.5 and 1 dyn/cm2 doubled the cellular delivery of photoimmunoconjugates. Fluid flow-mediated treatment with three different photosensitizer formulations (benzoporphyrin derivative, photoimmunoconjugates, and photoimmunoconjugate-coated liposomes) led to enhanced phototoxicity compared to static conditions. This study confirms the fundamental role of fluid flow-induced shear stress in the anti-cancer effects of photoimmunotherapy.

Local extensional flows promote long-range fiber alignment in 3D collagen hydrogels

Randomly oriented type I collagen (COL1) fibers in the extracellular matrix are reorganized by biophysical forces into aligned domains extending several millimeters and with varying degrees of fiber alignment. These aligned fibers can transmit traction forces, guide tumor cell migration, facilitate angiogenesis, and influence tissue morphogenesis. To create aligned COL1 domains in microfluidic cell culture models, shear flows have been used to align thin COL1 matrices (<50 µm in height) in a microchannel. However, there has been limited investigation into the role of shear flows in aligning 3D hydrogels (>130 µm). Here, we show that pure shear flows do not induce fiber alignment in 3D atelo COL1 hydrogels, but the simple addition of local extensional flow promotes alignment that is maintained across several millimeters, with a degree of alignment directly related to the extensional strain rate. We further advance experimental capabilities by addressing the practical challenge of accessing a 3D hydrogel formed within a microchannel by introducing a magnetically coupled modular platform that can be released to expose the microengineered hydrogel. We demonstrate the platform’s capability to pattern cells and fabricate multi-layered COL1 matrices using layer-by-layer fabrication and specialized modules. Our approach provides an easy-to-use fabrication method to achieve advanced hydrogel microengineering capabilities that combine fiber alignment with biofabrication capabilities.

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.

Laparoscopic Peritoneal Wash Cytology-Derived Primary Human Mesothelial Cells for In Vitro Cell Culture and Simulation of Human Peritoneum.

Peritoneal mucosa of mesothelial cells line the abdominal cavity, surround intestinal organs and the female reproductive organs and are responsible for immunological integrity, organ functionality and regeneration. Peritoneal diseases range from inflammation, adhesions, endometriosis, and cancer. Efficient technologies to isolate and cultivate healthy patient-derived mesothelial cells with maximal purity enable the generation of capable 2D and 3D as well as in vivo-like microfluidic cell culture models to investigate pathomechanisms and treatment strategies. Here, we describe a new and easily reproducible technique for the isolation and culture of primary human mesothelial cells from laparoscopic peritoneal wash cytology. We established a protocol containing multiple washing and centrifugation steps, followed by cell culture at the highest purity and over multiple passages. Isolated peritoneal mesothelial cells were characterized in detail, utilizing brightfield and immunofluorescence microscopy, flow cytometry as well as Raman microspectroscopy and multivariate data analysis. Thereby, cytokeratin expression enabled specific discrimination from primary peritoneal human fibroblasts. Raman microspectroscopy and imaging were used to study morphology and biochemical properties of primary mesothelial cell culture compared to cryo-fixed and cryo-sectioned peritoneal tissue.

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.

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.

In vitro “skin patch tests” for the detection of sensitization, allergy and relative potency assessment

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.

Computational analysis of integrated biosensing and shear flow in a microfluidic vascular model

Fluid flow and flow-induced shear stress are critical components of the vascular microenvironment commonly studied using microfluidic cell culture models. Microfluidic vascular models mimicking the physiological microenvironment also offer great potential for incorporating on-chip biomolecular detection. In spite of this potential, however, there are few examples of such functionality. Detection of biomolecules released by cells under flow-induced shear stress is a significant challenge due to severe sample dilution caused by the fluid flow used to generate the shear stress, frequently to the extent where the analyte is no longer detectable. In this work, we developed a computational model of a vascular microfluidic cell culture model that integrates physiological shear flow and on-chip monitoring of cell-secreted factors. Applicable to multilayer device configurations, the computational model was applied to a bilayer configuration, which has been used in numerous cell culture applications including vascular models. Guidelines were established that allow cells to be subjected to a wide range of physiological shear stress while ensuring optimal rapid transport of analyte to the biosensor surface and minimized biosensor response times. These guidelines therefore enable the development of microfluidic vascular models that integrate cell-secreted factor detection while addressing flow constraints imposed by physiological shear stress. Ultimately, this work will result in the addition of valuable functionality to microfluidic cell culture models that further fulfill their potential as labs-on-chips.

Microfluidic on-chip biomimicry for 3D cell culture: a fit-for-purpose investigation from the end user standpoint.

A plethora of 3D and microfluidics-based culture models have been demonstrated in the recent years with the ultimate aim to facilitate predictive in vitro models for pharmaceutical development. This article summarizes to date the progress in the microfluidics-based tissue culture models, including organ-on-a-chip and vasculature-on-a-chip. Specific focus is placed on addressing the question of what kinds of 3D culture and system complexities are deemed desirable by the biological and biomedical community. This question is addressed through analysis of a research survey to evaluate the potential use of microfluidic cell culture models among the end users. Our results showed a willingness to adopt 3D culture technology among biomedical researchers, although a significant gap still exists between the desired systems and existing 3D culture options. With these results, key challenges and future directions are highlighted.

Nanomaterial-based in vitro analytical system for diagnosis and therapy in microfluidic device

Nanomaterials have several advantages in detecting several biomaterials and in enhancing signals and their inherent effects. Thus, they have been widely applied in the biomedical fields such as biosensor and cancer therapeutics. Recently, the development of microfluidic technology has led to superior biological analysis systems to detect biomarkers related to diseases or serve as in vitro drug screening platform. In a microfluidic device, samples could be analyzed more accurately, rapidly and simply. In addition, it is possible to culture the cells in these microfluidic devices, therefore making the in situ analysis of secretomes easy. Nanomaterials can be easily applied in the microfluidic channels as capturing and signaling materials in order to improve the sensing or therapeutic property. In particular, nanomaterial integrated microfluidic cell culture model can replace in vivo disease models for nanotherapeutics screening. In this review, nanomaterial-based sensing systems, which include diverse organic and inorganic nanoparticles, are introduced with specific examples, including microfluidics integrated systems. Moreover, microfluidics derived nanomaterial analytic systems as in vitro 2 or 3-dimensional (2D or 3D) cell culture platform will be presented. We also highlight the future perspectives of the microfluidic-driven system as highly sensitive total analysis system with functional nanoparticles.

Pharmacokinetic profile that reduces nephrotoxicity of gentamicin in a perfused kidney-on-a-chip

Nephrotoxicity is often underestimated because renal clearance in animals is higher compared to in humans. This paper aims to illustrate the potential to fill in such pharmacokinetic gaps between animals and humans using a microfluidic kidney model. As an initial demonstration, we compare nephrotoxicity of a drug, administered at the same total dosage, but using different pharmacokinetic regimens. Kidney epithelial cell, cultured under physiological shear stress conditions, are exposed to gentamicin using regimens that mimic the pharmacokinetics of bolus injection or continuous infusion in humans. The perfusion culture utilized is important both for controlling drug exposure and for providing cells with physiological shear stress (1.0 dyn cm−2). Compared to static cultures, perfusion culture improves epithelial barrier function. We tested two drug treatment regimens that give the same gentamycin dose over a 24 h period. In one regimen, we mimicked drug clearance profiles for human bolus injection by starting cell exposure at 19.2 mM of gentamicin and reducing the dosage level by half every 2 h over a 24 h period. In the other regimen, we continuously infused gentamicin (3 mM for 24 h). Although junctional protein immunoreactivity was decreased with both regimens, ZO-1 and occludin fluorescence decreased less with the bolus injection mimicking regimen. The bolus injection mimicking regimen also led to less cytotoxicity and allowed the epithelium to maintain low permeability, while continuous infusion led to an increase in cytotoxicity and permeability. These data show that gentamicin disrupts cell–cell junctions, increases membrane permeability, and decreases cell viability particularly with prolonged low-level exposure. Importantly a bolus injection mimicking regimen alleviates much of the nephrotoxicity compared to the continuous infused regimen. In addition to potential relevance to clinical gentamicin administration regimens, the results are important in demonstrating the general potential of using microfluidic cell culture models for pharmacokinetics and toxicity studies.

Microfluidic cell culture model of the ocular fundus

Event Abstract Back to Event Microfluidic cell culture model of the ocular fundus Li Jiun Chen1, Shunichi Tsunajima1, Matsuhiko Nishizawa1, Nobuhiro Nagai2, Toshiaki Abe2 and Hirokazu Kaji1 1 Tohoku University, Bioengineering and Robotics, Japan 2 Tohoku University, Clinical Cell Therapy, United Centers for Advanced Research and Translational Medicine, Japan Introduction: Age-related macular disease (AMD) is a common eye condition and a leading cause of vision loss among people age 50 and above[1]. Simple tasks such as driving or reading become impossible without a functional central vision, which is impaired as the result of damages to the macula. The causes leading to AMD includes, but not limited to, family history, and changes in the cellular microenvironments. Wet form AMD is responsible for 90% of the blindness out of all AMD patients[1]; it is marked by the development of subretinal choroidal neovascularization and degeneration of retinal pigmental epithelial (RPE) cells[2]. Although there are many studies aiming at using microdevices to reproduce organ physiologies, knowledge of on-chip models of the eye is limited. Here, microfabrication technology is used to reconstruct ocular fundus tissues (OF) in vitro and to simulate neovascularization in the microfluidic devices. Materials and Methods: The microfluidic device was made up of 2 layers, on each a channel (width 500 µm; height 150 µm) was embedded. A porous membrane (thickness 6.5 µm, hole diameter 10 µm) was sandwiched between the layers. Microchannels and membranes were made of poly(dimethylsiloxane) (PDMS). Retinal pigment epithelial (RPE) cells were introduced into the upper channel of the device and cultured until they were confluent. Thereafter, human umbilical vein endothelial cells (HUVECs) were loaded into the lower channel. The device was turned upside down to allow cells to adhere onto the lower side of the membrane. RPE cells and HUVEC were dyed respectively with Cell TrackerTM green and red and cell growth was recorded under Real-Time Cultured Cell Monitoring System (Fig. 1,2). Before the aforementioned co-culture model, RPE were cultured alone on the upper channel and the VEGF concentration was collected and measured by ELIZA after replacing DME medium High Glucose (20 mM) with DME medium Low Glucose (3 mM). HUVECs were loaded into the lower channel and cultured alone. The upper channel was perfused with 50 ng/ml of VEGF-A165 and the number of HUVECs migrating to the upper channel was analyzed using the same monitoring system. Results and Discussion: Results show that RPE cells responded to the lowered glucose microenvironment by increasing VEGF secretion (58.8 % compared to the control). In addition, RPE cells exhibited a polar secretion of VEGF which is consistent to the cell physiology. The increased number of HUVEC migrating through the porous membrane in response to VEGF gradient signifies neovascularization. Comparing to the control, there was a 31.3 % increase of cell migration (Fig. 3). Conclusions: The physiology of RPE cells and HUVECs is reproduced in the microdevice. The preliminary results suggest that the alteration of the microenvironment induces responses of RPE cells and the significance of VEGF in neovascularization. In the near future, we anticipate studying responses of the co-culture model to hypoxia to characterize the AMD pathology. Figure 1. Schematic representation of the placement of cells. Figure 2. RPE cells were stained and imaged by fluorescent microscopy. Figure 3. Number of cells migrated through the porous membrane were counted and compared against the control.