A new protocol for the development of organoids based on molecular mechanisms in the developing newborn rat brain: Prospective applications in the study of Alzheimer’s disease

Abstract

Background

Brain organoids have emerged as powerful models for studying brain development and neurological disorders

Comparison with existing methods

Current models rely on stem cell isolation and differentiation using different growth factors. Thus, their composition varies according to the protocol followed.

New method

We developed a simple protocol to generate organoids from newborn rat whole brain. It is a one-step procedure that yields organoids of consistent composition. The whole brains from 3-day old pups were digested enzymatically. All isolated cells were seeded in culture plates using a basement membrane extract (BME) matrix as a scaffold and cultured in the presence of the appropriate medium.

Results

Hematoxylin-eosin staining of 28-day-old cultured domes revealed their structural integrity, while immunohistochemistry confirmed the presence of neurons, astrocytes, microglia, and progenitor stem cells in the structures. To assess whether these organoids can serve as a model to study brain physiopathology, and in particular neurodegenerative diseases such as Alzheimer’s disease (AD), we determined how these organoids respond upon their exposure to lipopolysaccharides (LPS), a potent neuroinflammatory factor. LPS-induced amyloid precursor protein (APP), tau protein and glial fibrillary acidic protein (GFAP) expression. Moreover, the intracellular levels of IL-1β and the extracellular levels of amyloid-beta (Aβ) were also elevated.

Conclusions

Therefore, this simple protocol results in the generation of functional brain organoids with a consistent structure, that requires no use of varying factors that may affect the structure and function of the produced organoids, thus providing a valuable tool for the study of the physiopathology of neurodegenerative disorders.