CN107779429A - A kind of tissue-derived fibroblast quick separating cultural method of application on human skin - Google Patents
A kind of tissue-derived fibroblast quick separating cultural method of application on human skin Download PDFInfo
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- CN107779429A CN107779429A CN201711281938.5A CN201711281938A CN107779429A CN 107779429 A CN107779429 A CN 107779429A CN 201711281938 A CN201711281938 A CN 201711281938A CN 107779429 A CN107779429 A CN 107779429A
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Abstract
The invention discloses a kind of tissue-derived fibroblast quick separating cultural method of application on human skin, this method will be grown up or the fresh prepuce tissues of the postoperative abandonment of children are cleaned after shearing, successively using dispase II solution and clostridiopetidase A IV solution at 37 DEG C, digested under conditions of water-bath concussion, again through skin histology block is collected by centrifugation, it is resuspended with culture medium, at 37 DEG C, CO2Volumetric concentration is to be cultivated in 5% incubator.Dispase II and clostridiopetidase A IV digestions temperature are brought up to 37 DEG C by the present invention by 4 DEG C, the activity of two kinds of enzymes is improved, two kinds of enzymes are made to increase with the contact area of corresponding substrate by water-bath concussion, be advantageous to the progress of enzymatic reaction, so as to substantially reduce the time of two kinds of enzymic digestions, the speed that fibroblast is separately cultured is improved；Enzyme is reduced simultaneously to histiocytic damage, improves the adherent rate of cell, and obtained primary fibroblast culture is passed on for 6~8 days.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of tissue-derived fibroblast of application on human skin is quickly divided
From cultural method.
Background technology
Fibroblast is most common cell in connective tissue, and from mesoderm, volume is larger, clear-cut, is
The fusiform or star topology of projection, it is main to synthesize extracellular matrix and collagen, to different degrees of cell degeneration, necrosis and tissue
The reparation of defect and bone wound has highly important effect.Fibroblast is widely present in human skin tissue, materials
It is convenient, it is easy to cultivate operation in vitro.Especially after the birth of iPS technologies, fibroblast, which turns into, induces various stem cells
Or the seed cell of adult cell, such as induced multi-potent stem cell (iPSCs), islet cells, cardiac muscle cell, nerve cell etc.,
Facilitate disease mechanisms exploration, drug test screening and the research of new treatment.
At present, the isolated culture method of human skin fibroblasts mainly has two classes：Enzyme digestion and tissue block method.Enzyme disappears
Change method is digested using trypsase, clostridiopetidase A etc. to the fell skin tissue after dual anti-cleaning, after removing tissue epidermis, is carried out
Centrifugation, it is resuspended and cultivates, obtains human skin fibroblasts；This method separates and the cycle of culture is longer, general 10~14 days
It can just pass on, and clostridiopetidase A has adverse effect to cell, causes the active not high of cell, adherent rate is low.Tissue block method will
Application on human skin tissue shear is broken into after small tissue blocks to be inoculated in culture dish and cultivated；This method is simple to operate, but tissue block adherent
Rate is relatively low, and cell climbs out of relatively slowly, can just be passed within general 20 days or so, and technical stability is poor.
Application publication number is that a kind of human skin fibroblasts separation and original are disclosed in CN106591223 patent of invention
For cultural method, this method is digested people's foreskin fritter overnight at 3~6 DEG C using trypsase, is carried out again after removing cuticula
Culture is until obtain human skin fibroblasts；This method avoid centrifugally operated process, effectively increases adherent rate, technique letter
It is single, but whole cycle is longer, the activity reduction of human skin fibroblasts.
The content of the invention
The technical problems to be solved by the invention are to be directed to above-mentioned the deficiencies in the prior art, there is provided a kind of application on human skin group
Knit source fibroblast quick separating cultural method.This method is successively using dispase II and clostridiopetidase A IV at 37 DEG C, water-bath
The fresh prepuce tissues of digestion adult or the postoperative abandonment of children under the conditions of concussion, substantially reduce the time of ferment treatment, reduce
Damage of the enzyme to cell, the adherent rate of cell is improved, the primary skin histology source Fibroblast cell-culture 6~8 of obtained people
It is passed on.
In order to solve the above technical problems, the technical solution adopted by the present invention is：A kind of application on human skin is tissue-derived into fiber finer
Born of the same parents' quick separating cultural method, it is characterised in that this method comprises the following steps：
Step 1: will be grown up or the fresh prepuce tissues of the postoperative abandonment of children clean 2~3 with containing dual anti-physiological saline
It is secondary, the adipose tissue of foreskin bottom surface is then removed with sterile scissors and tweezers, and it is 0.5cm to be cut into size2Foreskin fritter；
Step 2: the foreskin fritter obtained in step 1 is immersed in dispase II solution, 37 DEG C are subsequently placed in, water-bath shake
Once digested under conditions of swinging；
Step 3: the physiological saline for adding two volumes in postdigestive dispase II solution into step 2 is terminated and disappeared
Change, the tissue epidermis of foreskin fritter then removed with sterile scissors and tweezers, obtains dermal tissue, by the dermal tissue cut to
1mm3, then immerse in clostridiopetidase A IV solution, 37 DEG C are placed in, secondary digestion is carried out under conditions of water-bath concussion；
Step 4: the physiological saline for adding two volumes in postdigestive clostridiopetidase A IV solution into step 3 is terminated and disappeared
Change, then remove supernatant afterwards twice with physiological saline centrifuge washing, culture medium is added into centrifugation and is resuspended, then is transferred to culture
In ware, 37 DEG C are placed in, CO2Volumetric concentration is to be cultivated in 5% incubator；The quality of the centrifugation and the volume of culture medium
The ratio between be 3:7, the unit of quality is g, and the unit of volume is mL.
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 1
Described in dual anti-in physiological saline be penicillin and streptomysin, the concentration of the penicillin and streptomysin is 100U/mL.
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 2
Described in dispase II solution mass concentration be 0.4%.
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 2
Described in time for once digesting be 1h.
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 3
Described in clostridiopetidase A IV solution mass concentration be 0.2%.
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 3
Described in time of secondary digestion be 15min.
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 4
Described in the rotating speed that centrifuges be 1500rpm, time 5min.
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 4
Described in culture medium be the hyclone in the DMEM high glucose mediums containing hyclone and dual anti-DMEM high glucose mediums
Volume content be 15%, it is described it is dual anti-be penicillin and streptomysin, the concentration of the penicillin and streptomysin is 100U/
mL。
A kind of tissue-derived fibroblast quick separating cultural method of above-mentioned application on human skin, it is characterised in that step 4
Described in the process cultivated be：Fluid infusion is carried out after culture 24h, the partly amount that carries out is cultivated to the 2nd~3 day and changes liquid, cultivate to the 4th~5
It carries out full dose and changes liquid, cultivates to the 6th~8 day, when cell confluency degree is up to 80%~90%, that is, carries out had digestive transfer culture.
The present invention has advantages below compared with prior art：
1st, the present invention digests adult or children using dispase II and clostridiopetidase A IV under the conditions of 37 DEG C, water-bath concussion successively
The fresh prepuce tissues of postoperative abandonment, the temperature of digestion bring up to 37 DEG C by 4 DEG C, due to digest temperature raising, two kinds
The activity of enzyme is improved, and water-bath concussion makes the contact area increase of two kinds of enzymes and corresponding substrate, therefore two kinds of enzymes disappear
Changing product can be transferred in nutrient solution rapidly from digestion point, be advantageous to the progress of enzymatic reaction, so as to further shorten two
The time of kind enzymic digestion, improve speed and survival rate that fibroblast is separately cultured.
2nd, the dispase II moderate performances that use of the present invention, small to cellular damage, stability is strong, is not easy by temperature and pH
Influence, can effectively eliminate cell aggregation phenomenon, so as to remove tissue epidermis completely, improve fibroblastic purity；And glue
Protoenzyme IV has low enzymatic activity concurrently, larger to cell tissue damage, therefore shortens clostridiopetidase A IV digestions time, can effectively hydrolyze
Collagen and connective tissue, fibroblastic integrality after enzymolysis is maintained again, improves cell attachment during follow-up cultivation
Rate.
3rd, the fibroblast that the present invention obtains carries out multiple fluid infusion in incubation and changes liquid operation, is fibroblast
Growth provides nutrition in time, improves the speed of fibroblastic activity and propagation.
4th, the fibroblast purity that the present invention obtains is higher, and immunohistochemical staining is done for fibroblast from its P4,
Endochylema vimentin (Vimentin) is positive, and positive rate is higher.
Technical scheme is described in further detail below by drawings and examples.
Brief description of the drawings
Fig. 1 is the present inventor's skin histology source Fibroblast cell-culture growth conditions figure of the 3rd day.
Fig. 2 is the present inventor's skin histology source Fibroblast cell-culture growth conditions figure of the 5th day.
Fig. 3 is the present inventor's skin histology source Fibroblast cell-culture growth conditions figure of the 7th day.
Fig. 4 is dyeing of the P4 of the present invention for the tissue-derived fibroblastic endochylema vimentin SABC of application on human skin
Figure.
Fig. 5 is the colored graph of the endochylema vimentin SABC of control group squamous cell.
Embodiment
A kind of tissue-derived fibroblast quick separating cultural method of application on human skin, comprises the following steps in the present embodiment：
Step 1: taking the fresh prepuce tissues of adult or the postoperative abandonment of children, used in Biohazard Safety Equipment blue or green containing 100U/mL
The physiological saline of mycin and 100U/mL streptomysins is cleaned 2~3 times, and the fat of foreskin bottom surface is then removed with sterile scissors and tweezers
Fat tissue, and it is 0.5cm to be cut into size2Foreskin fritter；
Step 2: the foreskin fritter obtained in step 1 is put into the dispase II solution that mass concentration is 0.4%, so
After be placed in 37 DEG C, digest 1h under conditions of water-bath concussion；
Step 3: the physiological saline for adding two volumes in postdigestive dispase II solution into step 2 is terminated and disappeared
Change, the tissue epidermis of foreskin fritter then removed with sterile scissors and tweezers, obtains dermal tissue, by the dermal tissue cut to
1mm3, place into the clostridiopetidase A IV solution that mass concentration is 0.2%, be placed in 37 DEG C, digested under conditions of water-bath concussion
15min；
Step 4: the physiological saline for adding two volumes in postdigestive clostridiopetidase A IV solution into step 3 is terminated and disappeared
Change, be then 1500rpm in rotating speed, under conditions of the time is 5min, supernatant is removed afterwards twice with physiological saline centrifuge washing,
Culture medium is added into centrifugation to be resuspended, then is transferred in culture dish, is placed in 37 DEG C, CO2Volumetric concentration is in 5% incubator
Culture, fluid infusion after 24h is cultivated, cultivated to visible a small amount of fibroblast at the 2nd~3 day, carried out half amount and change liquid, cultivate to the 4th
Cell confluency degree carries out full dose and changes liquid up to 50%~60% at~5 days, cultivate to cell confluency degree at the 6th~8 day up to 80%~
90%, you can carry out Secondary Culture；The ratio between the quality of the centrifugation and the volume of culture medium are 3:7, the unit of quality is
G, the unit of volume is mL；The culture medium is containing hyclone and dual anti-DMEM high glucose mediums, the high sugar of the DMEM
The volume content of hyclone is 15% in culture medium, it is described it is dual anti-be penicillin and streptomysin, the penicillin and streptomysin
Concentration be 100U/mL.
Corresponding cell is taken behind the 3rd day in the tissue-derived Fibroblast cell-culture of application on human skin, the 5th day and the 7th day respectively,
The cell growth state of each cultivation period is observed after film-making, as a result.
Occur when Fig. 1, Fig. 2 and Fig. 3 can be seen that culture to the 3rd day fibroblast, in spindle shape, flat
Star, cultivate to fibroblast portion confluence at the 5th day, and metabolin increases, and cultivates to fibroblast height at the 7th day
Converge, and cluster arranges.
The tissue-derived fibroblast Secondary Culture process of application on human skin：It will cultivate to the Fibroblast cell-culture of the 6th~8 day
Liquid supernatant discarding, with brine twice afterwards add mass concentration be 0.25% trypsin solution (2mL/10cm culture dishes,
4mL/15cm culture dishes), then the condition at 37 DEG C digests 2min, adds isometric (volume content 10%) containing hyclone
DMEM high glucose mediums terminate digestion；Blow and beat culture dish inwall, adherent cell collecting repeatedly with pipette, then supernatant is transferred to
In centrifuge tube and add physiological saline, be 1500rpm in rotating speed, the time be 5min under conditions of once centrifuge, abandoning supernatant,
Physiological saline is added into a centrifugation cell is resuspended, and carry out cell count and secondary centrifuging, abandoning supernatant, to two
The DMEM high glucose mediums containing 10% (volume content) hyclone are added in secondary centrifugation cell is resuspended, and adjusted cell and hang
Liquid-tight degree is 5.0 × 104/ mL~7.5 × 104/ mL, then add 2mL cell suspension into 15cm culture dishes and 18mL contains
The DMEM high glucose mediums of 10% (volume content) hyclone, then be placed in 37 DEG C of incubators and cultivate, carried out every 3~4 days
Passage.
The tissue-derived fibroblastic endochylema vimentin SABC detection of application on human skin：P4 is cultivated for fell skin tissue
Source fibroblast simultaneously makes cell climbing sheet, when cell climbs the 60%~70% of full slide, takes out slide and is floated with PBS solution
Wash, then washed after fixing 15min with 95% ethanol, then block 12min with the methanol solution containing 3% hydrogen peroxide, it is molten with PBS
Liquid rinses 3 times；The anti-human vimentin of mouse is added dropwise on to cell climbing sheet and places 90min at room temperature as primary antibody, juxtaposition, uses PBS
Solution adds enzyme mark sheep anti mouse/rabbit igg as secondary antibody after rinsing 3 times, juxtaposition places 18min at room temperature, uses PBS solution again
DAB solution colour developing 5min is added after rinsing 3 times, rear mounting is redyed with haematoxylin；With on micro- sem observation cell climbing sheet into fibre
Cellular morphology is tieed up, as a result as shown in Figure 4.
From fig. 4, it can be seen that P4 is not colored for the tissue-derived fibroblastic nucleus of application on human skin, cytoplasm coloring.
Control group：Compareed from squamous cell, specific SABC detection process is same as above, thin with micro- sem observation
Squamous cell form on born of the same parents' creep plate, as a result as shown in Figure 5.
From fig. 5, it can be seen that the nucleus and cytoplasm of squamous cell are not colored.
Fig. 4 and Fig. 5 are compared it is known that the tissue-derived fibroblast of the application on human skin of quick separating culture of the present invention passes
To P4 generations, higher cytoactive and purity are still kept.
The manufacturer of the anti-human vimentin of mouse is Wuxi Origene Bio-tech Co., Ltd. in the present embodiment, product
Lot number is 17A10402.
The manufacturer of enzyme mark sheep anti mouse/rabbit igg is Wuxi Origene Bio-tech Co., Ltd., and batch number is
17872。
It is described above, only it is presently preferred embodiments of the present invention, not the present invention is imposed any restrictions.It is every according to invention skill
Any simple modification, change and equivalence change that art is substantially made to above example, still fall within technical solution of the present invention
Protection domain in.
Claims (9)
1. a kind of tissue-derived fibroblast quick separating cultural method of application on human skin, it is characterised in that this method includes following
Step：
Step 1: it will be grown up or the fresh prepuce tissues of the postoperative abandonment of children clean 2~3 times with containing dual anti-physiological saline, so
The adipose tissue of foreskin bottom surface is removed with sterile scissors and tweezers afterwards, and it is 0.5cm to be cut into size2Foreskin fritter；
Step 2: the foreskin fritter obtained in step 1 is immersed in dispase II solution, 37 DEG C are subsequently placed in, water-bath concussion
Under the conditions of once digested；
Step 3: the physiological saline for adding two volumes in postdigestive dispase II solution into step 2 terminates digestion, so
The tissue epidermis of foreskin fritter is removed with sterile scissors and tweezers afterwards, dermal tissue is obtained, the dermal tissue is cut to 1mm3,
Immerse again in clostridiopetidase A IV solution, be placed in 37 DEG C, secondary digestion is carried out under conditions of water-bath concussion；
Step 4: the physiological saline for adding two volumes in postdigestive clostridiopetidase A IV solution into step 3 terminates digestion, so
Supernatant is removed afterwards twice with physiological saline centrifuge washing afterwards, culture medium is added into centrifugation and is resuspended, then be transferred in culture dish,
37 DEG C are placed in, CO2Volumetric concentration is to be cultivated in 5% incubator；The ratio between the quality of the centrifugation and the volume of culture medium
For 3：7, the unit of quality is g, and the unit of volume is mL.
2. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, dual anti-in physiological saline described in step 1 is penicillin and streptomysin, and the concentration of the penicillin and streptomysin is equal
For 100U/mL.
3. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, the mass concentration of the solution of dispase II described in step 2 is 0.4%.
4. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, the time once digested described in step 2 is 1h.
5. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, the mass concentration of the solution of clostridiopetidase A IV described in step 3 is 0.2%.
6. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, the time of secondary digestion described in step 3 is 15min.
7. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, the rotating speed centrifuged described in step 4 is 1500rpm, time 5min.
8. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, culture medium described in step 4 is containing hyclone and dual anti-DMEM high glucose mediums, the high sugar cultures of the DMEM
The volume content of hyclone is 15% in base, it is described it is dual anti-be penicillin and streptomysin, the penicillin and streptomysin it is dense
Degree is 100U/mL.
9. the tissue-derived fibroblast quick separating cultural method of a kind of application on human skin according to claim 1, its feature
It is, the process cultivated described in step 4 is：Fluid infusion is carried out after culture 24h, the partly amount that carries out is cultivated to the 2nd~3 day and changes liquid, train
Support to the 4th~5 day progress full dose and change liquid, cultivate to the 6th~8 day, when cell confluency degree is up to 80%~90%, that is, carry out digestion biography
Generation.
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| CN109402048A (en) * | 2018-10-11 | 2019-03-01 | 浙江大学 | The method for building up of " humanized " arrhythmogenic right ventricular cardiomyopathy disease model |
| CN110438067A (en) * | 2019-08-22 | 2019-11-12 | 广东唯泰生物科技有限公司 | Human skin fibroblasts and preparation method thereof |
| CN112980769A (en) * | 2021-03-02 | 2021-06-18 | 纳美细胞科技有限公司 | Preparation method of autologous skin fibroblasts |
| CN113181219A (en) * | 2021-03-04 | 2021-07-30 | 纳美细胞科技有限公司 | Cell preparation for removing wrinkles and preparation method thereof |
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Application publication date: 20180309 |