Chinese scientists have created a mixture to safely freeze nerve tissue organoids
Researchers from China have developed a method for cryopreservation of cortical organoids using methylcellulose, ethylene glycol, dimethyl sulfoxide and Y27632 - a selective inhibitor of Rho-kinase. As reported in the journal Cell Reports Methods, the resulting mixture allows tissue preservation without disrupting neuronal cytoarchitectonics or functional activity. Notably, organoids from human brain tissue samples can be cryopreserved using this method: pathological features persist in them even after thawing.
More than 90 per cent of new drug candidates for brain diseases fail to pass clinical trials, also because of the difficulty of using them in animal models for humans. In recent years, organoid technology has partially addressed this limitation. Brain organoids have shown great potential for studying brain development, modelling diseases and developing new drugs.
However, organoids are long and expensive to culture, and there are technical problems associated with organoid storage that limit their use in biomedical research. Another approach to studying brain diseases is being developed: the study of fresh, viable human brain tissue with naturally occurring pathological features. However, due to limited availability and storage difficulties, this method is not widely used either.
This problem was addressed by a group of scientists led by Zhicheng Shao (Zhicheng Shao) from Fudan University, who developed a method of cryopreservation using a new medium. The researchers used methylcellulose, ethylene glycol, dimethyl sulfoxide and the selective Rho-kinase inhibitor Y27632, which is used to reprogramme cells, for cryopreservation.
Experiments showed that an organoid structure similar to the ventricular zone of the brain and multiple cortical layers are preserved during cryopreservation using the new method. Cortical organoids were frozen on day 28 and then continuously cultured for a further 3 weeks after thawing before immunostaining for cortical layer markers. At day 50, markers of ventricular zone structures of thawed organoids were fully preserved with normal morphology and neurite outgrowth. In addition, the scientists found preserved gene expression and neuronal activity in thawed organoids. These results were maintained both for organoids that were large in volume and mass and for organoids from other brain structures.
To expand the clinical application of the new cryopreservation technology, the scientists prepared brain organoids from induced pluripotent stem cells derived from patients with epilepsy and focal cortical dysplasia. These organoids were cryopreserved on day 28. From day 7 to 14 after thawing, axons rapidly enlarged to sizes greater than 200 micrometres. In addition, immunostaining of thawed organoids showed no abnormalities in the cell populations of neural progenitors and neuronal cells compared with normal cortical organoids.
RNA sequencing showed that cryopreservation altered the expression of 1334 genes. Most of these genes were expressed more actively in cryosolution and were responsible for nervous system development, neuronal and synapse protection. Moreover, genes that promote neurogenesis and neuronal maturation were more expressed in cryopreserved organoids.
According to the scientists, the cryopreservation method they developed could greatly alleviate the technical limitations of biomedical research due to the short-lived nature of brain organoids. Research could become more efficient and faster.
Cryopreservation is also important for transplantology to preserve donor organs for as long as possible.