IFlowPlate—A Customized 384‐Well Plate for the Culture of Perfusable Vascularized Colon Organoids

Abstract

Despite the complexity and structural sophistication that 3D organoid models provide, their lack of vascularization and perfusion limit the capability of these models to recapitulate organ physiology effectively. A microfluidic platform named IFlowPlate is engineered, which can be used to culture up to 128 independently perfused and vascularized colon organoids in vitro. Unlike traditional microfluidic devices, the vascularized organoid‐on‐chip device with an “open‐well” design does not require any external pumping systems and allows tissue extraction for downstream analyses, such as histochemistry or even in vivo transplantation. By optimizing both the extracellular matrix (ECM) and the culture media formulation, patient‐derived colon organoids are co‐cultured successfully within a self‐assembled vascular network, and it is found that the colon organoids grow significantly better in the platform under constant perfusion versus conventional static condition. Furthermore, a colon inflammation model with an innate immune function where circulating monocytes can be recruited from the vasculature, differentiate into macrophage, and infiltrate the colon organoids in response to tumor necrosis factor (TNF)‐ inflammatory cytokine stimulation is developed using the platform. With the ability to grow vascularized colon organoids under intravascular perfusion, the IFlowPlate platform could unlock new possibilities for screening potential therapeutic targets or modeling relevant diseases.

Engineering neurovascular organoids with 3D printed microfluidic chips

“…Here, we have demonstrated for the first time the possibility to control the interaction of endothelial cells, pericytes and organoids though the use of hPSCs and microfluidic technologies, in order to achieve an organoid vascularization process which is highly synchronized in time and space. The angiogenic sprouting and network formation observed in this platform are similar in size and branching architecture to those which have been reported with primary pericytes and ECs (11,35–38). An intriguing difference, however, is that primary cultures have been characterized by gel invasion of ECs first, followed by pericytes (38), whereas in our hPSC-based system simultaneous sprouting of ECs and pericytes is observed, reflecting possible PSC-specific characteristics or differences in developmental timing.…”

“…For example, subtractive removal of material from organoid tissue by sacrificial laser writing is a promising technique for creating perfusable channels (41), however the process of tissue ablation also has clear implications in altering tissue development. The use of a manufactured chip-like co-culture system on a re-engineered 384 well plate to study organoid-vasculature has also been recently proposed in the context of interaction between colon organoids and HUVECs and fibroblasts (11). This parallelized approach suggests that this technique could be useful for high throughput screening of drug candidates, however the use of such a standardized plate limits users flexibility in terms of the spatial control, which can be overcome using accessible 3D printing technology.…”

“…While these observations underscore the importance of vasculature in the development of human organoids, the use of an in-vivo host with endogenous vasculature is clearly not a practical approach for organoid vascularization, prompting a number of attempts at in-vitro vascularization. The most common approach has involved the co-culture of organoids, including liver, pancreas, colon, kidney and cerebral organoids with primary ECs, with human umbilical cord endothelial cells (HUVEC) being most frequently used (8)(9)(10)(11)(12). However HUVECs are already developmentally specified in contrast to hPSC-derived ECs, which can interact and specify the maturation of specific tissues.…”

Vascularized organoids on a chip: strategies for engineering organoids with functional vasculature

“…Very recently, Rajasekar et al engineered a microfluidic chamber, where patient-derived colon organoids were co-cultured within a self-assembled vascular network. 86 Similarly, they found ECs self-organized to form functional vessels in fibrin, but not in Matrigel, while colon organoids properly developed in Matrigel, but were unable to grow in fibrin. This dilemma was solved by co-culturing in a hydrogel mixture of fibrin with 10% Matrigel, which has been shown to support the development of both organoids and vasculature.…”

“…Even though Matrigel has also been utilized in developing various angiogenic assays, ECs embedded within Matrigel cannot self-organize into a perfusable vessel network, likely due to the difficulties in remodeling Matrigel by the ECs. 86 Instead, functional in vitro MVNs are generally grown within collagen or fibrin, where local matrix is remodeled by ECs and FBs and result in patent lumens, 15,17,67 and the initial matrix is gradually degraded and replaced by a cell-generated ECM. In the experimental design, the compatibility of supporting matrix must be optimized to ensure a successful co-culture.…”