CN104560875A - Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method - Google Patents
Method for separating and culturing trigeminal root ganglion neurons of mammal in vitro and application of method Download PDFInfo
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Abstract
The invention discloses a method for separating and culturing trigeminal root ganglion neurons of a mammal in vitro and application of the method, and belongs to the technical field of cell biology and neurobiology. The method comprises the following steps: placing the trigeminal root ganglion neurons of the mammal in a culture dish containing L-15 culture solution for washing treatment, cutting the trigeminal root ganglion neurons into pieces and then carrying out digestion treatment with type 11 collagenase, trypsin, and trypsin-EDTA in sequence, filtering with a cell strainer and then carrying out density gradient centrifugation for the filtrate, suspending and inoculating the obtained product to a wrapped cell culture device, and culturing with a basic neuronal medium. The method greatly reduces the damage to the neurons in the tissue processing process, removes the impurity contamination in the digestion process like cell debris and other cells, greatly improves the purity of the neurons, can obtain a million of pure trigeminal ganglion neurons, improves the yield and the quality of the trigeminal ganglion neurons, and is suitable for the in-vitro separation and culturing of the trigeminal root ganglion neurons of the mammal.
Description
Technical field
The present invention relates to a kind of In-vitro separation culture method and application of Mammals trigeminal ganglion neuron, belong to cytobiology and neurobiology technical field.
Background technology
Neuronic separation and Culture is the common technology of research Neuronal Growth Factor, neurite outgrowth, differentiation and esthesiophysiology etc.Although the neurone be separated before embryo or new born animal neurocyte formation cynapse wants relatively easy, Fetal neurons has different characteristics from mature neuron in electrophysiology, growth, regeneration etc.Therefore the cultivation of ripe neurocyte is very important in a lot of research.But support in process in culture of primary neurons and there is cell yield and the not high problem of quality always.
At present, existing trigeminal nerve cell primary culture technique mostly is collagenase digestion, and it is in culturing process, and some details is considered comprehensive not, as: there is no CO
2during balance regulator, the stability of nutrient solution is bad, and the impact for the treatment of processes on tissue or cell is comparatively large, comparatively large to cell injury, and digestion is thorough not, and contaminating cell is more, and cultivate the problems such as gained cell is impure, therefore the seed output and quality of cell is not high.
Summary of the invention
For solving the deficiencies in the prior art, the invention provides a kind of In-vitro separation culture method and application of Mammals trigeminal ganglion neuron, the technical scheme of employing is as follows:
The object of the present invention is to provide a kind of In-vitro separation culture method of Mammals trigeminal ganglion neuron, the method is placed in by Mammals trigeminal nerve tissue on the culture dish containing L-15 nutrient solution to carry out clean, digestion process is carried out with 11 Collagenase Types, trypsinase and trypsinase-EDTA successively after shredding, after the sieved filter of cell, density gradient centrifugation is carried out to filtrate, be inoculated in bag after suspension by good cell culture apparatus, cultivate with base neural substratum.
Described method steps is as follows:
1) the Mammals trigeminal nerve tissue of gained is placed on the culture dish containing L-15 nutrient solution and shreds after cleaning, obtain pre-treatment trigeminal nerve tissue;
2) by step 1) gained pre-treatment trigeminal nerve organizes and carries out digestion process with 11 Collagenase Types, trypsinase and trypsinase-EDTA successively, through neutralization, centrifugal and after suspending with the sieved elimination of cell except fragment of tissue, obtain cell suspending liquid;
3) step 2) gained cell suspending liquid, carries out density gradient centrifugation to filtrate, removes cell debris and heteroproteose cell, obtain purifying cells;
4) by step 3) the base neural substratum suspension of gained purifying cells, be then inoculated into and wrapped by good cell culture apparatus, cultivate with base neural substratum in incubator, cultivate 24-48h and obtain trigeminal ganglion neuron.
Step 1) described Mammals is Mammals except embryonic stage and introduction stage.
Preferably, step 1) described Mammals be 8-12 week age rat.
Step 2) described digestion process process is: (1) is by step 1) 200U 11 Collagenase Type that the pre-treatment trigeminal nerve tissue 5mL of gained dilutes through L-15 nutrient solution digests 90 minutes at 37 DEG C, during digestion, shake in every 10-15 minute once, enables collagenase fully and equably digest tissue; (2) concentration adding 500 μ L in mixture is the trypsinase of 0.25%, continues digestion 20 minutes in 37 DEG C of water-baths, the tissue of collected after centrifugation digestion and cell; (3) add the trypsinase-EDTA that 5mL dilutes through L-15 nutrient solution, after shaking up, continue digestion 20 minutes in 37 DEG C of water-baths; (4) add 10mL containing the neurobasal media of 10% foetal calf serum, in and digestive ferment, be centrifugally precipitated cell; (5) add the neurobasal media of 200 μ L containing 2%B27, suspension cell, then use the sieved filter cell suspension of the cell in 200 μm of apertures, carry out flushing cell sieve with the 2%B27 neurobasal media that contains of 2mL, obtain cell suspending liquid.
Step 3) described density gradient centrifugation is Optiprep density gradient centrifugation, process is: by OPTI and Nb+B27 nutrient solution successively according to 173:827, the volume ratio of 124:876,99:901,74:926 mixes, obtain four density gradient liquid, successively it is slowly added in the centrifuge tube of 15mL in order, then 2mL cell suspending liquid is slowly added to above density gradient liquid, the centrifugal 15min of 800g, discard two-layer liquid, collect remaining liq.
Step 4) described bag by culture apparatus be cultivate the day before yesterday, in culture apparatus, add aseptic Poly-L-Lysine Solution, wrap by 30 minutes, the poly-lysine that sucking-off is unnecessary, spending the night in Biohazard Safety Equipment, it is for subsequent use to dry.
Step 4) described suspension adds 10mL base neural substratum, the centrifugal 5min of 1000rpm, suspend with the neural nutrient solutions of 100 μ L after collecting cell;
The condition of described cultivation is 37 DEG C, 5%CO
2and saturated humidity.
Described method concrete steps are:
1) take out the trigeminal nerve of rat, be placed on the culture dish containing L-15 nutrient solution, then carry out clean, remove its hetero-organization sticked, shredded with eye scissors or scalpel, obtain pre-treatment trigeminal nerve tissue;
2) by step 1) gained pre-treatment trigeminal nerve tissue 5mL 200U 11 Collagenase Type of obtaining after the dilution of L-15 nutrient solution digests 90 minutes at 37 DEG C, during digestion, shake in every 10-15 minute once, enables collagenase fully and equably digest tissue;
3) to step 2) to add 500 μ L concentration in gained mixture be the trypsinase of 0.25%, continues digestion 20 minutes in 37 DEG C of water-baths, centrifugal, collect tissue and the cell of digestion, outwell supernatant liquor;
4) to step 3) add the trypsinase-EDTA that 5mL obtains after the dilution of L-15 nutrient solution in gained tissue and cell, shake up rear 37 DEG C of water-baths and continue digestion 20 minutes, add the neurobasal media of 10mL containing 10% foetal calf serum, in and digestive ferment, centrifugally be precipitated cell, discard supernatant liquor;
5) to step 4) add the neurobasal media of 200 μ L containing 2%B27 in gained sedimentation cell, suspension cell;
6) with the cell sieve filtration step 5 in 200 μm of apertures) gained suspension, with sieving containing 2%B27 base neural substratum flushing cell of 2mL, obtain cell suspending liquid;
7) by OPTI and Nb+B27 nutrient solution successively according to 173:827,124:876,99:901, the volume ratio of 74:926 mixes, and obtains four density gradient liquid, is slowly added in the centrifuge tube of 15mL by each gradient liquid successively in order, then 2mL cell suspending liquid is slowly added to above density gradient liquid, the centrifugal 15min of 800g, discards two-layer liquid, collects remaining liq;
8) to step 7) add 10mL base neural substratum, the centrifugal 5min of 1000rpm, collecting cell in gained liquid, suspend with the neural nutrient solutions of 100 μ L;
9) by step 8) be inoculated into step 9 gained uniform suspension) gained wrap in the cell culture apparatus of quilt, in incubator with base neural cultivate based on 37 DEG C, 5%CO
2, after cultivating 24-48h under the condition of saturated humidity, obtain trigeminal ganglion neuron;
The described cell cultures based devices having wrapped quilt, be in culture apparatus, add aseptic Poly-L-Lysine Solution in the day before yesterday of cultivating, processed 30 minutes of bag, wash out unnecessary poly-lysine, spending the night in Biohazard Safety Equipment, it is for subsequent use to dry.The method of the invention is applicable to the Isolation and culture of Mammals trigeminal ganglion neuron.Beneficial effect of the present invention:
1. present invention optimizes the digestion method of tissue, ban original protease digestion process, adopt collagenase and trypsinase simultaneous digestion, its digestive process and digestion time can make tissue digestion more thorough, do not use the mode of pressure-vaccum to carry out further cell dispersion simultaneously, the damage of cell is reduced greatly.
2. the present invention to clean outside organizer, digest and isolated cell process in employ L-15 substratum, can ensure there is no CO
2the stability of nutrient solution under the condition of balance regulator, even if provide one without CO
2balance adjustment, also can ensure to stablize, damage very little liquid environment to cell or tissue, stabilize the outside culture condition such as osmotic pressure, pH value, substantially reduces the impact on tissue or cell in treating processes.
3. the many processes of not taking out impurity of existing culture technique, first the present invention filters tissue after digestion, remove bulk impurity, the method enrichment of cell of recycling density gradient centrifugation, eliminate the interference of the impurity such as a large amount of cell debriss and other cell types produced in digestive process, make cultivation gained cell purer, having higher success rate of cell cultures.
4. utilize cultural method of the present invention can obtain about 1,000,000 purer trigeminal nerve cells, improve cell yield and quality, solve the deficiencies in the prior art, the research of each side such as pharmacology, electrophysiology, growth, regeneration and neurotoxicology can be met.
Accompanying drawing explanation
Fig. 1 trigeminal nerve cell phase microscope figure.
Fig. 2 trigeminal nerve cell fluorescence phase microscope figure.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not by the restriction of embodiment.
Embodiment 1:SD rat trigeminal cellular segregation is cultivated
1. bag is by culture plate (ware)
The day before yesterday of cultivating, add appropriate (covering at the bottom of ware) commercially available aseptic Poly-L-Lysine Solution in the culture plate (ware) of Xiang Yaoyong, wrap by about 30 minutes, the poly-lysine that sucking-off is unnecessary, spend the night in Biohazard Safety Equipment and dry, for subsequent use.
2. be separated the cell of trigeminal nerve tissue
The bilateral trigeminal nerve of the SD rat of the 150-250g of test is taken out, is placed in containing in cold Hank, s balanced salt solution, carries out clean, remove its hetero-organization adhered to, shredded with eye scissors or scalpel, obtain pre-treatment trigeminal nerve tissue.
First, digest with containing the HBSS of 1.5g/L XI-s collagenase, 37 DEG C, 30 minutes, then continue digestion 10 minutes with the DNase I of 20ug/ml, use in the DMEM-F12 substratum containing 10% foetal calf serum and digestive ferment, be centrifugally precipitated cell, discard supernatant liquor.
By DMEM-F12 substratum washing and precipitating 2 times, suspend, repeatedly blow and beat with pipettor with the DMEM-F12 nutrient solution containing 10% foetal calf serum, the cell of suspension is inoculated into and wraps by good culture apparatus, cultivates.
Embodiment 2: the Isolation and culture of rat trigeminal ganglion cell
1. bag is by culture plate (ware)
The day before yesterday of cultivating, add appropriate (covering at the bottom of ware) commercially available aseptic Poly-L-Lysine Solution in the culture plate (ware) of Xiang Yaoyong, wrap by about 30 minutes, the poly-lysine that sucking-off is unnecessary, spend the night in Biohazard Safety Equipment and dry, for subsequent use.
2. be separated the cell of trigeminal nerve tissue
Taken out by the trigeminal nerve of the 8-12 of test rat in age in week, be placed on the culture dish containing L-15 nutrient solution, said process operates on ice, then clean is carried out, remove its hetero-organization adhered to, shredded with eye scissors or scalpel, obtain pre-treatment trigeminal nerve tissue.
First, with the 200U ten one Collagenase Type process pre-treatment trigeminal nerve tissue of 5mL through the L-15 nutrient solution dilution gained of 4.5mL, digest 90 minutes at 37 DEG C, during digestion, shake in every 10-15 minute once, enables collagenase fully and equably digest tissue.Secondly, the concentration adding 500uL in mixture is the trypsinase of 0.25%, continues digestion 20 minutes, centrifugal step in 37 DEG C of water-baths, collects tissue and the cell of digestion, discards supernatant liquor.Finally, add the trypsinase-EDTA of 5mL through the L-15 nutrient solution dilution acquisition of 4.5mL, shake up rear 37 DEG C of water-baths and continue digestion 20 minutes, add the neurobasal media (neurobasal medium) of 10mL containing 10% foetal calf serum, in and digestive ferment, centrifugally be precipitated cell, discard supernatant liquor.
The neurobasal media (neurobasal medium) of 200 μ L containing 2%B27 is added in the sedimentation cell of above-mentioned steps gained, suspension cell, with the cell in 200 μm of apertures sieved filter gained suspension, rinse cell sieve with the 2%B27 neurobasal media (neurobasalmedium) that contains of 2mL, obtain 2.2mL cell suspending liquid.
3. the cell of purifying trigeminal nerve tissue
Utilize Optiprep density gradient centrifugation purifying cells.First density gradient is prepared with 4 1.5ml centrifuge tubes respectively according to table 1.
Table 1 density gradient liquid is prepared
Respectively with above four pipes of whirlpool device mixing.Slowly add in 15mL centrifuge tube by 1,2,3,4 in order respectively, finally 2mL cell suspension is also slowly added to above density gradient liquid.Then 800 centrifugal force under centrifugal 15 minutes, discard the upper two-layer liquid in centrifuge tube, collect remaining liq, then add 10mL base neural substratum (neurobasalmedium) in collected liquid, centrifugal 5min under 1000rpm, collecting cell.
4. the inoculation culture of cell
The cell the collected neural nutrient solution of 100 μ L suspends, and is inoculated in the culture apparatus prepared in step 1, uses base neural substratum (neurobasal medium) at 37 DEG C, 5%CO in incubator with suitable even concentration
2, cultivate under the condition of saturated humidity.The upgrowth situation (Fig. 1-2) of observation of cell after 24-48h.
The effect of embodiment 1 and embodiment 2 cultural method compared, comparative result is as shown in table 2:
The contrast of table 2 embodiment 1 and embodiment 2 cultural method effect
| Acquisition cell quantity/ | Cell purity | |
| Embodiment 1 | (2～3)×10 5 | The heteroproteose cell such as cell debris, spongiocyte is more |
| Embodiment 2 | (1～2)×10 6 | Cell debris, heteroproteose cell are less |
As can be seen from Table 2, utilize method provided by the present invention, (1 ~ 2) × 10 can be obtained
6individual cell, can reach 1,000,000 cells, and existing method only can obtain (2 ~ 3) × 10
5an only hundreds of thousands of cell, the inventive method obtains cell quantity and is significantly higher than existing method, and the inventive method obtains cell debris in cell, heteroproteose cell is less, and the heteroproteose cell such as cell debris, spongiocyte that traditional method obtains is more, therefore, the inventive method is obviously better than existing method in the purity etc. of the cell quantity cell obtained.The present invention is owing to controlling the outside culture condition such as osmotic pressure, pH value of vitro culture, have employed the method for collagenase and trypsinase simultaneous digestion, the damage to isolated cell is greatly reduced in organization processes, utilize the method enrichment of cell such as density gradient centrifugation, eliminate the interference of the impurity such as a large amount of cell debriss and other cell types produced in digestive process, improve cell yield and quality.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; can do various change and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. the In-vitro separation culture method of a Mammals trigeminal ganglion neuron, it is characterized in that: Mammals trigeminal nerve tissue is placed on the culture dish containing L-15 nutrient solution and carries out clean, digestion process is carried out with 11 Collagenase Types, trypsinase and trypsinase-EDTA successively after shredding, after the sieved filter of cell, density gradient centrifugation is carried out to filtrate, be inoculated in bag after suspension by good cell culture apparatus, cultivate with base neural substratum.
2. method according to claim 1, it is characterized in that, step is as follows:
1) the Mammals trigeminal nerve tissue of gained is placed on the culture dish containing L-15 nutrient solution and shreds after cleaning, obtain pre-treatment trigeminal nerve tissue;
2) by step 1) gained pre-treatment trigeminal nerve organizes and carries out digestion process with 11 Collagenase Types, trypsinase and trypsinase-EDTA successively, through neutralization, centrifugal and after suspending with the sieved elimination of cell except fragment of tissue, obtain cell suspending liquid;
3) step 2) gained cell suspending liquid, carries out density gradient centrifugation to filtrate, removes cell debris and heteroproteose cell, obtain purifying cells;
4) by step 3) the base neural substratum suspension of gained purifying cells, be then inoculated into and wrapped by good cell culture apparatus, cultivate with base neural substratum in incubator, cultivate 24-48h and obtain trigeminal ganglion neuron.
3. method according to claim 2, is characterized in that, step 1) described Mammals is Mammals except embryonic stage and introduction stage.
4. method according to claim 2, is characterized in that, step 1) described Mammals be 8-12 week age rat.
5. method according to claim 2, it is characterized in that, step 2) described digestion process process is: (1) is by step 1) 200U 11 Collagenase Type that the pre-treatment trigeminal nerve tissue 5mL of gained dilutes through L-15 nutrient solution digests 90 minutes at 37 DEG C, during digestion, shake in every 10-15 minute once, enables collagenase fully and equably digest tissue; (2) concentration adding 500 μ L in mixture is the trypsinase of 0.25%, continues digestion 20 minutes in 37 DEG C of water-baths, the tissue of collected after centrifugation digestion and cell; (3) add the trypsinase-EDTA that 5mL dilutes through L-15 nutrient solution, after shaking up, continue digestion 20 minutes in 37 DEG C of water-baths; (4) add 10mL containing the neurobasal media of 10% foetal calf serum, in and digestive ferment, be centrifugally precipitated cell; (5) add the neurobasal media of 200 μ L containing 2%B27, suspension cell, then use the sieved filter cell suspension of the cell in 200 μm of apertures, carry out flushing cell sieve with the 2%B27 neurobasal media that contains of 2mL, obtain cell suspending liquid.
6. method according to claim 2, it is characterized in that, step 3) described density gradient centrifugation is Optiprep density gradient centrifugation, process is: by OPTI and Nb+B27 nutrient solution successively according to 173:827,124:876,99:901, the volume ratio of 74:926 mixes, obtain four density gradient liquid, successively it is slowly added in the centrifuge tube of 15mL in order, then 2mL cell suspending liquid is slowly added to above density gradient liquid, the centrifugal 15min of 800g, discard two-layer liquid, collect remaining liq.
7. method according to claim 2, is characterized in that, step 4) described bag is the day before yesterday of cultivating by culture apparatus, aseptic Poly-L-Lysine Solution is added in culture apparatus, bag was by 30 minutes, and the poly-lysine that sucking-off is unnecessary, spending the night in Biohazard Safety Equipment, it is for subsequent use to dry.
8. method according to claim 2, is characterized in that, step 4) described suspension adds 10mL base neural substratum, the centrifugal 5min of 1000rpm, suspends with the neural nutrient solutions of 100 μ L after collecting cell; The condition of described cultivation is 37 DEG C, 5%CO
2and saturated humidity.
9. method according to claim 2, it is characterized in that, concrete steps are:
1) take out the trigeminal nerve of rat, be placed on the culture dish containing L-15 nutrient solution, then carry out clean, remove its hetero-organization sticked, shredded with eye scissors or scalpel, obtain pre-treatment trigeminal nerve tissue;
2) by step 1) gained pre-treatment trigeminal nerve tissue 5mL 200U 11 Collagenase Type of obtaining after the dilution of L-15 nutrient solution digests 90 minutes at 37 DEG C, during digestion, shake in every 10-15 minute once, enables collagenase fully and equably digest tissue;
3) to step 2) to add 500 μ L concentration in gained mixture be the trypsinase of 0.25%, continues digestion 20 minutes in 37 DEG C of water-baths, centrifugal, collect tissue and the cell of digestion, outwell supernatant liquor;
4) to step 3) add the trypsinase-EDTA that 5mL obtains after the dilution of L-15 nutrient solution in gained tissue and cell, shake up rear 37 DEG C of water-baths and continue digestion 20 minutes, add the neurobasal media of 10mL containing 10% foetal calf serum, in and digestive ferment, centrifugally be precipitated cell, discard supernatant liquor;
5) to step 4) add the neurobasal media of 200 μ L containing 2%B27 in gained sedimentation cell, suspension cell;
6) with the cell sieve filtration step 5 in 200 μm of apertures) gained suspension, with sieving containing 2%B27 base neural substratum flushing cell of 2mL, obtain cell suspending liquid;
7) by OPTI and Nb+B27 nutrient solution successively according to 173:827,124:876,99:901, the volume ratio of 74:926 mixes, and obtains four density gradient liquid, is slowly added in the centrifuge tube of 15mL by each gradient liquid successively in order, then 2mL cell suspending liquid is slowly added to above density gradient liquid, the centrifugal 15min of 800g, discards two-layer liquid, collects remaining liq;
8) to step 7) add 10mL base neural substratum, the centrifugal 5min of 1000rpm, collecting cell in gained liquid, suspend with the neural nutrient solutions of 100 μ L;
9) by step 8) be inoculated into step 9 gained uniform suspension) gained wrap in the cell culture apparatus of quilt, in incubator with base neural cultivate based on 37 DEG C, 5% CO
2, after cultivating 24-48h under the condition of saturated humidity, obtain trigeminal ganglion neuron;
The described cell cultures based devices having wrapped quilt, be in culture apparatus, add aseptic Poly-L-Lysine Solution in the day before yesterday of cultivating, processed 30 minutes of bag, wash out unnecessary poly-lysine, spending the night in Biohazard Safety Equipment, it is for subsequent use to dry.
10. method described in claim 1-9, is characterized in that, for the Isolation and culture of Mammals trigeminal ganglion neuron.
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| US20180016554A1 (en) * | 2015-03-30 | 2018-01-18 | Ajinomoto Co., Inc. | Human serum albumin-containing culture medium for growth of neural stem cells |
| CN111378613A (en) * | 2018-12-27 | 2020-07-07 | 深圳华大生命科学研究院 | Dissociation kit for amphibian cells |
| WO2022120916A1 (en) * | 2020-12-11 | 2022-06-16 | 深圳先进技术研究院 | Method for extracting synaptodendrosomes |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180016554A1 (en) * | 2015-03-30 | 2018-01-18 | Ajinomoto Co., Inc. | Human serum albumin-containing culture medium for growth of neural stem cells |
| CN111378613A (en) * | 2018-12-27 | 2020-07-07 | 深圳华大生命科学研究院 | Dissociation kit for amphibian cells |
| CN111378613B (en) * | 2018-12-27 | 2024-01-09 | 深圳华大生命科学研究院 | Amphibious animal cell dissociation kit |
| WO2022120916A1 (en) * | 2020-12-11 | 2022-06-16 | 深圳先进技术研究院 | Method for extracting synaptodendrosomes |
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