CN105950542A - Human skin epidermal cell culture medium and application thereof - Google Patents
Human skin epidermal cell culture medium and application thereof Download PDFInfo
- Publication number
- CN105950542A CN105950542A CN201610473310.4A CN201610473310A CN105950542A CN 105950542 A CN105950542 A CN 105950542A CN 201610473310 A CN201610473310 A CN 201610473310A CN 105950542 A CN105950542 A CN 105950542A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture medium
- application
- epidermis
- human skin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
- C12N2500/14—Calcium; Ca chelators; Calcitonin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/46—Amines, e.g. putrescine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Dermatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a human skin epidermal cell culture medium and application thereof. The culture medium is prepared by the following steps: mixing a K-SFM (keratinocyte serum-free medium), a DMEM (dulbecco's modified eagle medium) and a F12 culture medium according to the ratio of 2:1:1, and adding a BPE (bovine pituitary extract), an EGF (epidermal growth factor), an SCGF (stem cell growth factor), an FGF (fibroblast growth factor), a TGF-beta (transforming growth factor-beta), a VEGF (vascular endothelial growth factor), CaCl2, glutamine and a double-antibody. The culture medium is free of serum, and can be used for human skin epidermal cell primary culture and subculture. The human skin epidermal cell culture medium can obtain abundant epidermal cells by in-vitro culture by using only a small amount of patient skin tissues. Compared with the traditional epidermal cell culture medium, the human skin epidermal cell culture medium provided by the invention greatly shortens the time required by amplifying the same cell count, and satisfies the demands for clinical therapy.
Description
Technical field
The present invention relates to a kind of application on human skin cultured epidermal cell base and application thereof, belong to biomedical sector.
Background technology
Skin is the organ that people's bulk area is maximum, carries protection health, perspires, feels the functions such as cold and hot and pressure, makes internal each tissue and organ from the invasion and attack of physical property, mechanicalness, chemical and pathogenic microorganism.Skin can be caused damage by a lot of reasons such as genetic diseases, burn and scald, chronic cutaneous wound (such as diabetes), vitiligo, albinism, affects skin beauty, makes skin scar even lose physiological function.When skin is severely damaged, during such as the large area burn and scald of the degree of depth, skin can not carry out self-recovery again, can lose and antibacterial is resisted effect, causes a large amount of antibacterial to invade human body by skin, easily causes systemic infection to cause death.
The epithelial cell of application on human skin is epidermis cell, in addition to having general characteristics of epithelial cells, can secrete cutin, has the strongest keratinization feature, in this, as protective epithelium, completes the function of epidermis.Human epidermal cell plays vital effect in wound healing, the especially scarcity of Severe Burn Patients epidermis cell and its course of disease length, the generation of complication, the formation of later stage cicatrix all has substantial connection, and therefore application tissue engineering technique carries out the separation of epidermis cell in vitro, cultivates and preserve and be applied to the study hotspot that clinic is the most all burn worker.
At present, can be used for treating the maximum problem of the tissue engineering epidermis of all kinds of skin trauma be to provide enough epidermis quantity, in order to treat large-area traumatic wounds.Traditional epidermal cell culture base not only needs the interpolation factor of costliness, and its epidermis cell amplification rate cultivated is slow, can not meet the demand of burn and scald treatment in time, this factor has seriously fettered the tissue engineering epidermis clinical practice for the treatment of Acute skin trauma.
Summary of the invention
The constraint faced in clinical practice for tissue engineering epidermis, the invention provides a kind of application on human skin cultured epidermal cell base, this culture medium does not contains serum, have only to the least patient skin tissue, just can cultivate the substantial amounts of epidermis cell of acquisition in vitro, compared with tradition cultured epidermal cell base, substantially reduce the time required for amplification same cell quantity, meet the demand of clinical treatment.
To this end, the present invention uses technical scheme as follows, a kind of application on human skin cultured epidermal cell base, in terms of 500ml, each component and as follows with magnitude relation:
A. minimum necessary culture medium:
Cutin serum-free medium (K-sfm, keratinocyte serum-free medium) 250ml；
DMEM (Ca2+free) culture medium 125ml；
Ham ' s F-12 culture medium 125ml；
B.200mML-glutamine (L-Glutamine): final concentration 1.5mM；
C. cattle pituitary extract (Bovine Pituitary Extract, BPE): final concentration 25 μ g/ml；
D. epidermal growth factor (Epidermal Growth Factor, EGF): final concentration 0.2ng/ml；
E. stem cell factor (Stem Cell Growth Factors, SCGF): final concentration 30ng/ml；
F. fiber mother cell growth factor (Fibroblast growth factor, FGF): final concentration 1.5ng/ml；
G. transforming growth factor-β (Transforming growth factor-β, TGF-β): final concentration 0.8ng/ml；
H. VEGF (Vascular endothelial growth factor, VEGF): final concentration 10ng/ml；
I.100X Pen .-Strep (Penicillin/Streptomycin, PS): final concentration of 1X；
J.0.3M calcium chloride solution (CaCl2): final concentration of 0.4mM.
The each ingredient names of culture medium of the present invention and source are listed below:
| Nomenclature of drug | Company and article No. |
| K-sfm | Life Technologies catalog#17005-042042 |
| DMEM | Life Technologies catalog#21068-028 |
| Ham’s F-12medium | Life Technologies catalog#31765-092 |
| 200mM L-Glutamine | Life Technologies catalog#25030081 |
| BPE | ScienCell catalog#0713 |
| EGF | R&D systems catalog#236-EG-01M |
| Penicillin/Streptomycin | Life Technologies catalog#15140122 |
| SCGF | Peprotech#100-22B-10 |
| FGF | Millipore#341595 |
| TGF-β | Peprotech#500-P16Bt-25 |
| VEGF | Peprotech#AF-100-20-10 |
Compound method is:
Take K-SFM and DMEM and F12 proportionally 2:1:1 mixing, add the factor and make its final concentration reach: the BPE (cattle pituitary extract) of 25 μ g/ml, the EGF (epidermal growth factor) of 0.2ng/ml, the CaCl of 0.4mM2The SCGF (stem cell factor) of 30ng/ml, the FGF (fiber mother cell growth factor) of 1.5ng/ml, the TGF-β (transforming growth factor) of 0.8ng/ml, the VEGF (VEGF) of 10ng/ml, the glutamine (glutamine) of 1.5mM, 1xSAC is dual anti-, uses after mixing.
Culture medium of the present invention is primary and Secondary Culture for application on human skin epidermis cell, is particularly applicable to: 1. the required epidermal graft of burn wound's treatment is cultivated；2. cicatrix wound surface shaping and beauty；3. dermatosis treating medicine screening；4. the screening of cosmetics active ingredient and epidermis cell is had free of toxic effects for determining；5. determine and screen the impact on epidermal growth of some cytokine；6. other skin physiologies and dermatopathology research.
Described application on human skin epidermis cell collection be derived from fetal scalp, neonatal foreskin or adult scalp containing multi-functional epidermis and the tissue samples of hypodermal cell；Or from any tissue SOX2 gene expression Interstitial cell and from any tissue samples have the ability to be formed monoclonal epidermis cell and frost melt after still can have the ability to be formed the cell of application on human skin.
There is advantages that
1, the cultivating system of research preparation of the present invention is serum-free, is applied to human epidermal cell In vitro culture, and its effect compares without significant difference with there being serum.Dynamically observe under inverted microscope: serum-free group is similar to having serum group epidermal growth feature and the speed of growth.The comparison of ingredients of serum is complicated, and the not clear factor is various, and the change of somatomedin contained between each batch of serum and hormone is the biggest.Therefore, the culture medium of serum-free more can meet the prescription in clinical treatment for tissue engineering epidermis.
2, the cultured epidermal cell system that the present invention develops is not only suitable for passing on cultured epidermal cell, and is suitable for the cultivation of primary epidermis cell.The more common cultured epidermal cell base of country is cutin culture medium (the keratinocyte serum-free medium of serum-free now, K-sfm), the primary epidermal growth speed of this culture medium culturing is slow, the primary epidermal cell density cultivated is less, and is not easily formed epidermal graft.So, the primary epidermis cell separated only first has been used blood serum medium to cultivate by a lot of research workers, the most gradually changes K-sfm culture medium culturing after cell attachment well-grown into.For seeking to be similar to the condition of culture of physiological status, explore the cultivating system being suitable for primary epidermal growth, we are culture medium based on a small amount of K-sfm culture medium and the DMEM culture medium of non-calcium ions and F12 culture medium, with the addition of the complete medium of all kinds of factors composition serum-free that can promote Keratiocyte growth.Meanwhile, we with the addition of a certain amount of calcium ion in this culture medium, can promote the formation of cell clone and the adherent growth of cell, greatly facilitate the amplification rate of epidermis cell, saves the time required for cultivation, more reduces cost.
3, the cultured epidermal cell system that the present invention develops can make adult epidermal's cell of cultivation retain more preferable stem cell properties, and in the clinical transplantation of tissue engineering epidermis, the stem cell properties of epidermis cell is the strongest, and its growth healing ability is the best.Research shows, the cytokines such as stem cell factor and active component are for maintaining stem cell microenvironment, cell injury is repaired, and promote noble cells to regain differentiation capability to have greatly effect, wherein stem cell factor (SCGF) is for activating and maintain stem cells hyperplasia differentiation capability to have useful effect.Fiber mother cell growth factor (FGF) can promote that endothelioid cells is bred, and has preferable repair to damaging cells simultaneously.Transforming growth factor (TGF) is a kind of multifunctional protein for cell, can affect the functions such as the growth of various kinds of cell, differentiation, apoptosis and immunomodulating.Transforming growth factor-β (TGF-β) is a member of TGF family, can play its biological function by SMAD signal path and/or DAXX signal path.By the effect of the above-mentioned factor, can delay or reverse both ageing state, can also damage by repair cell simultaneously, improve cell proliferation, differentiation vigor.Therefore our culture medium can be that the clinical treatment of skin trauma brings the higher epidermis cell of stem cell properties.
Accompanying drawing explanation
Fig. 1 is the primary epidermis cell that embodiment 1 is cultivated containing serum epidermal cell culture base by tradition.
Fig. 2 is the primary epidermis cell of embodiment 1 the present inventor's epiderm skin culture medium culturing.
Fig. 3 is the primary epidermis cell that embodiment 2 K-sfm epidermal cell culture base is cultivated.
Fig. 4 is the primary epidermis cell of embodiment 2 the present inventor's skin epidermal cells culture medium culturing.
Fig. 5 be embodiment 3 K-sfm epidermal cell culture base cultivate pass on neonatal foreskin epidermis cell.
Fig. 6 is that embodiment 3 passes on neonatal foreskin epidermis cell with the present inventor's epiderm skin culture medium culturing.
Fig. 7 be embodiment 4 K-sfm epidermal cell culture base cultivate pass on adult's epidermal cells of prepuce.
Fig. 8 is that embodiment 4 passes on adult's epidermal cells of prepuce with the present inventor's epiderm skin culture medium culturing.
Fig. 9 is that the adult's epidermal cells of prepuce cultivated of two kinds of epidermal cell culture bases of embodiment 4 photo after immunofluorescence dyeing compares: wherein A is adult's epidermal cells of prepuce immunofluorescence dyeing result that K-sfm epidermal cell culture base is cultivated；B is adult's epidermal cells of prepuce immunofluorescence dyeing result of the present inventor's epiderm skin culture medium culturing.
Detailed description of the invention
Below in conjunction with concrete test method and accompanying drawing, technical scheme and produced technique effect thereof are further elaborated; the description below is merely to explain the present invention; but never in any form the present invention is any limitation as; based on present invention teach that any conversion or replacement made, belong to protection scope of the present invention.
Method used in the present invention if no special instructions, is this area conventional method.Test material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Example one: the present inventor's skin epidermal cells culture medium and tradition contain primary epidermis cell form and the comparison of cell quantity that serum epidermal cell culture base is cultivated
Material and method:
Tissue-derived: hospital surgical discards neonatal foreskin
Cell culture medium: tradition is containing serum epidermal cell culture base, the present inventor's epiderm skin culture medium
A) the fresh tissue samples gathered is disinfected process three minutes in alcohol, and then process twice with the PBS dual anti-containing 2 times, each 3 minutes.Tissue is soaked in and saves backup containing in 1 times of dual anti-F12 culture medium 4 DEG C.The tissue preserration time is not higher than 72 hours.
B) taking-up it is organized in immersion liquid, drip clean surplus liquid, slitting shape will be organized after weighing and performing record, and be laid in uniformly in 100mm culture dish, neutral protease (the Dispase that concentration is 2.5mg/ml is added in culture dish, neutral protease, grade II, neutral protease, also known as Bacillus polymyxa Neutral proteinase) enzyme liquid, every 5g skin histology adds 20ml enzyme liquid, makes each piece of skin histology all be completely submerged in enzyme liquid, by skin histology in 4 DEG C of static overnight incubation.
C) in Dispase, the neonatal foreskin skin of overnight incubation takes out from refrigerator, is carefully stripped down from corium by epidermis with pointed tweezers, proceeds to epidermis, in a new culture dish, drip clean surplus liquid, be cut into by tissue the most thick with scalpel.
D) add the phosphate buffer (PBS) that pancreas enzyme concentration is 0.05% and be used for the digestion of epidermis, every 5g skin histology adds 20ml above-mentioned enzyme liquid；Digesting 30 minutes at 37 DEG C, in digestion process, constantly shake mixes culture dish, shakes scattered by the tissue homogenate of agglomerate, becomes cell suspension；The DMEM containing 10%FBS of same volume is added to neutralize enzymic digestion liquid at the cell suspension digested.Blow and beat 20-30 time, it is to avoid blowout bubble.
E) the cell 100um filter after neutralizing filters.Filter process stirs solution with pipet so that it is filter completely.
F) regard how many tissue mass uses DMEM (containing the dual anti-) flush filter of appropriate (5～10ml) 10%FBS.
G) 1000rpm is centrifuged 5 minutes.Abandon supernatant.Often pipe addition 10mL is without the dual anti-resuspended precipitation of F12 culture medium, and 1000rpm is centrifuged 5 minutes.Count and make a record.
The epidermis cell of the separator well h) taking equal number is cultivated by two kinds of culture medium respectively, identical condition of culture (37 DEG C, 5% carbon dioxide) cultivate 5 days after observation of cell state.
Interpretation of result:
Fig. 2 is the primary epidermis cell cultivated containing serum epidermal cell culture base by tradition.Fig. 2 is with the primary epidermis cell of the present inventor's epiderm skin culture medium culturing.Be can be seen that by Fig. 1 and Fig. 2, the primary epidermis cell state cultivated with the application on human skin cultured epidermal cell base of our invention is compared with the primary epidermis cell state cultivated with the traditional epidermal cell culture base containing serum, cell density, cell shape size, all without significant difference, all can form complete epidermal graft.Therefore, we research and develop the application on human skin epidermal cell culture base of preparation and fully achieve traditional culture effect containing serum epidermal cell culture base.
Example two: use the present inventor's skin epidermal cells culture medium and tradition serum-free cutin culture medium (K-sfm) the primary epidermis cell form cultivated and the comparison of cell quantity
Material and method:
Tissue-derived: hospital surgical discards fetal scalp
Cell culture medium: K-sfm culture medium, the present inventor's epiderm skin culture medium
A) the fresh tissue samples gathered is disinfected process three minutes in alcohol, and then process twice with the PBS dual anti-containing 2 times, each 3 minutes.Tissue is soaked in and saves backup containing in 1 times of dual anti-F12 culture medium 4 DEG C.The tissue preserration time is not higher than 72 hours.
B) taking-up it is organized in immersion liquid, drip clean surplus liquid, slitting shape will be organized after weighing and performing record, and be laid in uniformly in 100mm culture dish, neutral protease (the Dispase that concentration is 2.5mg/ml is added in culture dish, neutral protease, grade II, neutral protease, also known as Bacillus polymyxa Neutral proteinase) enzyme liquid, every 5g skin histology adds 20ml enzyme liquid, makes each piece of skin histology all be completely submerged in enzyme liquid, by skin histology in 4 DEG C of static overnight incubation.
C) in Dispase, the embryo skin of overnight incubation takes out from refrigerator, is carefully stripped down from corium by epidermis with pointed tweezers, proceeds to epidermis, in a new culture dish, drip clean surplus liquid, be cut into by tissue the most thick with scalpel.
D) add the phosphate buffer (PBS) that pancreas enzyme concentration is 0.05% and be used for the digestion of epidermis, every 5g skin histology adds 20ml above-mentioned enzyme liquid；Digesting 30 minutes at 37 DEG C, in digestion process, constantly shake mixes culture dish, shakes scattered by the tissue homogenate of agglomerate, becomes cell suspension；The DMEM containing 10%FBS of same volume is added to neutralize enzymic digestion liquid at the cell suspension digested.Blow and beat 20-30 time, it is to avoid blowout bubble.
E) the cell 100um filter after neutralizing filters.Filter process stirs solution with pipet so that it is filter completely.
F) regard how many tissue mass uses DMEM (containing the dual anti-) flush filter of appropriate (5～10ml) 10%FBS.
G) 1000rpm is centrifuged 5 minutes.Abandon supernatant.Often pipe addition 10mL is without the dual anti-resuspended precipitation of F12 culture medium, and 1000rpm is centrifuged 5 minutes.Count and make a record.
The epidermis cell of the separator well h) taking equal number is cultivated by two kinds of culture medium respectively, identical condition of culture (37 DEG C, 5% carbon dioxide) cultivate 3 days after observation of cell state.
Interpretation of result:
Fig. 3 is the primary epidermis cell cultivated with K-sfm epidermal cell culture base.Fig. 4 is with the primary epidermis cell of the present inventor's skin epidermal cells culture medium culturing.Be can be seen that by the cell photo cultivated, the primary epidermis cell state that application on human skin cultured epidermal cell base after improveing with us is cultivated is compared with the primary epidermis cell state that K-sfm epidermal cell culture base is cultivated, growth phase is bigger with time cell density, cell state is more preferable, and is more likely formed the epidermal graft that formation is complete.
Example three: what employing the present inventor's epiderm skin culture medium and tradition serum-free cutin culture medium (K-sfm) were cultivated passes on neonatal foreskin epidermis cell form and the comparison of cell quantity
Material and method:
Cell: have passed through the neonatal foreskin epidermis cell of Secondary Culture
Culture medium: K-sfm culture medium, the present inventor's epiderm skin culture medium
A) the old culture fluid in culture bottle is removed.
B) every bottle of use 5～10ml PBS washes the serum once removing residual.
C) T75flask adds the 3ml 0.05% pancreatin containing EDTA in 37 degree of incubators, digest 10min.
D) examining under a microscope, if cell all comes off, every bottle adds the 10ml DMEM containing 10%FBS and neutralizes (can softly blow and beat several times), collected by whole cells in a 50ml centrifuge tube.
E) 1000rpm is centrifuged 5 minutes, abandons supernatant.
F) 10ml is added containing dual anti-F12 culture medium re-suspended cell.Counting.
G) 1000rpm is centrifuged 5 minutes, abandons supernatant.
H) epidermis cell is passed in the ratio of 1:3, observation of cell state after cultivating 3 days with the condition of culture (37 DEG C, 5% carbon dioxide) that two kinds of different culture medium are identical.
Interpretation of result:
Fig. 5 be K-sfm epidermal cell culture base cultivate pass on neonatal foreskin epidermis cell；Fig. 6 is the present inventor
Epiderm skin culture medium culturing pass on neonatal foreskin epidermis cell.Be can be seen that by the cell photo cultivated, compared with the epidermis cell state that passes on cultivated with the application on human skin cultured epidermal cell base of our invention passes on neonate epidermis cell state with K-sfm epidermal cell culture base is cultivated, growth phase is bigger with time cell density, cell state is more preferable, and is more likely formed the epidermal graft that formation is complete.Owing to neonate epidermis cell has stronger stem cell properties, after passing on, cell dryness is difficult to weaken, so the neonatal cells stem cell properties of two kinds of culture medium culturings is without larger difference.
Example four: what employing the present inventor's epiderm skin culture medium and tradition serum-free cutin culture medium (K-sfm) were cultivated passes on adult's epidermal cells of prepuce form and the comparison of cell quantity
Material and method:
Cell: have passed through adult's epidermal cells of prepuce of Secondary Culture
Culture medium: K-sfm culture medium, the present inventor's epiderm skin culture medium
A) cell density collects cell to when 90% with trypsin.Cell is forwarded to 15ml centrifuge tube counting.
B) 1000 the heart is left 4 minutes.
C) supernatant is sopped up, with culture medium re-suspended cell and to adjust cell concentration be 1 × 106Individual/ml.
D) in 24 orifice plates, every hole adds 3 × 105Individual cell, supplementing culture medium to 2ml.
E) 37 DEG C, CO2Incubator is cultivated 1～2 day.
F) sucking-off culture medium, every hole 500 μ l PBS 3 times.
G) every hole 4%PFA of 200～300 μ l fixes 20 minutes.
H) 4%PFA is sopped up, every hole 500 μ l PBS 3 times (in this step, the PBS cleaned for the last time can be stayed in hole, and save backup in 4 DEG C).
I) closing the cell cultivated with the PBS containing 5%NGS (sheep blood serum), be subsequently adding 5%NDS+0.2%TX-100, room temperature processes 30 minutes (every hole 500 μ l).
J) PBS is used 1 time, 500 μ l/ holes.
K) Anti-Cytokeratin 19antibody-Alexa is used488 (diluting with the PBS1:500 containing 5%NGS) incubated cell, is subsequently adding 5%NDS+0.2%TX-100, and room temperature processes 60 minutes (every hole 300 μ l).
L) PBS is used 3 times, 500 μ l/ holes.
M) 2～3 minutes (every hole 500 μ l) is hatched with Hirst fluorescent dye or DAPI (1:10000 dilutes, 4 DEG C of preservations) room temperature.
N) PBS is used 3 times, 500 μ l/ holes.
O) last cleanout fluid is stayed in hole
P) fluorescence microscope cell dyeing situation and cell quantity.
Interpretation of result:
Fig. 7 is to pass on adult's epidermal cells of prepuce with what K-sfm epidermal cell culture base was cultivated；Fig. 8 is to pass on adult's epidermal cells of prepuce with the present inventor's epiderm skin culture medium culturing；Fig. 9 is that the photo after immunofluorescence dyeing of the adult's epidermal cells of prepuce with two kinds of epidermal cell culture bases cultivations compares: wherein A is adult's epidermal cells of prepuce immunofluorescence dyeing result that K-sfm epidermal cell culture base is cultivated；B is adult's epidermal cells of prepuce immunofluorescence dyeing result of the present inventor's epiderm skin culture medium culturing.Be can be seen that by the cell photo cultivated, compared with the adult's epidermal cells of prepuce state that passes on cultivated with the novel application on human skin cultured epidermal cell base of our invention passes on epidermal cells of prepuce state of being grown up with K-sfm epidermal cell culture base is cultivated, after cell cultivates the identical time, our the culture medium cell density of invention is bigger, and noble cells is less, and little with cell density after adult epidermal's passage of K-sfm cultivation, and cell differentiation is the most serious.Cytokeratin 19 content in undifferentiated cell is more, we have found that, two kinds of cell states after immunofluorescence dyeing can be seen that, content by adult's epidermal cells of prepuce CK19 of novel culture medium cultivation is more much higher than the epidermis cell of K-sfm culture medium culturing, illustrating that adult epidermal's cell stem cell characteristic of the novel epidermis culture medium culturing that we invent is higher, more conducively clinical transplantation treats various skin traumas.
Claims (5)
1. an application on human skin cultured epidermal cell base, is characterized in that, in terms of 500ml, each component and consumption are as follows:
A. minimum necessary culture medium: cutin serum-free medium 250ml, DMEM culture medium 125ml,
Ham ' s F-12 culture medium 125ml；
B.200mML-glutamine: final concentration 1.5mM；
C. cattle pituitary extract: final concentration 25 μ g/ml；
D. epidermal growth factor: final concentration 0.2ng/ml；
E. stem cell factor: final concentration 30ng/ml；
F. fiber mother cell growth factor: final concentration 1.5ng/ml；
G. transforming growth factor-β: final concentration 0.8ng/ml；
H. VEGF: final concentration 10ng/ml；
I.100X Pen .-Strep: final concentration of 1X；
J.0.3M calcium chloride solution: final concentration of 0.4mM.
2. application on human skin cultured epidermal cell base described in claim 1 is primary at application on human skin epidermis cell and in Secondary Culture
Application.
3. application on human skin cultured epidermal cell base described in claim 2 is primary at application on human skin epidermis cell and in Secondary Culture
Application, it is characterized in that, described application on human skin epidermis cell collection is derived from fetal scalp, neonatal foreskin or adult
Scalp containing multi-functional epidermis and the tissue samples of hypodermal cell；Or the SOX2 gene from any tissue
The Interstitial cell expressed and have the ability to form monoclonal epidermis cell and frost from any tissue samples and melt
Still can have the ability to be formed the cell of application on human skin after change.
4. application on human skin cultured epidermal cell base described in Claims 2 or 3 is in application on human skin epidermis cell original cuiture
Application, is characterized in that, step is as follows:
A) the fresh skin histology sample gathered is disinfected in alcohol process three minutes, and then with dual anti-containing 2 times
PBS process twice, each 3 minutes；Tissue is soaked in containing 4 DEG C of guarantors in 1 times of dual anti-F12 culture medium
Deposit standby；
B) it is organized in immersion liquid taking-up, drips clean surplus liquid, after weighing and performing record, tissue is cut into length
Strip, is laid in 100mm culture dish uniformly, and adding concentration in culture dish is in 2.5mg/ml
Property protease, in every 5g skin histology add 20ml enzyme liquid, make each piece of skin histology all be completely submerged in
In enzyme liquid, by skin histology in 4 DEG C of static overnight incubation；
C) skin histology of overnight incubation takes out from refrigerator, is carefully peeled off from corium by epidermis with pointed tweezers
Come, proceed to epidermis, in a new culture dish, drip clean surplus liquid, be cut into uniformly by tissue with scalpel
Thick；
D) phosphate buffer that pancreas enzyme concentration is 0.05% is added for the digestion of epidermis, in every 5g skin histology
Add 20ml enzyme liquid；Digesting 30 minutes at 37 DEG C, in digestion process, constantly shake mixes culture dish, will be poly-
The tissue homogenate of group shakes scattered, becomes cell suspension；The cell suspension digested add same volume containing 10%FBS
DMEM neutralize enzymic digestion liquid, blow and beat 20-30 time, it is to avoid blowout bubble；
E) the cell 100um filter after neutralizing filters, and stirs solution with pipet, make in filter process
It filters completely；
F) with the appropriate DMEM flush filter containing dual anti-10%FBS；
G) 1000rpm is centrifuged 5 minutes, abandons supernatant, and often pipe adds 10mL without dual anti-F12 cultivation basic weight
Outstanding precipitation, 1000rpm is centrifuged 5 minutes, counts and make a record,
H) epidermis cell of separator well is taken respectively by culture medium 37 DEG C described in claim 1,5% carbon dioxide conditions
Under cultivate.
5. application on human skin cultured epidermal cell base described in Claims 2 or 3 is in application on human skin epidermis cell Secondary Culture
Application, is characterized in that, step is as follows:
A) take and have passed through the neonatal foreskin epidermis cell of Secondary Culture, the old culture fluid in culture bottle is removed；
B) every bottle of use 5～10ml PBS washes the serum once removing residual；
C) T75flask adds the 3ml 0.05% pancreatin containing EDTA in 37 degree of incubators, digest 10min；
D) examining under a microscope, if cell all comes off, every bottle adds the 10ml DMEM containing 10%FBS
Neutralize, whole cells are collected in a 50ml centrifuge tube；
E) 1000rpm is centrifuged 5 minutes, abandons supernatant；
F) 10ml is added containing dual anti-F12 culture medium re-suspended cell, counting；
G) 1000rpm is centrifuged 5 minutes, abandons supernatant；
H) epidermis cell is passed in the ratio of 1:3, with culture medium described in claim 1 37 DEG C, 5% carbon dioxide
Under the conditions of cultivate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610473310.4A CN105950542A (en) | 2016-06-24 | 2016-06-24 | Human skin epidermal cell culture medium and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610473310.4A CN105950542A (en) | 2016-06-24 | 2016-06-24 | Human skin epidermal cell culture medium and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105950542A true CN105950542A (en) | 2016-09-21 |
Family
ID=56903886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610473310.4A Pending CN105950542A (en) | 2016-06-24 | 2016-06-24 | Human skin epidermal cell culture medium and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105950542A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591220A (en) * | 2016-12-29 | 2017-04-26 | 深圳市永生原代细胞生物科技有限公司 | Human normal skin epithelial cell and use thereof |
| CN106591219A (en) * | 2016-12-22 | 2017-04-26 | 叶宗耀 | Culture medium for epidermal cells |
| CN106754640A (en) * | 2016-12-19 | 2017-05-31 | 江西宜信堂医疗科技有限公司 | A kind of cultured epidermal cell base and preparation method thereof |
| CN107129967A (en) * | 2017-06-18 | 2017-09-05 | 广东博溪生物科技有限公司 | A kind of serum free medium system for primary human tonsillar cell |
| CN107287153A (en) * | 2017-06-18 | 2017-10-24 | 广东博溪生物科技有限公司 | A kind of differential digestion method of human epidermal cell |
| CN108421034A (en) * | 2018-04-24 | 2018-08-21 | 济南磐升生物技术有限公司 | Application of the kallikrein 7 in skin wound healing |
| CN109364028A (en) * | 2018-12-14 | 2019-02-22 | 济南磐升生物技术有限公司 | The preparation method and application of the nanoparticle liposome of human skin cell's growth factor |
| CN110302426A (en) * | 2019-07-10 | 2019-10-08 | 山东大学 | A kind of stem cell skin adhesive piece and its preparation method and application |
| CN111337494A (en) * | 2020-03-27 | 2020-06-26 | 济南磐升生物技术有限公司 | Preparation method and application of artificial epidermis cosmetic detection kit |
| CN115227876A (en) * | 2022-07-28 | 2022-10-25 | 福建省海西细胞生物工程有限公司 | Preparation method of tissue engineering epidermis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101152580A (en) * | 2007-09-13 | 2008-04-02 | 陆洪光 | Method for producing tissue engineering skin outside of the body with scarifiskin stem cell |
| CN105219696A (en) * | 2014-07-04 | 2016-01-06 | 赫柏慧康生物科技无锡有限公司 | A kind of novel people's epidermal cell culture method |
| CA2968048A1 (en) * | 2014-10-29 | 2016-05-06 | R Bio Co., Ltd. | Medium composition for culturing stem cells |
| CN105602890A (en) * | 2016-03-01 | 2016-05-25 | 陕西博溪生物科技有限公司 | In-vitro skill model and keratinocyte cell long-acting culture solution |
-
2016
- 2016-06-24 CN CN201610473310.4A patent/CN105950542A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101152580A (en) * | 2007-09-13 | 2008-04-02 | 陆洪光 | Method for producing tissue engineering skin outside of the body with scarifiskin stem cell |
| CN105219696A (en) * | 2014-07-04 | 2016-01-06 | 赫柏慧康生物科技无锡有限公司 | A kind of novel people's epidermal cell culture method |
| CA2968048A1 (en) * | 2014-10-29 | 2016-05-06 | R Bio Co., Ltd. | Medium composition for culturing stem cells |
| CN105602890A (en) * | 2016-03-01 | 2016-05-25 | 陕西博溪生物科技有限公司 | In-vitro skill model and keratinocyte cell long-acting culture solution |
Non-Patent Citations (1)
| Title |
|---|
| 汪丽俐: "人表皮黑素细胞体外培养的条件探讨及鉴定", 《皮肤性病诊断学杂志》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754640A (en) * | 2016-12-19 | 2017-05-31 | 江西宜信堂医疗科技有限公司 | A kind of cultured epidermal cell base and preparation method thereof |
| CN106591219A (en) * | 2016-12-22 | 2017-04-26 | 叶宗耀 | Culture medium for epidermal cells |
| CN106591220A (en) * | 2016-12-29 | 2017-04-26 | 深圳市永生原代细胞生物科技有限公司 | Human normal skin epithelial cell and use thereof |
| CN107129967A (en) * | 2017-06-18 | 2017-09-05 | 广东博溪生物科技有限公司 | A kind of serum free medium system for primary human tonsillar cell |
| CN107287153A (en) * | 2017-06-18 | 2017-10-24 | 广东博溪生物科技有限公司 | A kind of differential digestion method of human epidermal cell |
| CN107287153B (en) * | 2017-06-18 | 2023-11-07 | 广东博溪生物科技有限公司 | Differential digestion method of human epidermal cells |
| CN108421034A (en) * | 2018-04-24 | 2018-08-21 | 济南磐升生物技术有限公司 | Application of the kallikrein 7 in skin wound healing |
| CN109364028A (en) * | 2018-12-14 | 2019-02-22 | 济南磐升生物技术有限公司 | The preparation method and application of the nanoparticle liposome of human skin cell's growth factor |
| CN110302426A (en) * | 2019-07-10 | 2019-10-08 | 山东大学 | A kind of stem cell skin adhesive piece and its preparation method and application |
| CN111337494A (en) * | 2020-03-27 | 2020-06-26 | 济南磐升生物技术有限公司 | Preparation method and application of artificial epidermis cosmetic detection kit |
| CN115227876A (en) * | 2022-07-28 | 2022-10-25 | 福建省海西细胞生物工程有限公司 | Preparation method of tissue engineering epidermis |
| CN115227876B (en) * | 2022-07-28 | 2023-08-29 | 福建省海西细胞生物工程有限公司 | Preparation method of tissue engineering epidermis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105950542A (en) | Human skin epidermal cell culture medium and application thereof | |
| CN101330935B (en) | Isolation and cultivation of stem/progenitor cells from the amniotic membrane of umbilical cord and uses of cells differentiated therefrom | |
| Supp et al. | Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| Labus et al. | Expression of Wnt genes in early wound healing | |
| CN101366728A (en) | Compositions for preventing or treating skin defects and methods of use thereof | |
| CN105079783B (en) | Pharmaceutical composition and its preparation method and application | |
| CN107217028A (en) | A kind of organization engineering skin containing appendicle and preparation method thereof | |
| CN106520676A (en) | Method for preparing human amniotic membrane epithelial cells from human placenta amnion and application thereof | |
| ATE517177T1 (en) | METHOD FOR CULTIVATION OF HUMAN MESENCHYMAL STEM CELLS, IN PARTICULAR FOR THE TREATMENT OF NON-HEALING BREAKS, AND BIOREACTOR FOR CARRYING OUT THIS CULTIVATION METHOD | |
| CN107254431A (en) | A kind of novel tissue engineering skin preparation method | |
| CN102172337B (en) | Tissue engineering skin with sebaceous gland-like structure and preparation method thereof | |
| CN108066750A (en) | Stem cell and its secretion are used to treat the new application of skin burn | |
| CN103893831B (en) | A kind of organotypic artificial skin, its preparation method and application | |
| CN105963795A (en) | Method for preparing tissue engineering epidermis based on collagen | |
| CN104971382A (en) | Adhesive bandage type artificial active tissue constructed by using culture solution without serum or bovine pituitary extracts and construction method of adhesive bandage type artificial active tissue | |
| CN101760447A (en) | Method for inducing and acclimating epidermal stem cells into nerve cells | |
| CN101875915B (en) | Method for inducing and acclimatizing epidermal stem cells into adipocytes | |
| CN105925524A (en) | Method for transforming human primary fibroblast into epidermic cell | |
| CN109722410B (en) | 3D full-layer skin model, culture medium for forming same and preparation method | |
| RU2384618C2 (en) | Method for making fibroblast-like cells of umbilical cord of newborn | |
| CN102552323A (en) | Medicine for accelerating skin repair and regeneration, preparation method thereof and application thereof | |
| AU2021105854A4 (en) | Cultivation method of porcine hair follicle stem cell | |
| CN105238740A (en) | In-vitro screening and culturing method for tissue engineering skin seed epidermal keratinocyte high-simulation in-vivo cell extracellular matrix attachment | |
| CN104561101B (en) | Methods and application of the 3p of MicroRNA 221 in epidermal cell is prepared |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wu Xunwei Inventor after: Xing Zhiqing Inventor before: Xing Zhiqing Inventor before: Wu Xunwei |
|
| COR | Change of bibliographic data | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160921 |