Method for separating, purifying and culturing naked mole rat neural stem cells
Through the culture method under specific conditions, the technical difficulties in the cultivation of naked mole rat neural stem cells were solved, and high-purity and good activity of neural stem cells were obtained, supporting their biological research and clinical applications.
Patent Information
- Application Number
- CN202510414218.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-08-08
Smart Images
Figure CN120442546A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of cell biology, in particular to a method for separating, purifying and culturing naked mole-rat neural stem cells. Background Art
[0002] The hippocampus plays a crucial role in short-term memory and learning (Brown PD, Ahluwalia MS, Khan OH, Asher AL, Wefel JS, Gondi V. 2018. Whole-Brain Radiotherapy for Brain Metastases: Evolution or Revolution? J Clin Oncol 36:483-491.). Physiologically, the dentate gyrus (DG) is a key region of hippocampal neurogenesis, where neural stem cells continuously self-renew through asymmetric division and differentiate into neurons and glial cells (Toda T, Gage FH. 2018. Review: Adult Neurogenesis Contributes to Hippocampal Plasticity. Cell Tissue Res 373:693-709.). Exposure to extreme environmental stresses, such as radiation damage and ischemia-hypoxia, can reduce or ablate hippocampal neurogenesis, leading to cognitive impairment. Therefore, in-depth understanding of the physiological functions and characteristics of neural stem cells will help clarify their functions and their role in the development and progression of neurological diseases.
[0003] Naked mole rats are long-lived, hypoxia-tolerant rodents. Their nervous system functions remain intact, both in hypoxia and in old age. Therefore, understanding how naked mole rats adapt to these extreme conditions, such as hypoxia and aging, is crucial for developing preventative strategies to combat these conditions.
[0004] At present, researchers have established a culture method for rat and mouse neural stem cells that are adapted to normoxic environments, but have not yet developed a culture method for naked mole-rat neural stem cells, which limits the further development and application promotion of naked mole-rat neural stem cells. Summary of the Invention
[0005] The purpose of the present invention is to establish a method for isolating, purifying and culturing naked mole-rat neural stem cells to obtain high-purity, good-condition naked mole-rat neural stem cells, and to provide strong technical support for in vitro research on the special characteristics of naked mole-rat neural stem cells and for effective drug development.
[0006] Existing methods for culturing neural stem cells in rats and mice cannot be used for the culture and purification of naked mole-rat neural stem cells, mainly because: 1) As a poikilothermic mammal, naked mole-rat's somatic cells have special metabolic requirements, resulting in different conditions for obtaining naked mole-rat somatic cells from those of rodents adapted to terrestrial life; 2) Naked mole-rat has long adapted to a humid and hot environment, and the temperature and humidity environment required for its somatic cell growth is also different from that of mice; 3) The embryonic development cycle of naked mole-rat is much longer than that of mice and rats, resulting in large differences in the selection of fetal mice of appropriate age.
[0007] Therefore, the key to establishing an in vitro culture system for naked mole-rat neural stem cells lies in finding the appropriate temperature, humidity, donor age, and a suitable separation and culture system.
[0008] The present invention provides a method for isolating, purifying and culturing naked mole-rat neural stem cells, comprising the following steps:
[0009] (A) Collection of naked mole-rat neural stem cells:
[0010] The subventricular zone (SVZ) brain tissue of naked mole rat fetuses at about 60 days of gestation was placed in HBSS on ice;
[0011] The brain tissue in the SVZ region was minced into 1 mm pieces. 3 After digestion in tissue digestion solution I at 32°C for 10 min, an equal volume of trypsin inhibitor (2 mg / ml) was added to terminate the digestion, and the cells were collected by centrifugation at 1000 rpm for 5 min;
[0012] The cells were then digested in tissue digestion solution II at 32°C for 10 min, an equal volume of trypsin inhibitor (2 mg / ml) was added to terminate the digestion, and the cells were collected by centrifugation at 1000 rpm for 5 min;
[0013] Finally, the cells were digested with tissue digestion solution III at 32°C for 5 min, and an equal volume of trypsin inhibitor (2 mg / ml) was added to terminate the digestion. The cells were then resuspended in growth medium I.
[0014] The tissue digestion solution I is a growth medium containing 0.125% (v / v) trypsin;
[0015] The tissue digestion solution II is a growth medium containing 0.7 mg / ml hyaluronidase;
[0016] The tissue digestion solution III is a growth medium containing 0.1 mg / ml DNase I;
[0017] The digestion stop solution is growth medium containing 2 mg / ml trypsin inhibitor;
[0018] (B) Culture of naked mole-rat neural stem cells:
[0019] The cells obtained in step A were cultured in a three-gas incubator with growth medium for 7 days, and the medium was replaced by half every 3 days;
[0020] The growth medium was DF12 medium containing 2% (v / v) B27 (minus vitamin A), 0.5% penicillin-streptomycin (units / ml), 2 mM / L glutamine (Sigma-Aldrich), 200 ng / ml heparan sulfate sodium salt from bovine kidney (Sigma-Aldrich), 20 ng / ml bFGF (Pepro Tech), and 20 ng / ml EGF;
[0021] The parameters of the three-gas incubator were set as follows: temperature controlled at 33±2°C, oxygen concentration at 15%, carbon dioxide concentration at 5±0.5%, and humidity at 96±2%.
[0022] Furthermore, in step A, naked mole-rat fetuses of about 60 days of gestation are isolated under sterile conditions, brain tissue from the SVZ region is obtained under a dissecting microscope, and the meninges are removed using microtweezers.
[0023] Furthermore, in step A, the nerve tissue was minced into 1 mm pieces using microscissors. 3 .
[0024] Furthermore, the parameters of the three-gas incubator described in step B are set as follows: temperature 33° C., oxygen concentration 15%, carbon dioxide concentration 5%, and humidity 96%.
[0025] The advantages and beneficial effects of the present invention are:
[0026] 1. Using a combination of digestive fluid and a culture medium containing multiple nutrient factors, researchers isolated, purified, and cultured neural stem cells from the SVZ of naked mole-rat fetuses, developing a culture method suitable for poikilothermic rodents. They found that cells were well maintained and able to grow at an oxygen concentration of 15%.
[0027] 2. The operation method is simple and has high repeatability;
[0028] 3. By using neural stem cell growth medium, the normal growth of neural stem cells is effectively guaranteed, and the purity of neural stem cells is guaranteed to reach more than 90%;
[0029] 4. The types and concentrations of additional growth factors are clear. Only 200ng / ml heparan sulfate sodium, 20ng / ml bFGF and 20ng / ml EGF need to be added to the neuron maintenance culture medium to ensure that neural stem cells maintain a high proliferation rate and functional status.
[0030] In summary, the method of the present invention can simply, efficiently and economically obtain a large number of naked mole-rat neural stem cells with normal functional activity, providing an important theoretical basis for further studying the special physiological functions of naked mole-rat neural stem cells, exploring the biological mechanisms therein and applying them in clinical related fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 These are neural stem cell spheres cultured in vitro (ordinary light microscope).
[0032] Figure 2 These are neural stem cell spheres cultured in vitro (identified by immunocytochemistry). DETAILED DESCRIPTION
[0033] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.
[0034] Example 1: Isolation, purification and culture conditions of naked mole-rat neural stem cells
[0035] Table 1. Effects of different heparan sulfate sodium concentrations on the survival of naked mole-rat neural stem cells
[0036]
[0037] Table 2. Effects of different bFGF concentrations on the growth rate of naked mole-rat neural stem cells
[0038]
[0039] Table 3. Effects of different EGF concentrations on the growth rate of naked mole-rat neural stem cells
[0040]
[0041] Example 2: Method for Isolating, Purifying and Cultivating Naked Mole Rat Neural Stem Cells
[0042] 1. Experimental Materials
[0043] Naked mole rats at about 60 days of gestation were provided by the Department of Experimental Animal Science, School of Basic Medical Sciences, Naval Medical University.
[0044] DNase I was purchased from Solarbio Biotechnology Co., Ltd. B27 (without vitamin A) and DF12 culture media were purchased from Thermo Fisher, glutamine, heparan sulfate sodium salt, and penicillin-streptomycin were purchased from Sigma-Aldrich, and bFGF and EGF were purchased from Pepro Tech.
[0045] The growth medium consisted of DF12 medium containing 2% (v / v) B27 (minus vitamin A), 0.5% penicillin-streptomycin (units / ml), 2 mM / L glutamine (Sigma-Aldrich), 200 ng / ml heparan sulfate sodium salt from bovine kidney (Sigma-Aldrich), 20 ng / ml bFGF (Pepro Tech), and EGF (Pepro Tech).
[0046] 2. Isolation, purification and culture of naked mole-rat neural stem cells
[0047] A. Collection of naked mole-rat neural stem cells:
[0048] Naked mole rat fetuses at about 60 days of gestation were isolated under sterile conditions, and SVZ tissues were obtained under a dissecting microscope and placed in HBSS in an ice bath.
[0049] The brain tissue in the SVZ region was minced into 1 mm pieces. 3 After digestion in Tissue Digestion Solution I at 32°C for 10 minutes, an equal volume of trypsin inhibitor (2 mg / ml) was added to terminate digestion, and cells were collected by centrifugation at 1000 rpm for 5 minutes. Tissue Digestion Solution I is preferably growth medium containing 0.125% trypsin.
[0050] The cells were then digested in Tissue Digestion Solution II at 32°C for 10 minutes. An equal volume of trypsin inhibitor (2 mg / ml) was added to terminate the digestion, and the cells were collected by centrifugation at 1000 rpm for 5 minutes. Tissue Digestion Solution II is preferably growth medium containing 0.7 mg / ml hyaluronidase.
[0051] Finally, digest the cells in Tissue Digestion Solution III at 32°C for 5 minutes, add an equal volume of trypsin inhibitor (2 mg / ml) to terminate the digestion, and resuspend the cells in Growth Medium I. The preferred Tissue Digestion Solution III is Growth Medium containing 0.1 mg / ml DNase I.
[0052] B. Cultivating naked mole-rat neural stem cells:
[0053] The cells obtained in step A were cultured in a tri-gas incubator using growth medium for 7 days, with half the medium replaced every 3 days. This constituted one cycle, and 2-3 cycles were performed.
[0054] The growth medium was DF12 medium containing 2% (v / v) B27 (minus vitamin A), 0.5% penicillin-streptomycin (units / ml), 2 mM / L glutamine (Sigma-Aldrich), 200 ng / ml heparan sulfate sodium salt from bovine kidney (Sigma-Aldrich), 20 ng / ml bFGF (Pepro Tech), and 20 ng / ml EGF (Pepro Tech).
[0055] The parameters of the three-gas incubator are set as follows: temperature is controlled at 33° C., oxygen concentration is 15%, carbon dioxide concentration is 5%, and humidity is 96%.
[0056] Example 2: Identification of naked mole-rat neural stem cells
[0057] Identification of naked mole-rat neural stem cells obtained in Example 1: The neural stem cells in serum-free culture medium were fixed with 4% paraformaldehyde and identified by combining morphological identification, immunohistochemistry and other methods.
[0058] 1. Cell morphology identification:
[0059] Naked mole-rat neural stem cells grown in suspension cultures exhibited an aggregated growth pattern, ultimately forming uniform spherical structures. Under an optical microscope, the spheres were denser in the center and less dense at the periphery.
[0060] 2. Immunocytochemical identification:
[0061] Immunofluorescence staining of neural stem cells using Sox2 antibody showed that the neural stem cell spheres were uniformly Sox2 positive.
[0062] According to the above experimental results, the naked mole-rat neural stem cells isolated and purified in the present invention have typical neural stem cell morphology, grow in suspension and aggregate into spheres, are Sox2 positive, and can maintain a good proliferation state in the growth culture medium.
[0063] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.
Claims
1. A method for isolating, purifying and culturing naked mole-rat neural stem cells, characterized in that: The following steps are involved: (A) Collection of naked mole-rat neural stem cells: The brain tissue of the SVZ region of naked mole rat fetuses at about 60 days of gestation was placed in HBSS in an ice bath; The brain tissue in the SVZ region was minced into 1 mm pieces. 3 After digestion in tissue digestion solution I at 32°C for 10 minutes, an equal volume of 2 mg / ml trypsin inhibitor was added to terminate digestion, and the cells were collected by centrifugation at 1000 rpm for 5 minutes; the tissue digestion solution I is growth medium containing 0.125% (v / v) trypsin; The cells were then digested in tissue digestion solution II at 32°C for 10 minutes, an equal volume of 2 mg / ml trypsin inhibitor was added to terminate the digestion, and the cells were collected by centrifugation at 1000 rpm for 5 minutes; the tissue digestion solution II was a growth medium containing 0.7 mg / ml hyaluronidase; Finally, the cells were digested in tissue digestion solution III at 32°C for 5 minutes, and an equal volume of trypsin inhibitor was added to terminate the digestion. The cells were then resuspended in growth medium I. The tissue digestion solution III consisted of growth medium containing 0.1 mg / ml DNase I. The digestion termination solution consisted of growth medium containing 2 mg / ml trypsin inhibitor. (B) Culture of naked mole-rat neural stem cells: The cells obtained in step A were cultured in a tri-gas incubator with growth medium for 7 days, with half the medium replaced every 3 days; this constituted one cycle, and 2-3 cycles were performed; The growth medium is DF12 medium containing 2% (v / v) B27 without vitamin A, 0.5% units / ml penicillin-streptomycin, 2 mM / L glutamine, 200 ng / ml heparan sulfate sodium salt from bovine kidney, 20 ng / ml bFGF and 20 ng / ml EGF; The parameters of the three-gas incubator were set as follows: temperature controlled at 33±2°C, oxygen concentration at 15%, carbon dioxide concentration at 5±0.5%, and humidity at 96±2%.
2. The method for isolating, purifying and culturing naked mole-rat neural stem cells according to claim 1, wherein: In step A, use microscissors to mince the nerve tissue into 1 mm pieces. 3 .
3. The method for isolating, purifying and culturing naked mole-rat neural stem cells according to claim 1, wherein: The three-gas incubator parameters described in step B were set as follows: temperature 33° C., oxygen concentration 15%, carbon dioxide concentration 5%, and humidity 96%.