CN112553144B - Separation and in-vitro culture method of mouse cochlear spiral organ - Google Patents

Abstract

The invention relates to a separation and in-vitro culture method of a mouse cochlear spiral organ. Gap connexin 30 and antioxidant phytic acid were used for in vitro culture of cochlear helices. The phytic acid and the rat tail gum are combined for use to prepare collagen gel for fixing a basement membrane, and the gap junction protein 30 is used as a supplement component of a culture medium, the cochlear helicopters of the mice are cultured in vitro, and the obtained helicopters hair cells are good in shape, good in activity and long in survival time. The steps of the method for separating the mouse cochlear spiral organ are simple and easy to implement and convenient to operate. The cochlear spiral organ is dyed by adopting an immunostaining marking Myo7a method in the culture process, the dyeing effect is good, and the growth state of hair cells can be clearly observed.

Description

Separation and in-vitro culture method of mouse cochlear spiral organ

Technical Field

The invention relates to the field of cell culture, in particular to a method for separating and culturing cochlear hair cells of mice in vitro.

Background

The basis of human hearing function is the auditory sense organ, the spiral organ, also known as the organ of Corti, present in the cochlea of the temporal bone. The spiral organ contains special sensory epithelial cells, also called hair cells, which can convert the acoustic energy transmitted to the basement membrane of the inner ear through the outer ear and middle ear into nerve impulses to transmit to the brain.

The inner ear is deeply embedded in the hard bone of the temporal bone rock, which makes it impossible to directly perform in vivo observation and photography, and also makes it difficult to take it out completely without being affected by the surrounding bone, which brings many difficulties to the study of the inner ear. However, the in vitro culture of the inner ear model system established by the in vitro tissue culture method allows many experimental studies that are subject to in vivo condition limitations or cannot be performed in vivo. In addition, since most of the inner ear lesions (e.g., ototoxic drugs, vocal damage, etc.) occur after birth and the inner ear tissues are very different between the individuals in the embryonic period and after birth, it is more practical to culture mature inner ear organs and use them for the study of inner ear diseases. Based on the anatomical characteristics of the inner ear, the culture method of the spiral organ tissue has unique superiority in the research of the inner ear, and is an ideal otology experiment modeling method.

In the in vitro culture test, a mouse is generally used as an experimental object, and in order to obtain a stable experimental material, the in vitro culture of the spiral organ containing the cochlear hair cells of the mouse is required, so that the in vitro cultured cells can be quickly obtained, and the biological activity of the spiral organ can be ensured under the in vitro condition. However, the cochlea of the mouse is small, and the difficulty of cell separation and culture is high, so that the method which is simple in separation, high in culture success rate and high in cell survival rate is very necessary.

The cochlear helical organ is attached to the basement membrane, the cochlear helical organ and the basement membrane are difficult to separate, the cochlear helical organ is not separated by in vitro tissue culture, and the in vitro culture of the cochlear helical organ is culture of the basement membrane containing the cochlear helical organ. The existing cochlear spiral organ in-vitro culture method comprises a three-dimensional culture method which is closer to the in-vivo environment than an adherence culture method. However, the existing three-dimensional culture method still has a plurality of defects, and the cochlear spiral organ cells obtained by the culture method have the problems of poor shape, poor activity, short survival time and the like. The maintenance time of the shape of inner ear hair cells of a cochlear spiral organ cultured in vitro by the traditional culture method is only 3-5 days, and the number of hair cells is gradually reduced and the shape of hair cell fiber bundles is gradually disappeared along with the extension of the culture time. Accordingly, there is a need in the art for improved cochlear auger stereotactic culture.

According to the search of the prior art, the gap junction protein 30 (CX 30, also called gap junction protein beta-6, the sequence information is shown in UniProtKB-095452) is expressed in a large quantity on the lateral wall and the spiral organ of the cochlea, human spiral ganglion cells (SG) also express CX30, and CX30 is highly expressed in the support cells of the human spiral organ (see: Zhanghongbud, etc., the specific expression of the human cochlear junction protein, published date 20151231). However, the prior art only discloses the expression of the gene in a screw machine, and does not disclose or suggest that the gap junction protein 30 expressed by the CX30 gene can be used for in vitro culture of the screw machine. Experiments show that the gap junction protein 30 is used as a culture medium supplement component and has an effect of prolonging the survival time of cells when used for culturing a mouse cochlear spiral organ in vitro.

In addition, the prior art shows that (see: Wang Elaeagnus mollis et al, the protective effect of alpha-lipoic acid on in-vitro mouse cochlear hair cell injury caused by kanamycin, published date 2013) the antioxidant alpha-lipoic acid has the protective effect on in-vitro mouse cochlear hair cell injury. The invention further screens other antioxidants with similar functions, and finds that the antioxidant phytic acid (inositol hexaphosphate) has the same benefits on the in vitro culture of the cochlear organ of the mouse. The phytic acid and the rat tail gum are combined for use to prepare collagen gel, a cochlea basement membrane containing a spiral organ is fixed in the collagen gel for in vitro culture, and cells obtained by culture have good shape, good activity and long survival time.

Disclosure of Invention

Aiming at the defects of the cochlear helical organ in vitro separation and culture method in the prior art, the invention aims to provide a separation and in vitro culture method of a cochlear helical organ of a newborn mouse, which can effectively separate and obtain a complete cochlear basilar membrane containing the cochlear helical organ, and the cells obtained by culture have good shape, good activity and long survival time by using gap connexin 30 and phytic acid in the culture process.

One aspect of the present invention provides a method for separating and culturing a cochlear spiral organ of a mouse, which is characterized by comprising the following steps:

(1) cutting off the skin of the head of the mouse after head breakage, cutting the head along the sagittal line of the skull by using an operation blade, transferring the head into a culture dish filled with sterile PBS, and removing brain tissues;

(2) removing the link between the cochlea and the temporal bone by using fine forceps, cutting off the connection between the cochlea and the vestibular tissue, taking out the cochlea, and transferring the cochlea to a culture dish filled with DMEM/F12 culture solution;

(3) stripping off the bone labyrinth from the bottom of the cochlea to the top of the cochlea to obtain a spiral ligament and a spiral organ which are wound around the snail shaft, clamping the bottom of the spiral ligament and the bottom of the spiral organ by using fine forceps, untwisting the spiral ligament and the bottom of the spiral organ from the snail shaft, and separating the spiral ligament from a basement membrane containing the cochlear spiral organ from the bottom to obtain the basement membrane containing the cochlear spiral organ;

(4) culture of cochlear spiral: dripping appropriate amount of collagen gel into the center of the culture dish, soaking the basement membrane into the collagen gel, and transferring the culture dish into CO₂Taking out the culture box after 20-30 minutes of incubation until the collagen gel is solidified into a gel block, fixing a substrate membrane in the gel block, adding a proper amount of serum-free culture solution into the culture dish to submerge the gel block, and finally putting back CO₂Culturing in an incubator, wherein the culture solution is replaced by a feed culture medium every other day for 7-10 days, wherein the collagen gel comprises rat tail gum and phytic acid with a final concentration of 50mM, and the feed culture medium is added with gap junction protein 30 with a final concentration of 20mM on the basis of the serum-free culture solution.

In a preferred embodiment, characterized in that the collagen gel is prepared by the following method: rat tail gum, 10 XBME and 2% Na₂CO₃The proportion is 9: 1:1, adding phytic acid with the final concentration of 50mM, uniformly mixing, and placing on ice to prevent coagulation.

In a preferred embodiment, the serum-free culture solution comprises 1 × DMEM 96.4%, 20% glucose 2.4%, penicillin G0.2%, and glutamine 1% by volume.

In a preferred embodiment, characterized in that said mouse is a mouse 3-5 days after birth.

In a preferred embodiment, characterized in that the DMEM/F12 culture solution in step (2) further comprises 1% by volume N2 feed, 2% by volume B27 feed, and ampicillin at a final concentration of 50. mu.g/ml; and (3) performing under a dissecting microscope.

In a preferred embodiment, characterized in that the number of culture days of the cochlear screw of step (4) is 10 days.

In a preferred embodiment, it is characterized in that CO in the step (4)₂The culture conditions in the incubator are 37 ℃ and 5% CO₂。

In a preferred embodiment, characterized in that said incubation time in said step (4) is 20 minutes.

In a preferred embodiment, the method is characterized in that the cochlear basilar membrane helix tissue and hair cell activity are observed by using an immunostaining marker Myo7a method in the culture stage of the cochlear helix.

In a preferred embodiment, wherein the immunostaining marker Myo7a method comprises:

1) removing culture solution from the cultured cochlear spiral organ, adding 2ml of 4% formalin fixing solution, standing at room temperature for 15 minutes, and washing with PBS;

2) adding 2ml of Triton x-100 solution prepared by 0.1% PBS, standing at room temperature for 15 minutes, and washing with PBS;

3) adding 2ml of BSA solution prepared by 2.5% PBS, standing at room temperature for 30 minutes, and washing with PBS;

4) primary antibody incubation of Rabbit anti-Myo7a 1:200, 2.5% BSA in PBS, overnight at 4 ℃;

5) washing the primary antibody with PBS, adding a secondary antibody which is Alexa Fluor 546 goat-anti Rabbit IgG 1:1000, 2.5% BSA in PBS, and standing at room temperature for 2 hours;

6) the secondary antibody was washed off with PBS, and then observed and photographed with a fluorescence microscope.

The invention achieves the following beneficial effects:

(1) the method for separating the mouse cochlear spiral organ is simple and easy to implement and convenient to operate.

(2) The invention innovatively uses the gap junction protein 30 and the antioxidant phytic acid for the in-vitro culture of the cochlear spiral organ. The phytic acid and the rat tail gum are combined for use to prepare collagen gel for fixing a basement membrane, and the gap junction protein 30 is used as a supplement component of a culture medium, the cochlear helicopters of the mice are cultured in vitro, and the obtained helicopters hair cells are good in shape, good in activity and long in survival time.

(3) The separation and in vitro culture method of the mouse cochlear spiral organ adopts the method of labeling Myo7a by immunostaining, has good dyeing effect, and can clearly observe the growth state of hair cells.

Drawings

Fig. 1 mouse cochlea.

Fig. 2a removes the peripheral bony cochlear membrane labyrinth.

Fig. 2b shows a cochlear screw with a spiral ligament.

FIG. 3 morphological micrographs of experiment 1 group after 24 hours of culture.

FIG. 4 microscopic photograph of MyO7a stained cochlear hair cells after 48 hours of culture in experiment 1 group (Scale bar: 20 μm).

FIG. 5 microscopic photograph of MyO7a staining at day 5 of group 4 culture (Scale bar: 20 μm).

FIG. 6 microscopic photograph of MyO7a staining at day 5 of group culture of experiment 1 (Scale bar: 20 μm).

FIG. 7 microscopic photograph of MyO7a staining at day 10 of group culture of experiment 1 (Scale bar: 20 μm).

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention. Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers. When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Example 1 separation of mouse cochlear augers

C57BL/6J mice 3-5 days after birth were selected as experimental material. Newborn mice were anesthetized on ice bath and sterilized with 70% alcohol. The head is cut off rapidly by a dissecting scissors, the skin of the head is cut off, and the head is cut along the sagittal line of the skull by an operation blade. The cells were transferred to a petri dish containing sterile PBS, and brain tissue was removed. The fine forceps are used for removing the link between the cochlea and the temporal bone, the connection between the cochlea and the vestibular tissue is cut off, the cochlea is taken out, and the obtained cochlea is shown in fig. 1. The isolated cochlea was transferred to a petri dish containing a culture medium of DMEM/F12 (Gibco/Invitrogen cat # 11320-033); + 1% N2 supplement (Gibco/Invitrogen, cat # 17502-048) + 2% B27 supplement (Gibco/Invitrogen, cat # 12587-010) + 50. mu.g/ml Ampicillin (Sigma, cat # A5354-10 ml)). The bony labyrinth is carefully stripped from the base of the cochlea to the apex, resulting in a spiral ligament and spiral around the modiolus, i.e. removal of the peripheral bony cochlear labyrinth, as shown in fig. 2 a. The spiral ligament and the bottom of the spiral organ are clamped by fine forceps and unscrewed from the modiolus to obtain the cochlear spiral organ with spiral ligament as shown in figure 2 b. The spiral ligament is separated from the basement membrane containing the cochlear helix from the bottom, and the basement membrane containing the cochlear helix is obtained.

Example 2 culture of mouse cochlear augers

(1) Reagent preparation

Preparation of serum-free culture solution: the volume percentage of each component is 1 × DMEM 96.4%, 20% glucose 2.4%, penicillin G0.2%, and glutamine 1%.

Preparation of a feed medium: gap junction protein 30 was added to the serum-free medium so that the final concentration was 20 mM.

Feed control medium: namely, the above serum-free culture medium without the gap junction protein 30.

Preparation of collagen gel: rat tail gum (purchased from BD), 10 XBME (purchased from Biowest, product number L0046-500) and 2% Na₂CO₃The proportion is 9: 1:1, adding phytic acid with the final concentration of 50mM, uniformly mixing, and placing on ice to prevent coagulation.

Preparation of collagen gel control 1: the procedure was the same as for the collagen gel described above except that phytic acid was not added.

(2) Culturing

Experiment 1: dropping appropriate amount of collagen gel into the center of the culture dish, soaking the basement membrane in the collagen gel, transferring the culture dish into CO₂The incubator was incubated for 20 minutes and removed, and the collagen gel was allowed to set into a gel mass in which the substrate membrane was immobilized. Adding appropriate amount of serum-free culture medium to submerge the gel block, and returning to 37 deg.C and 5% CO₂Culturing in an incubator, replacing the culture solution with a supplemented culture medium every other day, and culturing for 10 days.

Experiment 2: the culture procedure was identical to experiment 1, with the only difference that the feed medium was replaced with the feed control medium, i.e. with a serum-free medium without gap junction protein 30.

Experiment 3: the culture process was identical to experiment 1, with the only difference that the collagen gel was replaced with collagen gel control 1, i.e., a collagen gel containing no phytic acid was used for fixing the basement membrane.

Experiment 4: the culture process was identical to that of experiment 1, except that the supplemented medium was replaced with the supplemented control medium, the collagen gel was replaced with the collagen gel control 1, i.e., the supplemented medium was replaced with a serum-free culture solution without adding the gap junction protein 30, and the collagen gel without containing phytic acid was used to fix the basement membrane, and the culture process of experiment 4 was a conventional three-dimensional culture method of the prior art.

Example 3 morphological Observation of Spirometer tissue and Hair cells

After obtaining the basement membrane, the shape of the outline and the growth of the cells were observed under an inverted microscope after 24 hours of incubation in the above-described methods of experiment 1, experiment 2, experiment 3 and experiment 4 under each experimental group.

The results show that: as shown in fig. 3, experiment 1 has the best effect, the tissue outline of the basement membrane of the spiral organ is clear, and intracochlear and extracochlear hair cells and supporting cells of structures such as intracochlear and extracochlear hair cells and supporting cells can be seen under a high power microscope.

Example 4 Activity characterization of helical organ tissue and Hair cells

The cochlear basilar membrane organ tissue and hair cell activity were observed by immunostaining for Myo7 a.

The immunostaining reagents were as follows:

formalin fixation solution: sigma, # HT 501128-4L;

PBS：Corning, #21-040-CV；

TritonX-100：Sigma, #X100-100ML；

EDTA：Sigma, #E5134-250G；

BSA：Sigma, #A1933-25G；

Rabbit anti-Myo7a: Proteus Bioscience, #25-6790；

Alexa Fluor 546 Goat anti-Rabbit IgG: Invitrogen, #A11035。

an immunostaining step:

1) after 48 hours of culture in a cochlear spiral, removing the culture solution, adding 2ml of 4% formalin fixing solution, standing at room temperature for 15 minutes, and washing with PBS;

2) adding 2ml of 0.1% Triton x-100 (prepared by PBS) solution, standing at room temperature for 15 minutes, and washing with PBS;

3) adding 2ml of 2.5% BSA (PBS) solution, standing at room temperature for 30 minutes, and washing with PBS;

4) primary antibody incubation (Rabbit anti-Myo7a 1: 2002.5% BSA in PBS) at 4 ℃ overnight;

5) the primary antibody was washed with PBS, and a secondary antibody (Alexa Fluor 546 goat-anti Rabbit IgG 1:1000, 2.5% BSA in PBS) was added thereto and allowed to stand at room temperature for 2 hours;

6) the secondary antibody was washed off with PBS, and then observed and photographed with a fluorescence microscope.

As a result: in experiment 1, as shown in fig. 4, the outer hair cells and inner hair cells on the basement membrane of experiment 1 are clearly visible without defects.

Example 5 helical organ tissue and Hair cell survival time

In the culture process, the arrangement condition of cochlear helical organ tissues and hair cells is observed by microscopy under an inverted microscope. The culture method according to experiment 1-experiment 4 was followed, and the survival status of the organ tissue and hair cells was recorded at 3 days, 5 days, 8 days and 10 days of the culture, and the results are shown in Table 1.

TABLE 1 helical organ tissue and Hair cell survival status

(+++ represents cell survival and good morphology, + represents cell survival but poor morphology, -represents cell death)

Days of culture	Experiment 1	Experiment 2	Experiment 3	Experiment 4
					3 days	+++	+++	+++	+++
5 days	+++	+	+	+
					8 days	+	+	+	-
10 days	+	-	-	-

By staining with the immunostaining marker Myo7a and observing with a microscope, the culture method of experiment 4 showed that hair cells had been lost on day 5 of culture, and FIG. 5 showed that hair cells grew well without loss in experiment 1 group cultured for the same 5 days. Experiment 4 groups cells were dead by day 8 of culture. In the culture process by adopting the method of experiment 1, the growth inhibition of the epithelial cells at the edge of the basement membrane is obvious, the better shape of the inner ear hair cells can be maintained for at least one week, and the survival of the inner ear hair cells can be still realized after the culture for 10 days as shown in figure 7.

Claims (10)

1. A separation and in-vitro culture method of a mouse cochlear spiral organ is characterized by comprising the following steps:

(1) cutting off the skin of the head of the mouse after head breakage, cutting the head along the sagittal line of the skull by using an operation blade, transferring the head into a culture dish filled with sterile PBS, and removing brain tissues;

(2) removing the link between the cochlea and the temporal bone by using fine forceps, cutting off the connection between the cochlea and the vestibular tissue, taking out the cochlea, and transferring the cochlea to a culture dish filled with DMEM/F12 culture solution;

(3) stripping off the bone labyrinth from the bottom of the cochlea to the top of the cochlea to obtain a spiral ligament and a spiral organ which are wound around the snail shaft, clamping the bottom of the spiral ligament and the bottom of the spiral organ by using fine forceps, untwisting the spiral ligament and the bottom of the spiral organ from the snail shaft, and separating the spiral ligament from a basement membrane containing the cochlear spiral organ from the bottom to obtain the basement membrane containing the cochlear spiral organ;

(4) culture of cochlear spiral: dripping appropriate amount of collagen gel into the center of the culture dish, soaking the basement membrane into the collagen gel, and transferring the culture dish into CO₂Taking out the culture box after 20-30 minutes of incubation until the collagen gel is solidified into a gel block, fixing a substrate membrane in the gel block, adding a proper amount of serum-free culture solution into the culture dish to submerge the gel block, and finally putting back CO₂Culturing in an incubator, wherein the culture solution is replaced by a feed culture medium every other day for 7-10 days, wherein the collagen gel comprises rat tail gum and phytic acid with a final concentration of 50mM, and the feed culture medium is added with gap junction protein 30 with a final concentration of 20mM on the basis of the serum-free culture solution.

2. The method of claim 1, wherein the collagen gel is prepared by: rat tail gum, 10 XBME and 2% Na₂CO₃The proportion is 9: 1:1, adding phytic acid with the final concentration of 50mM, uniformly mixing, and placing on ice to prevent coagulation.

3. The method according to claim 1 or 2, wherein the serum-free culture solution comprises 1 × DMEM 96.4%, 20% glucose 2.4%, penicillin G0.2%, and glutamine 1% by volume.

4. The method of claim 1, wherein the mouse is a C57BL/6J mouse at 3-5 days after birth.

5. The method according to claim 1, wherein the DMEM/F12 culture solution in step (2) further comprises 1% by volume N2 feed, 2% by volume B27 feed, and a final concentration of 50 μ g/ml ampicillin; and (3) performing under a dissecting microscope.

6. The method of claim 1, wherein the number of days of culture of the cochlear screw of step (4) is 10 days.

7. The method of claim 1, wherein the CO in step (4)₂The culture conditions in the incubator are 37 ℃ and 5% CO₂。

8. The method of claim 1, wherein the incubation time in step (4) is 20 minutes.

9. The method according to any one of claims 1-2 and 4-8, wherein the cochlear basilar membrane helix tissue and hair cell activity are observed during the culture phase of the cochlear helix using immunostaining for the marker Myo7 a.

10. The method of claim 9, wherein the immunostaining marker Myo7a method comprises:

1) removing culture solution from the cultured cochlear spiral organ, adding 2ml of 4% formalin fixing solution, standing at room temperature for 15 minutes, and washing with PBS;

2) adding 2ml of Triton x-100 solution prepared by 0.1% PBS, standing at room temperature for 15 minutes, and washing with PBS;

3) adding 2ml of BSA solution prepared by 2.5% PBS, standing at room temperature for 30 minutes, and washing with PBS;

4) primary antibody incubation of Rabbit anti-Myo7a 1:200, 2.5% BSA in PBS, overnight at 4 ℃;

5) washing the primary antibody with PBS, adding a secondary antibody which is Alexa Fluor 546 goat-anti Rabbit IgG 1:1000, 2.5% BSA in PBS, and standing at room temperature for 2 hours;

6) the secondary antibody was washed off with PBS, and then observed and photographed with a fluorescence microscope.

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