The Organotypic Longitudinal Spinal Cord Slice Culture for Stem Cell Study

Abstract

The objective of this paper is to describe in detail the method of organotypic longitudinal spinal cord slice culture and the scientific basis for its potential utility. The technique is based on the interface method, which was described previously and thereafter was modified in our laboratory. The most important advantage of the presented model is the preservation of the intrinsic spinal cord fiber tract and the ventrodorsal polarity of the spinal cord. All the processes occurring during axonal growth, regeneration, synapse formation, and myelination could be visualized while being cultured in vitro for up to 4-5 weeks after the slices had been isolated. Both pups and adult animals can undergo the same, equally efficient procedures when going by the protocol in question. The urgent need for an appropriate in vitro model for spinal cord regeneration results from a greater number of clinical trials concerning regenerative medicine in the spinal cord injury and from still insufficient knowledge of the molecular mechanisms involved in the neuroreparative processes. The detailed method of organotypic longitudinal spinal cord slice culture is accompanied by examples of its application to studying biological processes to which both the CNS inhabiting and grafted cells are subjected.

In Vitro Models of Spinal Cord Injury

“…It is important to start the concrete experiments about 12-14 days after the beginning of OTS cultivation [24]. In some studies, the time of OTS culture before starting the actual experiment is shorter (1 week) [25,26]; so the mechanical damage caused by slicing disappears, and the cells can reach a more advanced developmental stage. In addition, this time period will allow the slices to acclimatize to the culturing conditions and mature.…”

“…Early postnatal periods (P0-P9) are ideally suited for culturing [26,39,42]. Since most diseases of the spinal cord concern the adults, some organotypic spinal cord slice culture models from grown rats were established [25,26].…”

“…The slice culture survives about 7 days maintaining proper parallel fiber tract architecture. Also, the constant high vitality and tissue organization are preserved up to 4-5 weeks in neonatal slices, while the vitality of adult slice cultures decreases significantly upon the first 5 days of cultivation [26]. On the contrary, spinal cord organotypic culture from adult mouse can be used to produce neurospheres that can be further differen-tiated into astrocytes and neuronal cells of different phenotypes.…”

Cell viability in three ex vivo rat models of spinal cord injury

“…; Sypecka et al. ). The organotypic slice culture (OSC) model appears to reproduce much of the post‐traumatic events following injury, including acute and secondary injury mechanisms.…”

“…Cell death is one of the consequences of traumatic SCI, affecting both neurons and supporting glial cells (Beattie et al 2002). Numerous studies have reported spinal cord tissue samples isolated from embryonic or newborn rats (Krassioukov et al 2002;Cho et al 2009;Kim et al 2010;Ravikumar et al 2012;Gerardo-Nava et al 2013Pinkernelle et al 2013;Hashemian et al 2014;Weightman et al 2014;Pohland et al 2015;Sypecka et al 2015). The organotypic slice culture (OSC) model appears to reproduce much of the post-traumatic events following injury, including acute and secondary injury mechanisms.…”