Cryopreservation of neuroectoderm on a pillar plate and in situ differentiation into human brain organoids

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80°C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 µm. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.