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Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures

Abstract

Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research.

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Figure 1: Surgical extraction of hDRG using ventral approach.
Figure 2: Cleaning of hDRG before dissociation.
Figure 3: Dissociated hDRG neurons over time in vitro.
Figure 4: Immunocytochemical analysis of cultured hDRG neurons.
Figure 5: Calcium imaging of human sensory neurons for the characterization of population responses to algogens and pruritogens.
Figure 6: Long-term culture with neurotrophic factors does not alter hDRG excitability.
Figure 7: Viral transduction of human sensory neurons for expression of optogenetic tools to manipulate neuronal firing.

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Acknowledgements

We thank the donors and their families for their generous donations to science. We thank Mid-America Transplant for providing access to donor tissue and their facilities, and especially P. Silva and J. Lemen for their tremendous help coordinating and performing hDRG extractions. We thank A. Vannucci and Y. Lin for helpful discussions and guidance in establishing the research collaboration with Mid-America Transplant. We thank O. Crisp from the Washington University Pathology Department for demonstrating DRG removal using the autopsy saw. We thank J. Sinn-Hanlon for generating the illustrations in Figure 1. We thank D. Brenner for helpful discussions on the development of extraction procedures. This work was supported by US National Institute of Neurological Disorders and Stroke grants R01NS042595 (R.W.G.), F31NS089130 (M.V.V.), F32NS076324 (S.D.), and T32GM108539 (B.A.C.) and T32GM007067-41 (M.Y.P.) from the US National Institute of General Medical Sciences.

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M.V.V., S.D., B.A.C. and K.D. developed the surgical approach. M.V.V., S.D., B.A.C. and T.D.S. optimized existing rodent culturing protocol to establish viable hDRG cultures. M.V.V., B.A.C., T.D.S., M.Y.P. and J.G.M. contributed to applications including immunocytochemistry, calcium imaging, electrophysiology and optogenetic experiments. All authors contributed to writing and editing the manuscript.

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Correspondence to Robert W Gereau IV.

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Valtcheva, M., Copits, B., Davidson, S. et al. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures. Nat Protoc 11, 1877–1888 (2016). https://doi.org/10.1038/nprot.2016.111

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