CN103881970A - Rat dermal mesenchymal stem cell culture method - Google Patents
Rat dermal mesenchymal stem cell culture method Download PDFInfo
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- CN103881970A CN103881970A CN201410085659.1A CN201410085659A CN103881970A CN 103881970 A CN103881970 A CN 103881970A CN 201410085659 A CN201410085659 A CN 201410085659A CN 103881970 A CN103881970 A CN 103881970A
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Abstract
The invention discloses a rat dermal mesenchymal stem cell culture method. The method comprises the steps of digesting skin tissue and keeping the skin tissue for a whole night; separating epidermis and dermis of the skin tissue; sticking a dermis tissue block in a cell culture dish and adhering in an inverted incubator at 37 DEG C for half an hour, and cultivating the tissue block through low-sugar DMEM and 10% of FBS; implementing conventional pancreatin digestion subculture when the dish is full of cells cultivated from the dermis tissue block to obtain dermal mesenchymal stem cells. The method disclosed by the invention is a novel method combining a skin tissue block adherent method and an enzyme digestion method, not only effectively avoids epidermal keratinocyte pollution caused by the skin tissue block adherent method but also avoids influence of a long-time pancreatic function on a cell growth state in the enzyme digestion method. The culture method disclosed by the invention provides possibility for further research on effects of mesenchymal stem cells in the skin tissue on skin development, growth and damage repair.
Description
Technical field
The invention belongs to the vitro culture field of cell, more specifically, the present invention relates to a kind of isolation cultivation method of rat corium interstital stem cell.
Background technology
Corium interstital stem cell (dermal fibroblasts) is as the histocyte of skin, there is very important biological function, be mainly reflected in the following aspects: 1. there is vital role as the stroma cell of dermis of skin to maintaining the balance of extracellular matrix and stablizing; 2. affect growth and the growth of epidermis by secrete cytokines; 3. regulate and control Skin appendages growth and the regeneration such as hair follicle; 4. can be used as the important sources of interstital stem cell, transplant and repair other histoorgans.The corium interstital stem cell of vitro culture is significant to interaction and the skin injury regeneration between corium and epidermis in research skin development process.
Existing corium interstital stem cell cell culture processes mainly contains two kinds: the direct adherent method of tissue block and Dispase(neutral protease) and trypsin digestion.
Tissue block adherent method is mainly by skin histology is cut into 1mm
3tissue block, then tissue block is affixed on and in culture dish, applies low sugar DMEM, 10%FBS nutrient solution is cultivated certain hour (have into fibrous cell and climb out of growth), the cell of growth is digested, because the primary corium trysinization time is shorter than epidermal keratinocyte, add the cell first digesting after pancreatin to be corium interstital stem cell, go down to posterity.This kind of method is to distinguish corium interstital stem cell and keratinocyte by the difference of trysinization time, and the corium interstital stem cell that separation and Culture obtains is mixed with epidermal keratinocyte, affects follow-up experimental study.Dispase(neutral protease) be skin histology to be cut into fine strip shape (width is approximately: 2mm with trypsin digestion, be about for: 1cm), the skin histology shearing is first spent the night with 4 ℃ of digestion of Dispase enzyme, the dermal tissue of separator well is shredded to the trysinization 30 minutes of rear application 0.25%, after by centrifugal 5 minutes of 800 rpms, postdigestive cell, add nutrient solution kind bottle to go down to posterity.Because this kind of method is through long trysinization, make the growth conditions of the dermal cell obtaining be subject to great impact, the data that such cell obtains for follow-up experimental study will be unstable.
As can be seen here, there is drawback in the cultural method of corium interstital stem cell at present, and corium interstital stem cell has vital role in the growth of skin histology and tissue repair regeneration, therefore obtain epidermic cell pollute few state again the establishment of good corium interstital stem cell cultural method be very important.
Summary of the invention
A kind of isolation cultivation method of new rat corium interstital stem cell is provided based on this this patent.
Cultivate an isolation cultivation method for rat corium interstital stem cell, comprise the step of getting rat back skin histology and be cut into fine strip shape, then by the skin histology shearing with digesting and spend the night in 4 ℃ of refrigerators of Dispase enzyme of 0.25%; With containing dual anti-PBS washing one time, then by the corium of skin histology and epidermis separately, dermal tissue is put into and contained dual anti-PBS washing and be once cut into afterwards 1mm
3tissue block; Dermal tissue piece is affixed in Tissue Culture Dish and is inverted adherent half an hour in 37 ℃ of incubators, rear application low sugar DMEM, and 10%FBS carries out the cultivation of tissue block; Dermal tissue piece is cultivated 48h and is climbed out of to having into fibrous cell after 96h, cover with capsule and can carry out the conventional had digestive transfer culture of pancreatin and cultivate and obtain corium interstital stem cell when climbing out of cell.
As concrete example, the present invention includes following steps:
(1) the disconnected neck of the birth rat suckling mouse of 1-2 days is put to death, puts into ethanol for disinfection and soak 5 minutes, after in Bechtop, take out, PBS washing three times in the large ware of aseptic 10cm;
(2) skin of clean ethanol for disinfection suckling mouse back of the body neck is cut off, caught hold of head and skin of back is firmly torn skin histology with ophthalmic tweezers, the skin of back of getting is put into and contained penicillin and the dual anti-PBS of Streptomycin sulphate soaks 5 minutes;
(3) the skin of back tissue of getting is cut into fine strip shape (width is approximately: 2mm, be about into: 1cm), by the skin histology shearing with digesting and spend the night in 4 ℃ of refrigerators of Dispase enzyme of 0.25%;
(4) the skin histology piece 0.25% Dispase enzymic digestion being spent the night is with containing dual anti-PBS washing one time, the corium of clamping respectively skin histology with ophthalmic tweezers and epidermis (epidermis relatively thin and become ground-glass-like) separate both, and dermal tissue is put into and contained dual anti-PBS washing and be once cut into afterwards 1mm
3tissue block;
(5), the dermal tissue piece obtaining in this step (4) is affixed in Tissue Culture Dish (3.5cm capsule) and is inverted adherent half an hour in 37 ℃ of incubators, rear application low sugar DMEM, 10%FBS carries out the cultivation of tissue block;
(6) dermal tissue piece is cultivated 48h and is climbed out of to having into fibrous cell after 96h, covers with capsule and can carry out the conventional had digestive transfer culture of pancreatin and cultivate and obtain corium interstital stem cell when climbing out of cell.
In an example, in step (1), application suckling mouse, as the skin-derived that separates corium interstital stem cell, utilizes primary cell state and the quantity that goes down to posterity that suckling mouse is cultivated obviously to increase therein.
Therein in an example, in step (3), skin histology is cut into fine strip shape (width is approximately: 2mm, be about into: 1cm), and the concentration of Dispase enzyme is the digestion best results of spending the night in 0.25%, 4 ℃ of refrigerator.
Therein in an example, in step (3) by skin histology application Dispase enzymic digestion to separate epidermis and corium, the present invention has taked the cultural method of the corium interstital stem cell that enzyme digestion and tissue mass cell culture combine new, this novel method, through the digestion of Dispase enzyme, separates epidermis can effectively to avoid the pollution of epidermal keratinocyte to corium interstital stem cell.
In an example, in step (5), Dispase enzymic digestion is cut into 1mm by separating epidermis dermal tissue afterwards therein
3tissue block carry out again the cultivation of tissue block, the corium interstital stem cell isolation cultivation method that the present invention has taked enzyme digestion and tissue mass cell culture to combine, this method is cut into 1mm dermal tissue
3tissue block carry out adherent culture, the impact of the long-time digestion of having avoided pancreatin on primary cell state.
Therein in an example, corium 1mm in step (5)
3tissue block be inverted adherent in incubator should be in 30min left and right, the time is too short adherent insecure, adherent overlong time tissue block is easily dry to be affected cell and climbs out of efficiency;
Therein in an example, dermal tissue piece is cultivated 48h and is climbed out of and can digest cell around in tissue block to there being into fibrous cell after 96h in step (6), the possibility of the heteroproteose cell such as this section of time-angle cell plastid growth is very low, and this kind of novel method avoided the pollution of other cells in guaranteeing primary cell culture quantity like this.
The present invention has done important improvement on the isolation cultivation method of original rat corium interstital stem cell, direct tissue mass cell culture and two enzyme (Dispase enzyme and pancreatin) digestion method are combined, utilize respectively the advantage of two kinds of methods, first digest with Dispase, face tissue is peeled off, can effectively avoid the pollution of epidermal keratinocyte, after carry out tissue block adherent cultivation and just avoided pancreatin to digest for a long time the damage to cell separating the skin histology of epidermis, make the state of cell better.
The present invention has set up a set of separation, cultivation and has identified the dry novel method of rat corium interstitial, confirm that the method has obtained the active corium interstital stem cell that more high purity is higher, to become the detection proved invention gained cell of fat differentiation capability be interstital stem cell for surface markers to gained cell of the present invention, skeletonization simultaneously, thereby be that further further investigation corium interstital stem cell propagation, differentiation and skin histology are grown, the changes of function in injury repairing in pathologic, physiologic situation provides may.
Accompanying drawing explanation
Fig. 1 is the method schematic diagram of this separation and Culture rat corium interstital stem cell.
Fig. 2 climbs out of into fibrous cell's (40 times) in inverted phase contrast microscope observation dermal tissue piece.
Fig. 3 is that inverted phase contrast microscope is observed the cell of separation and Culture of the present invention and the comparison of the cell state that enzyme digestion obtains (40 times); Wherein A-the present invention obtains corium interstital stem cell, and B-enzyme digestion obtains corium interstital stem cell.
Fig. 4 is the expression that RT-PCR detects separation and Culture cell of the present invention and direct tissue mass cell culture acquisition cell mesocuticle mark CK19.Wherein 1-the present invention obtains cell; The direct tissue block adherent method of 2-obtains cell.
Fig. 5 is the detection (CD29, CD44, CD90 and CD45) of drain cell instrument to separation and Culture corium interstital stem cell cell dryness surface markers of the present invention.
Fig. 6 is the osteogenic ability (100 times) that osteogenic induction detects invention separation and Culture corium interstital stem cell cell.
Fig. 7 is into the one-tenth fat ability (400 times) of fat induction detection invention separation and Culture corium interstital stem cell cell.
Concrete embodiment
The main raw that following instance uses and source are as follows respectively:
The suckling mouse in 1 day age (Jiangsu University's experimentation on animals center, this experiment is ratified by Ethics Committee of Jiangsu University) of SD rat;
0.25% trypsin Amresco); 0.25%Dispase neutral protease (Gibco); Autogamy phosphoric acid buffer PBS; Low sugar DMEM(Gibco); Foetal calf serum FBS(Gibco);
Tissue Culture Dish (3.5cm capsule) (corning); Tissue Culture Flask (corning);
CD44, CD29, CD90, the loss antibody of CD45 and IgG-FITC contrast;
Interstital stem cell becomes fat induced liquid (cyagen); Interstital stem cell osteogenic induction liquid (configuration voluntarily);
Below in conjunction with accompanying drawing and example in detail the present invention.
(1) in Jiangsu University's Experimental Animal Center buy in the same day 6 of the SD rat suckling mouses neck that breaks of being born put to death, put into ethanol for disinfection and soak 5 minutes, after in Bechtop, take out, put into the large ware PBS of aseptic 10cm washing three times;
(2) ethanol for disinfection soaked and cut off through the skin of the clean suckling mouse back of the body neck of PBS, catch hold of head and skin of back is slightly firmly torn skin of back tissue with ophthalmic tweezers, the skin of back of getting is put into and contained penicillin and the dual anti-PBS of Streptomycin sulphate soaks 5 minutes;
(3) subcutis such as subcutaneous lipids and blood vessel of the skin of back tissue of getting is rejected to clean (subcutis is rejected clean skin histology without obvious red blood vessel), after skin histology is cut into fine strip shape (width is approximately: 2mm, be about for: 1cm), skin histology that will shear immerses in 4 ℃ of refrigerators of Dispase enzyme of 0.25% and digests and spend the night; Also subcutis is rejected to clean skin histology and be cut into 1mm simultaneously
3tissue block, be affixed on the contrast as skin histology piece adherent method in Tissue Culture Dish (3.5cm capsule);
(4) the skin histology piece 0.25% Dispase enzymic digestion being spent the night is with containing dual anti-PBS washing one time, the corium of clamping respectively skin histology with ophthalmic tweezers and epidermis (epidermis relatively thin and become ground-glass-like) separate both, and the dermal tissue that separates epidermis is put into and contained dual anti-PBS and wash once;
(5) dermal tissue separation being obtained is cut into 1mm
3tissue block, then this tissue block is affixed in Tissue Culture Dish (3.5cm capsule) and is inverted adherent half an hour in 37 ℃ of incubators, rear application low sugar DMEM, 10%FBS carries out the cultivation of tissue block; Meanwhile apply 0.25% pancreatin dermal tissue is digested to 30min, centrifugal 5 minutes of 800 rpms of collecting cells obtain the contrast of cell kind bottle as enzyme digestion after centrifugal;
(6) dermal tissue piece is cultivated 48h and is climbed out of to having into fibrous cell after 96h, does next step evaluation (Fig. 2) when climbing out of Growth of Cells to carrying out the trysinization cultivation of going down to posterity after certain density.
Application inverted microscope carries out observation and comparison to the inventive method and enzyme digestion gained P2 for the growth conditions of corium interstital stem cell, result shows that the growth conditions of the inventive method gained cell is obviously better than enzyme digestion, the damage (Fig. 3) that can effectively avoid long enzymic digestion to cause cell;
Application RT-PCR technology is the detection (reference gene is: β-actin) for corium interstital stem cell mesocuticle index CK19 to the inventive method and the direct adherent method gained of skin histology piece P2, result shows because the inventive method is first with Dispase enzyme, face tissue to be peeled off, so the pollution of gained corium interstital stem cell mesocuticle keratinocyte of the present invention is obviously less than the direct adherent method of skin histology piece, has effectively avoided the skin histology piece with serious pollution drawback of direct adherent method keratinocyte (Fig. 4);
The primer sequence of CK19 and β-actin is following as table 1,
Table 1
Embodiment 3, the surface markers of corium interstital stem cell that separation and Culture of the present invention obtains and the evaluation of differentiation:
The surface markers of the corium interstital stem cell of streaming born of the same parents' art to separation and Culture of the present invention detects, and result shows gained cell CD29 of the present invention, and CD90 and CD44 strong positive and CD45 is substantially negative meet the surface markers (Fig. 5) of interstital stem cell;
Application skeletonization induced liquid carries out the research of osteogenic induction to the corium interstital stem cell of separation and Culture of the present invention, result shows that gained cell of the present invention has to the ability (Fig. 6) of osteocyte differentiation;
Be applied to fat induced liquid becomes fat induction research to the corium interstital stem cell of separation and Culture of the present invention, result shows that gained cell of the present invention has the ability (Fig. 7) to Adipocyte Differentiation;
The above example has only been expressed several implementation method of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction of the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, should be as the criterion with right protection book to protection of the present invention.
Claims (3)
1. the isolation cultivation method of a rat corium interstital stem cell, comprise the step of getting rat back skin histology and be cut into fine strip shape, it is characterized in that the skin histology shearing with digesting and spend the night in 4 ℃ of refrigerators of Dispase enzyme of 0.25%, with containing dual anti-PBS washing one time, again by the corium of skin histology and epidermis separately, dermal tissue is put into and contained dual anti-PBS washing and be once cut into afterwards 1mm
3tissue block; Dermal tissue piece is affixed in Tissue Culture Dish and is inverted adherent half an hour in 37 ℃ of incubators, rear application low sugar DMEM, and 10%FBS carries out the cultivation of tissue block; Dermal tissue piece is cultivated 48h and is climbed out of to having into fibrous cell after 96h, cover with capsule and can carry out the conventional had digestive transfer culture of pancreatin and cultivate and obtain corium interstital stem cell when climbing out of cell.
2. the isolation cultivation method of rat corium interstital stem cell according to claim 1, is characterized in that rat back skin histology derives from suckling mouse.
3. the isolation cultivation method of rat corium interstital stem cell according to claim 1, is characterized in that fine strip shape is width 2mm, long 1cm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104164406A (en) * | 2014-08-12 | 2014-11-26 | 江苏大学 | Mesenchymal stem cell in-vivo mutant tumor cell lung transitional cell strain B6 and application thereof |
| CN104263699A (en) * | 2014-09-19 | 2015-01-07 | 江苏华亿细胞组织工程有限公司 | Culture method for large-scale preparation of clinical treatment level dermal multipotent stem cells for cell transplantation |
| CN113881628A (en) * | 2021-11-16 | 2022-01-04 | 东莞再立健生物科技有限公司 | Culture method of primary stem cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101347640A (en) * | 2008-09-05 | 2009-01-21 | 西安交通大学 | Analogue model of skin with pigments for selecting stripping agent and construction method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164406A (en) * | 2014-08-12 | 2014-11-26 | 江苏大学 | Mesenchymal stem cell in-vivo mutant tumor cell lung transitional cell strain B6 and application thereof |
| CN104263699A (en) * | 2014-09-19 | 2015-01-07 | 江苏华亿细胞组织工程有限公司 | Culture method for large-scale preparation of clinical treatment level dermal multipotent stem cells for cell transplantation |
| CN113881628A (en) * | 2021-11-16 | 2022-01-04 | 东莞再立健生物科技有限公司 | Culture method of primary stem cells |
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Application publication date: 20140625 |