CN106497862A - A kind of mouse cerebellum granular cell is separated and cultural method - Google Patents
A kind of mouse cerebellum granular cell is separated and cultural method Download PDFInfo
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- CN106497862A CN106497862A CN201510560474.6A CN201510560474A CN106497862A CN 106497862 A CN106497862 A CN 106497862A CN 201510560474 A CN201510560474 A CN 201510560474A CN 106497862 A CN106497862 A CN 106497862A
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Abstract
The present invention provides a kind of mouse cerebellum granular cell and separates and cultural method, including step：A () disconnected neck puts to death mouse, peel off mouse cerebellum after alcohol disinfecting, be placed in precooling aseptic containing dual anti-PBS in, and rapid under disecting microscope carefully peel off meninx and blood vessel on cerebellum；B () digests 10 15min in 37 DEG C of incubators with preheating mixed enzyme solution；C () differential sedimentation is used in combination and differential attachment method obtains cerebellar granule cell；D, in () incubation, different time points add appropriate cytarabine to suppress the growth of non-neuronal cell.The mouse cerebellum granular cell that the present invention is provided is separated and cultural method can obtain high yield, survival rate height and the high mouse cerebellum granular cell of purity.
Description
Technical field
The invention belongs to cell biology, and in particular to a kind of mouse cerebellum granular cell is separated and cultivated
Method.
Background technology
Neuron is the most basic structure function unit of nervous system, and cerebellar granule cell is central nervous system
Middle rich content, a class neuron of small volume.In view of cerebellar neuron possesses, quantity is more, and it is easy to draw materials,
And Growth and Differentiation is similar to the characteristic of Cortical Neurons, therefore cerebellar granule neuron is usually used in ex vivo nerve
The research of the aspects such as unit grows, neural axon regenerates, nervous system injury reparation and clinical neuropharmacology.
At present, more about the in-vitro separation of cerebellar granule cell and the method for culture both at home and abroad, but due to non-nerve
The pollution of first cell makes the purity of the cerebellar granule cell of acquisition not high, and separating step is complicated and pancreatin disappears
The uncertainty for changing intensity causes the quantity and survival rate of institute's developing approach to reduce.
Content of the invention
The purpose of the present invention is to set up a kind of high acquisition yield, survival rate height and the high neonatal mouse cerebellum of purity
The method that granular cell is separately cultured.
Above-mentioned involved in order to solve the problems, such as, it is thin that the present invention takes following method to obtain mouse cerebellum particle
Born of the same parents：
The step of mouse cerebellum granular cell separation method, includes：A () disconnected neck puts to death mouse, after alcohol disinfecting
In mouse cerebellum is peeled off, be placed in precooling aseptic containing dual anti-PBS in, and fast under disecting microscope
Speed carefully peels off meninx and blood vessel on cerebellum；B the cerebellum after the stripping of () PBS, dissecting scissors shred group
The digestion of mixture slaking enzyme liquid is preheated after knitting block；C () blows and beats cell, differential sedimentation with the nutrient solution containing DnaseI
After take 200 mesh sieve net filtration of supernatant, supernatant is abandoned in centrifugation, and with the resuspended precipitation of complete medium, differential velocity adherent
It is inoculated in the coated culture dish of poly-l-lysine with suitable cell concentration；In (d) incubation, different
Time point adds appropriate cytarabine；E () is inoculated with 7d, add the D-Glucose of 5mM in culture medium,
Add once per 4d later.
The operation of separating mouse cerebellum must be carried out on ice.
Optional mouse is clear for the cerebellar tissue structure of 5-8 ages in days, and before there are a large amount of cerebellar granule cells
Body and the mouse of nerve cell, male and female.
Optional digestion digestion enzyme liquid used be in advance the 0.05% of 37 DEG C of preheatings pancreatin (m/v) and
The Papain enzyme mixation of 0.15-0.2% (m/v), digestion time is 10-15min；
Cell suspension after optionally differential sedimentation method is by piping and druming stands 8-10min；
The concentration of optional poly-l-lysine (PLL) is 50-80 μ g/mL, to guarantee neuron adhesion
Under the premise of, reduce toxicity of the poly-l-lysine (PLL) to neuron.
Optional differential velocity adherent method after adherent 20-30min, will not be pasted in 37 DEG C of incubators for cell suspension
The cell suspension of wall is transferred in new coated plate again, repeats differential velocity adherent once.
It is the potassium chloride containing 25mM and 2mM glutamine and 15% mouse in optional complete medium
The cell culture medium of serum.
The concentration of optional cytarabine is 5-10 μM, be separately added into when 24h, 48h is cultivated 5 μM Ah
Sugared cytidine, adds 10 μM of cytarabine when 72h.
Pancreas of the digestive juice from 0.05% for preheating in the mouse cerebellum granular cell separation method that the present invention is provided
The mixed liquor of the papain of enzyme (m/v) and 0.15-0.2% (m/v) on the one hand subtracts in order to vitellophag
The consumption of pancreatin is lacked, has reduced damage of the pancreatin to neuronal cell, improve the survival rate of cell, separately
On the one hand tedious steps and the papain digestion spent time of pancreatin substep digestion are saved.
The present invention also provides a kind of using differential sedimentation, differential velocity adherent and addition mitotic inhibitor joint
Method obtain the high cerebellar granule cell of purity.Carefully peel off under disecting microscope and remove meninx and blood vessel,
Spongiocyte and fibroblastic pollution is reduced, as Granule Neurons cell volume is little, and other are neural
First inclusion is relatively large, stands the cell suspension 8-10min after piping and druming, the tissue block for making some big and a part
The larger spongiocyte of volume is preferentially settled.Fibroblast and the adherent speed of spongiocyte, nerve
First cell attachment speed is slower, using the difference of adherent speed, will be not adherent after culture 20-30min after inoculation
Cell be transferred to new coated plate, be repeated once differential velocity adherent, further to remove fibroblast and glue
The pollution of cell plastid, obtains the high granular cell of purity.A small amount of nerve that the granular cell of original cuiture mixes
Spongiocyte can pass through to add a small amount of cytarabine in different time points, on the one hand suppress the life of spongiocyte
Long, on the other hand reduce the suppression to neuronal cell growth.
Further, the present invention promotes mouse from adding the nutrient solution of 15% mice serum as inoculation liquid
Granular cell adherent, reduce purchase improvement serum free medium cost.
The present invention solves the pollution of non-neuronal cell during neuronal cell is separately cultured, and makes acquisition
The purity of cerebellar granule cell is high, and the cerebellar granule cell yield height of acquisition, survival rate are high.
Description of the drawings
Fig. 1 is the neuronal cell (× 200) for being inoculated with 48h；
Fig. 2 is the neuronal cell (× 200) that cultivates 3 days；
Fig. 3 is the neuronal cell (× 200) that cultivates 8 days.
Specific embodiment
In order that the purpose of the present invention and advantage represent more pure and freshly, now specific embodiment is expanded on further.
Specific embodiment set forth herein is explained only for the present invention, is not intended to limit the present invention.
The present invention selects newborn 8 days SD mouse, separates cerebellar granule cell.Concrete operations are as follows：
1st, the mouse of newborn 8d is taken, and the neck that breaks is put to death.Fixed mouse, 75% alcohol-pickled 5min, from little
Skin and skull are cut off rapidly in mouse head both sides, start skull after mouse, expose cerebellum and part brain,
Whole cerebellum is gently peeled off down with tweezers to be placed in the PBS of precooling；
2nd, rapidly separate under microscope on ice and be attached to supracerebellar meninx and blood vessel, by stripping after
Cerebellum goes in new PBS and cleans 2 times；
3rd, cerebellar tissue, size about 1mm are shredded on ice with dissecting scissors3；
The 4th, the tissue for shredding is added the digestion mixed liquor of 4-5 times of volume of preheating, is digested in 37 DEG C
10-15min, every the reverse mixings of 5-10min once；The digestion mixed liquor is 0.05% pancreatin (m/v)
With the mixed liquor of the papain of 0.15-0.2% (m/v), the pancreatin of low concentration and low-cost pawpaw
Enzyme is used in combination, and not only reduces damage of the pancreatin to neuronal cell, increases the rate of recovery of cell, while
Shorten the time that papain digestion cell is used alone.
5th, the nutrient solution containing DNaseI is added to terminate digestion, piping and druming disperses cell, due to particle nerve
First cell volume is little, and the relatively large feature of other neuron inclusions, 8-10min is stood, is preferentially settled non-
Neuronal cell, draws suspension, adds the nutrient solution piping and druming containing DNaseI, stands and draw suspension, through 200
Mesh sieve net filtration abandons supernatant after 400-800r/min centrifugations, the neuronal cell higher to obtain purity；
6th, with the complete culture solution of the potassium chloride containing 25mM, 2mM glutamine and 15% mice serum
Re-suspended cell is precipitated, and is inoculated in the culture dish of poly-l-lysine, after 37 DEG C of culture 20-30min, due to
Most fibroblasts and spongiocyte are adherent, not adherent cell is transferred to new coated plate, is repeated
Differential velocity adherent, further reduces the pollution of non-neuronal cell, the neuronal cell high to obtain purity；
7th, 5 μM of cytarabine is separately added into when 24h, 48h is cultivated, and adds 10 μM when 72h
Cytarabine is suppressing the propagation of non-neuronal cell；
8th, complete medium is changed in 2d, 5d, 8d, cultivate the D- that 7d adds 5mM in culture medium
Glucose, adds once per 4d later.
9th, cell survival rate is calculated：The single cell suspension of preparation is taken, in microscope after 0.4% Trypan Blue
The lower ratio for counting pigmented cells (dead cell) and non-pigmented cells (living cells), wherein uncoloured thin
It is cell survival rate that born of the same parents' number accounts for the percentage of total cell number.
10th, NSE identifications neuron：Neuron is inoculated on the cover glass being positioned in 24 orifice plates, in vitro
Culture 6-8 days, takes out cover glass, and PBS is washed 3 times, each 5min.4% paraformaldehyde fixes 20min,
PBS is washed 3 times, is incubated 15min under 0.2% penetrating fluid Triton-100 room temperatures, and PBS washes 3 times, and 5%
BSA room temperatures close 1h, suck confining liquid.Neuronspecific enolase NSE antibody is added in 4 DEG C of mistakes
Night is incubated, and PBS washes 3 times.Two anti-igg (1: 100) of goat-anti rabbit FITC fluorescence is added dropwise, is put at room temperature
45min is incubated in wet box, PBS is washed 3 times, examined under a microscope after drying naturally.Negative control with
PBS substitutes NSE.NSE is positioned at kytoplasm and projection, with the kytoplasm that has green fluorescence and projection as positive knot
Really, the purity of neuronal cell accounts for the percentage of total cell number for positive cell number.
Up to more than 98%, cell purity is up to 97% for isolated mouse cerebellum granular cell survival rate in the present invention
More than.After basis of microscopic observation can see culture 48h, neuron grows the projection of some elongate curveds,
And connect into network；Cultivate the 3rd celestial axis and uprush long increasing slightly, iuntercellular aixs cylinder connects into fine-structure mesh, culture the 8th
Cell space is full bright, and cell process forms tight network.
Below the preferred embodiment of the invention is described in detail, but the invention is not limited
In above-described embodiment, all any modification, equivalent and improvement is made within the spirit and principles in the present invention
Deng should be included in protection scope of the present invention.
Claims (8)
1. a kind of mouse cerebellum granular cell is separated and cultural method, it is characterised in that including step：A () is broken
Neck puts to death mouse, and alcohol disinfecting is placed in precooling aseptic containing dual anti-PBS after mouse cerebellum is peeled off
In, and meninx and blood vessel on cerebellum is carefully peeled off rapidly under disecting microscope；B () PBS is peeled off
Cerebellum afterwards, dissecting scissors preheat the digestion of mixture slaking enzyme liquid after shredding tissue block；C () uses the training containing DnaseI
Nutrient solution blows and beats cell, takes 200 mesh sieve net filtration of supernatant after differential sedimentation, and supernatant is abandoned in centrifugation, and with training completely
The resuspended precipitation of foster base, differential velocity adherent are inoculated in the coated culture dish of poly-l-lysine with suitable cell concentration；
D, in () incubation, different time points add appropriate cytarabine；E () is inoculated with 7d, in culture medium
The D-Glucose of 5mM is added, is added once per 4d later.
2. mouse cerebellum granular cell according to claim 1 is separated and cultural method, it is characterised in that
Described mouse for 5-8 ages in days cerebellar tissue structure clear, and exist a large amount of cerebellar granule cell precursors and
The mouse of nerve cell, male and female.
3. mouse cerebellum granular cell according to claim 1 is separated and cultural method, it is characterised in that
Step b digestion digestion enzyme liquid used is that digestion time is in advance in the mixed liquor of 37 DEG C of preheatings
10-15min；The mixed liquor includes 0.05% pancreatin (m/v) and the pawpaw egg of 0.15-0.2% (m/v)
White enzyme.
4. mouse cerebellum granular cell according to claim 1 is separated and cultural method, it is characterised in that
The differential sedimentation of step c be by piping and druming after cell suspension stand 8-10min.
5. mouse cerebellum granular cell according to claim 1 is separated and cultural method, it is characterised in that
The concentration of the poly-l-lysine of step c is 50-80 μ g/mL.
6. mouse cerebellum granular cell according to claim 1 is separated and cultural method, it is characterised in that
The differential velocity adherent of step c be cell suspension in 37 DEG C of incubators after adherent 20-30min, will be not adherent
Cell suspension transfer in new coated plate again, repeat differential velocity adherent once.
7. mouse cerebellum granular cell according to claim 1 is separated and cultural method, it is characterised in that
Potassium chloride containing 25mM and 2mM glutamine and 15% mouse in the complete medium of step c
Serum.
8. mouse cerebellum granular cell according to claim 1 is separated and cultural method, it is characterised in that
The concentration of the cytarabine of step d is 5-10 μM, is separately added into 5 μM when 24h, 48h is cultivated
Cytarabine, adds 10 μM of cytarabine when 72h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475198A (en) * | 2017-08-21 | 2017-12-15 | 中国医学科学院医学生物学研究所 | A kind of tree shrew dopaminergic neuron cell isolated culture method |
-
2015
- 2015-09-07 CN CN201510560474.6A patent/CN106497862A/en active Pending
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| CN107475198A (en) * | 2017-08-21 | 2017-12-15 | 中国医学科学院医学生物学研究所 | A kind of tree shrew dopaminergic neuron cell isolated culture method |
| CN107475198B (en) * | 2017-08-21 | 2020-09-29 | 中国医学科学院医学生物学研究所 | Tree shrew dopaminergic neuron cell isolation and culture method |
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Application publication date: 20170315 |