Cryopreservation\nof Neuroectoderm on a Pillar Plate\nand <i>In Situ</i> Differentiation into Human Brain Organoids

Cryopreservation in cryovials extends cell storage at\nlow temperatures,\nand advances in organoid cryopreservation improve reproducibility\nand reduce generation time. However, cryopreserving human organoids\npresents challenges due to the limited diffusion of cryoprotective\nagents (CPAs) into the organoid core and the potential toxicity of\nthese agents. To overcome these obstacles, we developed a cryopreservation\ntechnique using a pillar plate platform. To demonstrate cryopreservation\napplication to human brain organoids (HBOs), early stage HBOs were\nproduced by differentiating induced pluripotent stem cells (iPSCs)\ninto neuroectoderm (NE) in an ultralow attachment (ULA) 384-well plate.\nThe NE was transferred and encapsulated in Matrigel on the pillar\nplate. The NE on the pillar plate was exposed to four commercially\navailable CPAs, including the PSC cryopreservation kit, CryoStor CS10,\n3dGRO, and 10% DMSO, before being frozen overnight at −80 °C\nand subsequently stored in a liquid nitrogen dewar. We examined the\nimpact of the CPA type, organoid size, and CPA exposure duration on\ncell viability post-thaw. Additionally, the differentiation of NE\ninto HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence\nstaining. The PSC cryopreservation kit proved to be the least toxic\nfor preserving the early stage HBOs on the pillar plate. Notably,\nsmaller HBOs showed higher cell viability postcryopreservation than\nlarger ones. An incubation period of 80 min with the PSC kit was essential\nto ensure optimal CPA diffusion into HBOs with a diameter of 400–600\nμm. These cryopreserved early stage HBOs successfully matured\nover 30 days, exhibiting gene expression patterns akin to noncryopreserved\nHBOs. The cryopreserved early stage HBOs on the pillar plate maintained\nhigh viability after thawing and successfully differentiated into\nmature HBOs. This on-chip cryopreservation method could extend to\nother small organoids, by integrating cryopreservation, thawing, culturing,\nstaining, rinsing, and imaging processes within a single system, thereby\npreserving the 3D structure of the organoids.