CN102994451A - Improved method for separating and culturing neurons - Google Patents
Improved method for separating and culturing neurons Download PDFInfo
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- CN102994451A CN102994451A CN2012105655161A CN201210565516A CN102994451A CN 102994451 A CN102994451 A CN 102994451A CN 2012105655161 A CN2012105655161 A CN 2012105655161A CN 201210565516 A CN201210565516 A CN 201210565516A CN 102994451 A CN102994451 A CN 102994451A
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Abstract
The invention provides an improved method for separating and culturing neurons. The method comprises the experimental steps of cerebral cortex tissue separation, trypsinization after mashing of histocytes, screen filtration and centrifugation, cell primary culture and neuron identification. The method has the advantages that the invention aims to improve the current method for separating and culturing neurons in vitro aiming at the problems in the prior art to enable the method to be simple and practicable; and the neurons obtained through culture grow well, have high purity and high yield and can meet the requirements of experiments of cell biology in neuroscience research.
Description
Technical field
The invention belongs to the animal cell culture technical field, be specifically related to a kind of neurone in-vitro separation and cultural method.
Research background
Neurone claims again neurocyte, is the fundamental unit that consists of the neural system structure and function.Neurone is the cell with long projection, and it is made of cell paste and cell process.At long aixs cylinder cover one deck sheath is arranged, form nerve fiber, the tiny branch of its end is called nerve ending.Cell paste is arranged in brain, spinal cord and neuroganglion, and cell process may extend in each Organ and tissue of whole body.Cell paste is the part that cell contains nuclear, and its shape size has very big difference, 4～120 microns of diameters.Nuclear is justified greatly, is positioned at cell central authorities, and chromatin is few, and kernel is obvious.Plaquelike chromidial substance (being once called as the Neil corpusculum) is arranged in the tenuigenin, also have many neurofibrils.Cell process is the elongated portion that is extended out by cell paste, can be divided into dendron and aixs cylinder again.Each neurone can have one or more dendron, and can accept stimulates and import excitement into cell paste.Each neurone only has an aixs cylinder, can be sent to another neurone or its hetero-organization to excitement from cell space, such as muscle or body of gland.
In nervous system development process, growth and cell that central nervous system has experienced cell quantity and volume are connected to form the change process.The neurone of differentiation just can not divide once generation again, and neuronic normal differentiation process is in a single day impaired, namely might affect a series of processes such as normal foundation of synaptic contact.To mechanism and the control thereof of neuronal damage, be the interested problems of people always.Along with the understanding to neuronal function, it becomes the focus of neurobiology area research gradually.But because neurone and other neurocyte and other histocytes mix existence in vivo, research and the application of its biological function have been hindered.Therefore need to carry out vitro culture and purifying to neurone, just can understand its morphological structure and biological function in depth.
Because neurone is a kind of well differentiated cell, division growth seldom after the animal birth, for other cell, neurone is at the external survival and growth that more is difficult to, and its cell space stretches out many not only long but also thin projections, easy its projection of damage in the process of drawing materials.In embryo development procedure, nervous system development early.After the birth, nervous system development is finished substantially, and a lot of neurones have grown projection this moment, and the cerebral tissue in this period that has drawn from is larger to neuronic damage.The neuron differentiation degree in embryo period is lower, and external viability is strong, and the contact between this neurone less in period, and the irreversible injury that causes in the process of drawing materials is less, is easy to successfully cultivate.
Cultural method and nutritional condition that the vitro culture neurone requires are all comparatively special.Present neuronic cultivation is divided into serum free culture system and serum-free culture, there is serum free culture system to provide cell cultures necessary nutritive ingredient, promote synthesizing of cell proliferation and DNA, but also for other neurocyte such as neurogliocyte, inoblast, neural stem cell provide abundant nutrition, but also so that the neurone of cultivating be difficult to separate.The serum-free culture technology is unfavorable for the growth of mitotic cell, can prevent the non-neuron hyper-proliferative, can improve the neurone purity of cultivation.Adopt the serum-free culture can be by some composition of interpolation, and optionally promote and control neuronic growth.
The patent documentation that has not yet to see relevant neurone in-vitro separation or cultural method is open, although neurone adopts conventional cell culture technology, can reach to a certain extent the purpose of cultivation, but exist and neurogliocyte, the problems such as difficulty that other heteroproteose cell such as inoblast is difficult to separate and vitro culture is survived can not satisfy the requirement of cell experiment.This chamber is with reference to the domestic and international neuronic method of culture of isolated.According to experimental study for many years, improved the extracorporeal culturing method of cerebral cortex neurons, set up a kind of neuronic method of stablizing, obtain reliably, for understanding neuronic structure and function, illustrate its effect research in cerebrovascular disease, nervous system disorders successful experimental model is provided.
Summary of the invention
The object of the invention is to the problem for the prior art existence, improve current in-vitro separation and cultivate neuronic method, make it simple, it is good to cultivate the neure growth that obtains, purity is high, and output is large, can satisfy the demand of Cell Biology Experiment in the Neuroscience Research.
Adopt following scheme to realize purpose of the present invention.A kind of in-vitro separation and cultivate neuronic method contains pallium separate tissue, histocyte and smashs 4 experimental procedures such as rear trysinization, screen filtration are centrifugal, the former culture of cell to pieces.Can comprise in addition the step that neurone is identified.
The pallium separate tissue
Pregnant mouse cervical vertebra dislocation causes death, and takes out the embryo under the aseptic condition, then gets tire mouse both sides hemicerebrum and put into culture dish under aseptic condition, peels off and remove cortex vascular tissue and pia mater.
Preferred version is: pregnant 15-17 days rat cervical vertebra dislocation causes death, the sterilization of 75% ethanol belly, scissors is cut off belly to both sides, take out the beading embryo under the aseptic condition, put into the culture dish that fills 1 * PBS, then sled is got tire mouse both sides hemicerebrum and is put into the culture dish that fills 1 * PBS in aseptic super clean bench, place in the culture dish that fills 1 * PBS with ophthalmic tweezers gripping animal bilateral pallium, carefully peel off and remove cortex vascular tissue and pia mater, pallium is moved in another culture dish that fills 1 * PBS again.
It should be noted that the dissection process must omnidistance carry out on ice.After purpose was to put to death female mouse from you, the temperature of the brain of tire mouse will be the fastest was reduced to 0 degree, helps to improve neuronic survival rate.In addition, in the dissection process, the tire mouse is until the process that cortex or hippocampus are shredded sometimes may need 2 hours, if sample is many.Must note ice bag of preparing more.Guarantee that your any process all carries out (except the digestion step) in the nutrient solution of ice bath.Can greatly improve like this survival rate of neuronal cell.When dissecting brain, human hank ' s liquid is arranged, dissect therein.Even but 0 degree, neurone is still carrying out largely metabolism.So the sugar-free environment of hank ' s liquid is very unfavorable.Suggestion is soaked brain in the dissection process with the high sugar of DMEM-or DMEM-F12 and horse serum, supplies with the metabolism of brain, and serum can suppress neural apoptosis.This wherein also has a problem, is exactly that the DMEM nutrient solution can become alkalescence in air.The use for laboratory L-15 nutrient solution that abroad has soaks the brain in the dissection process, because L-15 does not become alkalescence in the air again.
Histocyte is smashed rear trysinization to pieces
Under the aseptic condition, smash cerebral tissue to pieces and be rotten shape, then add the pancreatin of lower concentration, put 37 ℃ of incubator digestion 5-30 minute.Then cultivate first liquid with neurone and stop digestion, and continue to add first liquid and blow and beat gently, dispel until will organize all, form cell suspension.
Preferred version is: under the aseptic condition, be caught broken cerebral tissue with ophthalmic tweezers and be rotten shape, add concentration 0.1-0.125% pancreatin 2ml, 37 ℃ of incubators digested 10 minutes.After cultivating first liquid and stop digestion with neurone, continue to add neurone and cultivate first liquid and blow and beat gently, dispel until will organize all, form cell suspension.
It should be noted that except adopting pancreatin, can also adopt the method for papain combined DNA enzyme to digest.Papain be digestion after, the enzyme that survival rate is the highest.Papain is made into 1-3mg/ml concentration and digests, and adding simultaneously concentration is the DNA enzyme of 1-3mg/ml, each 1-2ml of add-on, and digestion time is 20-30 minute.The effect of DNA enzyme is, digests the DNA of ruptured cell, and DNA and the albumen having avoided discharging become entangled in the tissue block surface and hinders further digestion.Papain and DNA enzyme need now with the current in use.
The neurone that this programme is mentioned is cultivated first liquid formula:
| Foetal calf serum | 10% |
| The 200uM L-glutaminate | 1% |
| Two anti- | 1% |
| Neurobasal | 88% |
The neurone that this programme is mentioned is cultivated the solution that can be mixed with 50ml when first liquid is specifically used, and its prescription is:
| Foetal calf serum | 5.0ml |
| The 200uM L-glutaminate | 0.5ml |
| Two anti- | 0.5ml |
| Neurobasal | 44ml |
The neurone that this programme is mentioned is cultivated the maintenance medium prescription:
| The 200uM L-glutaminate | 1% |
| Two anti- | 1% |
| B27 | 2% |
| Neurobasal | 96% |
The neurone that this programme is mentioned is cultivated the solution that can be mixed with 50ml when maintenance medium is specifically used, and its prescription is:
| The 200uM L-glutaminate | 0.5ml |
| Two anti- | 0.5ml |
| B27 | 1.0ml |
| Neurobasal | 48ml |
The Neurobasal that mentions in this prescription is a kind of commercial substratum, has another name called the Neurobasal serum free medium, is to aim at the particular requirement of satisfying neuronal cell cultures and the basic medium of developing, and all can buy in conventional reagent company.What this programme used is the Neurobasal commercially available reagent of (1 *) concentration.But normal phenotype and the growth of this substratum long term maintenance neurocyte do not need the stellate cell trophoderm can keep highly purified neuronal cell population yet.The Neurobasal substratum is applicable to the long-term cultivation of fetus hippocampal neuron and other multiple axoneurons.The Neurobasal-A substratum is applicable to long-term cultivation and vigor newborn and the interior hippocampus of Central Nervous System of The Adult Rat and cortical neuron and keeps.
The B27 that mentions in this prescription also is a kind of commercial nerve growth additive, has another name called B27 Neuro Mix.Contain the required different somatomedins of neurocyte, specific antioxidant and the required special fatty acid of neurone.The important growth additive of cultivating neurocyte, and serum substitute.All can buy in conventional reagent company.What add in this programme is the B27 commercially available reagent of (50 *) concentration.Two anti-concentration in this prescription is Streptomycin sulphate 100U/ml, penicillin 100 U/ml.
Screen filtration is also centrifugal
After cell suspension filtered with micro-strainer, change in the centrifuge tube centrifugation over to.
Preferred version is: after cell suspension is filtered with 200 order micro-strainers, changes in the 15ml centrifuge tube, and centrifugal, 1000 rev/mins, 5 minutes.Practice shows that 200 order micro-strainers can effectively come neuronal cell and other impurity cellular segregation, play the purpose of initial gross separation, and the filter screen in other specification apertures are difficult to reach its effect.
Former culture
Centrifugal supernatant liquor is discarded, cultivate the resuspended centrifugation of first liquid with neurone after, be inoculated in the porous culture plate, put in the incubator and cultivate.Change liquid after 6-24 hour and use neurone cultivation maintenance medium instead, changed maintenance medium 1 time in later every 1-4 days, each half amount or full dose are changed liquid, can identify in Growth of Cells 3-10 days, are used for afterwards testing in 4-10 days.
Preferred version is: centrifugal supernatant liquor is discarded, cultivate the resuspended culture dish of putting into of first liquid with the 2ml neurone, adjusting cell density is 5 * 10
5Individual/ml, be inoculated in 24 holes of using in advance poly-lysine (PDL) coated, 96 well culture plates, insert 37 ℃, 5%CO
2Cultivate in the CO2gas incubator.All use the neurone that contains B27 after 12 hours instead and cultivate maintenance medium, full dose is changed the i.e. each 500 μ l/ holes of 24 orifice plates of liquid, 96 orifice plates, 100 μ l/ holes first, changed maintenance medium 1 time in per 2 days later on, each half amount is changed liquid, and Growth of Cells can be identified in 5 days, was used for afterwards testing in 6 days.
We study the porous culture plate that find to adopt take 6 orifice plates as best in addition, and namely 6 orifice plates of common orifice bore diameter 6cm are best.Neurone is cultivated and is allowed the problem of people's worries be, the situation of neuronal development is closely related with its density.Greater than the hole meeting of 6cm so that middle be difficult to the same with cell density on every side, cause in the middle of the plate and the edge ripening degree different, and this understands and bring very large interference to experiment.And too little words (96 holes or 128 holes) cell can tend to the centre to concentrate also can shine into grow inhomogeneous.So through groping to show that 6 orifice plates are optimal selections, adopt 6 orifice plate inoculating cells can regulate cell density.The orifice plate used of neuron culture must be coated with in advance in addition, and is commonly used with poly-lysine or collagen.Otherwise cell can't be adherent.
Neurone is identified
Employing Tuj1 antibody carries out the neurone in immunofluorescence experiment (or immunocytochemistry experiment) and the employing DAPI fluorescence dye evaluation culturing cell.
Cytoskeleton comprises microfilament, fiber in the intermediate filament, three kinds of cells of microtubule.Microtubule is the non-model structure that exists in all eukaryotic cells, and it is the cellularstructure that is assembled into tubulose by tubulin.Adopt Tuj1 antibody to carry out immunofluorescence experiment (or immunocytochemistry experiment) and DAPI fluorescence technique, visible significantly special neural network structure, as shown in Figure 3.Prove that this scheme is practical.
Tuj1 antibody has another name called Neuronal Class III β-Tubulin(neurone III beta tubulin) antibody, be with the tubulin that extracts in the rat brain as antigen, at last screening, the monoclonal antibody that obtains of purifying.Tuj1 antibody has extensively been confirmed only to identify the Neuronal Class III β-Tubulin of neurocyte, and the β of nonrecognition spongiocyte-Tubulin(β microtubule).Tuj1 antibody is used for the micro-tubular structure of neuronic immunostaining observation of cell, dyes micro-tubular structure and is green such as Fig. 1;
DAPI is 4', 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole) be a kind of fluorescence dye, can the double-stranded DNA of permeates cell membranes in nucleus be combined and bring into play the effect of mark, can produce the fluorescence stronger more than 20 times than DAPI self, compare with EB, its sensitivity of dyeing of double-stranded DNA is wanted high doubly a lot.Microscopically can be seen the cell of aobvious blue-fluorescence, the efficient of fluorescence microscope cell marking high (being almost 100 %), and viable cell had no side effect.DAPI dyeing is usually used in apoptosis and detects, and detects with fluorescence microscope or flow cytometer after the dyeing.DAPI also is usually used in common nucleus dyeing and the double-stranded DNA dyeing under some particular case.It is blue such as Fig. 2 that transfect cell nuclear is; Fig. 3 is that Fig. 1 and Fig. 2 merging form, visible significantly special neural network structure.
The present invention has following characteristics and advantage than prior art: 1) adopt the trysinization of short period of time, lower concentration during cellular segregation, reduced trysinization excessively to the damage of cell; 2) screen filtration is dispersed into individual cells with cell mass; 3) cultivate neurone with the special culture medium that contains Neurobasal and B27, the sufficient nutrient composition can be provided, alternative promotes neure growth and promotes that a little cell differentiation of nerve cord is grown formation neurone in the nervous tissue again, and the especially growth of neurogliocyte of energy establishment non-neuronal cell; 4) because the rear nervous system development of being born is finished substantially, and a lot of neurones have grown projection this moment, the cerebral tissue in this period that has drawn from is larger to neuronic damage, therefore tests the tire murine brain with pregnant 15-17 days, and is less to neuronic damage when drawing materials; 5) making the cover glass of cell climbing sheet must be coated with poly-lysine, otherwise that neurocyte is difficult for is adherent, also can fall sheet in the dyeing course; When 6) drawing materials pallium vitro culture neurone, divest the meninx cerebrovascular, to improve neurocyte purity, simultaneously, whole operating on the sled carried out as far as possible, and The faster the better for process, is beneficial to the vigor that keeps cell; 7) neurone cultivate first liquid and maintenance medium now with the current; 8) former culture neurone to the just can begin for experiment in 6 days; 9) aspect inhibition spongiocyte hyper-proliferative, this the research method of serum-free culture, do not use the cell division inhibitor cytosine arabinoside, itself also has in various degree restraining effect to neuronic growth, also because of the time point that adds cytosine arabinoside with and the short selection of long action time hold the inaccurate neuronal damage that may cause.
This improved neurone cultural method and experiment parameter are to grope to obtain according to experimental study for many years, and the neurone that adopts the method to obtain identifies that through immunohistochemical staining its purity reaches more than 90%, meets requirement of experiment fully.In addition, in the neurone culturing process, need to add a certain amount of mitotic inhibitor cytosine arabinoside, cytosine arabinoside can be suppressed to the propagation of fibrocyte and neurogliocyte preferably, but neuronic growth is had in various degree restraining effect.Cytosine arabinoside adds can cause a large amount of nerve cell apoptosis or necrosis too early, and the neurone productive rate is lower; If cytosine arabinoside used slow, existing a large amount of non-neuronal cell propagation reduces the neurone proportion; We have adopted the neurone of the Neurobasal serum-free that is added with B27 to cultivate maintenance medium in experiment, optionally promote neure growth.Serum-free medium is unfavorable for the growth of mitotic cell, prevent the non-neuronal cell hyper-proliferative, the neurone of cultivating meets requirement of experiment fully, its reliability scientific and experimental result belongs to leading domestic level, for neuroscience section research provides basic experimental model, for the factors such as nervous cell regenerating functional study and anoxic, medicine provide basic experimental model to the research of neurone impact.The neurone that my chamber adopted this method to cultivate has in the past found that to its functional study many known and unknown genes stress considerable change occur by Expression In The Process.
Experimental results show that present method can be used for the relatively fragile tissue such as tire mouse, new life's (being born 1 day) rat, mouse, cavy, the cultivation of cerebral cortex neurons etc., experimental program is consistent.But human for growing up, because brain cortical tissue is tougher, be difficult to achieve the goal with this method.But experiment shows the brain cortical tissue of the induced labor foetus of drawing materials, and adopts present method also can reach identical effect, and is the most suitable with gestational age 4 months.
Should note following operation in the experiment: when the cerebral tissue that draw materials (1) is used trysinization, hold the degree of digestion, can not digest excessively, can not digest again not exclusively, be generally less than 10 minutes, should be immediately cultivate first liquid with neurone and stop digestion, avoid digestion excessively and first vigor that affects the nerves, cause the cell quantity deficiency when incomplete and digest; (2) the piping and druming action wants soft, and better with the suction pipe piping and druming effect that capillary pipet piping and druming is thick than bore, the piping and druming number of times is suitable with about 20 times, otherwise can cause the neurone mortality.To slowly get liquid, liquid feeding along wall when (3) changing liquid, and change the amount of nutrient solution according to cell growth state and cell quantity.
Description of drawings
Fig. 1 is that laser confocal microscope amplifies 630 times of immunohistochemical stainings evaluations (it is green that micro-tubular structure is) of observing the neurone culturing cell
Fig. 2 is that laser confocal microscope amplifies 630 times of immunohistochemical stainings evaluations (transfect cell nuclear is blue) of observing the neurone culturing cell
Fig. 3 is that laser confocal microscope amplifies 630 times of immunohistochemical stainings evaluations (being formed by Fig. 1,2 merging) of observing the neurone culturing cell
Embodiment
Following content is the specific embodiment of a kind of neurone cultural method of the present invention, so that those skilled in the art can better understand the present invention and can be implemented.But need to prove, the inventive method never be confined to for embodiment.
Embodiment one: the cultivation of neurons of rats
(1) laboratory animal: 1 of the pregnant mouse of pregnant 15～17 days SD (the Xiangya Medical College, Zhongnan Univ Experimental Animal Center provides).
(2) main agents and instrument: Neurobasal (invitrogen company), trypsin AMERESCO company), foetal calf serum (Hangzhou folium ilicis chinensis company), neurone is cultivated first liquid (FBS, L-glutaminate, two anti-, Neurobasal prepares according to a certain percentage, now with the current), neurone is cultivated maintenance medium (L-glutaminate, two anti-, B27, Neurobasal prepares according to a certain percentage, now with the current), Tuj1 monoclonal antibody (Sigma company), DAPI (Sigma company), the rabbit anti-mouse igg of FITC mark (Vector company), PBS liquid (by the prescription self-control), L-glutaminate (gibco company), poly-lysine (the green skies), CO
2Incubator (FormaScientific company), inverted microscope (Olympus company), laser confocal microscope (LEICA company).
(3) former culture: get the pregnant mouse of pregnant 15～17 days SD, the cervical vertebra dislocation causes death.With the pregnant mouse belly of 75% ethanol disinfection, scissors is cut off belly to both sides, take out 5 of beading embryos, put into the culture dish that fills 1 * PBS liquid, after carefully cutting off skull with eye scissors under the aseptic condition, taking out the bilateral pallium with ophthalmic tweezers again places in the another one culture dish that fills 1 * PBS liquid, carefully peel off meninx and vascular tissue, again pallium is moved in another culture dish that fills an amount of 1 * PBS liquid, suck liquid as far as possible, be caught broken pallium with ophthalmic tweezers and be rotten shape, add 0.125% pancreatin 2ml, 37 ℃ of incubators digested 10 minutes.After cultivating first liquid and stop digestion with the 2ml neurone, continue to add neurone and cultivate first liquid and blow and beat gently, dispel until will organize all, form cell suspension.After the filtration of 200 order micro-strainers, change in the 15ml centrifuge tube, centrifugal, 1000 rev/mins, 5 minutes.Abandon supernatant, cultivate first liquid re-suspended cell with the 2ml neurone and put into culture dish, adjusting cell density is 5 * 10
5Individual/as ml) to be inoculated in advance with in 24 coated holes of PDL, 96 well culture plates, insert CO
2Cultivate in the incubator.All use the neurone that contains B27 after 10 hours instead and cultivate maintenance medium, full dose is changed the i.e. each 500 μ l/ holes of 24 orifice plates of liquid, 96 orifice plates, 100 μ l/ holes first, change later on neurone in per 2 days and cultivate maintenance medium 1 time, each half amount is changed liquid, and Growth of Cells can be identified in 5 days, was used for afterwards testing in 6 days.
(4) neuronic evaluation: Tuj1 antibody has extensively been confirmed only to identify neuronic Neuronal Class III β-Tubulin, and the β-Tubulin of nonrecognition spongiocyte.Tuj1 antibody is used for the micro-tubular structure of neuronic immunostaining observation of cell, dyes micro-tubular structure and is green; DAPI be a kind of can with the fluorescence dye of the powerful combination of DNA, can see through complete cytolemma, transfect cell nuclear is blueness.Primary neuron is pressed 5.0 * 10
5The density of individual/ml is planted in 24 well culture plates, places in advance the cover glass of coated PDL in the hole, 37 ℃, 5%CO
2Incubator is hatched, and changes liquid by above-mentioned different time, after 5 days, carries out fluorescent immunohistochemistry and identifies, fluorescence microscopy Microscopic observation dyeing situation is taken pictures under the laser confocal microscope.
(5) result:
Former culture result: when just inoculating, cell is rounded, and without projection, cell space is transparent.Cultivate 12h, cell is substantially adherent, and cell space is rounded or oval, and volume is little, and cell space is shinny, the visible significantly halation of periphery, and stereoscopic sensation is strong, and cell stretches out short and small projection.Cultivated 1 day, cell process increases, and its length is about 1 ~ 2 times of cell space.Cultivated 3 days, cell space obviously increases, rounded, ellipse or Polygons, and full, refractivity and stereoscopic sensation are strong, and projection rises appreciably, and form is various, visible single-stage, bipolar, multipole projection, more projection interconnects.When cultivating 5 days, neurosome begins to assemble, and projection links to each other and is the growth of network sample.Cultivated 6 days, cell space is assembled more obvious.Along with the prolongation of incubation time, Neuronal processes is crisscross, the origin of almost illegible projection.
Neuronic evaluation: cytoskeleton comprises microfilament, fiber in the intermediate filament, three kinds of cells of microtubule.Microtubule is the non-model structure that exists in all eukaryotic cells, and it is the cellularstructure that is assembled into tubulose by tubulin.Tuj1 antibody has extensively been confirmed only to identify the Neuronal Class III β-Tubulin of neurocyte, and the β-Tubulin of nonrecognition spongiocyte.Tuj1 antibody is used for the micro-tubular structure of the immunostaining observation of cell of neurocyte, dyes micro-tubular structure and is green such as Fig. 1; DAPI be a kind of can with the fluorescence dye of the powerful combination of DNA, can see through complete cytolemma, transfect cell nuclear is blueness such as Fig. 2; In the visible significantly special neural network structure of Fig. 3.Owing to cultivate 6 days later neurocyte, the neurone positive rate increases, but its cell space begins to assemble, even projection intermeshes, the origin of almost illegible projection, the neuron morphology of stained positive is not true to type, dye so get the cell of cultivating 5 days, find relatively typical case of neuronic form, positive rate is also higher.Identify that with immunohistochemical staining neurone purity reaches more than 90%, can satisfy the requirement of neurobiology experiment, so think that by the method cultured cortex neurons be feasible.(in former culture, adopt papain combined DNA enzyme to digest to replace trysinization, also can reach identical effect.Papain be digestion after, the enzyme that survival rate is the highest.Papain is made into 2.5mg/ml concentration and digests, and adding simultaneously concentration is the DNA enzyme of 2mg/ml, and digestion time is 20-30 minute.）
Embodiment two: other animal nerve units cultivate
Experimental results show that present method can be used for the relatively fragile tissue such as tire mouse, new life's (being born 1 day) rat, mouse, cavy, the cultivation of cerebral cortex neurons etc., experimental program is the same.But human for growing up, because brain cortical tissue is tougher, be difficult to achieve the goal with this method.But experiment shows the brain cortical tissue of the induced labor foetus of drawing materials, and adopts present method also can reach identical effect, and is the most suitable with induction of labour in second trimester gestational age 4 months, the too small or excessive experimental result that all affects of gestational age.Adopt the neurone purity of present method separation and Culture to reach more than 90%.
Claims (8)
1. a neurone separates and cultural method, and it may further comprise the steps: the pallium separate tissue; Histocyte is smashed rear enzymic digestion to pieces; Screen filtration and centrifugal step; And the former culture of cell; It is characterized in that: the step of the former culture of cell is: centrifugal supernatant liquor is discarded, cultivate the resuspended culture dish of putting into of first liquid with the 2ml neurone, adjusting cell density is 5 * 105/ml, is inoculated in advance with cultivating in coated 24 holes of poly-lysine or 96 well culture plates;
Cultivate and change liquid after 12 hours and use neurone instead and cultivate maintenance medium, full dose is changed liquid first;
Changed maintenance medium 1 time in per 2 days later on, each half amount is changed liquid;
Growth of Cells can be identified in 5 days, was used for afterwards testing in 6 days.
2. a kind of neurone according to claim 1 separates and cultural method, it is characterized in that: to change liquid long-pending be the each 500 μ l/ holes of 24 orifice plates or 96 orifice plates, 100 μ l/ holes to full dose first.
3. described a kind of neurone separates and cultural method according to claim 1-2, and it is characterized in that: described pallium separate tissue step is:
Pregnant 15-17 days rat cervical vertebra dislocation is caused death, the sterilization of 75% ethanol belly, scissors is cut off belly to both sides, take out the beading embryo under the aseptic condition, put into the culture dish that fills 1 * PBS, then sled is got tire mouse both sides hemicerebrum and is put into the culture dish that fills 1 * PBS in aseptic super clean bench, place in the culture dish that fills 1 * PBS with ophthalmic tweezers gripping animal bilateral pallium, carefully peel off and remove cortex vascular tissue and pia mater, pallium is moved in another culture dish that fills 1 * PBS again.
4. described a kind of neurone separates and cultural method according to claim 1-2, and it is characterized in that: described histocyte is smashed rear enzymatic digestion stage to pieces and is:
Under the aseptic condition, be caught broken cerebral tissue with ophthalmic tweezers and be rotten shape, add concentration 0.1-0.125% pancreatin 2ml, 37 ℃ of incubators digested 10 minutes; After cultivating first liquid and stop digestion with neurone, continue to add neurone and cultivate first liquid and blow and beat gently, dispel until will organize all, form cell suspension.
5. a kind of neurone according to claim 1 separates and cultural method, and it is characterized in that: histocyte is smashed the step of rear enzymic digestion to pieces, adopts the method for papain combined DNA enzyme to digest.
6. a kind of neurone according to claim 5 separates and cultural method, and it is characterized in that: histocyte is smashed the step of rear enzymic digestion to pieces: the papain concentration of adding is 1-3mg/ml, and add-on is 1-2ml.
7. a kind of neurone according to claim 5 separates and cultural method, and it is characterized in that: histocyte is smashed the step of rear enzymic digestion to pieces: the DNA enzyme concn of adding is 1-3mg/ml, and add-on is 1-2ml.
8. a kind of neurone according to claim 5 separates and cultural method, and it is characterized in that: histocyte is smashed the step of rear enzymic digestion to pieces: digestion time is 20-30 minute.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103789265A (en) * | 2014-02-21 | 2014-05-14 | 刘洛贤 | Method for efficiently separating and culturing hippocampal 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 |
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| CN113652403A (en) * | 2021-09-15 | 2021-11-16 | 复旦大学附属中山医院 | Primary neural cell plating method for high content drug screening |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789265A (en) * | 2014-02-21 | 2014-05-14 | 刘洛贤 | Method for efficiently separating and culturing hippocampal neurons |
| CN103789265B (en) * | 2014-02-21 | 2016-08-17 | 刘洛贤 | High efficiency separation and the method for hippocampal neuron |
| 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 |
| CN105695411A (en) * | 2016-03-22 | 2016-06-22 | 中国人民解放军第二军医大学 | Naked mole rat cortical neuron culture method |
| CN105754943A (en) * | 2016-03-22 | 2016-07-13 | 中国人民解放军第二军医大学 | Culture method of naked mole rat hippocampal neurons |
| CN105754943B (en) * | 2016-03-22 | 2019-07-02 | 中国人民解放军第二军医大学 | A kind of naked mole cultured hippocampal neuron method |
| CN105695411B (en) * | 2016-03-22 | 2019-07-02 | 中国人民解放军第二军医大学 | A kind of naked mole cortical neuron cultural method |
| CN105907717A (en) * | 2016-04-28 | 2016-08-31 | 王晓冰 | Primary mouse or rat neuron isolation and culture method |
| CN113652403A (en) * | 2021-09-15 | 2021-11-16 | 复旦大学附属中山医院 | Primary neural cell plating method for high content drug screening |
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