CN105907701A - Method for cultivating dermal fibroblasts of mice or rats in separation manner - Google Patents
Method for cultivating dermal fibroblasts of mice or rats in separation manner Download PDFInfo
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- CN105907701A CN105907701A CN201610277788.XA CN201610277788A CN105907701A CN 105907701 A CN105907701 A CN 105907701A CN 201610277788 A CN201610277788 A CN 201610277788A CN 105907701 A CN105907701 A CN 105907701A
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- 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
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- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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Abstract
The invention discloses a method for cultivating dermal fibroblasts of mice or rats in a separation manner. The method has the advantages that treated dermal tissues are creatively washed and digested by the aid of shaking tables, accordingly, bloodstain and impurities can be effectively removed, the surfaces of the tissues are free of epidermal residues, the inner surfaces of the tissues are free of red blood streak and residues of adipose tissues, and the high fibroblast separation purification degree can be guaranteed; the fibroblasts obtained by means of digestion are high in activity and easy to attach to walls and have little cell debris, and a novel idea and the novel method can be provided for cultivating the fibroblasts in the separation manner; specially prepared complex enzymatic digestion liquid is applied to the method for cultivating the primary fibroblasts of the mice or the rats in the separation manner, the dermal tissues are digested under the condition of the temperature of 4 DEG C overnight (for 4-10 h), accordingly, problems of low cell activity and poor yield due to excessive digestion and excessive beating on tissues can be solved as compared with the traditional single-collagenase preheating digestion modes, and the dermal tissues can be uniformly and thoroughly digested.
Description
Technical field
The present invention relates to the cultivation of a kind of mice or rat cell, particularly relate to the body of a kind of dermal fibroblast
Outer isolated culture method.
Background technology
Fibroblast is derived from mesoblastic mesenchymal cell.Owing to fibroblast is Cell culture invitro
Time be easiest to the cell that obtains, therefore be widely used in the research of cytology and molecules.Further, since fibroblast
The adaptability of dimension cell is stronger, therefore can tolerate various operation, such as: transgenic and micro-note
Penetrate research.Generally, fibroblast can secrete a kind of non-specific cell epimatrix, this extracellular
Substrate is widely present in I type and type III collagen protein.There is proof to show, derive from the one-tenth fiber of Different Organs
Cell has the difference of essence.When wound healing, dermal fibroblast experienced by from propagation, migrates in opposite directions
The transformation of phase is reinvented in contraction, conformation.It addition, when inflammatory stimulus, fibroblast can secrete substantial amounts of transparent
Matter acid.
At present, substantial amounts of researcher has been had to carry out the fibroblastic cultivation of mice/rat dermal
With the research in terms of pharmacological characteristics, using tissue mass cell culture to cultivate research previously more.Its shortcoming is group
The primitive cell culture time knitting block cultural method is long, and the most each piece has a cell eruption, therefore passage
Number of times and the final cell number obtained are less, and cell category is impure, make to continue in-depth study further and are limited
System.
Summary of the invention
It is an object of the invention to, the defect existed for prior art, it is provided that one is different from culture-based method
Mice or the fibroblastic cultural method of rat dermal, be allowed to simple, cultivate the mice obtained or big
Mus dermal fibroblast is purer, well-grown, and cell proliferation is fast, can repeatedly pass on, and cell yield and depositing
Motility rate is all greatly improved.
In order to realize above-mentioned technical purpose, the technical measures that the present invention is taked are:
A kind of mice or the fibroblastic isolated culture method of rat dermal, it is characterised in that include following step
Rapid:
Step 1: mice or the extraction of rat skin tissue
With shears clip mice or the skin graft of rat, then remove subcutaneous fat and connective tissue, and by skin graft
Cut to size be 4-5cm2；
Step 2: the stripping of skin corium and the washing of dermal tissue
Skin graft step 1 obtained is immersed in II type Dispase enzyme, tears epidermis the most afterwards off, will residue
Dermal tissue put in transparent bag, be charged with PBS solution, be placed on after sealing on shaking table and slowly shake
Dynamic washing, until dermal tissue becomes white；
Step 3: enzymic digestion
Dermal tissue step 2 obtained is put in transparent bag, is charged with compound enzyme Digestive system, after sealing
Being placed on shaking table and slowly shake digestion, wherein compound enzyme Digestive system includes the I type that mass ratio is 1:1:1:1
Collagenase, II Collagenase Type, IV Collagenase Type and Dispase enzyme；
Step 4: separation and Culture fibroblast
The digestion reaction in step 3 is terminated, then by one-tenth fiber complete for digestion with the cell culture medium containing serum
Cell crosses 70um screen cloth, adds the PBS solution of volume equity afterwards, i.e. obtains the mice of purification after being centrifuged
Or rat dermal fibroblast, then with cell culture medium resuspended after be positioned over 37 DEG C, 5%CO2Thin
Born of the same parents' incubator is cultivated.
In order to optimize technique scheme further, the technical measures that the present invention is taked also include:
Preferably, the shaking table in above-mentioned steps 2 and step 3 is side-sway shaking table.
Preferably, the transparent bag in above-mentioned steps 2 and step 3 can also replace with plastic casing.
Preferably, in above-mentioned steps 2, II type Dispase enzyme concentration is 1mg/ml.
Preferably, in above-mentioned steps 3, in compound enzyme Digestive system, the concentration of each enzyme is 1mg/ml.
Preferably, in above-mentioned steps 3, enzymic digestion process is 4 DEG C of compound enzyme Digestive systems digestion 4-10h of use.
Preferably, in above-mentioned steps 4 cell culture medium containing 20% hyclone, 1% mycillin
DMEM culture medium, specifically, at original cuiture, passes on and recover first day, with containing 20% tire cattle
Serum, the DMEM culture medium of 1% mycillin.At All Other Times, with containing 10% hyclone, 1% blue or green chain
The DMEM culture medium of mycin.
Preferably, in above-mentioned steps 4, centrifugal condition is 400g, centrifugal 5 minutes.
Another aspect of the present invention also provides for the separation and Culture of the Primary mouse according to above-mentioned or rat fibroblast
Mice that method is cultivated and rat fibroblast.
The inventive method compared with prior art has the advantage that
1. preparation method is easy, consistent, reproducible；
2. use compound enzyme digestion method to obtain single dermal fibroblast, it is to avoid piece of tissue previously
The cell concentration that method obtains is few, the shortcoming that cell is impure, and uses compound enzyme to replace single collagenase,
Avoid and the problem that the cytoactive caused is low, obtain rate variance is excessively blown and beaten in excessive tissue digestion；
3. use primary, pass on the 1st day and recover the 1st day increase serum-concentration mode, can be greatly
Improve the adherent rate of cell, improve cytoactive and yield further；
4. the use shaking table washing dermal tissue that the present invention is initiative, can effectively remove bloodstain and impurity, tissue
Surface does not has epidermis to remain, and inner face does not has the blood streak and the residual of fatty tissue of redness yet, it is ensured that
The High Purity degree that fibroblast separates；
5. the use shaking table digestion dermal tissue that the present invention is initiative, the fibroblasts that digestion obtains is high,
The most adherent, cell debris is few, it is provided that a kind of new thinking of separation and Culture fibroblast and method.
Accompanying drawing explanation
Fig. 1 is the microphotograph of the rat fibroblast shooting that the isolated culture method of the present invention obtains
(10X)；
Detailed description of the invention
Below by specific embodiment, the present invention is carried out detailed and concrete introduction, so that being better understood from this
Bright, but following embodiment is not limiting as the scope of the invention.
Embodiment 1
The present embodiment uses rat, uses the isolated culture method of the present invention to be separated into fibrocyte, concrete steps
As follows:
1. the extraction of rat skin tissue
First neonate rat (freezing 10 minutes or cervical dislocation) is put to death, alcohol-pickled 5 minutes.Newborn
Mice need not lose hair or feathers, if adult rat needs first to lose hair or feathers with hairclipper before essence of steeping in wine, exposes skin.With
Shears clip skin graft, subcutaneous fat that clip of trying not is too much and connective tissue.The skin histology leaching that will take off
Bubble, in the pre-cooling PBS containing 2% mycillin, is put and is kept on ice.Treat that all tissue samplings are complete, system
One processes.Under anatomic microscope, carefully remove subcutaneous fat and connective tissue with eye scissors, and by institute
Skin graft is had to cut little to 4～5cm2.Skin graft can not ether big, in order to II type Dispase enzyme can fully permeate
Between corium and epidermis.
2. the stripping of skin corium
The skin graft of subcutaneous fat and connective tissue will be divested, has been immersed in the II type Dispase enzyme of 1mg/ml,
4 degree of refrigerator overnight.2nd day, take out tissue from refrigerator, under anatomic microscope, carefully delay with aseptic nipper
Slowly tear epidermis off.Due to the effect of Dispase enzyme, the epidermis of skin can separate easily with corium, therefore
The epidermis of each piece of skin graft is intactly torn as far as possible.Remaining dermal tissue puts into the ice containing 2% mycillin
It is placed on cyclic washing on shaking table in PBS, removes bloodstain and impurity, clarify to cleaning mixture.At dermal tissue
Reason is successfully masked as, and dermal tissue bleaches color, and surface does not has epidermis remain, inner face do not have the red blood streak and
The residual of fatty tissue.
3. enzymic digestion
The dermal tissue bleached after processing shreds, and adds homemade compound enzyme (type i collagen enzyme+II Collagen Type VI
Enzyme+IV Collagenase Type+Dispase enzyme=1:1:1:1), the initial concentration of 4 kinds of enzymes is 1mg/ml.4
At DEG C, shaking table digests 7h.Last slight piping and druming, less than 10 times, crosses 70um screen cloth.
4. centrifugal purification
Taking the cell filtrate after sieving, add the PBS solution of equity according to volume, 400g is centrifuged 5 minutes,
The mice dermal fibroblast of purification can be obtained, then expect blue living cell counting number with 0.2%.
5. culture medium preparation
In order to promote fibroblastic adherent efficiency, at original cuiture, pass on and recover first day, with containing
20% hyclone, the DMEM culture medium of 1% mycillin.At All Other Times, with containing 10% hyclone,
The DMEM culture medium of 1% mycillin.
6. two dimension is cultivated
By resuspended by above-mentioned culture medium to mice or the rat dermal fibroblast of purification, prepared corium becomes fiber finer
Born of the same parents' re-suspension liquid, joins in T25 culture bottle and cultivates, and inoculum density is 5 × 105/ bottle.It is placed in 37 DEG C,
5%CO2Incubator in cultivate.Every culture medium more renewed for 3 days, every day is thin in basis of microscopic observation
Intracellular growth situation, reaches 70%～80% to cell degrees of fusion, carries out Secondary Culture.
7. Secondary Culture
When cell degrees of fusion reaches 70%～80%, every bottle of cell adds 0.25% pancreas enzyme-EDTA 1ml, 37
DEG C incubator is hatched digestion 2 minutes.Basis of microscopic observation, when cell rounding, gap increase, with containing blood
Clear culture medium terminates digestion, and is blown and beaten by cell.400g is centrifuged 5 minutes, it is thus achieved that cell add again
Enter containing 20% hyclone, the DMEM culture medium of 1% mycillin, be inoculated in T25 culture bottle, connect
Planting density is 5 × 105/ bottle.2nd day, replace medium to containing 10% hyclone, 1% mycillin
DMEM culture medium, is placed in 37 DEG C, 5%CO2Incubator in continue amplification cultivation.Within every 3 days, more renew
Culture medium, every day is in basis of microscopic observation cell growth status.
8. cellular morphology is observed
The fibroblast form of the primary separation and Culture of basis of microscopic observation, as shown in Figure 1.
9. other indexs of correlation measure
Antibacterial and fungal contamination situation, cell contamination situation, statistics Cell viability etc. is measured during cultivation.
Comparative example
Comparative example uses traditional piece of tissue digestion method, separates after obtaining dermal tissue, peels off and remove other
Subcutaneous fat and connective tissue, use and rinse containing dual anti-PBS, under anatomic microscope, use aseptic tweezer
Son carefully tears epidermis off lentamente.Shred under sterile working, under the conditions of 37 DEG C, stand with II collagenase and disappear
Change 2h, after terminating reaction, use buffer piping and druming tissue, cell dispersion, cross screen cloth, be centrifuged washing,
Then carrying out resuspended and recentrifuge, finally the most resuspended with the liquid containing serum free culture system, isolated mice becomes fiber
Cell, after cell counting, joins in T25 culture bottle and cultivates, and inoculum density is 5 × 105/ bottle, puts
In 37 DEG C, 5%CO2Incubator in carry out cultivating and observing and measure index of correlation.
The above embodiments and comparative example experiment, applicant carried out many experiments, and carried out index of correlation
Comparison and data statistics.As shown in table 1:
Table 1
Applicant uses mice to carry out testing several times simultaneously, and experimental result is identical with mice, it was demonstrated that the present invention
Method authentic and valid to the good result of the skeletal muscle separation and Culture of rat and mice.
In sum, the Primary mouse of the present invention or the isolated culture method of rat fibroblast, easy, skill
Art is stable, reproducible；Compound enzyme digestion method is used to obtain single dermal fibroblast, it is to avoid both
The cell concentration that past tissue block method obtains is few, the shortcoming that cell is impure, and uses compound enzyme to replace single collagen
Enzyme, it is to avoid the problem that the cytoactive caused is low, obtain rate variance is excessively blown and beaten in excessive tissue digestion；Use
Primary, pass on the 1st day and recover the 1st day increase serum-concentration mode, the patch of cell can be greatly enhanced
Wall rate, improves cytoactive and yield further.Additionally, the present invention initiative wash dermal tissue with shaking table,
Can effectively remove bloodstain and impurity, tissue surface does not has epidermis to remain, and inner face does not has the blood streak and the fat of redness yet
The residual of tissue, it is ensured that the High Purity degree that fibroblast separates.Digest dermal tissue with shaking table, digest
The fibroblasts arrived is high, and the most adherent, cell debris is few, it is provided that separation and Culture fibroblast is a kind of
New thinking and method.
Being described in detail the specific embodiment of the present invention above, but it is intended only as example, the present invention is also
It is not restricted to particular embodiments described above.To those skilled in the art, any the present invention is carried out
Equivalent modifications and substitute the most all among scope of the invention.Therefore, without departing from the spirit of the present invention and model
Enclose lower made impartial conversion and amendment, all should contain within the scope of the invention.
Claims (10)
1. a mice or the fibroblastic isolated culture method of rat dermal, it is characterised in that comprise the following steps:
Step 1: mice or the extraction of rat skin tissue
With shears clip mice or the skin graft of rat, then remove subcutaneous fat and connective tissue, and skin graft is cut to
Size is 4-5cm2；
Step 2: the stripping of skin corium and the washing of dermal tissue
Skin graft step 1 obtained is immersed in II type Dispase enzyme, tears epidermis the most afterwards off, by remaining very
Skin tissue is put in transparent bag, is charged with PBS solution, is placed on shaking table and slowly shakes washing after sealing,
Until dermal tissue becomes white；
Step 3: enzymic digestion
Dermal tissue step 2 obtained is put in transparent bag, is charged with compound enzyme Digestive system, places after sealing
Slowly shaking digestion on shaking table, wherein compound enzyme Digestive system includes the type i collagen that mass ratio is 1:1:1:1
Enzyme, II Collagenase Type, IV Collagenase Type and Dispase enzyme；
Step 4: separation and Culture fibroblast
The digestion reaction in step 3 is terminated, then by fibroblast complete for digestion with the cell culture medium containing serum
Cross 70um screen cloth, add the PBS solution of volume equity afterwards, after being centrifuged, i.e. obtain mice or the rat of purification
Dermal fibroblast, then with cell culture medium resuspended after be positioned over 37 DEG C, 5%CO2Cell culture incubator
In cultivate.
A kind of mice the most according to claim 1 or the fibroblastic isolated culture method of rat dermal, it is special
Levying and be, the shaking table in described step 2 and step 3 is side-sway shaking table.
A kind of mice the most according to claim 1 or the fibroblastic isolated culture method of rat dermal, it is special
Levying and be, the transparent bag plastic casing in described step 2 and step 3 replaces.
A kind of mice the most according to claim 1 or the fibroblastic isolated culture method of rat dermal, it is special
Levying and be, in described step 2, II type Dispase enzyme concentration is 1mg/ml.
A kind of mice the most according to claim 1 or the fibroblastic isolated culture method of rat dermal, it is special
Levying and be, in described step 3, in compound enzyme Digestive system, the concentration of each enzyme is 1mg/ml.
A kind of mice the most according to claim 1 or the fibroblastic isolated culture method of rat dermal, it is special
Levying and be, in described step 3, enzymic digestion process is for using 4 DEG C of compound enzyme Digestive system digestion 4-10h.
A kind of mice the most according to claim 1 or the fibroblastic isolated culture method of rat dermal, it is special
Levying and be, in described step 4, cell culture medium is containing 20% hyclone, 1% dual anti-DMEM culture medium.
A kind of mice the most according to claim 7 or the fibroblastic isolated culture method of rat dermal, its
It is characterised by, dual anti-for mycillin in described step 4.
A kind of mice the most according to claim 1 or the fibroblastic isolated culture method of rat dermal, it is special
Levying and be, in described step 4, centrifugal condition is 400g, centrifugal 5 minutes.
10. one kind separates training according to the mice described in claim 1-9 any one or rat dermal are fibroblastic
Mice that breeding method is cultivated or rat fibroblast.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610277788.XA CN105907701A (en) | 2016-04-28 | 2016-04-28 | Method for cultivating dermal fibroblasts of mice or rats in separation manner |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610277788.XA CN105907701A (en) | 2016-04-28 | 2016-04-28 | Method for cultivating dermal fibroblasts of mice or rats in separation manner |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257353A (en) * | 2019-05-22 | 2019-09-20 | 中国人民解放军第四军医大学 | The method that application on human skin digests complex enzyme and separates skeptophylaxis cell Treg cell from people on a small quantity full pachydermia |
| CN110305857A (en) * | 2019-05-22 | 2019-10-08 | 中国人民解放军第四军医大学 | The method that mouse skin digests complex enzyme and separates skeptophylaxis cell Treg cell from mouse skin |
| CN110747159A (en) * | 2019-11-12 | 2020-02-04 | 武汉普诺赛生命科技有限公司 | Mouse or rat kidney fibroblast cell separation and subculture method |
| CN113621555A (en) * | 2021-08-18 | 2021-11-09 | 郑州源创吉因实业有限公司 | Preparation method of skin fibroblast |
-
2016
- 2016-04-28 CN CN201610277788.XA patent/CN105907701A/en active Pending
Non-Patent Citations (3)
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| 卫俊霞: "人皮肤成纤维细胞的生物学特性以及在同种异体脱细胞真皮上的培养研究", 《第四军医大学硕士学位论文》 * |
| 王孟丽 等: "人皮肤成纤维细胞分离培养的研究", 《心血管康复医学杂志》 * |
| 车鹏程 等: "用于修复组织缺损的人真皮成纤维细胞体外培养", 《中国煤炭工业医学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257353A (en) * | 2019-05-22 | 2019-09-20 | 中国人民解放军第四军医大学 | The method that application on human skin digests complex enzyme and separates skeptophylaxis cell Treg cell from people on a small quantity full pachydermia |
| CN110305857A (en) * | 2019-05-22 | 2019-10-08 | 中国人民解放军第四军医大学 | The method that mouse skin digests complex enzyme and separates skeptophylaxis cell Treg cell from mouse skin |
| CN110747159A (en) * | 2019-11-12 | 2020-02-04 | 武汉普诺赛生命科技有限公司 | Mouse or rat kidney fibroblast cell separation and subculture method |
| CN113621555A (en) * | 2021-08-18 | 2021-11-09 | 郑州源创吉因实业有限公司 | Preparation method of skin fibroblast |
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