Generation of Human Naïve Pluripotent Stem Cell-Derived Blastoids in Thermoformed Microwell Platforms

Due to the inaccessibility of early human embryos for large, robust studies, many questions regarding the mechanisms of early embryogenesis remain. To address these questions, multiple research groups have developed human stem cell-based models of the pre-implantation blastocyst stage. These models, known as blastoids, mimic several key processes that natural blastocysts undergo as they prepare to implant into the uterine wall. One of the main advantages of blastoids is their scalability, making them suitable for both screenings and molecular studies. To leverage this advantage, we established a protocol for the parallel formation and culture of blastoids in thermoformed microwell arrays. Thin-walled thermoformed microwell platforms allow for uniform, large-scale generation of blastoids and enable in situ high-resolution imaging through both widefield and confocal microscopy. Here we present a step-by-step protocol for the culture of blastoids in thermoformed microwell platforms.