CN110199985A - A kind of preparation method of neuron frozen stock solution - Google Patents
A kind of preparation method of neuron frozen stock solution Download PDFInfo
- Publication number
- CN110199985A CN110199985A CN201910517771.0A CN201910517771A CN110199985A CN 110199985 A CN110199985 A CN 110199985A CN 201910517771 A CN201910517771 A CN 201910517771A CN 110199985 A CN110199985 A CN 110199985A
- Authority
- CN
- China
- Prior art keywords
- neuron
- culture
- stock solution
- cell
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of preparation methods of neuron frozen stock solution, comprising the following steps: samples, digests, preparing cell suspension, freezing and recovery step；Glutamine is not contained in neuron frozen stock solution used and culture medium.It in the preparation method of above-mentioned neuron frozen stock solution, is frozen after primary neuron is mixed with frozen stock solution of the invention, can reduce neuron and freeze damage, neuron motility rate is high after recovery, and activity is strong.Further, the method for sampling separation nerve fiber of the invention is efficient and convenient, avoids neure damage and cell contamination, improves sampling efficiency and be conducive to subsequent digestion process.
Description
Technical field
The invention belongs to histocyte culture technique fields, and in particular to a kind of preparation method of neuron frozen stock solution.
Background technique
In the research around nervous system, primary be separately cultured of neuron is very important in vitro study, is used for
The various physiological functions of Studies On Neuronal, due to nerve fiber obtain limitation, about neuron it is primary separation be mainly
It is carried out on rodent mammal.For example, being usually used in the primary isolated rodent of neuron has tire mouse, newborn rat.So
And since neuronal cell lacks proliferative capacity, after primary primary separation, it is not suitable for long-term surviving, growth, differentiation in vitro, no
Conducive to going on smoothly for research；In addition, for primary neuron, pessimal stimulation after pole particularly sensitive to external environment
It is easily dead, so typically current now to separate flesh tissue culture, this just leads in the primary cell experiment in vitro of nerve fiber
Caused when studying primary neuron, due to goal in research materials limitation, such as tire mouse, newborn rat obtain be not easy, the period
Longer, synthesis leads to increasing for cost research cycle.
Cell freezing technology has deeply been widely applied as a kind of effective ways for saving cell in field of biology.It grinds
Study carefully personnel to be frequently necessary to frozen cell and save, for subsequent experimental or clinical use.Traditional cryogenic freezing Techniques of preserving is
It is put into cryopreservation tube after cell suspension being added in dimethyl sulfoxide and serum, after -80 DEG C of refrigerators slowly freeze, by cryopreservation tube
It is placed in liquid nitrogen and saves, cell is made to be temporarily disengaged from growth conditions and save its cell characteristics, quick-thawing when needing.And
And a certain amount of cell is moderately saved, it can prevent from making cell because the cell cultivated is contaminated or other accidents
Kind is lost, cell conservation is played the role of.In addition to this it is possible to bought using the form of cell cryopreservation, post increasing, exchange and
Transport certain cells.The glycerol or dimethyl sulfoxide (DMSO) of protective agent final concentration 5%-15% are added when cell cryopreservation, can make
Freezing point of solution reduces, and under the conditions of slow freezing, intracellular water is appeared, and is reduced ice crystal and is formed, to avoid cellular damage.
Method using " slow freeze is melted fastly " can preferably guarantee cell survival.
Primary neuron is frozen according to Cryopreservation Technology, can not only solve separation costs, also uses sample to maximizing
Material.However it is existing when freezing primary neuron as frozen stock solution using dimethyl sulfoxide, serum mixed liquor, often to neuron
It causes to freeze damage, can not continue normally to survive after causing neuron to be recovered.Cryopreservation mainly has the damage that cell generates
The following aspects: cell mobility reduces, and cell membrane, which is talked, leads to the further damage of cell, it is suppressed that Na+/K+ATP enzyme,
Cause cell expansion；Lead to meronecrosis apoptosis；Generate oxygen radical.How the damage of the cryopreservation of primary neuron is mitigated
Wound, the survival rate and activity for improving neuron after recovering are this field urgent problems to be solved.
Summary of the invention
The present invention mentions aiming at the problem that traditional cryopreservation methods not can avoid low neuron cryopreservation resuscitation motility rate, poor activity
For a kind of preparation method of neuron frozen stock solution.
A kind of preparation method of neuron frozen stock solution provided by the invention, comprising the following steps:
Sampling: take the nerve fiber of isolated mammalian, shred be placed in the first centrifuge tube with 800~1200rpm from
4~6min of the heart removes centrifuged supernatant；
Digestion: adding pancreatin, the sedimentation cell in first centrifuge tube blown open in the first centrifuge tube of Xiang Suoshu, extraction is heavy
Shallow lake cell is placed in digestion culture dish, and into the digestion culture dish, additional pancreatin, is placed in 37 DEG C for the digestion culture dish
In incubator after 8~12min, planted medium is added into the digestion culture dish and terminates digestion；
It prepares cell suspension: by the mixture in the digestion culture dish after cell sieve filters, the second centrifuge tube is added
In, 4~6min is centrifuged with 800~1200rpm, removes supernatant after centrifugation, primary neuron is made；
It freezes: freeze-stored cell liquid being made after the primary neuron is mixed with frozen stock solution, the freeze-stored cell liquid is set
In cryopreservation tube, freezed after freezing 1.0~2.0h in -20 DEG C of refrigerators in -80 DEG C of refrigerators；
Recovery: the cryopreservation tube being quickly placed into instant in 37 DEG C of water-baths, and the freeze-stored cell liquid after melting is with 800
~1200rpm is centrifuged 4~6min, removes supernatant after centrifugation, is made and freezes rear neuron, freezes rear neuron by described and is transferred to
Planted medium or recovery medium culture；
Glutamine is not contained in the neuron frozen stock solution, the planted medium and the recovery medium.
In one of them embodiment, the nerve fiber includes hippocampal tissue, cerebral cortex, appointing in cerebral tissue
It anticipates one kind.
It is further comprising the steps of before the cryopreservation step in one of them embodiment: by the primary nerve
It is first to be mixed with the planted medium, 4~6min is centrifuged with 800~1200rpm, removes supernatant after centrifugation.
In one of them embodiment, in the recovery step, rear neuron will be frozen and be transferred to recovery medium culture
Before, it is further comprising the steps of:
Rear neuron will be frozen to mix with recovery medium, 4~6min is centrifuged with 800~1200rpm, after centrifugation in removal
Clear liquid.
In one of them embodiment, the planted medium includes the component of following volumes number: 36.53~
45.87 parts of DMEM culture mediums, 4.50~5.50 parts of horse serums and 2.70~3.30 parts of fetal calf serums.
In one of them embodiment, the neuron frozen stock solution includes the component of following volumes number: 45.00~
55.00 parts of neuronal cultures, 40.50~49.50 part of 10% bovine serum albumin(BSA) and 4.50~5.50 parts of dimethyl sulfoxides.
In one of them embodiment, the neuronal culture includes the component of following volumes number:
45.00~55.00 parts of Neurobasal A culture mediums or Neurobasal culture medium and 0.90~1.10 part of B27
Cell culture additive；The Neurobasal A culture medium or Neurobasal culture medium are to have sugar-type.
In one of them embodiment, in the recovery step, rear neuron will be frozen and be transferred to planted medium training
Culture 4h is supported, then is changed to neuronal culture culture.
In one of them embodiment, the recovery medium includes the component of following volumes number: 45.00~
55.00 parts of neuronal cultures, 45.00~55.00 parts of nerve stem cell culture mediums, 0.90~1.10 part of 10%BSA and
0.90~1.10 part of 100 × nerve growth factor；
The nerve stem cell culture medium includes the component of following volumes number: 41.50~49.50 parts of DMEM/F12 cultures
Base, 0.90~1.10 part of basic fibroblast growth factor, 0.90~1.10 part of epithelical cell growth factor, 0.90~1.10
Part B27 cell culture additive, 0.45~0.55 part of N2 cell culture additive and 0.45~0.55 part are dual anti-.
In the preparation method of above-mentioned neuron frozen stock solution, frozen after primary neuron is mixed with frozen stock solution of the invention,
It can reduce neuron and freeze damage, neuron motility rate is high after recovery, and activity is strong.Further, sampling of the invention separates mind
Method through organizing is efficient and convenient, avoids neure damage and cell contamination, improves sampling efficiency and is conducive to subsequent digestion
Processing.
Detailed description of the invention
Fig. 1 to Fig. 4 is the immunofluorescence dyeing result figure of the embodiment of the present invention 1；
Fig. 5 to Fig. 8 is the immunofluorescence dyeing result figure of the embodiment of the present invention 2；
Fig. 9 is the immunofluorescence dyeing result figure of comparative example 1 of the present invention；
Figure 10 is the immunofluorescence dyeing result figure of comparative example 2 of the present invention；
Figure 11 is the immunofluorescence dyeing result figure of comparative example 3 of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment pair
The present invention is further described.It should be appreciated that the specific embodiments described herein are only used to explain the present invention, but simultaneously
It is not used in the restriction present invention.
The present invention also provides a kind of preparation methods of neuron frozen stock solution, comprising the following steps:
Sampling: take the nerve fiber of isolated mammalian, shred be placed in the first centrifuge tube with 800~1200rpm from
4~6min of the heart removes centrifuged supernatant；
Digestion: adding pancreatin into the first centrifuge tube, the sedimentation cell in the first centrifuge tube blown open, and extraction sedimentation cell is set
In digestion culture dish, digestion culture dish is placed in 8~12min in 37 DEG C of incubators by the additional pancreatin into digestion culture dish
Afterwards, planted medium is added into digestion culture dish and terminates digestion；
It prepares cell suspension: the mixture in culture dish will be digested after cell sieve filters, with 800~1200rpm centrifugation 4
~6min removes supernatant after centrifugation, and primary neuron is made；
It freezes: freeze-stored cell liquid being made after primary neuron is mixed with frozen stock solution of the invention, freeze-stored cell liquid is set
In cryopreservation tube, freezed after freezing 1.0~2.0h in -20 DEG C of refrigerators in -80 DEG C of refrigerators；
Recovery: cryopreservation tube is quickly placed into it is instant in 37 DEG C of water-baths, freeze-stored cell liquid after melting with 800~
1200rpm is centrifuged 4~6min, removes supernatant after centrifugation, is made and freezes rear neuron, will freeze rear neuron and be transferred to recovery training
Support base culture or planted medium culture.
In the preparation method of above-mentioned neuron frozen stock solution, frozen after primary neuron is mixed with frozen stock solution of the invention,
It can reduce neuron and freeze damage, neuron motility rate is high after recovery, and activity is strong.Further, sampling of the invention separates mind
Method through organizing is efficient and convenient, avoids neure damage and cell contamination, improves sampling efficiency and is conducive to subsequent digestion
Processing.
Specifically, in recovery step, culture medium used by recovering can be selected according to the purpose of recovery, i.e., according to nerve
The purpose selection of member culture carries out recovery culture using recovery medium or selection planted medium carries out recovery culture.It selects multiple
When Soviet Union's culture medium carries out recovery culture, can recover the product turned out containing neuron and star spongiocyte, positive cell
Number total amount reaches 70% or more；When planted medium being selected to carry out recovery culture, it can recover and turn out the product containing neuron,
The positive cell number of neuron reaches 90% or more.
Optionally, nerve fiber includes hippocampal tissue, cerebral cortex, any one in brain tissue.
It is further comprising the steps of before cryopreservation step as a kind of optional embodiment: by primary neuron and plantation
Culture medium mixing is centrifuged 4~6min with 800~1200rpm, removes supernatant after centrifugation.Planted medium is utilized before freezing
It is centrifuged again after being mixed with primary neuron, helps to purify Yuan Dynasty's neuron, avoid the wherein objects such as pancreatin of digestion step remaining
Matter, when influence freezes or environment when recovery leads to neure damage.
As a kind of optional embodiment, in recovery step, will freeze before rear neuron is transferred to recovery medium culture,
It is further comprising the steps of: rear neuron will be frozen and mixed with recovery medium, 4~6min, centrifugation are centrifuged with 800~1200rpm
After remove supernatant.It is centrifuged again after being mixed using planted medium with primary neuron before recovery, helps to purify the Yuan Dynasty
Neuron, avoids wherein that remaining frozen stock solution, environment when influencing recovery lead to neure damage.
As a kind of optional embodiment, planted medium includes the component of following volumes number: 36.53~45.87 parts
DMEM culture medium, 4.50~5.50 parts of horse serums and 2.70~3.30 parts of fetal calf serums.Wherein, DMEM (Dulbecco's
Modified eagle medium) culture medium is a kind of culture medium containing various amino acid and glucose, what the present invention selected
What DMEM culture medium was free from L-Glutamine has sugar-type culture medium.Horse serum (Horse employed in planted medium
Serum, HS) and fetal calf serum (Fetal bovine serum, FBS) be common cell culture additive.Although DMEM
Culture medium is common cell culture medium, but present invention research has been surprisingly found that, is combined using DMEM culture medium with HS and FBS
Better culture effect can be obtained by being used as when planted medium afterwards, using the neuron of the planted medium culture of the component proportion
Activity is stronger.
Preferably, planted medium includes the component of following volumes number: 40.00~43.40 parts of DMEM culture mediums, 4.90
~5.10 parts of HS and 2.90~3.10 part of FBS.It is highly preferred that planted medium includes the component of following volumes number: 41.70
Part DMEM culture medium, 5.00 parts of HS, 3.00 parts FBS and 0.30 part it is dual anti-.
Neuron frozen stock solution provided by the invention includes the component of following volumes number: 45.00~55.00 parts of neuron trainings
Support base, 40.50~49.50 part of 10% bovine serum albumin(BSA) and 4.50~5.50 parts of dimethyl sulfoxides；Neuron frozen stock solution is not
Contain glutamine.
Wherein, bovine serum albumin(BSA) (Albumin from bovine serum, BSA), CAS 9048-46-8, also known as
Fifth component is one of cow's serum albumin, includes 583 amino acid residues, molecular weight 66.430kDa, isoelectric point
It is 4.7.In frozen stock solution of the invention, BSA can protect neuronal cell, can mitigate adverse environment to the damage of neuronal cell
Wound can be such that freezing point of solution reduces using the effect of dimethyl sulfoxide, and under the conditions of slow freezing, intracellular water is appeared, and is subtracted
Ice crystal is lacked to be formed, to avoid cellular damage, and has been avoided that dimethyl sulfoxide concentration is excessively high, freeze-stored cell has been generated serious
Toxic effect leads to protein denaturation.Further, it does not contain in above-mentioned neuron frozen stock solution and is generally added in general culture medium
Glutamine, for containing glutamine, can less cytotoxicity, neuron viability is higher after recovery.It can
Selection of land, 10%BSA are the aqueous solution that BSA mass concentration is 10%.
Preferably, neuron frozen stock solution includes the component of following volumes number: 49.00~51.00 parts of neuronal cultures,
44.50~45.50 parts of 10%BSA and 4.90~5.10 part of dimethyl sulfoxides.It is highly preferred that neuron frozen stock solution includes following
The component of volume parts: 50.00 parts of neuronal cultures, 45.00 parts of 10%BSA and 5.00 part of dimethyl sulfoxides.Research hair
Existing, the collaboration that cooperates between the component of neuron frozen stock solution plays a role, if a certain constituent content is too high or too low, can lead
The death or activity for causing neuron reduce.For example, the toxicity of cell is then excessive to lead to nerve when dimethyl sulfoxide too high levels
It is first dead.For another example, neuronal culture is with the volume ratio of 10% bovine serum albumin with 1:(0.85~0.95) it is advisable, this is because
Although bovine serum albumin can protect neuron injury-free, when its individualism, does not have protection or maintains neuron
The condition of survival is suitable for the ratio for deploying neuronal culture and bovine serum albumin therefore, it is necessary to pass through, between the two to coordinate
Effect, thus provide most beneficial for neuron environment.
As a kind of optional embodiment, neuronal culture includes the component of following volumes number: 45.00~55.00
Part Neurobasal A or Neurobasal culture medium and 0.90~1.10 part of B27 cell culture additive.Wherein,
Neurobasal A culture medium new life and adult neurons culture, Neurobasal culture medium for before birth with the nerve of fetus
Member culture, Neurobasal A culture medium and Neurobasal culture medium are all free of L-Glutamine, Pidolidone or asparagus fern ammonia
Acid.Corresponding Neurobasal A culture medium or Neurobasal culture medium can be selected according to neuronal origin.B27 cell culture
Additive is Ying Geen biotech firm (Engreen Biosystem Co., Ltd) according to the newest optimization of relevant cell culture feature
The serum-free cell culture additive of research and development.
Preferably, neuronal culture includes the component of following volumes number: 45.00~55.00 parts of Neurobasal A
Or Neurobasal culture medium, 0.90~1.10 part of B27 cell culture additive and 0.27~0.33 part are dual anti-.Dual anti-is green
Streptomysin mixed liquor, it is special that mycillin mixed liquor (100X) (Penicillin-Streptomycin Solution) is dual anti-
Door can be directly appended in cell culture fluid for cell culture.In Pen .- Strep solution (100X), penicillin
Content be 10000U/mL, the content of streptomysin is 10mg/mL.The working concentration of preferred penicillin in cell culture fluid
Working concentration for 100U/mL, streptomysin is 0.1mg/mL, and the concentration of mycillin is finally 100 units/mL.It is added dual anti-
It can inhibit bacterial growth, avoid cell contamination.When in use, dual anti-to configure as needed, or purchase it is commercially available it is dual anti-at
Product.It is highly preferred that neuronal culture includes the component of following volumes number: there is sugar-type Neurobasal A culture medium 50.00
Part, 1.00 parts of B27 cell culture additive and 0.30 part dual anti-.
As a kind of optional embodiment, the neuronal culture in frozen stock solution of the present invention is cultivated using NeurobasalA
Base, Neurobasal A culture medium are to have sugar-type.
As a kind of optional embodiment, in recovery step, rear neuron will be frozen and be transferred to planted medium culture training
4h is supported, then is changed to neuronal culture culture.
As a kind of optional embodiment, optional recovery medium provided by the invention includes the group of following volumes number
Point: 45.00~55.00 parts of neuronal cultures, 45.00~55.00 parts of nerve stem cell culture mediums, 0.90~1.10 part 10%
BSA and 0.90~1.10 part of 100 × nerve growth factor.Wherein, nerve growth factor (Nerve growth factor,
It NGF) is a kind of protein, the growth and development of NGF adjustable surrounding and axoneuron maintains the survival of neuron, has
Neurotrophic and rush enation double biological function.
Preferably, recovery medium includes the component of following volumes number: 49.00~51.00 parts of neuronal cultures,
49.00~51.00 parts of nerve stem cell culture mediums, 0.95~1.05 part of 10%BSA and 0.95~1.05 part of 100 × NGF.More
Preferably, recovery medium includes the component of following volumes number: 50.00 parts of neuronal cultures, 50.00 parts of neural stem cell
Culture medium, 1.0 parts of 10%BSA and 1.0 part of 100 × NGF.
As a kind of optional embodiment, above-mentioned nerve stem cell culture medium includes the component of following volumes number: 41.50
~49.50 parts of DMEM/F12 culture mediums, 0.90~1.10 part of basic fibroblast growth factor, 0.90~1.10 part of epidermis are thin
The intracellular growth factor, 0.90~1.10 part of B27 cell culture additive, 0.45~0.55 part of N2 cell culture additive and 0.45
~0.55 part dual anti-.Wherein, basic fibroblast growth factor (bFGF) belongs to FGF family, is the rush containing 155 amino acid
Mitotic cationic polypeptide, molecular weight are 16~18.5KD, can promote to include mesenchymal cell, neuroderm and blood vessel
A series of proliferation of cells including endothelial cell, bFGF also have potential Angiogensis activity in vivo.In addition, bFGF
It is an important composition ingredient in embryonic stem cell medium, cell is made to keep undifferentiated shape in serum free medium
State.BFGF is the mitogenesis original of Deiter's cells and Schwann cell as neurotrophic factor.The irritating mind of bFGF
Non- mitotic activity through spongiocyte such as promotes the release of migration and the fibrinolytic enzyme activity agent of astroglia；Adjust glue
The expression of cell plastid fibrillary acidic protein (GFAP) and the synthesis of glutamic acid and S-100 albumen；Change the allusion quotation of astroglia
The cellular processes and membrane structure of type；Promote the proliferation of astroglia and forms fibrous shape；Also it can promote and dash forward less
The proliferation of spongiocyte, and increase the content of its myelin GAP-associated protein GAP and lipoid.In vitro when developing approach, bFGF can prolong
The survival of a variety of maincenters and peripheral neurons in long culture solution stimulates the synthesis of choline acetylase and the growth of protrusion.Have
It reports and bFGF is added in the tire mouse hippocampal neuron of culture, neuron can be made to increase and its Neurite elongation at live time.?
BFGF10~30pgmL-1 is added in the tire mouse hippocampal neuron of culture, prolongs the former neuron life that can only be survived 5~7 days
14 days long, number increases by 4 times；When concentration increases to 200~500pgmL-1, former 30 μm of protrusion can be made to extend to 100 μ
m.After bFGF is added in the Schwann cell of culture, 5%~10% cell enters division stage.BFGF is to the embryo mouse in culture
The frontal region of brain, top area, corpus straitum, the cholinergic neuron of thalamus and dopaminergic, GABAergic neuron, rat are small
The all nutritious and facilitations such as Cerebral cortex neuron, sympathetic ganglion cell, chicken embryo spinal cord Anterior Horn Neurons.There are also to nerve by bFGF
The Proliferation, Differentiation of precursor acts on.After research finds that bFGF is added in the neurons of rats of culture, there is the division of cholinergic composition
And it is proliferated.In addition adjusting of the division by bFGF of neuroblast is also observed, Disproportional segregation axon process is grown
The neuron behaviors such as growth cone, neurotransmitter synthesis, the transhipment of mediator vesicle.In addition, bFGF can also be raw by its rush blood vessel
The development of nervous centralis and peripheral nervous system is influenced at effect.Epithelical cell growth factor (Epidermal Growth
Factor, EGF) also known as people oligopeptides -1, it is a kind of intracorporal active material of people, the active peptides being made of 53 amino, by
The tyrosine phosphorylation for stimulating epidermal growth factor receptor reaches repairing hyperplasia skin surface cell, its biggest characteristic is that
It can promote the Proliferation, Differentiation of cell, to replace aging and dead cell with newborn cell.N2 cell culture additive
It is that Ying Geen biotech firm (Engreen Biosystem Co., Ltd.) researches and develops according to the newest optimization of relevant cell culture feature
Serum-free cell culture additive.
Preferably, nerve stem cell culture medium includes the component of following volumes number: 44.50~45.50 parts of DMEM/F12
Culture medium, 0.95~1.05 part of bFGF, 0.95~1.05 part of EGF, 0.95~1.05 part of B27 cell culture additive, 0.48~
0.52 part of N2 cell culture additive and 0.48~0.52 part it is dual anti-.
Preferably, nerve stem cell culture medium includes the component of following volumes number: 45.00 parts of DMEM/F12,1.00 parts
BFGF, 1.00 parts of EGF, 1.00 parts of B27 cell culture additives, 0.50 part of N2 cell culture additive and 0.50 part are dual anti-.
It compares for ease of description, in following embodiment of the present invention and comparative example, used frozen stock solution and recovery training
Feeding base can using on the basis of the planted medium of following proportion, neuronal culture and nerve stem cell culture medium further
Configuration.It should be noted that for the ease of comparing, following planted medium, neuronal culture and Culture of neural stem cells
Base fixed use preferably matches in proportion, and in other proportions of culture medium of the present invention, the present invention is also carried out
Largely experiments have shown that being also able to achieve the purpose of the present invention, details are not described herein again compares for other proportioning effects.
Embodiment 1
(1) Preparatory work of experiment: poly-D-lysine be coated with 4 24 orifice plates, 26 orifice plates, 37 DEG C overnight after remove coating buffer, blow
Wind 2h is until drying；After sterile washing 2 times, added with the hole DMEM culture medium 0.3mL/ of sugar-type, DMEM is extracted out before cell seeding
Culture medium, 24 orifice plates are changed to 500 μ L planted mediums, and 6 orifice plates are changed to 2mL planted medium；Disinfection of surgical equipment, including cut
Son, tweezers and fiber tweezer.
(3) microscope is sterilized, the animal to be tried of this implementation is anaesthetized --- 5 days mouse embryos.
(4) it samples: cutting off mouse embryo skull, take out brain tissue, remove blood vessel and meninx in the DMEM culture medium of Yu Bingleng,
It is extracted out after brain tissue is shredded, is placed in the first centrifuge tube of 15mL and 5min is centrifuged with 1000rpm, remove centrifuged supernatant.
(5) it digests: adding 0.25% pancreatin 6.0mL into the first centrifuge tube, the sedimentation cell in the first centrifuge tube is blown open,
Extraction sedimentation cell is placed in digestion culture dish, and the additional 0.25% pancreatin 1.0mL into digestion culture dish, will digest culture dish
It is placed in 37 DEG C of incubators after 10min, plantation culture 2mL is added into digestion culture dish and terminates digestion.
(6) it prepares cell suspension: the mixture extraction digested in culture dish being placed in the second centrifuge tube of 15mL, is disappeared at this time
Changing cell in culture dish in the pasty state, needs to blow and beat when extracting, as far as possible dispels cell；Cell in second centrifuge tube is crossed into cell
2mL planted medium is added into the second centrifuge tube that cell is added and washes out in the second centrifuge tube remaining cell and meticulous for sieve
Born of the same parents' sieve, draws back the cell that cell sieve obtains is crossed twice in centrifuge tube, is centrifuged 5min with 1000rpm, removes supernatant after centrifugation,
Primary neuron is made.
(7) it plants: 5.00mL planted medium being added into the centrifuge tube of obtained primary neuron, under piping and druming 30 or so,
Until not having packed cell, 500 μ L planted medium containing cell is planted into 24 holes good after Preparatory work of experiment step process
In plate, 1.00mL planted medium containing cell is planted in 6 orifice plates good after Preparatory work of experiment step process；It is used after 4h
The mode that half amount changes liquid is changed to neuronal culture.
It freezes: freeze-stored cell liquid being made after primary neuron obtained is mixed with 10mL frozen stock solution, by freeze-stored cell liquid
It is placed in cryopreservation tube, is freezed after freezing 1.5h in -20 DEG C of refrigerators in -80 DEG C of refrigerators.
(8) it recovers: cryopreservation tube being quickly placed into instant in 37 DEG C of water-baths, the freeze-stored cell liquid after melting is with 1000rpm
It is centrifuged 5min, removes supernatant after centrifugation, is made and freezes rear neuron, rear neuron will be frozen is transferred in planted medium and cultivate
4h, then it is changed to neuronal culture culture.
(9) immunofluorescence dyeing is tested: the neuron after recovery is carried out immunofluorescence dyeing experiment (primary antibody: Tub-M
1:200GFAP-R 1:200 secondary antibody: GM488GR568 1:500), experimental result is as shown in Figures 1 to 4.
Wherein, planted medium, neuronal culture used by embodiment 1, the proportion of frozen stock solution are as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 50.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 5.00mL
Dimethyl sulfoxide.
Shown in please referring to Fig.1 to Fig.4, through known to measuring and calculating: Tub/DAPI=14/15 ≈ 90%, GFAP/DAPI=0/15 ≈
0%, Tub positive cell number account for 90%, GFAP positive cell number and account for 0%.As calculated result it is found that using made from embodiment 1
It is high that frozen stock solution freezes rear neuronal cell motility rate, and as shown in Figure 2, the pericaryon for culture of recovering is mellow and full, the protrusion of stretching
It is thick long, illustrate that neuronal activity is stronger.It is demonstrated experimentally that utilizing dedicated neuron culture after planted medium culture 4h of the invention
Base continues to cultivate, the neuron of the only target of recovery, anastral type spongiocyte, convenient for individually studying neuron, and
Just gradually there is the phenomenon that apoptosis after 20 days in the neuron cultivated of recovering, substantially prolonged the Motility service life of neuron.
Embodiment 2
Embodiment 2 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to recovery culture
Base culture.Planted medium used by embodiment 2, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Nerve stem cell culture medium volume components proportion: 45.00mL DMEM/F12,1.00mLbFGF, 1.00mLEGF,
1.00mLB27,0.50mLN2,0.50mL are dual anti-.
Frozen stock solution volume components proportion: 50.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 5.00mL
Dimethyl sulfoxide.
Recovery medium volume components proportion: 50.00mL neuronal culture, 50.00mL nerve stem cell culture medium,
100 × NGF of 1.0mL 10%BSA and 1.0mL.
By embodiment 2 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), experimental result is as shown in Fig. 5 to Fig. 8, through known to measuring and calculating: Tub/DAPI=15/138
≈ 11%, GFAP/DAPI=81/138 ≈ 59%, Tub positive cell number accounts for 11%, GFAP positive cell number and accounts for 59%.By upper
Calculated result is stated it is found that freezing the total motility rate height of rear cell using frozen stock solution made from embodiment 2, and by Fig. 6 and 7 it is found that recovery
The pericaryon of culture is mellow and full, and the protrusion of stretching is slightly grown, and illustrates that neuronal activity is stronger, and star spongiocyte is also largely deposited
It is living, convenient for studying the interaction of star spongiocyte and neuron.And it is demonstrated experimentally that through recovery culture neuron and
Just gradually there is the phenomenon that apoptosis after star spongiocyte 20 days, extends neuron and star spongiocyte is deposited in vitro
Time living.
Embodiment 3
Embodiment 3 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to plantation culture
Base culture.Planted medium used by embodiment 3, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 45.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 4.50mL
Dimethyl sulfoxide.
By embodiment 3 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), 92%, GFAP positive cell number, which is accounted for, through measuring and calculating Tub positive cell number accounts for 0%.Recovery
The pericaryon of culture is mellow and full, and the protrusion of stretching is slightly grown, and neuronal activity is stronger, after neuron 20 days of culture of recovering
Gradually there is the phenomenon that apoptosis.
Embodiment 4
Embodiment 4 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to plantation culture
Base culture.Planted medium used by embodiment 4, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 55.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 5.50mL
Dimethyl sulfoxide.
By embodiment 4 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), 90%, GFAP positive cell number, which is accounted for, through measuring and calculating Tub positive cell number accounts for 0%.Recovery
The pericaryon of culture is mellow and full, and the protrusion of stretching is slightly grown, and neuronal activity is stronger, after neuron 20 days of culture of recovering
Gradually there is the phenomenon that apoptosis.
Embodiment 5
Embodiment 5 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to plantation culture
Base culture.Planted medium used by embodiment 5, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 45.00mL neuronal culture, 10% bovine serum albumin of 49.50mL, 4.50mL
Dimethyl sulfoxide.
By embodiment 5 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), 93%, GFAP positive cell number, which is accounted for, through measuring and calculating Tub positive cell number accounts for 0%.Recovery
The pericaryon of culture is mellow and full, and the protrusion of stretching is slightly grown, and neuronal activity is stronger, after neuron 20 days of culture of recovering
Gradually there is the phenomenon that apoptosis.
Embodiment 6
Embodiment 6 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to plantation culture
Base culture.Planted medium used by embodiment 6, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 55.00mL neuronal culture, 10% bovine serum albumin of 40.50mL, 5.50mL
Dimethyl sulfoxide.
By embodiment 6 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), 91%, GFAP positive cell number, which is accounted for, through measuring and calculating Tub positive cell number accounts for 0%.Recovery
The pericaryon of culture is mellow and full, and the protrusion of stretching is slightly grown, and neuronal activity is stronger, after neuron 20 days of culture of recovering
Gradually there is the phenomenon that apoptosis.
Embodiment 7
Embodiment 7 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to recovery culture
Base culture.Planted medium used by embodiment 7, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Nerve stem cell culture medium volume components proportion: 45.00mL DMEM/F12,1.00mLbFGF, 1.00mLEGF,
1.00mLB27,0.50mLN2,0.50mL are dual anti-.
Frozen stock solution volume components proportion: 50.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 5.00mL
Dimethyl sulfoxide.
Recovery medium volume components proportion: 45.00mL neuronal culture, 50.00mL nerve stem cell culture medium,
100 × NGF of 1.0mL 10%BSA and 1.0mL.
By embodiment 7 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), calculated, freezes the total motility rate of rear cell and reach 72%, Tub positive cell number and GFAP
Positive cell number ratio is 10:62, and the pericaryon for culture of recovering is mellow and full, and the protrusion of stretching is slightly grown, and illustrates neuronal activity
It is relatively strong, star spongiocyte also large number of viable, convenient for studying the interaction of star spongiocyte and neuron.And test card
It is bright, just there is through the neuron of recovery culture and gradually the phenomenon that apoptosis after star spongiocyte 20 days, extends nerve
The time that member and star spongiocyte are survived in vitro.
Embodiment 8
Embodiment 8 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to recovery culture
Base culture.Planted medium used by embodiment 8, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Nerve stem cell culture medium volume components proportion: 45.00mL DMEM/F12,1.00mLbFGF, 1.00mLEGF,
1.00mLB27,0.50mLN2,0.50mL are dual anti-.
Frozen stock solution volume components proportion: 50.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 5.00mL
Dimethyl sulfoxide.
Recovery medium volume components proportion: 55.00mL neuronal culture, 50.00mL nerve stem cell culture medium,
100 × NGF of 1.0mL 10%BSA and 1.0mL.
By embodiment 8 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), calculated, freezes the total motility rate of rear cell and reach 77%, Tub positive cell number and GFAP
Positive cell number ratio is 12:65, and the pericaryon for culture of recovering is mellow and full, and the protrusion of stretching is slightly grown, and illustrates neuronal activity
It is relatively strong, star spongiocyte also large number of viable, convenient for studying the interaction of star spongiocyte and neuron.And test card
It is bright, just there is through the neuron of recovery culture and gradually the phenomenon that apoptosis after star spongiocyte 20 days, extends nerve
The time that member and star spongiocyte are survived in vitro.
Embodiment 9
Embodiment 9 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to recovery culture
Base culture.Planted medium used by embodiment 9, neuronal culture, nerve stem cell culture medium, frozen stock solution, recovery training
The proportion for supporting base is as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Nerve stem cell culture medium volume components proportion: 45.00mL DMEM/F12,1.00mLbFGF, 1.00mLEGF,
1.00mLB27,0.50mLN2,0.50mL are dual anti-.
Frozen stock solution volume components proportion: 50.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 5.00mL
Dimethyl sulfoxide.
Recovery medium volume components proportion: 55.00mL neuronal culture, 50.00mL nerve stem cell culture medium,
100 × NGF of 1.0mL 10%BSA and 1.0mL.
By embodiment 9 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), calculated, freezes the total motility rate of rear cell and reach 76%, Tub positive cell number and GFAP
Positive cell number ratio is 15:61, and the pericaryon for culture of recovering is mellow and full, and the protrusion of stretching is slightly grown, and illustrates neuronal activity
It is relatively strong, star spongiocyte also large number of viable, convenient for studying the interaction of star spongiocyte and neuron.And test card
It is bright, just there is through the neuron of recovery culture and gradually the phenomenon that apoptosis after star spongiocyte 20 days, extends nerve
The time that member and star spongiocyte are survived in vitro.
Comparative example 1
Comparative example 1 difference from example 1 is that, in recovery step, will freeze rear neuron be transferred to recovery culture
Base culture.Planted medium used by comparative example 1, neuronal culture, frozen stock solution, the proportion of recovery medium are as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 50.00mL neuronal culture, 10% bovine serum albumin of 45.00mL, 5.00mL
Dimethyl sulfoxide.
Recovery medium volume components proportion: 50.00mL neuronal culture, 0.50mL10%BSA, 0.5mL100 ×
NGF。
By comparative example 1 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), the nerve of survival is not detected as shown in figure 9, display the failure of an experiment in experimental result
Member.As it can be seen that recovery medium plays a significant role the recovery for freezing rear neuron, not only different recovery mediums recovery
Cell category it is different, and have an effect on the survival of neuron.
Comparative example 2
Embodiment 2 difference from example 1 is that, in recovery step, a half bore use planted medium culture 4h
After change neuronal culture culture, another half bore is using planted medium and adds 0.1 μ L10000 × RA, 10 μ L100 × NGF
Neuronal culture culture is changed after culture 4h.Planted medium used by comparative example 2, neuronal culture, frozen stock solution are matched
Shown in such as:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 45.00mL10% bovine serum albumin, 5.00mL dimethyl sulfoxide.
By comparative example 2 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), experimental result is as shown in Figure 10, shows the failure of an experiment, the nerve of survival is not detected
Member.Compared with Example 1, comparative example 2 is due to the frozen stock solution difference of use, and cellular damage is even dead when neuron being caused carefully to freeze
It dies, neuron cannot survive when recovering culture.
Comparative example 3
Comparative example 3 difference from example 1 is that, in recovery step, a half bore use recovery medium culture, separately
One half bore is using recovery medium and adds 0.1 μ L10000 × RA, 10 μ L100 × NGF culture.Plantation used by comparative example 3
Culture medium, neuronal culture, nerve stem cell culture medium, frozen stock solution, the proportion of recovery medium are as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Nerve stem cell culture medium volume components proportion: 45.00mL DMEM/F12,1.00mLbFGF, 1.00mLEGF,
1.00mLB27,0.50mLN2,0.50mL are dual anti-.
Frozen stock solution volume components proportion: 45.00mL10% bovine serum albumin, 5.00mL dimethyl sulfoxide.
Recovery medium volume proportion: 45.00mL neuronal culture, 5.00mL nerve stem cell culture medium,
2.5mL10%BSA.
By comparative example 3 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), experimental result shows the failure of an experiment as shown in figure 11, and the nerve of survival is not detected
Member.
Compared with Example 1, the frozen stock solution that comparative example 3 uses is different, even if using recovery medium modified,
It cannot make neuron recovery survival.
Comparative example 4
Comparative example 4 difference from example 1 is that, in sampling procedure, cut off mouse embryo skull, take out brain tissue,
Blood vessel and meninx are removed in ice-cold D-Hank ' s and is rinsed twice, are shredded.
By comparative example 4 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), it shows the failure of an experiment, the neuron of survival is not detected.
Comparative example 5
Comparative example 5 difference from example 1 is that, in recovery step, a half bore use planted medium culture 4h
After change neuronal culture culture, another half bore is using planted medium and adds 0.1 μ L10000 × RA, 10 μ L100 × NGF
Neuronal culture culture is changed after culture 4h.Planted medium used by comparative example 4, neuronal culture, frozen stock solution are matched
Shown in such as:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Frozen stock solution volume components proportion: 45.00mL neuronal culture, 5.00mL dimethyl sulfoxide.
By comparative example 5 recover after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), it shows the failure of an experiment, the neuron of survival is not detected.
Comparative example 6
6 embodiment 1 of comparative example the difference is that, in recovery step, will freeze rear neuron be transferred to recovery medium training
It supports.Planted medium, neuronal culture, frozen stock solution, the proportion of recovery medium of the use of comparative example 6 are as shown:
Planted medium volume components proportion: 41.70mL DMEM, 5.00mL HS, 3.00mL FBS, 0.30mL are dual anti-.
Neuronal culture volume components proportion: 50.00mL Neurobasal A, 1.00mL B27,0.30mL are dual anti-.
Nerve stem cell culture medium volume components proportion: 45.00mL DMEM/F12,1.00mLbFGF, 1.00mLEGF,
1.00mLB27,0.50mLN2,0.50mL are dual anti-.
Frozen stock solution volume components proportion: 450mL neuronal culture, 5.00mL dimethyl sulfoxide.
Recovery medium volume components proportion: 50.00mL neuronal culture, 50.00mL nerve stem cell culture medium,
100 × NGF of 1.0mL 10%BSA and 1.0mL.
By comparative example 6 revive after neuron carry out immunofluorescence dyeing experiment (primary antibody: Tub-M 1:200GFAP-R 1:
200 secondary antibodies: GM488GR568 1:500), it shows the failure of an experiment, the neuron of survival is not detected.As it can be seen that recovery medium pair
Play a significant role in the recovery for freezing rear neuron, not only the cell category of different recovery mediums recovery is different, and
Have an effect on the survival of neuron.
Further calculate Tub positive cell number and primary mind that primary neuron is frozen culture of recovering after a certain period of time
Ratio through the member directly Tub positive cell number of plantation culture, can obtain freezing neuron survival rate, Examples 1 and 2 are surveyed respectively
The neuron survival rate that neuron freezes 1 month, 6 months, 12 months and 24 months is determined, the results are shown in Table 1.
1 embodiment of table, 1 difference freezes the cell survival rate of duration
As can be seen from Table 1, frozen stock solution of the invention can make neuron keep surviving in longer time, convenient for experiment
The preservation of research and sample.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (9)
1. a kind of preparation method of neuron frozen stock solution, which is characterized in that the preparation method of the neuron frozen stock solution include with
Lower step:
Sampling: take the nerve fiber of isolated mammalian, shred be placed in the first centrifuge tube with 800~1200rpm centrifugation 4~
6min removes centrifuged supernatant；
Digestion: adding pancreatin, the sedimentation cell in first centrifuge tube blown open in the first centrifuge tube of Xiang Suoshu, extraction precipitating is thin
Born of the same parents are placed in digestion culture dish, and into the digestion culture dish, additional pancreatin, is placed in 37 DEG C of cultures for the digestion culture dish
In case after 8~12min, planted medium is added into the digestion culture dish and terminates digestion；
It prepares cell suspension: by the mixture in the digestion culture dish after cell sieve filters, being added in the second centrifuge tube, with
800~1200rpm is centrifuged 4~6min, removes supernatant after centrifugation, and primary neuron is made；
It freezes: freeze-stored cell liquid being made after the primary neuron is mixed with frozen stock solution, the freeze-stored cell liquid is placed in jelly
It deposits in pipe, is freezed after freezing 1.0~2.0h in -20 DEG C of refrigerators in -80 DEG C of refrigerators；
Recovery: the cryopreservation tube is quickly placed into it is instant in 37 DEG C of water-baths, the freeze-stored cell liquid after melting with 800~
1200rpm is centrifuged 4~6min, removes supernatant after centrifugation, is made and freezes rear neuron, freezes rear neuron by described and is transferred to kind
Plant culture medium or recovery medium culture；
Glutamine is not contained in the neuron frozen stock solution, the planted medium and the recovery medium.
2. the preparation method of neuron frozen stock solution according to claim 1, which is characterized in that the nerve fiber includes sea
Horse tissue, cerebral cortex, any one in cerebral tissue.
3. the preparation method of neuron frozen stock solution according to claim 2, which is characterized in that the cryopreservation step it
Before, it is further comprising the steps of: the primary neuron is mixed with the planted medium, with 800~1200rpm centrifugation 4~
6min removes supernatant after centrifugation.
4. the preparation method of neuron frozen stock solution according to claim 2, which is characterized in that, will in the recovery step
It freezes before rear neuron is transferred to recovery medium culture, further comprising the steps of:
Rear neuron will be frozen to mix with recovery medium, 4~6min is centrifuged with 800~1200rpm, removes supernatant after centrifugation
Liquid.
5. the preparation method of neuron frozen stock solution according to claim 2, which is characterized in that the planted medium includes
The component of following volumes number:
36.53~45.87 parts of DMEM culture mediums, 4.50~5.50 parts of horse serums and 2.70~3.30 parts of fetal calf serums.
6. the preparation method of neuron frozen stock solution according to claim 5, which is characterized in that the neuron frozen stock solution packet
Include the component of following volumes number:
45.00~55.00 parts of neuronal cultures, 40.50~49.50 part of 10% bovine serum albumin(BSA) and 4.50~5.50 parts
Dimethyl sulfoxide.
7. the preparation method of neuron frozen stock solution according to claim 6, which is characterized in that the neuronal culture packet
Include the component of following volumes number:
45.00~55.00 parts of Neurobasal A culture mediums or Neurobasal culture medium and 0.90~1.10 part of B27 cell
Cultivate additive；The Neurobasal A culture medium or Neurobasal culture medium are to have sugar-type.
8. the preparation method of neuron frozen stock solution according to claim 7, which is characterized in that in the recovery step,
Rear neuron will be frozen and be transferred to planted medium culture culture 4h, then be changed to neuronal culture culture.
9. according to claim 1 to the preparation method of neuron frozen stock solution described in 7 any one, which is characterized in that described multiple
Soviet Union's culture medium includes the component of following volumes number: 45.00~55.00 parts of neuronal cultures, 45.00~55.00 parts of nerves
Stem cell media, 0.90~1.10 part of 10%BSA and 0.90~1.10 part of 100 × nerve growth factor；
The nerve stem cell culture medium includes the component of following volumes number: 41.50~49.50 parts of DMEM/F12 culture mediums,
0.90~1.10 part of basic fibroblast growth factor, 0.90~1.10 part of epithelical cell growth factor, 0.90~1.10 part
B27 cell culture additive, 0.45~0.55 part of N2 cell culture additive and 0.45~0.55 part are dual anti-.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910517771.0A CN110199985B (en) | 2019-06-14 | 2019-06-14 | Preparation method of neuron cryopreservation liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910517771.0A CN110199985B (en) | 2019-06-14 | 2019-06-14 | Preparation method of neuron cryopreservation liquid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110199985A true CN110199985A (en) | 2019-09-06 |
| CN110199985B CN110199985B (en) | 2021-12-14 |
Family
ID=67792910
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910517771.0A Active CN110199985B (en) | 2019-06-14 | 2019-06-14 | Preparation method of neuron cryopreservation liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110199985B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112889812A (en) * | 2021-01-29 | 2021-06-04 | 内蒙古大学 | Application of epicatechin, cell cryopreservation liquid and cell cryopreservation method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU7308491A (en) * | 1990-11-08 | 1992-06-11 | Cryolife, Inc. | Cryoprotective agent |
| CN102578078B (en) * | 2012-02-02 | 2013-06-19 | 温州医学院附属第二医院 | Frozen stock solution for nerve cells and freezing storage method |
| CN104542576B (en) * | 2015-01-24 | 2016-05-25 | 中南大学 | The cryopreserving liquid of a kind of NSC and using method thereof |
| CN105454222B (en) * | 2016-02-02 | 2018-10-30 | 河南省银丰生物工程技术有限公司 | A kind of cells frozen storing liquid and its preparation method and application |
| CN107996559B (en) * | 2018-01-03 | 2021-03-16 | 吉林省拓华生物科技有限公司 | Neural stem cell cryopreservation liquid and using method thereof |
-
2019
- 2019-06-14 CN CN201910517771.0A patent/CN110199985B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112889812A (en) * | 2021-01-29 | 2021-06-04 | 内蒙古大学 | Application of epicatechin, cell cryopreservation liquid and cell cryopreservation method |
| CN112889812B (en) * | 2021-01-29 | 2022-02-01 | 内蒙古大学 | Application of epicatechin, cell cryopreservation liquid and cell cryopreservation method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110199985B (en) | 2021-12-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2669304C (en) | Use of a composition contaning human umbilical cord blood-derived mesenchymal stem cell for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells | |
| JP2003521910A (en) | Isolation and transplantation of retinal stem cells | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| CN101330935A (en) | Isolation and cultivation of stem/progenitor cells from the amniotic membrane of umbilical cord and uses of cells differentiated therefrom | |
| EP2446892A2 (en) | Isolated adult pluripotent stem cells and method for isolating and cultivating same | |
| KR101686315B1 (en) | A method for differentiation of tonsil-derived mesenchymal stem cell into schwann cells | |
| EP1812560B1 (en) | Retina-specific cells differentiated in vitro from bone marrow stem cells, the production thereof and their use | |
| CN102899285A (en) | Method for differentiation of embryonic stem cells into nerve cells through in vitro induction | |
| Baehr et al. | Growth of adult rat retinal ganglion cell neurites on astrocytes | |
| Cameron et al. | Post-thaw viability and functionality of cryopreserved rat fetal brain cells cocultured with Sertoli cells | |
| Mao et al. | Skin-derived precursor cells promote angiogenesis and stimulate proliferation of endogenous neural stem cells after cerebral infarction | |
| Hu et al. | Tissue culture of adult human retinal ganglion cells | |
| Adler | Preparation, enrichment, and growth of purified cultures of neurons and photoreceptors from chick embryos and from normal and mutant mice | |
| RU2409663C1 (en) | Method for producing dedifferentiated cells of adult retinal pigment eye epithelium | |
| CN110199985A (en) | A kind of preparation method of neuron frozen stock solution | |
| Dutt et al. | RPE-secreted factors: influence differentiation in human retinal cell line in dose-and density-dependent manner | |
| US11439730B2 (en) | Cell sheet construct for neurovascular reconstruction and manufacture thereof | |
| CN107164325B (en) | The preparation method and kit of the oligodendroglia in the source MSCs | |
| WO2001076507A2 (en) | Use of oxygen carriers to improve grafted cell survival in neural transplantation | |
| CN110338188A (en) | A kind of neuron frozen stock solution and neuron freeze, method for resuscitation | |
| Petite et al. | Cryopreserved neuronal cells in long-term cultures of dissociated rat cerebral cortex: survival and morphometric characteristics as revealed by immunocytochemistry | |
| CN107338216A (en) | Artificial epidermal graft, its preparation method and application | |
| Johnson | Primary cultures of sympathetic ganglia | |
| Pinson | Neonatal rat heart muscle cells | |
| CN103343108B (en) | Method of promoting differentiation of rat neural stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |