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. 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 days, and are amenable to electrophysiological recording, calcium imaging, and viral gene transfer. The entire process of extraction and culturing can be completed in less than 7 hours, and 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 provides an invaluable resource for improving translational research.

Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch

“…Thus, we obtained viable sensory neurons from human cadaveric donors and stimulated these cells with IL-4 (Valtcheva et al, 2016). Similar to our observations in mice (Figure 1D), we found that a subset of human sensory neurons directly responded to IL-4 (Figure 1H).…”

“…After hDRG extraction, fat, dura, and connective tissues were removed as previously described (Valtcheva et al, 2016). The hDRG were subsequently stored in RNAlater (Sigma-Aldrich) at −80°C until RNA isolati on was performed.…”

Group II mGluRs suppress hyperexcitability in mouse and human nociceptors

“…and prepared as described previously [14]. Detailed protocols for the preparation and experimental use of human sensory neurons are available [47]. Briefly, the DRGs were dissected to remove connective tissue and fat.…”

Primary Postnatal Dorsal Root Ganglion Culture from Conventionally Slaughtered Calves

“…Loss of pseudounipolarity has been described also in cultured DRG neurons from rat, mice and rabbit and have been attributed to different factors including the removal of satellite cells, which prevent dendritic formation in vivo [8], the type of GF supplementation [57] and activation of transcription factors associated to the regenerative state of dissociated neurons [58]. As in DRG cultures from other species [47, 59] postnatal bovine DRG neurons still had significant numbers of ensheathing cells attached, which does not support the removal of satellite cells as the cause for multipolarity. In contrast, neurite outgrowth, arborisation and elongation were modulated under certain GF supplementations suggesting that presence of GF is involved in neuronal morphology and loss of neuronal pseudounipolarity.…”