Poster M15 - Development of an in vitro CAM model for perfusion of vascularized kidney organoids

Introduction: Organoids are an important model used to study organogenesis, disease, drug efficacy, and genetic disorders. However, a major limitation to the use of organoids as a model is the lack of perfused vasculature. Without perfusion the organoid relies on passive diffusion for transport of nutrients and waste, resulting in necrosis within the organoid core, limiting its growth and development. The most reliable current method to achieve intralumenal perfusion of organoid vasculature is in vivo engraftment within densely vascularized tissues such as the Chicken Chorioallantoic Membrane (CAM). This leads to ingrowth and perfusion of the host vasculature throughout the organoid which stabilizes and enhances the organoid vasculature and increases organoid size. Unfortunately, in vivo transplantation limits use of the organoids for functional assays, imaging, and other in vitro applications. The development of an easy-to-use in vitro CAM model is needed to combine the benefits of in vivo implantation with the accessibility and control of in vitro culture models. To meet this need, we have developed a microphysiological system (MPS) capable of providing controllable intralumenal perfusion through a self-assembled capillary bed anastomosed with a vascularized organoid. This system is constructed using readily available and low-cost materials and equipment to maximize translation to other researchers. Additionally, minimal specialized techniques are required for the setup and use of this system to allow ease of use among researchers without prior experience with similar MPS models.

Materials and

Methods: The MPS is composed of two components: (1) the Polydimethylsiloxane (PDMS)-glass device containing the hydrogel with embedded cellular components and (2) a laser-cut acrylic cassette housing to seal the open-topped PDMS device and provide fluidic connection points for perfusion. (Fig 1AB). Each PDMS device contains a center well connected to 2 outer media reservoirs via 0.25 mm diameter microchannels patterned within the PDMS, which also serve as guide channels to position molds used to pattern the deterministic vessels within the hydrogel. The perfusable vascular plexus was created using a gel-in-gel method to contain the self-assembled network locally to the area of the organoid. The cellular hydrogel was either collagen (6 µL, 4 mg/mL) containing 3x106 cells/mL human umbilical vascular endothelial cells (HUVEC) or varying collagen-Matrigel mixtures that was placed into an acellular collagen (30 µL, 4 mg/mL) hydrogel. After hydrogel polymerization, the vessel molds were removed and subsequent microchannels were seeded with HUVECs. Endothelial self-assembly occurred after 7 days to form perfusable capillary beds and human induced pluripotent stem cell (hiPSC) derived kidney organoids with RFP-tagged tubulin were added to the gel surface and cultured for up to 14 days. Immunofluorescence staining for VE-cadherin was then performed on the co-cultured CAM model. Vascular perfusion was verified by tracking 500 nm fluorescent microspheres and morphology of vascular plexus formation within collagen and Matrigel composite hydrogels was assessed from 3D confocal micrographs of F-actin fluorescence staining using the TubeAnalyst (IRB Barcelona) ImageJ macro.

Results, Conclusions, and Discussions: We have developed a modular system to provide controllable intralumenal perfusion through a self-assembled vascular network and engrafted vascularized organoid. The in vitro CAM model presented here is stable for at least 14 days in culture without degradation of the vascular network. This methodology reliably generates dense self-assembled vascular networks connected to both patterned vessels and engrafts with organoids. The opened-top design of the PDMS device enabled engraftment of the kidney organoids with an established capillary bed (Fig 1CD). Anastomosis and perfusion of the self-assembled vascular plexus and organoid from the large, patterned vessels were quantified by calculating the corrected total fluorescence of fluorescent microspheres perfused through the system at multiple locations within and adjacent to the vasculature. Both hydrostatic pressure and syringe pump-driven flow resulted in the perfusion of fluorescent microspheres through the patterned inlet vessel, throughout the integrated self-assembled plexus, and out of the outlet vessel. The choice of hydrogel composition is an important consideration for many engineered tissues as different ECM compositions can elicit specific biochemical responses. Additionally, many organoid culture protocols use Matrigel as a culture substrate. We, therefore, quantified the average total vessel length, area percentage, and number of branches of the vascular networks grown in 100:0, 90:10, and 80:20 collagen I to Matrigel ratio hydrogels (Fig 1EF). We found decreased vascular network formation in the 80:20 collagen to Matrigel hydrogels; however, perfusion was able to be achieved in self-assembled capillary beds created with all hydrogel compositions. This methodology developed to create a perfusable vascular plexus that is compatible with other hydrogel systems, synthetic or naturally derived, and can be tailored to the organoid-specific need.
Conclusion
We have created an easy-to-use and accessible model to enable engraftment of vascularized organoids into a robust perfusable in vitro capillary bed. The modularity of this system and open-top design of the PDMS component provides flexibility in timing for establishing the vascular network, engraftment of the organoids, and application of perfusion through the system. Moreover, the construction techniques and materials used are low-cost and readily available, allowing wide adoption among researchers.

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