(L-460) Differentiation of Human Kidney Organoids Within a Microfluidic Chip Promotes Podocyte-Endothelial Cell Interaction

Introduction::
Combining kidney organoids with microfluidic chips has promise as a powerful tool to improve organoid maturation, vascularization, and create a highly tunable environment for drug testing or disease modeling. Organoids can be transferred into microfluidic channels after differentiation, but this risks irreversibly damaging the newly formed structures or inducing their dedifferentiation upon attachment in the channel. Thus, we have developed a protocol to carry out kidney organoid differentiation entirely within a microfluidic device.




Materials and Methods::
A positive-relief mold was generated using a stereolithography 3D printer to make a PDMS device with multiple channels separated by micro-pillars, which could accommodate a physiological flow rate. Then, we optimized our standard kidney organoid protocol for the microfluidic chip by altering the ECM surface coating, undifferentiated human pluripotent stem cell seeding density, and timing of media exchanges to support the much smaller growth environment. Following these modifications, we identified several organoids in each device with brightfield microscopy by day 18 of differentiation and fixed them for immunostaining.




Results, Conclusions, and Discussions::
The presence of kidney-specific cells and structures was confirmed by confocal microscopy after fixing and staining for podocyte, proximal tubule, and endothelial cells. The organization of these organoids was comparable to those made in 24-well plates indicating that we have successfully optimized our standard differentiation protocol for a microfluidic platform. Increased endothelial cells were observed in chip-differentiated organoids, compared to 24-well plates. The endothelial cells integrated into organoids in 3D and demonstrated a tendency to interact with the basal membrane of podocytes which was not observed in 24-wells. These results suggest that the environment created by our microfluidic chips does not have a negative impact on kidney organoid differentiation. To the contrary, this new environment led to a more in vivo-like interaction between the endothelial cells and podocytes within our organoids.



This protocol eliminates transfer steps during the differentiation process by creating a stable environment for organoid development within our microfluidic chips. In addition, the confined growth environment of the microfluidic chips or the intermittent shear stress from media changes may be inducing endothelial cell differentiation and podocyte-endothelial cell interactions. The endothelial interactions observed in organoids grown in these devices provides a strong foundation for future vascularization work.




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