CN110338188A - A kind of neuron frozen stock solution and neuron freeze, method for resuscitation - Google Patents
A kind of neuron frozen stock solution and neuron freeze, method for resuscitation Download PDFInfo
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- CN110338188A CN110338188A CN201910516679.2A CN201910516679A CN110338188A CN 110338188 A CN110338188 A CN 110338188A CN 201910516679 A CN201910516679 A CN 201910516679A CN 110338188 A CN110338188 A CN 110338188A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a kind of neuron frozen stock solution, the component including 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；Neuron frozen stock solution does not contain glutamine.Above-mentioned neuron frozen stock solution; bovine serum albumin(BSA) can protect neuronal cell; damage of the adverse environment to neuronal cell can be mitigated, freezing point of solution can be made to reduce using the effect of dimethyl sulfoxide, under the conditions of slow freezing; intracellular water appears; reduce ice crystal to be formed, to avoid cellular damage, and is avoided that dimethyl sulfoxide concentration is excessively high; serious toxic effect is generated to freeze-stored cell, leads to protein denaturation.In addition, without containing the glutamine generally added in general culture medium in above-mentioned neuron frozen stock solution, for containing glutamine, can less cytotoxicity, neuron viability is higher after recovery.
Description
Technical field
The invention belongs to histocyte culture technique fields, and in particular to a kind of neuron frozen stock solution and neuron freeze,
Method for resuscitation.
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, one of key factor are just to provide a kind of suitable frozen stock solution.
Summary of the invention
The present invention not can avoid neuron and freeze damage for traditional frozen stock solution, cause neuron deposit recovery motility rate it is low,
The problem of poor activity, a kind of neuron frozen stock solution is provided and neuron freeze, method for resuscitation.
A kind of neuron frozen stock solution provided by the invention, 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 diformazans
Base sulfoxide；The neuron frozen stock solution does not contain glutamine.
The neuronal culture includes the component of following volumes number in one of the embodiments: 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 neuronal culture further includes the component of following volumes number in one of the embodiments: 0.27 part~
0.33 part dual anti-.
The Neurobasal A culture medium is to have sugar-type in one of the embodiments,.
The present invention also endured a kind of neuron freeze, method for resuscitation, the neuron freezes, method for resuscitation includes following step
It is rapid:
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；
Prepare cell suspension: by the mixture in the digestion culture dish after cell sieve filters, with 800~1200rpm
It is centrifuged 4~6min, removes supernatant after centrifugation, primary neuron is made；
It freezes: being made and freeze after the primary neuron is mixed with the frozen stock solution as described in claim 1 to 5 any one
Cell liquid is deposited, the freeze-stored cell liquid is placed in cryopreservation tube, in -80 DEG C of refrigerators after freezing 1.0~2.0h in -20 DEG C of refrigerators
Interior freezing；
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
Recovery medium culture or planted medium culture.
It is further comprising the steps of in one of the embodiments, before the cryopreservation step: by the primary neuron
It is mixed with the planted medium, 4~6min is centrifuged with 800~1200rpm, removes supernatant after centrifugation.
In one of the embodiments, in the recovery step, will freeze rear neuron be transferred to recovery medium culture or
It is further comprising the steps of before planted medium culture: rear neuron will be frozen and mixed with recovery medium, with 800~
1200rpm is centrifuged 4~6min, removes supernatant after centrifugation.
In one of the embodiments, the nerve fiber include hippocampal tissue, it is cerebral cortex, any one in brain tissue
Kind.
The planted medium includes the component of following volumes number in one of the embodiments: 36.53~45.87
Part DMEM culture medium, 4.50~5.50 parts of horse serums and 2.70~3.30 parts of fetal calf serums.
In one of the embodiments, in the recovery step, rear neuron will be frozen and be transferred to planted medium culture
4h is cultivated, then is changed to neuronal culture culture.
Above-mentioned neuron frozen stock solution, including neuronal culture, bovine serum albumin(BSA) and dimethyl sulfoxide, ox blood are pure
Albumen can protect neuronal cell, can mitigate damage of the adverse environment to neuronal cell, can utilize dimethyl sulfoxide
Effect reduces freezing point of solution, and under the conditions of slow freezing, intracellular water is appeared, and reduces ice crystal and is formed, to avoid thin
Cellular damage, and it is avoided that dimethyl sulfoxide concentration is excessively high, serious toxic effect is generated to freeze-stored cell, leads to albumen qualitative change
Property.In addition, without containing the glutamine generally added in general culture medium in above-mentioned neuron frozen stock solution, compared to containing paddy ammonia
For amide, can less cytotoxicity, neuron viability is higher after recovery.
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.
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 Neurobasal A
Base, Neurobasal A culture medium are to have sugar-type.
The present invention also provides a kind of neurons to freeze, method for resuscitation, 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.
Above-mentioned neuron freezes, in method for resuscitation, freezes after primary neuron is mixed with frozen stock solution of the invention, can
It reduces neuron and freezes damage, neuron motility rate is high after recovery, and activity is strong.Further, sampling of the invention separates neural group
The method knitted is efficient and convenient, avoids neure damage and cell contamination, improves sampling efficiency and is conducive to subsequent digestion process.
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.
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.
Optionally, nerve fiber includes hippocampal tissue, cerebral cortex, any one in brain tissue.
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-.
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 is added in the tire mouse hippocampal neuron of culture-1, prolong 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-1When, former 30 μm of protrusion can be made to extend to 100 μm.
After bFGF is added in the Schwann cell of culture, 5%~10% cell enters division stage.BFGF is to the embryo mouse brain in culture
Frontal region, top area, corpus straitum, the cholinergic neuron of thalamus and dopaminergic, GABAergic neuron, rat cerebellum
The all nutritious and facilitations such as cortical neuron, sympathetic ganglion cell, chicken embryo spinal cord Anterior Horn Neurons.There are also before to nerve by bFGF
The Proliferation, Differentiation of body cell acts on.After research finds that bFGF is added in the neurons of rats of culture, there is the division of cholinergic composition simultaneously
Proliferation.In addition adjusting of the division by bFGF of neuroblast, the growth birth of Disproportional segregation axon process is also observed
The neuron behaviors such as long cone, neurotransmitter synthesis, the transhipment of mediator vesicle.In addition, bFGF can also be by its Angiogensis
It acts on to influence the development of nervous centralis and peripheral nervous system.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-R1:200 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 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 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
5 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 5 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 5 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 (10)
1. a kind of neuron frozen stock solution, which is characterized in that 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 diformazans
Base sulfoxide；The neuron frozen stock solution does not contain glutamine.
2. neuron frozen stock solution according to claim 1, which is characterized in that the neuronal culture includes following volumes
The component of number: 45.00~55.00 parts of Neurobasal A culture mediums or Neurobasal culture medium and 0.90~1.10 part
B27 cell culture additive.
3. neuron frozen stock solution according to claim 2, which is characterized in that the neuronal culture further includes with lower body
The component of product number: 0.27 part~0.33 part dual anti-.
4. neuron frozen stock solution according to claim 2 or 3, which is characterized in that the Neurobasal A culture medium is
There is sugar-type.
5. a kind of neuron freezes, method for resuscitation, which is characterized in that the neuron freezes, method for resuscitation the following steps are included:
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；
Prepare cell suspension: by the mixture in the digestion culture dish after cell sieve filters, with 800~1200rpm centrifugation 4
~6min removes supernatant after centrifugation, and primary neuron is made；
It freezes: being made and frozen carefully after the primary neuron is mixed with the frozen stock solution as described in claim 1 to 5 any one
The freeze-stored cell liquid is placed in cryopreservation tube by cytosol, in cold in -80 DEG C of refrigerators after freezing 1.0~2.0h in -20 DEG C of refrigerators
Freeze；
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, by it is described freeze rear neuron be transferred to it is multiple
Soviet Union's culture medium culture or planted medium culture.
6. neuron according to claim 5 freezes, method for resuscitation, which is characterized in that before the cryopreservation step, also
The following steps are included: the primary neuron is mixed with the planted medium, 4~6min is centrifuged with 800~1200rpm,
Supernatant is removed after centrifugation.
7. neuron according to claim 5 freezes, method for resuscitation, which is characterized in that in the recovery step, will freeze
Neuron is transferred to before recovery medium culture or planted medium culture afterwards, further comprising the steps of: will freeze rear neuron
It is mixed with recovery medium, 4~6min is centrifuged with 800~1200rpm, removes supernatant after centrifugation.
8. neuron according to claim 5 freezes, method for resuscitation, which is characterized in that the nerve fiber includes hippocampus
Tissue, cerebral cortex, any one in brain tissue.
9. neuron according to claim 5 freezes, method for resuscitation, which is characterized in that the planted medium include with
The component of lower volume number: 36.53~45.87 parts of DMEM culture mediums, 4.50~5.50 parts of horse serums and 2.70~3.30 parts
Fetal calf serum.
10. being frozen according to neuron described in claim 5 to 9 any one, method for resuscitation, which is characterized in that described multiple
In step of reviving, rear neuron will be frozen and be transferred to planted medium culture culture 4h, then be changed to neuronal culture culture.
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| CN105454222A (en) * | 2016-02-02 | 2016-04-06 | 河南省银丰生物工程技术有限公司 | Cell cryopreservation liquid and preparation method and application thereof |
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| CN105454222A (en) * | 2016-02-02 | 2016-04-06 | 河南省银丰生物工程技术有限公司 | Cell cryopreservation liquid and preparation method and application thereof |
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