consistent generation of human vascular organoids using microwell technology

hiPSC-Derived Vascular Organoids for Drug Screening and Disease Modeling

In vitro microphysiological models, including organoid and tissue-on-a-chip systems, have emerged over the past decade1,2,3. These three-dimensional (3D), human cell-derived systems address the limitations in conventional two-dimensional (2D) cell culture and animal models, such as the lack of many components in the physiological microenvironment and genetic differences across species, respectively. They provide more physiological insights and are more suitable for disease modeling and drug screening than conventional models. Among the microphysiological models, organoids derived from human induced pluripotent stem cells (hiPSCs) have been proven to recapitulate the architectural features of native human tissues effectively and have been developed to mimic different human organs, including the brain4,5, kidney6, liver7,8&#160;and retina9. Here, we particularly focus on the generation of vascular organoids from hiPSCs and outline a streamlined protocol that aims to minimize the variations between different organoid samples and batches, thereby improving the overall reproducibility.
Vasculature plays a crucial role in maintaining physiological homeostasis across all human organs. The formation of vasculature involves the processes of vasculogenesis and angiogenesis, orchestrated through the interactions between endothelial and mural (e.g., pericytes and vascular smooth muscle cells) cells and influenced by various stimuli10. Penninger, Wimmer&#160;and colleagues developed the first vascular organoid generation method11,12 that mimics these two processes. With the organoids generated from this method, their group12 and us13,14 have demonstrated the potential of vascular organoids for modeling vascular malfunctions in diseases. For example, in our recent works13,14, distinct phenotypes were observed, such as vessel sprouting and mural cell composition variations, between disease-mimicking vascular organoids and their wild-type counterparts. These examples highlight that the vascular&#160;organoid model could serve as a platform for drug screening by mimicking disease-related vascular malfunctions.
Built upon the initial vascular organoid generation works11,12, a revised protocol is presented that includes the use of microwell to facilitate homogenous embryoid body (EB) formation, a critical factor in minimizing the variations often seen within the same organoid generation batch. Additionally, the CEPT cocktail, a combination of chroman 1, emricasan, polyamines&#160;and the integrated stress response inhibitor (trans-ISRIB)15, is employed&#160;to improve the viability of hiPSCs and differentiated cells during the EB formation process. This modification is mainly to reduce the variations between different organoid samples and batches. Uniform vascular organoids can be produced with this roughly two-week-long protocol, where vasculogenesis is induced to help endothelium organize into primitive tubular networks on Day 1, followed by angiogenesis induction on Day 5 to develop complex vascular networks in organoids. On Day 12-15, the generated organoids should show mature vascular networks composed of both endothelium and mural cells.

Author Spotlight: Improving Reproducibility in Vascular Organoids Using ROCK Inhibitors and Microwell Confinement

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

This study introduces a modified method for scalable production of homogeneous vascular organoids from human IPSCs. By using novel ROCK inhibitors and the microwell confinement, the approach enhances reproducibility and minimize batch-to-batch variations, making it suitable for downstream morphological analysis and disease modeling applications. Single-cell sequencing technologies have been extensively utilized in organoid research to unravel the genomic, epigenomic, and the transcriptomic heterogeneity and dynamics within cellular populations.3D imaging technologies such as tissue clearing and the light-sheet microscopy facilitate rapid, high-resolution imaging of cellular morphology and the tissue architecture in a three-dimensional context. The throughput and the standardization are key challenges in organoid research. Manual handling is labor-intensive and limits the throughput, making robotic automation a promising solution.Standardization of organoid culture, including IPSC quality control and the differentiation protocol optimization can significantly enhance reproducibility and minimize variations between batches and cell lines.

Consistent Generation of Human Vascular Organoids Using Microwell Technology

To begin, place a 50 milliliter tube on ice and add collagen one solution along with other components sequentially. Shake the tube to mix thoroughly. Using a P10 pipette, add one molar sodium hydroxide solution dropwise while shaking the mix solution to adjust the pH to 7.3.Add 1.25 milliliters of basement membrane matrix to the five milliliter collagen one mixture and mix the solution gently. Next place a 12-well plate on ice for two minutes. Using wide bore tips, add the collagen one basement membrane matrix mixture slowly to the 12-well plate.Place the 12-well plate in the 37 degree Celsius incubator for two hours to solidify the first layer of hydrogel. Keep the remaining collagen one basement membrane matrix mixture on ice for later use. Then transfer approximately 60 cell aggregates into a 1.5 milliliter microcentrifuge tube.And cool the tube on ice for five minutes. Using a P200 pipette tip, carefully remove the medium from the tube. Add 1.5 milliliters of the collagen one basement membrane mixture, dropwise to the cell aggregates, and gently mix them using a wide bore P200 pipette tip to resuspend.Transfer the plate with the first layer of hydrogel from the incubator to the biosafety cabinet. Add 1.5 milliliters of the aggregate suspension onto the cured hydrogel layer and gently shake the plate to distribute the aggregates evenly. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for two hours.Prewarm vascular differentiation medium two in a 37 degrees Celsius water bath for 20 minutes. Add two milliliters of medium to each well and culture the cells at 37 degrees Celsius with 5%carbon dioxide. Change the medium every three days with fresh pre-warmed vascular differentiation medium two.Organoids expressed CD31, ERG1, and PDGFR beta, indicating the presence of vascular endothelial cells and parasites. Vascular networks were visualized via whole mount immunofluorescence microscopy after tissue clearing. Mature organoids stained on day 17 showed vascular networks encapsulating the spheroids within the gel block.Organoids retrieved on day 28 had vascular networks wrapping around the spheroids instead of radially expanding.

consistent-generation-human-vascular-organoids-using-microwell

Research

JoVE Journal

Bioengineering

80 vues. Pour commencer, placez un tube de 50 millilitres sur de la glace et ajoutez du collagène dans une solution ainsi que d’autres composants séquentiellement. Secouez le tube pour bien mélanger. À l’aide d’une pipette P10, ajoutez une solution molaire d’hydroxyde de sodium goutte à goutte tout en agitant la solution de mélange pour ajuster le pH à 7,3.Ajoutez 1,25 millilitre de matrice de membrane basale au mélange de cinq millilitres de co...