CN106834205A - A kind of Mouse Kidney sertoli cell is separated and cultural method - Google Patents
A kind of Mouse Kidney sertoli cell is separated and cultural method Download PDFInfo
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- CN106834205A CN106834205A CN201510906695.4A CN201510906695A CN106834205A CN 106834205 A CN106834205 A CN 106834205A CN 201510906695 A CN201510906695 A CN 201510906695A CN 106834205 A CN106834205 A CN 106834205A
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The present invention provides a kind of mouse kidney sertoli cell and separates and cultural method, including step：A the yellow Jackets of () intraperitoneal injection 2% put to death mouse, mouse kidney tissue is taken out after alcohol disinfecting, and it is aseptic containing washing by soaking in dual anti-PBS to be placed in precooling, and peels off removing kidney peplos；B after () mechanical dissociation renal tissue, preheating mixed enzyme solution digestion, mixing complete culture solution terminates digestion；C () obtains kidney sertoli cell using the method for difference sieving, sertoli cell is inoculated in the cell bottle after treatment and is cultivated；(d) will cultivate 3d cell digested through pancreatin after screen filtration again, supernatant is abandoned in centrifugation, in the resuspended cell bottle being inoculated in after treatment；E () cellular immunofluorescence identifies kidney podocytic process cell.The Mouse Kidney sertoli cell that the present invention is provided is separated and cultural method is reproducible, easy to operate, and can obtain the Mouse Kidney sertoli cell that yield is high, purity is high.
Description
Technical field
The invention belongs to cell biology, and in particular to a kind of Mouse Kidney sertoli cell isolation and culture of cell
Method.
Background technology
Kidney sertoli cell (podocyte), i.e. capsula glomeruli visceral layer epithelial cell, it is attached to GBM (GBM)
Outermost, the structure together with glomerular endothelial cells (glomerular endothelial cells) and capillary endothelium
It is the last of filtration membrane to form slit diaphragm into the molecular barriers and electrostatic barrier of glomerular filtration membrane, between podocytic process
One barrier.The damage of sertoli cell plays an important role in albuminuria develops, and many is thin with foot
The renal glomerular disease of cellular damage, may progress to chronic renal failure.To the physiological property and its cell of sertoli cell
The research of injury response mechanism can further appreciate that the pathogenesis of albuminuria and renal glomerular disease.But by
In Renal Podocytes be a kind of terminally differentiated cells, complex structure, it is special to position, in vitro culture it is primary thin
Born of the same parents can hardly breed, and the cellular genome of the sertoli cell strain for immortalizing changes, it is impossible to accurate anti-
Kidney physiology and pathological conditions are reflected, confidence level is not high, therefore, the present invention is intended to provide it is a kind of reproducible,
Mouse Kidney sertoli cell easy to operate is separated and cultural method, and can obtain the mouse that yield is high, purity is high
Kidney sertoli cell, for research Podocytes in Renal Tissue mechanism provides rational experiment basis.
The content of the invention
The purpose of the present invention is to set up a kind of reproducible, easy to operate, obtains that yield is high, that purity is high is small
The method of mouse kidney sertoli cell cell separation culture.
Above-mentioned involved in order to solve the problems, such as, it is thin that the present invention takes following method to obtain Mouse Kidney sertoli cell
Born of the same parents：
The step of Mouse Kidney sertoli cell cell isolation method, includes：A the yellow Jackets of () intraperitoneal injection 2% put to death mouse,
Mouse kidney tissue is taken out after alcohol disinfecting, it is aseptic containing washing by soaking in dual anti-PBS to be placed in precooling,
And peel off removing kidney peplos；After (b) mechanical dissociation renal tissue, preheating mixed enzyme solution digestion, complete culture solution
Terminate digestion；C () obtains kidney sertoli cell using the method for difference sieving, sertoli cell is inoculated in thin after treatment
Cultivated in born of the same parents' bottle；(d) will cultivate 3d cell digested through pancreatin after screen filtration again, centrifugation abandons supernatant, weight
In the outstanding cell bottle being inoculated in after treatment；E () cellular immunofluorescence identifies kidney podocytic process cell.
Optional mouse is the mouse of body weight 22 ± 3gg, 6-8 week old.
Optional digestion digestion enzyme liquid used is in advance in 37 DEG C of 0.1%-0.2% (m/v) IV Collagenase Types of preheating
With the enzyme mixation of 0.05%-0.1% (m/v) II Collagenase Types, digestion time is 20-30min；
Optional mixing complete culture solution be containing the dual anti-DMEM/F12 of 15% mice serum and 1% and
RPMI1640 (1: 1~1: 1.5)；
The method of optional differential sieving is that will terminate postdigestive cell suspension to sequentially pass through 80 mesh, 100 mesh,
The screen cloth of 400 mesh, collects the cell on the filtered solution and 400 eye mesh screens of 400 mesh respectively, and supernatant is abandoned in centrifugation,
Complete culture solution is resuspended；
The processing method of optional cell bottle is 3-5 μ g/cm2Cell after I types rat tail collagen protein coating
Bottle；
Optional purification process is after cultivating the cell of 3d through Trypsin Induced, again by 400 purposes
Screen cloth, collects filtrate, and supernatant, resuspended inoculation are abandoned in centrifugation；
Digestive juice is from preheating in the Mouse Kidney sertoli cell cell isolation method that the present invention is provided
0.1%-0.2% (m/v) IV Collagenase Types and 0.05%-0.1% (m/v) II Collagenase Type mixed liquors are used to digest carefully
Born of the same parents, not only avoid using the pancreatin larger to cellular damage, and small through the postdigestive kidney of collagen enzyme mixation
Ball major part has removed Bowman capsules, also improve the adherent rate of cell, shorten cell it is adherent when
Between.
The present invention provides a kind of method separating mouse kidney sertoli cell first digested after screen cloth, mixes through clostridiopetidase A
After enzyme liquid digestion, the time that sertoli cell is climbed out of from glomerulus shortens, and postdigestive cell suspension is through three
Layer screen filtration and the method purified mouse kidney sertoli cell for sieving again, it is simple to operate, and compared with magnetic bead sorting
Cost savings it is a lot, and improve the purity and yield of cell.
The present invention provides a kind of reproducible, and Mouse Kidney sertoli cell easy to operate is separated and cultural method, and
And obtain Mouse Kidney sertoli cell yield is high, purity is high.
Brief description of the drawings
The cell picture (100 ×) of Fig. 1 cultures 7d；
Fig. 2 cultures 7d cells picture (200 ×)；
Fig. 3 cellular immunofluorescences identify picture.
Specific embodiment
In order that the purpose of the present invention and advantage represent more pure and freshly, now specific embodiment is expanded on further.
Specific embodiment set forth herein is explained only for the present invention, is not intended to limit the present invention.
The mouse of present invention selection 6-8 week old, separates kidney sertoli cell.Concrete operations are as follows：
1st, the mouse of 6-8 week old is taken, the yellow Jackets of intraperitoneal injection 2% put to death mouse, fixed mouse, 75%
Alcohol disinfecting after take out mouse kidney tissue, be placed in that precooling is aseptic to wash containing being soaked in dual anti-PBS
Wash；
2nd, on ice in promptly removed under microscope suprarenal coating, haemocyte and connective tissue etc. attachment
Thing, cleans 2 times during the kidney after stripping is gone into new PBS；
3rd, renal tissue, size about 1mm are shredded with dissecting scissors machinery on ice3The fragment of left and right；
4th, the tissue that will be shredded adds the digestion mixed liquor of 4-5 times of volume of preheating, in 37 DEG C of digestion
20-30min, it is reverse per 10min to mix once；The digestion mixed liquor includes 0.1%-0.2% (m/v) IV types
Clostridiopetidase A and 0.05%-0.1% (m/v) II Collagenase Types；
5th, the mixing containing 10%FBS and 1% dual anti-RPMI160 and DMEM/F12 is added to cultivate completely
Liquid terminates digestion, and piping and druming disperses cell, will terminate postdigestive cell suspension and sequentially passes through 80 mesh, 100
Mesh, the screen cloth of 400 mesh collects the cell on the filtered solution and 400 eye mesh screens of 400 mesh, 1500rpm/min respectively
Centrifugation 5min, abandons supernatant；
6th, precipitated with mixing complete culture solution re-suspended cell, be inoculated in 3-5 μ g/cm2I type rat tail collagen protein bags
In cell bottle by after, 37 DEG C, the CO2 of 5% (m/v), cultivate in the incubator of saturated humidity；
7th, mixing complete culture solution is changed after culture 12h, visible cell is adherent under microscope, and cell is in flat
Flat sample；
8th, after culture 3d, after cell by degrees of fusion up to 90% is through Trypsin Induced, again through 400 mesh
Screen filtration, collects filtrate, and 1500rpm/min centrifugation 5min abandon supernatant, and mixing complete culture solution is resuspended,
It is inoculated in the cell bottle after coating, 37 DEG C, the CO2 of 5% (m/v), cultivate in the incubator of saturated humidity；
9th, cellular immunofluorescence identification：By in kidney podocytic process cell kind to the cover glass being positioned in 24 orifice plates,
In vitro culture 6-8 days, takes out cover glass, and PBS is washed 3 times, each 5min.4% paraformaldehyde fixes 20min,
PBS is washed 3 times, and 0.2% penetrating fluid Triton-100 is incubated 15min at room temperature, and PBS washes 3 times, 5%BSA
Room temperature closes 1h, sucks confining liquid.Add Nephrin antibody in 4 DEG C of night incubations, PBS is washed 3 times.
The anti-igg (1: 100) of sheep anti mouse PE fluorescence two is added dropwise, is placed in 45min, PBS are incubated in wet box at room temperature
Wash 3 times, examined under a microscope after drying naturally.
Mouse Kidney podocytic process adherence rate isolated in the present invention is higher, survival rate up to more than 98%, carefully
Born of the same parents' purity is up to more than 97%.
Below the preferred embodiment to the invention is described in detail, but the invention is not limited
It is all to make any modification, equivalent and improvement within the spirit and principles in the present invention in above-described embodiment
Deng should be included within the scope of the present invention.
Claims (7)
1. a kind of Mouse Kidney sertoli cell isolation and culture of cell method, it is characterised in that including step：A the yellow Jackets of () intraperitoneal injection 2% put to death mouse, mouse kidney tissue is taken out after alcohol disinfecting, and it is aseptic containing washing by soaking in dual anti-PBS to be placed in precooling, and peels off removing kidney peplos；B after () mechanical dissociation renal tissue, preheating mixed enzyme solution digestion, mixing complete culture solution terminates digestion；C () obtains kidney sertoli cell using the method for difference sieving, sertoli cell is inoculated in the cell bottle after treatment and is cultivated；D () will cultivate cell screen cloth purifying again after pancreatin digestion of 3d, supernatant is abandoned in centrifugation, in the resuspended cell bottle being inoculated in after treatment；E () cellular immunofluorescence identifies kidney podocytic process cell.
2. Mouse Kidney sertoli cell isolation and culture of cell method according to claim 1, it is characterised in that described mouse is 22 ± 3g, the mouse of 6-8 week old.
3. Mouse Kidney sertoli cell isolation and culture of cell method according to claim 1, it is characterised in that step b digestion digestion enzyme liquid used is that digestion time is 20-30min in advance in 37 DEG C of mixed liquors for preheating；The mixed liquor includes 0.1%-0.2% (m/v) IV Collagenase Types and 0.05%-0.1% (m/v) II Collagenase Types.
4. Mouse Kidney sertoli cell isolation and culture of cell method according to claim 1, it is characterised in that it is containing 15% mice serum and 1% dual anti-DMEM/F12 and RPMI1640 (1: 1~1: 1.5) to mix complete culture solution in the step b.
5. Mouse Kidney sertoli cell isolation and culture of cell method according to claim 1, it is characterized in that, the method of the step c differences sieving is that will terminate postdigestive cell suspension to sequentially pass through 80 mesh, 100 mesh, the screen cloth of 400 mesh, the cell on the filtered solution and 400 eye mesh screens of 400 mesh is collected respectively, and supernatant is abandoned in centrifugation, and complete culture solution is resuspended.
6. Mouse Kidney sertoli cell isolation and culture of cell method according to claim 1, it is characterised in that the processing method of cell bottle is 3-5 μ g/cm in the step c2Cell bottle after I types rat tail collagen protein coating.
7. Mouse Kidney sertoli cell isolation and culture of cell method according to claim 1, it is characterised in that the purification process of the step d is cell by 3d is cultivated after Trypsin Induced, again by the screen cloth of 400 mesh, filtrate is collected, supernatant, resuspended inoculation are abandoned in centrifugation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402041A (en) * | 2017-08-16 | 2019-03-01 | 江苏齐氏生物科技有限公司 | A kind of people's renal cells isolation and culture method |
| CN113684174A (en) * | 2021-08-26 | 2021-11-23 | 山西省人民医院 | Preparation method of human kidney podocyte |
| CN115322956A (en) * | 2022-05-26 | 2022-11-11 | 上海墨卓生物科技有限公司 | Kit for dissociation of different tissues and dissociation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105087467A (en) * | 2015-08-30 | 2015-11-25 | 中国医学科学院基础医学研究所 | Preparation method of kidney sertoli cells and special mediums thereof |
-
2015
- 2015-12-07 CN CN201510906695.4A patent/CN106834205A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087467A (en) * | 2015-08-30 | 2015-11-25 | 中国医学科学院基础医学研究所 | Preparation method of kidney sertoli cells and special mediums thereof |
Non-Patent Citations (2)
| Title |
|---|
| 柯堂山等: ""大鼠足细胞原代培养及观察"", 《中国医学工程》 * |
| 陶于洪等: ""大鼠肾小球足细胞的原代培养与鉴定"", 《四川大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109402041A (en) * | 2017-08-16 | 2019-03-01 | 江苏齐氏生物科技有限公司 | A kind of people's renal cells isolation and culture method |
| CN113684174A (en) * | 2021-08-26 | 2021-11-23 | 山西省人民医院 | Preparation method of human kidney podocyte |
| CN115322956A (en) * | 2022-05-26 | 2022-11-11 | 上海墨卓生物科技有限公司 | Kit for dissociation of different tissues and dissociation method thereof |
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Application publication date: 20170613 |