CN105695410A - Naked mole rat microglial cell culture method - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
The invention relates to the technical field of cell biology and in particular relates to a separation, purification and culture method of naked mole rat microglial cells. The microglial cells are separated from cerebral cortices of newly-born naked mole rats and are purified and cultured by comprehensively utilizing a plurality of types of culture mediums, and a reasonable culture method applicable to poikilothermal rodent mammal naked mole rat microglial cells is explored. The method provided by the invention can be used for simply, conveniently, efficiently and economically obtaining a lot of naked mole rat microglial cells with normal functional activity, and cells can still keep biological characteristics under an in-vivo state in an in-vitro environment through culture under a low-oxygen condition, so that special physiological functions of the naked mole rat microglial cells can be conveniently and directly researched in a pure in-vitro cell culture model, and furthermore, an important theoretical basis is provided for exploring a biological mechanism and applying the biological mechanism to clinically relative fields.
Description
Technical field:
The present invention relates to technical field of cell biology, be specifically related to separation purification and the cultural method of a kind of cell, particularly to separation purification and the cultural method of a kind of naked mole microglia。
Background technology:
Microglia (Microglia) is the important component of central nervous system's immunologic function, plays an important role in the adjustment of central nervous system's stable state。Microglia pays close attention to the microenvironment that it is residing, will activate once be upset。Research finds, the activation of microglia all be can be observed in the central nervous system disease such as senile dementia, multiple sclerosis, amyotrophic lateral sclerosis side institute sclerosis。The activation of microglia plays important role in central nervous system injury, by the repair process after suppressing or promoting the activation of microglia likely to reach regulation and control central nervous system injury, this also becomes scientific research personnel's research and development for the regulation and control of microglia function thus the key areas repaired after regulating central nervous system injury。
Naked mole (Heterocephalusglaber) is a kind of wild animal being distributed in the ground such as Africa Somalia, Kenya, Ethiopia, belongs to Mammalia, Rodentia, Bathyergidae, naked mole genus, naked mole kind on zootaxy position。Naked mole is the rodent of the longest-lived found up to now, up to more than 28 years, is about 7-9 times of normal rats or mouse life。In the life-span of its many decades, do not find that it has senile dementia, there is also can keep good nervous function (JudyC.Triplett in the nervous system disease such as parkinson, AntonellaTramutola, AaronSwomley, JessimeKirk, KellyGrimes, KaitilynLewis, MirandaOrr, KarlRodriguez, JianCai, JonB.Klein, MarziaPerluigi, RochelleBuffenstein, D.AllanButterfield.Age-relatedchangesintheproteostasisne tworkinthebrainofthenakedmole-rat:Implicationspromotingh ealthylongevity.BiochimBiophysActa.2015；1852 (10): 2213-24.)。And microglia is as the cell of performance immunologic function important in central nervous system, it is possible to play an important role in monitoring its cerebral tissue microenvironment。But due to extremely complex in body microenvironment, it is not easy to directly observe immunoloregulation function (the DoraBritesandAdelaideFernandes.NeuroinflammationandDepre ssion:MicrogliaActivation of microglia at body, ExtracellularMicrovesiclesandmicroRNADysregulation.Front CellNeurosci.2015,9:476.doi:10.3389/fncel.2015.00476.)。It is thus desirable to set up in vitro naked mole microglia model to make up the defect observed at body。At present, research worker has built up out rat, In vitro culture strategy (the Boza-SerranoA of the rodent microglias such as mice, ReyesJF, ReyNL, LefflerH, BoussetL, NilssonU, BrundinP, VeneroJL, BurguillosMA, DeierborgT.TheroleofGalectin-3in α-synuclein-inducedmicroglialactivation.ActaNeuropatholCom mun.2014, 2:156.), cultural method is mainly newborn mice cortical tissue and separates, machinery shreds and chemical method digestion, cell primary is cultivated, strike four experimental procedures such as method of hitting culture bottle；Wherein cell primary cultivates the calf serum that used medium is DMEM culture fluid and final concentration of 20%；Wherein passage cultivates the hyclone that used medium is DMEM culture medium and final concentration of 10%。
But in prior art, no matter mice or the microglia separation purification of rat and cultural method are not all suitable for naked mole, and current domestic and foreign literature there is no the relevant report about naked mole microglia separation purification and cultural method。
Summary of the invention:
It is an object of the invention to provide the separation purification of a kind of naked mole microglia and cultural method, the naked mole microglia that purity is high, state is good is obtained with convenient, efficient, set up the offer technical support of naked mole microglia for external, lay the foundation for the polarization of the naked mole microglia of in vitro study, activation, phagocytosis, other immunoloregulation functions and the medicine of microglia relevant disease or the screening of target molecule simultaneously。
We have tried out current mice, the microglia separation purification of rat and cultural method, obtain, found that all cannot cultivate, the naked mole microglia that purity is high, state is good, and the quantity of naked mole microglia propagation is also less。
Cultivation about naked mole microglia is still not set up out feasible experimental technique, we analyze its reason, mainly: 1) naked mole is a kind of poikilothermal animal, the temperature of its somatic cell isolated culture needs to be groped, and its inconstant organism metabolism speed causes that its somatic cell in-vitro culture medium is different from common rat or mice；2) existence of naked mole is in comparatively dark moist environment, and humidity is had higher requirement by its somatic cultivation；3) naked mole has been already adapted to the environment of extraneous hypoxia, so the animal that the oxygen concentration in its somatic cell isolated culture environment is different from normal oxygen environment lives, and need to grope。
Therefore, most important in the cultivation of naked mole microglia grope to suitable temperature, humidity and oxygen concentration, and grope to applicable culture medium。
For reaching this purpose, the isolation and purification method of the naked mole microglia of the present invention comprises the following steps:
A, collect naked mole cerebral cortex cell mixing:
Separate birth 1-7 Tian Luo mole brain cortical tissue, tissue is shredded, after adding the mixing of tissue digestion liquid, puts it in three gas incubators and digest；It is subsequently adding mixed nerve cell culture medium and terminates digestion, after centrifugal segregation supernatant, by resuspended for cell precipitation and blow and beat into single cell suspension and plant in culture bottle；Described culture bottle is the T25 culture bottle not being coated hypothallus；
Described tissue digestion liquid, it is possible to for conventional enzyme of proteolysis Digestive system, for instance trypsin, papain etc., be aided with DNA enzymatic I to release the cell aggregation caused by DNA fragmentation adhesion。
Preferred tissue digestion liquid: containing the mixed liquor of the pancreatin that mass concentration is 0.25% and final concentration 0.1mg/mlDNA enzyme I。
B, naked mole cerebral cortex cell mixing cultivation:
The cell mixing obtained by step A, is incubated in three gas incubators with mixed nerve cell culture medium and cultivates 30--45 days；After cell mixing is inoculated, the method taking half amount to change liquid in every 10 days changes mixed nerve cell culture medium；
C, the separation of naked mole microglia and purification are cultivated:
The cell mixing isothermal vibration cultivated in step B is cultivated, constant temperature 35 ± 2 DEG C, concussion cultured cells suspension is placed in the culture dish not being coated with left-handed poly-D-lysine and makes cell attachment more than 10 minutes, very easily adherent microglia is made to be affixed on bottom plate, weak vibrations plate, to remove other adherent cell conditioned medium loosely, is changed and is continued in fresh mixed nerve cell culture medium to cultivate；
Described microglia culture medium, it is preferred to add the Neurobasal (namely the volume ratio of Neurobasal:B27 is 50:1) that percentage by volume is 2%B27。
Described mixed nerve cell culture medium, it is possible to for conventional containing serum mixed nerve cell culture fluid, for instance low sugar DMEM etc.。Preferred mixed nerve cell culture medium is the low sugar DMEM containing 10% percentage by volume hyclone。
The parameter of three described gas incubators is set to: temperature controls at 35 ± 2 DEG C, and oxygen concentration is 5%, and gas concentration lwevel is 5 ± 1%, and humidity is 96 ± 2%。
Further, in step A, take out raw 1-7 days naked mole brains, divest selective separating tissue after meninges, with microscissors, tissue shear is broken to 1mm3, after adding the mixing of tissue digestion liquid, put it in three gas incubators and digest。It is subsequently adding mixed nerve cell culture medium and terminates digestion, after centrifugal segregation supernatant, by resuspended for cell precipitation and blow and beat into single cell suspension and plant in the T25 culture bottle not being coated hypothallus。
Further, in step A, described tissue digestion liquid is composition is: pancreatin mass concentration is 0.25%, and DNA enzymatic I concentration is 0.1mg/ml, and digestion time is digest 30 minutes at 37 DEG C。
Further, in step A, described parameter of noncentricity is 500rpm under room temperature condition, 5 minutes。
Further, in step B, cultivate 42 days with neurocyte mixed culture medium being incubated in three gas incubators。
Further, in step C, concussion incubation keeps cultivate bottleneck and screws to reduce the exchange of bottle inner air and outer air。
Further, in step C, described constant-temperature table major parameter is set to: temperature is 35 DEG C, and rotating speed is 150rpm。
Further, in step C, described concussion incubation time is 4 hours
Further, in step C, until when needing microglia functional study, then within 30 minutes, it is replaced by microglia in advance and cultivates and cultivate based in three gas incubators。
Further, in step C, the cell attachment time is 15 minutes。
Further, the parameter of described three gas incubators is set to: temperature controls at 35 ± 2 DEG C, and oxygen concentration is 5%, and gas concentration lwevel is 5 ± 1%, and humidity is 96 ± 2%。
The beneficial effects of the present invention is: 1. the multiple culture medium of comprehensive use separates from newborn naked mole cerebral cortex and purification cultivation microglia, has found out the reasonable cultural method of the Rodents mammal naked mole microglia being suitable to alternating temperature。When we have found that 5% oxygen concentration, cell state can be kept preferably, and can keep increasing；2. operational approach is simple, has higher repeatability, it is possible to obtain the microglia of purity more than 98%；3. by using the Neurobasal serum-free medium of the B27 adding 2%, it is possible to the effective propagation ensureing microglia and vigor, also allow for the follow-up pharmacology experiment to microglia and study。
In sum, the inventive method can be easy, efficient, economical the normal naked mole cortical neuron of a large amount of functional activity of acquisition, cultivation under hypoxia condition ensure that the biological characteristics that this cell still can be maintained under body state in ex vivo environment, consequently facilitating directly study the special physiological function of naked mole microglia in pure in-vitro cell culture model further, thus for exploring biological mechanism therein and being applied to the theoretical foundation that clinical relevant provides important。
Accompanying drawing illustrates:
Fig. 1 is the mixed nerve cell of In vitro culture；
Fig. 2 is the microglia of external mixing under ordinary optical microscope；Wherein A is microglia under 10 times of common light microscopics, and B is microglia under 20 times of light microscopics；
Fig. 3 is that the microglia immunocytochemical stain that purification is cultivated is identified, the amalgamation result of wherein A is Iba1, B to be Hoechst, C be Iba1 and Hoechst two kinds dyeing picture。
Detailed description of the invention:
Below in conjunction with the drawings and specific embodiments, the invention will be further described。Should be understood that following example are merely to illustrate the present invention not for limiting the scope of the present invention。
Embodiment 1: naked mole microglia separation purification and cultivation
1, experiment material
The naked mole of cleaning grade of birth 1-7 is provided by Second Military Medical University, PLA's Experimental Animal Center。Newborn naked mole is placed in-20 DEG C of slabs carry out anesthesia and be placed in 75% ethanol immersion 10min, then break end, and with penicillin 100U/ml and the naked mole head surface of 0.1mg/ml streptomycin mixed liquor wiping, then cut off skin of head and skull, brain is peeled off in the dissection liquid being placed in pre-cooling。
DNA enzymatic I is purchased from Solarbio bio tech ltd, the available from Sigma such as trypsin, mycillin mixed liquor, DMEM, low sugar DMEM, source, Australia hyclone, Neurobasal culture medium, the B27 serum-free interpolation factor etc. are purchased from ThermoFisherScientific company, and culture dish, T25 and T75 culture bottle is purchased from Corning company。
Mixed nerve cell culture media component is the hyclone that low sugar DMEM culture medium contains that volume fraction is 10%, and oligodendrocyte precursor cells pure medium is add the Neurobasal culture medium that volume fraction is 2%B27。
2, naked mole oligodendrocyte precursor cells separation purification and cultural method:
1. collect naked mole cerebral cortex epoxy glue cell plastid: take out 2, raw 1-7 days naked mole brains, obtain cortical tissue after divesting meninges and be placed in 1ml and dissect in liquid, with microscissors, tissue shear is broken to 1mm3,Add Digestive system (mass fraction be 0.25% pancreatin 1ml, the wherein final concentration of 0.1mg/ml of DNA enzymatic I) mixing after, 30min is digested in 37 DEG C, then digestion is terminated with mixed nerve cell culture medium, when 500rpm centrifugal 5 minutes to remove supernatant, then add resuspended for cell precipitation and carefully blow and beat into single cell suspension (trying one's best avoids bubble to produce) in 5ml mixed nerve cell culture medium。Subsequently by single cell suspension according to 1 × 106Density kind is in the T25 culture bottle being coated with left-handed poly-D-lysine, and every bottle of culture fluid volume is 5ml。
2. the cultivation of naked mole cerebral cortex epoxy glue cell plastid: the cell that will obtain in 1., cultivates 42 days in three gas incubators with mixed nerve cell culture medium。After cell is inoculated, within every 7 days, take half amount to change liquid method and change culture medium。
3. the separation of naked mole microglia and purification are cultivated: by the epoxy glue cell plastid cultivated in 2. (as shown in Figure 1, circular is microglia) shake by the rotating speed of 150rpm in 35 DEG C of isothermal vibration shaking tables and cultivate 4 hours, concussion incubation keeps cultivate bottleneck and screws to reduce the exchange of bottle inner air and outer air。Carefully draw supernatant, in the centrifugal 10min of 500rpm, abandon supernatant and by Neurobasal/B27 (Neurobasal:B27=50:1) culture medium fresh for 5ml by resuspended for cell one-tenth single cell suspension, then according to 1 × 106Density kind is in the culture dish not being coated with hypothallus or culture bottle, and by the cell kind in supernatant in the culture dish being coated with left-handed poly-D-lysine。After it is adherent, undertaken cultivating (Neurobasal:B27=50:1) 24 hours by Neurobasal/B27 culture medium, the little glial precursor cell of purification can be obtained, it is incubated at 35 ± 2 DEG C, oxygen concentration is 5%, gas concentration lwevel is 5 ± 1%, and humidity is in the three gas incubators of 96 ± 2%。
Embodiment 2: the qualification of naked mole microglia
Adopt the qualification of the naked mole microglia of embodiment 1 gained: utilize 4% paraformaldehyde that the microglia being in proliferated culture medium is fixed, and combining form is identified, immunity refines the methods such as chemistry and identifies。
1, morphocytology is identified:
Authentication method is (ChungWS, ClarkeLE, WangGX described in document such as, StaffordBK, SherA, ChakrabortyC, JoungJ, FooLC, ThompsonA, ChenC, SmithSJ, BarresBA.AstrocytesmediatesynapseeliminationthroughMEGF1 0andMERTKpathways.Nature, 2013,504 (7480): 394-400.)。
Result is as in figure 2 it is shown, under light microscopic, microglia cell space is rounded or oval, and having no around cell space time unactivated has projection。
2, immunocytochemistry is identified:
Described in authentication method such as document (E,AriasC.Cholesterol-inducedastrocyteactivationisassociatedwithincreasedamyloidprecursorproteinexpressionandprocessing.Glia2015,63:2010–2022.)。
Result is as shown in Figure 3, the microglia of differential velocity adherent purification is carried out creep plate experiment, after serum-free medium is cultivated 24 hours, utilize 4% paraformaldehyde to be fixed by cell, then utilize the antibody of anti-microglia surface specific antigen Iba1 to carry out immunocytochemical stain。Result shows, in serum-free medium, cell can keep Iba1 positive (green fluorescence)。
According to above-mentioned experimental result, the naked mole microglia of separation purification of the present invention possesses typical little glial precursor cellular morphology, positive in Iba1, can be held round or ellipsoidal structure time unactivated。And good vegetative state can be kept in serum-free medium。
Below the preferred embodiment of the invention has been illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement, these equivalent modification or replacement under the premise without prejudice to the invention spirit and be all contained in the application claim limited range。
Claims (10)
1. a naked mole microglia cultural method, comprises the following steps:
A, collect naked mole cerebral cortex cell mixing:
Separate birth 1-7 Tian Luo mole brain cortical tissue, tissue is shredded, after adding the mixing of tissue digestion liquid, puts it in three gas incubators and digest；It is subsequently adding mixed nerve cell culture medium and terminates digestion, after centrifugal segregation supernatant, by resuspended for cell precipitation and blow and beat into single cell suspension and plant in culture bottle；
B, naked mole cerebral cortex cell mixing cultivation:
The cell mixing obtained by step A, cultivates 30--45 days in three gas incubators with mixed nerve cell culture medium；After cell mixing is inoculated, the method taking half amount to change liquid in every 10 days changes mixed nerve cell culture medium；
C, the separation of naked mole microglia and purification are cultivated:
The cell mixing isothermal vibration cultivated in step B is cultivated, constant temperature 35 ± 2 DEG C, concussion cultured cells suspension is placed in the culture dish not being coated with left-handed poly-D-lysine and makes cell attachment more than 10 minutes, removes cell conditioned medium, change and fresh mixed nerve cell culture medium continues cultivate；
Until during to microglia functional study, within 20-40 minute, being replaced by microglia cultivation in advance and cultivate based in three gas incubators；
Described microglia culture medium, is the Neurobasal of 2%B27 for adding percentage by volume；
The parameter of three described gas incubators is set to: temperature controls at 35 ± 2 DEG C, and oxygen concentration is 5%, and gas concentration lwevel is 5 ± 1%, and humidity is 96 ± 2%。
2. the naked mole microglia cultural method of one according to claim 1, it is characterised in that in step A, described tissue digestion liquid is mass concentration 0.25% pancreatin and mixed liquor that concentration is 0.1mg/mlDNA enzyme I。
3. the naked mole microglia cultural method of one according to claim 1, it is characterised in that described mixed nerve cell culture medium is the low sugar DMEM containing percentage by volume 10% hyclone。
4. the naked mole microglia cultural method of one according to claim 1, it is characterised in that in step A, takes out raw 1-7 days naked mole brains, divests selective separating tissue after meninges, with microscissors, tissue shear is broken to 1mm3, after adding the mixing of tissue digestion liquid, put it in three gas incubators and digest；Digestion condition is digest 30 minutes at 37 DEG C。
5. the naked mole microglia cultural method of one according to claim 1, it is characterised in that in step A, described parameter of noncentricity is 500rpm under room temperature condition, 5 minutes。
6. the naked mole microglia cultural method of one according to claim 1, it is characterised in that in step B, closes culture medium culturing with mixed neurocyte and cultivates 42 days in three gas incubators。
7. the naked mole microglia cultural method of one according to claim 1, it is characterised in that in step C, the cell attachment time is 15 minutes。
8. the naked mole microglia cultural method of one according to claim 1, it is characterised in that in step C, keeps cultivating bottleneck and screws to reduce the exchange of bottle inner air and outer air in concussion incubation；Described constant-temperature table major parameter is set to: temperature is 35 DEG C, and rotating speed is 150rpm。
9. the naked mole microglia cultural method of one according to claim 1 or 8, it is characterised in that in step C, described concussion incubation time is 4 hours。
10. the naked mole microglia cultural method of one according to claim 1, it is characterised in that the parameter of described three gas incubators is set to: temperature controls at 35 ± 2 DEG C, and oxygen concentration is 5%, and gas concentration lwevel is 5 ± 1%, and humidity is 96 ± 2%。
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| CN109722419A (en) * | 2017-10-29 | 2019-05-07 | 复旦大学 | A method of oligodendrocyte precursor cells are obtained and cultivated using micro nerve fiber |
| CN111925989A (en) * | 2020-09-14 | 2020-11-13 | 苏州良辰生物医药科技有限公司 | Culture method of microglia |
| CN113924362A (en) * | 2019-03-29 | 2022-01-11 | 新加坡科技研究局 | Microglial-rich brain organoids |
| CN114480282A (en) * | 2022-01-17 | 2022-05-13 | 安宁市第一人民医院 | Method for establishing oxidative stress response cell model of neurodegenerative disease caused by proinflammatory cytokines |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109722419A (en) * | 2017-10-29 | 2019-05-07 | 复旦大学 | A method of oligodendrocyte precursor cells are obtained and cultivated using micro nerve fiber |
| CN109722419B (en) * | 2017-10-29 | 2023-12-01 | 复旦大学 | Method for obtaining and culturing oligodendrocyte precursor cells by utilizing trace nerve tissue |
| CN113924362A (en) * | 2019-03-29 | 2022-01-11 | 新加坡科技研究局 | Microglial-rich brain organoids |
| CN111925989A (en) * | 2020-09-14 | 2020-11-13 | 苏州良辰生物医药科技有限公司 | Culture method of microglia |
| CN114480282A (en) * | 2022-01-17 | 2022-05-13 | 安宁市第一人民医院 | Method for establishing oxidative stress response cell model of neurodegenerative disease caused by proinflammatory cytokines |
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