Hydrogel Skin-Covered Neurons Self-Assembled with Gustatory Cells for Selective Taste Stimulation

Abstract

Many technical challenges exist in the co-culture of multiple types of cells, including medium optimization, cell-to-cell connection, and selective data acquisition of cellular responses. Particularly, mixed cellular responses limit the precise interpretation of intercellular signal transduction. Here, we report the formation of an agarose gel skin on neurons closely assembled with gustatory cells to selectively stimulate gustatory cells by retarding the diffusion of tastants to neurons. The signal transmission, triggered by denatonium benzoate, from gustatory cells to neurons was monitored using intracellular calcium ion concentrations. The agarose gel skin efficiently suppressed the direct transfer of tastants to neurons, decreasing the number of responsive neurons from 56 to 13% and the number of calcium ion signals per neuron from multiple to single. The assembly of neurons with gustatory cells induced the high level of neuronal responses through taste signal transduction from gustatory cells to neurons. However, the calcium ion signal peaks of free neurons coated with agarose gel were much shorter and weaker than those of neurons closely assembled with gustatory cells. This work demonstrated that agarose gel skin is a simple, fast, and effective means to increase the signal selectivity of cellular responses in the co-culture of multiple types of cells.

Artificial Taste Buds: Bioorthogonally Ligated Gustatory–Neuronal Multicellular Hybrids Enabling Intercellular Taste Signal Transmission

“…The time gap of Ca 2+ signals in the gustatory− neuronal cell hybrids prepared by the click chemistry was 10− 40 s but 25−75 s in the DNA-assisted cell assemblies. 27 Overall, Ca 2+ signaling analysis indicates that the clickchemistry-assisted gustatory−neuronal cell hybrids exhibit a higher number and frequency of signal transmission with the prolonged signal transmission activity.…”

“…Primary gustatory and neuronal cells were isolated and cultured according to our previous reports. 26,27 Briefly, hippocampal neurons were taken from the brain of rat embryos and cultured on poly-D-lysine (PDL) and laminin-coated surfaces. About 6− 8% of astrocytes are known to exist in the murine hippocampus, 34 but the growth of non-neuronal cells was effectively suppressed in the absence of growth factors as previously demonstrated.…”

“…The different signaling characteristics were in line with our previous works. 26,27 The co-cultured gustatory−neuronal hybrid cell clusters exhibited distinctive responses with multiple peaks.…”

“…The duration of signaling, which is the total time between the first and last signals, in the neurons assembled with gustatory cells was much longer than that of the monocultured neurons: ∼300 s for click-chemistry-assisted multicellular assemblies but <150 s for DNA-assisted ones. 27 The average time gap between signal peaks indicates the rate of exchanging Ca 2+ concentrations, corresponding to the signal transmission rate from gustatory cells to neurons. The neurons assembled with gustatory cells had a much shorter peak-to-peak time gap than the unbound neurons, indicating the fast taste signal transmission.…”

“…30−32 Our recent works successfully tracked taste signal transmission between gustatory cells and neurons by generating a DNA-assisted synaptic gap. 26,27 However, the signal frequency and transmission speed were very low due to the limitation of DNA hybridization for cellular assembly. Both gustatory and neuronal cells were modified with a DNA−lipid conjugate, consisting of a single-stranded oligonucleotide (ssODN, 20 bases), poly(ethylene glycol) (PEG), and a phospholipid, as a linker between the cells.…”