Abstract
Organotypic brain culture is an experimental tool widely used in neuroscience studies. One major drawback of this technique is reduced neuronal survival across time, which is likely exacerbated by the loss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into the commonly-used, standard semi-permeable membrane culture methodology which has significantly enhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states: the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removes waste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Using immunohistochemistry and electrophysiology, our system was found superior to standard techniques in preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provide evidence for the first time that the LDT can be preserved in organotypic slice culture, and further, our technical improvements of adding a flow system, which likely enhanced perfusion to the slice, were associated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypic experiments focused on chronic stimulations and multielectrode recordings in the LDT, as well as enhance neuronal survival in slice cultures originating from other brain regions.
Highlights
Cell culture techniques that allow in-vitro investigations are essential tools for studying cellular processes, and have been used extensively, among other applications, in examination of neuropharmacological effects of stimulation of receptors by natural and synthetic compounds
Our team has designed a flow system for the culture of organotypic mouse brain stem slices, which was shown in validation tests to improve cell survival for up to 14 days, according to those established by the cell survival tests (MTT, DAPI/propidium iodide (PI)), electrophysiology and immunohistochemistry
Our results indicated that the constant circulation of culture medium at a low speed was associated with a longer lifespan of cells of the brain stem in organotypic culture
Summary
Cell culture techniques that allow in-vitro investigations are essential tools for studying cellular processes, and have been used extensively, among other applications, in examination of neuropharmacological effects of stimulation of receptors by natural and synthetic compounds. Organotypic culture of brain slices is an ex-vivo technique, which can be used to promote long-term neuronal survival, thereby allowing mid- to long-term manipulations in a preparation in which the gross cytoarchitecture of neuronal cell groups remains preserved. The technique of organotypic culturing of brain slices was first described in the late 1940s, in which slices were cultured on glass coverslips using the roller-tube method[5] This opened the door for deeper investigative neurobiological studies and promoted the development of technical improvements which resulted in adequate neuronal survival, preservation of tissue architecture, as well as persistent neuronal interactions with other cell groups, such as glial cells, e.g. astrocytes[6]. As slices in organotypic culture do not have a circulatory system that facilitates the entry of nutrients and the exit of tissue waste[12], perfusion systems must be utilised which allow regular change of the culture medium with a turnover time of at most 2 days
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
