CN108060116A - A kind of extraction isolated culture method of tire mouse endothelial progenitor cells - Google Patents
A kind of extraction isolated culture method of tire mouse endothelial progenitor cells Download PDFInfo
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Abstract
The invention discloses a kind of isolated culture methods of tire mouse lung derived endothelial progenitor cells, it is impregnated including the uterus of becoming pregnant of pregnant mouse is put into penicillin and the dual anti-precooling PBS of streptomysin, in the plate for taking out tire mouse degree of being put into penicillin and the dual anti-precooling PBS of streptomysin, washing；It takes out lung tissue to be put into PBS and crush, with 0.25% pancreatin digestion step disrupting tissue of the above, with the DMEM medium cultures containing 10%FBS, centrifuges, take its precipitation, hanged with 2 culture mediums of EBM containing growth factor and the serum of 2%FBS, with 106/ ML cell concentrations are inoculated in 6 orifice plates, are placed in constant incubator and are cultivated, and when primary cell growth fusion is up to 80 90%, with 0.25% pancreatin had digestive transfer culture culture, until homogeneous form is presented in cell, complete culture.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of endothelial progenitor cells extract isolated culture method, special
It is not the extracting and developing and cultural method for being related to the endothelial progenitor cells that a kind of source is tire mouse.
Background technology
Endothelial progenitor cells (ENDOTHELIAL PROGENITOR CELLS, referred to as " EPCS ") are that vascular endothelial precursor is thin
Born of the same parents, energy directed differentiation are ripe endothelial cell, also known as Angioblast.During body ischemic, EPCS can be mobilized from marrow into
Enter peripheral blood, reach ischemic tissue and participate in angiogenesis.In vitro culture and the EPCS of amplification inputs is internal, it improves in Xun Huan
The quality and quantity of EPCS can promote the angiogenesis of ishemic part.Therefore, EPCS is migrated to treat ischemic blood vessels disease
One new approaches of disease.
Extraction for endothelial progenitor cells, document report mainly acquisition Cord blood or birth after all ages and classes animal
Marrow, peripheral blood, by density-gradient centrifugation method obtain mononuclearcell, be then inoculated in the coated blake bottle of particular matter or
External evoked culture is carried out in culture dish.
The key of endothelial progenitor cells clinical practice be seek its preferably it is tissue-derived, but endothelial progenitor cells raise, separation
Method with culture is there are larger difference, and obtained endothelial progenitor cells quantity is few, amplification efficiency and cell Proliferation are slower.And
The present invention takes new available sources to extract endothelial progenitor cells, that is, uses the lung tissue of tire mouse to be tissue-derived, is that endothelium ancestral is thin
Extraction separation, the culture of born of the same parents provides a kind of new practicable method.
The content of the invention
It is an object of the invention to provide a kind of extraction isolated culture method of tire mouse lung derived endothelial progenitor cells, the present invention
Method use the lung tissue of tire mouse as the new sources of endothelial progenitor cells, it is extracted, separation, culture obtain endothelial progenitor cells.
A kind of extracting and developing cultural method of tire mouse lung derived endothelial progenitor cells of the present invention, comprises the following steps：
(1) lungs of tire mouse are taken, are put into containing in the dual anti-precooling PBS of 500U-1000U penicillin and streptomysin, and will
Lung tissue crushes；
(2) with the disrupting tissue in pancreatin digestion step (1)；
(3) after having digested, with the DMEM medium cultures 15-18H containing FBS；
(4) non-adherent cell is then taken out, centrifuges, takes its precipitation；
(5) precipitation of step (4) with the EBM-2 culture mediums containing growth factor and the serum of FBS has been hanged, has been inoculated with concentration
In orifice plate, it is placed in constant incubator and cultivates；
(6) per changing within 2-4 days a not good liquor, when primary cell growth fusion reaches 80-90%, with pancreatin had digestive transfer culture culture,
Lasting passage is until cell is presented homogeneous form and completes culture.
The method of the invention described above, the tire mouse, the method obtained include：(1) beading sample is taken out after pregnant mouse is anaesthetized
It becomes pregnant uterus, is put into containing being impregnated in the dual anti-precooling PBS of 500U-1000U penicillin and streptomysin；(2) by after immersion by
After the umbilical cord tissue of connection tire mouse navel and placenta is isolated in pregnant uterus, tire mouse is put into containing 500U-1000U high intensity penicillin
In the plate of the precooling PBS dual anti-with streptomysin, tire mouse is obtained after washing.
In a particular embodiment, a kind of extraction isolated culture method of tire mouse lung derived endothelial progenitor cells of the invention leads to
Embodiments below realization is crossed, is comprised the following steps：
(1) beading sample will be taken out after the anesthesia of pregnant mouse to become pregnant uterus, be put into containing 500U-1000U penicillin and strepto-
It is impregnated in the dual anti-precooling PBS of element；
(2) after the umbilical cord tissue that the uterus of becoming pregnant after immersion is isolated to connection tire mouse navel and placenta, tire mouse, which is put into, to be contained
Have in the penicillin of each 500U-1000U and the plate of the dual anti-precooling PBS of streptomysin, washing；
(3) lungs of tire mouse are taken out, are put into containing in the dual anti-precooling PBS of 500U-1000U penicillin and streptomysin, and
Lung tissue is crushed；
(4) with the disrupting tissue in 0.25% pancreatin digestion step (3)；
(5) after having digested, with the DMEM medium cultures 16H containing 10%FBS；
(6) non-adherent cell is then taken out, centrifuges, takes its precipitation；
(7) precipitation of step (6) has been hanged with the EBM-2 culture mediums containing growth factor and the serum of 2%FBS, with 106/
ML cell concentrations are inoculated in 6 orifice plates, are placed in 37 DEG C, 5%CO2It is cultivated in constant incubator；
(8) not good liquor is changed within every 3 days, when primary cell growth fusion is up to 80-90%, with 0.25% pancreatin had digestive transfer culture
Culture, lasting passage is until cell is presented homogeneous form and completes culture.
In the above-described embodiment, method of the invention, in step (1), the immersion, soaking time is 3-5 minutes, institute
State the pregnant mouse that pregnant mouse reaches 15-19 days for pregnant age；Step is washed described in (2), and washing times are 2 times；It is broken described in step (3)
Broken tissue, broken tissue size is 0.5-1.0MM3；The digestion of pancreatin described in step (4) is to use substep digestion method, often
Step is placed in being incubated 10MIN in 37 DEG C of cell incubators, until tissue digestion completely；It is centrifuged described in step (6) and is
1000RPM, 5MIN.
In one embodiment, a kind of extracting and developing cultural method of tire mouse lung derived endothelial progenitor cells, including with
Lower step：
(1) pregnant age is reached to the pregnant mouse of 15-19 days, 10% chloral hydrate anesthesia is given, 75% disinfection wine is immediately placed in after anesthesia
It is impregnated 15 minutes in essence, moves into iuntercellular, four limbs are fixed by pregnant mouse abdomen upward.
(2) cut with Sterile ophthalmic and successively cut off pregnant mouse abdominal skin and mucous layer with tweezers, taken out beading sample and become pregnant son
Palace is put into containing being impregnated in the dual anti-precooling PBS of 500U-1000U high intensity penicillin and streptomysin, then moves into super-clean bench.
(3) uterus is cut off with eye scissors, removes amniotic fluid, pick net rete layer, isolate the navel of connection tire mouse navel and placenta
Band tissue, tire mouse is put into the plate containing the dual anti-precooling PBS of 500U-1000U high intensity penicillin and streptomysin, is washed
2 times.
(4) lungs of tire mouse are carefully taken out with eye scissors and tweezers, be put into containing 500U-1000U high intensity penicillin and
In the plate of the dual anti-precooling PBS of streptomysin, and it is shredded one by one.
(5) with the tissue in 0.25% pancreatin digestion step (4), using substep digestion method, 37 DEG C of cells are placed in per this
10MIN is incubated in incubator, until tissue digestion completely.
(6) after having digested, with the DMEM culture mediums containing 10%FBS, 16H is cultivated.
(7) and then afterwards non-adherent cell, 1000RPM, 5MIN are taken out, centrifugation takes its precipitation.
(8) precipitation in step (7) has been hanged with the EBM-2 culture mediums containing growth factor and serum (2%FBS), with
106/ ML cell concentrations are inoculated in 6 orifice plates, are placed in 37 DEG C, 5%CO2It is cultivated in constant incubator.
(9) change a not good liquor within every 3 days after, when primary cell growth fusion is up to 80-90%, digested with 0.25% pancreatin
Secondary culture, lasting passage is until homogeneous form, completion culture is presented in cell.
The endothelial progenitor cells that the method culture of the present invention obtains are identified that identification method is as follows：
1) endothelial progenitor cells morphological observation,
2) cellular immunofluorescence identification EPCS surface markers:CD34, CD133, CD31, VEGFR-2 antigen,
3) flow cytometry identification EPCS surface markers：CD34, CD133, CD31, VEGFR-2 antigen,
4) laser co-focusing detection EPCS phagocytic functions：It absorbs DIL-ACLDL and combines FITC-UEA-1 abilities,
5) the external official jargons of EPCS form experiment：Matrigel.
The endothelial progenitor cells that the method culture of the present invention is obtained carry out growing multiplication ability detection：Such as CCK8.
The method of the invention described above, wherein, in step (1) the pregnant age of pregnant mouse be advisable with 15-19 days, pregnant age is too small, tire mouse
Not yet ripe, lungs are smaller, and separation is more difficult；In step (2), (3), (4) by separated in conceptus uterus, tire mouse, lung
Tissue is preferably put into containing immersion in the dual anti-antibiotic P BS of high intensity and washs：Dual anti-penicillin and the applicable concentration of streptomysin are equal
For 500U-1000U, soaking time is 5-10 minutes；In step (6), (8), dual anti-not only effective suppression of 500U-1000U concentration
Bacterium, and there is no any side effect to cell；
The method of the invention described above, step shred lung tissue to 0.5-1.0MM3 in (4), and tissue block is smaller, and pancreatin disappears
Change is more complete, and the efficiency that cell climbs out of after inoculation is higher；Postdigestive tissue suspension is uniformly spread in the orifice plate in step (6),
Culture medium is advisable on a small quantity.It is more to cultivate base unit weight, the suspension of fine tissue block is not easy adherent, substantially reduces primary cell yield；
The method of the present invention establishes a kind of extracting and developing of tire mouse lung derived endothelial progenitor cells, the method for culture.It should
Method has the advantage of following 6 aspects：
(1) it is simple and practicable, it is workable；
(2) amplification efficiency of cell is high, and visible cell climbs out of after organizing adherent 24-48H, generally grows 8-12 days feasible originals
Generation digestion.It is different when tire mouse is individual, cell continuously reach P5-P7 for when morphologic appearance it is homogeneous, it is special by immunophenotype, growth
Point, Multidirectional Differentiation etc. confirm this law and are separately cultured the cell of acquisition as endothelial progenitor cells；
(3) multiplication capacity of cell is strong, still has endothelial progenitor cells activity after reaching for 5 generations, maintains multiplication and differentiation potency
Power, therefore it is preferable to use cell P2-P5 generations after purification, it is seen that available cell quantity is more sufficient in experiment；
(4) marrow, peripheral blood, the endothelial progenitor cells extracting and developing culture skill of Cord Blood-Derived with being widely used at present
The advantages of art is compared, the endothelial progenitor cells in tire mouse lung source is：Separation method is easy, and amplification efficiency is high, multiplication capacity is abnormal
By force, the generation time is long, and cell concentration is big；Especially in terms of CCK8 detects cell Proliferation, the proliferation activity of tire mouse source endothelial progenitor cells
Apparently higher than bone marrow, peripheral blood, Cord Blood-Derived endothelial progenitor cells.
(5) preferably new cell derived is provided for transplantation of Endothelial Progenitor Cells.
In conclusion a kind of extracting and developing cultural method of tire mouse lung derived endothelial progenitor cells of the present invention is with larger
Application prospect and value.
Description of the drawings
The endothelial progenitor cells form that Fig. 1 is separately cultured；
Endothelial progenitor cells strongly expressed CD34, CD133, CD31, VEGFR-2 that Fig. 2 is separately cultured out；
The phagocytic function experimental result for the endothelial progenitor cells that Fig. 3 is separately cultured；
Fig. 4 tests Testing and appraisal EPCS into pipe ability as a result, forming tube-like structures into pipe in vitro；
Lung that Fig. 5 embodiments 2 are separately cultured, the heart, liver source endothelial progenitor cells 1D forms.
Specific embodiment
Main material used in following instance and source difference are as follows：
Cleaning grade SD rats are (female, male) from Medical University Of Chongqing's Experimental Animal Center, 0.25% trypsase
(AMRESCO companies), autogamy phosphate buffer PBS, DMEM culture medium are purchased from HYCLONE companies, and hyclone FBS is purchased from
GIBCO companies, Tissue Culture Dish (diameter 10CM), 6 orifice plate of cell culture are purchased from CORNING companies；CD34、CD133、CD31、
The streaming antibody of VEGF-R2, IGG-FITC and IGG-PE control is purchased from BD companies, the purchase of alkaline phosphatase (ALP) staining kit
From Shanghai sun biotech firm, oil red O is the same as purchased from SIGMA companies.
With reference to specific embodiment, the present invention will be further described.The person that is not specified actual conditions in example, according to normal
Conditions Condition is advised to carry out.
The extracting and developing of 1 tire mouse lung derived endothelial progenitor cells of embodiment, culture
Implementation steps are as follows：
(1) in the adult cleaning grade SD rats (female/male) of Medical University Of Chongqing's Experimental Animal Center purchase health, in rat
During oestrus, by 2 female mices and 1 male mouse in same mouse case overnight, early morning on next day checks female mice introitus, visually sees sperm
Bolt person represents to become pregnant, and starts to calculate pregnant age (the 0th day).If checking feminine gender, continue to mate until checking positive.
(2) pregnant age is reached to the pregnant mouse of 15-19 days, 10% chloral hydrate anesthesia is given, 75% disinfection wine is immediately placed in after anesthesia
It is impregnated 15 minutes in essence, moves into iuntercellular, four limbs are fixed by pregnant mouse abdomen upward.
(3) cut with Sterile ophthalmic and successively cut off pregnant mouse abdominal skin and mucous layer with tweezers, fully expose son of becoming pregnant
Beading appearance palace with eye scissors and tweezers is taken out, is put into dual anti-containing 500U-1000U high intensity penicillin and streptomysin by palace
Precooling PBS in impregnate, then move into super-clean bench.
(4) uterus is cut off with Sterile ophthalmic, removes amniotic fluid, pick net rete layer, isolate connection tire mouse navel and placenta
Umbilical cord tissue, each tire mouse (containing placenta, number 10-16 is differed) is separated, tire mouse is put into containing 500U-
In the plate of 1000U high intensity penicillin and the dual anti-precooling PBS of streptomysin, wash 2 times.
(5) lungs of tire mouse are carefully taken out with eye scissors and tweezers, be put into containing 500U-1000U high intensity penicillin and
In the plate of the dual anti-precooling PBS of streptomysin, and it is shredded one by one to the fine tissue block of 0.5-1.0MM3.
(6) with the tissue in 0.25% pancreatin digestion step (4), using substep digestion method, often walk and be placed in 37 DEG C of cells
10MIN is incubated in incubator, until tissue digestion completely.
(7) after terminating digestion, with the DMEM culture mediums containing 10%FBS, 16H is cultivated.
(8) non-adherent cell, 1000RPM, 5MIN are taken out after 16H, centrifugation takes its precipitation.
(9) precipitation into step (8) has been hanged with the EBM-2 culture mediums containing growth factor and serum (2%FBS), with
106/ ML cell concentrations are inoculated in 6 orifice plates, are placed in 37 DEG C, 5%CO2It is cultivated in constant incubator.
(10) a culture solution is changed within every 3 days after, when primary cell growth fusion is up to 80-90%, with 0.25% pancreas
Enzymic digestion secondary culture, lasting passage is until homogeneous form, completion culture is presented in cell.And do following identification or verification.
Verify example 1
The endothelial progenitor cells morphological observation that the present invention is separately cultured：
(1) the separated tire mouse lung derived endothelial progenitor cells of 1 method of embodiment are used：Primary (P0 generations) cell is adherent in organizing
It is climbed out of after 24-48H, continues culture 3-4 days, observation visible cell form is inhomogenous under inverted microscope, there is spindle shape, polygonal
Shape also has circular (see Fig. 1)；
(2) according to embodiment 1 described in propagating method, and fertilization mouse individual difference influence, cell reach P2-P2 for when
It is observed under inverted microscope, it is seen that relatively uniform spindle shape is presented in cell.It is more than generation continuously to reach 5, cellular morphology is without bright
It is aobvious to change (see Fig. 1).
(3) morphological outcomes of tire mouse lung derived endothelial progenitor cells show：In the cell that the present invention is separately cultured out possesses
The spindle shape Morphological Features and adherent growth of skin progenitor cells, the characteristic passed on repeatedly.
Verify example 2
The Immunophenotype analysis for the endothelial progenitor cells that the present invention is separately cultured：
(1) the tire mouse lung derived endothelial progenitor cells (such as P2 generations) that 1 method of selection example separation passage obtains, 0.25%
It is counted after pancreatin digestion, is packed as 6 pipes by 1 × 105/ pipe, PBS is washed 1 time, then cell precipitation is resuspended with 50 Μ L PBS；
(2) the corresponding streaming antibody of 1 Μ L is separately added into every pipe：CD34、CD133、CD31、VEGFR-2、FITC-
IGG, PE-IGG, 4 DEG C are protected from light incubation 30 minutes；
(3) PBS is washed 1 time, in flow cytomery and is analyzed.
(4) tire mouse lung derived endothelial progenitor cells Immunophenotype analysis the result shows that：The cell that the present invention is separately cultured out
Strongly expressed CD34, CD133, CD31, VEGFR-2 meet the surface markers feature of endothelial progenitor cells (see Fig. 2).
Verify example 3
The phagocytic function analysis for the endothelial progenitor cells that the present invention is separately cultured：
(1) the tire mouse lung derived endothelial progenitor cells (such as P2 generations) that 1 method of selection example separation passage obtains, 0.25%
It is counted after pancreatin digestion, is inoculated on glass slide and carries out cell climbing sheet.
(2) DIL-ACLDL and FITC-UEA-1 is given to be incubated 12H.
(3) fluorescing matter is observed under laser confocal microscope.
(4) tire rat heart endothelial progenitor cell phagocytic function analysis result shows：Carry out DIL-ACLDL, FITC-UEA-
1 double fluorescent stainings, cell is observed under laser confocal microscope, excitated red fluorescence but also can excite green fluorescence,
Composite diagram is considered as EPCS for yellow fluorescence.Red fluorescence is AC-LDL positive cells, and green is positive thin for UEA-1
Born of the same parents, double positive cells are EPCS (such as Fig. 3).
Verify example 4
The segment dislocation capability analysis for the endothelial progenitor cells that the present invention is separately cultured：
(1) the tire mouse lung derived endothelial progenitor cells (such as P2 generations) that 1 method of selection example separation passage obtains, 0.25%
It is counted after pancreatin digestion.
(2) inoculating cell after the general plate of MATRIGL matrigels is given.
(3) tire mouse lung derived endothelial progenitor cells segment dislocation capability analysis the result shows that：It can be seen that the cell after cultivating 14 days
Tube-like structures can be formed, meet the endothelium feature (such as Fig. 4) of endothelial progenitor cells.
Verify example 5
The multiplication capacity analysis for the endothelial progenitor cells that the present invention is separately cultured：
(1) the tire mouse lung derived endothelial progenitor cells (such as P2 generations) that 1 method of selection example separation passage obtains, 0.25%
Cell precipitation is resuspended with quantitative culture medium after pancreatin digestion, cell count simultaneously adjusts concentration as 1 × 104/ML；
(2) cell suspension inoculation is taken, per 100 Μ L of hole, it is thin to be placed in 37 DEG C of constant temperature, 5%CO2 saturated humidities in 96 orifice plates
It is cultivated in born of the same parents' incubator；
(3) 3 hole cells are taken daily, it is indigested per hole with row cell count respectively after quantitative 0.25% pancreatin digestion
The every 3 days full doses of cell change liquid 1 time；
(4) continuous counter 3 days, and draw growth curve chart；
Example described above only expresses several preferable implementations of the present invention, and description is more specific and detailed, but
Protection scope of the present invention is not limited with above-mentioned implementation.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, several modification and improvement can also be made, these belong to the present invention's
Protection domain.
The extraction of embodiment 2 tire mouse lung, heart and liver source endothelial progenitor cells, which is separately cultured, compares
It is as follows to extract isolated culture method：
(1) the pregnant mouse of SD are taken, are impregnated immediately with 75% ethyl alcohol after anesthesia, iuntercellular is moved into, rat four limbs is fixed with pin
In on the cystosepiment of work top.
(2) abdomen is splitted, it is careful to take out uterus and tire mouse, it moves into super-clean bench, tire mouse is removed into placenta, amniotic fluid, amnion
Deng being placed in tire mouse in the plate containing dual anti-precooling PBS.
(3) PBS is washed 2 times.The internal organs such as careful heart, lungs, the liver for taking out tire mouse.
(4) classification of each internal organs is shredded, each personal 0.25% pancreatin digests each fragment of tissue, using substep digestion method, directly
It is complete to tissue.
(5) after terminating digestion, heart, lung tissue, the cell in liver source are with the DMEM medium cultures containing 10%FBS
16H。
(6) non-adherent cell, 1000RPM, 5MIN, centrifugation are each taken out.
(7) 6 orifice plates are inoculated in 106/ML cell concentrations with the EBM-2 culture mediums containing growth factor and serum (2%FBS)
In, it puts 37 DEG C, cultivate in 5%CO2 constant incubators, respectively obtain heart, lung tissue, the endothelial progenitor cells in liver source.
The heart obtained, lung tissue, the endothelial progenitor cells (EPCS) in liver source are cultivated by the method progress of verification example 1
Skin progenitor morphology is observed.It turns out that liver is worst, it is hardly adherent.From the point of view of form, lung source endothelial progenitor cells compare
It is good, heart it is adherent more, but there are part heteroproteose cell, and the cellular morphology in heart source and EPCs less as, it is too long a bit, see
Fig. 5 shows that lung source endothelial progenitor cells are optimal.
Claims (10)
1. a kind of isolated culture method of tire mouse lung derived endothelial progenitor cells, comprises the following steps：
(1) lungs of tire mouse are taken, are put into containing in the dual anti-precooling PBS of 500U-1000U penicillin and streptomysin, and by lung group
It knits broken；
(2) with the disrupting tissue in pancreatin digestion step (1)；
(3) after having digested, with the DMEM medium cultures 15-18 containing FBS it is small when；
(4) non-adherent cell is then taken out, centrifuges, takes its precipitation；
(5) precipitation of step (4) with the EBM-2 culture mediums containing growth factor and the serum of FBS has been hanged, has been inoculated in orifice plate,
It is placed in constant incubator and cultivates；
(6) per a not good liquor is changed within 2-4 days, when primary cell growth fusion is up to 80-90%, with pancreatin had digestive transfer culture culture, continue
Passage is until cell is presented homogeneous form and completes culture.
2. the method as described in claim 1, the tire mouse is included：(1) beading sample will be taken out after the anesthesia of pregnant mouse to become pregnant son
Palace is put into containing being impregnated in the dual anti-precooling PBS of 500U-1000U penicillin and streptomysin；(2) by the uterus of becoming pregnant after immersion
After the umbilical cord tissue for isolating connection tire mouse navel and placenta, tire mouse is put into containing 500U-1000U high intensity penicillin and strepto-
In the plate of the dual anti-precooling PBS of element, tire mouse is obtained after washing.
3. method as claimed in claim 2, step is washed described in (2), and washing times are 2 times.
4. method as claimed in claim 1, the pancreatin described in step (2) is 0.25% pancreatin, and DMEM culture mediums contain in step (3)
10%FBS, when incubation time is 16 small.
5. the method as described in claim 1, it is 1000RPM to be centrifuged described in step (4), 5MIN, the EBM-2 in step (5)
Culture medium contains 2%FBS.
6. the method as described in claim 1, the inoculation of step (5), cell concentration 106/ ML contains 5%CO in insulating box2、
Temperature is 37 DEG C, a not good liquor is changed within every 3 days in step (6), the concentration of pancreatin is 0.25%.
7. the method as described in claim 1, the digestion of pancreatin described in step (2) is using substep digestion method, often walks and is placed in
10MIN is incubated in 37 DEG C of cell incubators, until tissue digestion completely.
8. the method as described in claim 1, step is impregnated described in (1), and soaking time is 3-5 minutes.
9. the method as described in claim 1, it is the pregnant mouse of 15-19 days in pregnant age that pregnant mouse described in step (1), which is,.
10. the method as described in claim 1, disrupting tissue described in step (1), broken tissue size is 0.5-
1.0MM3。
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