This study introduces a modified method for scalable production of homogeneous vascular organoids from human iPSCs by using novel ROCK inhibitors and microwell confinement. The approach enhances reproducibility and minimizes 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 the dynamics within cellular populations.
3D imaging technologies, such as tissue-clearing and 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 it 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. To begin, place a 50 milliliter tube on ice and add Collagen 1 solution, along with other components, sequentially. Shake the tube to mix thoroughly.
Using a P10 pipette, add 1 molar sodium hydroxide solution drop-wise while shaking the mixed solution to adjust the pH to 7.3. Add 1.25 milliliters of basement membrane matrix to the five milliliter Collagen 1 mixture and mix the solution gently. Next, place a 12 well plate on ice for two minutes.
Using wide bore tips, add the Collagen 1 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 1 basement membrane matrix mixture on ice for later use.
Then, transfer approximately 60 cell aggregates into a 1.5 milliliter micro centrifuge 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 1 basement membrane mixture drop-wise 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 2 in a 37 degree 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 2.
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.
