CN106754716B - Naked mole rat Schwann cell culture method - Google Patents
Naked mole rat Schwann cell culture method Download PDFInfo
- Publication number
- CN106754716B CN106754716B CN201610919420.9A CN201610919420A CN106754716B CN 106754716 B CN106754716 B CN 106754716B CN 201610919420 A CN201610919420 A CN 201610919420A CN 106754716 B CN106754716 B CN 106754716B
- Authority
- CN
- China
- Prior art keywords
- cell culture
- culture medium
- naked mole
- cells
- mole rat
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/02—Atmosphere, e.g. low oxygen conditions
-
- 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
- C12N2501/135—Platelet-derived growth factor [PDGF]
-
- 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
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of cell biology, in particular to a separation, purification and culture method of naked mole rat Schwann cells. The invention comprehensively uses a plurality of culture mediums to separate and purify and culture the Schwann cells from the DRG ganglia of the mole glaber and discloses a reasonable culture method of the mole glaber and Schwann cells suitable for temperature variation. The method can simply, efficiently and economically obtain a large amount of naked mole snow bloom cells with normal functional activity, and the culture under the hypoxia condition can ensure that the cells can still keep the biological characteristics under the physical state in the in vitro environment, so that the method is convenient for further researching the special physiological functions of the naked mole snow bloom cells in a pure in vitro cell culture model directly, and provides an important theoretical basis for exploring the biological mechanism and applying the biological mechanism in the clinical relevant fields.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a method for separating, purifying and culturing cells, and particularly relates to a method for separating, purifying and culturing naked mole rat snowmans cells.
Background
Schwann cells (Schwann cells) are myelinating sheath cells of the spinal cord, which surround the myelin sheath structure formed by neuronal axons and have a trophic, supportive and protective effect on neurons, and the presence of myelin sheaths ensures the rate of information transfer between neurons. Immature schwann cells can be stimulated by the microenvironment to again initiate differentiation. Myelin damage is commonly associated with diseases such as spinal cord transection injuries, amyotrophic lateral sclerosis and the like (Billiards SS, Haynes RL, Folkert RD, Borenstein NS, Tractenserg FL, Rowitch DH, Ligon KL, Volpe JJ, Kinney HC.2008.Myelin antigens with out oligodendrocyte loss in permanent vertebral Leukomalacia. brain 18: 153-.
Naked mole rat is a kind of alternating temperature mammal, in the not ventilative cave that oxygen concentration is only about 6% can keep the intact and normal life activity of brain structure and function, especially oxygen consumption higher snow wang cell that has already formed the myelin sheath. Therefore, the research on the special hypoxia tolerance capability of the Schwann cells in the spinal cord of the naked mole rat has important guiding significance for the treatment of Schwann cell injury in spinal cord injury diseases. Since the microenvironment in vivo is very complex, the functional change of schwann cells and their adaptation mechanism in hypoxic environment cannot be directly observed in vivo, and the results in vivo may be easily affected by other cellular components and structures in the microenvironment. Therefore, it is necessary to establish an isolated naked mole rat schwann cell model to compensate for the defect observed in vivo.
Immature spinal cord derived Schwann cells have strong division and proliferation capacity, are easy to culture in vitro, and can obtain mature cells with myelinating capacity by a method of inducing differentiation of the immature spinal cord derived Schwann cells. At present, researchers have searched for methods of inducing rat Schwann cells derived from sciatic nerve tissue and rat spinal cord DRG ganglion derived Schwann cells culture (Wang Y, Zhou S, Xu H, Yan S, Xu D, Zhang Y. Up-regulation of NF45 corrlates with Schwann cell promotion after neural neuron 2015May; 56(1): 216-27.).
However, in the prior art, the methods for separating, purifying and culturing the Schwann cells of both mice and rats are not suitable for naked mole rats, and at present, no relevant report about the methods for separating, purifying and culturing the Schwann cells of the naked mole rats exists in domestic and foreign documents.
Disclosure of Invention
The invention aims to provide a separation, purification and culture method of naked mole rat snow-bloom cells, which is used for conveniently and efficiently obtaining the naked mole rat snow-bloom cells with high purity and good state, providing technical support for establishing the naked mole rat snow-bloom cells in vitro, and simultaneously providing powerful guarantee for researching the differentiation, migration and hypoxia tolerance capability of the naked mole rat snow-bloom cells in vitro, describing the hypoxia tolerance mechanism of the naked mole rat snow-bloom cells and screening corresponding therapeutic drugs or target molecules.
We try out the existing separation, purification and culture methods of the Schwann cells of mice and rats, and find that the naked mole Schwann cells with high purity and good state can not be obtained by culture, and the proliferation quantity of the naked mole Schwann somatic cells is less.
No feasible experimental method has been established for culturing naked mole rat schwann cells, and the reasons for this are analyzed mainly as follows: 1) naked mole rat is a kind of variable temperature animal, the temperature of its somatic cell culture in vitro is to be groped, and its inconstant rate of body metabolism causes its somatic cell culture medium in vitro to be different from ordinary rat or mouse; 2) naked mole rat lives in a dark and humid environment, and the culture of somatic cells of the naked mole rat has high requirements on humidity; 3) naked mole rats have been adapted to an external hypoxic environment, so the oxygen concentration in the somatic cell ex vivo culture environment is different from that of animals living in an normoxic environment, and need to be groped.
Therefore, it is most important to fumble the proper temperature, humidity and oxygen concentration in the culture of naked mole rat schwann cells, and to fumble the proper culture medium.
In order to achieve the above objects, the present invention provides a naked mole rat Schwann cell culture method, comprising the steps of:
A. collecting naked mole rat DRG mixed nerve cells:
separating DRG ganglia of naked mole spinal cord in embryo stage of 58-65 days, cutting DRG ganglia into pieces, adding tissue digestive juice I, mixing, digesting, and digesting with tissue digestive juice II; then terminating digestion by using a mixed nerve cell culture medium, centrifuging to remove supernatant, and then resuspending and blowing the precipitate into single cell suspension in the mixed nerve cell culture medium; seeding the single cell suspension in a sterile culture plate (preferably a 24-well plate) which is pre-coated with a matrix layer;
the tissue digestive juice I can be conventional proteolytic enzyme digestive juice, such as collagenase type I and collagenase type II, and is supplemented with DNase I to relieve cell aggregation caused by adhesion of DNA fragments. Preferably, the tissue digest I is a mixture containing 0.15% collagenase and 0.03mg/ml DNase I.
The tissue digestive juice II can be conventional proteolytic enzyme digestive juice, such as trypsin, papain and the like, and is supplemented with DNase I to relieve cell aggregation caused by adhesion of DNA fragments. The preferred tissue digest II is a mixture containing 0.25% pancreatin and 0.03mg/ml DNase I.
The matrix layer can be conventional L-polylysine, D-polylysine, collagen, lamin, etc. The preferred matrix layer is a matrix layer of levorotatory polylysine.
B. Culture of naked mole rat DRG mixed nerve cells:
culturing the DRG mixed nerve cells obtained in the step A in a three-gas culture box by using a mixed nerve cell culture medium for 24 hours, then replacing a fresh mixed nerve cell culture medium, and then replacing the culture medium every 3 days; culturing for 25 days, changing to a Schwann cell purification culture medium for culturing, and updating the culture medium every three days.
The mixed nerve cell culture medium can be a conventional serum-containing mixed nerve cell culture solution, such as low-sugar DMEM and the like. Preferably low-sugar DMEM medium containing 15% by volume of imported fetal bovine serum.
The Schwann cell purification culture medium is Neurobasal containing 2% B27 by volume fraction and is added with PDGFa with 0.01mg/ml by mass volume fraction.
The parameters of the three-gas incubator are set as follows: the temperature is controlled at 35 +/-2 ℃, the oxygen concentration is 6%, the carbon dioxide concentration is 5 +/-2%, and the humidity is 96 +/-2%.
Preferably, in step A, the method for isolating DGR tissue from the dorsal root ganglion of a naked mole rat with an embryonic period of 58-65 days is as follows: taking female naked mole rat pregnant for 58-65 days, and placing in CO2Immediately after the asphyxiation treatment, it was sterilized by soaking in 75% ethanol, then placed in a sterile glass plate, carefully dissected and its uterus containing fetal rats was removed intact, the uterine membranes were cut open and fetal rats were carefully removed. The dorsal spinal column of the fetal rat is cut along the trend of the dorsal spinal column of the fetal rat, the vertebral canal and the intervertebral foramen are exposed under a body type microscope, the round and bright spinal ganglia in the outer hidden pits of the vertebral bodies at two sides are removed one by using the microscope forceps, and the fascia on the surfaces of the ganglia is stripped as much as possible.
Preferably, in step A, the ganglion tissue is minced to 1mm by using microshear3Adding tissue digestive juice I, mixing, digesting at 37 deg.C for 30min, and digesting with tissue digestive juice II at 37 deg.C for 30 min. Digestion was then stopped with 2ml of mixed neural cell culture medium. The cells were then centrifuged and the cell pellet was collected for resuspension into a single cell suspension.
Preferably, in step B, the mixed nerve cell culture medium is used for 24 hours, after the nerve cell culture medium is completely attached to the wall, the fresh mixed nerve cell culture medium is replaced, and then the culture medium is replaced every 3 days.
Preferably, the parameters of the three-gas incubator are set as follows: the temperature was controlled at 35 deg.C, oxygen concentration was 6%, carbon dioxide concentration was 5%, and humidity was 96%.
Preferably, in step A, the L-polylysine concentration is 0.01 mg/ml.
Preferably, in step B, the centrifugation parameters are set to 1000rpm for 5 minutes at room temperature.
Preferably, in step B, the cells are cultured in mixed nerve cell culture medium in a three-atmosphere incubator for 31 days.
The invention has the beneficial effects that: A. the culture of the Schwann cells is separated and purified from the dorsal ganglion of the nude mole rat, and a reasonable culture method of the nude mole Schwann cells of rodent mammals suitable for temperature change is developed. We found that the cell state can be better maintained and can be kept increasing under the condition of 6% oxygen concentration; B. the operation method is simple, the repeatability is high, and the cell purity can reach more than 98%. In 15% concentration fetal bovine serum, the schwann cells in the spinal ganglia maintain a high proliferation rate, and due to this proliferation advantage and the lack of neuronal trophic factors, the growth of neurons in the spinal ganglia is affected resulting in a gradually decreasing proportion of neurons, while the proportion of schwann cells gradually increases and reaches the highest purity.
Chinese patent document CN105695409A discloses a naked mole oligodendrocyte precursor cell culture method, but the naked mole oligodendrocyte precursor cell is obtained from brain tissue without connective tissue, and the schwann cell is obtained from DRG ganglia of lateral crypts of two side vertebral bodies, each DRG ganglia diameter is not more than 1mm, and is wrapped by a layer of connective tissue membrane, this layer of connective tissue membrane can not be stripped by mechanical method, and there are neurons from brain tissue in the separation and digestion method. Therefore, in digesting the DRG ganglia, it is necessary to first digest the connective tissue on the surface of the DRG ganglia with a mixture containing 0.1% collagenase and 0.06mg/ml dnase I, i.e. digestive juice I. Then, the inside of the DRG ganglia is digested with the digestive fluid II, which enables efficient digestion of the DRG ganglia to increase the amount of cells obtained. In addition, the CCK8 was used to test the cell proliferation rate, and it was found that the optimal oxygen concentration for naked mole Schwann cells to grow is different from that of oligodendrocyte precursor cells, which can maintain a high proliferation rate at 6% oxygen concentration (as shown in Table 1).
TABLE 1 Effect of different oxygen concentrations on the proliferation Rate of naked mole Schwann cells (OD450)
Note: p < 0.01, and 5% O2And (4) comparing the concentrations.
In conclusion, the method can simply, efficiently and economically obtain a large number of naked mole rat Schwann cells with normal functional activity, and the culture under the hypoxia condition can ensure that the cells can still keep the biological characteristics under the physical state in the in vitro environment, so that the special physiological functions of the naked mole rat Schwann cells can be further researched directly in a pure in vitro cell culture model, and an important theoretical basis is provided for exploring the biological mechanism and applying the biological mechanism to the clinical relevant fields.
Drawings
FIG. 1 shows Schwann cells cultured in vitro at 200-fold magnification, with a scale of 20 μm.
FIG. 2 shows immunochemical staining of cultured cells in vitro, which are marked with S100 staining, wherein the scale is 20 μm.
FIG. 3 shows the identification of Schwann cell by immunocytochemical staining of cultured cells purified in vitro, wherein the cells are marked with S100 staining and the scale is 20 μm.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1: naked mole rat snow vigorous cell separation, purification and culture
1. Experimental Material
Naked mole rat with embryo stage of 58-65 days is provided by laboratory animal center of China people liberation military and second military medical university. DNase I was obtained from Solambio Biotechnology, Inc., trypsin, L-polylysine, PDGFa, and penicillin mixture was obtained from Sigma, DMEM, Low-sugar DMEM, Australian-derived fetal bovine serum was obtained from Thermo Fisher Scientific, Inc., and Petri dishes, T25, and T75 flasks were obtained from Corning.
The mixed nerve cell culture medium is a low-sugar DMEM medium containing 15% imported fetal bovine serum by volume percentage.
The Schwann cell purification culture medium is Neurobasal containing 2% B27 by volume fraction and PDGFa with 0.01mg/ml by mass volume fraction is added.
2. Naked mole rat Schwann cell separation, purification and culture method
A. Collecting naked mole rat schwann cells: collecting DRG ganglia of naked mole spinal cord of 58-65 days in embryo stage, cutting DRG ganglia, adding tissue digestive juice I (mixture of 0.25% pancreatin and 0.03mg/ml DNase I), mixing, digesting at 37 deg.C for 30min, and digesting at 37 deg.C for 30min with tissue digestive juice II (mixture of 0.15% collagenase and 0.03mg/ml DNase I); then terminating digestion by using 2ml of mixed nerve cell culture medium, centrifuging to remove supernatant, and then resuspending and blowing the precipitate into single cell suspension in the mixed nerve cell culture medium; according to 1 × 106The culture medium is densely planted in T25 culture bottles coated with L-polylysine, and the volume of the culture medium in each bottle is 4 ml;
B. culture of naked mole rat schwann cells: the cells obtained in A were cultured for 20 days in a mixed nerve cell culture medium three-atmosphere incubator. From the cell inoculation, every 3 days, half of the liquid change method to replace fresh mixed nerve cell culture medium. After 20 days, the culture medium was changed to a Schwann cell purification medium (Neurobasal: B27: 50:1, and PDGFa with a final mass-volume fraction of 0.01mg/ml was added thereto) to obtain purified Schwann cells, which were cultured in a three-gas culture chamber at 35 deg.C, an oxygen concentration of 6%, a carbon dioxide concentration of 5%, and a humidity of 96%
Example 2: identification of naked mole rat Schwann cells
Identification of naked mole rat Schwann cells using the results of example 1: fixing Schwann cells in a proliferation culture medium by using 4% paraformaldehyde, and identifying by combining methods such as morphological identification and immune refining chemistry.
1. Cell morphology identification:
identification methods are described in the literature references (wenking Yang, Lin Xiao, Cui Li, Xiuyun Liu, Mingdong Liu, Qi Shao, Dan Wang, Aijun Huang and Cheng he. tip30inhibition of cell differentiation of cytological characterization of cells 201563 (4): 684-98.).
As shown in FIG. 2, under the optical microscope, the Schwann cell body is circular or elliptical, the refractive index of the cell body is strong, a circle of distinct halo is around the cell body, and two or three-pole protrusions extend from the cell body to the periphery. After culturing in the Schwann cell purification culture medium for 24 hours, the cells can still maintain the two-pole or three-pole processes.
2. And (3) carrying out immunocytochemistry identification:
identification methods are described in the literature references (wenking Yang, Lin Xiao, Cui Li, Xiuyun Liu, Mingdong Liu, Qi Shao, Dan Wang, Aijun Huang and Cheng he. tip30inhibition of cell differentiation of cytological characterization of cells 201563 (4): 684-98.).
As a result, as shown in fig. 3, the schwann cells purified by differential velocity adherence were subjected to a slide plate climbing experiment, cultured in a proliferation medium for 24 hours, fixed with 4% paraformaldehyde, and then subjected to immunocytochemical staining with an antibody against schwann cell surface specific antigen S100. The results showed that the cells were able to maintain the S100 positive state in the schwann cell purification medium.
According to the above experimental results, the isolated and purified naked mole rat Schwann cells of the present invention have typical morphology of Schwann cells, are S100 positive, have typical bipolar or tripolar processes, and can maintain a good proliferation state in a purified culture medium.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (8)
1. A naked mole rat Schwann cell culture method is characterized by comprising the following steps:
A. collecting naked mole rat DRG mixed nerve cells:
separating DRG ganglia of naked mole spinal cord in embryo stage of 58-65 days, cutting DRG ganglia into pieces, adding tissue digestive juice I, mixing, digesting, and digesting with tissue digestive juice II; then terminating digestion by using a mixed nerve cell culture medium, centrifuging to remove supernatant, and then resuspending and blowing the precipitate into single cell suspension in the mixed nerve cell culture medium; seeding the single cell suspension in a sterile culture plate which is coated with a matrix layer in advance;
the tissue digestive juice I is a mixed solution containing 0.15% collagenase and 0.03mg/ml DNase I;
the tissue digestive juice II is a mixed solution containing 0.25 percent of pancreatin and 0.03mg/ml of DNase I;
the matrix layer is a matrix layer of levorotatory polylysine;
B. culture of naked mole rat DRG mixed nerve cells:
culturing the DRG mixed nerve cells obtained in the step A in a three-gas culture box by using a mixed nerve cell culture medium for 24 hours, then replacing a fresh mixed nerve cell culture medium, and then replacing the culture medium every 3 days; culturing for 25 days, changing to a Schwann cell purification culture medium for culturing, and updating the culture medium every three days;
the mixed nerve cell culture medium is a low-sugar DMEM culture medium containing 15% of imported fetal calf serum in volume percentage;
the Schwann cell purification culture medium is Neurobasal containing 2% B27 by volume fraction and is added with PDGFa with 0.01mg/ml by mass volume fraction;
the parameters of the three-gas incubator are set as follows: the temperature is controlled at 35 +/-2 ℃, the oxygen concentration is 6%, the carbon dioxide concentration is 5 +/-2%, and the humidity is 96 +/-2%.
2. The naked mole rat Schwann cell culture method according to claim 1, wherein in step A, ganglion tissue is minced to 1mm using micro scissors3Adding tissue digestive juice I, mixing, digesting at 37 deg.C for 30min, and digesting with tissue digestive juice II at 37 deg.C for 30 min.
3. The naked mole rat snowman cell culture method according to claim 1, wherein in step a, digestion is terminated with 2ml of mixed nerve cell culture medium.
4. The naked mole rat snowwang cell culture method according to claim 1, wherein in the step B, the mixed neural cell culture medium is cultured for 24 hours, and after it is completely attached, the fresh mixed neural cell culture medium is replaced, and thereafter the culture medium is replaced every 3 days.
5. A naked mole rat schwann cell culture method according to claim 1, wherein the parameters of the triple gas incubator in step B are set as: the temperature was controlled at 35 deg.C, oxygen concentration was 6%, carbon dioxide concentration was 5%, and humidity was 96%.
6. The naked mole rat snowmobile cell culture method according to claim 1, wherein the concentration of the levopolylysine in step a is 0.01 mg/ml.
7. The naked mole rat snowman cell culture method according to claim 1, wherein the centrifugation parameters in step a are set to 1000rpm for 5 minutes at room temperature.
8. The naked mole rat snowmobile cell culture method according to claim 1, wherein in step B, the culture is performed in a three-atmosphere culture chamber for 31 days using a mixed neural cell culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610919420.9A CN106754716B (en) | 2016-10-21 | 2016-10-21 | Naked mole rat Schwann cell culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610919420.9A CN106754716B (en) | 2016-10-21 | 2016-10-21 | Naked mole rat Schwann cell culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754716A CN106754716A (en) | 2017-05-31 |
| CN106754716B true CN106754716B (en) | 2021-01-26 |
Family
ID=58972429
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610919420.9A Active CN106754716B (en) | 2016-10-21 | 2016-10-21 | Naked mole rat Schwann cell culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754716B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502593B (en) * | 2017-09-29 | 2021-01-12 | 南方医科大学 | Extraction, purification and culture method of Schwann cells |
| CN107779436A (en) * | 2017-10-27 | 2018-03-09 | 南京中医药大学 | The structure of peripheral neuropathy cell model caused by chemotherapy and its detection method |
| CN108103013B (en) * | 2018-01-26 | 2020-08-04 | 河北医科大学第四医院 | Enzyme digestion method primary culture and identification method for smooth muscle cells of esophageal-gastric junction |
| CN111378613B (en) * | 2018-12-27 | 2024-01-09 | 深圳华大生命科学研究院 | Amphibious animal cell dissociation kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695408A (en) * | 2016-03-22 | 2016-06-22 | 中国人民解放军第二军医大学 | Naked mole rat astroglia cell culture method |
-
2016
- 2016-10-21 CN CN201610919420.9A patent/CN106754716B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695408A (en) * | 2016-03-22 | 2016-06-22 | 中国人民解放军第二军医大学 | Naked mole rat astroglia cell culture method |
Non-Patent Citations (2)
| Title |
|---|
| SAhobrts reeponrtce of histamine-induced itch in the African naked mole-rat and "rescue" by Substance P;Ewan St John Smith1 et al.;《molecular pain》;20101231;第1-6页 * |
| 背根神经节来源的雪旺细胞的离体培养;陈益人;《神经损伤与功能重建》;20070731;摘要，权利要求1，说明书第3段、第32段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754716A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754716B (en) | Naked mole rat Schwann cell culture method | |
| KR101293364B1 (en) | Method for reconstructing tissue engineered human corneal endothelium | |
| Medina-Rodríguez et al. | Protocol to isolate a large amount of functional oligodendrocyte precursor cells from the cerebral cortex of adult mice and humans | |
| Kim et al. | Isolation and culturing of glioma cancer stem cells | |
| JP2016537022A (en) | Method of differentiating into hepatocytes using universal stem cells derived from mesenchymal stem cells | |
| Najafzadeh et al. | Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury | |
| Wang et al. | The neural stem cell properties of PKD2L1+ cerebrospinal fluid-contacting neurons in vitro | |
| Zhu et al. | Isolation and Long‐Term Expansion of Functional, Myelinating Oligodendrocyte Progenitor Cells from Neonatal Rat Brain | |
| CN112608878B (en) | In-vitro cochlear micro-organ functional unit and three-dimensional construction method and application thereof | |
| CN105695409B (en) | A kind of naked mole oligodendrocyte precursor cells cultural method | |
| Serra et al. | Novel culture strategy for human stem cell proliferation and neuronal differentiation | |
| CN105695408B (en) | A kind of naked mole Astrocytes Primary Cultivation | |
| Espinosa‐Jeffrey et al. | Culture system for rodent and human oligodendrocyte specification, lineage progression, and maturation | |
| CN106635990B (en) | Method for primary culture of dorsal root ganglion satellite glial cells | |
| RU2645255C1 (en) | Method for obtaining of biosafe culture of mesenchimal stem cells from human chorionic villae | |
| CN115354029A (en) | Method for producing neural organoid and neural organoid | |
| CN106520697B (en) | Naked mole rat DRG neuron culture method | |
| JP2016537021A (en) | Method for differentiating into adipocytes using universal stem cells derived from mesenchymal stem cells | |
| Huang et al. | Generation of human epidermis-derived mesenchymal stem cell-like pluripotent cells (hEMSCPCs) | |
| CN104130975B (en) | The neuron cell and its preparation method and application in human adipose-derived stem cells source | |
| CN112852715A (en) | Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells | |
| CN105695411B (en) | A kind of naked mole cortical neuron cultural method | |
| CN110862966A (en) | Method for constructing three-dimensional engineered neural tissue, three-dimensional engineered neural tissue and application thereof | |
| CN108441475B (en) | Method for culturing mesonasal concha-derived olfactory ensheathing cells | |
| KR101671882B1 (en) | Method for Differentiating Pluripotent Stem Cell induced from adipose-derived Mesenchymal Stem Cell into Neuron |
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 | ||
| CB03 | Change of inventor or designer information |
Inventor after: Cui Shufang Inventor after: Zhao Shanmin Inventor after: Xu Chen Inventor after: Li Zhoutong Inventor after: Yang Wenjing Inventor after: Ni Jian Inventor after: Sun Wei Inventor after: Cheng Jishuai Inventor after: Lin Lifang Inventor after: Cong Wei Inventor after: Cai Liping Inventor after: Yu Chenlin Inventor before: Cui Shufang Inventor before: Xu Chen Inventor before: Li Zhoutong Inventor before: Yang Wenjing Inventor before: Sun Wei Inventor before: Cheng Jishuai Inventor before: Lin Lifang Inventor before: Cong Wei Inventor before: Cai Liping Inventor before: Yu Chenlin Inventor before: Zhao Shanmin |
|
| CB03 | Change of inventor or designer information | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210108 Address after: 200433 No. 800 Xiang Yin Road, Shanghai, Yangpu District Applicant after: THE SECOND MILITARY MEDICAL University Applicant after: SHANGHAI PULMONARY Hospital Address before: 200433 No. 800 Xiang Yin Road, Shanghai, Yangpu District Applicant before: THE SECOND MILITARY MEDICAL University |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |