Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures

Published in Neuroscience
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Neural cell dissociation protocols have been conducted in rodent, which ensures a quick process and a high yield. However, translation of preclinical findings may be greatly required by direct validation in tissue of primates. The ability to interrogate neural cell from adult primates will greatly advance our understanding of the primate brain, especially with evidence to suggest that many phenotypic and functional differences exist between the primate brain and rodent model systems. Due to ethical constraints, it is difficult to obtain human brain tissue. The cynomolgus macaque (Macaca fascicularis) has been widely used as a promising model to study multiple pathological conditions in humans. The ability to characterize and study neural cells isolated directly from the macaque brain would greatly promote the research in human neuroscience. However, significant challenges such as accessibility of macaque brain tissue and the lack of a robust neural cell isolation and culture protocol have hampered its progress.

To achieve high-yield and viability neural cells from adult and aged macaques, we have incorporated knowledge accumulated from multiple fields. The macaques are perfused with a cold anticoagulating and oxygenated perfusion solution to remove hematocytes. A cocktail of small-molecule inhibitors is added during perfusion and dissociation to protect neural cells from cytotoxicity by blocking receptors or channels. We also utilized adult brain slicing protocols from in vitro patch-clamping, in which brains are sectioned within a neural protective recovery solution to enhance neural survival. This approach helps to disentangle the neural cells and avoids cell damage by shear force. Using this vastly improved neural cell dissociation pipeline, we obtained single-cell suspensions with excellent cell viability rates (>90%) from the adult and aged macaque brains. In our recent work published on Nature Protocols, we describe a simple and reproducible method for the isolation and culture of functional adult and aged macaque brain cells.

When combined with single-cell profiling techniques, the approach allows an unbiased and comprehensive mapping of cell states in the adult and aged macaque brain, which is needed to advance our understanding of human cognitive and neurological diseases. When combined with single-cell profiling techniques, the approach allows an unbiased and comprehensive mapping of cell states in the adult and aged macaque brain, which is needed to advance our understanding of human cognitive and neurological diseases.

Importantly, the major advantage of this protocol is that our approach is able to yield viable and high-density neural progenitor cells (NPCs), neurons, and immature cells (OPCs, immature astrocytes and immature granule cells).

Isolation of primary cells from the hippocampus provides an opportunity to study neurogenesis, particularly capturing the neurogenesis process. Our protocol can be achieved at different stages and state NPCs (RGL, IPC and NB). To further confirm the dissociation of NPCs from the hippocampus of adult macaques, we have undertaken studies on culture of NPCs from adult macaque hippocampus. Furthermore, our work on primary hippocampal cells from adult macaque has been applied to compare the species discrepancies for NPCs. There is also the potential to identify neurogenesis mechanisms, perhaps increase neurogenesis to replace cells lost in particular diseases.

Furthermore, our protocol will be informative to investigators interested in immature cell types that may be missed through analysis of single-nucleus RNA-sequencing approaches. This protocol resulted in an increased number of NPCs and immature cells from the hippocampus, which revealed the primate-specific physiological properties of the neuron populations and the molecular cascade during adult neurogenesis.

In addition, our primary cell isolation protocol was adapted for high-yield neurons. Isolation of high-quality living neurons from the adult and aged is one of the major bottlenecks for the broad application. We applied the optimised pipeline for single-cell dissociation to achieve a limited amount of hematocytes and a neuron ratio that is comparable with that seen in vivo. We have captured rare cell types for further interrogations of the macaque brain using this approach in our published work on Nature Communications, such as novel excitatory neuron types HPCAL1 NPY and RORB OSTN. The HPCAL1 NPY expresses genes for dopamine receptor DRD3 and neuromodulator NPY, potentially important in sensory modulation and plasticity. The RORB OSTN is an anthropoid primate-specific, activity-induced excitatory neuron population. The finding of these populations in our neural cells enable detailed interrogation of the fundamental molecular and cellular of brain function.

Summary

Isolation of high-yield and viability neurons and NPCs from adult and aged macaque brains is a prerequisite for various omics sequencing and establishment of cell models in vitro. Our protocol is extremely useful for future understanding of human health, development and disease.

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