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. 2026 Mar 11;9(4):925–933. doi: 10.1021/acsptsci.5c00663

A Simple and Robust Microfluidic Glomerular Filtration Barrier-on-a-Chip Platform for Investigating Drug-Induced Nephrotoxicity

Yeo Jin Hwang †, Chaewon Jin ‡, Hongsoo Choi §, Kyeong-Min Lee ‡,*, Jin-Young Kim ‡,∥,⊥,*
PMCID: PMC13077489  PMID: 41988366

Abstract

The glomerulus is the filtering unit of the kidney, and the glomerular filtration barrier (GFB) is responsible for filtering waste, retaining plasma protein, and maintaining fluid balance. Drug-induced nephrotoxicity is characterized by dysfunction of the GFB and is a main obstacle in the new therapeutic screening process. This paper presents the simple and robust SLAS (Society of Laboratory Automation and Society) standard format-based microfluidic GFB-on-a-chip (GFBoC). We formed and cultured the GFB by aligning human glomerular mesangial cells (gMCs), podocytes, and glomerular endothelial cells (gECs) on each side of a conventional transwell membrane as the glomerular basement membrane (GBM). This provides facile loading/unloading of the GFB transwell into the microfluidic chip, enabling its cultivation under pump-/tubing-less perfusion flow and additional off-chip bioanalysis. It also allows various and multiple experiments in parallel in a conventional incubator at moderate operation complexity. Glomerular selective permeability of the GFB was characterized by filtration and leakage of the representative macromolecule, albumin, via the GFB, while the drug doxorubicin affected the GFB during cultivation in the GFBoC. This demonstrated that the GFBoC has potential as a simple, robust, and efficient platform for the multiple testing of nephrotoxicity and kidney disease drugs in parallel.

Keywords: microfluidic chip, glomerular filtration barrier (GFB), drug-induced nephrotoxicity, organ-on-a-chip, modular transwell system, tubing/pump-less perfusion system

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Kidneys are crucial for filtering out waste products from the body and maintaining fluid balance. − The glomerulus is the key filtration unit of the kidney, which is comprised of a bundle of capillaries. This blood ultrafiltration occurs at the level of the glomerular filtration barrier (GFB), which is a critical part of the glomerulus; this allows water and small solutes to pass while holding circulating macromolecules (e.g., albumin, large plasma proteins, etc.) in the blood. Such selective transport of the GFB is attributed to the three-dimensional (3D) footprint-like structure of the glomerular epithelial cell (podocyte) and the glomerular endothelial cells (gECs), which are separated by the glomerular basement membrane (GBM). Intercellular crosstalk between these components is important for the effective functioning of the GFB. Damage to the GFB causes severe glomerular dysfunction associated with a loss of selective ultrafiltration capacity, resulting in proteinuria (leakage of macromolecules such as albumin and plasma protein into the urine) by increasing glomerular permeability, which is a common symptom of kidney diseases such as diabetes, hypertension, and progressive renal diseases. , Recent studies have underlined the importance of harmonizing all the GFB components that are vital for its proper functioning. However, the specifics of their association remain unclear. −

Drug-induced nephrotoxicity is inevitable and frequently observed, and remains a major hurdle in pharmacotherapy and drug development , as it limits drug dosages due to side effects. Therefore, it is important to investigate the pathophysiology and mechanism of nephrotoxicity in GFB to support the development of safer drugs and treatments. Such studies have been conducted with mostly two-dimensional cell culture (2DCC) tools and animal models. Advanced three-dimensional cell culture (3DCC) tools have also been recently proposed. However, they have chronic limitations in providing and recapitulating the physiological environment of GFB in vivo to sustain gene expression and function in the kidney. , 2DCC mostly focuses on single types of cells in monolayer and lacks cell-to-cell interactions, tissue-like architecture, and perfusion conditions. Furthermore, animal experiments are expensive, cumbersome, time-consuming, and, importantly, not human cells.

Due to advances in microengineering and microfluidic technologies, the microphysiological system (MPS)known as “organ-on-a-chip” (OoC)has emerged as a promising in vitro model for better mimicking the microphysiological environment compared to conventional methods. − By incorporating microfluidic systems and advanced biomaterials, microfluidic devices recreate the complex microenvironment of tissues and organs, allowing for the study of physiological and pathological processes in well-controlled and in vivo-like experimental conditions. − This offers various advantages. First, it enables the compensation and reduction of animal experimentation, therefore addressing ethical concerns and promoting a more humane approach to research. Second, microfluidic platforms allow for the real-time monitoring of cellular responses and dynamic interactions within the cell microenvironment, which enhances our understanding of organ functions and their response to various stimulations. − Lastly, more precise control over experimental conditions in OoC technology facilitates the development of targeted therapies and drug screening, fostering advancements in personalized medicine. As such, the ability of OoCs to mimic various tissues and organs has been investigated, and studies have been conducted to develop an in vitro GFB model for drug-induced nephrotoxicity and advanced therapeutic strategies for kidney disease research. , In addition, innovative microfabrication techniques and biomimetic materials have been employed to construct 3D microenvironments that mimic the physical and biochemical cues of the native glomerulus. , Achieving physiological relevance regarding barrier permeability, cellular interactions, and dynamic responses to hemodynamic forces represents a key milestone in the maturation of glomerulus-on-a-chip technology. , However, prior research has relied on rather complex internal structures, fabrication processes, and operation systems. Those studies have typically focused on recapitulating an individual organ such as the GFB, but they have mostly not been designed for facile usability, high-throughput screening (HTS), and commercial production. Issues have also arisen because it is often difficult to form the GFB and unload/reload it for further typical analysis and subsequent culturing. Previous studies have also not been particularly compatible with SLAS (Society for Laboratory Automation and Screening) standard-based screening tools (e.g., 24, 48, 96 wells, etc.) and conventional cell culture incubators. However, these features are critical for the commercialization and automation of the developed in vitro system, its integration with existing techniques, and its scale-up to the HTS platform for efficient and reliable drug evaluation. Therefore, it is important to recapitulate the microphysiological environments of the GFB in a device while maintaining its simple and robust use and ensuring its versatility and compatibility with SLAS-based HTS tools.

In this study, we developed a simple, robust microfluidic GFB-on-a-chip (GFBoC) platform based on a 24-well format, which is one of the most widely used SLAS standard formats (Figure ). The GFB was formed in a conventional transwell insert, which allowed it to be easily and repeatedly loaded into and unloaded from the chip. Transwell inserts have a thin, porous membrane at the bottom, allowing the cultivation of different cells on each side. They enable membrane sandwich-based 3D cell coculturing and easily facilitate cellular membrane formation for barrier-related experiments. , The GFB transwells inserted into the microfluidic chip were cultured under media perfusion flowing through the microfluidic channels using a gravity-based pump-/tubing-less perfusion system. The GFBoC platform provided a simple operation and was robust enough to even be used by technicians and laboratories with no microtechnology experience. The simple structural design and rapid prototyping fabrication process of the GFBoC offers a straightforward translation to an injection molding process, which is the representative commercial manufacturing process, with only minor modifications. We also characterized the GFB formation in the chip platform by measuring membrane permeability relative to the cultured cell combination and various concentrations of the anticancer drug doxorubicin. With the presented GFBoC platform, various and multiple experiments can be conducted in parallel in a conventional incubator at moderate operation complexity. The platform also has the potential to realize complex interconnections between the different tissue or organ models within it for more comprehensive drug testing.

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Design for the glomerulus filtration barrier-on-a-chip (GFBoC). The developed chip mimics the structure of cells constituting the glomerulus, the glomerular epithelial cell (podocyte), glomerular mesangial cells (gMCs), and glomerular endothelial cells (gECs) using typical transwell inserts for filtration. The chip features a microfluidic channel structure that allows media to circulate, enabling interactions between inserts in each channel.

Experimental Section

Development of Microfluidic Chip

In this study, the developed microfluidic chip follows conventional 24-well plate specifications, with a spacing of 19.2 mm pitch between wells. It has four channels, each containing four compartments where the transwell inserts are easily plugged in and out. In addition, there are two open media reservoirs on both ends of the microfluidic channel for the facile exchange and sampling of media. The width and height of the microfluidic channels connecting the wells were designed as 3 mm and 100 μm, respectively (Figure a).

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Layout of the GFBoC: (a) Schematic overview of the GFBoC containing four channels in a conventional 24-well plate format; each channel comprises four transwell insert compartments interconnected by a straight perfusion microfluidic channel. Open medium reservoirs are located at both ends of each channel, allowing for media exchange and sampling. (b) A cross-section of the transwell insert compartment and a close-up of the cellular membrane of the inset. The cells are cultured adherently on both sides of the membrane, and media flows through the microfluidic channel, uniting with the cells on the bottom side of the inset. The yellow arrow represents the selective transfer of biomolecules through the membrane. (c) The process of connecting the developed transwell insert to the chip. Glomerular endothelial cells (gECs) are seeded on the lower side, then podocytes and glomerular mesangial cells (gMCs) are seeded on the upper side, followed by incubation (i). Each insert is placed into the compartment and connected to the chip (ii). Finally, the chip is tilted to provide a flowing media environment via the microfluidic channel (iii). (d) The tilting tower system (TTS) is operable inside an incubator. It tilts left and right to generate media flow within the GFBoC. Cell viability (e) and TEER (f) of GFB composing cells during a 7-day culture period. *P < 0.01 and **P < 0.001 vs day 1, # P < 0.01 and ## P < 0.001 vs N/C. N/C, nothing seeded cells. Data are from three independent experiments, and the values represent the mean ± standard error of the mean. Statistical significance between groups was analyzed using Student’s t test, and p-values were reported at the levels of <0.001, < 0.01, and <0.05.

Figure b presents a cross-sectional view of the chip and an enlarged schematic of the interior of the inset and the microfluidic channel. The diameter of the inset compartment is 9 mm and is designed to prevent media leakage around the 9.15 mm outer diameter of the transwell insert that is loaded into the device during the operation of the device. The device incorporates microfluidic channels to connect the loaded inserts, allowing for media perfusion and interaction through flow. The perfusion channel has a height of 100 μm and a width of 3 mm. The device enables the cultivation of different cell types on the upper and lower sides of the membrane in the transwell insert, facilitating substance movement through membrane pores. The microfluidic device was fabricated using the standard soft lithography technique, , as described in Supporting Information 1 and Figure S1.

Cell Culture

The human CiGEnC (gECs), human CIHGM-1 (gMCs), and human CIHP-1 (podocyte) cells were purchased from Ximbio (London, UK). The gECs were cultured in 1× EBMTM-2 basal medium (Lonza, Switzerland) with growth factors as supplied, except vascular endothelial growth factor (VEGF). The podocyte and gMC cells were grown in Roswell Park Memorial Institute 1640 medium (Hyclone, USA) containing 10% fetal bovine serum, insulin 1 mg/mL, transferrin 0.55 mg/mL, 0.67 μg/mL sodium selenite, 100 U/mL penicillin, and 100 μg/mL streptomycin (Welgene, Korea). These cells were then incubated in a humidified incubator at 5% CO2 and 33 °C under sterile conditions.

Loading of GFB Transwell Inserts into the Chip with the Parallelized Perfusion System

The GFBoC platform consists of the GFB transwell inserts, a microfluidic channel chip that mimics biofluidic flow, and a tilting tower system (TTS) to generate the media perfusion and operate multiple GFBoCs in parallel. Figure c illustrates the process of forming the GFB in the transwell inserts and loading them into the GFBoC. For culturing renal cells on both sides of the transwell membrane, 6.5 mm diameter polyester membranes with 0.4 μm pore inserts (Corning, Corning, NY, USA) were coated with 0.1 mg/mL collagen type IV from human placenta (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 2h as a cell adhesion layer. After incubation, the insets were carefully aspirated, dried, and sterilized by ultraviolet (UV) sterilization. The gECs (1.6 × 104 cells/70 μL) were first seeded and incubated overnight on the lower side of the membrane, and then the podocyte and gMC mixtures (8 × 103 cells/100 μL) were seeded and incubated overnight on the upper side of the membrane; both incubations were at 33 °C in a conventional incubator (Figure c­(i)). Prepared inserts were sequentially inserted into the compartments of the chip. Cell detachment due to pressure was avoided by gently rotating the insets clockwise while inserting them into the compartments (ii). After inserting all four inserts in a row into a single microfluidic channel, 600 μL of media was added to one open reservoir at the end of the channel; this then flowed through the microfluidic channel, directly uniting with the gECs on the lower side of the transwell membrane (iii). Figure d shows the TTS for generating gravity-driven flow via the microfluidic channel between two open reservoirs; by tilting the chip forward and backward, the TTS offers a pump- and tubing-less perfusion system. The system is waterproofed to operate inside a normal incubator, while the controller allows for angle, speed, and time adjustments outside the incubator. The detailed experimental setup is described in Supporting Information 2 and Figure S2.

Fluorescent Staining with CellTracker and Confocal Imaging

To distinguish between the human kidney cells, the gMCs, podocytes, and gECs in the GFB transwell insert were stained with fluorescent dyes of three different colorsdeep red, blue-CMAC, and green-CMFDA, respectively (CellTracker Dye, Invitrogen, USA)according to the manufacturer’s instructions. The stained cells were washed with phosphate-buffered saline (PBS, Hyclone, USA) three times and then seeded in the upper or lower side of the transwell inserts and incubated overnight. Fluorescence images were captured with a 100× objective lens of ImageXpress Micro Confocal (Molecular Devices, USA) in the range of 21 μm per 0.5 μm and were detected by MetaXpress software Version 6.5.4.532 (Molecular Devices). To determine cell viability following doxorubicin treatment, fluorescence intensity was analyzed using the ImageJ program (National Institutes of Health, USA).

Measurement of Glomerular Permeability

The permeability of the GFB cultured in the GFBoC was characterized by the albumin passage from the media in the microfluidic channels and reservoirs into the transwell insert through the gMCs, podocytes, and gECs on the membrane. First, human albumin (5 mg/dL, Sigma-Aldrich) was added to the reservoirs and cultured in the GFBoC with the perfusion flow via the microfluidic channel for 2 days. The albumin concentration of the insets and reservoirs was then measured and calculated as the average value of the supernatants of four transwell inserts and two reservoirs, respectively, with interconnected perfusion channels. Glomerular permeability was presented by the albumin concentration ratio of the insets to the reservoirs. The human albumin concentration in the media was quantitatively measured using a human albumin enzyme-linked immunosorbent assay kit (Bethyl Laboratories, Inc., Montgomery, Texas, USA) according to the manufacturer’s instructions.

Drug-Induced Nephrotoxicity and Cell Viability

The indicated dose (0, 0.3, 0.7 μM) of doxorubicin (Sigma-Aldrich) was prepared in the media and cultured in the GFBoC for 2 days. To identify doxorubicin-induced nephrotoxicity on the GFB, the glomerular permeability and cell viability were measured via the albumin concentration ratio and fluorescence of the cells, as described above. Cell viability was determined by an adenosine triphosphate (ATP)-based detection kit (CellTiter-Glo 2.0 cell viability assay kit, Promega, USA), according to the manufacturer’s instructions. The ready-to-use reagent was added both inside (100 μL) and outside (500 μL) of the inset, and the luminescence of these mixtures was measured using the GloMax Navigator and GloMax Navigator program Version 3.1.0 (Promega).

Results and Discussion

Fabrication of the Microfluidic Chip for Formation and Cultivation of the GFB

Figure shows the fabricated 24-well-based microfluidic chip containing four microfluidic perfusion channels. Each channel includes four transwell insert compartments, where typical transwell inserts can be easily plugged in and out. In addition, there are two media open reservoirs at both ends of the chip, which provide direct access to a conventional pipet for the straightforward exchange and sampling of media, and also for gravity-driven flow by tilting. The photographs in Figure a–c depict the chip before (a) and after (b,c) facile coupling with inserts. The multiple channels allow simultaneous experimentation with different conditions in a single chip. Furthermore, multiple chips can be operated in parallel by loading them onto the TTS system (see Supporting Information 2 and Figure S2). Blue dye was introduced into the microfluidic channel and media reservoir, while red dye was added inside the transwell insert (Figure d). This illustrates that the media in the channel and the inset are independent spaces that are separated by the inset’s membrane.

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Photographs of the fabricated glomerular filtration barrier-on-a-chip (GFBoC). Microfluidic channels were filled with various colored dyes for visualization: (a) Top view before, (b) overhead view, (c) isometric view, and (d) side view after a conventional transwell insert connection and an enlarged photograph of the inset. Characterization of the GFB cultured in the chip: (e) 2D and 3D fluorescence images of the GFBoC; the GFBoC consists of podocytes (green), gMCs (blue), and gECs (deep red) (magnification × 100). Cell viability (f) and TEER (g) of GFB composing cells during a 7-day culture period. Data are from three independent experiments, and the values represent the mean ± standard error of the mean. *P < 0.01 and **P < 0.001 vs day 1, # P < 0.01 and ## P < 0.001 vs N/C. N/C, nothing seeded cells. (h) Glomerular permeability of the GFBoC as a function of the consisting cells and culturing conditions; glomerular permeability was expressed as a ratio of human albumin concentration in the media in inserts to reservoirs. # P < 0.05 and ## P < 0.01 vs N/C on static, *P < 0.05 and **P < 0.01 vs N/C on perfusion. N/C, nothing seeded cells; P, podocyte; gMC, glomerular mesangial cells; gEC, glomerular endothelial cells. Data are from three independent experiments, and the values represent the mean ± standard error of the mean. Statistical significance between groups was analyzed using Student’s t test, and p-values were reported at the levels of <0.001, < 0.01, and <0.05.

In the developed GFBoC, cells were cultured with and without the media flow to investigate the effect of the media perfusion on cell cultivation, as described in Supporting Information 3. The results showed that when cultured under perfusion conditions in the chip, 60% more cells proliferated, with a greater number of live cells being observed compared to the static conditions (Figure S3).

Development and Validation of the GFBoC

To develop an in vitro GFBoC, the three cells comprising the glomerulus, gMCs, podocytes, and gECs were cultured stereotypically in transwells, as described in the Experimental Section and Figure .

To identify the culture conditions of kidney cells in the chip for developing the GFB, fluorescent dye-stained kidney cells were seeded on both sides of the transwell membrane, and their morphological change was observed using confocal microscopy. When the transwells forming the GFB were loaded into the compartments of the device and then cultured under microfluidic perfusion conditions for 2 days, the morphology of the cells in the GFBoC was maintained well compared to those grown in a commercial 24-well plate (Figure e, Supporting Information 4, and Figure S4). Deep red gECs were cultured in the bottom layer of the device with the flow; these cells exhibited a more elongated spindle shape compared to commercial 24-well plate culture conditions. Green-CMFDA podocytes and blue-CMAC gMCs cultured in the top surface of the transwell membrane reflected the homogeneity of distribution of both cell types and maintained a shape appropriate for the characteristics of each cell (Figure e). During the long-term culture of GFBoC, cell viability gradually increased from day 1 to day 7 (Figure f). To evaluate barrier integrity of GFBoC during long-term culture, TEER measurements were conducted. TEER values increased by approximately 20 Ω·cm2 on day 1 compared to the N/C (50 Ω·cm2) and subsequently ranged between 67 to 87 Ω·cm2 from day 1 to day 7 (Figure g). These results showed that the membrane sandwich-based GFB model was formed within the transwell insert of the GFBoC and successfully cultured and simulated the glomerulus structure of the human kidney.

The GFBoC was successfully produced by forming the GFB transwell inserts and culturing them in the 24-well-based format microfluidic chip. Since a 24-well is one of the SLAS standard formats, the GFBoC is compatible with conventional bioanalysis tools and automated equipment and allows cell observation under a typical microscope. In addition, the use of commercial transwell inserts simplifies user handling during experiments, enabling easy insertion and removal according to experimental needs without significant damage to cells.

Control of the Perfusion Flow Rate

During the cultivation of GFB, tilting the GFBoC induces a flow between the two open reservoirs at both ends of the microfluidic channel without the need for an external pump and tubing for media perfusion. The flow rate is determined by the height difference of the reservoirs, which generates a hydrostatic pressure. As described in Table , the flow rates in the GFBoC were investigated with five different angles from 4–40° on the TTS. While 600 μL of the media was flushed from the upper to the lower reservoir, the transferred volume of the flowed media was measured over time until there was complete drainage in the top reservoir. The flow rate increased from 15 to 200 μL/min as a function of the tilting angle. In this experiment, a 15 μL/min flow rate was chosen from various tested flow rates to minimize shear stress on the cells while providing sufficient media circulation volume during cultivation. It is not recommended to move the overall media volume by tilting because it can result in bubbles at the media inlets and microfluidic channels, which could disturb the media perfusion when the reservoirs are completely emptied. The TTS provides a simple and robust control of the perfusion flow with multiple GFBoCs in parallel. Furthermore, open reservoirs provide sufficient gas exchange.

1. Flow Rate Measurements According to Tilting Angle.

tilting angle (°) 4 10 20 30 40
flow rate (μL/min) 15 46 117 171 200
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Permeation Evaluation of Culture in the GFBoC

An important function of the glomerulus is to selectively filter molecules and proteins by size and charge, thus maintaining the body’s electrolyte and pH balance and blood homeostasis. Plasma protein, such as albumin, is retained in the bloodstream, while water and small solutes are excreted into the urine. Therefore, we assessed the glomerular permeability of the GFBoC by measuring the passage of albumin from the reservoir and microfluidic channel to the inset across the GFB. When gMCs + podocytes + gECs were cocultured in the chip with the flow, the passage of albumin was reduced, and glomerular permeability was significantly decreased by a maximum of three times, compared to the membrane alone or gECs alone, or podocytes + gMCs cultured in the chip (Figure h, Supporting Information 5, and Figure S5b). However, when the GFB was cultured without perfusion flow in the GFBoC, albumin transport did not significantly occur regardless of the GFB existence and cell combination (Figure h, and Supporting Information 5, and Figure S5a). These results illustrate that the perfusion flow is important to mimic the transport of molecules and proteins through the GFB, and indeed, glomerular permeability was significantly decreased with the GFB composed of gECs + gMCs + podocytes on a chip. This means that the GFBoC platform properly simulates the physiological function of the GFB, which can prevent the plasma proteins from being transported across the gEC layer and retain them in the perfusion flow under the gEC layer. We confirmed that the fabricated GFBoC-like human glomerulus functionally mimics the GFB by preventing albumin leakage.

Evaluation of Drug-Induced Nephrotoxicity and Albuminuria in the GFBoC

Proteinuria is routinely used in clinical practice to assess renal injury through damage to the GFB. , To demonstrate the utility of the GFBoC for screening drug-induced glomerular injury, we exposed and cultured the GFB in the chip platforms to various concentrations of doxorubicin for 2 days. It was observed that the glomerular permeability of the GFBoC was significantly affected and changed under drug-induced glomerular injury conditions when the GFBoC was cultured under perfusion conditions. In the GFBoC with flow, glomerular permeability was dose-dependently increased up to about two times by treatment with doxorubicin. In contrast, albumin passage from the channels and reservoirs to inserts was not displayed, regardless of the doxorubicin concentration in the GFBoC without flow (Figure a, Supporting Information 6, and Figure S6). Figure b,c show the fluorescent images and intensity of each cell component of the GFB as a function of the doxorubicin concentration. In the gECs on the lower side of the GFBoC, the spindle shape was shortened, and dislodged cells were increased by doxorubicin treatment. Similarly, the detached cells were dose-dependently increased by doxorubicin in the podocytes and gMCs on the upper side of the GFBoC. In addition, cell viability in the GFBoC decreased by doxorubicin treatment in a dose-dependent manner, as depicted in Figure d. These findings demonstrate that drug-induced nephrotoxicity in the GFBoC platform simulated the proteinuria of renal injury, in which the doxorubicin induced damage to the GFB and significantly increased albumin leakage from the reservoir to the inset across the GFB. In terms of ATP based viability, typical ATP assays cause bulk lysis of all cells in the transwell, and thus it is difficult to selectively lysate cells and measure ATP levels for individual cell types in our coculture. For this reason, we relied on cell-type–resolved fluorescence readouts (Figure b–c) to present differential viability across the three cell populations. It is important to study glomerular permeability for drug screening, and it is necessary to develop an in vitro GFB simulation model. In this study, the GFBoC restricted the passage of albumin under orchestrated cells, which was validated only under perfusion conditions. Additionally, we constructed a doxorubicin-induced nephrotoxicity model and confirmed that the glomerular permeability increases due to renal injury on the GFBoC. Hence, the GFBoC platform is appropriate for assessing new drug development and nephrotoxicity testing.

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Glomerular permeability of the GFBoC in doxorubicin-induced nephrotoxicity. 0 to 0.7 μM of doxorubicin was treated to both insert and reservoir of the GBoC for 2 days at static or perfusion conditions. (a) Glomerular permeability of the GFBoC using measurement of albumin passage from the reservoir to insert of the GFBoC. Drug responses were characterized because it can affect the differential microenvironments such as drug diffusion and uptake into cells under static versus perfusion. Data presents the glomerular permeability by the ratio of albumin concentration of insert and reservoir. *P < 0.01 vs N/C and **P < 0.05 vs 0 μM of doxorubicin. (b) Fluorescence GFBoC image. Fluorescent-stained podocytes (green), gMCs (blue), and gECs (deep red) in the GFBoC were incubated with 0 to 0.7 μM of doxorubicin for 2 days in the perfusion condition. (c) Fluorescence intensity graph of the GFBoC. Quantification fluorescence data were normalized to the control (=1) and expressed as the fold-in decrease relative to the control. *P < 0.01, **P < 0.001, and # P < 0.05 vs 0 μM of doxorubicin. (d) Cell viability of GFB composing cells using ATP measurement. *P < 0.05 vs 0 μM of doxorubicin. Data in bar graphs were normalized to the control (=1) and expressed as the fold decreased relative to the 0 μM doxorubicin. Data are from three independent experiments, and the values represent the mean ± standard error of the mean. Statistical significance between groups was analyzed using Student’s t test, and p-values were reported at the levels of <0.001, <0.01, and <0.05.

Discussion

For the GFBoC, 15 μL/min, the lowest of the tested flow rates, was selected to minimize shear. The corresponding wall shear stress is ∼0.16 dyn·cm–2 with our chamber geometry. This value is within the subdyne low-shear range commonly targeted in kidney/glomerulus-on-a-chip studies (e.g., ∼0.2 dyn·cm–2 in human proximal-tubule chips; ∼0.3 dyn·cm–2 reported as physiological shear for proximal-tubule epithelium). , As shown in Figure S4, cells cultured under 15 μL·min–1 perfusion exhibited no apparent damage and displayed an elongated morphology, consistent with an appropriate, noninjurious mechanical environment. Notably, the applied shear can be tuned to match a desired physiological target by adjusting the tilting angle and microchannel dimensions.

For more comprehensive characterization of cells cultured in the transwell, additional quantitative analysessuch as measurement of cell shape index, circularity, or aspect ratiocould provide further insights into morphological changes under various culturing conditions in the GFBoC.

Compared to recently published glomerulus-on-chip models, , our GFBoC platform offers several unique advantages: compatibility with SLAS-standard 24-well plates, the use of loadable transwell inserts for easy handling and parallelization, and a tubing- and pump-less perfusion system. These features provide operational simplicity, scalability (e.g., 96/384 well plate formats), and make the platform particularly robust and suitable for high-throughput drug screening applications.

To better mimic the physiological environment and functionality of the kidney and to enable more comprehensive studies, future development of the GFBoC platform should incorporate additional features such as immune components, mechanical stretch, and systemic hormonal influences, which have been identified as important mechanisms contributing to drug-induced nephrotoxicity in vivo.

Conclusion

In conclusion, we developed a simple and robust 24-well/transwell format-based microfluidic GFBoC platform for simulation of the filtration role of glomerulus and efficient drug testing. The GFB was successfully formed in a conventional transwell insert using podocytes, gMCs, and gECs. The macromolecule albumin was significantly filtered by the GFB while being cultured under perfusion in the chip. It was also characterized by glomerular permeability, which was increased as a function of the drug (doxorubicin) concentration because of drug-induced nephrotoxicity that resulted in damage to the GFB in the chip and albumin leakage. This demonstrates that the GFBoC can be a potential application model in glomerular pathophysiology and drug-induced nephrotoxicity studies.

The GFBoC platform offers various benefits compared with existing drug testing methods: (1) compatibility with SLAS format-based technologies (24-well/transwell format in this study), (2) facile and repeatable loading and unloading of the GFB transwell into or from the chip, (3) simple and robust media perfusion and operation in a conventional incubator using a pump-/tubing-less flow system, (4) convenient sampling and media exchange, (5) execution of multiple experiments in parallel without increasing system complexity, (6) straightforwardly translatable to the representative commercial manufacturing process, (7) an injection molding, with minor modification, and (8) flexibility in the formation of various organ and tissue models, such as skin, lung, muscle, liver, heart, etc., and the configuration of their interconnection. The developed GFBoC not only provides an environment where different wells can interact through microfluidic channels using commercial inserts, but it also creates a flow environment through TTS in a simple and robust manner, offering a more in vivo-like in vitro setting.

As a result, the GFBoC will help to advance current in vitro models and could also contribute to the compensation and reduction of animal experiments for renal research, as it can be used with multiorgan or tissue network formats, such as “body-on-a-chip” configurations, in drug discovery and chemical safety testing studies.

Supplementary Material

pt5c00663_si_001.pdf (695.8KB, pdf)

Acknowledgments

This work was supported by DGIST projects 23-BT-06, 24-NT-01, NRF-2021M3F7A1082275, and 2021R1A2C1011314 from the National Research Foundation of Korea, funded by the Ministry of Science, ICT of the Republic of Korea.

Glossary

Abbreviations

GFB

glomerular filtration barrier

GFBoC

glomerular filtration barrier-on-a-chip

gMCs

glomerular mesangial cells

gECs

glomerular endothelial cells

GBM

glomerular basement membrane

SLAS

Society for Laboratory Automation and Screening

OoC

organ-on-a-chip

MPS

microphysiological system

2DCC

two-dimensional cell culture

3DCC

three-dimensional cell culture

TTS

tilting tower system

PBS

phosphate-buffered saline

ATP

adenosine triphosphate

TEER

trans-epithelial electrical resistance

VEGF

vascular endothelial growth factor

UV

ultraviolet

ELISA

enzyme-linked immunosorbent assay

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00663.

  • Additional experimental details, materials, methods, and results, including photographs; fabrication of the microfluidic channel device; tilting tower system (TTS) setting; cell culture performance testing in static and perfusion conditions; membrane sandwich-based GFB model with human kidney cells; albumin passage resistance of the GFBoC in static and perfusion conditions; functional test of GFBoC with nephrotoxic or non-nephrotoxic drug; and doxorubicin-induced nephrotoxicity of the GFBoC in static and perfusion conditions (PDF)

#.

Y.J.H. and C.J. contributed equally to this work. Y.J.H.: Designed and performed the biological experiment, analyzed the data, and wrote the paper. C.J.: Designed and performed the device-related experiment, analyzed the data, and wrote the paper. K.-M.L. and J.-Y.K.: Wrote the paper, reviewed the manuscript, and supervised the study. H.C.: Reviewed the manuscript.

The authors declare no competing financial interest.

References

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