Method for separating inner ear FZD10 positive glial cells

Abstract

The invention relates to the field of cell separation and purification, in particular to a method for separating inner ear FZD10 positive glial cells. According to the method for separating the inner ear FZD10 positive glial cells, the inner ear FZD10 positive glial cells are subjected to fluorescence labeling by virtue of transgenic mouse hybridization, so that purified inner ear glial cells can be conveniently obtained subsequently by virtue of flow sorting. According to a separation and purification technology adopted by the method, the glial cells can be obtained to the maximum extent, the activity of the cells can be kept, the purity of the separated FZD10 positive glial cells reaches 98% or above, the requirements of follow-up experiments can be met, analysis and purification process is simple and rapid, and the method is very suitable for follow-up experiment research.

Description

technical field [0001] The invention relates to the field of cell separation and purification, in particular to a method for isolating inner ear Frizzled10 (FZD10) positive glial cells. Background technique [0002] Deafness is a major global public health problem. About 500 million people around the world suffer from disabling hearing loss, accounting for 6.5% of the world's total population, and there is an upward trend. Among them, patients with sensorineural deafness accounted for about 65%. Sensorineural deafness refers to the location of the lesion in the cochlea and the auditory pathway behind it, which is mainly caused by the degeneration of cochlear hair cells and spiral neurons. Unlike conductive deafness caused by external auditory canal or middle ear lesions, a considerable part of sensorineural deafness is often permanent and difficult to reverse. The current treatment for sensorineural hearing loss mainly adopts cochlear implantation. Cochlear implants play ...

AI Technical Summary

Problems solved by technology

[0004] However, due to the complex anatomical structure of the cochlea and the limitations of research techniques, there is still a lack of methods for the isolation and purification of cochlear glial cells.

The first step in the isolation and purification of glial cells is the digestion and separation of cochlear modioli. In most previous literatures, the introduction of the digestion method of cochlear modioli in the inner ear is not perfect, such as the type, concentration, and digestion of enzymes used for digestion. Conditions, cell pipetting methods, etc. are not described in detail (Petitpre et al., 2018)

Sun et al. used a two-step digestion method in the digestion process of the snail shaft, and the digestion time of each step was as long as 30 minutes, and there were many kinds of reagents used, and the process was cumbersome

[0005] In addition, in terms of purification of glial cells, previous studies could only isolate whole modioli cells and study their differentiation into neurons

However, the cells in the modiolus include neurons, glial cells, fibroblasts and other cell types, which makes it difficult to study the proliferation and differentiation characteristics of glial cells

Some researchers take advantage of the characteristics that neurons cannot proliferate and pass on, and remove the neurons in the isolated cochlear modiolus cells by first cultured into spheres for more than three generations. Greatly prolong the experiment time and reduce the activity of glial cells to a certain extent

In addition, since fibroblasts in the inner ear also have the ability to proliferate, this method does not remove modioli fibroblasts very well (McLean, McLean, Eatock, & Edge, 2016)

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