Increasing cellular lifespan with a flow system in organotypic culture of the Laterodorsal Tegmentum (LDT)

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.

A living ex vivo platform for functional, personalized brain cancer diagnosis

“…To generate this rapid, high-fidelity ex vivo testing platform, we first assessed OBSC quality and reproducibility, optimizing the process using quantitative fluorescence-based imaging methods. After piloting several assays, we adapted an established nuclear permeability assay using propidium iodide 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 (PI; catalog #P4170; Sigma-Aldrich) to quantify cell death within OBSCs. This method provided a large dynamic range to detect nuanced differences between healthy and unhealthy OBSCs, with higher fluorescence values indicating more dead cells ( Figure 1 A).…”

Glutamatergic Mechanisms in Glioblastoma and Tumor-Associated Epilepsy

“…Here, we show that C6-bearing slices based on brain tissue from 6–8 days-old Fischer rats with C6 cells also represent a feasible tool to investigate the network activity ( Figure 3 A). A high synaptic synchrony of cortical neurons that declines as the incubation progresses was estimated, which was expected for slice cultures without glioma [ 183 ].…”

Devices and techniques used to obtain and analyze three‐dimensional cell cultures

“…This approach was used to demonstrate the long-time survival of the latero-dorsal tegmental (LDT) nucleus of brain stem nucleus. 31 F I G U R E 3 Culture devices which integrate microfluidics technology include, droplet, centrifugal, lab-on-achip, organ-on-a-chip, digital, microelectromechanical system (MEMS), PDMS-based and paperbased platforms. Overall, these devices enable easy fabrication, increased sustenance, increased sensitivity to external stimuli, and recapitulation of dynamic cellular microenvironment.…”

“…The incorporation of automated tube flow system into a conventional semi‐permeable membrane enhanced perfusion of oxygen and nutrient supply, waste removal, and maintenance of temperature. This approach was used to demonstrate the long‐time survival of the latero‐dorsal tegmental (LDT) nucleus of brain stem nucleus 31 …”