CN104694475A - Novel neural stem cell culture additive, screening method and application of additive and culture medium utilizing additive - Google Patents
Novel neural stem cell culture additive, screening method and application of additive and culture medium utilizing additive Download PDFInfo
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- CN104694475A CN104694475A CN201410817677.4A CN201410817677A CN104694475A CN 104694475 A CN104694475 A CN 104694475A CN 201410817677 A CN201410817677 A CN 201410817677A CN 104694475 A CN104694475 A CN 104694475A
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Abstract
The invention provides a novel neural stem cell culture additive. The novel neural stem cell culture additive comprises the following components in final use concentration: 1mg/ml of bovine serum albumin, 50ng/ml of biotin, 1mg/ml of L-carnitine, 0.5mcg/ml of ethanol amine, 7.5mcg/ml of D(+)-galactose, 0.1 micromole of glutathione and 30nmol of sodium selenite. The novel neural stem cell culture additive is based on a scientific design, is simple in formula, low in cost, economic and reasonable, the multiplication of neural stem cells can be promoted, and therefore, the differentiation from the neural stem cells to gliocyte is reduced; the novel neural stem cell culture additive can be applied to aspects of culture of neural stem cell, particularly of the culture of neural stem cells and neural precursor cells of all mammals including human beings and is extremely wide in use range.
Description
Technical field
The invention belongs to cell technology field, relate to a kind of cell cultures additive, especially a kind of new type nerve stem cell is cultivated additive and screening method, applies and utilize the substratum of this additive.
Background technology
Stem cell (stem cell) is the primitive undifferentiated cells in health, and neural stem cell (neural stem cell, and neural precursor (mother) cell (neural precursor cell NSC), NPC is the neural stem cell of Primary Differentiation) be neural stem cell.Because it is at treatment nervous system disorders and the wide prospect of the application in damaging, studies and clinical application in recent years by leaps and bounds develops.And the application of nerve trunk system the most primary be successfully can cultivate in vitro and obtain to breed neural stem cell.Because nerve is the most complicated in body and the tissue of topnotch differentiation, the cultivation difficulty of neural stem cell is high, extremely easy Spontaneous Differentiation in culturing process, thus loses characteristic and the function of stem cell soon.So the optimization of substratum is necessary, be also very difficult.
The simplest nerve stem cell culture medium is at basic medium DMEM and DMEM/F 12 (1:1) and Prostatropin (bFGF) at present, adds N2 and cultivate additive in Urogastron (EGF).The comparison of ingredients of N2 additive is simple, containing the necessary 0.025 mg/ml Regular Insulin of Growth of Cells, 0.1 mg/ml Transferrins,iron complexes and 20 micromole's progesterone, 100 micromole's putrescine, and 30 nanomole Sodium Selenites (Bottenstein and Sato).But because wherein total protein content is low, and lack the demand thing of some Growth of Cells, therefore can not meet the user demand of existing Culture of neural stem cells completely.
At present, solve N2 and cultivate additive Problems existing, the most widely used is on the basis of above-mentioned nerve stem cell culture medium, add B27 cultivate additive.But, B27 additive is originally for developing approach designs (Brewer et al.), its composition is quite complicated, and comprise Kendall compound, Vogan-Neu and three iodo-L-thyronines (T3), these compositions have been proved to be as causing neural stem cell (neural stem cell, and the composition that breaks up of neural precursor (mother) cell (neural precursor cell, NPC) NSC).Also have some B27 compositions be not needed for neural stem cell, or provide by N2.
Wherein, above-mentioned B27 additive component is as follows: vitamin H, L-carnitine, cholesterol, Kendall compound, thanomin, D (+)-semi-lactosi, gsh, Yelkin TTS, linolic acid, linolenic acid, phosphatidylcholine, progesterone, putrescine, Vogan-Neu, retinyl acetate, Sodium Selenite, three iodo-L-thyronines (T3), DL-alpha-tocopherol, bovine serum albumin, catalase, Regular Insulin, superoxide-dismutase, Transferrins,iron complexes.
By retrieval, not yet find the patent publication us relevant to patent application of the present invention.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of design science is provided, simple, with low cost, economical rationality of filling a prescription, and can cell proliferation of nerve cord be promoted and reduce its new type nerve stem cell to spongiocyte differentiation and cultivate additive and screening method, apply and utilize the substratum of this additive.
To achieve these goals, the technical solution adopted in the present invention is as follows:
A kind of new type nerve stem cell cultivates additive, and its moiety and final utilization concentration are:
Bovine serum albumin 1 mg/ml, vitamin H 50 ngs/ml, L-carnitine 1 mcg/ml, thanomin 0.5 mcg/ml, D (+)-semi-lactosi 7.5 mcg/ml, gsh 0.1 micromole and Sodium Selenite 30 nanomole.
Utilize new type nerve stem cell as above to cultivate the substratum of additive, add new type nerve stem cell in described substratum and cultivate additive.
And, its moiety and ratio as follows:
The synthetic medium of DMEM substratum and DMEM/F 12 substratum, wherein the volume ratio of DMEM and DMEM/F 12 is 1:1;
N2 cultivates additive, and N2 cultivates composition and the working concentration of additive: 0.025 mg/ml Regular Insulin, 0.1 mg/ml Transferrins,iron complexes, 20 micromole's progesterone, 100 micromole's putrescine and 30 nanomole Sodium Selenites;
10 ngs/ml of Prostatropins and 20 ngs/ml of Urogastrons;
And above-mentioned new type nerve stem cell cultivates additive.
New type nerve stem cell as above cultivates the application of additive in culture of neural stem cells neural.
And described neural stem cell is mammiferous neural stem cell.
New type nerve stem cell as above cultivates a screening method for additive, and step is as follows:
(1) select B27 to cultivate in additive to become to be grouped into a simplified culture additive formulations, the final utilization concentration of this formula contains bovine serum albumin 1 mg/ml, vitamin H 50 ngs/ml, L-carnitine 1 mcg/ml, thanomin 0.5 mcg/ml, D (+)-semi-lactosi 7.5 mcg/ml, gsh 0.1 micromole and Sodium Selenite 30 nanomole; Then on the basis of minimum medium, compare this simplified culture additive and B27 cultivate additive to the promoter action of Neural Stem Cells ' Growth; On the basis of simplified culture additive, add composition in other B27 afterwards more one by one whether have additive effect to measure it;
(2) cell cultures:
1) pallium (e12.5-13.5) of fetal mice between the gestation 12.5 days and 13.5 of drawing materials is dissected;
2) DMEM washing cultivates rear nervous tissue once, and in 10 units per ml papoids and 4 mg/ml DNA enzymatic solution, 37 DEG C digest 45 minutes;
3) 5-7 time is blown and beaten lightly with dispersion tissue with suction pipe after adding 4 mg/ml DNA enzymatic;
4) centrifugal 5 minutes at 1000 revs/min, settling flux cleans in without the Hanks balanced salt solution of calcium magnesium; In triplicate, the single cell suspension made is measured cell density with blood-counter system after Trypan Blue;
5) by 5 × 10
4cell suspension in the neural stem cell perfect medium containing novel simplified culture additive, is inoculated in 24 well culture plates by the density of/square centimeter; Tissue Culture Plate is coated 15 ngs/ml of poly-ornithines first, are then the fibronectins of 3 ngs/ml; At 37 DEG C of 5%CO
2cultivate in water-jacket typ CO2gas incubator;
6) every two heaven-made total amount 3/4ths substratum change liquid, until when cell covers with culture plate, generally need 6-7 days, namely can be used to carry out
3h-thymidine incorporation methods measures mouse neural stem cells propagation;
7) in order to the ratio in deciding with immunocytochemical stain to cultivate after various differentiation shared by cell, need all to use fresh culture instead when cell covers with culture plate and remove Prostatropin and Urogastron becomes neurone and spongiocyte to impel differentiation of stem cells; Immunocytochemical stain is made after within four days, fixing with 4% paraformaldehyde afterwards;
(3)
3h-thymidine incorporation methods measures mouse neural stem cells propagation:
First in nutrient solution, 0.5 nanocurie/milliliter is added
3h-thymidine also hatches 24 hours; With the DNA10 minute of 10% trichloroacetic acid precipitation cell, after room temperature and 10% Tricholroacetic Acid wash twice, at room temperature extract DNA sample 10 minutes with 0.2 molar sodium hydroxide, described DNA sample is containing the DNA of 4 milligrams of % choppings; On liquid scintillation counter, isotropic substance exit dose is measured with after liquid scintillation solution mixing;
(4) immunocytochemical stain decides the ratio of various cell in cultivation
With 4% paraformaldehyde room temperature immobilized-cell culture 25 minutes, wash three times by phosphate-buffered saline (PBS); For the dyeing of intracellular antigen, broken cell film 30 minutes in 0.1%Triton X-100, then hatches 30 minutes to block non-specific binding at 5% Normal Goat Serum; Incubated at room 2 hours; Use antibody be the anti-tubulin of mouse monoclonal for contaminating neurone, 1:750 dilute, the anti-GFAP of rabbit polyclonal is for contaminating astroglia cell (1:400); The anti-O4 of mouse monoclonal is for contaminating oligodendrocyte and precursor cell (1:10) thereof; After washing three times with PBS, in biotin labeled sheep anti-mouse igg two is anti-, hatch 1 hour; After washing three times with PBS, hatch 30 minutes at Streptavidin alkaline phosphatase enzyme solution; Finally use substrate B CIP/NBT Show Color; Observe under inverted microscope and counting;
The ratio of described various cell is cell quantity ratio;
(5) choose the simplified culture additive formulations with the strongest promotion cell proliferation of nerve cord, obtain the screening method that new type nerve stem cell cultivates additive.
And, in described step (1) in the composition of minimum medium be: DMEM and DMEM/F 12 (volume ratio is 1:1) synthetic medium, adds N2 and cultivates additive and 10 ngs/ml of Prostatropins and 20 ngs/ml of Urogastrons;
Wherein, described N2 cultivates additive and is diluted in synthetic medium from commercially concentrated solution by 1:100 volume ratio.
Advantage of the present invention and positively effect are:
1, additive design science of the present invention, simple, with low cost, economical rationality of filling a prescription, and can cell proliferation of nerve cord be promoted and reduce it to spongiocyte to break up, this cultivation additive can be applied in culture of neural stem cells neural aspect, comprise all mammiferous neural stem cell of people and the cultivation of neural precursor, use range is very extensive.
2, when additive of the present invention is to Culture of neural stem cells, on the basis of adding N2, this cultivation additive has the strongest promotion cell proliferation of nerve cord effect, and cell proliferation ratio only has during N2 and exceeds 30%, and its effect is better than complexity and the B27 of costliness (Fig. 1).B27 ratio only has during N2 and only exceeds inappreciable 5% by contrast, and does not have significant difference; On N2 and this cultivation additive basis, increase by three iodo-L-thyronines (T3) or vitamin-E have restraining effect to Neural Stem Cells ' Growth, and other B27 composition comprises Kendall compound, Vogan-Neu, resin acid, gsh, catalase+superoxide-dismutase (SOD) then acts on without obvious interpolation.These results prove that N2 adds that simplified culture additive is enough to meet the needs of neural stem cell height growth, and the B27 that there is no need to add other cultivates the composition of additive, has therefore saved cost.
3, when additive of the present invention is to Culture of neural stem cells, on the basis of N2, this cultivation additive significantly increases the neuronic ratio of ratio (45.9 compare to 31.2%) in the neural stem cell differentiating rear cell colony of cultivation shared by neurone, and reduce the ratio (22.3 compare to 34.4%) of astroglia cell, the ratio no significant difference of few branch spongiocyte, this cultivation additive is pointed out not only to promote cell proliferation of nerve cord, and more protect its stem cell properties, reduce the tendency of neural stem cell Spontaneous Differentiation astrocytoblast in culturing process, this characteristic is that culture of neural stem cells neural institute is in demand.
Accompanying drawing explanation
Fig. 1 is
3h-thymidine incorporation methods measures stem cells hyperplasia results contrast figure; Data represent mean number ± standard error .N=6; Result shows additive of the present invention and more more promotes stem cells hyperplasia with N2, and statistically has significant difference (* * * P<0.001).
Embodiment
Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The equipment used in the present invention, if no special requirements, is equipment conventional in this area; The method used in the present invention, if no special requirements, is method conventional in this area.
The N2 additive used in the present invention, B27 cultivate additive, the minimum medium of Culture of neural stem cells all can purchased from biotechnology (LifeTech).
A kind of new type nerve stem cell cultivates additive, and its moiety and final utilization concentration are:
Bovine serum albumin 1 mg/ml, vitamin H 50 ngs/ml, L-carnitine 1 mcg/ml, thanomin 0.5 mcg/ml, D (+)-semi-lactosi 7.5 mcg/ml, gsh 0.1 micromole and Sodium Selenite 30 nanomole.
The application of above-mentioned new type nerve stem cell cultivation additive can be as follows:
What the first step will be done is the neural stem cell perfect medium of preparation containing novel simplified culture additive.This step is applied to all following four embodiments.So be not just repeated in embodiments.
1. be first the storage liquid * of the formulated 100 times concentrated (100X) according to novel simplified culture additive, first each composition be dissolved in the pure deionized water of cell cultures level one by one, finally dissolve in albumin.Be divided in the cryopreservation tube of 5 milliliters ,-20 DEG C of preservations after filtering with 0.22 micron sterile filter.Dissolve 37 DEG C of water-baths before each use.
* note: moiety and the final utilization concentration of above-mentioned novel simplified culture additive are:
Bovine serum albumin 1 mg/ml, vitamin H 50 ngs/ml, L-carnitine 1 mcg/ml, thanomin 0.5 mcg/ml, D (+)-semi-lactosi 7.5 mcg/ml, gsh 0.1 micromole and Sodium Selenite 30 nanomole.
2. the neural stem cell perfect medium of preparation containing novel simplified culture additive: be mixed into concentrated N2 additive (volume ratio 1:100 dilution) in DMEM and DMEM/F 12 (volume ratio 1:1) synthetic medium successively, concentrated novel simplified culture additive (volume ratio 1:100 dilution), 10 ngs/ml of Prostatropins (FGF) and 20 ngs/ml of Urogastrons (EGF).
So cultivate the substratum of additive containing above-mentioned new type nerve stem cell, its moiety and ratio as follows:
The synthetic medium of DMEM substratum and DMEM/F 12 substratum, wherein the volume ratio of DMEM and DMEM/F 12 is 1:1;
N2 cultivates additive (dilution 1:100), and N2 cultivates composition and the working concentration of additive: 0.025 mg/ml Regular Insulin, 0.1 mg/ml Transferrins,iron complexes, 20 micromole's progesterone, 100 micromole's putrescine and 30 nanomole Sodium Selenites;
10 ngs/ml of Prostatropins and 20 ngs/ml of Urogastrons;
And above-mentioned new type nerve stem cell cultivates additive.
Embodiment 1
The brain of cultivator embryonic origin and Spinal Neural Stem Cells and neural precursor (mother) cell.
1) through strict test sieve gather except common transmissible disease and obtain pregnant woman written permission after, the embryo between the pregnant 8-10 week obtaining artificial abortion source, dissects draw materials pallium and myeloid tissue under dissecting microscope.
2) DMEM washs nervous tissue 3 times, adds in 4 mg/ml DNA enzymatic solution at 10 units per ml papoids, and 37 DEG C digest 45 minutes.
3) 5-7 time is blown and beaten lightly with dispersion tissue with suction pipe after adding 4 mg/ml DNA enzymatic;
4) centrifugal 5 minutes at 1000 revs/min, settling flux cleans in without the Hanks balanced salt solution (HBSS) of calcium magnesium.In triplicate, the single cell suspension made is measured cell density with blood-counter system after Trypan Blue;
5) by 5 × 10
4cell suspension in the neural stem cell perfect medium containing novel simplified culture additive, is inoculated in porous culture plate or culturing bottle by the density of/square centimeter.Cell culture container is coated 15 ngs/ml of poly-ornithines all in advance, are then the fibronectins of 3 ngs/ml.At 37 DEG C, 5%CO
2cultivate in water-jacket typ CO2gas incubator;
6) every two heaven-made total amount 3/4ths substratum change liquid, until when cell covers with culture plate, Secondary Culture or carry out low-temperature storage again after namely can be used to carry out various experiment or digestion.
Embodiment 2
Neural stem cell and neural precursor (mother) cell in organ culture (having another name called tissue culture) grownup myeloid tissue
1) get rid of common transmissible disease after obtain the written permission of the family members of the deceased after, dissect and to draw materials donor spinal cord, the crosscut spinal cord that breaks is that 2-3mm is thick, then nervous tissue is cut under dissecting microscope the tissue block of 2-3 cubic millimeter further.
2) add PBS and clean the neural stem cell perfect medium added afterwards containing novel simplified culture additive for three times.Transfer in uncoated plastics plate, at 37 DEG C, 5%CO
2cultivate 7 days in water-jacket typ CO2gas incubator, namely can disperse, cultivate to prepare adherent cell dispersion through papain digestion, piping and druming.Also can fix by direct formalin, after section, do immunohistochemical staining research,
Embodiment 3
The cell dispersion that goes down to posterity of the mammalian neural stem cells of all embryos and adult origin and neural precursor (mother) cell is cultivated.
1) washing cell dispersion without the Hanks balanced salt solution (HBSS) of calcium magnesium cultivates after 3 times, adds in 4 mg/ml DNA enzymatic solution at 10 units per ml papoids, 37 DEG C of digestion 10 minutes;
2) 5-7 time is blown and beaten lightly with dispersion tissue with suction pipe after adding 4 mg/ml DNA enzymatic;
3) centrifugal 5 minutes at 1000 revs/min, settling flux cleans in without the Hanks balanced salt solution (HBSS) of calcium magnesium.In triplicate, the single cell suspension made is measured cell density with blood-counter system after Trypan Blue;
4) by 5 × 10
4cell suspension in the neural stem cell perfect medium containing novel simplified culture additive, is inoculated in porous culture plate or culturing bottle by the density of/square centimeter.Cell culture container is coated 15 ngs/ml of poly-ornithines all in advance, are then the fibronectins of 3 ngs/ml.At 37 DEG C, 5%CO
2cultivate in water-jacket typ CO2gas incubator;
5) every two heaven-made total amount 3/4ths substratum change liquid, until when cell covers with culture plate, Secondary Culture or carry out low-temperature storage again after namely can be used to carry out various experiment or digestion.
New type nerve stem cell of the present invention cultivates the relevant screening method of additive and detected result and checking:
The applicant is first according to the analysis to B27 composition, select in B27 to become to be grouped into a simplified culture additive formulations, the final utilization concentration of this formula contains bovine serum albumin 1 mg/ml, vitamin H 50 ngs/ml, L-carnitine 1 mcg/ml, thanomin 0.5 mcg/ml, D (+)-semi-lactosi 7.5 mcg/ml, gsh 0.1 micromole and Sodium Selenite 30 nanomole.Then on the basis of minimum medium, compare this simplified culture additive and B27 cultivate additive to the promoter action of Neural Stem Cells ' Growth.On the basis of simplified culture additive, add composition in other B27 afterwards more one by one whether have additive effect to measure it.
* note: the composition of minimum medium is that DMEM and DMEM/F 12 (mass ratio is 1:1) synthetic medium adds N2 additive (milliliter 1:100 dilutes *) and 10 ngs/ml of Prostatropins (FGF) and 20 ngs/ml of Urogastrons (EGF) (all from Life Sciences)
* note: N2 cultivates composition and the working concentration of additive: 0.025 mg/ml Regular Insulin, 0.1 mg/ml Transferrins,iron complexes, 20 micromole's progesterone, 100 micromole's putrescine, and 30 nanomole Sodium Selenites.
One, cell cultures:
1) pallium (e12.5-13.5) of fetal mice between the gestation 12.5 days and 13.5 of drawing materials is dissected;
2) DMEM washing cultivates rear nervous tissue once, and in 10 units per ml papoids and 4 mg/ml DNA enzymatic solution, 37 DEG C digest 45 minutes;
3) 5-7 time is blown and beaten lightly with dispersion tissue with suction pipe after adding 4 mg/ml DNA enzymatic;
4) centrifugal 5 minutes at 1000 revs/min, settling flux cleans in without the Hanks balanced salt solution (HBSS) of calcium magnesium.In triplicate, the single cell suspension made is measured cell density with blood-counter system after Trypan Blue;
5) by 5 × 10
4cell suspension in the neural stem cell perfect medium containing novel simplified culture additive, is inoculated in 24 well culture plates by the density of/square centimeter.Tissue Culture Plate is coated 15 ngs/ml of poly-ornithines first, are then the fibronectins of 3 ngs/ml.At 37 DEG C, 5%CO
2cultivate in water-jacket typ CO2gas incubator;
6) every two heaven-made total amount 3/4ths substratum change liquid, until when cell covers with culture plate, generally need 6-7 days, namely can be used to carry out
3h-thymidine incorporation methods measures the mensuration of mouse neural stem cells propagation.
7) in order to the ratio in deciding with immunocytochemical stain to cultivate after various differentiation shared by cell, need all to use fresh culture instead when cell covers with culture plate and remove Prostatropin (FGF) and Urogastron (EGF) becomes neurone and spongiocyte to impel differentiation of stem cells.Immunocytochemical stain is made after within four days, fixing with 4% paraformaldehyde afterwards.
Two,
3h-thymidine incorporation methods measures mouse neural stem cells propagation:
1) be integrated into when thymidine is cell fission DNA replication dna in gene, therefore when
3when H-thymidine joins in substratum, measure in cell
3the isotopic content of H gets final product the rate of propagation of reacting cells.Concise and to the point working method first in nutrient solution, adds 0.5 nanocurie/milliliter
3h-thymidine also hatches 24 hours.With the DNA10 minute of 10% trichoroacetic acid(TCA) (TCA) sedimentation cell, after room temperature and 10% Tricholroacetic Acid wash twice, at room temperature extract DNA sample (DNA containing 4 milligrams of % choppings) 10 minutes with 0.2 molar sodium hydroxide.On liquid scintillation counter, isotropic substance exit dose is measured with after liquid scintillation solution mixing.
Three, immunocytochemical stain decides the ratio of various cell in cultivation
With 4% paraformaldehyde room temperature immobilized-cell culture 25 minutes, PBS washs three times.For the dyeing of intracellular antigen, broken cell film 30 minutes in 0.1%Triton X-100, then hatches 30 minutes to block non-specific binding at 5% Normal Goat Serum.Primary antibodie is incubated in room temperature 2 hours.The main antibody used is the anti-tubulin of mouse monoclonal for contaminating neurone (TuJ1,1:750 dilute, Berkeley clonal antibody company), and the anti-GFAP of rabbit polyclonal is for contaminating astroglia cell (1:400, DAKO); The anti-O4 of mouse monoclonal is for contaminating oligodendrocyte and precursor cell (1:10, Boehringer Mannheim) thereof.After washing three times with PBS, in biotin labeled sheep anti-mouse igg two is anti-, hatch 1 hour.After washing three times with PBS, hatch 30 minutes at Streptavidin alkaline phosphatase enzyme solution.Finally use substrate B CIP/NBT Show Color.Observe under inverted microscope and counting.
After testing, result shows:
1, simplified culture additive has the strongest promotion cell proliferation of nerve cord:
3h-thymidine incorporation methods measures stem cells hyperplasia result and represents (see Fig. 1): on the basis of N2, simplified culture additive has the strongest promotion cell proliferation of nerve cord effect, cell proliferation ratio only has during N2 and exceeds 30%, and its effect is better than complexity and the B27 of costliness.B27 ratio only has during N2 and only exceeds inappreciable 5% by contrast, and does not have significant difference.The cultivation additive basis of N2 and simplification increases by three iodo-L-thyronines (T3) or vitamin-E has restraining effect to Neural Stem Cells ' Growth, and other B27 composition comprises Kendall compound, Vogan-Neu, resin acid, gsh, catalase+superoxide-dismutase (SOD) then acts on without obvious interpolation.These results display N2 adds that simplified culture additive is enough to meet the needs of neural stem cell height growth, and the B27 that there is no need to add other cultivates the composition of additive.
2, immunocytochemical stain result display simplified culture additive increases neuronic generation:
Detected result is in table 1, result in table 1 represents: on the basis of N2, simplified culture additive significantly increases the ratio (45.9 compare to 31.2%) in the neural stem cell differentiating rear cell colony of cultivation shared by neurone, and reduces the ratio (22.3 compare to 34.4%) shared by astroglia cell.The ratio no significant difference of few branch spongiocyte.Prompting simplified culture additive not only promotes cell proliferation of nerve cord, and more protects its stem cell properties, reduces the tendency of neural stem cell Spontaneous Differentiation astrocytoblast in culturing process.This characteristic is that culture of neural stem cells neural institute is in demand.
Table 1 immunocytochemical stain result display simplified culture additive increases neuronic generation table
Data represent mean number ± standard error .N=12, * P<0.05
Arrogate all powers to oneself and sell company's (biotechnology of B27, LifeTech) B27 of a remodeling is in recent years started selling, it is not containing Vogan-Neu and retinyl acetate: two kinds of known compositions that can cause neural stem cell premature differentiation, but remain other numerous compositions all shows without effect in mensuration of the present invention, the even growth (T3 and vitamin-E) of T suppression cell.So a just very small change, fundamentally do not change the characteristic of B27.
Although this simplified culture additive sets up based on on a large amount of Research foundations of mouse neural stem cells, in the application for many years of the applicant, prove that it is effective equally for the neural stem cell in cultivator and rat source.Therefore can infer that its range of application should comprise all mammiferous neural stem cell.Especially far reaching facts have proved that it is applicable to comprise embryonic origin and adult, the vitro culture of the neural stem cell of the people of brain and spinal cord and neural precursor (mother) cell.Lift one below and be applied to the neural stem cell of cultivator embryonic origin brain and spinal cord and the example of neural precursor (mother) cell:
1. be first the storage liquid of the formulated 100 times dense (100X) according to simplified culture additive, first each composition be dissolved in the pure deionized water of cell cultures level one by one, finally dissolve in albumin.Be divided in the cryopreservation tube of 5 milliliters ,-20 DEG C of preservations after filtering with 0.22 micron sterile filter.Dissolve 37 DEG C of water-baths before each use.
2. prepare neural stem cell perfect medium: in DMEM and DMEM/F 12 (volume ratio 1:1) synthetic medium, be mixed into N2 additive (volume ratio 1:100 dilution) successively, simplified culture additive (volume ratio 1:100 dilution), 10 ngs/ml of Prostatropins (FGF) and 20 ngs/ml of Urogastrons (EGF).
3. the brain of cultivator embryonic origin and Spinal Neural Stem Cells and neural precursor (mother) cell, complete in the aseptic operating platform of all operations all in the facility that GMP is qualified.
1) through strict test sieve gather except common transmissible disease and obtain pregnant woman written permission after, the embryo between the pregnant 8-10 week obtaining artificial abortion source, dissects draw materials pallium and myeloid tissue under dissecting microscope;
2) DMEM washs nervous tissue 3 times, adds in 4 mg/ml DNA enzymatic solution at 10 units per ml papoids, and 37 DEG C digest 45 minutes.The cell having started to go down to posterity only need digest 10 minutes;
3) 5-7 time is blown and beaten lightly with dispersion tissue with suction pipe after adding 4 mg/ml DNA enzymatic;
4) centrifugal 5 minutes at 1000 revs/min, settling flux cleans in without the Hanks balanced salt solution (HBSS) of calcium magnesium.In triplicate, the single cell suspension made is measured cell density with blood-counter system after Trypan Blue;
5) by 5 × 10
4cell suspension in the neural stem cell perfect medium containing novel simplified culture additive, is inoculated in porous culture plate or culturing bottle by the density of/square centimeter.Cell culture container is coated 15 ngs/ml of poly-ornithines all in advance, are then the fibronectins of 3 ngs/ml.At 37 DEG C, 5%CO
2cultivate in water-jacket typ CO2gas incubator;
6) every two heaven-made total amount 3/4ths substratum change liquid, until when cell covers with culture plate, Secondary Culture or carry out low-temperature storage again after namely can be used to carry out various experiment or digestion.
Claims (7)
1. new type nerve stem cell cultivates an additive, it is characterized in that: its moiety and final utilization concentration are:
Bovine serum albumin 1 mg/ml, vitamin H 50 ngs/ml, L-carnitine 1 mcg/ml, thanomin 0.5 mcg/ml, D (+)-semi-lactosi 7.5 mcg/ml, gsh 0.1 micromole and Sodium Selenite 30 nanomoles/milliliter.
2. utilize new type nerve stem cell as claimed in claim 1 to cultivate the substratum of additive, it is characterized in that: add new type nerve stem cell in described substratum and cultivate additive.
3. according to claim 2ly utilize new type nerve stem cell to cultivate the substratum of additive, it is characterized in that: its moiety and ratio as follows:
The synthetic medium of DMEM substratum and DMEM/F 12 substratum, wherein the volume ratio of DMEM and DMEM/F 12 is 1:1;
N2 cultivates additive, and N2 cultivates composition and the working concentration of additive: 0.025 mg/ml Regular Insulin, 0.1 mg/ml Transferrins,iron complexes, 20 micromole's progesterone, 100 micromole's putrescine and 30 nanomole Sodium Selenites;
10 ngs/ml of Prostatropins and 20 ngs/ml of Urogastrons;
And above-mentioned new type nerve stem cell cultivates additive.
4. a new type nerve stem cell as claimed in claim 1 cultivates the application of additive in culture of neural stem cells neural.
5. new type nerve stem cell according to claim 4 cultivates the application of additive in culture of neural stem cells neural, it is characterized in that: described neural stem cell is mammiferous neural stem cell and neural precursor.
6. new type nerve stem cell as claimed in claim 1 cultivates a screening method for additive, it is characterized in that: step is as follows:
(1) select B27 to cultivate in additive to become to be grouped into a simplified culture additive formulations, the final utilization concentration of this formula contains bovine serum albumin 1 mg/ml, vitamin H 50 ngs/ml, L-carnitine 1 mcg/ml, thanomin 0.5 mcg/ml, D (+)-semi-lactosi 7.5 mcg/ml, gsh 0.1 micromole and Sodium Selenite 30 nanomoles/milliliter; Then on the basis of minimum medium, compare this simplified culture additive and B27 cultivate additive to the promoter action of Neural Stem Cells ' Growth; On the basis of simplified culture additive, add composition in other B27 afterwards more one by one whether have additive effect to measure it;
(2) cell cultures:
1) pallium (e12.5-13.5) of fetal mice between the gestation 12.5 days and 13.5 of drawing materials is dissected;
2) DMEM washing cultivates rear nervous tissue once, and in 10 units per ml papoids and 4 mg/ml DNA enzymatic solution, 37 DEG C digest 45 minutes;
3) 5-7 time is blown and beaten lightly with dispersion tissue with suction pipe after adding 4 mg/ml DNA enzymatic;
4) centrifugal 5 minutes at 1000 revs/min, settling flux cleans in without the Hanks balanced salt solution of calcium magnesium; In triplicate, the single cell suspension made is measured cell density with blood-counter system after Trypan Blue;
5) by 5 × 10
4cell suspension in the neural stem cell perfect medium containing novel simplified culture additive, is inoculated in 24 well culture plates by the density of/square centimeter; Tissue Culture Plate is coated 15 ngs/ml of poly-ornithines first, are then the fibronectins of 3 ngs/ml; At 37 DEG C, 5%CO
2cultivate in water-jacket typ CO2gas incubator;
6) every two heaven-made total amount 3/4ths substratum change liquid, until when cell covers with culture plate, generally need 6-7 days, namely can be used to carry out
3h-thymidine incorporation methods measures the mensuration of mouse neural stem cells propagation;
7) in order to the ratio in deciding with immunocytochemical stain to cultivate after various differentiation shared by cell, need all to use fresh culture instead when cell covers with culture plate and remove Prostatropin and Urogastron becomes neurone and spongiocyte to impel differentiation of stem cells; Immunocytochemical stain is made after within four days, fixing with 4% paraformaldehyde afterwards;
(3)
3h-thymidine incorporation methods measures mouse neural stem cells propagation:
First in nutrient solution, 0.5 nanocurie/milliliter is added
3h-thymidine also hatches 24 hours; With the DNA10 minute of 10% trichloroacetic acid precipitation cell, after room temperature and 10% Tricholroacetic Acid wash twice, at room temperature extract DNA sample 10 minutes with 0.2 molar sodium hydroxide, described DNA sample is containing the DNA of 4 milligrams of % choppings; On liquid scintillation counter, isotropic substance exit dose is measured with after liquid scintillation solution mixing;
(4) immunocytochemical stain decides the ratio of various cell in cultivation
With 4% paraformaldehyde room temperature immobilized-cell culture 25 minutes, PBS washs three times; For the dyeing of intracellular antigen, broken cell film 30 minutes in 0.1%Triton X-100, then hatches 30 minutes to block non-specific binding at 5% Normal Goat Serum; Incubated at room 2 hours; Use antibody be the anti-tubulin of mouse monoclonal for contaminating neurone, 1:750 dilute, the anti-GFAP of rabbit polyclonal is for contaminating astroglia cell (1:400); The anti-O4 of mouse monoclonal is for contaminating oligodendrocyte and precursor cell (1:10) thereof; After washing three times with PBS, in biotin labeled sheep anti-mouse igg two is anti-, hatch 1 hour; After washing three times with PBS, hatch 30 minutes at Streptavidin alkaline phosphatase enzyme solution; Finally use substrate B CIP/NBT Show Color; Observe under inverted microscope and counting;
The ratio of described various cell is cell quantity ratio;
(5) choose the simplified culture additive formulations with the strongest promotion cell proliferation of nerve cord, obtain the screening method that new type nerve stem cell cultivates additive.
7. new type nerve stem cell according to claim 6 cultivates the screening method of additive, it is characterized in that: in described step (1) in the composition of minimum medium be: DMEM and DMEM/F 12 (volume ratio is 1:1) synthetic medium, adds N2 and cultivates additive and 10 ngs/ml of Prostatropins and 20 ngs/ml of Urogastrons;
Wherein, described N2 cultivates additive and is diluted in synthetic medium from commercially concentrated solution by 1:100 volume ratio.
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| CN113528440A (en) * | 2021-07-05 | 2021-10-22 | 四川大学华西第二医院 | Separation and culture method of mouse oligodendrocyte precursor |
| WO2022077784A1 (en) * | 2020-10-16 | 2022-04-21 | 武汉睿健医药科技有限公司 | Chemical culture system and use thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567638A (en) * | 2016-03-01 | 2016-05-11 | 赛百慷（上海）生物技术股份有限公司 | Additive for neural stem cells |
| WO2022077784A1 (en) * | 2020-10-16 | 2022-04-21 | 武汉睿健医药科技有限公司 | Chemical culture system and use thereof |
| CN113528440A (en) * | 2021-07-05 | 2021-10-22 | 四川大学华西第二医院 | Separation and culture method of mouse oligodendrocyte precursor |
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