Cryopreservation of Neuroectoderm on a Pillar Plate and In Situ Differentiation into Human Brain Organoids

类有机物 神经外胚层 支柱 低温保存 细胞生物学 人脑 生物 纳米技术 生物医学工程 材料科学 神经科学 中胚层 胚胎干细胞 胚胎 医学 生物化学 工程类 结构工程 基因
作者
Mona Zolfaghar,Prabha Acharya,Pranav Joshi,Na Young Choi,Sunil Shrestha,L. Vinod Kumar Reddy,Soo Yeon Kang,Minseong Lee,Moo‐Yeal Lee
出处
期刊:ACS Biomaterials Science & Engineering [American Chemical Society]
标识
DOI:10.1021/acsbiomaterials.4c01383
摘要

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 demonstrate cryopreservation application to human brain organoids (HBOs), early stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NE) in an ultralow attachment (ULA) 384-well plate. The NE was transferred and encapsulated in Matrigel on the pillar plate. The NE on the pillar plate was exposed to four commercially available CPAs, including the 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 the CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of NE into 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 the early stage HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability postcryopreservation than larger ones. An incubation period of 80 min 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 noncryopreserved 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.
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