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A well plate-based multiplexed platform for incorporation of organoids

A well plate-based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature


Owing to their high spatiotemporal precision and adaptability to different host cells, organ-on-a-chip systems are showing great promise in drug discovery, developmental biology studies, and disease modeling. However, many current micro-engineered biomimetic systems are limited in technological application because of culture media mixing that does not allow direct incorporation of techniques from stem cell biology, such as organoids. Here, they describe a detailed alternative method to cultivate millimeter-scale functional vascularized tissues on a biofabricated platform, termed ‘integrated vasculature for assessing dynamic events’, that enables facile incorporation of organoid technology. Utilizing the 3D stamping technique with a synthetic polymeric elastomer, a scaffold termed ‘AngioTube’ is generated with a central microchannel that has the mechanical stability to support a perfusable vascular system and the self-assembly of various parenchymal tissues. They demonstrate an increase in user familiarity and content analysis by situating the scaffold on a footprint of a 96-well plate. Uniquely, the platform can be used for the facile connection of two or more tissue compartments in series through a common vasculature. Built-in micropores enable the studies of cell invasion involved in both angiogenesis and metastasis. They describe how this protocol can be applied to create both vascularized cardiac and hepatic tissues, metastatic breast cancer tissue, and personalized pancreatic cancer tissue through the incorporation of patient-derived organoids. Platform assembly to populating the scaffold with cells of interest into perfusable functional vascularized tissue will require 12–14 d and an additional 4 d if pre-polymer and master molds are needed.




Dimensions of the InVADE platform and AngioTube bioscaffolds.

a, Image of the InVADE platform that depicts the custom-designed base plate features. b, Schematic showing the dimension of the tissue chambers of the Long and Cantilever versions of the AngioTube bioscaffolds. c, Schematics showing the dimensions of the central lumen and micro-holes of the AngioTube bioscaffolds. d, The three-well–based microfluidic system connected by a single AngioTube bioscaffold and gravity-driven fluid flow driven by hydrostatic pressure gradient.


For several decades, the use of immortalized human cell lines on a Petri dish and animal models has dominated preclinical screening of promising drug therapeutics. In practice, the high failure rate of preclinical compounds in clinical stages suggests that current methodologies in early-stage drug development do not adequately recapitulate drug response in human biology and pathophysiology. Most drug candidate failures are attributed to inaccurate modeling of the adsorption, distribution, metabolism, and elimination of the therapeutic in 3D human tissue, or toxic side effects on a non-targeted organ, such as cardiac and renal toxicity. This contributes significantly to a ‘drying pipeline’ of drug development, reflected in a decreasing number of novel first-in-class drugs approved each year by the US Food and Drug Administration. The development of a novel drug is not a stand-alone act; the convergence of researchers from different fields to develop an accurate in vitro drug screening model that can mirror human physiology in an integrated manner is essential for advancing this development pipeline.


The development of effective models has been recently accelerated by the derivation of relevant cell sources. From the discovery of human-induced pluripotent stem cells by Shinya Yamanaka’s group to the organoid technology pioneered by Hans Clevers’ team, drug screening strategies are beginning to utilize more human-relevant cell sources, moving away from the use of immortalized cell culture and animal models. The capability of stem cells to differentiate and spontaneously self-assemble into organ-level architecture may enable a higher predictive power of drug responses. However, in most organoid systems, there is no direct ability to control flow inputs and outputs; nor are their predictable routes to study cell migration involved in processes such as angiogenesis or cancer metastasis. The primary readout for drug efficacy or toxicity is often limited to the change in a number of viable metabolically active cells or gene expression, with a lack of built-in readouts to inform tissue function over time. Furthermore, the lack of a stromal microenvironment and a vascular system in an organoid approach may limit the predictive capacity. This motivates the development of 3D tissue models with active readout capability that provide routes for the control of flow inputs and outputs akin to in vivo conditions.


In recent years, advances in microfabrication techniques have provided a solid foundation for platform engineers to develop relevant screening models to predict reliable drug efficacy and drug toxicity. Leveraging this approach, systems with tighter spatiotemporal precision can be manufactured to reproduce key features of tissue microenvironments, including tissue-tissue interfaces and vascular perfusion relevant to pharmacokinetics. On the basis of these engineering approaches, Ingber et al. ignited the field of organ-on-a-chip engineering, focusing on 3D models that recapitulate human physiology, which hold relevance in the study of response to drugs or hazardous substances. With the convergence of soft/photolithography and microfluidic technologies, replica-molding patterns of interest with the use of elastomeric materials allows for the generation of compartmentalized 3D microfluidic systems with two to three microchannels upon capping onto a glass or another elastomeric substrate. In this close-capped design, a subsection of an intact organ, such as the vascular and epithelial interface, can be replicated.


Polydimethylsiloxane (PDMS) is often the choice of material for rapid-prototyping these micro-engineered biomimetic systems, because of the low cost, biocompatibility, transparency for optical monitoring, and flexibility as an on-plate detection tool. In addition, PDMS exhibits high gas permeability, allowing human cells to remain viable within close-capped (irreversibly sealed) polymeric microchannels. However, over-relying on PDMS as a material for prototyping the organ-on-a-chip design has produced some limitations. Because of its high gas permeability, PDMS-based micro devices such as microfluidic channels are susceptible to bubble formation, thus necessitating the incorporation of a bulky bubble trap system for maintenance of relevant circulation. PDMS has also been shown to absorb small hydrophobic compounds from the culture medium, limiting its application in pharmacokinetic studies.


Although advantageous in diverse applications, a close-capped design also presents some technical challenges. To drive continuous media recirculation that mimics vascular perfusion and small molecule diffusion, an external pumping system is necessary. This complicates translation to high throughput workflows common in the pharmaceutical industry. Because close-capped devices are manufactured by irreversibly bonding PDMS with channel features onto a piece of glass or a separate PDMS mold, fluid handling is similarly limited. The inaccessibility of the parenchymal space of a tissue interface limits the use of screening or profiling assays. Furthermore, interface designs control the development of cell–cell interactions, perhaps neglecting how cells remodel and function in 3D environments. The use of a close-capped organ-on-a-chip system makes it difficult to recapitulate the differentiation of patient cells into organoids on the microdevices, thus limiting their use in studies of disease progression over the cultivation time. This may affect the overall predictive power of biomimetic microdevices on a drug’s efficacy in the human body. Therefore, there is a motivation to optimize these limitations and to enhance the user-friendliness in organ-on-a-chip applications, including the choice of base material, fluid handling, tissue stability as a 3D model, and design adaptability.


They recently introduced a new approach to engineering organ-on-a-chip microdevices with fabrication of microchannels from a synthetic polymeric elastomer that can act as a stand-alone bioscaffold template to provide the stability of a perfusable vascular network and support the remodeling of dense 3D parenchymal tissue. Importantly, this approach allows for facile incorporation of organoids into an organ-on-a-chip system through individual compartmentalization of the vascular and tissue systems with limited fluid transfer between them. Leveraging novel 3D stamping fabrication to construct hollow microstructured scaffolds, they have developed AngioChip, a multi-channel microfluidic network, and AngioTube, a single-channel microfluidic system. To increase the permeability of small and large molecules and allow for trans-endothelial cell migration, they incorporated patterned micro-holes and materials-generated nanopores on the sidewalls of the bioscaffold constructs. This protocol highlights the capability and usability of the AngioTube bioscaffold in a ready-to-use 96-well plate platform: integrated vasculature for assessing dynamic events (InVADE).


The development of the AngioTube bioscaffold and assembly into the InVADE platform relies on a user-friendly multi-material approach to develop a functional organ-on-a-chip platform. A single AngioTube bioscaffold spans three consecutive wells on a custom-designed polystyrene base plate intended to house 20-mm-scale functional vascularized tissues. The middle well is the tissue chamber and connects to an inlet chamber and outlet chamber through the perfusable bioscaffold. After bonding the base plate to a bottomless 96-well plate structure, fluid circulation within the bioscaffolds is achieved through the gravity-driven flow without the use of an external pump. To drive continuous circulation within the bioscaffold, the InVADE platform is situated on a programmable rocker system, a method that was previously employed successfully by others. The open-well design significantly simplifies experimental setup and facilities flexibility in fluid handling for tissue seeding and screening assays.


The InVADE platform is manufactured entirely on the basis of cell culture–friendly material polystyrene, addressing adsorption concerns in PDMS-based microdevices. The use of a synthetic elastic material to construct the bioscaffold provides mechanical stability for endothelium, which is needed in a perfusable luminal space to generate a vascular microenvironment. The surrounding parenchymal space allows for organ-specific cell seeding in a hydrogel matrix and allows for appropriate cell remodeling into dense 3D tissue. Perhaps most notably, this platform allows for the linking of multiple organ compartments in series for assessment of interactions. For example, to reveal the entire cancer metastasis cascade, the InVADE platform can be used to track cancer cell migration and provide better compartmentalization for each organ type with minimal concern regarding media use. This highlights a potential opportunity in tissue mechanistic studies or pharmacokinetics, appreciating the importance of multiorgan interactions1.


1.Lai, B.F.L., Lu, R.X.Z., Davenport Huyer, L. et al. A well plate–based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature. Nat Protoc (2021). https://doi.org/10.1038/s41596-020-00490-1