CN109517795A - A method of obtaining pig stem cell of neural crest - Google Patents
A method of obtaining pig stem cell of neural crest Download PDFInfo
- Publication number
- CN109517795A CN109517795A CN201811522213.5A CN201811522213A CN109517795A CN 109517795 A CN109517795 A CN 109517795A CN 201811522213 A CN201811522213 A CN 201811522213A CN 109517795 A CN109517795 A CN 109517795A
- Authority
- CN
- China
- Prior art keywords
- stem cell
- culture
- neural crest
- cell
- culture solution
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides, bases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Abstract
The present invention relates to a kind of methods for obtaining pig stem cell of neural crest, pig neck fat group is taken to be woven in chopping and stationary culture in culture solution, isolated primary pig stem cell of neural crest, by originally culture, the amplification cultivation of pig stem cell of neural crest and etc. obtain pig stem cell of neural crest.Pass through a large number of experiments and optimization, set up a set of effective pig stem cell of neural crest isolation and purification method, the method of the present invention separation stem cell manipulation is simple, reproducible, high survival rate, after external 10 amplification cultivations, cell still has stronger stem cell properties, can meet the needs of experimental study, is easy to cultivate, stem cell of neural crest not only can be rapidly and efficiently obtained, and the stem cell obtained has the faster proliferation speed of growth and stronger differentiation potential.It improves stem cell and carries out the speed and efficiency repaired in therapeutic process to neurodevelopment and damaged nerve tissue again.The successful implementation of this method can lay a solid foundation for the clinical application of stem cell of neural crest, and be possible to carry out developmental research as stem cell of neural crest drug.
Description
Technical field
The present invention relates to a kind of stem cell acquisition methods, the especially extraction of acquisition pig stem cell of neural crest and culture side
Method.
Background technique
Neural crest be come out from nerve channel back outside migration vertebrate embryos early stage be located at nerve channel and ectoderm it
Between two longitudinal cell bands.During biological development, the nerve of neural crest cell migration generates the thin of a variety of mammals
Born of the same parents' type such as neuron, Deiter's cells or melanocyte.It is divided into the head zone of the neural crest cell of vertebrate
Ectoderm and mesoblastema.Neuroectodermal cells between ectoderm and neural furrow rim are constituted on the outside of the back of nerve channel
The two banded cell ropes arranged in parallel with nerve channel, extend to tail portion from midbrain plane.Thus the neural crest moved out
Stem cell is widely distributed.From the perspective of stem cell biology, neural crest is the ideal source of multipotent adult stem cells.
Stem cell of neural crest and its derivative spread it is outer, in, interior three germinal layers, be present in the difference of all ages and classes tissue
Position, such as body early embryo, adult hair follicle, enteric nervous system position.Stem cell of neural crest can be sent out in various adult tissues
Existing, there is extremely strong plasticity.Ex vivo nerve ridge stem cell shows as the stem cell of multipotency self-renewing, can be divided into more
Kind cell.Stem cell of neural crest can stable massive amplification in vitro.In addition, either chicken embryo chimera, or by Wnt1,
The cell lineage tracer that Sox10, tPA, Cre recombinase drive the nerve cell of PO protein promoter label to be formed, shows
Neural crest derived stem cells have the potential of Multidirectional Differentiation.
Stem cell of neural crest quantity is sufficient, it is from a wealth of sources, show stronger Proliferation, Differentiation ability, be not related to law human relations
Reason, without apparent rejection, at present increasingly by the attention of researcher.The NSC system established at present is most
From mouse, and there is apparent species variations between mouse and people, and for disease model, pig is more closer than mouse
People, the stem cell of neural crest model of pig is used to do drug screening, human diseases is studied, the effect of stem-cell therapy is more excellent
Show, but not yet occur the stem cell of neural crest isolation technics of pig and method in the prior art, it influences subsequent experimental and is ground with treatment
Study carefully.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of methods for efficiently obtaining pig stem cell of neural crest.
In order to solve the above technical problems, the technical solution adopted by the present invention is that: it is a kind of obtain pig stem cell of neural crest side
Method, comprising steps of
Step 1, the separation of pig stem cell of neural crest,
Take I-type collagen be placed in culture dish apply sample it is uniform, by above-mentioned culture dish be placed in 37-37.5 DEG C, 5% carbon dioxide train
It supports stand-by in case；The pig neck fat group that will acquire, which is woven in culture solution, shreds；Using culture solution by culture dish surface wettability；It will
The pig neck fat tissue of chopping is placed in culture solution, covers the coverslip of sterilizing；Culture solution is supplemented, in 37-37.5 DEG C, 5% 2
Stationary culture in carbonoxide incubator, the free cell colony out of adipose tissue is primary stem cell of neural crest；
Step 2, originally culture,
Continuation is cultivated in culture solution, and the 4th culture day, which inhales, abandons cell culture fluid；Dropwise addition phosphate buffer embathes adherent thin
Born of the same parents inhale and abandon cleaning solution, and Accutase digestive juice is added, and 37-37.5 DEG C of digestion to 80% or more attached cell falls off；Culture is added
Liquid terminates digestion, and cell is reached other culture dishes；
Step 3, the amplification cultivation of pig stem cell of neural crest,
It is inhaled when the cell density of originally culture reaches 90% or more and abandons culture solution, PBS cleaning solution embathes attached cell, inhales to abandon and wash
Liquid is washed, Accutase digestive juice is added, 37-37.5 DEG C of digestion to 80% or more patch attached cell falls off, culture solution termination is added and disappears
Change, carries out sub-bottle and pass on amplification cultivation.
Above-mentioned steps one, Step 2: culture solution described in step 3 is the DMEM culture solution of the fetal calf serum of 5%-15%.
Further, Step 1: Step 2: culture solution described in step 3 is containing final concentration 1mmol/L pyruvic acid
Sodium, 0.05mg/ml uridine, 50U/ml penicillin, 50U/ml streptomysin and 10% fetal calf serum DMEM culture solution.
Further, in step 1, take Ι collagen type 10-20 μ L in 35 millimeters of culture dish, with disposable scraper plate
It applies sample and hangs even, be placed in 37 DEG C, be stand-by after 1h in 5% carbon dioxide incubator.
Further, in step 1, after supplementing culture solution, the stationary culture one week in 37 DEG C, 5% carbon dioxide incubator,
Replace fresh culture solution；A culture solution is replaced weekly；After continuing culture 3-4 weeks, the free cell colony out of adipose tissue
As primary stem cell of neural crest.
Further, in step 2, Accutase digestive juice is added, it is 37 DEG C digestion 5-10 minutes to 80% or more adherent thin
Born of the same parents fall off.
Further, in step 3, add Accutase digestive juice, it is 37 DEG C digestion 5-10 minutes to 80% or more adherent thin
Born of the same parents fall off.
The present invention provides a kind of method of effective acquisition pig stem cell of neural crest, separation including pig stem cell of neural crest,
Originally culture, the amplification cultivation of pig stem cell of neural crest and etc..Pass through a large number of experiments and optimization, it is established that a set of effective pig
Stem cell of neural crest isolation and purification method, the method for the present invention separation stem cell manipulation is simple, reproducible, high survival rate, through body
After outer 10 amplification cultivations, cell still has stronger stem cell properties, can meet the needs of experimental study, is easy to cultivate,
The stem cell that not only can rapidly and efficiently obtain stem cell of neural crest, and obtain has the faster proliferation speed of growth and stronger
Differentiation potential.It improves stem cell and carries out the speed and effect repaired in therapeutic process to neurodevelopment and damaged nerve tissue again
Rate.The successful implementation of this method can lay a solid foundation for the clinical application of stem cell of neural crest, and be possible to it
Developmental research is carried out as stem cell of neural crest drug.
Detailed description of the invention
Fig. 1 is 40 times of morphology pictures of 1st generation pork fat source stem cell of neural crest；
Fig. 2 is the 5th generation pork fat source 40 times of morphology pictures of stem cell of neural crest；
Fig. 3 is the 7th generation pork fat source 40 times of morphology pictures of stem cell of neural crest；
Fig. 4 is the 10th generation pork fat source 40 times of morphology pictures of stem cell of neural crest；
Fig. 5 is pork fat source stem cell of neural crest Oct4 genetic immunization fluorescent staining picture；
Fig. 6 is pork fat source stem cell of neural crest Sox2 genetic immunization fluorescent staining picture；
Fig. 7 is pork fat source stem cell of neural crest Sox10 genetic immunization fluorescent staining picture；
Fig. 8 is the present invention from pork fat source stem cell of neural crest, at osteogenic induction the 21st day, carries out alizarin to the cell induced
The picture of red colouring；
Fig. 9 is that the present invention is immunized the cell induced from pork fat source stem cell of neural crest at osteogenic induction the 21st day
The picture of fluorescent staining；
Figure 10 is the present invention from pork fat source stem cell of neural crest, Adipogenic induction the 8th day, the cell induced is immunized
The picture of fluorescent staining；
Figure 11 is the present invention from pork fat source stem cell of neural crest, Adipogenic induction the 8th day, carries out oil red to the cell induced
The picture of dyeing；
Figure 12 is that the present invention exempts from the cell induced from pork fat source stem cell of neural crest at chondrocyte induction the 21st day
The picture of epidemic disease fluorescent staining；
Figure 13 is the present invention from pork fat source stem cell of neural crest, at chondrocyte induction the 21st day, carries out HE to the cell induced
The picture of dyeing.
Specific embodiment
A kind of a kind of method for acquisition pig stem cell of neural crest that typical embodiment provides of the present invention, comprising steps of
Step 1, the separation of pig stem cell of neural crest,
Take I-type collagen be placed in culture dish apply sample it is uniform, by above-mentioned culture dish be placed in 37-37.5 DEG C, 5% carbon dioxide train
It supports stand-by in case, can guarantee that pig stem cell of neural crest is stablized adherent, and can be improved adherent speed using above-mentioned I-type collagen
Degree.The pig neck fat group that will acquire, which is woven in culture solution, shreds, and experiment Adipose Tissue takes its normal growth pig Cervical Vessels
It is abundant to locate tissue, operation is not killed in experimentation.Using culture solution by culture dish surface wettability；By the pig neck of chopping
Adipose tissue is placed in culture solution, covers the coverslip of sterilizing；Culture solution is supplemented, in 37-37.5 DEG C, 5% carbon dioxide incubator
Middle stationary culture, the free cell colony out of adipose tissue is primary stem cell of neural crest.
Step 2, originally culture,
Continuation is cultivated in culture solution, and the 4th culture day, which inhales, abandons cell culture fluid；Dropwise addition phosphate buffer embathes adherent thin
Born of the same parents inhale and abandon cleaning solution, and Accutase digestive juice is added, and 37-37.5 DEG C of digestion to 80% or more attached cell falls off；Culture is added
Liquid terminates digestion, and cell is reached other culture dishes.
Step 3, the amplification cultivation of pig stem cell of neural crest,
It is inhaled when the cell density of originally culture reaches 90% or more and abandons cell culture fluid, PBS cleaning solution embathes attached cell, inhales
Cleaning solution is abandoned, Accutase digestive juice is added, 37-37.5 DEG C of digestion to 80% or more patch attached cell falls off, and complete culture solution is added
Digestion is terminated, sub-bottle is carried out and passes on amplification cultivation.
Above-mentioned steps one, Step 2: culture solution described in step 3 is the DMEM culture solution of the fetal calf serum of 5%-15%.
Preferably, complete culture solution described in above-mentioned steps be containing final concentration 1mmol/L Sodium Pyruvate, 0.05mg/ml uridine,
The DMEM culture solution of 50U/ml penicillin, 50U/ml streptomysin and 10% fetal calf serum.
In a preferred embodiment, it in step 1, takes Ι collagen type 10-20 μ L in 35 millimeters of culture dish, uses
Disposable scraper plate applies sample and hangs even, is placed in 37 DEG C, is stand-by after 1h in 5% carbon dioxide incubator.
In a preferred embodiment, quiet in 37 DEG C, 5% carbon dioxide incubator after supplementing culture solution in step 1
Culture one week is set, fresh culture solution is replaced；A culture solution is replaced weekly；After continuing culture 3-4 weeks, adipose tissue is free out
Cell colony be primary stem cell of neural crest.
In a preferred embodiment, in step 2, Accutase digestive juice, 37 DEG C of digestion 5-10 minutes to 80% are added
The above attached cell falls off.
In a preferred embodiment, in step 3, add Accutase digestive juice, 37 DEG C of digestion 5-10 minutes to 80% with
Upper patch attached cell falls off.
The stem cell of neural crest obtained according to the present invention plays important work in neurodevelopment and reparation damaged nerve tissue
With.Injured cerebral tissue can be repaired and be replaced to stem cell of neural crest transplanting by rebuilding partial loop and function；Also it can be used as
Genophore, the gene therapy for intracranial tumors and other neurological diseases；Furthermore stem cell of neural crest can also be used to judge
Drug effect and drug toxicity.
Claimed technical solution is described further below by some embodiments.But embodiment
It is to be used to explain the present invention embodiment, without departing from the range of present subject matter, the scope of the present invention is not by the implementation
The restriction of example.Unless otherwise specifically stated, material therefor, reagent can be obtained from the commercially produced product of this field in embodiment.
Following embodiment experiment Adipose Tissue used is derived from the abundant place's tissue of normal growth pig Cervical Vessels, tissue extraction
It is washed away by 75% ethyl alcohol, washes away 3 times for subsequent experimental through phosphate buffer.In implementation process of the present invention, there is no kills to deposit
The operation of live hog.
Embodiment 1
One, the separation of pig stem cell of neural crest:
Take 10 μ L of Ι collagen type in the culture dish of 35mm, it is even with disposable scraper plate painting sample extension, it is placed in 37 DEG C, 5% titanium dioxide
It is stand-by after 1h in carbon incubator；Pig neck fat tissue is drilled through by small-sized skin puncher, is washed away through 75% ethyl alcohol, takes centre not
The tissue impregnated through alcohol, phosphate buffer wash away 3 times, shred in the DMEM culture solution of 10% fetal calf serum；It takes
The DMEM culture solution of the fetal calf serum of 2 drops 10% is added in the culture dish of 35mm；The Adipose Tissue of chopping is placed in the culture solution
In, the coverslip of sterilizing is covered, with the reverse side flicking of tweezers, is allowed to contact with bottom, whether microscopically observation has connective group
It knits.In 37 DEG C, 5% carbon dioxide incubator after stationary culture 1h, 1ml culture solution is added, added-time pipette tips contact coverslip；?
37 DEG C, in 5% carbon dioxide incubator after stationary culture 1h, continuously add 1ml culture solution；In 37 DEG C, 5% carbon dioxide incubator
Middle stationary culture after a week, replaces fresh culture solution；A culture solution is replaced weekly；After continuing culture 3-4 weeks, adipose tissue
Free cell colony out is primary stem cell of neural crest.
Two, originally culture:
Continuation is cultivated in the DMEM culture solution of 10% fetal calf serum, and the 4th culture day, which inhales, abandons cell culture fluid；Phosphoric acid is added dropwise
It is multiple that salt buffer embathes attached cell, inhales and abandons cleaning solution, adds the Accutase digestive juice of 1ml, 37 DEG C of digestion 5 minutes or so are extremely
80% or more attached cell falls off；3 times of DMEM culture solutions containing 10% fetal calf serum are added and terminate digestion, cell is reached into 35mm training
Support ware.
Three, the amplification cultivation of pig stem cell of neural crest:
It is inhaled when the cell density of originally culture reaches 90% or more and abandons cell culture fluid, it is more that PBS cleaning solution embathes attached cell
It is secondary, it inhales and abandons cleaning solution, add the Accutase digestive juice of 1ml, 37 DEG C of digestion 5 minutes or so to 80% or more attached cell falls off.Add
Enter 3 times of complete nutrient solutions and terminate digestion, is passed on 1:3, cell is reached into 35mm culture dish.
Culture solution described in above step use containing final concentration 1mmol/L Sodium Pyruvate, 0.05mg/ml uridine,
The DMEM culture solution of 50U/ml penicillin, 50U/ml streptomysin and 10% fetal calf serum.
Embodiment 2
One, the separation of pig stem cell of neural crest:
Take 20 μ L of Ι collagen type in the culture dish of 35mm, it is even with disposable scraper plate painting sample extension, it is placed in 37.5 DEG C, 5% dioxy
Change stand-by after 1h in carbon incubator；Pig neck fat tissue is drilled through by small-sized skin puncher, is washed away through 75% ethyl alcohol, takes centre
Without the tissue that alcohol impregnated, phosphate buffer washes away 3 times, shreds in the DMEM culture solution of 10% fetal calf serum；It takes
The DMEM culture solution of the fetal calf serum of 2 drops 10% is added in the culture dish of 35mm；The Adipose Tissue of chopping is placed in the culture solution
In, the coverslip of sterilizing is covered, with the reverse side flicking of tweezers, is allowed to contact with bottom, whether microscopically observation has connective group
It knits.In 37.5 DEG C, 5% carbon dioxide incubator after stationary culture 1h, 1ml culture solution is added, added-time pipette tips contact coverslip；
In 37.5 DEG C, 5% carbon dioxide incubator after stationary culture 1h, 1ml culture solution is continuously added；In 37.5 DEG C, 5% carbon dioxide
Stationary culture after a week, replaces fresh culture solution in incubator；A culture solution is replaced weekly；After continuing culture 3-4 weeks, rouge
The free cell colony out of fat tissue is primary stem cell of neural crest.
Two, originally culture:
Continuation is cultivated in the DMEM culture solution of 10% fetal calf serum, and the 4th culture day, which inhales, abandons cell culture fluid；Phosphoric acid is added dropwise
It is multiple that salt buffer embathes attached cell, inhales and abandons cleaning solution, adds the Accutase digestive juice of 1ml, and 37.5 DEG C digest 5 minutes or so
It falls off to 80% or more attached cell；3 times of DMEM culture solutions containing 10% fetal calf serum are added and terminate digestion, cell is reached into 35mm
Culture dish.
Three, the amplification cultivation of pig stem cell of neural crest:
It is inhaled when the cell density of originally culture reaches 90% or more and abandons cell culture fluid, it is more that PBS cleaning solution embathes attached cell
It is secondary, it inhales and abandons cleaning solution, add the Accutase digestive juice of 1ml, 37.5 DEG C of digestion 5 minutes or so to 80% or more attached cell falls off.
3 times of complete nutrient solutions are added and terminate digestion, is passed on 1:3, cell is reached into 35mm culture dish.
Culture solution is containing final concentration 1mmol/L Sodium Pyruvate, 0.05mg/ml uridine, 50U/ml penicillin, 50U/ml
The DMEM culture solution of streptomysin and 10% fetal calf serum.
Embodiment 3
Difference with documents 1 is, Step 1: containing final concentration 1mmol/L pyruvic acid Step 2: using in step 3
Sodium, 0.05mg/ml uridine, 50U/ml penicillin, 50U/ml streptomysin and 5% fetal calf serum DMEM culture solution.
Embodiment 4
Difference with documents 1 is, Step 1: containing final concentration 1mmol/L pyruvic acid Step 2: using in step 3
Sodium, 0.05mg/ml uridine, 50U/ml penicillin, 50U/ml streptomysin and 15% fetal calf serum DMEM culture solution.
It is detection and the qualification result of the pig stem cell of neural crest of embodiment 1 below.
1. the Immunofluorescence test of pig stem cell of neural crest
Cell is washed twice with PBS, every time washing 5 minutes.Cell is fixed 15 minutes with 4% paraformaldehyde at room temperature, with
It uses 0.25%Triton X-100 permeabilization 15 minutes afterwards.After washing three times, with the PBS closing cell for containing 10% lowlenthal serum
30 minutes, and be incubated overnight at 4 DEG C with primary antibody, the cell of positive staining is observed with the secondary antibody of suitable fluorescent marker.It is pure
After the pig stem cell of neural crest immunofluorescence dyeing of change, confocal microscopy endonuclear albumen OCT4, Sox2,
Corresponding position where Sox10 (such as Fig. 5-Fig. 7), it was demonstrated that the pig nerve of feature is efficiently enriched using the method for embodiment 1
Ridge stem cell.
2. the Osteoblast Differentiation of pig stem cell of neural crest is identified
In each hole that sterility cover is slid to 24 orifice plates of insertion, 0.5ml(1ug/ml is smeared in every hole) fibronectin 3 hours 37
It is spare at DEG C.Pig stem cell of neural crest is separated with Accutase enzyme, it is rear to add the neutralization reaction of grown cultures liquid.With 1000rpm from
Make cell precipitation within the heart 10 minutes, so that cell is suspended with culture solution (containing 10% fetal calf serum, 1% penicillin/streptomycin), reach 7.4
× 104 cells/ml density.0.5ml cell suspending liquid is added in each hole of 24 orifice plates, at 37 DEG C, 5% carbon dioxide is trained
It supports and is incubated in case.It is adherent when reaching 50 ~ 70% after culture in 1 ~ 2 day, culture solution is replaced with Osteoblast Differentiation culture solution, is placed on
Continue to support in incubator.Liquid is changed with the fresh Osteoblast Differentiation culture solution of 0.5ml within every 4 days.After differentiation in 21 days, with containing 4% poly first
The PBS of aldehyde is fixed, and is saved and is dyed for immunostaining or alizarin red agent (ARS), to assess the deposition of osteocyte richness calcium.Fig. 8 is pig
Stem cell of neural crest changes at cellular morphology after osteogenic induction, it is seen that apparent Mineral nodules, it can be by its shape with Alizarin red staining
At calcium tubercle dye red.Fig. 9 is the picture that the osteoblast that stem cell of neural crest induces carries out immunofluorescence dyeing.
3. breaking up at rouge for pig stem cell of neural crest is identified
Sterile cover plate is placed in each hole of 24 orifice plates.Pig stem cell of neural crest and culture dish are divided using Accutase enzyme
From rear to add in culture solution and endonuclease reaction.With 1000rpm, centrifugation makes cell precipitation for 10 minutes, and addition culture solution (contains 10% tire
Cow's serum, 1% penicillin/streptomycin) so that cell is suspended again, make cell concentration 7.4 × 10 in culture solution4/ml.It will
0.5ml cell suspending liquid is assigned in each hole of 24 orifice plates.It is answered by cell after culture in 24 hours 100% adherent.If not yet
Have and reach, then should be carried out within every 2-3 days changing liquid with culture solution, until 100% adherent.Culture solution is removed from each hole afterwards, rouge is added
Fat breaks up culture solution, incubates in 5% carbon dioxide incubator at 37 DEG C.Every 4 days primary with culture solution is changed.By induction differentiation to the 8th
It tissue culture plate takes out, and discards culture solution, is gently washed 2 ~ 3 times with the PBS of 1.01 mm, pH 7.4, with 10% formal
Woods calcium liquid fixes cell 60min, inhales and abandons fixer, is cleaned 3 times with PBS, and oil red dye liquor is added in cell surface, dyes at room temperature
30min abandons dye liquor, and is rinsed 3 times with PBS, and culture plate is placed under inverted microscope and is observed.Figure 11 is pig stem cell of neural crest
Cellular morphology changes after adipogenic induction, it is seen that for most cells full of red oil droplet vacuole, Figure 10 is pig stem cell of neural crest
The cell induced carries out the picture of immunofluorescence dyeing.
4. the cartilage differentiation of pig stem cell of neural crest
D-MEM/F-12 basic culture solution is supplemented with 1:100 ratio in freshly prepared cartilage differentiation culture solution.With Accutase enzyme
Pig stem cell of neural crest is separated, it is rear to add the neutralization reaction of grown cultures liquid.By in culture solution 2.5 × 105A cell is transferred to
In 15ml conical pipe.Cell precipitation is made in 5 minutes with 200 × g centrifugation at room temperature.Culture solution is removed, with 0.5ml cartilage cell point
Changing culture solution makes cell suspend again, and 200 × g centrifugation makes its precipitating in 5 minutes at room temperature.Culture solution is not removed, and will be managed
Cap, which is unscrewed, swaps gas.The cell precipitated in pipe is cultivated in 5% carbon dioxide incubator at 37 DEG C.Using at
Chondrocyte induction culture solution culture, the fresh induction broth of replacement in every 2-3 days, detection is at cartilage differentiation result after 3 weeks.Figure 13
It is pig stem cell of neural crest into HE coloration result after chondrocyte induction.Figure 12 be the cartilage cell that induces of pig stem cell of neural crest into
The picture of row immunofluorescence dyeing.
Claims (6)
1. a kind of method for obtaining pig stem cell of neural crest, which is characterized in that comprising steps of
Step 1, the separation of pig stem cell of neural crest,
Take I-type collagen be placed in culture dish apply sample it is uniform, by above-mentioned culture dish be placed in 37-37.5 DEG C, 5% carbon dioxide train
It supports stand-by in case；The pig neck fat group that will acquire, which is woven in culture solution, shreds；Using culture solution by culture dish surface wettability；It will
The pig neck fat tissue of chopping is placed in culture solution, covers the coverslip of sterilizing；Culture solution is supplemented, in 37-37.5 DEG C, 5% 2
Stationary culture in carbonoxide incubator, the free cell colony out of adipose tissue is primary stem cell of neural crest；
Step 2, originally culture,
Continuation is cultivated in culture solution, and the 4th culture day, which inhales, abandons cell culture fluid；Dropwise addition phosphate buffer embathes adherent thin
Born of the same parents inhale and abandon cleaning solution, and Accutase digestive juice is added, and 37-37.5 DEG C of digestion to 80% or more attached cell falls off；Culture is added
Liquid terminates digestion, and cell is reached other culture dishes；
Step 3, the amplification cultivation of pig stem cell of neural crest,
It is inhaled when the cell density of originally culture reaches 90% or more and abandons culture solution, PBS cleaning solution embathes attached cell, inhales to abandon and wash
Liquid is washed, Accutase digestive juice is added, 37-37.5 DEG C of digestion to 80% or more patch attached cell falls off, culture solution termination is added and disappears
Change, carries out sub-bottle and pass on amplification cultivation；
Above-mentioned steps one, Step 2: culture solution described in step 3 is the DMEM culture solution of the fetal calf serum of 5%-15%.
2. according to the method described in claim 1, it is characterized by: Step 1: Step 2: culture solution described in step 3 is
Contain final concentration 1mmol/L Sodium Pyruvate, 0.05mg/ml uridine, 50U/ml penicillin, 50U/ml streptomysin and 10% tire ox
The DMEM culture solution of serum.
3. method according to claim 1 or 2, it is characterised in that: in step 1, take Ι collagen type 10-20 μ L in 35
In the culture dish of millimeter, sample is applied with disposable scraper plate and hangs even, 37 DEG C is placed in, is stand-by after 1h in 5% carbon dioxide incubator.
4. according to the method described in claim 3, it is characterized by: in step 1, after supplementing culture solution, in 37 DEG C, 5% dioxy
Change in carbon incubator stationary culture one week, replaces fresh culture solution；A culture solution is replaced weekly；After continuing culture 3-4 weeks,
The free cell colony out of adipose tissue is primary stem cell of neural crest.
5. according to the method described in claim 4, it is characterized by: Accutase digestive juice, 37 DEG C of digestion are added in step 2
It falls off to 80% or more attached cell within 5-10 minutes.
6. according to the method described in claim 5, it is characterized by: adding Accutase digestive juice, 37 DEG C of digestion 5- in step 3
Attached cell is pasted to 80% or more within 10 minutes to fall off.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811522213.5A CN109517795A (en) | 2018-12-13 | 2018-12-13 | A method of obtaining pig stem cell of neural crest |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811522213.5A CN109517795A (en) | 2018-12-13 | 2018-12-13 | A method of obtaining pig stem cell of neural crest |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109517795A true CN109517795A (en) | 2019-03-26 |
Family
ID=65796063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811522213.5A Pending CN109517795A (en) | 2018-12-13 | 2018-12-13 | A method of obtaining pig stem cell of neural crest |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109517795A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007030469A2 (en) * | 2005-09-06 | 2007-03-15 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Transplantable cell growth niche and related compositions and methods |
| CN106190967A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of autologous fat derived stem cell is for the preparation method of beautifying and antisenility |
| CN107475200A (en) * | 2017-08-08 | 2017-12-15 | 浙江大学 | A kind of separation, culture and the differentiation method of the stem cell of neural crest in DRGs source |
| CN108753693A (en) * | 2018-05-07 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of stem cell of neural crest preparation and its application |
-
2018
- 2018-12-13 CN CN201811522213.5A patent/CN109517795A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007030469A2 (en) * | 2005-09-06 | 2007-03-15 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Transplantable cell growth niche and related compositions and methods |
| CN106190967A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of autologous fat derived stem cell is for the preparation method of beautifying and antisenility |
| CN107475200A (en) * | 2017-08-08 | 2017-12-15 | 浙江大学 | A kind of separation, culture and the differentiation method of the stem cell of neural crest in DRGs source |
| CN108753693A (en) * | 2018-05-07 | 2018-11-06 | 广州沙艾生物科技有限公司 | A kind of stem cell of neural crest preparation and its application |
Non-Patent Citations (1)
| Title |
|---|
| 张迪等: "胎盘间充质干细胞研究进展", 《安徽医学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Quattrocelli et al. | Mouse and human mesoangioblasts: isolation and characterization from adult skeletal muscles | |
| CN101748096A (en) | Sub totipotential stem cell and preparation method and application thereof | |
| CN107022521A (en) | Decidua vera tissue freezing, the method recovered and be separately cultured mescenchymal stem cell | |
| KR102291271B1 (en) | Cardiac organoids, method for preparing the same and method for evaluating drug toxicity using the same | |
| CN105861428B (en) | A kind of induced fibroblast transdifferentiation is the induced medium and its application of cardiac muscle cell | |
| CN105039247B (en) | It is a kind of for induce stem cell at cartilage differentiation preparation and preparation method and application | |
| CN106282100A (en) | A kind of method of the purification tissue-derived Preadipocyte In Vitro of cattle fetal skeletal muscle | |
| CN104946590A (en) | Method for inducing Muse cells in adult bone marrow into neural precursor cells (NPCs) | |
| RU2433172C2 (en) | Method of obtaining homogenous population of stem cells and its application | |
| CN107475179A (en) | The separation of mouse synovial cell a kind of and cultural method | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| CN105106240B (en) | A kind of stem cell medicine and its purposes in vascular interventional treatment cerebral apoplexy | |
| CN108753711A (en) | A kind of method that human pluripotent stem cells induction generates mescenchymal stem cell | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN106350480A (en) | Method for purifying myoblast derived from bovine fetus skeletal muscle tissue | |
| CN106119191A (en) | A kind of placental villi plate mescenchymal stem cell and clinicization preparation method | |
| CN103396985A (en) | Method for inducing differentiation of human umbilical cord mesenchymal stem cells into hepatocytes and applications | |
| CN106399248A (en) | Method for inducing transdifferentiation of fibroblasts to nerve cells | |
| CN111575227B (en) | Method for establishing human-derived diabetic cardiomyopathy model | |
| CN108324993A (en) | A kind of stem cell complex, preparation method and the application of induction hair regeneration | |
| CN109852581A (en) | A kind of method for promoting stem cell to break up at rouge and its agents useful for same or kit | |
| CN108486039A (en) | The method that small molecule induction human adipose-derived stem cell is divided into interstitial glands | |
| CN105670989A (en) | Kit and method for effectively inducing somatic cell phenotype reprogramming | |
| CN103382458A (en) | Culture solution and method for inducing mesenchymal stem cells to differentiate into glomerular mesangial cells | |
| CN107164325B (en) | The preparation method and kit of the oligodendroglia in the source MSCs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190326 |