1. A method for separating inner ear FZD10 positive glial cells is characterized by comprising the following specific steps:

(1) taking out FZD10-creER +/-/Rosa26R-tdTomato +/-double positive suckling mice of the 2 th to 4 th days after birth, dissecting out a cochlea, removing the volute to expose the internal structure of the cochlea, tearing off peripheral spiral ligaments and removing a Kitiki device, transferring the residual spiral shaft part into a centrifugal tube containing precooled Hanks liquid, and then centrifuging at 1000-1500rpm for 5-10 minutes;

(2) after the centrifugation in the step (1) is finished, removing the supernatant Hanks liquid, adding an enzymolysis liquid, placing the mixture into a cell culture box at 37 ℃ for digestion for 7-15 minutes, flicking the bottom of the centrifuge tube at intervals of two minutes to enable cochlear tissues to be more fully contacted with the enzymolysis liquid, adding an isovolumetric DMEM/F12 culture medium containing 10% FBS to stop the digestion, and centrifuging at 1500rpm for 5 minutes;

(3) after the centrifugation in the step (2) is finished, removing a supernatant, adding a fresh culture solution, holding a centrifugal tube to incline by 30 degrees, slightly blowing and beating by using a micropipettor, avoiding introducing air bubbles as much as possible, and blowing and beating for 90-120 times until no conglobate tissue can be seen by naked eyes;

(4) and (3) removing a small amount of undispersed and complete cell masses by passing the cell suspension through a 40-micron cell filter, collecting the single cell suspension, performing flow sorting, and separating to obtain the FZD10 positive glial cells.

2. The method for separating inner ear FZD10 positive glial cells according to claim 1, wherein the centrifugation in step (1) is: centrifuge at 1500rpm for 5 minutes.

3. The method for separating FZD 10-positive glial cells from inner ear of claim 1, wherein the digestion time in step (2) is 10 minutes.

4. The method for separating FZD 10-positive glial cells of the inner ear according to claim 1, wherein the enzymatic hydrolysate of step (2) comprises: 1mL of the enzymatic hydrolysate contained 10 Kunits DNAse, 1 mg-5 mg of collagenase, and the balance Hanks solution.

5. The method for separating FZD 10-positive glial cells of the inner ear according to claim 4, wherein the enzymatic hydrolysate of step (2) comprises: 1mL of the enzymatic hydrolysate contained 10 Kunits of DNAse, 4mg of collagenase, and the balance of Hanks solution.

6. The method for separating FZD 10-positive glial cells from inner ear of claim 1, wherein the fresh culture solution in step (3) consists of: DMEM/F12 medium was supplemented with N2: 10. mu.L/mL, B27: 0. mu.L/mL, ampicillin: 1 μ L/mL, human basic fibroblast growth factor hfgf was added prior to use: 10 ng/mL, insulin-like growth factor-1: 50 ng/mL, epidermal growth factor EGF: 20 ng/mL, heparan sulfate HS: 50 ng/mL.

7. The method for separating FZD 10-positive glial cells of inner ear of claim 1, wherein a 70 μm nozzle is used in the flow sorting process, the flow rate is adjusted to 5000 events/s, and a 561nm yellow-green laser is used for target sorting.

Background

Deafness is a major public health problem worldwide, and about 5 million people worldwide suffer from disabled hearing loss disorders, accounting for 6.5% of the world's general population, and rising in trend. Wherein the proportion of the patients with sensorineural deafness is about 65 percent. Sensorineural deafness refers to the location of the affected area in the cochlea and the auditory pathways behind the cochlea, which are mainly caused by the deciduation of cochlear hair cells and spiral neurons. Unlike conductive deafness caused by external auditory meatus or middle ear lesions, a significant portion of sensorineural deafness is often permanent and difficult to reverse. Currently, the treatment for sensorineural deafness mainly adopts the mode of artificial cochlea transplantation. The cochlear implant plays a role in treating hearing loss by directly electrically stimulating neurons in the inner ear, and thus the therapeutic effect of the cochlear implant largely depends on the number and quality of spiral neurons surviving in the inner ear. Recent studies on the protection and regeneration of helical neurons have been very hot.

There is a group of glial cells that have common developmental origins with neurons, including both schwann cells and satellite cells, and it has been found that these cells have the ability to self-proliferate and can be transformed into neurons in vitro and in vivo. Therefore, the method for obtaining purified cochlear glial cells from inner ear tissues to study the proliferation and neuron differentiation processes of the glial cells has great significance for inner ear regeneration studies.

However, due to the complicated anatomy of the cochlea and the limitations of research techniques, there is still no description of the method for isolating and purifying the cochlear glial cells. The first step in glial cell isolation and purification is cochlear modiolus digestion, and in most of the previous literature, the description of methods for digesting the modiolus in the inner ear is not complete, e.g., the description of the types of enzymes used for digestion, concentrations, digestion conditions, cell beating patterns, etc. is not exhaustive (Petitpre et al, 2018). Sun et al, used a two-step digestion procedure with digestion time of 30min per step, with more reagents and a cumbersome procedure.

In addition, only whole modiolar cells were isolated and studied for differentiation into neurons in previous studies with respect to the purification of glial cells. However, cells in the modiolus include neurons, glial cells, fibroblasts and other cell types, which makes it difficult to study the proliferation and differentiation properties of glial cells. Researchers use the characteristic that neurons can not be propagated and passaged, and remove neurons in isolated modiolar cells by first performing more than three generations of spherulization culture, however, since the spherulization requires 4 to 6 days, the method greatly prolongs the experimental time and reduces the activity of glial cells to a certain extent. Furthermore, because fibroblasts in the inner ear also have proliferative capacity, this method does not provide good removal of cochlear fibroblasts (McLean, easock, & Edge, 2016).

Therefore, there is a need for a simple and rapid method for separating and purifying inner ear glial cells to meet the demand for inner ear glial cell research. The difficulty in separating and purifying glial cells is mainly due to two reasons, one is that the inner ear has a complex anatomical structure: glial cells are located within the modiolus of the inner ear. The inner ear is located in the temporal bone petrography, and includes a complex and delicate double-layer labyrinth structure: outer bony labyrinth and inner membranous labyrinth. The cochlea is located in the front of the inner ear labyrinth and has a spiral structure of two and a half circles. The bony modiolus is located at the center of the cochlear capsule labyrinth and is conical. The spiral plate surrounds the bone on the worm shaft, rises spirally from the bottom to the top and extends into the bony worm pipe. Therefore, the cochlea modiolus is deep in position and hard in sclerotin, and the anatomy of the cochlea has certain technical difficulty. On the other hand, neurons, glial cells, fibroblasts and the like are closely associated in the modiolus, and the mixture of the above cells is obtained after the conventional separation and digestion of the modiolus tissue, but not only the glial cells. Glial cells in the cochlea include both schwann cells and satellite cells. The schwann cells attach tightly to the projections of the neurons to form a myelin sheath structure, which allows all nerve fibers to be surrounded by myelin sheaths. Satellite cells are distributed around and surround the cell body of neurons. Therefore, it is impossible to obtain pure glial cells directly after cochlear isolation.

Disclosure of Invention

Aiming at the problems in the prior art, the FZD10 positive glial cells in the inner ear are subjected to fluorescent labeling by utilizing transgenic mouse hybridization, so that purified inner ear glial cells can be conveniently obtained by flow sorting. The separation and purification technology adopted by the invention can obtain the glial cells to the maximum extent and keep the activity of the cells, the purity of the sorted FZD10 positive glial cells reaches more than 98%, the requirement of subsequent experiments can be met, the analysis and purification process is simple and quick, and the method is very suitable for subsequent experimental research.

The specific technical scheme of the method for separating the FZD10 positive glial cells of the inner ear comprises the following steps:

(1) taking out FZD10-creER +/-/Rosa26R-tdTomato +/-double positive suckling mice of 2 to 4 days after birth, placing the mice in a biological safety cabinet for operation after disinfection, dissecting out a cochlea, removing the volute to expose the internal structure of the cochlea, tearing off peripheral spiral ligaments and removing a Kitiki device, transferring the residual scroll part into a centrifuge tube containing Hanks liquid precooled in a refrigerator at 4 ℃, and then centrifuging at 1000-1500rpm for 5-10 minutes, preferably 1500-rpm for 5 minutes. Too high a centrifugation speed may cause cell damage, and a decrease in the centrifugation speed may increase the required centrifugation time, may cause incomplete cell sedimentation to affect the amount of cell harvest, and may delay the experiment time to affect cell activity.

Preferably, the dissecting of the cochlea specifically includes: taking 20 left and right suckling mice, removing heads, splitting from the midsagittal of the skull, removing brain parenchyma, cutting off bilateral temporal bones where the cochlea is located, quickly placing the temporal bones into a culture dish containing cold Hanks liquid, and carefully dissecting the cochlea under a dissecting microscope. The dissection time is controlled within 40 minutes as much as possible.

(2) And (2) after the centrifugation in the step (1) is finished, removing the supernatant Hanks liquid, adding an enzymolysis liquid, placing the mixture into a cell culture box at 37 ℃ for digestion for 7-15 minutes, flicking the bottom of the centrifuge tube at intervals of two minutes to enable cochlear tissues to be more fully contacted with the enzymolysis liquid, adding an isovolumetric DMEM/F12 culture medium containing 10% FBS to stop the digestion, and centrifuging at 1500rpm for 5 minutes.

The preferred digestion time is 10 minutes.

The enzymolysis solution comprises the following components: 1mL of enzymolysis solution contains 10 Kunits DNAse, 1 mg-5 mg of collagenase and the balance Hanks solution; the optimal collagenase content is 3 mg.

The dosage of the enzymolysis liquid is as follows: approximately 20 mice cochlea added 1ml of the enzymatic hydrolysate.

(3) And (3) after the centrifugation in the step (2) is finished, removing the supernatant, adding a fresh culture solution, holding the centrifuge tube to incline by 30 degrees, slightly blowing and beating by using a micropipettor, avoiding introducing air bubbles as much as possible, and blowing and beating for 90-120 times until no conglobation tissue can be seen by naked eyes.

The fresh culture solution comprises the following components: DMEM/F12 medium was supplemented with N2 (10. mu.L/mL), B27 (20. mu.L/mL), ampicillin (1. mu.L/mL), and human basic fibroblast growth factor (hBFGF, 10 ng/mL), insulin-like growth factor-1 (IGF-1, 50 ng/mL), epithelial cell growth factor (EGF, 20 ng/mL), heparan sulfate (HS, 50 ng/mL) prior to use.

The specific blowing operation is as follows: the 1mL micropipette was adjusted to 900 μ L, the tip of the gun was extended to the bottom of the liquid, gently whipped to avoid introducing air bubbles as much as possible, and whipped about 100 times until no clumped tissue was visible to the naked eye.

(4) And (3) removing a small amount of undispersed and complete cell masses by passing the cell suspension through a 40-micron cell filter, collecting the single cell suspension, performing flow sorting, and separating to obtain the FZD10 positive glial cells.

The specific flow type sorting process comprises the following steps:

1) carrying out ultraviolet disinfection and sterilization on the environment before flow type sorting, and operating the machine-mounted sorting process strictly according to a sterile technology;

2) firstly, adding 75% ethanol into a sheath liquid cylinder to wash and sterilize the sheath liquid cylinder, adding 1mL of 75% ethanol into a sample inlet tube, placing the sample inlet tube on a sample loading frame, operating for 20 minutes, taking down the sample loading tube containing the 75% ethanol, changing the sample loading tube into a sample loading tube containing 1mL of sterile PBS, and continuously operating twice to clean the residual 75% ethanol in a pipeline;

3) diluting the single cell suspension to 1000/mu L to improve the sorting yield, placing a sample loading tube containing the single cell suspension in a sample loading port, and placing two 15mL centrifuge tubes containing 10 mL culture medium at a collection tube interface to respectively collect FZD10 positive cells and FZD10 negative cells;

4) during the sorting, a 70 μm nozzle was used, the flow rate was adjusted to 5000 events/s, and yellow-green laser light of 561nm was used for the target sorting. The sorting process took approximately 30 minutes.

The FZD10 positive glial cells separated by the method are purified cochlear FZD10 positive glial cells, wherein the number of the glial cells accounts for more than 95% of the total number of the cells, and the cell inoculation survival rate is high, so that the method can be used for subsequent culture and audiological research.

The FZD10-creER +/-/Rosa26R-tdTomato +/-double positive P3 stage suckling mice used in the application can be obtained by the conventional technology in the field, and the following method can be specifically adopted:

(1) inserting a coding gene of cre enzyme and an estrogen receptor (estrogen receptor) gene into a frizzled10 promoter to construct a conditional transgenic mouse model FZD10-creERT2 heterozygous mouse;

(2) hybridizing FZD10-creERT2 heterozygous mice with Rosa26R-tdTomato homozygous mice, and obtaining double-positive FZD10-creER +/-/Rosa26R-tdTomato +/-mice, wherein all descendant mice develop normally;

(3) the stomach is perfused at the rate of 0.75mg tamoxifen (tamoxifen) given to each gram of mice, and according to the action principle of a cre-loxp system, cre recombinase in FZD10 positive cells is activated to cut off stop codes among loxp sites, so that the FZD10 positive cells and daughter cells thereof are marked by red fluorescent protein tomato.

The invention is original by adopting 1mL of Hanks solution containing collagenase with the concentration of 1-5 mg/mL and 10 Kunits/mL DNAse as the enzymolysis solution. Conventional cell separation was performed by using 0.25% pancreatin (example 4) for digestion, and the tissue cell yield was low and the damage to cell activity was greater when the pancreatin was used for digestion. In the application, 1mL of Hanks solution containing 1-5 mg/mL of collagenase and 10 Kunits/mL of DNAse is digested, the collagenase (especially 3 mg/mL of collagenase) at the concentration can digest the modiolution of the modiolution tissue mildly and has high cell yield, undegraded tissue components can form jelly adhesion cells in the process of digesting and dispersing the collagenase, the dispersion of the cells is influenced, and the DNase is added to decompose the substances and disperse the adhered cells.

The dissected scroll is placed into the solution and then placed in a 37 ℃ cell incubator (Heal Force, HF 240) to digest for 7-15 minutes (preferably 10 minutes), during which the bottom of the centrifuge tube is flicked every two minutes to make the cochlear tissue more fully contact with the enzymatic hydrolysate. The digestion time and related operation are important steps of the invention, although the operation seems to be simple, the digestion is an important step in the separation process, and if the digestion is insufficient for a short time, the cochlear cells are damaged for a long time.

After digestion, the centrifuge tube is held and inclined for 30 degrees, a 1mL micropipette is adjusted to 900 mu L of volume, the gun head is slightly blown and beaten after extending into the bottom of the liquid, and air bubbles are prevented from being introduced as much as possible. About 100 strokes down to no visible clumping of tissue. This operation is a key step in obtaining discrete single cells. Wherein in the blowing process, the blowing is carried out after the volume of the 1mL liquid transfer machine is adjusted to 900 mu L, which is an operation key point for avoiding introducing bubbles. The number of blows is about 100, less than 90 tissues can not be dispersed sufficiently, and more than 120 tissues can seriously damage the activity of cells.

In conclusion, the invention has the following beneficial effects:

I. the tissue digestion and blow beating method adopted by the invention can digest and disperse tissues to the maximum extent and obtain single cell suspension, and simultaneously avoids serious damage to the cell activity of the cochlear tissue.

II, the experimental process is simple and convenient to operate, the reagent required for digesting the cochlear tissue only comprises collagenase, DNAse, Hanks liquid and a serum-containing culture medium, the process of obtaining the single-cell suspension after the cochlear tissue is dissected out is carried out according to four steps of digestion-termination digestion-centrifugation-blowing and beating, the experimental process is shortened as much as possible in time, the experimental steps can be controlled within 20min, and the cell activity is guaranteed to the maximum extent.

And III, the sorted cells are purified cochlear FZD10 positive glial cells, and the immune staining proves that the percentage of the glial cells is more than 98 percent, and the glial cells hardly contain other cochlear cell types such as neurons or fibroblasts and the like. Subsequent accurate audiological studies can be performed using the obtained purified FZD10 positive glial cells due to the elimination of other cell types.

Drawings

FIG. 1 is a representation of the inoculated cells before and after sorting by flow after vortex elimination into single cells and a representation of the inoculated cells after sorting by flow in example 1;

FIG. 2 is a comparison of different fluorescent staining of cells after flow sorting in example 1;

wherein:

in fig. 1, reference numeral 1 is a flow sorting front mirror field of view; reference numeral 2 is the fluoroscopic field before flow sorting; reference numeral 3 is the flow sorting rear mirror field of view; the label 4 shows that the total number of cells in the field is basically consistent after flow sorting, thus the number of cells in the field is basically consistent before and after flow sorting, the spiral shaft is digested by enzymolysis and blown into single cells and then passes through a 40-micron cell filter, the spiral shaft tissue is digested into single cells after inoculation, and labels 1 and 2 show that the tomato positive cells only account for a very small proportion of the number of the spiral shaft cells before flow sorting; reference numerals 3 and 4 illustrate that more than 98% of the cells obtained after flow sorting were totato-labeled FZD10 positive cells.

In fig. 2, from left to right:

DAPI: all cells with DAPI-labeled nuclei;

tomato: FZD10 positive cells labeled with immunofluorescent protein tomato;

FZD 10: adding an antibody of FZD10 to mark positive cells of FZD 10;

merge: the combined graph of the first three graphs DAPI, Tomato, FZD 10;

it can be seen that more than 98% of the tomato-positive cells obtained after sorting showed positive staining of FZD10, confirming that purified glial cell populations were obtained after flow sorting.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention. Except as otherwise noted, the following examples were carried out using conventional techniques.

The following examples several pairs of FZD10-creER +/-/Rosa26R-tdTomato +/-double positive P3 stage suckling mice in the ratio were obtained using the following method:

(1) inserting a coding gene of cre enzyme and an estrogen receptor (estrogen receptor) gene into a frizzled10 promoter to construct a conditional transgenic mouse model FZD10-creERT2 heterozygous mouse;

(2) hybridizing FZD10-creERT2 heterozygous mice with Rosa26R-tdTomato homozygous mice, and obtaining double-positive FZD10-creER +/-/Rosa26R-tdTomato +/-mice, wherein all descendant mice develop normally;

(3) injecting tamoxifen (tamoxifen) 0.75mg per gram of mouse, and according to the action principle of cre-loxp system, cre recombinase in FZD10 positive cells is activated to cut off stop codes among loxp sites, so that FZD10 positive cells and daughter cells thereof are marked by red fluorescent protein tomato.

The specific flow sorting process in each example and comparative example was:

flow sorting procedure and parameters

1) The environment of the flow type sorting room is irradiated by ultraviolet rays half an hour before the flow type sorting, and the operation of the machine-mounted sorting process is strictly carried out according to the aseptic technique.

2) Firstly, adding 75% ethanol into a sheath liquid cylinder to wash and sterilize the sheath liquid cylinder, adding 1mL of 75% ethanol into a sample inlet tube, placing the sample inlet tube on a sample loading frame, running for 20 minutes, taking off the sample loading tube containing the 75% ethanol, changing the sample loading tube into a sample loading tube containing 1mL of sterile PBS, and continuously running twice to clean the residual 75% ethanol in the pipeline.

3) The single cell suspension was diluted to a concentration of 1000/μ L to increase the sorting yield. The loading tube containing the single cell suspension is placed at the loading port, and two 15mL centrifuge tubes containing 10 mL culture medium are placed at the interface of the collection tube to collect FZD10 positive cells and FZD10 negative cells respectively.

4) During the sorting, a 70 μm nozzle was used, the flow rate was adjusted to 5000 events/s, and yellow-green laser light of 561nm was used for the target sorting. The sorting process took approximately 30 minutes.

In the following examples:

a biosafety cabinet, sourced from Thermo Fisher Scientific, model 1300;

hanks liquid, sourced from Solarbio, model H1045;

collagenase, from Sigma, model C5138;

DNAse, sourced Stem Cell Technologies, model 07473;

10% FBS, sourced Ausbian, model VS 500T;

DMEM/F12 medium, source gibco;

b27, source invitrogen, model 17504044;

n2 from invitrogen, model 17502048;

ampicillin, sourced from Solarbio, type a 7490;

human basic fibroblast growth factor, from Sigma, model F0291;

insulin-like growth factor-1, from Sigma, type I8779;

epithelial cell growth factor, source Sigma, model E9644;

heparan sulfate, sourced from Sigma, model H4777;

pancreatin-EDTA, source Gibco, model 25200072;

FZD10 antibody, derived proteintech, model 18175-1-AP;

DAPI, sourced from Thermo Fisher Scientific, model D1306;

laminin, source Merck, model L2020;

cell filters from Falcon, model 352340;

flow cytometric sorter, source BD, model FACSAria iii;

cell culture box, sourced from Heal Force, model HF 240.

Example 1

(1) Taking 20 postnatal 3-day-old FZD10-creER +/-/Rosa26R-tdTomato +/-double-positive suckling mice, spraying alcohol for disinfection, placing the mice in a biological safety cabinet for operation, removing the heads of the mice, splitting the mice from the midsagittal of the cranium, removing parenchyma of the brain, cutting off bilateral temporal bones where the cochlea is located, quickly placing the temporal bones into a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under a dissecting microscope, removing the volute to expose the internal structure of the cochlea, carefully tearing off peripheral spiral ligaments and removing a kirt organ, and transferring the residual spiral shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.

(2) Preparing an enzymolysis liquid required by digesting the scroll in advance: 1mL of the enzymatic hydrolysate contained 10 Kunits of DNAse and 3mg of collagenase, and the balance Hanks solution. After centrifugation is finished, the supernatant Hanks solution is discarded, 1mL of enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 10 minutes, the bottom of the centrifuge tube is flicked at intervals of two minutes so that cochlear tissues are more fully contacted with the enzymolysis solution, the same volume of DMEM/F12 culture medium containing 10% FBS is added to stop digestion, and the mixture is centrifuged at 1500rpm for 5 minutes.

(3) And (3) after the centrifugation in the step (2) is finished, removing a supernatant, adding a fresh culture solution, holding a 15mL centrifuge tube with the left hand, inclining by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, slightly blowing and beating, and avoiding introducing air bubbles as much as possible. Blow and beat 100 times until no conglomeration tissue can be seen with naked eyes.

The fresh culture solution is as follows: DMEM/F12 medium was supplemented with N2 (10. mu.L/mL), B27 (20. mu.L/mL), ampicillin (1. mu.L/mL), and human basic fibroblast growth factor (hBFGF, 10 ng/mL), insulin-like growth factor-1 (IGF-1, 50 ng/mL), epithelial cell growth factor (EGF, 20 ng/mL), heparan sulfate (HS, 50 ng/mL) prior to use.

(4) The cell suspension was passed through a 40 μm cell filter to remove a small amount of undispersed and incomplete cell clumps, and the single cell suspension was collected and immediately flow sorted.

The total number of modiolus unicells separated by the digestion and single cell separation method is 200 ten thousand, and the number of FZD10 positive cells obtained after separation is 7 ten thousand.

Cell culture and identification after flow sorting:

1) FZD10 positive cells were seeded at a concentration of 50/μ L in laminin-coated 48-well culture plates (laminin was coated by dissolving in PBS at a ratio of 1: 500), and 200 μ L, i.e., 10000 cells, were seeded per well. Place the Petri dish in a 5% CO solution₂At 37 ℃ for 24 hours.

2) Counting the number of adherent cells under a light mirror and calculating the adherent survival rate of the cells; the FZD10 positive glial cell rate was calculated using cell counts and immunofluorescent staining.

After the culture is finished, the number of adherent cells in each hole of the culture plate is 7890-8350 by counting under a light microscope, and the average number is 8047, so the adherent survival rate of the cells cultured at this time is 8047/10000 × 100% = 80.47%.

The immunofluorescence staining uses a primary antibody which is an anti-FZD 10 antibody, adds a corresponding secondary antibody and DAPI to incubate, uses a confocal microscope to observe after mounting, and respectively counts the number of FZD10 positive cells and the number of DAPI positive cells in each culture well. The number of the FZD10 positive cells in each hole is 7050-7960, the average 7487, the number of the DAPI positive cells is 7210-8100 and the average 7637, so that the FZD10 positive glial cell rate of the cells cultured at this time is 7487/7637 x 100% = 98.04%.

The FZD10 positive glial cells separated by the method are purified cochlear FZD10 positive glial cells, wherein the number of the FZD10 positive glial cells accounts for 98.04% of the total cells, and the cell adherence survival rate reaches 80.47%, so that the FZD10 positive glial cells can be used for subsequent culture and audiological research.

Example 2

(1) The method comprises the steps of taking 20 FZD10-creER +/-/Rosa26R-tdTomato +/-double positive suckling mice two days after birth, spraying alcohol for disinfection, placing the mice in a biological safety cabinet for operation, removing the heads of the mice, splitting the mice from the median sagittal of the skull, removing brain parenchyma, cutting off bilateral temporal bones where the cochlea is located, quickly placing the temporal bones into a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under a dissecting microscope, removing the spiral casing to expose the internal structure of the cochlea, carefully tearing off peripheral spiral ligaments and removing a kirt organ, and transferring the residual spiral shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.

(2) Preparing an enzymolysis liquid required by digesting the scroll in advance: 1mL of the enzymatic hydrolysate contained 10 Kunits of DNAse and 1mg of collagenase, and the balance Hanks solution. After centrifugation is finished, the supernatant Hanks solution is discarded, 1mL of enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 15 minutes, the bottom of the centrifuge tube is flicked at intervals of two minutes so that cochlear tissues are more fully contacted with the enzymolysis solution, the same volume of DMEM/F12 culture medium containing 10% FBS is added to stop digestion, and the mixture is centrifuged at 1000rpm for 10 minutes.

(3) And (3) after the centrifugation in the step (2) is finished, removing a supernatant, adding a fresh culture solution, holding a 15mL centrifuge tube with the left hand, inclining by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, slightly blowing and beating, and avoiding introducing air bubbles as much as possible. Blow 120 times until no clumped tissue is visible to the naked eye.

The fresh culture solution is as follows: DMEM/F12 medium was supplemented with N2 (10. mu.L/mL), B27 (20. mu.L/mL), ampicillin (1. mu.L/mL), and human basic fibroblast growth factor (hBFGF, 10 ng/mL), insulin-like growth factor-1 (IGF-1, 50 ng/mL), epithelial cell growth factor (EGF, 20 ng/mL), heparan sulfate (HS, 50 ng/mL) prior to use.

(4) The cell suspension was passed through a 40 μm cell filter to remove a small amount of undispersed and incomplete cell clumps, and the single cell suspension was collected and immediately flow sorted.

The total number of modiolus unicells separated by the digestion and single cell separation method is 150 ten thousand, and the number of FZD10 positive cells obtained after separation is 5 ten thousand.

Cell culture and identification after flow sorting:

1) FZD10 positive cells were seeded at a concentration of 50/μ L in laminin-coated 48-well culture plates (laminin was coated by dissolving in PBS at a ratio of 1: 500), and 200 μ L, i.e., 10000 cells, were seeded per well. Place the Petri dish in a 5% CO solution₂At 37 ℃ for 24 hours.

2) Counting the number of adherent cells under a light mirror and calculating the adherent survival rate of the cells; the FZD10 positive glial cell rate was calculated using cell counts and immunofluorescent staining.

After the culture is finished, the number of adherent cells in each hole of the culture plate is 6200-7600 counted under a light microscope, and 7037 adherent cells are averagely counted, so that the adherent survival rate of the cells cultured at this time is 7037/10000 x 100% = 70.37%.

The immunofluorescence staining uses a primary antibody which is an anti-FZD 10 antibody, adds a corresponding secondary antibody and DAPI to incubate, uses a confocal microscope to observe after mounting, and respectively counts the number of FZD10 positive cells and the number of DAPI positive cells in each culture well. The number of the FZD10 positive cells in each well is 5450-6830, average 6290, the number of the DAPI positive cells is 5620-6900, and average 6423, so that the FZD10 positive glial cell rate of the cells cultured at this time is 6290/6423 x 100% = 97.92%.

The FZD10 positive glial cells separated by the method are purified cochlear FZD10 positive glial cells, wherein the number of the FZD10 positive glial cells accounts for 97.92% of the total cells, and the cell adherence survival rate is 70.37%.

Example 3

(1) The method comprises the steps of taking 20 FZD10-creER +/-/Rosa26R-tdTomato +/-double positive suckling mice three days after birth, spraying alcohol for disinfection, placing the mice in a biological safety cabinet for operation, removing the heads of the mice, splitting the mice from the median sagittal of the skull, removing brain parenchyma, cutting off bilateral temporal bones where the cochlea is located, quickly placing the temporal bones into a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under a dissecting microscope, removing the spiral casing to expose the internal structure of the cochlea, carefully tearing off peripheral spiral ligaments and removing a Kitik machine, and transferring the residual spiral shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.

(2) Preparing an enzymolysis liquid required by digesting the scroll in advance: 1mL of the enzymatic hydrolysate contained 10 Kunits of DNAse and 5mg of collagenase, and the balance Hanks solution. After centrifugation is finished, the supernatant Hanks solution is discarded, 1mL of enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 7 minutes, the bottom of the centrifuge tube is flicked at intervals of two minutes so that cochlear tissues are more fully contacted with the enzymolysis solution, the same volume of DMEM/F12 culture medium containing 10% FBS is added to stop digestion, and the mixture is centrifuged at 1500rpm for 5 minutes.

(3) And (3) after the centrifugation in the step (2) is finished, removing a supernatant, adding a fresh culture solution, holding a 15mL centrifuge tube with the left hand, inclining by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, slightly blowing and beating, and avoiding introducing air bubbles as much as possible. Blow and beat for 90 deg.C until no conglomeration tissue is visible to the naked eye.

The fresh culture solution is as follows: DMEM/F12 medium was supplemented with N2 (10. mu.L/mL), B27 (20. mu.L/mL), ampicillin (1. mu.L/mL), and human basic fibroblast growth factor (hBFGF, 10 ng/mL), insulin-like growth factor-1 (IGF-1, 50 ng/mL), epithelial cell growth factor (EGF, 20 ng/mL), heparan sulfate (HS, 50 ng/mL) prior to use.

(4) The cell suspension was passed through a 40 μm cell filter to remove a small amount of undispersed and incomplete cell clumps, and the single cell suspension was collected and immediately flow sorted.

The total number of modiolus unicells separated by the digestion and single cell separation method is 200 ten thousand, and the number of FZD10 positive cells obtained after separation is 7 ten thousand.

Cell culture and identification after flow sorting:

1) FZD10 positive cells were seeded at a concentration of 50/μ L in laminin-coated 48-well culture plates (laminin was coated by dissolving in PBS at a ratio of 1: 500), and 200 μ L, i.e., 10000 cells, were seeded per well. Place the Petri dish in a 5% CO solution₂At 37 ℃ for 24 hours.

2) Counting the number of adherent cells under a light mirror and calculating the adherent survival rate of the cells; the FZD10 positive glial cell rate was calculated using cell counts and immunofluorescent staining.

After the culture is finished, the number of adherent cells in each hole of the culture plate is 5500-6400 and 5933 on average under a light microscope, so the adherent survival rate of the cells cultured this time is 5933/10000 × 100% = 59.33%.

The immunofluorescence staining uses a primary antibody which is an anti-FZD 10 antibody, adds a corresponding secondary antibody and DAPI to incubate, uses a confocal microscope to observe after mounting, and respectively counts the number of FZD10 positive cells and the number of DAPI positive cells in each culture well. The number of FZD10 positive cells in each well is 4740-.

The FZD10 positive glial cells separated by the method are purified cochlear FZD10 positive glial cells, wherein the number of the FZD10 positive glial cells accounts for 97.90% of the total cells, and the cell adherence survival rate is 59.33%.

Comparative example 1

(1) The method comprises the steps of taking 20 FZD10-creER +/-/Rosa26R-tdTomato +/-double positive suckling mice three days after birth, spraying alcohol for disinfection, placing the mice in a biological safety cabinet for operation, removing the heads of the mice, splitting the mice from the median sagittal of the skull, removing brain parenchyma, cutting off bilateral temporal bones where the cochlea is located, quickly placing the temporal bones into a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under a dissecting microscope, removing the spiral casing to expose the internal structure of the cochlea, carefully tearing off peripheral spiral ligaments and removing a Kitik machine, and transferring the residual spiral shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.

(2) After centrifugation is finished, the supernatant Hanks solution is discarded, 1mL of enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 10 minutes, the bottom of the centrifuge tube is flicked at intervals of two minutes so that cochlear tissues are more fully contacted with the enzymolysis solution, the same volume of DMEM/F12 culture medium containing 10% FBS is added to stop digestion, and the mixture is centrifuged at 1500rpm for 5 minutes.

The enzymolysis solution comprises the following components: 1mL of the enzymatic hydrolysate contained 10 Kunits DNAse and 0.25% of pancreatin-EDTA, and the balance Hanks solution.

(3) And (3) after the centrifugation in the step (2) is finished, removing a supernatant, adding a fresh culture solution, holding a 15mL centrifuge tube with the left hand, inclining by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, slightly blowing and beating, and avoiding introducing air bubbles as much as possible. Blow and beat 100 times until no conglomeration tissue can be seen with naked eyes. The fresh culture solution is as follows: DMEM/F12 medium was supplemented with N2 (10. mu.L/mL), B27 (20. mu.L/mL), ampicillin (1. mu.L/mL), and human basic fibroblast growth factor (hBFGF, 10 ng/mL), insulin-like growth factor-1 (IGF-1, 50 ng/mL), epithelial cell growth factor (EGF, 20 ng/mL), heparan sulfate (HS, 50 ng/mL) were added prior to use.

(4) The cell suspension was passed through a 40 μm cell filter to remove a small amount of undispersed and incomplete cell clumps, and the single cell suspension was collected and immediately flow sorted.

The total number of modiolus unicells separated by the digestion and single cell separation method is 150 ten thousand, and the number of FZD10 positive cells obtained after separation is 5 ten thousand.

Cell culture and identification after flow sorting:

1) FZD10 positive cells were seeded at a concentration of 50/μ L in laminin-coated 48-well culture plates (laminin was coated by dissolving in PBS at a ratio of 1: 500), and 200 μ L, i.e., 10000 cells, were seeded per well. Place the Petri dish in a 5% CO solution₂Culturing in a cell culture box at 37 DEG CAnd culturing for 24 hours.

2) Counting the number of adherent cells under a light mirror and calculating the adherent survival rate of the cells; the FZD10 positive glial cell rate was calculated using cell counts and immunofluorescent staining.

After the culture is finished, the number of adherent cells in each hole of the culture plate is 4830-5250, and the average is 5096, so the adherent survival rate of the cells cultured this time is 5096/10000 × 100% = 50.96%.

The immunofluorescence staining uses a primary antibody which is an anti-FZD 10 antibody, adds a corresponding secondary antibody and DAPI to incubate, uses a confocal microscope to observe after mounting, and respectively counts the number of FZD10 positive cells and the number of DAPI positive cells in each culture well. The number of FZD10 positive cells in each well is 4630-4980, average 4830, the number of DAPI positive cells is 4700-5100, and average 4937, so that the FZD10 positive glial cell rate of the cells cultured at this time is 4830/4937 x 100% = 97.84%.

The FZD10 positive glial cells separated by the method are purified cochlear FZD10 positive glial cells, wherein the number of the FZD10 positive glial cells accounts for 97.84% of the total cells, and the cell adherence survival rate is only 50.96%.