Neurobiology and Cultivation of Olfactory Receptor Neurons on a Chip - Bioelectronic Nose: Integration of Biotechnology and Nanotechnology - page 99

6.3 Current Knowledge Regarding in vitro ORN

Cultivating Methods

As mentioned, the initial attempt of the ORN cultivation was conducted in 1984 by
Noble et al. [ 44 ]. Before this attempt, cell suspension techniques had been widely
used for dissociation of intact ORNs. Although these techniques did not provide
sufficient quality ORNs for olfactory-specific experiments, they did contribute to
the development of advanced ORN cultivating methods. In 1991, Ronnett et al. in-
troduced a primary ORN cultivation method that provided almost pure ORNs [ 45 ].
However, ORNs cultured using this method rarely survived beyond culture day 7,
prompting researchers to attempt the engineering of ORN cell lines.

6.3.1 Development of ORN Cultivation Methods

Before ORN primary culture was introduced, several attempts to collect pure popu-
lations of ORNs had been made. Initial efforts pioneered by Kowen et al. in 1966
involved dispersing olfactory cells using mechanical, chemical, and enzymatic
treatments to make cell suspensions [ 46 ]. Hirsch and Margolis attempted to use
similar methods with some modification, primarily the dissociation of cells by cen-
trifugation with a bovine serum albumin gradient [ 47 ]. These pioneering attempts
were critical, as they represented some of the earliest steps in the progression to
biological artificial electric nose development. However, the ORN yield achieved
using these techniques was too low to advance biomimetic artificial nose develop-
ment. Another method employed to dissociate OE cells used N-ethylmaleimide.
Unfortunately, this method also deactivated the electrical excitability of cells and
therefore could not be used to contribute to future studies.
Initial ORN cultivation methods used only isolated OE in vitro . In 1984, Noble
et al. employed purified astrocytes to support OE neurons [ 44 ]. Gonzales et al.
also succeeded in cultivating OE cells in 1985 [ 48 ]. The results of these studies
allowed not only for measurable growth of olfactory neurons in vitro , but also for
more advanced study of the mechanisms of olfactory signal transduction. However,
these ORN cultivation methods were suboptimal, as cultivation of isolated OE cells
without ORN purification provided ORN yields unsuitable for biochemical experi-
ments. Additionally, cultivated cells were associated with poor survival times. Fur-
thermore, the heterogeneous cell populations derived using these methods impaired
the ability to study olfactory signal transduction with precision.
Ronnett et al. established a cultivation method which yielded a nearly pure popu-
lation of ORNs in 1991 [ 45 ]. This method consisted of mechanical disruption, en-
zymatic dissociation, and a series of filtrations, followed by incubation in medium
conducive to neuronal survival. Thus, this technique overcame shortcomings of
other methodologies regarding purity and yield of ORNs in culture. Most impor-
tantly, the development of this primary ORN culture, which could respond to odor-
ous chemicals and evoke electronic signals, allowed for further research regarding
olfactory signal transduction at the cellular and molecular levels.