The next critical step in generating a physiologically relevant in vitro kidney model involves seeding dissociated tubuloid-derived cells into the OrganoPlate 3-Lane, a microfluidic multi-chip platform that enables the formation of perfused, three-dimensional tubular structures. This protocol outlines the precise procedure for preparing and loading the microfluidic chip with extracellular matrix (ECM) gel and single-cell suspensions to generate functional tubules under controlled flow conditions.
The process begins with preparation of the collagen I-ECM gel. A mixture is freshly prepared on ice by combining 1 M HEPES, 37 mg/mL NaHCO₃, and 5 mg/mL collagen I in a 1:1:8 ratio. For a full OrganoPlate 3-Lane (40 chips), at least 100 µL of this solution is made. The mixture must remain cold throughout handling to prevent premature polymerization. Once ready, 2 µL of the gel is dispensed into each ECM inlet well (columns 1, 4, 7, 10, 13, 16, 19, 22; rows B, E, H, K, N) using an 8-channel electronic pipette. The pipette tip is gently placed on top of the well opening to allow capillary forces to draw the gel into the microfluidic channel without air entrapment. After dispensing, the plate is incubated at 37°C for 15 minutes in a humidified incubator to allow gel polymerization.
Following gel solidification, 30 µL of HBSS is added to each gel inlet to prevent drying. The plate is then placed in a static position in the incubator until cell seeding. Prior to seeding, a single-cell suspension is prepared from passaged tubuloids. Cells are first dissociated using dispase II (1 mg/mL) for 30 minutes at 37°C to dissolve the BME/Matrigel. After centrifugation, the pellet is resuspended in Accutase with Y-27632 (10 µM) and mechanically sheared ~15 times with a flame-polished Pasteur pipette (~0.5 mm tip diameter). Incubation continues for another 30–45 minutes, with periodic checks under a microscope to confirm complete dissociation into single cells. Once a homogeneous suspension is achieved, cells are centrifuged, resuspended in expansion medium, and counted using a hemocytometer. The final concentration is adjusted to 10 × 10⁶ cells/mL.
Cell seeding is performed using a repeating single-channel pipette. Two microliters of the cell suspension (containing approximately 20,000 cells) are added to the inlet of the top medium perfusion channel (columns 1, 4, 7, 10, 13, 16, 19, 22; rows A, D, G, J, M).Bmi-1 Antibody medchemexpress To ensure consistent cell numbers across chips, the pipette is reloaded every 5–10 wells.Galactosidase β Antibody Autophagy Cell-free control chips are included by adding 2 µL of expansion medium instead of cell suspension. After seeding, the plate is placed at a 75° angle in a humidified incubator (37°C, 5% CO₂) for 5 hours to allow cell attachment to the ECM gel surface. During this time, the plate remains stationary to facilitate adhesion.
Once attachment is complete, 50 µL of warm expansion medium is added to each inlet and outlet of the top and bottom perfusion channels. Care is taken to avoid introducing air bubbles, which can disrupt flow.PMID:34993311 The plate is then transferred to a modified rocker platform (OrganoFlow), set at a 7° angle with an 8-minute rocking interval. This setup generates passive, gravity-driven bidirectional flow through the microfluidic channels, creating physiological shear stress (mean shear rate ~0.13 dyne/cm²) and continuous medium refreshment. Flow induction initiates within minutes and is maintained throughout culture.
Tubule formation is monitored daily via phase contrast microscopy. By day 7, confluent tubular structures lined with polarized epithelial cells are visible along the ECM gel interface. These structures exhibit leak-tight barrier integrity and functional transport capabilities, making them suitable for downstream assays such as transepithelial electrical resistance (TEER) measurement, fluorescent probe diffusion, and transporter activity testing. Medium is refreshed 2–3 times per week using an aspiration system, with complete replacement to maintain optimal culture conditions.
This seeding strategy enables high-throughput generation of physiologically relevant tubular models with minimal cell input, scalability, and compatibility with real-time imaging and multiplexed readouts. The resulting tubuloid-on-a-chip system provides a robust platform for studying renal function, drug toxicity, and personalized disease modeling in a dynamic, human-relevant environment.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com