A kind of nerve stem cell culture medium and cultural methodTechnical field
The present invention relates to field of biotechnology more particularly to a kind of nerve stem cell culture mediums and cultural method.
Background technique
Neural stem cell (neural stem cell, NSC) is the nerve with height self-renewing and proliferative capacityPrecursor.It has multinomial differentiation potential, can be divided into neuron, astroglia and oligodendroglia under certain conditionCell has the potential for treating a variety of the nervous system diseases, especially has in terms for the treatment of neurodegenerative disease and damage very wellApplication prospect, have become the research hotspot in neural field.In order to promote the clinical research and application of neural stem cell, developThe culture medium for being suitable for neural stem cell amplification in vitro culture is most important.Currently, the culture medium for neural stem cell is universalThere is a problem of that complicated component, growth rate are slow, it is difficult to obtain enough cell quantities.Therefore, it is necessary to which developing a kind of can haveEffect improves the culture medium of neural stem cell amplification in vitro efficiency.
Summary of the invention
Technical problems based on background technology, the invention proposes a kind of nerve stem cell culture medium and culture sidesMethod, the ingredient of the culture medium is simple, clear, is free of animal source component, can effectively improve the amplification in vitro efficiency of neural stem cell,To be quickly obtained sufficient amount of neural stem cell, be conducive to the clinical research and application of neural stem cell.
A kind of nerve stem cell culture medium, including basal medium and additive；The basal medium is DMEM/F12Culture medium, the additive include: insulin, transferrins, basic fibroblast growth factor, epidermal growth factor, sulphurOctanoic acid, glutathione, vitamin C, ginsenoside Rb1, salviandic acid A, resveratrol.
Preferably, the additive is final concentration of: insulin 5-10 μ g/mL, transferrins 10-15 μ g/mL, alkalinity atFibroblast growth factor 10-15ng/mL, epidermal growth factor 20-30ng/mL, lipoic acid 20-30ng/mL, glutathione1.5-2.5 μ g/mL, vitamin C 3-5 μ g/mL, ginsenoside Rb1 2-3 μ g/mL, salviandic acid A 0.5-1 μ g/mL, resveratrol1-1.5μg/mL。
It is highly preferred that the additive is final concentration of: 8 μ g/mL of insulin, 14 μ g/mL of transferrins, basic fibroblastGrowth factor-21 3ng/mL, epidermal growth factor 22ng/mL, lipoic acid 25ng/mL, 2 μ g/mL of glutathione, vitamin C4 μ g/mL, 2.2 μ g/mL of ginsenoside Rb1,0.8 μ g/mL of salviandic acid A, 1.2 μ g/mL of resveratrol.
A kind of cultural method of neural stem cell, using above-mentioned nerve stem cell culture medium culture.
Preferably, which includes:
S1, it is shredded after cleaning brain tissue with PBS with operating scissors, digests 20-30min with the pancreatin of 0.02-0.03%, useSuction pipe is gently blown and beaten with dispersion tissue, and DMEM/F12 culture medium is then added and terminates digestion, is centrifuged at 1000-1200r/min4-5min；
S2, the cell inoculation for obtaining step S1 are trained in tissue culture plate with the nerve stem cell culture mediumIt supports, the half amount culture medium of replacement in every 2-3 days is digested after cell reaches 90% degrees of fusion, passed in 1:3 ratio.
Preferably, the inoculum density in the step S2 is 2-5 × 104/cm2。。
Preferably, the tissue culture plate is 12 holes or 24 porocyte culture plates.
Preferably, the condition of culture is 37 DEG C, CO2Concentration 5%.
Preferably, the digestion uses 0.125% pancreatin digestive juice.
Preferably, the tissue culture plate first carries out coating processing with the poly-D-lysine of 0.1mg/mL before inoculation.
Beneficial effects of the present invention are as follows:
Nerve stem cell culture medium of the invention is basic culture medium with DMEM/F12 culture medium, insulin, transferrins,Basic fibroblast growth factor, epidermal growth factor, lipoic acid, glutathione, vitamin C, ginsenoside Rb1, red phenolSour A, resveratrol are additive.Wherein, DMEM/F12 culture medium provides the necessary nutrient of cell growth；Alkalinity is at fibreDimension Porcine HGF, epidermal growth factor can promote the proliferation of cell, growth；Insulin, transferrins, lipoic acid can improveCellular uptake, utilization, the efficiency for converting nutritional ingredient, to promote the growth metabolism of cell；Lipoic acid, glutathione, dimension lifePlain C, ginsenoside Rb1, salviandic acid A, resveratrol cooperation can be played the role of anti-inflammatory, oxidation resistant, to protect cell, dropLow cellular damage；By the ginsenoside Rb1 of suitable concentration, salviandic acid A, resveratrol cooperation has synergy, can not onlyPlay the role of cell repair, moreover it is possible under the premise of not generating toxicity, inhibiting effect to neural stem cell, more effectively activatePI3K/AKT signal path raises the expression of PI3K, AKT albumen, to greatly enhance the proliferative capacity of neural stem cell.
In conclusion nerve stem cell culture medium and cultural method of the invention can effectively improve the increasing of neural stem cellSpeed is grown, increasing substantially its amplification in vitro efficiency to be quickly obtained sufficient amount of neural stem cell may advantageously facilitate mindClinical research and application through stem cell.
Specific embodiment
In the following, technical solution of the present invention is described in detail by specific embodiment.
Embodiment 1
A kind of nerve stem cell culture medium, ingredient are as follows: DMEM/F12 culture medium, the insulin of final concentration of 5 μ g/mL,The transferrins of final concentration of 10 μ g/mL, the basic fibroblast growth factor of final concentration of 10ng/mL are final concentration ofThe epidermal growth factor of 20ng/mL, the lipoic acid of final concentration of 20ng/mL, the glutathione of final concentration of 1.5 μ g/mL are dense eventuallyVitamin C of the degree for 3 μ g/mL, the ginsenoside Rb1 of final concentration of 2 μ g/mL, the salviandic acid A of final concentration of 0.5 μ g/mL, eventuallyConcentration is the resveratrol of 1 μ g/mL.
Embodiment 2
A kind of nerve stem cell culture medium, ingredient are as follows: DMEM/F12 culture medium, the insulin of final concentration of 8 μ g/mL,The transferrins of final concentration of 14 μ g/mL, the basic fibroblast growth factor of final concentration of 13ng/mL are final concentration ofThe epidermal growth factor of 22ng/mL, the lipoic acid of final concentration of 25ng/mL, the glutathione of final concentration of 2 μ g/mL, final concentrationFor the vitamin C of 4 μ g/mL, the ginsenoside Rb1 of final concentration of 2.2 μ g/mL, the salviandic acid A of final concentration of 0.8 μ g/mL, endConcentration is the resveratrol of 1.2 μ g/mL.
Embodiment 3
A kind of nerve stem cell culture medium, ingredient are as follows: DMEM/F12 culture medium, the insulin of final concentration of 10 μ g/mL,The transferrins of final concentration of 15 μ g/mL, the basic fibroblast growth factor of final concentration of 15ng/mL are final concentration ofThe epidermal growth factor of 30ng/mL, the lipoic acid of final concentration of 30ng/mL, the glutathione of final concentration of 2.5 μ g/mL are dense eventuallyVitamin C of the degree for 5 μ g/mL, the ginsenoside Rb1 of final concentration of 3 μ g/mL, the salviandic acid A of final concentration of 1 μ g/mL are dense eventuallyDegree is the resveratrol of 1.5 μ g/mL.
Embodiment 4
A kind of cultural method of neural stem cell, by newborn mice break neck put to death, take out brain, peel off meninx, it is clear with PBSIt is shredded after washing with operating scissors, digests 20min with 0.02% pancreatin, gently blown and beaten with suction pipe with dispersion tissue, be then addedDMEM/F12 culture medium terminates digestion, is centrifuged 4min at 1000r/min, and it is 2 × 10 that the cell of acquisition, which is pressed inoculum density,4/cm2It is inoculated in and is carried out in coating treated 24 porocyte culture plates with the poly-D-lysine of 0.1mg/mL, in embodiment 1Nerve stem cell culture medium is in 37 DEG C, CO2It is cultivated under the conditions of concentration 5%, the half amount culture medium of replacement in every 2 days is reached to cellTo after 90% degrees of fusion, is digested with 0.125% pancreatin digestive juice, passed in 1:3 ratio.
Embodiment 5
A kind of cultural method of neural stem cell, by newborn mice break neck put to death, take out brain, peel off meninx, it is clear with PBSIt is shredded after washing with operating scissors, digests 20min with 0.02% pancreatin, gently blown and beaten with suction pipe with dispersion tissue, be then addedDMEM/F12 culture medium terminates digestion, is centrifuged 4min at 1000r/min, and it is 2 × 10 that the cell of acquisition, which is pressed inoculum density,4/cm2It is inoculated in and is carried out in coating treated 24 porocyte culture plates with the poly-D-lysine of 0.1mg/mL, in embodiment 2Nerve stem cell culture medium is in 37 DEG C, CO2It is cultivated under the conditions of concentration 5%, the half amount culture medium of replacement in every 2 days is reached to cellTo after 90% degrees of fusion, is digested with 0.125% pancreatin digestive juice, passed in 1:3 ratio.
Embodiment 6
A kind of cultural method of neural stem cell, by newborn mice break neck put to death, take out brain, peel off meninx, it is clear with PBSIt is shredded after washing with operating scissors, digests 20min with 0.02% pancreatin, gently blown and beaten with suction pipe with dispersion tissue, be then addedDMEM/F12 culture medium terminates digestion, is centrifuged 4min at 1000r/min, and it is 2 × 10 that the cell of acquisition, which is pressed inoculum density,4/cm2It is inoculated in and is carried out in coating treated 24 porocyte culture plates with the poly-D-lysine of 0.1mg/mL, in embodiment 3Nerve stem cell culture medium is in 37 DEG C, CO2It is cultivated under the conditions of concentration 5%, the half amount culture medium of replacement in every 2 days is reached to cellTo after 90% degrees of fusion, is digested with 0.125% pancreatin digestive juice, passed in 1:3 ratio.
Comparative example 1
A kind of nerve stem cell culture medium, ingredient are as follows: DMEM/F12 culture medium, the insulin of final concentration of 5 μ g/mL,The transferrins of final concentration of 10 μ g/mL, the basic fibroblast growth factor of final concentration of 10ng/mL are final concentration ofThe epidermal growth factor of 20ng/mL, the lipoic acid of final concentration of 20ng/mL, the glutathione of final concentration of 1.5 μ g/mL are dense eventuallyDegree is the vitamin C of 3 μ g/mL, the salviandic acid A of final concentration of 0.5 μ g/mL, the resveratrol of final concentration of 1 μ g/mL.Above-mentioned mindDifference through stem cell media and embodiment 1 is only are as follows: is free of ginsenoside Rb1.
A kind of cultural method of neural stem cell, by newborn mice break neck put to death, take out brain, peel off meninx, it is clear with PBSIt is shredded after washing with operating scissors, digests 20min with 0.02% pancreatin, gently blown and beaten with suction pipe with dispersion tissue, be then addedDMEM/F12 culture medium terminates digestion, is centrifuged 4min at 1000r/min, and it is 2 × 10 that the cell of acquisition, which is pressed inoculum density,4/cm2It is inoculated in and is carried out in coating treated 24 porocyte culture plates with the poly-D-lysine of 0.1mg/mL, with above-mentioned nerve cordCell culture medium is in 37 DEG C, CO2It is cultivated under the conditions of concentration 5%, the half amount culture medium of replacement in every 2 days reaches 90% to cellAfter degrees of fusion, is digested with 0.125% pancreatin digestive juice, passed in 1:3 ratio.
Comparative example 2
A kind of nerve stem cell culture medium, ingredient are as follows: DMEM/F12 culture medium, the insulin of final concentration of 5 μ g/mL,The transferrins of final concentration of 10 μ g/mL, the basic fibroblast growth factor of final concentration of 10ng/mL are final concentration ofThe epidermal growth factor of 20ng/mL, the lipoic acid of final concentration of 20ng/mL, the glutathione of final concentration of 1.5 μ g/mL are dense eventuallyDegree is the vitamin C of 3 μ g/mL, the ginsenoside Rb1 of final concentration of 2 μ g/mL, the resveratrol of final concentration of 1 μ g/mL.It is above-mentionedThe difference of nerve stem cell culture medium and embodiment 1 is only are as follows: is free of salviandic acid A.
A kind of cultural method of neural stem cell, by newborn mice break neck put to death, take out brain, peel off meninx, it is clear with PBSIt is shredded after washing with operating scissors, digests 20min with 0.02% pancreatin, gently blown and beaten with suction pipe with dispersion tissue, be then addedDMEM/F12 culture medium terminates digestion, is centrifuged 4min at 1000r/min, and it is 2 × 10 that the cell of acquisition, which is pressed inoculum density,4/cm2It is inoculated in and is carried out in coating treated 24 porocyte culture plates with the poly-D-lysine of 0.1mg/mL, with above-mentioned nerve cordCell culture medium is in 37 DEG C, CO2It is cultivated under the conditions of concentration 5%, the half amount culture medium of replacement in every 2 days reaches 90% to cellAfter degrees of fusion, is digested with 0.125% pancreatin digestive juice, passed in 1:3 ratio.
Comparative example 3
A kind of nerve stem cell culture medium, ingredient are as follows: DMEM/F12 culture medium, the insulin of final concentration of 5 μ g/mL,The transferrins of final concentration of 10 μ g/mL, the basic fibroblast growth factor of final concentration of 10ng/mL are final concentration ofThe epidermal growth factor of 20ng/mL, the lipoic acid of final concentration of 20ng/mL, the glutathione of final concentration of 1.5 μ g/mL are dense eventuallyDegree is the vitamin C of 3 μ g/mL, the ginsenoside Rb1 of final concentration of 2 μ g/mL, the salviandic acid A of final concentration of 0.5 μ g/mL.OnState the difference of nerve stem cell culture medium and embodiment 1 only are as follows: be free of resveratrol.
A kind of cultural method of neural stem cell, by newborn mice break neck put to death, take out brain, peel off meninx, it is clear with PBSIt is shredded after washing with operating scissors, digests 20min with 0.02% pancreatin, gently blown and beaten with suction pipe with dispersion tissue, be then addedDMEM/F12 culture medium terminates digestion, is centrifuged 4min at 1000r/min, and it is 2 × 10 that the cell of acquisition, which is pressed inoculum density,4/cm2It is inoculated in and is carried out in coating treated 24 porocyte culture plates with the poly-D-lysine of 0.1mg/mL, with above-mentioned nerve cordCell culture medium is in 37 DEG C, CO2It is cultivated under the conditions of concentration 5%, the half amount culture medium of replacement in every 2 days reaches 90% to cellAfter degrees of fusion, is digested with 0.125% pancreatin digestive juice, passed in 1:3 ratio.
Comparative example 4
A kind of cultural method of neural stem cell, by newborn mice break neck put to death, take out brain, peel off meninx, it is clear with PBSIt is shredded after washing with operating scissors, digests 20min with 0.02% pancreatin, gently blown and beaten with suction pipe with dispersion tissue, be then addedDMEM/F12 culture medium terminates digestion, is centrifuged 4min at 1000r/min, and it is 2 × 10 that the cell of acquisition, which is pressed inoculum density,4/cm2It is inoculated in and is carried out in coating treated 24 porocyte culture plates with the poly-D-lysine of 0.1mg/mL, with conventional nerveStem cell complete medium is in 37 DEG C, CO2It is cultivated under the conditions of concentration 5%, the half amount culture medium of replacement in every 2 days is reached to cellTo after 90% degrees of fusion, is digested with 0.125% pancreatin digestive juice, passed in 1:3 ratio.
Wherein, conventional neural stem cell complete medium ingredient is as follows: DMEM/F12 culture medium, commercially available N2 additive(Gibco, article No. 17502-048), basic fibroblast growth factor, epidermal growth factor.Wherein DMEM/F12 culture mediumVolume ratio with N2 additive is 100:1, the final concentration of 10ng/mL and epidermal growth of basic fibroblast growth factorThe final concentration of 20ng/mL of the factor.
Test example 1
When the P3 of embodiment 4-6 and comparative example 1-4 reach 90% degrees of fusion for cell, group of cells quantity is measured.KnotFruit is as shown in table 1:
1 cell proliferation test result of table
It can be seen that culture medium of the invention has the effect of promoting nerve stem cell proliferation well.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and itsInventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.