CN104560875B - A kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application - Google Patents
A kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application Download PDFInfo
- Publication number
- CN104560875B CN104560875B CN201410826389.5A CN201410826389A CN104560875B CN 104560875 B CN104560875 B CN 104560875B CN 201410826389 A CN201410826389 A CN 201410826389A CN 104560875 B CN104560875 B CN 104560875B
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- tissue
- gained
- mammal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention discloses a kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application, belong to cell biology and Neurobiology technical field.Method provided by the present invention carries out cleaning treatment for mammal trigeminal neuralgia tissue is placed on the culture dish containing the nutrient solutions of L 15, digestion process is carried out after shredding with 11 Collagenase Types, trypsase and trypsase EDTA successively, density gradient centrifugation is carried out to filtrate after cell sieve filtering, it is inoculated in the cell culture apparatus being coated with, is cultivated with base neural culture medium after suspension.The inventive method greatly reduces the damage to cell in organization processes, eliminate a large amount of impurity such as cell fragment and other cell types interference in digestion process, substantially increase the purity of cell, million purer trigeminal neuralgia cells can be obtained, improve cell yield and quality, it is adaptable to the Isolation and culture of mammal trigeminal ganglion neuron.
Description
Technical field
The present invention relates to a kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application, belong to cell
Biology and Neurobiology technical field.
Background technology
Being separately cultured for neuron, is research Neuronal Growth Factor, neurite outgrowth, differentiation and esthesiophysiology
Deng common technology.Although the neuron before separation embryo or the formation cynapse of new born animal nerve cell wants relatively easy one
A bit, but Fetal neurons and mature neuron have different characteristics in terms of electrophysiology, development, regeneration.Therefore into
The culture of ripe nerve cell is very important in many researchs.But existed always during culture of primary neurons is supported thin
The problem of born of the same parents' yield and not high quality.
At present, existing trigeminal neuralgia cell primary culture technique is generally collagenase digestion, and it has in incubation
A little details consider not comprehensive enough, such as：In no CO2The stability of nutrient solution is bad during balance regulator, and processing procedure is to group
Knit or the influence of cell is larger, larger to cellular damage, digestion is not thorough enough, and contaminating cell is more, culture gained cell is impure
The problems such as, therefore the yield and quality of cell is not high.
The content of the invention
To solve the deficiencies in the prior art, the invention provides a kind of in-vitro separation of mammal trigeminal ganglion neuron
Cultural method and application, the technical scheme of use are as follows：
It is an object of the invention to provide a kind of In-vitro separation culture method of mammal trigeminal ganglion neuron, the party
Method is that mammal trigeminal neuralgia tissue is placed on the culture dish containing L-15 nutrient solutions to carry out cleaning treatment, after shredding successively
Digestion process is carried out with 11 Collagenase Types, trypsase and trypsase-EDTA, line density is entered to filtrate after cell sieve filtering
It is inoculated in the cell culture apparatus being coated with, is cultivated with base neural culture medium after gradient centrifugation, suspension.
Methods described step is as follows：
1) the mammal trigeminal neuralgia tissue of gained is placed in after being cleaned on the culture dish of the nutrient solution containing L-15 and shredded, obtained
Trigeminal neuralgia tissue must be pre-processed；
2) by step 1) gained pretreatment trigeminal neuralgia tissue successively with 11 Collagenase Types, trypsase and tryptose
Enzyme-EDTA carries out digestion process, removes fragment of tissue with cell sieve filtering after neutralizing, centrifuging and suspend, obtains cell outstanding
Supernatant liquid；
3) step 2) gained cell suspending liquid, density gradient centrifugation is carried out to filtrate, cell fragment and heteroproteose cell is removed, obtains
Obtain purifying cells；
4) by step 3) the base neural culture medium suspension of gained purifying cells, it is then seeded into coated good cell
In culture apparatus, cultivated in incubator with base neural culture medium, culture 24-48h obtains trigeminal ganglion neuron.
Step 1) mammal is mammal in addition to embryonic period, embryonic phase and introduction stage.
Preferably, step 1) mammal be 8-12 week old rat.
Step 2) the digestion process process is：(1) by step 1) obtained by pretreatment trigeminal neuralgia tissue with 5mL through L-
The Collagenase Types of 200U 11 of 15 nutrient solutions dilution shook once per 10-15 minutes during digestion, made in being digested 90 minutes at 37 DEG C
Clostridiopetidase A can sufficiently and evenly digest tissue；(2) concentration that 500 μ L are added into mixture is 0.25% tryptose
Enzyme, continues to digest 20 minutes, the tissue and cell of collected after centrifugation digestion in 37 DEG C of water-baths；(3) 5mL is added through L-15 nutrient solutions
Trypsase-the EDTA of dilution, shakes up and continues to digest 20 minutes after 37 DEG C of water-baths；(4) add 10mL and contain 10% hyclone
Neurobasal media, neutralize digestive ferment, centrifugation obtain sedimentation cell；(5) 200 neurobasal medias of the μ L containing 2%B27 are added, are hanged
Floating cell, then with the cell sieve filtration cell suspension in 200 μm of apertures, be rinsed carefully with 2mL neurobasal media containing 2%B27
Born of the same parents sieve, and obtain cell suspending liquid.
Step 3) density gradient centrifugation is Optiprep density gradient centrifugations, process is：OPTI and Nb+B27 is trained
Nutrient solution is successively according to 173:827,124:876,99:901,74:926 volume ratio is mixed, and obtains four density gradient liquid,
It is added slowly to successively in 15mL centrifuge tube in order, 2mL cell suspending liquids is then added slowly to density gradient liquid
Above, 800g centrifuges 15min, discards two layers of liquid, collects remaining liq.
Step 4) the coating culture apparatus is the previous day in culture, and sterile poly-D-lysine is added into culture apparatus
Solution, is coated with 30 minutes, suctions out unnecessary poly-D-lysine, is dried overnight in Biohazard Safety Equipment standby.
Step 4) it is described suspension be add 10mL base neural culture mediums, 1000rpm centrifugation 5min, collect cell after use
100 μ L nerve nutrient solutions suspend；
The condition of the culture is 37 DEG C, 5%CO2And saturated humidity.
Methods described is concretely comprised the following steps：
1) trigeminal neuralgia of rat is taken out, is placed on the culture dish of the nutrient solution containing L-15, then carries out cleaning treatment, is removed
Its hetero-organization sticked, is shredded with eye scissors or scalpel, obtains pretreatment trigeminal neuralgia tissue；
2) by step 1) gained pretreatment trigeminal neuralgia tissue diluted with 5mL through L-15 nutrient solutions after obtained 200U 11
Collagenase Type digests 90 minutes at 37 DEG C, was shaken once per 10-15 minutes during digestion, enables clostridiopetidase A sufficiently and evenly
Digest tissue；
3) to step 2) gained mixture in add 500 μ L concentration be 0.25% trypsase, in 37 DEG C of water-baths continue
Digestion 20 minutes, centrifugation collects the tissue and cell of digestion, outwells supernatant；
4) to step 3) gained tissue and cell in add 5mL through L-15 nutrient solutions dilute after obtain trypsase-
EDTA, 37 DEG C of water-baths continue to digest 20 minutes after shaking up, and add the neurobasal media that 10mL contains 10% hyclone, neutralize digestion
Enzyme, centrifugation obtains sedimentation cell, discards supernatant；
5) to step 4) gained sedimentation cell in add 200 neurobasal medias of the μ L containing 2%B27, suspension cell；
6) with the cell sieve filtration step 5 in 200 μm of apertures) gained suspension, with the 2mL culture medium of base neural containing 2%B27
Cell sieve is rinsed, cell suspending liquid is obtained；
7) by OPTI and Nb+B27 nutrient solutions successively according to 173:827,124:876,99:901,74:926 volume ratio is entered
Row mixing, obtains four density gradient liquid, each gradient liquid is added slowly in 15mL centrifuge tube successively in order, then will
2mL cell suspending liquids are added slowly to above density gradient liquid, 800g centrifugation 15min, are discarded two layers of liquid, are collected raffinate
Body；
8) to step 7) gained liquid in add 10mL base neural culture mediums, 1000rpm centrifugation 5min, collect cell,
Suspended with the neural nutrient solutions of 100 μ L；
9) by step 8) gained uniform suspension be inoculated into step 9) obtained by coated cell culture apparatus in,
In incubator 37 DEG C, 5%CO are based on base neural culture2, trident god is obtained under conditions of saturated humidity after culture 24-48h
Through ganglion cell；
The coated cell culture based devices, are to add sterile poly into culture apparatus in the previous day of culture to rely
Propylhomoserin solution, coating processing 30 minutes, washes out unnecessary poly-D-lysine, is dried overnight in Biohazard Safety Equipment standby.This hair
Bright methods described is applied to the Isolation and culture of mammal trigeminal ganglion neuron.Beneficial effect of the present invention：
1. present invention optimizes the digestion method of tissue, banned original protease digestion process, using clostridiopetidase A and
Trypsase simultaneous digestion, its digestion process and digestion time can make tissue digestion ratio more thoroughly, while without using the side of pressure-vaccum
Formula carrys out further cell dispersion, and the damage to cell is greatly reduced.
2. the present invention has used L-15 culture mediums, energy during to organizing external cleaning, digestion and separation cell
Enough ensure no CO2Even if under conditions of balance regulator the stability of nutrient solution there is provided one without CO2Balance adjustment,
It can guarantee that stabilization, the liquid environment of very little damaged to cell or tissue, stabilize the outside condition of culture such as osmotic pressure, pH value, greatly
The big influence reduced in processing procedure to tissue or cell.
3. not taking out the process of impurity more than existing culture technique, the present invention is filtered to tissue after digestion first, is gone
Except bulk impurity, the method enrichment of cell of density gradient centrifugation is recycled, the substantial amounts of cell produced in digestion process is eliminated
The interference of the impurity such as fragment and other cell types, make cell obtained by culture purer, and cell culture has higher success rate.
4. million or so purer trigeminal neuralgia cells can be obtained using cultural method of the present invention, cell production is improved
Amount and quality, solve the deficiencies in the prior art, can meet pharmacology, electrophysiology, development, regeneration and neurotoxicology
Etc. the research of each side.
Brief description of the drawings
Fig. 1 trigeminal neuralgia cell phase contrast microscope figures.
Fig. 2 trigeminal neuralgia cell fluorescence phase contrast microscope figures.
Embodiment
With reference to specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Embodiment 1：SD rat trigeminal cell separation cultures
1. it is coated with culture plate (ware)
Commercially available sterile of appropriate (covering ware bottom) is added in the previous day of culture, in Xiang Yaoyong culture plate (ware)
Poly-L-Lysine Solution, be coated with 30 minutes or so, suction out unnecessary poly-D-lysine, dried overnight in Biohazard Safety Equipment,
It is standby.
2. separate the cell of trigeminal neuralgia tissue
The bilateral trigeminal neuralgia of the 150-250g of experiment SD rats is taken out, is placed in containing cold Hank, s balance salt is molten
In liquid, cleaning treatment is carried out, its hetero-organization of adhesion is removed, is shredded with eye scissors or scalpel, pretreatment trident is obtained
Nerve fiber.
First, digested with the HBSS of the clostridiopetidase As of XI-s containing 1.5g/L, 37 DEG C, 30 minutes, then with 20ug/ml DNase
I continues to digest 10 minutes, and with the DMEM-F12 culture mediums containing 10% hyclone and digestive ferment, centrifugation obtains sedimentation cell,
Discard supernatant.
Precipitation is cleaned with DMEM-F12 culture mediums 2 times, suspended with the DMEM-F12 nutrient solutions containing 10% hyclone, with shifting
Liquid device is blown and beaten repeatedly, and the cell of suspension is inoculated into the culture apparatus being coated with, and is cultivated.
Embodiment 2：The Isolation and culture of rat trigeminal ganglion cell
1. it is coated with culture plate (ware)
Commercially available sterile of appropriate (covering ware bottom) is added in the previous day of culture, in Xiang Yaoyong culture plate (ware)
Poly-L-Lysine Solution, be coated with 30 minutes or so, suction out unnecessary poly-D-lysine, dried overnight in Biohazard Safety Equipment,
It is standby.
2. separate the cell of trigeminal neuralgia tissue
The trigeminal neuralgia of the 8-12 week old rats of experiment is taken out, is placed on the culture dish of the nutrient solution containing L-15, it is above-mentioned
Process is operated on ice, then carries out cleaning treatment, is removed its hetero-organization of adhesion, is cut with eye scissors or scalpel
It is broken, obtain pretreatment trigeminal neuralgia tissue.
First, with the Collagenase Types of the 200U 11 processing pretreatment trident obtained by L-15 nutrient solutions dilutions of the 5mL through 4.5mL
Nerve fiber, digests 90 minutes at 37 DEG C, was shaken once per 10-15 minutes during digestion, enables clostridiopetidase A abundant and uniform
Ground digests tissue.Secondly, the concentration that 500uL is added into mixture is 0.25% trypsase, is continued in 37 DEG C of water-baths
Digestion 20 minutes, centrifugation step, collects the tissue and cell of digestion, discards supernatant.Finally, L-15 trainings of the 5mL through 4.5mL is added
Trypsase-the EDTA that nutrient solution dilution is obtained, 37 DEG C of water-baths continue to digest 20 minutes after shaking up, and add 10mL and contain 10% tire ox blood
Clear neurobasal media (neurobasal medium), neutralizes digestive ferment, and centrifugation obtains sedimentation cell, discards supernatant.
Neurobasal media (neurobasals of the 200 μ L containing 2%B27 is added into the sedimentation cell obtained by above-mentioned steps
Medium), suspension cell, with the cell sieve filtering gained suspension in 200 μm of apertures, with 2mL neurobasal media containing 2%B27
(neurobasal medium) rinses cell sieve, obtains 2.2mL cell suspending liquids.
3. purify the cell of trigeminal neuralgia tissue
Utilize Optiprep density-gradient centrifugation method purifying cells.Matched somebody with somebody respectively according to table 1 with 4 1.5ml centrifuge tubes first
Density gradient processed.
The density gradient liquid of table 1 is prepared
Mixed and four managed above with whirlpool device respectively.1,2,3,4 are slowly added in 15mL centrifuge tubes in order respectively, finally
2mL cell suspensions are also added slowly to above density gradient liquid.Then centrifuged 15 minutes under 800 centrifugal force, discard centrifugation
Upper two layers of liquid in pipe, collects remaining liq, then the addition 10mL base neural culture mediums into collected liquid
5min is centrifuged under (neurobasal medium), 1000rpm, cell is collected.
4. the inoculated and cultured of cell
The cell of collection is suspended with the neural nutrient solutions of 100 μ L, and the training prepared in step 1 is inoculated in appropriate even concentration
Support in device, with base neural culture medium (neurobasal medium) in 37 DEG C, 5%CO in incubator2, saturated humidity
Under conditions of cultivated.The upgrowth situation (Fig. 1-2) of cell is observed after 24-48h.
The effect of embodiment 1 and the cultural method of embodiment 2 is compared, comparative result is as shown in table 2：
The contrast of the embodiment 1 of table 2 and the cultural method effect of embodiment 2
| Acquisition cell quantity/ | Cell purity | |
| Embodiment 1 | (2~3) × 105 | The heteroproteose cells such as cell fragment, spongiocyte are more |
| Embodiment 2 | (1~2) × 106 | Cell fragment, heteroproteose cell are less |
From table 2 it can be seen that using method provided by the present invention, (1~2) × 10 can be obtained6Individual cell, up to hundred
Ten thousand cells, and existing method is only capable of obtaining (2~3) × 105, only hundreds of thousands cell, the inventive method obtains cell quantity
It is significantly higher than cell fragment, heteroproteose cell in existing method, and the inventive method acquisition cell less, and what conventional method was obtained
The heteroproteose cells such as cell fragment, spongiocyte are more, therefore, and the inventive method is in terms of the purity of the cell quantity cell of acquisition
It is substantially better than existing method.The outside condition of culture such as osmotic pressure, pH value of the present invention due to controlling in vitro culture, employs glue
The method of protoenzyme and trypsase simultaneous digestion, greatly reduces to a point cellifugal damage in organization processes, utilizes
The method enrichment of cell such as density gradient centrifugation, eliminates the substantial amounts of cell fragment produced in digestion process and other cell classes
The interference of the impurity such as type, improves cell yield and quality.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this
The people of technology, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection of the present invention
What scope should be defined by claims is defined.
Claims (9)
1. a kind of In-vitro separation culture method of mammal trigeminal ganglion neuron, it is characterised in that：By mammal trident
Nerve fiber, which is placed on the culture dish containing L-15 nutrient solutions, carries out cleaning treatment, is diluted successively with through L-15 nutrient solutions after shredding
11 Collagenase Types, trypsase and through L-15 nutrient solutions dilute trypsase-EDTA carry out digestion process, cell be sieved through
Density gradient centrifugation is carried out to filtrate after filter, is inoculated in after suspension in the cell culture apparatus being coated with, uses base neural culture
Base is cultivated.
2. method according to claim 1, it is characterised in that step is as follows：
1) the mammal trigeminal neuralgia tissue of gained is placed in after being cleaned on the culture dish of the nutrient solution containing L-15 and shredded, obtain pre-
Handle trigeminal neuralgia tissue；
2) by step 1) gained pretreatment trigeminal neuralgia tissue successively with 11 Collagenase Types, trypsase and trypsase-
EDTA carries out digestion process, and fragment of tissue is removed with cell sieve filtering after neutralizing, centrifuging and suspend, and obtains cell suspension
Liquid；
3) step 2) gained cell suspending liquid, density gradient centrifugation is carried out to filtrate, cell fragment and heteroproteose cell is removed, obtained pure
Change cell；
4) by step 3) the base neural culture medium suspension of gained purifying cells, it is then seeded into coated good cell culture
In device, cultivated in incubator with base neural culture medium, culture 24-48h obtains trigeminal ganglion neuron.
3. method according to claim 2, it is characterised in that step 1) mammal is except embryonic period, embryonic phase and introduction stage
Mammal in addition.
4. method according to claim 2, it is characterised in that step 1) mammal be 8-12 week old rat.
5. method according to claim 2, it is characterised in that step 2) the digestion process process is：(1) by step 1)
The Collagenase Types of 200U 11 that the pretreatment trigeminal neuralgia tissue of gained is diluted with 5mL through L-15 nutrient solutions at 37 DEG C in digesting 90
Minute, shaken once per 10-15 minutes during digestion, clostridiopetidase A is sufficiently and evenly digested tissue；(2) into mixture
The concentration for adding 500 μ L is 0.25% trypsase, continues to digest 20 minutes in 37 DEG C of water-baths, the group of collected after centrifugation digestion
Knit and cell；(3) trypsase-EDTA that 5mL dilutes through L-15 nutrient solutions is added, shakes up and continue to digest 20 after 37 DEG C of water-baths
Minute；(4) neurobasal media that 10mL contains 10% hyclone is added, neutralizes digestive ferment, centrifugation obtains sedimentation cell；(5) add
Enter 200 neurobasal medias of the μ L containing 2%B27, suspension cell, then with the cell sieve filtration cell suspension in 200 μm of apertures, use 2mL
Neurobasal media containing 2%B27 be rinsed cell sieve, obtain cell suspending liquid.
6. method according to claim 2, it is characterised in that step 3) density gradient centrifugation is Optiprep density
Gradient centrifugation, process is：By OPTI and Nb+B27 nutrient solutions successively according to 173:827,124:876,99:901,74:926 body
Product obtains four density gradient liquid, is added slowly to successively in 15mL centrifuge tube in order than being mixed, and then will
2mL cell suspending liquids are added slowly to above density gradient liquid, 800g centrifugation 15min, are discarded two layers of liquid, are collected raffinate
Body.
7. method according to claim 2, it is characterised in that step 4) the coating culture apparatus is in the previous of culture
My god, sterile Poly-L-Lysine Solution is added into culture apparatus, is coated with 30 minutes, unnecessary poly-D-lysine is suctioned out, in biology
Dried overnight in safety cabinet standby.
8. method according to claim 2, it is characterised in that step 4) suspension is to add 10mL base neural cultures
Base, 1000rpm centrifugation 5min are collected after cell with the neural nutrient solution suspensions of 100 μ L；The condition of the culture be 37 DEG C, 5%
CO2And saturated humidity.
9. method according to claim 2, it is characterised in that concretely comprise the following steps：
1) trigeminal neuralgia of rat is taken out, is placed on the culture dish of the nutrient solution containing L-15, then carries out cleaning treatment, removal is sticked
Its hetero-organization, shredded with eye scissors or scalpel, obtain pretreatment trigeminal neuralgia tissue；
2) by step 1) gained pretreatment trigeminal neuralgia tissue diluted with 5mL through L-15 nutrient solutions after the obtained type glue of 200U 11
Protoenzyme digests 90 minutes at 37 DEG C, was shaken once per 10-15 minutes during digestion, clostridiopetidase A is sufficiently and evenly digested
Tissue；
3) to step 2) gained mixture in add 500 μ L concentration be 0.25% trypsase, in 37 DEG C of water-baths continue digest
20 minutes, centrifugation collected the tissue and cell of digestion, outwells supernatant；
4) to step 3) obtained trypsase-EDTA after 5mL dilutes through L-15 nutrient solutions is added in gained tissue and cell, shake
37 DEG C of water-baths continue to digest 20 minutes after even, add the neurobasal media that 10mL contains 10% hyclone, neutralize digestive ferment, centrifugation
Sedimentation cell is obtained, supernatant is discarded；
5) to step 4) gained sedimentation cell in add 200 neurobasal medias of the μ L containing 2%B27, suspension cell；
6) with the cell sieve filtration step 5 in 200 μm of apertures) gained suspension, is rinsed with the 2mL culture medium of base neural containing 2%B27
Cell sieve, obtains cell suspending liquid；
7) by OPTI and Nb+B27 nutrient solutions successively according to 173:827,124:876,99:901,74:926 volume ratio is mixed
Close, obtain four density gradient liquid, each gradient liquid is added slowly in 15mL centrifuge tube successively in order, it is then that 2mL is thin
Born of the same parents' suspension is added slowly to above density gradient liquid, 800g centrifugation 15min, is discarded two layers of liquid, is collected remaining liq；
8) to step 7) gained liquid in add 10mL base neural culture mediums, 1000rpm centrifugation 5min, collect cell, with 100
μ L nerve nutrient solutions suspend；
9) by step 8) gained uniform suspension be inoculated into step 9) obtained by coated cell culture apparatus in, culture
In case 37 DEG C, 5%CO are based on base neural culture2, gasserian ganglion is obtained after culture 24-48h under conditions of saturated humidity
Cell；
The coated cell culture based devices, are that sterile poly-D-lysine is added into culture apparatus in the previous day of culture
Solution, coating processing 30 minutes, washes out unnecessary poly-D-lysine, is dried overnight in Biohazard Safety Equipment standby.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410826389.5A CN104560875B (en) | 2014-12-26 | 2014-12-26 | A kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410826389.5A CN104560875B (en) | 2014-12-26 | 2014-12-26 | A kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104560875A CN104560875A (en) | 2015-04-29 |
| CN104560875B true CN104560875B (en) | 2017-10-20 |
Family
ID=53077955
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410826389.5A Expired - Fee Related CN104560875B (en) | 2014-12-26 | 2014-12-26 | A kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104560875B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107429234A (en) * | 2015-03-30 | 2017-12-01 | 味之素株式会社 | Nerve stem cell proliferation culture medium containing human serum albumins |
| CN111378613B (en) * | 2018-12-27 | 2024-01-09 | 深圳华大生命科学研究院 | Amphibious animal cell dissociation kit |
| CN114621921A (en) * | 2020-12-11 | 2022-06-14 | 深圳先进技术研究院 | Method for extracting neurite contact body |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102809596A (en) * | 2012-08-21 | 2012-12-05 | 辉源生物科技（上海）有限公司 | Method for recording T type calcium channel current by separating and cultivating newborn rat cortical neural cells |
-
2014
- 2014-12-26 CN CN201410826389.5A patent/CN104560875B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102809596A (en) * | 2012-08-21 | 2012-12-05 | 辉源生物科技（上海）有限公司 | Method for recording T type calcium channel current by separating and cultivating newborn rat cortical neural cells |
Non-Patent Citations (3)
| Title |
|---|
| Acid Activation of Trpv1 Leads to an Up-Regulation of Calcitonin Gene-related Peptide Expression in Dorsal Root Ganglion Neurons via the CaMK-CREB Cascade: A Potential Mechanism of Inflammatory Pain;Masako Nakanishi等;《Molecular Biology of the Cell》;20100801;第21卷;2568-2577页 * |
| Intrinsic Innate Immunity Fails To Control Herpes Simplex Virus and Vesicular Stomatitis Virus Replication in Sensory Neurons and Fibroblasts;Pamela C. Rosato等;《Journal of Virology》;20140612;第88卷(第17期);第9992页右栏第3段 * |
| P2Y2受体对大鼠三叉神经节神经元介导瞬时外向钾电流的电压门控性钾通道的抑制作用研究;李娜;《中国博士学位论文全文数据库医药卫生科技辑》;20120915(第 09 期);正文第14-15页研究方法第（一）部分 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104560875A (en) | 2015-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105969720B (en) | A kind of Human vascular endothelial's cell culture fluid and its cultural method | |
| WO2020103192A1 (en) | Method for extracting exosome | |
| CN104560875B (en) | A kind of In-vitro separation culture method of mammal trigeminal ganglion neuron and application | |
| CN108728413A (en) | A kind of preparation of human pluripotent stem cells source Human RPE Cells in Vitro and amplification cultivation method | |
| CN101735983B (en) | Method for separating and purifying oligodendrocyte precursor cells | |
| US20210355439A1 (en) | Differentiation medium and method for preparing oligodendrocyte precursor | |
| CN107142242B (en) | Isolated culture method of naked mole rat skeletal muscle myoblasts | |
| CN104087550A (en) | Culture method of rat myocardial cell | |
| CN110747159A (en) | Mouse or rat kidney fibroblast cell separation and subculture method | |
| CN110628706B (en) | Method for extracting and culturing embryonic neural stem cells in vitro and preparation of culture medium | |
| JP2018526025A (en) | Method for preparing olfactory nerve sheath cells | |
| CN113462639A (en) | Method for separating fibroblasts from human induced pluripotent stem cells and application of method | |
| CN105695409B (en) | A kind of naked mole oligodendrocyte precursor cells cultural method | |
| CN114807008B (en) | Preparation method and application of tomato leaf protoplast single-cell suspension | |
| CN111004776A (en) | Method for separating and culturing equine skeletal muscle satellite cells | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| CN110396496B (en) | Culture method and application of duck small intestine epithelial cells | |
| CN112280733B (en) | Amniotic stem cell extraction method | |
| CN106497863A (en) | A kind of separation of cornea of rats endothelial cell, purifying and cultural method | |
| CN109666642B (en) | Method for in vitro separation and purification of oligodendrocyte precursor cells of tree shrew cerebral cortex | |
| CN105969733B (en) | Induction differentiation hPSCs is the method for glutaminergic neurons | |
| CN113801847A (en) | Improved astrocyte culture method | |
| CN113583938A (en) | Method for forming islet-like structure by islet cells differentiated by in vitro induced stem cells | |
| CN113186155A (en) | High-efficiency culture method of primary cells of sheep embryonic skeletal muscle | |
| Mii et al. | Isolation of chromosomes from cell suspension cultures of Vicia hajastana |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20171020 Termination date: 20181226 |