CN102533654A - Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof - Google Patents
Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses culture solution for primary culture of newly born rat hippocampal neuron and a method for primarily culturing the rat hippocampal neuron. The culture solution is formed by adding 1,6 diphosphonic acid fructose, fructose and nutrition additive into base culture solution Neurobasalmedium. The method for primarily culturing the rat hippocampal neuron comprises the following steps: 1 preparing hippocampal neuron culture solution; 2 drawing materials and digesting the materials; 3 preparing single cell suspension; and 4 conducting inoculating and cultivating. The method for primarily culturing the rat hippocampal neuron is simple, convenient and capable of obtaining hippocampal neuron with good growth state and high purity by adopting the culture solution and has important application value and economical value.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of neonate rat hippocampal neuron nutrient solution of former generation.
Background technology
Neurone is the fundamental unit of neural system 26S Proteasome Structure and Function, also is the good material of research neural system physiology, pathomechanism and various neurotransmitter function.Neuronal quantity is comparatively concentrated with distribution in the hippocampal tissue, and the hippocampal neuron vitro culture is one of important means of research neural system structure, function and pathology, physiological change.Basically all be to add serum in the existing hippocampal neuron nutrient solution, cultivate hippocampal neuron at DMEM, F12 substratum.Serum has increased non-neuron rate of propagation such as spongiocyte, has influenced the purity of hippocampal neuron, and neurocyte is had toxicity in various degree, and owing to complicated component in the serum, experiment is produced certain influence or interference.The major issue that neurone is cultivated is to guarantee that neuronic purity is with active.
Chinese patent CN1966674A discloses a kind of spider axoneure nutrient solution, and the nutrient solution that this invention is adopted has added the 200ml calf serum; Chinese patent CN1171991C discloses " cultural method of human nerve stem cell "; This invention nutrient solution is by DMEM and the F12 basic culture solution to mix at 1: 1; Add Regular Insulin, L-glutaminate, hydrochloric acid tetramethylenediamine, sodium selenide, HTrf, HYDROCORTISONE INJECTIONS, Progesterone, add the 5-20% fresh serum in addition; Therefore, there is the growth that can not effectively reduce spongiocyte in prescription and the method with the epineural nutrient solution, the problem of assurance neurone purity equally.Even added the purity that inhibition spongiocyte splitted medicines such as cytosine arabinoside can improve neurocyte in the nutrient solution, still, the adding of cytosine arabinoside also has certain restraining effect to nerve growth, has a strong impact on the neuronal activity of being cultivated.
Although serum-free culture occurs; Improved the purity that neurone is cultivated; Reduced the growth of spongiocyte, but institute's cultured rat hippocampal neurone has still that survival rate is low, cell poor growth and cynapse form shortcomings such as few, can not satisfy the requirement of pair cell further experiment.At present the additive of serum-free medium all contain the research and development of Invitrogen (GIBCO) company neuronal cultured solution B27 or (with) N2, its staple is multivitamin and trace element.Although improved the purity of neuronal cell cultures; Effectively reduced the growth of spongiocyte; But still exist to cultivate enough energy are provided for hippocampal neuron, particularly evident under anaerobic environment, cause lethal injury more easily; Influenced the growth conditions of hippocampal neuron, thereby caused that cell survival rate is low, cell poor growth and cynapse form problems such as few.Through increasing the method for glucose dosage; The result can be worse, and glucose produces a large amount of lactic acid as energy substance in anaerobic glycolysis and piles up, and also can reduce the pH value of nutrient solution; Increase osmotic pressure; The infringement neurocyte causes nerve growth inhibition in the substratum, has also increased the chance that neurocyte pollutes simultaneously.
Summary of the invention
Technical problem to be solved by this invention provides a kind of nutrient solution with purity height, hippocampal neuron that growth conditions is good and preparation method thereof, and the method for utilizing this nutrient solution to cultivate hippocampal neuron is provided.
For solving the problems of the technologies described above, the technical scheme that the present invention takes is that a kind of hippocampal neuron nutrient solution, this nutrient solution are in basic culture solution, to add 1,6 hexose diphosphates, fructose and nutritional additive; Add 1,6 hexose diphosphates of 1-10mmol and the fructose of 2-20mmol in every liter of basic culture solution, wherein said basic culture solution is Neurobasal Medium.
Said 1,6 hexose diphosphates and fructose mass concentration ratio are 1: 2.The nutritional additive that adds following component in every liter of basic culture solution: 10-20 μ g Prostatropin; 6-15mg ATP; The 30-50U coenzyme A, 0.1-1g amphotericin B, 5-50mg Regular Insulin; 0.42-0.83mg ferrous sulfate, 5-50mg HTrf and 20-60mg vitamin mixture.Vitamin mixture is the mixture of vitamin H, choline chloride 60, VA, folic acid, inositol, VITMAIN B1, Wei ShengsuB2, Y factor, cobalamin and vitamin PP.Wherein, commercialization basic culture solution Neurobasal Medium of the present invention (GIBCO company).Adopt the membrane filtration degerming of 0.22 μ m afterwards.The nutrient solution that configures places 4 ℃ of storages.
In the nutrient solution of the present invention 1, the 6-hexose diphosphate be can be in the cellular metabolism of molecular level adjusted some enzymic activitys, recover and improve cellular metabolism, alleviate cell injury, can stimulate circulation, it is synthetic to increase ATP.Fructose obviously is superior to glucose as the energy ingredient of vitro culture; The glucolytic key distinction of fructose glycolysis and grape is in fructose glycolysis process; The fructose oxidative phosphorylation is F-1-P (fructose 1-phosphate)---a glycolysis-precursor substance; Can under anoxic condition, ATP be provided, keep the ionic pump of cytolemma, so can effectively protect the integrity and the function of keeping cytoactive of cytolemma.And can not produce F-1-P in the glucose metabolism process.The present invention adds 1, and the mixture of 6-hexose diphosphate and fructose has effect preferably, 1; 6-hexose diphosphate and fructose have significant synergy; Can significantly increase the vigor of hippocampal neuron, the ATP cell especially is provided under anoxic condition as usual, guarantee that neuronic purity is with active.
Nutritional additive comprises Prostatropin in the said nutrient solution of the present invention, Regular Insulin, ferrous sulfate, HTrf, ATP, coenzyme A, amphotericin B and vitamin mixture.
Regular Insulin is important blood serum substituting composition in the non-blood serum low density culture medium for animal cell that provides among the present invention.Regular Insulin can promote the synthetic of glycogen and lipid acid, promotes the synthetic of RNA, protein and lipid simultaneously.Regular Insulin can be Recombulin, ox source Regular Insulin or people source Regular Insulin, and the insulin type factor also can be used.The ferrous sulfate that uses among the present invention provides the cell growth required ferrous ion as substratum, can be used as the alternative composition of Transferrins,iron complexes simultaneously.The Transferrins,iron complexes that uses among the present invention mainly is the main channel that cell obtains trace element, has the function that promotes that Regular Insulin plays a role simultaneously.The Transferrins,iron complexes that uses in this invention can be reorganization, the people source or Niu Yuan etc., ironic citrate also can play same effect in addition.The VITAMINs that uses among the present invention provides essential vitamins as the growth metabolism of cell, promotes the cell growth and prolongs cytoactive, reduces the accumulation of meta-bolites when nutritive substance is provided.VITAMINs used in the present invention is the vitamin mixture of sterile packed.Working concentration adopts corresponding substratum dilution to get final product with reference to saying the bright book recommended density of commercially available product.ATP is as energy matter, when decomposing, emits lot of energy, can competent energy all be provided for each process of cell cultures, improves the metabolic function of cellular enzymes, promotes the amplification of cell and goes down to posterity.Coenzyme A can promote tricarboxylic circulation, for the amplification of cell provides energy with going down to posterity.The working concentration of amphotericin B is at 0.1-1g/L.
The present invention also provides a kind of method of former primary cultures of rat hippocampal neurons, has following steps:
1. preparation is like the former foster nutrient solution of being commissioned to train of the described hippocampal neuron of one of claim 1-5;
2. draw materials, digest: the neonate rat broken end is got brain, and passivity is dialled from going out cerebral tissue on ice, removes tunica vasculose, complete taking-up hippocampal tissue; Shred the fritter of written treaty 1mm3, adding is the papain of 2-4mg/mL with organizing isopyknic concentration approximately, mixing, 37 ℃ of digestion 5-15min.Stop digestion with containing 10% foetal calf serum DMEM/F12 substratum, and add 10 μ lDNase I and open cotton-shaped glutinous company, suction pipe is blown and beaten the tissue block cell dispersion gently.
3. prepare single cell suspension: 2. cell suspension is through 400 order nylon net filters with step, and 1000r/min is centrifugal, and 10min removes supernatant, and the DMEM/F12 that adds 10% foetal calf serum is resuspended, and transferring cell density is the single cell suspension of 1～5 * 105 cells/mL concentration;
4. inoculate, cultivate: the single cell suspension that 3. step is prepared adds in the culture plate of poly-l-lysine, places 37 ℃, the cultivation of 5%CO2 incubator; To contain 10% foetal calf serum DMEM/F12 substratum behind the 24h originally and change to the former foster nutrient solution of being commissioned to train of hippocampal neuron that 1. step prepares, every separated 2d changes liquid 1 time, changes half nutrient solution at every turn.
The beneficial effect that the present invention can reach is following:
(1) utilizes nutrient solution of the present invention to carry out the hippocampal neuron vitro culture, obtained hippocampal neuron single relatively, that purity is high (purity >=more than 90%).Here it is owing to 1, and 6-hexose diphosphate and fructose are enough energy that hippocampal neuron is cultivated to be provided, and promoted the activity of hippocampal neuron;
(2) utilize nutrient solution of the present invention to carry out the hippocampal neuron vitro culture, improved neuronal survival effectively, and neuronic growth conditions is good, physiological property stable, shows that this cultural method can satisfy the requirement of experiment in vitro;
(3) utilize nutrient solution of the present invention to carry out the hippocampal neuron vitro culture, compare with traditional cultural method, the time of cultured hippocampal neurons in vitro survival obtains prolonging (high purity, highly active Hippocampal Neuron Cells can be kept more than 16 days) greatly;
(4) nutrient solution hippocampal neuron nutrient solution compound method of the present invention is simple, and is repeatable strong, easy to operation.
Embodiment
The prescription and the preparation method of embodiment 1 hippocampal neuron nutrient solution
According to the recipe configuration nutrient solution 1-9 in the following table:
The preparation method of above-mentioned nutrient solution 1-7 is following:
A. get among 1,6 hexose diphosphates, the fructose adding basic culture solution Neurobasal Medium by said proportioning, stir;
B. the Prostatropin, Regular Insulin, ferrous sulfate, HTrf, amphotericin B, the vitamin mixture that add above-mentioned amount;
C. transfer pH 7.4-8.0 with the centinormal 1 sodium hydroxide of 1-10;
D. laminar flow cell culture chamber suction filtration sterilization, 4 ℃ store for future use, and add the ATP and the coenzyme A of said amount before the use again.The method of embodiment 2 former primary cultures of rat hippocampal neurons
Have following steps:
1. preparation is like the former foster nutrient solution of being commissioned to train of embodiment 1 described hippocampal neuron;
2. draw materials, digest: the neonate rat broken end is got brain, and passivity is dialled from going out cerebral tissue on ice, removes tunica vasculose, complete taking-up hippocampal tissue; Shred the fritter of written treaty 1mm3, adding is the papain of 2-4mg/mL with organizing isopyknic concentration approximately, mixing, 37 ℃ of digestion 5-15min.Stop digestion with containing 10% foetal calf serum DMEM/F12 substratum, and add 10 μ lDNase I and open cotton-shaped glutinous company, suction pipe is blown and beaten the tissue block cell dispersion gently.
3. prepare single cell suspension: 2. cell suspension is through 400 order nylon net filters with step, and 1000r/min is centrifugal, and 10min removes supernatant, and the DMEM/F12 that adds 10% foetal calf serum is resuspended, and transferring cell density is the single cell suspension of 1～5 * 105 cells/mL concentration;
4. inoculate, cultivate: the single cell suspension that 3. step is prepared adds in the culture plate of poly-l-lysine, places 37 ℃, the cultivation of 5%CO2 incubator; To contain 10% foetal calf serum DMEM/F12 substratum behind the 24h originally and change to the former foster nutrient solution of being commissioned to train of hippocampal neuron that 1. step prepares, every separated 2d changes liquid 1 time, changes half nutrient solution at every turn.
The morphological observation and the function test of embodiment 3 former primary cultures of rat hippocampal neurons
Under inverted microscope, observe, rat hippocampus neurocyte inoculation 6～8h begins adherent, and single dispersion is rounded.Adherent basically fully behind the 24h, it is flat that cell begins to become, and begin to grow 1～2 projection.Behind the 3d, neuronal quantity further increases, and projection further increases, and neurogliocyte also begins rapid division growth, and neurone forms a slice supporting layer down.The 10d neuron state is good, and synapse is reached maturity, and forms tangible neural network.Be cultured to 14d, though cell number reduces to some extent, cell reaches optimal state, around the cell space tangible halation is arranged, and cell is the fullest.Cultivate 17d, along with the further prolongation of incubation time, cell begins to occur degenerating, coming off.The death of neurocyte, visible apoptotic body appear in DMEM/F12+20% calf serum group and Neurobasal medium+2%B27 group.DMEM/F12+20% calf serum experimental group is cultured to 24d, and neurocyte is all dead, degeneration, the neurocyte vestiges of visible sex change.
Experimental group pericaryon of the present invention is full, and nuclear is big, and projection rises appreciably, and connects intensively, forms the nerve fiber network of complicacy, positive cell (purity) >=90%.Be significantly higher than DMEM/F12+20% calf serum group and Neurobasal Medium+2%B27 group; DMEM/F12+20% calf serum group and Neurobasal Medium+2%B27 group cell quantity are less relatively, and the only a few cell grows projection.Use the image analyzer analysis, the result sees table 1-4.Of the present invention group of neurosome area, major diameter, minor axis all are significantly higher than DMEM/F12+20% calf serum group and Neurobasal Medium+2%B27 group (p＜0.01), and showing of the present invention group nerve growth to be grown has promoter action.
The neuronic cell viability of table 1 cultured rat hippocampal
Annotate:
*Expression P＜0.01 expression difference is extremely remarkable.
5mmol/L 1 in the nutrient solution 4, and 6-hexose diphosphate and 10mmol/L fructose have best effect.
Table 2 cultured rat hippocampal neurone MTT
Can find out by table 1; Organize relatively with DMEM/F12+20% calf serum group, Neurobasal Medium+2%B27; The present invention's (nutrient solution 1) group, the present invention's (nutrient solution 5) group OD value all obviously increase, and show that nutrient solution of the present invention can promote the propagation of neurocyte, improve neural activity.
Table 3 cultured rat hippocampal neurone LDH burst size
Annotate:
*Expression p＜0.01 expression difference is extremely remarkable.
Table 4 group cell space diameter and projection length are measured
Annotate:
*Expression P＜0.01 expression difference is extremely remarkable.
The mensuration of embodiment 4 neurocyte survival rates
Neuronal cultured solution of the present invention plays obvious facilitation to neuron survival; Neuron survival rate when cultivating 14 days; Compare with DMEM/F12+20% calf serum, Neurobasal Medium+2%B27 group, DMEM/F12+20% calf serum group, Neurobasal Medium+2%B27 group survival rate are respectively 65%, 69%., and invent 1 group and invention 2
Group is respectively 81%, 85%, and the present invention obviously promotes neuron survival, and difference is (p＜0.01) extremely significantly.
The mensuration of embodiment 5 different incubation time neurone purity
The different incubation time neurone of table 5 purity (%) and neuron morphology
Explain:
++ ++, neuron density is high, is evenly distributed, and cell space is thick
+++, neurone is more, the form typical case
++, neurone is dispersed in
+, neurone once in a while
The result shows, the refreshing neurone mass mortality of DMEM/F12+20% calf serum group and Neurobasal Medium+2%B27 group after 12 days; And the neuronic survival of the present invention's (nutrient solution 2) group is high, and it is vigorous grow, and good neurone purity and typical neuron morphology were still arranged on the 16th day.
Conclusion: it is surplus that the external current that lives forever most of former generation hippocampal neuron that the inventive method is cultivated reaches January; The neuron morphology typical case; Hippocampal neuron purity >=90%; And neuronic growth conditions is good, physiological property stable, can satisfy the requirement of experiment in vitro, can be used as the former foster good nutrient solution of being commissioned to train of hippocampal neuron.
Claims (8)
1. former foster nutrient solution of being commissioned to train of neonate rat hippocampal neuron is characterized in that: this nutrient solution is for adding 1,6 hexose diphosphates, fructose and nutritional additive in basic culture solution; Wherein, add 1,6 hexose diphosphates of 1-10mmol, the fructose of 2-20mmol in every liter of basic culture solution, wherein said basic culture solution is Neurobasal Medium.
2. the former foster nutrient solution of being commissioned to train of neonate rat hippocampal neuron according to claim 1 is characterized in that said 1,6 hexose diphosphates and fructose mass concentration ratio are 1:2.
3. the former foster nutrient solution of being commissioned to train of neonate rat hippocampal neuron according to claim 1; It is characterized in that adding in every liter of basic culture solution the nutritional additive of following component: 10-20 μ g Prostatropin, 6-15mg ATP, 30-50U coenzyme A; 0.1-1g amphotericin B; 5-50mg Regular Insulin, 0.42-0.83mg ferrous sulfate, 5-50mg HTrf and 20-60mg vitamin mixture.
4. the former foster nutrient solution of being commissioned to train of neonate rat hippocampal neuron according to claim 3; It is characterized in that vitamin mixture is the mixture of vitamin H, choline chloride 60, VA, folic acid, inositol, VITMAIN B1, Wei ShengsuB2, Y factor, cobalamin and vitamin PP.
5. the former foster nutrient solution of being commissioned to train of neonate rat hippocampal neuron according to claim 4 is characterized in that in said every liter of basic culture solution, vitamin mixture is made up of each component of following content,
Vitamin H 0.001-0.01mg/L
Choline chloride 60 5-20mg/L
VA 2-10mg/L
Folic acid 0-5 mg/L
Inositol 5-20 mg/L
VITMAIN B1 2-10 mg/L
Wei ShengsuB2 0.1-0.5mg/L
Y factor 2-10mg/L
Cobalamin 0.2-1.0 mg/L
Vitamin PP 2-5 mg/L.
6. the preparation method of the former foster nutrient solution of being commissioned to train of neonate rat hippocampal neuron according to claim 1 is characterized in that, comprises the steps,
A: get among 1,6 hexose diphosphates, the fructose adding basic culture solution Neurobasal Medium by said proportioning, stir;
B: in above-mentioned solution, add nutritional additive;
C: transfer pH 7.4-8.0 with the centinormal 1 sodium hydroxide of 1-10;
D: the sterilization of laminar flow cell culture chamber suction filtration, 4 ℃ store for future use;
E: the ATP and the coenzyme A that add said amount at last.
7. the preparation method of the former foster nutrient solution of being commissioned to train of neonate rat hippocampal neuron according to claim 1 is characterized in that, adds the nutritional additive of following component in every liter of basic culture solution: 10-20 μ g Prostatropin; 6-15mg ATP; The 30-50U coenzyme A, 0.1-1g amphotericin B, 5-50mg Regular Insulin; 0.42-0.83mg ferrous sulfate, 5-50mg HTrf and 20-60mg vitamin mixture.
8. neonate rat hippocampal neuron cultured method of former generation is characterized in that having following steps:
1. preparation is like the former foster nutrient solution of being commissioned to train of the described hippocampal neuron of one of claim 1-5;
2. draw materials, digest: the neonate rat broken end is got brain, and passivity is dialled from going out cerebral tissue on ice, removes tunica vasculose, complete taking-up hippocampal tissue; Shred the fritter of written treaty 1mm3; Add is the papain of 2-4 mg/mL with organizing isopyknic concentration approximately; Mixing, 37 ℃ of digestion 5-15min stop digestion with containing 10% foetal calf serum DMEM/F12 substratum; And add 10 μ l DNase I and open cotton-shaped glutinous company, suction pipe is blown and beaten the tissue block cell dispersion gently;
3. prepare single cell suspension: 2. cell suspension is through 400 order nylon net filters with step, and 1000r/min is centrifugal, and 10min removes supernatant, and the DMEM/F12 that adds 10% foetal calf serum is resuspended, and transferring cell density is the single cell suspension of 1～5 * 105 cells/mL concentration;
4. inoculate, cultivate: the single cell suspension that 3. step is prepared adds in the culture plate of poly-l-lysine, places 37 ℃, the cultivation of 5%CO2 incubator; To contain 10% foetal calf serum DMEM/F12 substratum behind the 24h originally and change to the former foster nutrient solution of being commissioned to train of hippocampal neuron that 1. step prepares, every separated 2d changes liquid 1 time, changes half nutrient solution at every turn.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210023408.1A CN102533654B (en) | 2012-02-02 | 2012-02-02 | Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof |
| PCT/CN2012/075162 WO2013113196A1 (en) | 2012-02-02 | 2012-05-08 | Culture medium for primary culture of hippocampus neurons of a neonatal rat and preparation method and use thereof |
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| CN201210023408.1A CN102533654B (en) | 2012-02-02 | 2012-02-02 | Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2013113196A1 (en) * | 2012-02-02 | 2013-08-08 | Sun Jing | Culture medium for primary culture of hippocampus neurons of a neonatal rat and preparation method and use thereof |
| CN103789267A (en) * | 2014-02-21 | 2014-05-14 | 刘洛贤 | Improved primary culture method for hippocampal neurons |
| CN103805565A (en) * | 2014-02-21 | 2014-05-21 | 温州医科大学附属第二医院、育英儿童医院 | Hippocampal neuron separation and primary culture method and reagent |
| CN103898007A (en) * | 2014-02-21 | 2014-07-02 | 刘洛贤 | Bifidobacterium strain with activity |
| CN104611294A (en) * | 2015-02-11 | 2015-05-13 | 中国人民解放军第三军医大学第一附属医院 | In-vitro culture method of visual cortex neuron |
| CN104818251A (en) * | 2015-05-20 | 2015-08-05 | 妙顺（上海）生物科技有限公司 | In-vitro separation culture method for hippocampal neurons of adult rat |
| CN105695411A (en) * | 2016-03-22 | 2016-06-22 | 中国人民解放军第二军医大学 | Naked mole rat cortical neuron culture method |
| CN105754943A (en) * | 2016-03-22 | 2016-07-13 | 中国人民解放军第二军医大学 | Culture method of naked mole rat hippocampal neurons |
| CN112391342A (en) * | 2020-12-07 | 2021-02-23 | 山东省齐鲁干细胞工程有限公司 | Efficient recovery method for umbilical cord blood stem cells |
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| CN103789268B (en) * | 2014-02-21 | 2016-04-27 | 刘洛贤 | A kind of method of separation and Culture hippocampal neurons and special culture solution thereof |
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| CN102533654B (en) * | 2012-02-02 | 2014-01-29 | 温州医学院附属第二医院 | Culture solution for primary culture of newly born rat hippocampal neuron and preparation method and application thereof |
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2012
- 2012-02-02 CN CN201210023408.1A patent/CN102533654B/en not_active Expired - Fee Related
- 2012-05-08 WO PCT/CN2012/075162 patent/WO2013113196A1/en active Application Filing
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013113196A1 (en) * | 2012-02-02 | 2013-08-08 | Sun Jing | Culture medium for primary culture of hippocampus neurons of a neonatal rat and preparation method and use thereof |
| CN103805565B (en) * | 2014-02-21 | 2016-03-09 | 温州医科大学附属第二医院、育英儿童医院 | Hippocampal neuron is separated and primary culture method and reagent |
| CN103805565A (en) * | 2014-02-21 | 2014-05-21 | 温州医科大学附属第二医院、育英儿童医院 | Hippocampal neuron separation and primary culture method and reagent |
| CN103898007A (en) * | 2014-02-21 | 2014-07-02 | 刘洛贤 | Bifidobacterium strain with activity |
| CN103789267A (en) * | 2014-02-21 | 2014-05-14 | 刘洛贤 | Improved primary culture method for hippocampal neurons |
| CN103789267B (en) * | 2014-02-21 | 2016-05-11 | 刘洛贤 | A kind of improved primary hippocampal neurons method |
| CN104611294A (en) * | 2015-02-11 | 2015-05-13 | 中国人民解放军第三军医大学第一附属医院 | In-vitro culture method of visual cortex neuron |
| CN104818251A (en) * | 2015-05-20 | 2015-08-05 | 妙顺（上海）生物科技有限公司 | In-vitro separation culture method for hippocampal neurons of adult rat |
| CN104818251B (en) * | 2015-05-20 | 2018-08-21 | 妙顺（上海）生物科技有限公司 | Hippocampus of adult rat neuron In-vitro separation culture method |
| CN105695411A (en) * | 2016-03-22 | 2016-06-22 | 中国人民解放军第二军医大学 | Naked mole rat cortical neuron culture method |
| CN105754943A (en) * | 2016-03-22 | 2016-07-13 | 中国人民解放军第二军医大学 | Culture method of naked mole rat hippocampal neurons |
| CN105695411B (en) * | 2016-03-22 | 2019-07-02 | 中国人民解放军第二军医大学 | A kind of naked mole cortical neuron cultural method |
| CN105754943B (en) * | 2016-03-22 | 2019-07-02 | 中国人民解放军第二军医大学 | A kind of naked mole cultured hippocampal neuron method |
| CN112391342A (en) * | 2020-12-07 | 2021-02-23 | 山东省齐鲁干细胞工程有限公司 | Efficient recovery method for umbilical cord blood stem cells |
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| WO2013113196A1 (en) | 2013-08-08 |
| CN102533654B (en) | 2014-01-29 |
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