A method for the simultaneous isolation and culture of neural cells and astrocytes from non-human primate cryopreserved tissue

Abstract

The application belongs to the field of cell biology, and particularly relates to a method for synchronously isolating and culturing neural cells and astrocytes from non-human primate frozen tissues. The method comprises the following steps: taking non-human primate embryonic cerebral cortex tissues, rinsing, cutting and then storing in liquid nitrogen; after resuscitation, using papain, collagenase IV and DNase I for joint digestion to prepare a single cell suspension; using the characteristic that neural cells adhere to the wall earlier than astrocytes, after differential adhesion separation, the adherent cells are purified and cultured in a screening culture medium containing cytosine arabinoside to obtain high-purity neural cells, and the non-adherent cells are expanded in an astrocyte culture medium to obtain astrocytes. The application realizes synchronous acquisition of two kinds of primary cells from the same frozen tissue, significantly improves the utilization rate of tissues, breaks through the dependence on fresh samples, and the obtained neural cells have good activity and high purity, the astrocytes can be stably passaged, and are suitable for neuroscience research and drug screening.

Description

Technical Field

[0001] This invention belongs to the field of cell biology, specifically relating to a method for simultaneously isolating and culturing nerve cells and astrocytes from cryopreserved non-human primate tissues. Background Technology

[0002] The isolation and culture of primary neural cells and astrocytes are key techniques for elucidating brain function mechanisms, constructing disease models, and screening drugs in neuroscience research. Primary cells are derived directly from living tissues, thus preserving their natural structure and functional characteristics in vivo to the greatest extent possible, making them ideal in vitro models for simulating the physiological and pathological states of the central nervous system.

[0003] However, current methods for simultaneously obtaining high-purity neural cells and astrocytes still face core technological bottlenecks: traditional methods typically optimize the isolation of only a single cell type, making it difficult to achieve efficient and high-purity simultaneous isolation of both cell types in a single experiment. Furthermore, the strategy of separating and culturing them separately leads to significant resource waste. For example, during the enrichment of neural cells, glial cells often need to be removed, resulting in a large number of astrocytes being discarded directly as byproducts.

[0004] On the other hand, current neuroscience research primarily relies on rodent models. However, due to significant differences in evolutionary levels, the results of rodent model studies cannot be directly extrapolated to primates. Non-human primates (such as monkeys) are closer to humans in terms of evolutionary relationships, neural structure, and cognitive function, and are gradually becoming important model animals for neuroscience research. In rodents, primary nerve cells are typically obtained using a "use-as-you-go" approach. However, primates are large, costly, and subject to strict ethical requirements; continuing this approach would lead to significant resource waste and severely limit its widespread application.

[0005] Therefore, there is an urgent need in the field for a method that can efficiently and synchronously isolate and culture nerve cells and astrocytes from non-human primate tissues. Summary of the Invention

[0006] This invention aims to address the deficiencies and shortcomings of existing technologies by providing a method for simultaneously isolating and culturing neural cells and astrocytes from cryopreserved non-human primate tissues. This method achieves efficient isolation and simultaneous culture of primary neural cells and astrocytes from cryopreserved tissues, overcoming the dependence of traditional methods on fresh samples. Furthermore, it can simultaneously obtain two cell types—highly active and pure mammalian neural cells and glial cells—from the same brain region, significantly conserving valuable animal resources.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a method for simultaneously isolating and culturing nerve cells and astrocytes from cryopreserved non-human primate tissues, comprising the following steps: (1) Take the cerebral cortex tissue of a non-human primate embryo, rinse it in pre-cooled rinsing solution, remove the rinsing solution, cut the tissue into pieces, add cryopreservation solution and freeze it. (2) After the frozen tissue is thawed, it is rinsed in the rinsing solution. After removing the rinsing solution, digestion solution is added for digestion. Digestion is terminated with astrocyte culture medium. The tissue is pipetted into a single-cell suspension, centrifuged, and then resuspended in astrocyte culture medium. (3) Inoculate the cell suspension obtained in step (2) into a culture container that has been coated and let it stand for culture. Then, transfer the culture supernatant containing non-adherent cells to a new culture container for astrocyte culture. Add the adherent nerve cells to the nerve cell selection medium for culture, and then replace it with nerve cell maintenance medium for continued culture.

[0008] This invention utilizes the characteristic that nerve cells adhere to the substrate earlier than astrocytes, combined with the in vitro proliferation capability of astrocytes, to simultaneously obtain high-purity nerve cells and astrocytes in a single experiment, significantly improving tissue utilization and avoiding waste of samples and resources.

[0009] Preferably, the non-human primate is a cynomolgus monkey, and the gestational age of the cynomolgus monkey embryo is 50-100 days.

[0010] The inventors have experimentally confirmed that gestational age has a crucial impact on the separation effect of this method—embryos with a gestational age of 60 days yield nerve cells with high purity and good growth status; while embryos with a gestational age of 120 days contain more glial cells and have poorer nerve cell growth status. Therefore, a gestational age range of 50-100 days is preferred, and 60 days is even more preferred.

[0011] Preferably, the cryopreservation solution in step (1) comprises dimethyl sulfoxide, fetal bovine serum and basal culture medium; the volume concentration of dimethyl sulfoxide in the cryopreservation solution is 5-15% (more preferably 10%), the volume concentration of fetal bovine serum is 10-90% (more preferably 20%), and the remainder is basal culture medium (such as Neurobasal basal culture medium).

[0012] Preferably, in step (1), the cryopreservation is performed by programmed cooling to -80°C and then transferring to liquid nitrogen for storage; the cooling program is -1°C / minute.

[0013] Preferably, the digestive solution in step (2) is a mixed solution of papain, collagenase IV and DNase I, and the solvent used is a rinsing solution.

[0014] The digestion solution formulation is one of the key parameters that has a crucial impact on the final separation effect in this invention. Traditional methods often use pancreatic enzymes or single papain for digestion, which has the following drawbacks: (1) Pancreatic enzyme digestion is relatively vigorous, nerve cells are relatively fragile, and pancreatic enzyme digestion causes significant damage to nerve cell activity and neurite development; (2) Papain causes less damage to nerve cells, but its digestive capacity is weak, resulting in poor separation effect on glial cells, incomplete digestion, and low tissue utilization. This invention uses a composite formulation of papain combined with collagenase IV, which can simultaneously ensure the survival rate of nerve cells and the full digestion of tissues; and the addition of DNase can effectively degrade the DNA released during digestion, prevent cell aggregation, and improve the quality of single-cell suspension.

[0015] Preferably, the concentration of papain in the digestive fluid is 10-50 U / mL (more preferably 20 U / mL), the concentration of collagenase IV is 0.1-2 mg / mL (more preferably 0.5 mg / mL), and the concentration of DNase I is 0.05-0.2 mg / mL (more preferably 0.1 mg / mL).

[0016] Preferably, the static culture time in step (3) is 4-6 hours, more preferably 4 hours. This time window is crucial to ensure that nerve cells adhere fully to the culture vessel while astrocytes have not yet adhered extensively.

[0017] Preferably, the matrix used for the coating treatment is selected from at least one of poly-L-lysine, Laminin polymer, and Matrigel matrix gel. Poly-L-lysine (e.g., 100 µg / mL) is preferred.

[0018] Preferably, the nerve cell screening medium in step (3) comprises Neurobasal basal medium, B27 additive, and cytarabine; the volume concentration of B27 additive in the nerve cell maintenance medium is 1-5%, and the concentration of cytarabine is 1-10 μM. Cytarabine can effectively inhibit the proliferation of glial cells and ensure the purity of nerve cells.

[0019] Preferably, the nerve cell maintenance culture medium in step (3) comprises Neurobasal basal medium and B27 additive, wherein the volume concentration of B27 additive in the nerve cell maintenance culture medium is 1-5%.

[0020] Preferably, the astrocyte culture medium in step (2) and / or step (3) comprises DMEM basal medium, fetal bovine serum and bFGF, wherein the volume concentration of fetal bovine serum in the astrocyte culture medium is 5-15% (more preferably 10%) and the concentration of bFGF is 4-20 ng / mL (more preferably 10 ng / mL).

[0021] Preferably, the rinsing solution in step (1) is selected from at least one of D-Hank's buffer, PBS buffer, DPBS buffer, physiological saline, and Hibernate-A basal medium, with D-Hank's buffer being preferred. Penicillin-streptomycin antibiotics (100×) may be added to the rinsing solution to prevent contamination.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) Achieving synchronous separation: This invention achieves the synchronous separation of nerve cells and astrocytes from cryopreserved non-human primate brains, overcoming the technical bottleneck that traditional methods can only separate single cell types.

[0023] (2) Breaking the dependence on fresh samples: This invention uses frozen tissue as the starting material, breaking the dependence of traditional methods on fresh samples, making the utilization of primate resources more flexible and efficient, and greatly saving precious animal resources.

[0024] (3) Improve tissue utilization: Two cell types can be obtained from the same tissue sample at the same time, avoiding the waste of resources in the traditional method of "choosing one and discarding the other".

[0025] (4) Excellent cell quality: The nerve cells obtained by the method of the present invention have high purity and good growth status; astrocytes can be stably expanded and passaged, and the purity after passage can reach close to 100%.

[0026] (5) Wide range of applications: This invention is not only applicable to cynomolgus monkeys, but can also be extended to other non-human primates, and has broad prospects for scientific research applications. Attached Figure Description

[0027] Figure 1 The image shows a morphological comparison of cynomolgus monkey nerve cells under white light (on day 5 of culture). The left image shows nerve cells cultured after digestion with the digestive solution of this invention, and the right image shows nerve cells cultured after digestion with 0.25% trypsin digestive solution.

[0028] Figure 2 Comparison of nerve cells cultured for 12 days after digestion with the digestive solution of this invention in cynomolgus monkey embryos of different gestational ages. The left image shows a 60-day gestational age cynomolgus monkey embryo, and the right image shows a 120-day gestational age cynomolgus monkey embryo.

[0029] Figure 3 Comparison of the purity of nerve cells cultured on day 5 and day 8 after digestion and isolation with different digestive solutions.

[0030] Figure 4 The morphology and purity of astrocytes at different amplification passages are shown, with DAPI labeling cell nuclei (representing the total number of cells) and GFAP labeling astrocytes.

[0031] Figure 5 The images show fluorescently stained cynomolgus monkey nerve cells (cultured for 5 days), with DAPI labeling the cell nuclei, GFAP labeling astrocytes, and Tuj1 labeling the nerve cells. Detailed Implementation

[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments.

[0033] Unless otherwise specified, all reagents used in the examples are conventional reagents available in the art and can be purchased commercially. Experimental procedures not specifically described in the examples are conventional procedures in the art or can be understood or known by those skilled in the art based on their prior knowledge or common general knowledge.

[0034] Example 1 This embodiment provides a method for simultaneously isolating and culturing nerve cells and astrocytes from cryopreserved tissue of cynomolgus monkeys, comprising the following steps: 1. Active cryopreservation of cynomolgus monkey cerebral cortex tissue: (1) Take the brain of a 60-day-old cynomolgus fetus and place it in a 4°C pre-cooled rinsing solution (D-Hank's buffer, containing penicillin-streptomycin (100×)) and rinse 3 times. The cerebral cortex is then separated under a dissecting microscope. (2) Transfer the cerebral cortex to a sterile culture dish, place it on ice, and cut it into pieces about 1 mm in diameter with ophthalmic scissors. 3 Small pieces.

[0035] (3) Add 5 times the volume of cryopreservation solution (Neurobasal basal medium:DMSO:FBS = 70%:10%:20%), mix well and then evenly dispense into cryopreservation tubes; (4) Place the cryovial in a programmed cooling instrument and adjust the cooling program to -1℃ / minute until the temperature drops to -80℃. Then quickly transfer it to liquid nitrogen for long-term storage.

[0036] 2. Digestion and cell separation of cryopreserved tissues: (1) Adjust the water bath to 37°C, take out the frozen tissue from the liquid nitrogen, quickly transfer it to the water bath, and shake the cryotube until it thaws; (2) Transfer the tissue along with the cryopreservation solution to a 15 mL centrifuge tube, add 5 times the volume of washing solution, centrifuge at 200×g at room temperature, and discard the supernatant; (3) Add 5 times the volume of digestion solution (D-Hank's buffer containing 20 U / mL papain, 0.5 mg / mL collagenase IV and 0.1 mg / mL DNase I), transfer the tissue to a 6 cm cell culture dish, and place it in a 37°C, 5% CO2 cell culture incubator for 15 minutes to digest; (4) Take out and shake every 5 minutes until the tissue is milky. Add 2 times the volume of astrocyte culture medium (DMEM basal medium containing 10% FBS, with 10 ng / mL bFGF added) to stop digestion. Then gently blow it into a single cell suspension using a Pasteur tube. (5) Pass the cells through a 40μm cell sieve to remove large particles of impurities. Centrifuge the sieved cells at 200×g at room temperature, discard the supernatant, and resuspend them in astrocyte culture medium.

[0037] 3. Purification and culture of nerve cells: (1) Coat a 6cm culture dish with 100µg / mL polylysine overnight, then rinse the culture dish three times with sterile water and air dry in a biosafety cabinet for later use. (2) The single-cell suspension obtained in step 2 was seeded into a 6cm culture dish at an appropriate density, and the astrocyte culture medium was added to 4mL. The dish was then placed in a cell culture incubator and cultured for 4 hours. (3) Gently shake the culture dish and transfer the non-adherent cells to a new centrifuge tube for later use (for astrocyte culture). Replace the adherent cells with neural cell selection medium (Neurobasal medium + 2% B27 additive + 5μM cytarabine) and culture in a cell culture incubator for 3 days. (4) Discard the nerve cell selection medium, rinse twice with PBS, and then add nerve cell maintenance medium (Neurobasal medium + 2% B27 additive) to continue culturing. Change the medium every 3 days.

[0038] 4. Astrocyte culture (1) The non-adherent cell suspension obtained in step 3 was seeded at an appropriate density in a 6cm culture dish coated with 100µg / mL poly-L-lysine; (2) Add astrocyte culture medium to a suitable volume, place it in an incubator for culture, replace the culture medium with fresh medium every 3 days, and passage can be performed when the cell fusion density reaches more than 80%.

[0039] Example 2 This embodiment provides a method for simultaneously isolating and culturing nerve cells and astrocytes from cryopreserved tissue of cynomolgus monkeys, comprising the following steps: 1. Active cryopreservation of cynomolgus monkey cerebral cortex tissue: (1) Take the brain of a 50-day-old cynomolgus fetus and place it in a 4°C pre-cooled rinsing solution (D-Hank's buffer, containing penicillin-streptomycin antibiotics (100×)) and rinse it 3 times. The cerebral cortex is then separated under a dissecting microscope. (2) Transfer the cerebral cortex to a sterile culture dish, place it on ice, and cut it into pieces about 1 mm in diameter with ophthalmic scissors. 3 Small pieces.

[0040] (3) Add 5 times the volume of cryopreservation solution (Neurobasal basal medium:DMSO:FBS = 55%:5%:40%), mix well and then evenly dispense into cryovials; (4) Place the cryovial in a programmed cooling instrument and adjust the cooling program to -1℃ / minute until the temperature drops to -80℃. Then quickly transfer it to liquid nitrogen for long-term storage.

[0041] 2. Digestion and cell separation of cryopreserved tissues: (1) Adjust the water bath to 37°C, take out the frozen tissue from the liquid nitrogen, quickly transfer it to the water bath, and shake the cryotube until it thaws; (2) Transfer the tissue along with the cryopreservation solution to a 15 mL centrifuge tube, add 5 times the volume of washing solution, centrifuge at 200×g at room temperature, and discard the supernatant; (3) Add 5 times the volume of digestion solution (D-Hank's buffer containing 10 U / mL papain, 2 mg / mL collagenase IV and 0.2 mg / mL DNase I), transfer the tissue to a 6 cm cell culture dish, and place it in a 37°C, 5% CO2 cell culture incubator for 15 minutes to digest; (4) Take out and shake every 5 minutes until the tissue becomes milky. Add 2 times the volume of astrocyte culture medium (DMEM basal medium containing 5% FBS, with 20 ng / mL bFGF added) to stop digestion. Then gently blow the mixture into a single-cell suspension using a Pasteur tube. (5) Pass the cells through a 40μm cell sieve to remove large particles of impurities. Centrifuge the sieved cells at 200×g at room temperature, discard the supernatant, and resuspend them in astrocyte culture medium.

[0042] 3. Purification and culture of nerve cells: (1) Use 5ug / cm one day in advance 2 Laminin polymer was coated on 6cm culture dishes overnight, then the culture dishes were rinsed three times with sterile water and air-dried in a biosafety cabinet for later use. (2) The single-cell suspension obtained in step 2 was seeded into a 6cm culture dish at an appropriate density, and the astrocyte culture medium was added to 4mL. The dish was then placed in a cell culture incubator and cultured for 6 hours. (3) Gently shake the culture dish and transfer the non-adherent cells to a new centrifuge tube for later use (for astrocyte culture). Replace the adherent cells with neural cell selection medium (Neurobasal medium + 1% B27 additive + 10μM cytarabine) and culture in a cell culture incubator for 3 days. (4) Discard the nerve cell selection medium, rinse twice with PBS, and then add nerve cell maintenance medium (Neurobasal medium + 1% B27 additive) to continue culturing. Change the medium every 3 days.

[0043] 4. Astrocyte culture (1) The non-adherent cell suspension obtained in step 3 was seeded at an appropriate density in a 5ug / cm² medium. 2 Laminin polymer-coated 6cm culture dish; (2) Add astrocyte culture medium to a suitable volume, place it in an incubator for culture, replace the culture medium with fresh medium every 3 days, and passage can be performed when the cell fusion density reaches more than 80%.

[0044] Example 3 This embodiment provides a method for simultaneously isolating and culturing nerve cells and astrocytes from cryopreserved tissue of cynomolgus monkeys, comprising the following steps: 1. Active cryopreservation of cynomolgus monkey cerebral cortex tissue: (1) Take the brain of a 100-day-old cynomolgus fetus and place it in a 4°C pre-cooled rinsing solution (D-Hank's buffer, containing penicillin-streptomycin antibiotics (100×)) and rinse it 3 times. The cerebral cortex is then separated under a dissecting microscope. (2) Transfer the cerebral cortex to a sterile culture dish, place it on ice, and cut it into pieces about 1 mm in diameter with ophthalmic scissors. 3 Small pieces.

[0045] (3) Add 5 times the volume of cryopreservation solution (Neurobasal medium:DMSO:FBS = 75%:15%:10%), mix well and then evenly dispense into cryovials; (4) Place the cryovial in a programmed cooling instrument and adjust the cooling program to -1℃ / minute until the temperature drops to -80℃. Then quickly transfer it to liquid nitrogen for long-term storage.

[0046] 2. Digestion and cell separation of cryopreserved tissues: (1) Adjust the water bath to 37°C, take out the frozen tissue from the liquid nitrogen, quickly transfer it to the water bath, and shake the cryotube until it thaws; (2) Transfer the tissue along with the cryopreservation solution to a 15 mL centrifuge tube, add 5 times the volume of washing solution, centrifuge at 200×g at room temperature, and discard the supernatant; (3) Add 5 times the volume of digestion solution (D-Hank's buffer containing 50 U / mL papain, 0.1 mg / mL collagenase IV and 0.05 mg / mL DNase I), transfer the tissue to a 6 cm cell culture dish, and place it in a 37°C, 5% CO2 cell culture incubator for 15 minutes to digest; (4) Take out and shake every 5 minutes until the tissue is milky. Add 2 times the volume of astrocyte culture medium (DMEM basal medium containing 15% FBS, with 4 ng / mL bFGF added) to stop digestion. Then gently blow it into a single cell suspension using a Pasteur tube. (5) Pass the cells through a 40μm cell sieve to remove large particles of impurities. Centrifuge the sieved cells at 200×g at room temperature, discard the supernatant, and resuspend them in astrocyte culture medium.

[0047] 3. Purification and culture of nerve cells: (1) One day in advance, coat a 6cm culture dish with Matrigel matrix and leave it overnight. Then rinse the culture dish three times with sterile water and air dry it in a biosafety cabinet for later use. (2) The single-cell suspension obtained in step 2 was seeded into a 6cm culture dish at an appropriate density, and the astrocyte culture medium was added to 4mL. The dish was then placed in a cell culture incubator and cultured for 6 hours. (3) Gently shake the culture dish and transfer the non-adherent cells to a new centrifuge tube for later use (for astrocyte culture). Replace the adherent cells with neural cell selection medium (Neurobasal medium + 5% B27 additive + 1μM cytarabine) and culture in a cell culture incubator for 3 days. (4) Discard the nerve cell selection medium, rinse twice with PBS, and then add nerve cell maintenance medium (Neurobasal medium + 5% B27 additive) to continue culturing. Change the medium every 3 days.

[0048] 4. Astrocyte culture (1) The non-adherent cell suspension obtained in step 3 was seeded at an appropriate density in a 6cm culture dish coated with Matrigel. (2) Add astrocyte culture medium to a suitable volume, place it in an incubator for culture, replace the culture medium with fresh medium every 3 days, and passage can be performed when the cell fusion density reaches more than 80%.

[0049] Test Example 1: Effects of different digestive juices on nerve cell morphology and purity The same batch of cryopreserved cynomolgus brain tissue was digested using the digestion solution of this invention (D-Hank's buffer containing 20 U / mL papain, 0.5 mg / mL collagenase IV, and 0.1 mg / mL DNase I) and the 0.25% trypsin digestion solution (D-Hank's buffer containing 0.25% trypsin), with the remaining steps being the same (see Example 1 for details). Cell morphology was observed under a white light microscope on day 5 of culture. Figure 1 ), and the purity of nerve cells was measured on days 5 and 8 of culture, respectively. Figure 3 ).

[0050] Figure 1 The results showed that nerve cells obtained using the digestive solution of this invention had plump cell bodies and well-developed processes; while nerve cells obtained using pancreatic enzyme digestive solution had shrunken cell bodies, sparse processes, and even breakage. This indicates that the digestive solution formulation of this invention causes significantly less damage to nerve cells than the traditional pancreatic enzyme digestion method, and can better maintain the activity and normal morphology of nerve cells.

[0051] Figure 3 The results showed that the purity of nerve cells obtained using the digestive solution of this invention was significantly higher than that of the single pancreatic enzyme digestive solution group. This indicates that the synergistic use of papain, collagenase IV, and DNase I can more fully digest tissue, release more nerve cells, and maintain cell viability, thereby improving the final purity of nerve cells.

[0052] Test Example 2: The Effect of Different Gestational Ages on the Efficacy of Neural Cell Culture Cerebral cortex tissues from cynomolgus monkey embryos at 60 and 120 days gestation were collected and digested, separated, and cultured using the method of this invention (see Example 1 for details). The growth status of nerve cells was observed on day 12 of culture. Figure 2 ).

[0053] Figure 2 The results showed that the neural cells obtained from embryos with a gestational age of 60 days had high purity, good cell morphology, and excellent growth status; while the cells obtained from embryos with a gestational age of 120 days contained more glial cells and had poor neural cell growth status. This indicates that gestational age is a key parameter affecting the separation effect of this invention, with 50-100 days being preferred, and about 60 days being more preferred.

[0054] Test Example 3: Identification of the purity of astrocytes The astrocytes isolated by the method of the present invention (see Example 1) were passaged and cultured. Cells from different passages (P0, P1, P2) were subjected to immunofluorescence staining. DAPI was used to label the cell nuclei (total cell count), and GFAP was used to label the astrocytes (…). Figure 4 ).

[0055] Figure 4The results showed that the primary (P0) astrocytes already contained a high proportion of GFAP-positive cells; after passage to P1 and P2, the proportion of GFAP-positive cells was close to 100%, indicating that the astrocytes isolated by the method of the present invention can achieve extremely high purity after simple passage without the need for complicated further purification steps.

[0056] Test Example 4: Identification of Neural Cell Purity The nerve cells cultured for 5 days using the method of the present invention (see Example 1) were subjected to immunofluorescence staining. DAPI was used to label the cell nuclei (total cell count), Tuj1 was used to label the nerve cells, and GFAP was used to label the astrocytes. Figure 5 ).

[0057] Figure 5 The results showed that Tuj1-positive cells (neural cells) were the absolute majority in the field of view, while GFAP-positive cells (astrocytes) were very few, indicating that the method of the present invention can obtain high-purity neural cell cultures.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for simultaneously isolating and culturing nerve cells and astrocytes from cryopreserved non-human primate tissues, characterized in that, Includes the following steps: (1) Take the cerebral cortex tissue of a non-human primate embryo, rinse it in pre-cooled rinsing solution, remove the rinsing solution, cut the tissue into pieces, add cryopreservation solution and freeze it. (2) After the frozen tissue is thawed, it is rinsed in the rinsing solution. After removing the rinsing solution, digestion solution is added for digestion. Digestion is terminated with astrocyte culture medium. The tissue is pipetted into a single-cell suspension, centrifuged, and then resuspended in astrocyte culture medium. (3) Inoculate the cell suspension obtained in step (2) into a coated culture container and let it stand for culture. Then, transfer the culture supernatant containing non-adherent cells to a new culture container for astrocyte culture. Add the adherent nerve cells to the nerve cell selection medium for culture, and then replace it with nerve cell maintenance medium for continued culture. The digestive solution mentioned in step (2) is a mixed solution of papain, collagenase IV and DNase I.

2. The method as described in claim 1, characterized in that, The non-human primate is the cynomolgus monkey, and the gestational age of the cynomolgus monkey embryo is 50-100 days.

3. The method as described in claim 1, characterized in that, The cryopreservation solution in step (1) contains dimethyl sulfoxide, fetal bovine serum and basal culture medium; the volume concentration of dimethyl sulfoxide in the cryopreservation solution is 5-15%, the volume concentration of fetal bovine serum is 10-90%, and the remainder is basal culture medium.

4. The method as described in claim 1, characterized in that, The concentration of papain in the digestive fluid is 10-50 U / mL, the concentration of collagenase IV is 0.1-2 mg / mL, and the concentration of DNase I is 0.05-0.2 mg / mL.

5. The method as described in claim 1, characterized in that, The static incubation time in step (3) is 4-6 hours; the substrate used for the coating treatment is selected from at least one of polylysine, Laminin polymer and Matrigel matrix gel.

6. The method as described in claim 1, characterized in that, The nerve cell screening medium in step (3) contains Neurobasal basal medium, B27 additive and cytarabine; the volume concentration of B27 additive in the nerve cell maintenance medium is 1-5% and the concentration of cytarabine is 1-10 μM.

7. The method as described in claim 1, characterized in that, The nerve cell maintenance culture medium in step (3) contains Neurobasal basal medium and B27 additive, and the volume concentration of B27 additive in the nerve cell maintenance culture medium is 1-5%.

8. The method as described in claim 1, characterized in that, The astrocyte culture medium in step (2) and / or step (3) comprises DMEM basal medium, fetal bovine serum and bFGF, wherein the volume concentration of fetal bovine serum in the astrocyte culture medium is 5-15% and the concentration of bFGF is 4-20 ng / mL.

9. The method as described in claim 1, characterized in that, The rinsing solution in step (1) is selected from at least one of D-Hank's buffer, PBS buffer, DPBS buffer, physiological saline and Hibernate-A basal medium.

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