CN105907717A - Primary mouse or rat neuron isolation and culture method - Google Patents
Primary mouse or rat neuron isolation and culture method Download PDFInfo
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- CN105907717A CN105907717A CN201610277888.2A CN201610277888A CN105907717A CN 105907717 A CN105907717 A CN 105907717A CN 201610277888 A CN201610277888 A CN 201610277888A CN 105907717 A CN105907717 A CN 105907717A
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Abstract
The invention discloses a primary mouse or rat neuron isolation and culture method. Biopsy needles are used for extracting treated tissues innovatively to enable sampling to target objective cells directly, so that directness and effectiveness are achieved, mixing of surface envelope cells and bacteria is avoided ingeniously, cell and bacterial contamination is reduced and objective cell purity is improved greatly. Specially prepared compound enzyme digestive juice avoids the problems of low cell viability and poor yield caused by excessive digestion and blowing of the tissues and is capable of digesting more evenly and thoroughly as compared with preheated digestion of traditional single collagenase. Pre-adherence and an approach of adding Laminin in an early stage of primary culture can promote neuron adherence, neural synapse growth and neural network forming greatly so as to further increase yield and long-term survival rate of neurons.
Description
Technical field
The present invention relates to technical field of cell culture, particularly relate to a kind of Primary mouse or rat neuronal cell
Isolated culture method.
Background technology
Central nervous system is by two big class cellularities, it may be assumed that neuron and neurogliocyte.Neuron is big
The structure of brain, function and kilonem.Although there is the biggest difference in the size and shape of neuron, but all
Neuron is owned by common Morphological Characteristics, it may be assumed that constitute extremely complex internet.As nerveous system
Uniting main signal unit, neuron belongs to a kind of dynamically depolarized cells.Human brain has 1 × 1011Individual neuron,
Each neuron can at least contact other 10000 neurons.
At present, substantial amounts of researcher has been had to carry out cultivation and the pharmacology of mice/neurons of rats
Research in terms of characteristic, uses trypsin digestion to cultivate research previously more.It is disadvantageously, pancreatin is to nerve
The injury of unit is very big, is difficult to control digestion time and the dynamics of piping and druming, and the result being easily caused is often, cell
Vigor is poor, and dead cell is the most, and attached cell is few, and the glial cell mixed is the most.
Summary of the invention
The present invention solves a kind of high-purity that the problems referred to above of the prior art propose, high motility rate, high activity with
Time oligosaprobic Primary mouse or the isolated culture method of rat neuronal cell.
In order to realize above-mentioned technical purpose, the technical measures that the present invention is taked are:
A kind of Primary mouse or the isolated culture method of rat neuronal cell, comprise the following steps:
Step 1: mice or the acquisition of rat brain tissue and pretreatment
Mice or rat are breaked end and obtains cerebral tissue, after in vitro cerebral tissue PBS solution washing,
It is put on ice for stand-by；
Step 2: take neuronal cell
Use biopsy needle to thrust the cerebral tissue after step 1 processes, cut neuronal cell；
Step 3: enzymic digestion
Neuronal cell step 2 obtained is placed in compound enzyme Digestive system digestion, wherein in compound enzyme Digestive system
Including type i collagen enzyme, II Collagenase Type, IV Collagenase Type and Dispase enzyme that mass ratio is 1:1:1:1；
Step 4: separation and Culture neuronal cell
Step 3 is digested complete Skeletal Muscle Cell and crosses 70um screen cloth, be centrifuged washing, then carry out resuspended also
Recentrifuge, hangs with pre-adhere-wall culture basic weight the most again, changes to neuron cultivation after the most pre-adherent 4h
Base, cell is positioned over 37 DEG C, 5%CO2Incubator in cultivate.
Preferably, the biopsy needle used in above-mentioned steps 2 is semi-automatic or full-automatic biopsy needle.
Preferably, in above-mentioned steps 3, in compound enzyme Digestive system, the concentration of each enzyme is 1mg/ml.
Preferably, in above-mentioned steps 3 enzymic digestion process for use 4 DEG C of compound enzyme Digestive systems digestion 4-20h or
37 DEG C of compound enzyme Digestive system digestion 10min-2h.
Preferably, in above-mentioned steps 4, pre-adhere-wall culture base is the DMEM/F12 containing Laminin and 10%FBS
Culture medium.
Preferably, in above-mentioned steps 4, in pre-adhere-wall culture base, Laminin concentration is 1ug/ml.
Preferably, in above-mentioned steps 4, neuronal culture is containing B27, the Neurobasal of L-glutaminate
Culture medium.
Preferably, in above-mentioned steps 4, centrifugal condition is 400g, centrifugal 5 minutes.
Another aspect of the present invention also provides for the separation and Culture of the Primary mouse according to above-mentioned or rat neuronal cell
Mice that method is cultivated and rat neuronal cell.
The present invention uses technique scheme, compared with prior art, has the following technical effect that
(1) what the present invention was initiative have employed the tissue after process taked by biopsy needle so that sampling is directed to purpose
Cell, not only directly effectively, avoids being mixed into of surface-coating cell and antibacterial simultaneously dexterously,
While reducing cell, germ contamination, substantially increase the purity of purpose cell, when separating cell
Can be close to 100%；
(2) Primary mouse of the present invention or the isolated culture method of rat neuronal cell, uses compound enzyme digestion method
Obtain single neuron, it is to avoid excessive tissue is digested by conventional trypsin digestion, excessively blows
Beat the problem that the cytoactive caused is low, obtain rate variance, in particular for neuron is this, ischemia is lacked
Oxygen is than more sensitive histiocyte.And use 4 DEG C of condition digested overnight (4-20h), compare tradition single
The collagenase preheating digestion of one, not only avoids and excessive tissue digestion is excessively blown and beaten the cell caused
Activity is low, the problem of rate variance, and digest the most thorough；
(3) use the pre-adherent and mode of original cuiture addition 1ug/ml Laminin in early days, can greatly promote
Enter adherent, the growth of nerve synapse and the formation of neutral net of neuron, thus improve further
The yield of neuron and long-term surviving rate；
(4) preparation method is easy, consistent, reproducible.
In sum, the Primary mouse of the present invention or the isolated culture method of rat neuronal cell, it is possible to obtain
High-purity, high motility rate, the high activity Primary mouse that pollution risk is extremely low simultaneously or rat neuronal cell, cultivate
Cell viability high, there is bigger dissemination.
Accompanying drawing explanation
Fig. 1 is the microphotograph of the rat neuronal cell shooting that the isolated culture method of the present invention obtains
(10X)；
Fig. 2 is the microphotograph of the rat neuronal cell shooting that the isolated culture method of the present invention obtains
(20X)；
Detailed description of the invention
Below by specific embodiment, the present invention is carried out detailed and concrete introduction, so that being better understood from this
Bright, but following embodiment is not limiting as the scope of the invention.
Embodiment 1
The embodiment of the present invention 1 provides the isolated culture method of a kind of primary rat neuronal cell, concrete steps
As follows:
1. the acquisition of rat brain tissue
First new life (within being born 48 hours) rat (freezing 10 minutes or cervical dislocation), wine are put to death
Essence is soaked 5 minutes, and in super-clean bench, broken end takes brain.Whole process will be carried out on ice.The cerebral tissue that will take off
It is placed in the culture dish filling pre-cooling PBS, washs 3 times, reject surface residual bloodstain, be immediately placed in containing 2%
In the pre-cooling L-15 culture medium of mycillin, putting and be kept on ice, treat that all tissue samplings are complete, unification is entered
Row processes.
2. take neuronal cell
Use biopsy needle to thrust the cerebral tissue after step 1 processes, cut neuronal cell.
3. enzymic digestion
The cerebral cortex bleached after processing shreds, and adds homemade compound enzyme (type i collagen enzyme+II Collagen Type VI
Enzyme+IV Collagenase Type+Dispase enzyme=1:1:1:1), the initial concentration of 4 kinds of enzymes is 1mg/ml.37 degree
Digesting 30 minutes, somewhat blow and beat several times, according to circumstances, 37 degree digest 30 minutes again, the most slightly blow
Beat less than 5 times, cross 70um screen cloth.
4. separation and Culture neuronal cell
Step 3 is digested complete Skeletal Muscle Cell and crosses 70um screen cloth, be centrifuged washing, then carry out weight
Outstanding and recentrifuge, the particularity cultivated due to neuron, separate the single neuron suspension obtained need through
One pre-adherent process.That is: in the DMEM/F12 culture medium containing 10%FBS, pre-adherent 4 little
Time.After 4 hours, change to neuronal culture, it may be assumed that containing B27 (1:50), L-glutaminate (0.5um/ml)
Neurobasal culture medium.Cell is positioned over 37 DEG C, 5%CO2Incubator in cultivate.
5. two dimension is cultivated
By the neurons of rats of purification, first hang with pre-adhere-wall culture basic weight, be seeded in gelatin pretreatment overnight
Cultivating in T25 culture bottle, inoculum density is 5 × 105/ bottle.In order to promote that neuron is the most adherent, add
Enter the Laminin of 1ug/ml.It is placed in 37 degree, 5%CO2Incubator in cultivate.After 4 hours,
Abandon pre-adhere-wall culture base, be replaced by neuronal culture, be simultaneously introduced the Laminin of 1ug/ml.It is placed in 37
Degree, 5%CO2Incubator in cultivate.The most every neuronal culture more renewed for 3 days, every day in
Basis of microscopic observation cell growth status, according to circumstances, can add the cytosine arabinoside of final concentration of 10umol/L,
The non-neuronal growths such as suppression glial cell.
6. cellular morphology is observed
The cellular morphology of basis of microscopic observation primary separation and Culture neuronal cell, as shown in Figure 1-2.
7. other indexs of correlation measure
Antibacterial and fungal contamination situation, cell contamination situation, statistics Cell viability etc. is measured during cultivation.
In the neurons of rats cultural method of the present invention, cerebral tissue used, compound enzyme, cell culture medium,
Cytokine and hyclone etc., all should meet sterility requirements.
Comparative example
Comparative example uses conventional methods, and after the acquisition of rat brain tissue, under anatomic microscope, uses
Aseptic nipper carefully peels off cerebral cortex, and whole process will be carried out on ice.Further, in the PBS of pre-cooling,
Carefully divest pia mater encephali and the blood streak, completely strip as far as possible, in order to avoid mantle brain deficiency stays, cause substantial amounts of one-tenth fiber finer
Born of the same parents' hypertrophy.After being stripped clean, wash 2-3 time with the PBS of pre-cooling.Cerebral cortex processes and is successfully masked as,
Cerebral tissue becomes white, and outer surface does not has any blood streak to remain.Then the cerebral cortex bleached after processing is cut
Broken, add enzymatic solution, 37 degree digest 30 minutes, somewhat blow and beat several times, and according to circumstances, 37 degree disappear again
Changing 30 minutes, then slight piping and druming is less than 5 times, crosses 70um screen cloth.Take the cell filtrate after sieving,
The PBS, 400g that add equity according to volume are centrifuged 5 minutes, can obtain mice or the rat of purification
Unit, then expects blue living cell counting number with 0.2%.After cell counting, join in T25 culture bottle and carry out
Cultivating, inoculum density is 5 × 105/ bottle, is placed in 37 degree, 5%CO2Incubator in carry out cultivating and observing and
Measure index of correlation.
The above embodiments and comparative example experiment, applicant carried out many experiments, and carried out index of correlation
Comparison and data statistics.As shown in table 1:
Table 1
Applicant uses mice to carry out testing several times simultaneously, and experimental result is identical with rat, it was demonstrated that the present invention
Method authentic and valid to the good result of the neuron separation and Culture of rat and mice.
In sum, the Primary mouse of the present invention or the isolated culture method of rat neuronal cell, use special
Compound enzyme carry out long-term ingestion so that obtaining cell total amount big, separating degree is high, and motility rate is high；Due to collection side
The difference of formula, the contamination probability pole of neuronal cell antibacterial, fungus and other cells that the method for the present invention obtains
Low, Secondary Culture can also be carried out simultaneously, provide good for the correlational study based on cultivating by neuron
Cell cultivates solution.
Being described in detail the specific embodiment of the present invention above, but it is intended only as example, the present invention is also
It is not restricted to particular embodiments described above.To those skilled in the art, any the present invention is carried out
Equivalent modifications and substitute the most all among scope of the invention.Therefore, without departing from the spirit of the present invention and model
Enclose lower made impartial conversion and amendment, all should contain within the scope of the invention.
Claims (10)
1. a Primary mouse or the isolated culture method of rat neuronal cell, it is characterised in that include following step
Rapid:
Step 1: mice or the pretreatment of rat brain tissue
After the cerebral tissue of mice or isolated rat is washed by PBS solution, it is put on ice for stand-by；
Step 2: take neuronal cell
Use biopsy needle to thrust the cerebral tissue after step 1 processes, cut neuronal cell；
Step 3: enzymic digestion
Neuronal cell step 2 obtained is placed in compound enzyme Digestive system digestion, and wherein compound enzyme Digestive system includes
Mass ratio is the type i collagen enzyme of 1:1:1:1, II Collagenase Type, IV Collagenase Type and Dispase enzyme；
Step 4: separation and Culture neuronal cell
Step 3 is digested complete Skeletal Muscle Cell and crosses 70um screen cloth, be centrifuged washing, then carry out resuspended also
Recentrifuge, hangs with pre-adhere-wall culture basic weight the most again, changes to neuron cultivation after the most pre-adherent 4h
Base, cell is positioned over 37 DEG C, 5%CO2Incubator in cultivate.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat neuronal cell, its
Being characterised by, the biopsy needle used in described step 2 is semi-automatic or full-automatic biopsy needle.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat neuronal cell, its
Being characterised by, in described step 3, in compound enzyme Digestive system, the concentration of each enzyme is 1mg/ml.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat neuronal cell, its
Being characterised by, in described step 3, enzymic digestion process is for using 4 DEG C of compound enzyme Digestive system digestion 4-20h.
A kind of Primary rat the most according to claim 1 or the isolated culture method of Mouse Gastric Mucous Membrane epithelial cell,
It is characterized in that, in described step 5, compound enzyme digestion process is digestion 10min-2h under the conditions of 37 DEG C.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat neuronal cell, its
Being characterised by, in described step 4, pre-adhere-wall culture base is the DMEM/F12 training containing Laminin and 10%FBS
Support base.
A kind of Primary mouse the most according to claim 6 or the isolated culture method of rat neuronal cell, its
Being characterised by, in described step 4, in pre-adhere-wall culture base, Laminin concentration is 1ug/ml.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat neuronal cell, its
Being characterised by, in described step 4, neuronal culture is the Neurobasal training containing B27, L-glutaminate
Support base.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat neuronal cell, its
Being characterised by, in described step 4, centrifugal condition is 400g, centrifugal 5 minutes.
10. one kind according to the Primary mouse described in any one in claim 1-9 or rat neuronal cell point
The mice cultivated from cultural method or rat neuronal cell.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610277888.2A CN105907717A (en) | 2016-04-28 | 2016-04-28 | Primary mouse or rat neuron isolation and culture method |
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| CN201610277888.2A CN105907717A (en) | 2016-04-28 | 2016-04-28 | Primary mouse or rat neuron isolation and culture method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113388581A (en) * | 2021-07-05 | 2021-09-14 | 中国人民解放军海军军医大学第一附属医院 | Primary cell culture method for sleeve gastrectomy mouse vagus nerve |
| CN118325838A (en) * | 2024-04-25 | 2024-07-12 | 中国农业大学 | Isolation and primary culture method of adult mouse stomach inter-muscular neurons |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1435187A (en) * | 2002-02-01 | 2003-08-13 | 北京科宇联合干细胞生物技术有限公司 | Neural stem cell preparation, preparing method thereof and use of same |
| CN102978162A (en) * | 2012-12-24 | 2013-03-20 | 黄柏胜 | Neuron separation and culture method and reagent |
| CN102994451A (en) * | 2012-12-24 | 2013-03-27 | 黄柏胜 | Improved method for separating and culturing neurons |
| CN104611294A (en) * | 2015-02-11 | 2015-05-13 | 中国人民解放军第三军医大学第一附属医院 | In-vitro culture method of visual cortex neuron |
-
2016
- 2016-04-28 CN CN201610277888.2A patent/CN105907717A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1435187A (en) * | 2002-02-01 | 2003-08-13 | 北京科宇联合干细胞生物技术有限公司 | Neural stem cell preparation, preparing method thereof and use of same |
| CN102978162A (en) * | 2012-12-24 | 2013-03-20 | 黄柏胜 | Neuron separation and culture method and reagent |
| CN102994451A (en) * | 2012-12-24 | 2013-03-27 | 黄柏胜 | Improved method for separating and culturing neurons |
| CN104611294A (en) * | 2015-02-11 | 2015-05-13 | 中国人民解放军第三军医大学第一附属医院 | In-vitro culture method of visual cortex neuron |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113388581A (en) * | 2021-07-05 | 2021-09-14 | 中国人民解放军海军军医大学第一附属医院 | Primary cell culture method for sleeve gastrectomy mouse vagus nerve |
| CN113388581B (en) * | 2021-07-05 | 2023-03-07 | 中国人民解放军海军军医大学第一附属医院 | Primary cell culture method for sleeve gastrectomy mouse vagus nerve |
| CN118325838A (en) * | 2024-04-25 | 2024-07-12 | 中国农业大学 | Isolation and primary culture method of adult mouse stomach inter-muscular neurons |
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