CN113403277B - Method for separating FZD10 positive glial cells of inner ear - Google Patents
Method for separating FZD10 positive glial cells of inner ear Download PDFInfo
- Publication number
- CN113403277B CN113403277B CN202110717916.9A CN202110717916A CN113403277B CN 113403277 B CN113403277 B CN 113403277B CN 202110717916 A CN202110717916 A CN 202110717916A CN 113403277 B CN113403277 B CN 113403277B
- Authority
- CN
- China
- Prior art keywords
- cells
- fzd10
- glial cells
- positive
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/105—Insulin-like growth factors [IGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/90—Polysaccharides
- C12N2501/91—Heparin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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. The application utilizes the hybridization of the transgenic mice to obtain fluorescent markers for FZD10 positive glial cells in the inner ear, thereby facilitating the subsequent acquisition of purified inner ear glial cells by flow sorting. The separation and purification technology adopted by the invention can obtain glial cells to the maximum extent and maintain the activity of the cells, the purity of the FZD10 positive glial cells after separation reaches more than 98%, the requirements of subsequent experiments can be met, and the analysis and purification process is concise and rapid and is very suitable for the subsequent experimental study.
Description
Technical Field
The invention relates to the field of cell separation and purification, in particular to a method for separating inner ear Frizzled10 (FZD 10) positive glial cells.
Background
Deafness is a global major public health problem, and there is an upward trend in about 5 hundred million people worldwide suffering from disabled hearing loss disorders, accounting for 6.5% of the world's general population. Wherein the sensorineural deafness is about 65%. Sensorineural deafness refers to auditory pathways in which the diseased site is located in and behind the cochlea, and is mainly caused by the disintegration of cochlear hair cells and spiral neurons. Unlike conductive deafness caused by lesions of the external auditory canal or middle ear, a significant portion of sensorineural deafness tends to be permanent and relatively difficult to reverse. Currently, artificial cochlea transplantation is mainly adopted for treating sensorineural deafness. The artificial cochlea plays a role in treating hearing loss by directly generating electric stimulation to neurons in the inner ear, so that the treatment effect of the artificial cochlea is greatly dependent on the number and quality of spiral neurons surviving in the inner ear. In recent years, studies on the protection and regeneration of spiral neurons have been very hot.
There is a class of glial cells in the cochlea that are developmentally co-derived with neurons, including schwann cells and satellite cells, and studies have found that such cells have self-proliferation capacity and are capable of being transformed into neurons in culture in vitro and in vivo. Therefore, the purified cochlear glial cells are obtained from the inner ear tissue, so that the research on the proliferation of the glial cells and the differentiation process of the glial cells into neurons is of great significance to the research on inner ear regeneration.
However, due to the complicated anatomical structure of cochlea and the limitations of research technology, there is still a lack of description of methods for separation and purification of cochlear glial cells. The first step in glial cell separation and purification is the digestion separation of the cochlea snail, and in most of the previous literature, the digestion method of the snail in the inner ear is not well described, for example, in terms of the type of enzyme used for digestion, concentration, digestion conditions, cell blowing mode, etc. (Petitpre et al, 2018). In the digestion process of the worm shaft, sun et al adopts a two-step digestion method, wherein the digestion time of each step is up to 30 minutes, and the adopted reagents are more in variety and the process is complicated.
In addition, in terms of purification against glial cells, all the snail cells could be isolated in the previous study and their differentiation into neurons was studied. However, cells in the snail shaft include various cell types such as neurons, glial cells, fibroblasts, etc., which makes it difficult to study the proliferation and differentiation properties of glial cells. By utilizing the characteristic that neurons cannot proliferate and passaged, researchers firstly carry out more than three generations of balling culture to remove neurons in separated snail shaft cells, however, the method greatly prolongs the experiment time and reduces the activity of glial cells to a certain extent because one generation of balling requires 4 to 6 days. In addition, this approach does not remove the snail's fibroblasts well because the fibroblasts in the inner ear also have proliferative capacity (McLean, eatock, & Edge, 2016).
There is thus a need for a simple and rapid method for isolating and purifying inner ear glial cells that meets the needs of inner ear glial cell research. The difficulty in separating and purifying glial cells is mainly caused by two reasons, namely, the complex anatomy of the inner ear: glial cells are located in the snail shaft of the inner ear. The inner ear is located in the temporal bone and comprises a complex and precise double-layer labyrinth structure: an outer bony labyrinth and an inner membranous labyrinth. The cochlea is positioned at the front part of the inner ear labyrinth and is in a spiral structure with two turns and one half. The bony snail shaft is positioned at the middle of the cochlear membrane labyrinth and is conical. The worm shaft surrounds the bone screw plate, rises spirally from the bottom to the top and extends into the bone worm tube. Therefore, the cochlea worm shaft is deep in position, the bone is hard, and the dissection has certain technical difficulty. On the other hand, neurons, glial cells, fibroblasts and the like are closely associated in the snail shaft, and a mixture of the above cells is obtained after the snail shaft tissue is conventionally separated and digested, not only glial cells. Glial cells in the cochlea include both schwann cells and satellite cells. Numerous schwann cells adhere tightly to the processes of neurons to form myelin structures, so that all nerve fibers are myelinated. While satellite cells are distributed around the cell bodies of neurons, surrounding the cell bodies of neurons. Therefore, it is impossible to obtain pure glial cells after direct separation of cochlea.
Disclosure of Invention
Aiming at the problems in the prior art, the method utilizes the hybridization of the transgenic mice to enable FZD10 positive glial cells in the inner ear to obtain fluorescent markers, thereby being convenient for obtaining purified inner ear glial cells through flow separation later. The separation and purification technology adopted by the invention can obtain glial cells to the maximum extent and maintain the activity of the cells, the purity of the FZD10 positive glial cells after separation reaches more than 98%, the requirements of subsequent experiments can be met, and the analysis and purification process is concise and rapid and is very suitable for the subsequent experimental study.
The specific technical scheme of the method for separating the inner ear FZD10 positive glial cells comprises the following steps:
(1) FZD10-creer+/-/Rosa26R-tdTomato +/-biscationic rats at 2 to 4 days of birth are taken, sterilized and placed in a biosafety cabinet for operation, cochlea is dissected, peripheral spiral ligaments are torn off and the catfish's device is removed after removal of the internal structure of the cochlea exposed by the volute, the remaining snail shaft portion is transferred to a centrifuge tube containing Hanks liquid pre-cooled in a refrigerator at 4 ℃, and centrifuged at 1000-1500rpm for 5-10 minutes, preferably 1500rpm for 5 minutes. Too high a centrifugal speed can cause cell damage, and a decrease in the centrifugal speed can prolong the required centrifugal time, can cause incomplete cell sedimentation to affect the cell acquisition amount, and can delay the experimental time to affect the cell activity.
Preferably, the cochlea specific operations are dissected as: about 20 suckling mice are taken to remove heads and split from the middle sagittal of the skull to remove brain parenchyma, bilateral temporal bones where cochlea is located are cut off, and the cochlea is rapidly placed in a culture dish containing cold Hanks liquid and carefully dissected under a dissecting microscope. The dissection time is controlled to be less than 40 minutes as much as possible.
(2) And (3) discarding the supernatant Hanks liquid after the centrifugation in the step (1), adding an enzymolysis liquid, placing in a cell culture box at 37 ℃ for digestion for 7 to 15 minutes, flicking the bottom of a centrifuge tube every two minutes during the period to enable cochlear tissues to be in full contact with the enzymolysis liquid, adding an equal volume of DMEM/F12 culture medium containing 10% FBS for stopping digestion, and centrifuging at 1500rpm for 5 minutes.
The preferred digestion time is 10 minutes.
The enzymolysis liquid comprises the following components: 1mL of enzymolysis liquid contains DNAse of 10 Kunits, collagenase of 1 mg-5 mg and Hanks liquid of the rest; the optimal collagenase content is 3 mg.
The consumption of the enzymolysis liquid is as follows: about 20 mice cochlea was added with 1ml of enzymatic hydrolysate.
(3) And (3) discarding the supernatant after the centrifugation in the step (2), adding fresh culture solution, holding the centrifuge tube at an inclination of 30 ℃, lightly blowing by adopting a micropipette, and avoiding introducing bubbles as much as possible, blowing under 90-120 ℃ until no agglomerated tissue is seen by the naked eye.
The fresh culture solution comprises the following components: n2 (10. Mu.L/mL), B27 (20. Mu.L/mL), ampicillin (1. Mu.L/mL) were added to DMEM/F12 medium, and human basic fibroblast growth factor (hBFG, 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.
The specific blowing operation is as follows: the 1mL micropipette was adjusted to 900 μl and the gun tip was extended into the bottom of the liquid, gently blown and as much as possible avoiding introducing air bubbles, about 100 a down to no agglomerated tissue visible to the naked eye.
(4) And (3) removing a small amount of undispersed complete cell clusters from the cell suspension through a 40-mu m cell filter, collecting single cell suspension, and carrying out flow separation to obtain FZD10 positive glial cells.
The specific flow type sorting process comprises the following steps:
1) Ultraviolet sterilization is carried out on the environment before the flow separation, and the separation process of the machine is strictly operated 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 injection tube, placing the sample injection tube on a sample loading frame, running for 20 minutes, taking down a sample loading tube containing 75% ethanol, replacing the sample loading tube with a sample loading tube containing 1mL sterile PBS, and continuing to run for two times to clean the residual 75% ethanol in a pipeline;
3) Diluting the single-cell suspension to a concentration of 1000 per mu L to improve the sorting yield, placing a loading tube containing the single-cell suspension at a loading port, and placing two 15mL centrifuge tubes containing 10 mL culture medium at the interface of the loading tube to collect FZD10 positive and FZD10 negative cells respectively;
4) In the sorting process, a 70 μm nozzle is used, the flow rate is adjusted to 5000 events/s, and a 561nm yellow-green laser is used for sorting targets. The sorting process takes about 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 cell number, and the survival rate of cell inoculation is high, so that the FZD10 positive glial cells can be used for subsequent culture and audiology research.
The FZD10-creER+/-/Rosa 26R-tdTomato+/-double positive P3 phase mice used in the present application can be obtained by the conventional technique in the art, and specifically, the following method can be adopted:
(1) Inserting a gene encoding cre enzyme and an estrogen receptor (estrogen receptor) gene under the promoter of frizzled10 to construct a conditional transgenic mouse model FZD10-creERT2 heterozygous mouse;
(2) Crossing the FZD10-creERT2 heterozygous mice with the Rosa26R-tdTomato homozygous mice, and obtaining double positive FZD10-creER+/-/Rosa26R-tdTomato +/-mice by using all offspring mice to develop normally;
(3) Gastric lavage was performed at a rate of 0.75mg tamoxifen (tamoxifen) per gram of mice, and based on the principle of action of the cre-loxp system, cre recombinase was activated in FZD 10-positive cells to cleave off stop codes between loxp sites, allowing FZD 10-positive cells and their daughter cells to be labeled with red fluorescent protein, tolato.
The invention is the invention which uses Hanks solution of 1mL containing collagenase with concentration of 1-5 mg/mL and DNAse with concentration of 10 Kunits/mL as enzymolysis liquid. Conventional cell separations use 0.25% pancreatin (example 4) for digestion, which gives low yields of cells from tissues and greater damage to cell viability. In the application, 1mL contains collagenase with concentration of 1-5 mg/mL and DNAse with concentration of 10 Kunits/mL for digestion, collagenase (especially collagenase with concentration of 3 mg/mL) can digest the snail shaft tissue mildly and has higher cell yield, undegraded tissue components can form jelly adhesion cells in the process of digestion and dispersion of collagenase, and the DNase can decompose the substances to disperse the adhered cells.
The dissected snail shaft was placed in the solution and then placed in a 37 ℃ cell incubator (Heal Force, HF 240) for digestion for 7-15 minutes (preferably 10 minutes) during which time the bottom of the centrifuge tube was flicked every two minutes to allow more complete contact of the cochlear tissue with the enzymatic hydrolysate. This digestion time and related operations are important steps in the present invention, and although seemingly simple operations, digestion is an important step in the separation process, and too short a time is insufficient to digest, and too long a time will cause damage to cochlear cells.
After digestion is completed, the centrifuge tube is held at an inclination of 30 degrees, a 1mL micropipette is adjusted to a capacity of 900 mu L, a gun head is gently blown after extending into the bottom of the liquid, and air bubbles are prevented from being introduced as much as possible. At about 100 f, no agglomerated tissue was visible to the naked eye. This operation is a critical step in obtaining discrete single cells. Wherein, the 1mL pipettor is adjusted to the volume of 900 mu L for blowing in the blowing process, which is an operation key point for avoiding introducing bubbles. The number of blows is about 100, tissues below 90 cannot be sufficiently scattered, and cell activity is seriously damaged if the number exceeds 120.
In summary, the invention has the following beneficial effects:
I. the tissue digestion and blowing method adopted by the invention can digest and disperse tissues to the maximum extent and obtain single cell suspension, and meanwhile, serious damage to the activity of cochlear tissue cells is avoided.
And II, the experimental process is simple and convenient to operate, reagents required for digesting the cochlear tissue only comprise collagenase, DNAse, hanks liquid and serum-containing culture medium, the process of obtaining single cell suspension after dissecting the cochlear tissue is carried out according to four steps of digestion, digestion termination, centrifugation and blowing, the experimental process is shortened as much as possible in time, and the experimental steps can be controlled within 20min, so that the cell activity is ensured to the greatest extent.
And III, the sorted cells are purified cochlear FZD10 positive glial cells, and the immune staining proves that the glial cells account for more than 98 percent and hardly contain other cochlear cell types such as neurons or fibroblasts. Since other cell types are removed, a subsequent accurate audiological study can be performed using the obtained purified FZD 10-positive glial cells.
Drawings
FIG. 1 is a schematic representation of cells inoculated before and after flow sorting after the worm shaft had been digested into single cells in example 1;
FIG. 2 is a comparison of the different fluorescent staining of cells after flow sorting in example 1;
wherein:
in fig. 1, reference numeral 1 denotes a flow sorting front mirror field of view; reference numeral 2 is a fluoroscopic field of view before flow sorting; reference numeral 3 is the field of view of the flow sorting rear mirror; the total number of cells in the field of view after the flow sorting is basically consistent with that of the fluorescent screen after the flow sorting, and the snail shaft is digested by enzymolysis and blown into single cells and then passes through a 40 mu m cell filter, and the snail shaft tissue is digested into single cells after inoculation, and the field views with the numbers 1 and 2 indicate that the total number of the positive cells of the total number of the snail shaft is only a small proportion of the number of the positive cells of the total number of the positive cells of the total number of the positive cells; the visual field charts of reference numerals 3 and 4 indicate that 98% or more of the cells obtained after flow sorting are all FZD 10-positive cells labeled with a key.
In fig. 2, from left to right, the following are in order:
DAPI: all cells of the nuclei were DAPI-labeled;
and (3) a step of Tomato: FZD 10-positive cells labeled with immunofluorescent protein cyto;
FZD10: adding an antibody to FZD10 to label positive cells of FZD 10;
merge: a combined graph of the first three graphs DAPI, tomo, FZD 10;
from this, it was found that more than 98% of the total number of the cells obtained after sorting showed positive staining with FZD10, confirming that the purified glial cell population was obtained after flow sorting.
Detailed Description
The above-described aspects of the present invention will be described in further detail by way of the following embodiments, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention. The following examples were carried out using conventional techniques, except as specifically described.
The FZD10-creer+/-/Rosa26R-tdTomato +/-double positive P3 phase rats in several comparative examples below were obtained using the following procedure:
(1) Inserting a gene encoding cre enzyme and an estrogen receptor (estrogen receptor) gene under the promoter of frizzled10 to construct a conditional transgenic mouse model FZD10-creERT2 heterozygous mouse;
(2) Crossing the FZD10-creERT2 heterozygous mice with the Rosa26R-tdTomato homozygous mice, and obtaining double positive FZD10-creER+/-/Rosa26R-tdTomato +/-mice by using all offspring mice to develop normally;
(3) The stomach is perfused with tamoxifen (tamoxifen) at a rate of 0.75 to mg tamoxifen per gram of mice, and cre recombinase is activated in FZD10 positive cells to excise stop codes between loxp sites according to the principle of action of cre-loxp system, so that FZD10 positive cells and sub-cells thereof are labeled with red fluorescent protein.
The specific flow sorting process in each example and comparative example is:
flow sorting step and parameters
1) The first half hour of the flow separation carries out ultraviolet irradiation on the environment of a flow separation room, and the separation process of the machine is strictly operated according to the 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 injection tube, placing the sample injection tube on a sample loading frame, running for 20 minutes, taking down the sample loading tube containing 75% ethanol, replacing the sample loading tube with the sample loading tube containing 1mL sterile PBS, and continuing to run for two times to clean the residual 75% ethanol in the pipeline.
3) Single cell suspensions were diluted to a concentration of 1000/μl to increase the sorting yield. The loading tube containing the single cell suspension was placed in the loading port and two 15mL centrifuge tubes containing 10 mL medium were placed at the collection tube interface to collect FZD10 positive and FZD10 negative cells, respectively.
4) In the sorting process, a 70 μm nozzle is used, the flow rate is adjusted to 5000 events/s, and a 561nm yellow-green laser is used for sorting targets. The sorting process takes about 30 minutes.
In the following examples:
biological safety cabinet, source Thermo Fisher Scientific, model 1300;
hanks' solution, from Solarbio, model H1045;
collagenase, source Sigma, model C5138;
DNAse, source Stem Cell Technologies, model 07473;
10% fbs, from Ausbian, model VS500T;
DMEM/F12 medium, source gibco;
b27, source invitrogen, model 17504044;
n2 source invitrogen, model 17502048;
ampicillin, from Solarbio, model a7490;
human basic fibroblast growth factor, derived from Sigma, model F0291;
insulin-like growth factor-1, source Sigma, model I8779;
epithelial cell growth factor, derived from Sigma, model E9644;
heparan sulfate, source Sigma, model H4777;
pancreatin-EDTA, source Gibco, model 25200072;
FZD10 antibody, protein of origin, model 18175-1-AP;
DAPI, source Thermo Fisher Scientific, model D1306;
laminin, source Merck, model L2020;
cell filters, source Falcon, model 352340;
a flow cytometer from BD, model FACSAria III;
cell incubator, source Heal Force, model HF240.
Example 1
(1) Taking 20 FZD10-creER+/-/Rosa 26R-tdTomato+/-double-positive rats of 3 days old after birth, placing the rats in a biosafety cabinet for operation after alcohol spray sterilization, removing the head of the rats and performing sagittal cleavage from the middle of the skull, removing brain parenchyma, shearing off bilateral temporal bones of the cochlea, rapidly placing the cochlea in a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under an dissecting microscope, carefully tearing off peripheral spiral ligaments and removing the Kotikoff's device after the internal structure of the cochlea is exposed, and transferring the rest of the snail shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail shaft tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.
(2) Preparing enzymolysis liquid required by digestion of the worm shaft in advance: 1mL of the enzymatic hydrolysate contains DNAse of 10 Kunits and collagenase of 3mg, and the balance is Hanks solution. After centrifugation, the supernatant Hanks solution is discarded, 1mL enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 10 minutes, the bottom of a centrifuge tube is flicked every two minutes during the period to make cochlear tissues fully contacted with the enzymolysis solution, an equal volume of DMEM/F12 culture medium containing 10% FBS is added for stopping digestion, and centrifugation is carried out at 1500rpm for 5 minutes.
(3) And (3) discarding the supernatant after the centrifugation in the step (2), adding fresh culture solution, tilting a left-hand 15mL centrifuge tube by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, lightly blowing and avoiding introducing bubbles as much as possible. The agglomerated tissue was not visible to the naked eye under blowing 100.
The fresh culture solution is as follows: n2 (10. Mu.L/mL), B27 (20. Mu.L/mL), ampicillin (1. Mu.L/mL) were added to DMEM/F12 medium, and human basic fibroblast growth factor (hBFG, 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 small amounts of undispersed complete cell clusters, and the single cell suspension was collected and immediately flow sorted.
The total number of the single cells of the worm shaft obtained by the digestion and single cell separation method is 200 ten thousand, and the number of the 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/μl in laminin coated 48-well plates (laminin was coated by dissolving in PBS at a ratio of 1:500) with 200 μl, i.e., 10000 cells, per well. The dishes were placed in a medium containing 5% CO 2 Is cultured in a cell culture incubator at 37℃for 24 hours.
2) Counting the number of adherent cells under a light microscope and calculating the survival rate of the adherent cells; FZD10 positive glial cell rate was calculated using cell counting and immunofluorescent staining.
After the culture is finished, the number of adherent cells in each hole of the culture plate is 7890-8350 and is 8047 on average, so that the adherent survival rate of the cultured cells is 8047/10000×100% = 80.47%.
Immunofluorescent staining was performed using primary antibody as anti-FZD 10 antibody, adding corresponding secondary antibody and DAPI for incubation, and after sealing, confocal microscopy was used for observation, and the number of FZD10 positive cells and DAPI positive cells in each culture well were counted, respectively. The number of FZD10 positive cells in each well is 7050-7960, average 7487, dapi positive cells is 7210-8100, average 7637, so that the rate of FZD10 positive glial cells in the cells cultured this time is 7487/7637×100% =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 percent of the total cell number, the survival rate of cell adhesion reaches 80.47 percent, and the FZD10 positive glial cells can be used for subsequent culture and audiology research.
Example 2
(1) Taking 20 FZD10-creER+/-/Rosa26R-tdTomato +/-double-positive rats after two days of birth, placing the rats in a biosafety cabinet for operation after alcohol spray sterilization, removing head and sagittal cleavage from the middle of the skull, removing brain parenchyma, shearing off bilateral temporal bones of the cochlea, rapidly placing the cochlea in a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under an dissecting microscope, carefully tearing off peripheral spiral ligaments and removing the Kotikoff's device after the internal structure of the cochlea is removed, and transferring the remaining snail shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail shaft tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.
(2) Preparing enzymolysis liquid required by digestion of the worm shaft in advance: 1mL of the enzymatic hydrolysate contains DNAse of 10 Kunits and 1mg of collagenase, and the balance is Hanks solution. After centrifugation, the supernatant Hanks solution is discarded, 1mL enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 15 minutes, the bottom of a centrifuge tube is flicked every two minutes during the period to make cochlear tissues fully contacted with the enzymolysis solution, an equal volume of DMEM/F12 culture medium containing 10% FBS is added for stopping digestion, and the mixture is centrifuged at 1000rpm for 10 minutes.
(3) And (3) discarding the supernatant after the centrifugation in the step (2), adding fresh culture solution, tilting a left-hand 15mL centrifuge tube by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, lightly blowing and avoiding introducing bubbles as much as possible. No agglomerated tissue was visible to the naked eye under blow 120.
The fresh culture solution is as follows: n2 (10. Mu.L/mL), B27 (20. Mu.L/mL), ampicillin (1. Mu.L/mL) were added to DMEM/F12 medium, and human basic fibroblast growth factor (hBFG, 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 small amounts of undispersed complete cell clusters, and the single cell suspension was collected and immediately flow sorted.
The total number of the single cells of the worm shaft obtained by the digestion and single cell separation method is 150 ten thousand, and the number of the 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/μl in laminin coated 48-well plates (laminin was coated by dissolving in PBS at a ratio of 1:500) with 200 μl, i.e., 10000 cells, per well. The dishes were placed in a medium containing 5% CO 2 Is cultured in a cell culture incubator at 37℃for 24 hours.
2) Counting the number of adherent cells under a light microscope and calculating the survival rate of the adherent cells; FZD10 positive glial cell rate was calculated using cell counting and immunofluorescent staining.
After the culture is finished, the number of adherent cells in each hole of the culture plate is 6200-7600 and the average number of adherent cells is 7037, so that the adherent survival rate of the culture cells is 7037/10000×100% = 70.37%.
Immunofluorescent staining was performed using primary antibody as anti-FZD 10 antibody, adding corresponding secondary antibody and DAPI for incubation, and after sealing, confocal microscopy was used for observation, and the number of FZD10 positive cells and DAPI positive cells in each culture well were counted, respectively. The number of FZD10 positive cells in each well is 5450-6830, average 6290, dapi positive cells is 5620-6900, average 6423, so that the rate of FZD10 positive glial cells in the cells cultured this time is 6290/6423×100% =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 cell number, and the survival rate of cell adhesion is 70.37%.
Example 3
(1) Taking 20 FZD10-creER+/-/Rosa26R-tdTomato +/-double-positive rats after three days of birth, placing the rats in a biosafety cabinet for operation after alcohol spray sterilization, removing the head of the rats and performing sagittal cleavage from the middle of the skull, removing brain parenchyma, shearing off bilateral temporal bones of the cochlea, rapidly placing the cochlea in a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under an dissecting microscope, carefully tearing off peripheral spiral ligaments and removing the Kotikoff's device after the internal structure of the cochlea is removed, and transferring the remaining snail shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail shaft tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.
(2) Preparing enzymolysis liquid required by digestion of the worm shaft in advance: 1mL of the enzymatic hydrolysate contains DNAse of 10 Kunits and collagenase of 5mg, and the balance is Hanks solution. After centrifugation, the supernatant Hanks solution is discarded, 1mL enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 7 minutes, the bottom of a centrifuge tube is flicked every two minutes during the period to make cochlear tissues fully contacted with the enzymolysis solution, an equal volume of DMEM/F12 culture medium containing 10% FBS is added for stopping digestion, and centrifugation is carried out at 1500rpm for 5 minutes.
(3) And (3) discarding the supernatant after the centrifugation in the step (2), adding fresh culture solution, tilting a left-hand 15mL centrifuge tube by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, lightly blowing and avoiding introducing bubbles as much as possible. The ball of tissue was not visible to the naked eye under blow 90.
The fresh culture solution is as follows: n2 (10. Mu.L/mL), B27 (20. Mu.L/mL), ampicillin (1. Mu.L/mL) were added to DMEM/F12 medium, and human basic fibroblast growth factor (hBFG, 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 small amounts of undispersed complete cell clusters, and the single cell suspension was collected and immediately flow sorted.
The total number of the single cells of the worm shaft obtained by the digestion and single cell separation method is 200 ten thousand, and the number of the FZD10 positive cells obtained after separation is 7 ten thousand.
Cell culture and identification after flow sorting:
1) FZD 10-positive cells were seeded at 50/μl concentration into laminin-coated 48 Kong PeiIn the plates (laminin was coated in PBS at a ratio of 1:500), 200 μl/well of 10000 cells were plated. The dishes were placed in a medium containing 5% CO 2 Is cultured in a cell culture incubator at 37℃for 24 hours.
2) Counting the number of adherent cells under a light microscope and calculating the survival rate of the adherent cells; FZD10 positive glial cell rate was calculated using cell counting and immunofluorescent staining.
After the culture is finished, the number of adherent cells in each hole of the culture plate is 5500-6400 and the average number of adherent cells is 5933, so that the adherent survival rate of the culture cells is 5933/10000×100% = 59.33%.
Immunofluorescent staining was performed using primary antibody as anti-FZD 10 antibody, adding corresponding secondary antibody and DAPI for incubation, and after sealing, confocal microscopy was used for observation, and the number of FZD10 positive cells and DAPI positive cells in each culture well were counted, respectively. The number of FZD10 positive cells in each well at this time is 4740-6030, average 5427, dapi positive cells is 4850-6130, average 5543, so that the rate of FZD10 positive glial cells in the cells cultured at this time is 5427/5543 x 100% = 97.90%.
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 cell number, and the survival rate of cell adhesion is 59.33%.
Comparative example 1
(1) Taking 20 FZD10-creER+/-/Rosa26R-tdTomato +/-double-positive rats after three days of birth, placing the rats in a biosafety cabinet for operation after alcohol spray sterilization, removing the head of the rats and performing sagittal cleavage from the middle of the skull, removing brain parenchyma, shearing off bilateral temporal bones of the cochlea, rapidly placing the cochlea in a culture dish containing cold Hanks liquid, carefully dissecting the cochlea under an dissecting microscope, carefully tearing off peripheral spiral ligaments and removing the Kotikoff's device after the internal structure of the cochlea is removed, and transferring the remaining snail shaft part into a 15ml centrifuge tube containing the cold Hanks liquid. A15 mL centrifuge tube containing the snail shaft tissue was placed in a centrifuge and centrifuged at 1500rpm for 5 minutes.
(2) After centrifugation, the supernatant Hanks solution is discarded, 1mL enzymolysis solution is added, the mixture is placed in a cell culture box at 37 ℃ for digestion for 10 minutes, the bottom of a centrifuge tube is flicked every two minutes during the period to make cochlear tissues fully contacted with the enzymolysis solution, an equal volume of DMEM/F12 culture medium containing 10% FBS is added for stopping digestion, and centrifugation is carried out at 1500rpm for 5 minutes.
The enzymolysis liquid comprises the following components: 1mL of enzymolysis liquid contains DNAse of 10 Kunits, 0.25% of pancreatin-EDTA, and the balance of Hanks liquid.
(3) And (3) discarding the supernatant after the centrifugation in the step (2), adding fresh culture solution, tilting a left-hand 15mL centrifuge tube by 30 degrees, adjusting a 1mL micropipette to 900 mu L, extending a gun tip into the bottom of the liquid, lightly blowing and avoiding introducing bubbles as much as possible. The agglomerated tissue was not visible to the naked eye under blowing 100. The fresh culture solution is as follows: n2 (10. Mu.L/mL), B27 (20. Mu.L/mL), ampicillin (1. Mu.L/mL) were added to DMEM/F12 medium, and human basic fibroblast growth factor (hBFG, 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 small amounts of undispersed complete cell clusters, and the single cell suspension was collected and immediately flow sorted.
The total number of the single cells of the worm shaft obtained by the digestion and single cell separation method is 150 ten thousand, and the number of the 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/μl in laminin coated 48-well plates (laminin was coated by dissolving in PBS at a ratio of 1:500) with 200 μl, i.e., 10000 cells, per well. The dishes were placed in a medium containing 5% CO 2 Is cultured in a cell culture incubator at 37℃for 24 hours.
2) Counting the number of adherent cells under a light microscope and calculating the survival rate of the adherent cells; FZD10 positive glial cell rate was calculated using cell counting and immunofluorescent staining.
After the culture is finished, the number of adherent cells in each hole of the culture plate is 4830-5250 and is 5096 on average, so that the adherent survival rate of the cultured cells is 5096/10000×100% = 50.96%.
Immunofluorescent staining was performed using primary antibody as anti-FZD 10 antibody, adding corresponding secondary antibody and DAPI for incubation, and after sealing, confocal microscopy was used for observation, and the number of FZD10 positive cells and DAPI positive cells in each culture well were counted, respectively. The number of FZD10 positive cells in each well is 4630-4980, average 4830, dapi positive cells is 4700-5100, average 4937, so that the cell FZD10 positive glial cell rate in this culture is 4830/4937×100% =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 cell number, and the survival rate of cell adhesion is only 50.96%.
Claims (6)
1. A method for isolating FZD 10-positive glial cells of the inner ear, comprising the specific steps of:
(1) Taking FZD10-creER+/-/Rosa 26R-tdTomato+/-double-positive milk mice of 2 to 4 days old after birth, dissecting cochlea, removing spiral ligament at periphery and removing the Kotiva device after the internal structure of the cochlea is exposed, transferring the rest snail shaft part into a centrifuge tube containing precooled Hanks liquid, and centrifuging at 1000-1500rpm for 5-10 minutes;
(2) Discarding supernatant Hanks liquid after centrifugation in the step (1), adding an enzymolysis liquid, placing in a cell culture box at 37 ℃ for digestion for 7 to 15 minutes, flicking the bottom of a centrifuge tube every two minutes during the period to enable cochlear tissues to be in full contact with the enzymolysis liquid, adding an equal volume of DMEM/F12 culture medium containing 10% FBS, stopping digestion, and centrifuging at 1500rpm for 5 minutes;
(3) Discarding the supernatant after the centrifugation in the step (2), adding fresh culture solution, holding the centrifuge tube at an inclination of 30 ℃, lightly blowing by adopting a micropipette and avoiding introducing bubbles as much as possible, blowing under 90-120 ℃ until no agglomerated tissue is seen by the naked eye;
(4) Removing a small amount of undispersed complete cell clusters from the cell suspension through a 40 mu m cell filter, collecting single cell suspension, carrying out flow separation, and separating to obtain FZD10 positive glial cells;
the enzymolysis liquid in the step (2) comprises the following components: 1mL of enzymolysis liquid contains DNAse of 10 Kunits, collagenase of 1 mg-5 mg and Hanks liquid in balance.
2. The method for isolating inner ear FZD 10-positive glial cells according to claim 1, wherein the centrifugation operation in step (1) is: centrifugation at 1500rpm for 5 minutes.
3. The method for isolating inner ear FZD 10-positive glial cells according to claim 1, wherein the digestion time in step (2) is 10 minutes.
4. The method for isolating inner ear FZD 10-positive glial cells according to claim 1, wherein the enzymatic hydrolysate in step (2) consists of: 1mL of the enzymatic hydrolysate contained 10 Kunits DNAse, 4mg collagenase, and the balance Hanks solution.
5. The method for isolating inner ear FZD 10-positive glial cells according to claim 1, wherein the fresh culture solution in step (3) consists of: adding N2 into the DMEM/F12 culture medium: 10. mu L/mL, B27: 0. mu L/mL, ampicillin: 1. mu.L/mL, human basic fibroblast growth factor hBFG was added prior to use: 10 ng/mL, insulin-like growth factor-1: 50 ng/mL, epithelial cell growth factor EGF:20 ng/mL, heparan sulfate HS:50 ng/mL.
6. The method for separating inner ear FZD 10-positive glial cells according to claim 1, wherein the specific flow sorting process uses a 70 μm nozzle, the flow rate is adjusted to 5000 events/s, and the target sorting is performed using 561nm yellow-green laser.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110717916.9A CN113403277B (en) | 2021-06-28 | 2021-06-28 | Method for separating FZD10 positive glial cells of inner ear |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110717916.9A CN113403277B (en) | 2021-06-28 | 2021-06-28 | Method for separating FZD10 positive glial cells of inner ear |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113403277A CN113403277A (en) | 2021-09-17 |
| CN113403277B true CN113403277B (en) | 2023-05-16 |
Family
ID=77679611
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110717916.9A Active CN113403277B (en) | 2021-06-28 | 2021-06-28 | Method for separating FZD10 positive glial cells of inner ear |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113403277B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747104A (en) * | 2011-04-21 | 2012-10-24 | 中国科学院上海生命科学研究院 | Non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method |
| CN109844098A (en) * | 2016-06-20 | 2019-06-04 | 荷兰皇家科学院 | Improved differentiation method |
| CN111727203A (en) * | 2017-12-19 | 2020-09-29 | 瑟罗泽恩公司 | WNT surrogate molecules and uses thereof |
-
2021
- 2021-06-28 CN CN202110717916.9A patent/CN113403277B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747104A (en) * | 2011-04-21 | 2012-10-24 | 中国科学院上海生命科学研究院 | Non-exogenous induced factor integrated porcine induced pluripotent stem cell and its construction method |
| CN109844098A (en) * | 2016-06-20 | 2019-06-04 | 荷兰皇家科学院 | Improved differentiation method |
| CN111727203A (en) * | 2017-12-19 | 2020-09-29 | 瑟罗泽恩公司 | WNT surrogate molecules and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| "Frizzled-9+ Supporting Cells Are Progenitors for the Generation of Hair Cells in the Postnatal Mouse Cochlea;Shasha Zhang et al.;《Frontiers in Molecular Neuroscience》;20190731;第12卷;第3页左栏最后1段 * |
| Expression of Wnt receptors in adult spiral ganglion neurons frizzled 9 localization at growth cones of regenerating neurites";S. M. SHAH et al.;《Neuroscience》;20091231;第164卷(第2期);第482页左栏第4段，第483页图3 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113403277A (en) | 2021-09-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113564100B (en) | Method for extracting and purifying brain microvascular endothelial cells | |
| CN111467373A (en) | Dental pulp stem cell exosome preparation, preparation method and application thereof | |
| CN105238738A (en) | Isolated culture method of piglet myocardial fibroblasts | |
| CN112063583A (en) | Method for efficiently separating and extracting adipose-derived mesenchymal stem cells from adipose tissue | |
| CN116034987A (en) | Tissue and cell preservation solution, cell separation method and application thereof | |
| CN105695410B (en) | A kind of naked mole microglia cultural method | |
| Ou et al. | An efficient method for the isolation and cultivation of hypothalamic neural stem/progenitor cells from mouse embryos | |
| CN104726403A (en) | Method for separating and culturing human placental-derived mesenchymal stem cell | |
| KR20090008784A (en) | Method to isolate and culture of mesenchymal stem cells from human tissues and thier cells efficiently using low-intensity ultrasound | |
| CN113403277B (en) | Method for separating FZD10 positive glial cells of inner ear | |
| CN116836934B (en) | Osteosarcoma organoid culture solution, culture reagent combination and culture method | |
| CN110628706B (en) | Method for extracting and culturing embryonic neural stem cells in vitro and preparation of culture medium | |
| CN105695409B (en) | A kind of naked mole oligodendrocyte precursor cells cultural method | |
| CN111088215A (en) | Method for in vitro isolated culture of Mongolian horse testicular support cells | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| CN107058225B (en) | Compound induction culture medium and method for inducing umbilical cord mesenchymal stem cells into neuron-like cells by adopting culture medium | |
| CN113186155B (en) | High-efficiency culture method of primary cells of sheep embryonic skeletal muscle | |
| CN106497863A (en) | A kind of separation of cornea of rats endothelial cell, purifying and cultural method | |
| CN111733133B (en) | Method for promoting differentiation and growth of epidermal stem cells | |
| CN109439614B (en) | Exosome preparation for maintaining and restoring hair papilla cell stem property | |
| CN108949679B (en) | Soft tissue construction method of synovial mesenchymal stem cells and application thereof | |
| CN105176812A (en) | Stem cell globe cutting and collecting device and stem cell globe cutting and separating passage method | |
| CN112852715A (en) | Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells | |
| CN111304162A (en) | In vitro culture method of Schwann cells and fibroblasts of sensory/motor nerves | |
| CN105695411B (en) | A kind of naked mole cortical neuron cultural method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |