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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session A

Poster J11 - A 3D Kidney Microphysiological System with Expandable and Modular Nephron Segments

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Chronic kidney disease (CKD) affects 10% of people across the globe. CKD is characterized by renal fibrosis and increased extracellular matrix (ECM) deposition, which impairs localized cell-cell and cell-ECM interactions. Furthermore, tubular injury and maladaptive regeneration increases myofibroblast differentiation, amplifying ECM production. To elucidate CKD mechanisms, it is essential to build in vitro models that represent the tubulointerstitium, include a tailorable ECM, and permit the study of communication across diverse nephron cell populations. Microphysiological systems (MPS) allow for the integration of ECM hydrogels and fluid flow within a controlled environment. While multiple kidney-on-a-chip systems have been created, focus is often on recapitulating a singular nephron region. To investigate cell-cell interactions in the context of CKD, there is a need for an in vitro kidney model that connects nephron regions such as the collecting duct, proximal and distal convoluted tubules, and vasculature within the ECM microenvironment. However, kidney and endothelial cell types have unique growth timescales and ECM requirements, making coculture and anastomosis between regions difficult when seeded in a single platform. To address this, we have developed a microphysiological kidney device with stackable tissue components, allowing independent culture of nephron regions before modular assembly. These components are created from ECM hydrogel layers, which are open to air/fluid from both the top and bottom of the tubule-containing hydrogel. Our lumen device layers are perfusable and can be expanded with parallel channels to facilitate network formation and tubulogenesis via self-assembly, permitting anastomosis and signaling between regions once combined.

Materials and

Methods:  Three iterations of the modular lumen device were developed, to establish ECM layer components and system perfusability. Version 1 (V1) consists of a polydimethylsiloxane (PDMS) device plasma bonded to a coverslip, with an open-face design. Version 2 (V2) is a base-free PDMS structure, open through its depth, allowing the suspended collagen layer to be accessible from both the top and bottom (Figure 1A) to construct layered modular systems (Figure 1B). The third MPS version is composed of a silicone ‘insert’, consisting of two vinyl-cut silicone sheets sealed with double-sided tape, clamped inside an acrylic cassette (Figure 1C) to facilitate perfusion. All versions were treated with polydopamine for collagen-substrate attachment. A 120 µm diameter acupuncture needle (V1 and V2) or 152 µm diameter nylon fishing line (V3) was used to mold the microchannel within the rat tail collagen-I hydrogel (4 mg/mL). Due to the nylon fishing line flexibility in V3, the microchannel diameter was quantified at 1 mm intervals along the channel length to validate the consistency of this fabrication technique. Four devices were measured using ImageJ and analyzed with Welch’s ANOVA test and multiple comparisons. Leak-free perfusion through V3 with and without collagen was validated by syringe pump perfusion of PBS colored with food dye for 24 hrs at 10 µL/min. Madin-Darby canine kidney (MDCKs) epithelial cells were seeded through the media wells (V1 and V2) or fluid ports of the cassette lid (V3) and cultured for 3 days. Epithelial tissues were stained for F-actin with phalloidin-647.

Results, Conclusions, and Discussions: We have established a novel kidney MPS with cell culture within ECM hydrogel layers, which can be modularly assembled to connect nephron regions. V1 developed the lumen culture model, to study epithelial-ECM interactions. V2 modified the tubule culture device to generate a modular ECM unit (Figure 1A), enabling the stacked integration of device tissue layers (Figure 1B). This permits subsequent anastomosis and signaling across nephron regions. Version 3 established a simple modular perfusable system. All iterations were successfully designed and constructed to maintain a microchannel within the collagen-I bulk gel (Figure 1DE). Minimal variation in channel diameter was noted (p < 0.05) using monofilament line (Figure 1F) on par with a rigid needle mold with a mean standard deviation of 30.04 µm in width over an 8 mm long channel. Therefore, our process forms microchannels of consistent diameter, both across device replicates and along individual channel lengths. Seeded MDCKs generated a confluent monolayer surrounding the lumenized channel after 3 days of culture in V1 (Figure 1GH). Flow (10 µl/min) tests demonstrated successful perfusion of the epithelial tube and no leakage over 24 hours (Figure 1E). This flow rate results in a shear stress of 1.61 dyn/cm², representing the maximum proximal tubule fluid shear stress in kidney disease. The intraluminal perfusability enables the tuning of filtration pressures within the device, to investigate how tubulointerstitium injury and inflammation contributes to ECM remodeling, and subsequently alters cell communication in CKD. The device perfusion also permits the analysis of filtrate output composition and can mimic flows that guide kidney development, informing differentiation and morphology.

Conclusions
Our modular kidney microphysiological system enables independent cell-specific culture of nephron region layers, which can be modularly assembled for connectivity and signaling across nephron tissue regions. The system uses accessible materials and supports long-term intraluminal perfusion to replicate healthy and pathological fluid shear stress which can induce fibrosis and CKD following tubule injury. Moreover, our lumen culture design can be expanded to construct multiple patterned channels within a single layer, facilitating anastomosis within and across nephron region layers via self-assembly of epithelial and vascular tissue.

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