Abstract
Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.
Highlights
Peripheral nerve injuries are common with approximately 9,000 cases in the UK occurring each year in a predominantly young and working population[1]
Schwann cells (SC)-like Adipose-derived stem cells (ASC) are able to produce growth factors and chemokines[19] which can improve the ability of Dorsal root ganglia (DRG) neurons to sprout neurites when they are released in the culture medium
This protocol (Figure 1) illustrates the procedure to perform a direct co-culture of SC-like ASC and DRG neurons, during which neuronal cells get in direct contact with previously seeded SC-like ASC
Summary
Peripheral nerve injuries are common with approximately 9,000 cases in the UK occurring each year in a predominantly young and working population[1]. Despite microsurgical nerve repair techniques, normal restoration of function is unattainable with resulting impaired hand sensation, reduced motor function and frequent pain and cold intolerance[2] Such injuries have a profound and permanent impact on the patient and their ability to perform activities of daily living, with less than 60 % returning to work[3]. The nerve is divided into proximal and distal stumps; the proximal stump being the point from which the regenerative process takes place, whilst the distal stump undergoes Wallerian degeneration whereupon the SC detach from the injured axons, de-differentiate and proliferate This is fundamental towards removing myelin debris and preparing the distal stump for nerve re-generation 4,5. SC form the new myelin sheath wrapping the regenerated axons, but sensory and motor function is only partially restored[8]
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