CN111411081A - Construction method of human neural stem cell bank - Google Patents
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Abstract
The invention discloses a method for constructing a human neural stem cell bank, which comprises the following steps: the method comprises the following steps: collecting human neural stem cells; step two: culturing primary cells, identifying the primary neural stem cells, carrying out passage and partial cryopreservation on the primary cells; step three: purifying, conveying and expanding culture of the neural stem cells; step four: identifying neural stem cells, cooling by a program, and coding and warehousing. The method has simple steps and operation, and the obtained, cultured and cryopreserved human neural stem cells have high purity, strong multiplication capacity and high recovery efficiency, and the problems of preparation, production, storage and the like of the neural stem cells are solved.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of biology, in particular to a method for constructing a human neural stem cell bank.
[ background of the invention ]
Neural stem cells are stem cells with multidirectional differentiation potential, can be self-renewed for a long time, and can be differentiated into neurons, astrocytes and oligodendrocytes under certain conditions. The neural stem cell contains nestin and Musashi protein which are used as main markers of the neural stem, thereby marking and identifying the neural stem cell. Cells under the neural stem obtained by in vitro separation and culture are both positive to Nestin and Musashi protein.
The neural stem cells enrich effective treatment means for treating intracranial diseases due to the characteristics of the neural stem cells, and particularly have unique advantages in the aspects of treating Parkinson's disease, Alzheimer's disease, spinal cord injury, ischemic brain injury and the like. Therefore, we propose a method for constructing a human neural stem cell bank.
[ summary of the invention ]
The invention mainly aims to provide a method for constructing a human neural stem cell bank, which can effectively solve the problems in the background technology.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for constructing a human neural stem cell bank comprises the following steps:
the method comprises the following steps: collecting human neural stem cells;
step two: culturing primary cells, identifying the primary neural stem cells, carrying out passage and partial cryopreservation on the primary neural stem cells;
step three: purifying, conveying and expanding culture of the neural stem cells;
step four: identifying neural stem cells, cooling by a program, and coding and warehousing.
Further, the source of the primary neural stem cells in the first step is mammalian neural stem cells, the primary neural stem cells are obtained by surgically separating waste brain tissues including frontal cortex, midbrain and hippocampal brain tissues, repeatedly cutting in ophthalmology, rinsing in DEME/F12 (volume ratio: 1:1), passing through a 200-mesh filter screen, and centrifuging to obtain a primary neural stem cell suspension.
Further, the step of using the serum-free medium and the culture process after digestion counting, resuspending and adjusting the density to 1 × 105/ml, culturing in an ultra-low adsorption medium at 37 ℃ in an environment of 5% CO2, wherein the serum-free medium is composed of DMEM/F12, B27, N2, and neurotrophic factors BFGF, EGF, &lTtTtransfer = L "&gTt L &lTt/T &gTt IF, and has no animal serum components.
Further, digesting and passaging the expanded primary neural stem cells until passage reaches 5 th, identifying the cell purity to be more than 95%, using the cells as seed cells, freezing and storing the seed cells according to a program for convenient storage, and when the diameter of the expanded primary neural stem cells exceeds 300 mu m, digesting and conveying the cells to the neural stem cells of the P1 generation; digesting the P1 generation neural stem cells, inoculating the digested cells into an ultra-low adsorption culture bottle, placing the bottle in an environment with the temperature of 37 ℃ and the CO of 5% 2 for culturing until passage, repeatedly digesting the bottle, inoculating the bottle and culturing the bottle until the P5 generation neural stem cells are obtained; detecting the cell characteristics and heterologous substances of the neural stem cells of the P5 generation, wherein the cell characteristic identification comprises flow identification and immunofluorescence, the flow identification is used for identifying the neural stem cell surface markers Nestin and Musashi, the purity of the neural stem cell surface markers is detected to be more than 95%, the differentiation characteristics of the neural stem cells are identified by a fluorescence immunization method, namely the differentiation results of neurons, astrocytes and oligodendrocytes, the heterologous substances are mainly the detection of microorganisms, and comprise sterility detection, mycoplasma detection and endotoxin detection, the negative result is qualified, and the cells are frozen and stored as seed cells after the detection is qualified.
Step four, counting the seed cells, performing suspension culture without serum culture medium, digesting and passaging the cells when the cells in the culture flask are fused to 80% -90%, and repeating the steps until the cells are passaged for P9; and (4) freezing and storing the neural stem cells in a liquid nitrogen tank by adopting uniform coding according to a programmed cooling mode.
Further, the serum-free medium is prepared from DMEM/F12 (volume ratio l: l), 7 g/L glucose, 4 mmo/L glutamine, 5 mmol/L Hepes buffer solution, 5 mu g/ml insulin, 100 mu g/ml transferrin, 20 nmol/L progesterone, 100 mu g/ml putrescine, 30 nmol/L sodium selenide, 5 mu g/ml heparin and sodium bicarbonate, and the pH value is adjusted to be 7.2-7.4.
Further, the primary neural stem cell culture method is characterized in that continuous culture is carried out for 10-14 days, observation is carried out every 24 hours during the period, the cell growth state is confirmed, and liquid change is carried out every 48-72 hours; the culture method of the P1 generation neural stem cells comprises the steps of changing liquid every 48-72 hours and continuously culturing for 10-14 days.
Compared with the prior art, the invention has the following beneficial effects: the method has the advantages of simple steps and operation, high purity of the obtained, cultured and cryopreserved human neural stem cells, strong proliferation capacity and high recovery efficiency, and solves the problems of preparation, production, storage and the like of the neural stem cells.
Drawings
FIG. 1 is a schematic overall flow chart of the method for constructing the human neural stem cell bank of the present invention;
FIG. 2 is a schematic view of the morphology of suspension-cultured neural stem cells in a microscope according to the method for constructing a human neural stem cell bank of the present invention.
FIG. 3 is a schematic diagram showing the flow identification and detection results of the method for constructing a human neural stem cell bank of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Transferring the humanized cerebral cortex and buffer solution obtained by material taking into one or more 15/50ml centrifuge tubes by using a sterile disposable 3ml dropper, standing for 5 minutes to ensure that a brain tissue block is fully precipitated to the bottom of the tube, sucking and removing supernatant, then performing sterile filtration by using a sterile injector and a 0.22mm filter membrane, directly filtering into the centrifuge tubes, wherein 5ml of digestive juice is required in each 15ml centrifuge tube, then slightly shaking, placing the centrifuge tubes into a 37 ℃, digesting in a 5% carbon dioxide incubator, manually shaking once every 5 minutes, digesting for 30 minutes, centrifuging 300g, 5 minutes and 20 ℃. And finally, sucking and removing the supernatant to 50 mu l by using a sterile disposable 3ml dropper, sucking 950 mu l of buffer solution by using a 1ml gun head, adding the buffer solution into a centrifuge tube, and gently blowing and sucking for 10-15 times to form a single cell suspension.
Inoculation: 20ml of prepared neural stem cell culture solution is added into a low-adsorption T75 culture bottle, the cell density of the obtained suspension is counted and the final cell inoculation density of 200 cells/ul is prepared, the cell suspension is uniformly mixed, a pipette gun is used for blowing and beating under the liquid level again, the single cell suspension with the calculated volume is taken out and rapidly added into a low-adsorption T75 culture bottle, the container is gently shaken from different directions, the cells are uniformly distributed in the culture solution, the inoculated T75 culture bottle is rapidly placed into a carbon dioxide culture box, the culture temperature is 37 ℃, and the concentration of carbon dioxide is 5%.
The serum-free culture medium is prepared from DMEM/F12 (volume ratio l: l), 7 g/L glucose, 4 mmo/L glutamine, 5 mmol/L Hepes buffer solution, 5 mu g/ml insulin, 100 mu g/ml transferrin, 20 nmol/L progesterone, 100 mu g/ml putrescine, 30 nmol/L sodium selenide, 5 mu g/ml heparin and sodium bicarbonate, wherein the pH value is adjusted to be 7.2-7.4.
Example 1:
primary cell culture, namely performing cell culture solution replacement every 2 days after primary cell inoculation culture, and basically performing the following operations: and taking out the T75 culture bottle from the carbon dioxide incubator, putting the culture bottle into a biological safety cabinet, taking out 2ml of the prepared neural stem cell culture solution nutrition supplementing solution by using a 2ml sterile pipette, quickly adding the prepared neural stem cell culture solution nutrition supplementing solution into the low-adsorption T75 culture bottle, and slightly shaking from different directions to uniformly distribute the cells. And quickly putting the low-adsorption T75 culture bottle added with the supplementary solution back to a carbon dioxide incubator, wherein the culture temperature is 37 ℃, and the concentration of carbon dioxide is 5%.
Harvesting the cells, culturing the neural stem cells for 7-10 days, observing the growth state of the cells, sucking the neurospheres and culture solution in a low-adsorption T75 culture bottle into a 50ml centrifugal tube by using a sterile pipette, centrifuging at 100g and 4 ℃ for 10min to ensure that the neurospheres are fully precipitated to the bottom of the tube, sucking the supernatant as much as possible, and putting the sucked supernatant into a sterile container to obtain the primary neural stem cells.
Preparation of P1-P5 generation neural stem cells: primary cell culture, counting, digestion, washing, passage and culture, identification, split charging and freezing storage.
Passage and culture of primary neural stem cells: injecting prepared 10ml of sterile nerve digestive juice into a centrifuge tube filled with primary neural stem cell balls by using a sterile syringe; sealing, standing for 3 minutes, adding the prepared degerming nervous tissue digestive juice, putting into a carbon dioxide incubator at 37 ℃ for 30 minutes (shaking manually for 5 seconds every 10 minutes), centrifuging at 4 ℃ and 300g by using a low-temperature centrifuge for 10 minutes; sucking and removing the supernatant by using a sterile disposable pipette, adding 5ml of DMEM/F12 into the pipette by using a pipette gun and a pipette, flushing the pipette by blowing for 2 to 3 times up and down to separate tissues, and centrifuging the tissues for 10 minutes at 4 ℃ by using a low-temperature centrifuge and 300 g; adding 5ml of hibernationmedia again for washing for three times; and (3) sucking the supernatant by using a sterile disposable pipette, adding 5ml of DMEM (DMEM) to enable the cell sediment to be uniformly suspended, and taking care that bubbles are not generated to enable the cell sediment to be uniformly suspended for subsequent counting operation.
Cell counting: the single cell suspensions were counted according to Standard protocols for cell Density measurements.
Digestion and harvest: sucking neurospheres and culture solution in a low adsorption T75 culture bottle into a 50ml centrifuge tube by using a sterile pipette, centrifuging for 10min at 100g and 4 ℃ to ensure that the neurospheres fully sink to the bottom of the tube, sucking supernatant as much as possible, putting the sucked supernatant into a sterile container, marking, and storing in a refrigerator at 4 ℃ for later use.
Washing: injecting the prepared sterile nerve digestive juice into a centrifuge tube filled with a P2 neurosphere by using a sterile syringe, sealing the centrifuge tube, standing for 3 minutes, adding the sterilized tissue digestive juice, putting the sterilized tissue digestive juice into a carbon dioxide incubator at 37 ℃ for acting for 10-50 minutes, centrifuging at 4 ℃ at low temperature for 300g, and centrifuging for 10 minutes. Sucking the supernatant with a sterile disposable pipette, adding 5ml DMEM/F12 with a high-power pipettor and a pipette, flushing, blowing and beating for 2-3 times up and down to separate tissues, centrifuging at 4 ℃ and 300g for 5-30 minutes by a low-temperature centrifuge. 10ml of hibernationmedia was added again for washing, and washing was carried out three times in total.
Passage and culture: calculating the volume of the cell suspension to be inoculated according to the cell density of the suspension obtained by counting, adding 10-30ml of neural stem cell culture solution into three low-adsorption T75 culture bottles, uniformly mixing the cell suspension by using a sterile disposable pipette, blowing and beating under the liquid level by using a pipette with a proper range, taking out the single cell suspension with the calculated volume, quickly adding the single cell suspension into a low-adsorption T75 culture bottle, slightly shaking from different directions to uniformly distribute the cells in the culture solution, quickly putting the inoculated low-adsorption T75 culture bottle into a carbon dioxide incubator, wherein the culture temperature is 37 ℃, and the concentration of carbon dioxide is 5%.
After the P2 cells are inoculated and cultured, according to the growth condition and the cell state of the cells, taking out a T75 culture bottle from a carbon dioxide incubator every 2-3 days according to the standard operation program for the culture of the neural stem cells, putting the T75 culture bottle into a biological safety cabinet, taking out 2ml of prepared nutrient supplement solution for the culture of the neural stem cells by using a 2ml sterile pipette, slightly shaking the bottle from different directions to uniformly distribute the cells, and quickly putting the T75 culture bottle with the supplement solution added into the carbon dioxide incubator at the culture temperature of 37 ℃ and the carbon dioxide concentration of 5 percent to obtain the P2 cell generation.
And (3) culturing the cells from the generation P2 to the generation P5, namely inoculating the digested cells from the generation P2 into a T75 culture bottle according to the density of 1 × 105 cells/ml, wherein the culture process and the digestion process are the same as those of the cells from the generation P1, digesting when the cells grow to more than 80%, counting, passaging and the like until the cells from the generation P5 are obtained.
Identifying neural stem cells: identifying the phenotype, differentiation capacity and safety indexes according to a method specified in the industry; after being qualified, the product can be frozen and stored.
Cell subpackaging and freezing: after the cell purity is over 95 percent, sucking neurospheres and culture solution in a low-adsorption T75 culture bottle into a 50ml centrifugal tube by using a sterile suction tube, centrifuging at 100g and 4 ℃ for 10ml to ensure that the neurospheres are fully precipitated at the bottom of the tube, sucking supernatant as much as possible, putting the sucked supernatant into a sterile container, marking and storing in a refrigerator at 4 ℃ for later use; performing programmed cooling by using a programmed cooling instrument, and placing cells to be cryopreserved in the programmed cooling instrument to be below-80 ℃ according to a standard cryopreservation program; and (4) program cooling procedure: 0.25/min at 4 ℃ to-10 ℃; 0.5 ℃/min at-10 ℃ to-40 ℃; -40 ℃ to-80 ℃ 1/min, -80 ℃ below 2 ℃/min.
Putting the cells into a warehouse, taking out the freezing storage box after freezing storage, transferring the freezing storage box into a liquid nitrogen tank for storage
Preparation of P6-P9 generation neural stem cells:
and (3) recovering the cells: the frozen cells are taken out from the liquid nitrogen tank, put on a recovery frame in an electric heating constant temperature water tank (the water temperature is kept at about 40 degrees), and quickly shaken for rewarming. When the solid state is changed into the liquid state, the recovery is completed, the centrifugal tube mouth is sealed by a parafilm, the standing is carried out for 3 minutes, the prepared degerming nervous histochemical digestion solution is added, the mixture is put into a carbon dioxide incubator at 37 ℃ for 5 to 60 minutes, and a low-temperature centrifuge is used for centrifuging for 10 minutes at 4 ℃ and 300 g; sampling, counting and washing, removing supernatant with a sterile disposable pipette, adding 1-10ml of the supernatant with a pipette and a pipette, washing with DMEM/F12, blowing and beating for 2-3 times up and down to separate tissues, and centrifuging for 10 minutes at 4 ℃ by using a low-temperature centrifuge at 300 g; adding 1-10ml hibernation medium again for washing for three times; the supernatant was discarded with a sterile disposable pipette.
Carrying out passage and culture; inoculating 1:3 into a low-adsorption T75 culture bottle, adding 5-50ml of neural stem cell culture solution, uniformly mixing cell suspension with a sterile disposable pipette, blowing the cell suspension under the liquid level with a pipette with a proper range, taking out the single cell suspension with the calculated volume, rapidly adding into a low-adsorption T75 culture bottle, and rapidly placing into a carbon dioxide culture box, wherein the culture temperature is 37 ℃, and the carbon dioxide concentration is 5%
Freezing and storing the neural stem cells: the cryopreservation was performed according to the above method.
Example 2:
passage of seed cells: after the P5 cells are inoculated and cultured, taking out a T75 culture bottle from a carbon dioxide culture box every 2-3 days according to the growth condition of the cells and the appearance of a culture solution, putting the culture bottle into a biological safety cabinet, taking out 1-5ml of prepared neural stem cell culture nutrient supplement solution by using a 2ml sterile pipette, quickly adding the prepared neural stem cell culture nutrient supplement solution into a low-adsorption T75 culture bottle, slightly shaking the culture bottle from different directions to uniformly distribute the cells, and quickly putting the low-adsorption T75 culture solution with the added supplement solution back into the carbon dioxide culture box, wherein the culture temperature is 37 ℃ and the carbon dioxide concentration is 5%.
Digestion: culturing the neural stem cells to 7-10 generations, observing the growth state of the cells, reaching the plateau stage, and carrying out subculture operation according to the Neurosphere digestion standard operating procedure, wherein 6 bottles of T75 are used for 1: and 3, continuously subculturing, and freezing and storing the rest according to the standard operation procedure of the neural stem cells.
Cell counting: the cells were blown into single neural stem cells, and 10. mu.l was counted on an automatic cell counter.
And (3) passage and culture, namely inoculating the digested single neural stem cell according to the concentration of 0.1-10 × 105/ml for continuous culture, uniformly mixing and culturing in a T75 bottle, putting into a carbon dioxide incubator, digesting and carrying out passage on the cell when the cell in the culture bottle is fused to 80-90%, and repeating the steps until the cell is subjected to passage for P9.
Cell subpackaging and freezing: after the cell purity is judged to be more than 95%, sucking neurospheres and culture solution in a low-adsorption T75 culture bottle into a 50ml centrifugal tube by using a sterile suction tube, centrifuging at 100g and 4 ℃ for 10ml to ensure that the neurospheres are fully precipitated to the bottom of the tube, sucking supernatant as much as possible, putting the sucked supernatant into a sterile container, marking, storing the supernatant in a 4 ℃ refrigerator for later use, carrying out program cooling by using a program cooling instrument, and putting cells to be frozen in the program cooling instrument to be below-80 ℃ according to a standard freezing procedure; and (4) program cooling procedure: 0.25/min at 4 ℃ to-10 ℃; 0.5 ℃/min at-10 ℃ to-40 ℃; -40 ℃ to-80 ℃ 1/min, -80 ℃ below 2 ℃/min.
The neural stem cells frozen in the examples 1 and 2 were stored in liquid nitrogen for 1 month, 6 months and 12 months, respectively, and the survival rate after recovery of the cells and the ratio of the number of the cells cultured for 24 hours after recovery to the number of the frozen cells were compared, and the time required for the first passage after recovery was obtained.
Claims (7)
1. A method for constructing a human neural stem cell bank comprises the following steps:
the method comprises the following steps: collecting human neural stem cells;
step two: culturing primary cells, identifying the primary neural stem cells, carrying out passage and partial cryopreservation on the primary neural stem cells;
step three: purifying, conveying and expanding culture of the neural stem cells;
step four: identifying neural stem cells, cooling by a program, and coding and warehousing.
2. The method of claim 1, wherein the method comprises the steps of: the primary neural stem cells in the first step are derived from mammalian neural stem cells, the primary neural stem cells are obtained by surgically separating waste brain tissues including frontal cortex, midbrain and hippocampal brain tissues, repeatedly cutting in ophthalmology, rinsing in DEME/F12 (volume ratio: 1:1), filtering with a 200-mesh filter screen, and centrifuging to obtain a primary neural stem cell suspension.
3. The method for constructing a human neural stem cell bank as set forth in claim 1, wherein the step of using a serum-free medium and a culture process digestion counting is followed by resuspending, adjusting the density to 1 × 105/ml, and culturing in an ultra-low adsorption medium at 37 ℃ in a 5% CO2 environment, wherein the serum-free medium is composed of DMEM/F12, B27, N2, and neurotrophic factors BFGF, EGF, &lTtTtransfer = L "&gTt L &/T &TtTgIF, and has no animal serum components.
4. The method of claim 1, wherein the method comprises the steps of: step three, digesting and passaging the expanded primary neural stem cells until passage to 5 th generation, identifying the cell purity to be more than 95%, using the cells as seed cells, freezing and storing the seed cells according to a program for convenient storage, and when the diameter of the expanded primary neural stem cells exceeds 300 mu m, digesting and conveying the cells to the neural stem cells of P1 generation; digesting the P1 generation neural stem cells, inoculating the digested cells into an ultra-low adsorption culture bottle, placing the bottle in an environment with the temperature of 37 ℃ and the CO of 5% 2 for culturing until passage, repeatedly digesting the bottle, inoculating the bottle and culturing the bottle until the P5 generation neural stem cells are obtained; detecting the cell characteristics and heterologous substances of the neural stem cells of the P5 generation, wherein the cell characteristic identification comprises flow identification and immunofluorescence, the flow identification is used for identifying the neural stem cell surface markers Nestin and Musashi, the purity of the neural stem cell surface markers is detected to be more than 95%, the differentiation characteristics of the neural stem cells are identified by a fluorescence immunization method, namely the differentiation results of neurons, astrocytes and oligodendrocytes, the heterologous substances are mainly the detection of microorganisms, and comprise sterility detection, mycoplasma detection and endotoxin detection, the negative result is qualified, and the cells are frozen and stored as seed cells after the detection is qualified.
5. The method of claim 1, wherein the method comprises the steps of: step four, counting seed cells, carrying out suspension culture in a serum-free culture medium, digesting and passaging the cells when the cells in the culture flask are fused to 80-90%, and repeating the steps until the cells are passaged for P9 generations; and (4) freezing and storing the neural stem cells in a liquid nitrogen tank by adopting uniform coding according to a programmed cooling mode.
6. The method for constructing a human neural stem cell bank according to claim 3, wherein the serum-free medium is prepared from DMEM/F12 (volume ratio l: l), 7 g/L glucose, 4 mmo/L glutamine, 5 mmo/L Hepes buffer, 5 μ g/ml insulin, 100 μ g/ml transferrin, 20 nmol/L progesterone, 100 μ g/ml putrescine, 30 nmol/L sodium selenide, 5 μ g/ml heparin, sodium bicarbonate, and the pH value is adjusted to 7.2-7.4.
7. The method of claim 4, wherein the method comprises the steps of: the primary neural stem cell culture method comprises the steps of continuously culturing for 10-14 days, observing every 24 hours during the continuous culture, confirming the growth state of cells, and changing liquid every 48-72 hours; the culture method of the P1 generation neural stem cells comprises the steps of changing liquid every 48-72 hours and continuously culturing for 10-14 days.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013918A (en) * | 2012-11-30 | 2013-04-03 | 陆华 | Method for separating and subculturing primary neural stem cell of human embryo midbrain |
| CN103451153A (en) * | 2013-05-22 | 2013-12-18 | 栾佐 | Establishment method and application of clinical-grade neural stem cell line |
| CN104152488A (en) * | 2014-08-15 | 2014-11-19 | 济南干细胞再生与转化医学研究院 | Construction method of human nerve stem cell bank |
| CN106619722A (en) * | 2016-12-05 | 2017-05-10 | 上海安集协康生物技术股份有限公司 | Neural stem cell injection for treating brain damage disease |
-
2019
- 2019-01-07 CN CN201811464989.6A patent/CN111411081A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013918A (en) * | 2012-11-30 | 2013-04-03 | 陆华 | Method for separating and subculturing primary neural stem cell of human embryo midbrain |
| CN103451153A (en) * | 2013-05-22 | 2013-12-18 | 栾佐 | Establishment method and application of clinical-grade neural stem cell line |
| CN104152488A (en) * | 2014-08-15 | 2014-11-19 | 济南干细胞再生与转化医学研究院 | Construction method of human nerve stem cell bank |
| CN106619722A (en) * | 2016-12-05 | 2017-05-10 | 上海安集协康生物技术股份有限公司 | Neural stem cell injection for treating brain damage disease |
| WO2018103406A1 (en) * | 2016-12-05 | 2018-06-14 | 上海安集协康生物技术股份有限公司 | Neural stem cell injection for treating brain damage diseases and preparation method and use method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 王佳等: "颅内动脉瘤手术废弃脑组织中成人自体神经干细胞的培养与鉴定", 《中国组织工程研究》 * |
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Application publication date: 20200714 |