Abstract
The low number of neural progenitor cells (NPCs) present in the adult and aged primate brains represents a challenge for generating high-yield and viable in vitro cultures of primary brain cells. Here we report a step-by-step approach for the fast and reproducible isolation of high-yield and viable primary brain cells, including mature neurons, immature cells and NPCs, from adult and aged macaques. We describe the anesthesia, transcardial perfusion and brain tissue preparation; the subsequent microdissection of the regions of interest and their enzymatic dissociation, leading to the separation of single cells. The cell isolation steps of our protocol can also be used for routine cell culturing, in particular for NPC expansion and differentiation, suitable for studies of hippocampal neurogenesis in the adult macaque brain. The purified primary brain cells are largely free from myelin debris and erythrocytes, paving the way for multiple downstream applications in vitro and in vivo. When combined with single-cell profiling techniques, this 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. The neural cell isolation protocol requires 4 h and a team of four to six users with expertize in primary brain cell isolation to avoid tissue hypoxia during the time-sensitive steps of the procedure.
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Data availability
The data supporting the findings of this study are available within the article, the primary supporting research paper of macaque hippocampus20. The scRNA-seq datasets shown in Figs. 4–6 are publicly available at ArrayExpress under the accession code E-MTAB-12399. Source data are provided with this paper.
Code availability
The code for processing the datasets is available as jupyter notebooks at GitHub (https://github.com/leitang607/macaque_Neural_cell).
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Acknowledgements
We gratefully acknowledge C. Xu for language editing and proofreading. This research was supported by grants from the National Key R&D Program of China (2018YFA0108300), the Natural Science Foundation of China (81961128021, 81870682, 82201231 and 32270864), the National Key R&D Program of China (2022YEF0203200), the Major Project on Brain Science and Brain-Like Computing of the Ministry of Science and Technology of China (2021ZD0200103), the Basic and Applied Basic Research Foundation of Guangdong Province (2023A1515011593), the Guangdong Provincial Key R&D Programs (2018B030335001 and 2018B030337001), the Science and Technology Program of Guangzhou (202007030010 and 202007030011), the China Postdoctoral Science Foundation (2022M713609) and Science and Technology Planning Projects of Guangzhou City (2019A1515012033).
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Contributions
S.L. conceived and supervised the protocol. S.L., J.-R.W. and D.X. contributed to the development of the protocol and wrote the manuscript. L.T., N.X., R.L., Y.S., Z.X., X.S. J.G. and M.X. helped with protocol optimization.
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S.L. and J.-R.W. have developed the patent for this work (no. ZL202111114666.6, Sun Yat-sen University Zhongshan Ophthalmic Center).
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Key references using this protocol
Wei, J. R. et al. Nat. Commun. 13, 6902 (2022): https://doi.org/10.1038/s41467-022-34590-1
Hao, Z. Z. et al. Nat. Neurosci. 25, 805–817 (2022): https://doi.org/10.1038/s41593-022-01073-x
Xiao, D. et al. Nat. Commun. 9, 2865 (2018): https://doi.org/10.1038/s41467-018-05209-1
Extended data
Extended Data Fig. 1 Representative figures and electrophysiological recordings demonstrating the viability of acute brain slices in the adult macaque cortex.
a, Representative images were acquired from the cortex in acute slices from adult macaques. Scale bar, 20 μm. b, The single whole-cell patch-clamp electrophysiological recordings of pyramidal and interneuron neurons showed a multi-action potential firing cell. Pyr, pyramidal neuron; InN, inhibitory neurons.
Extended Data Fig. 2 Primary culture of cells and labeling assessments from the hippocampus of adult macaque monkeys.
a, b, The neurospheres are formed from cells cultured from the hippocampus of adult macaque monkeys. Three weeks after cell seeding, NPCs elucidated spindle morphology in a. Thirty days after cell seeding in b. D, days. Scale bars, a, 40 μm; b, 160 μm. c, d, NPCs view in P1 and P4 passages; P, passages. Scale bars, c, 80 μm; d, 60 μm. e–j, Cultured NPCs form proliferating neurospheres positive for NESTIN, MKI67, PAX6, VIMENTIN, SOX4 and HMGB2. Scale bars, 40 μm.
Extended Data Fig. 3 Preparation of fire-polishing glass Pasteur pipettes.
a, The glass pipette is heated in the top part of the flame and spun evenly. b, The heated pipette is quickly drawn out. c, The sharply drawn-out edge is quickly heated to form a smooth and round edge. d–f, Example of three fire-polishing glass Pasteur pipettes with 150-μm, 350-μm, 650-μm bores. Scale bars, 200 μm.
Supplementary information
Supplementary Video 1
The morphologies of recorded neurons.
Supplementary Video 2
The morphologies of recorded neurons.
Source data
Source Data Figs. 7 and 8
Statistical source data for Figs. 7g and 8c.
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Wei, JR., Xiao, D., Tang, L. et al. Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures. Nat Protoc 18, 1930–1957 (2023). https://doi.org/10.1038/s41596-023-00820-z
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DOI: https://doi.org/10.1038/s41596-023-00820-z
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