CN106676060A - Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration - Google Patents
Gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration Download PDFInfo
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- CN106676060A CN106676060A CN201710013729.6A CN201710013729A CN106676060A CN 106676060 A CN106676060 A CN 106676060A CN 201710013729 A CN201710013729 A CN 201710013729A CN 106676060 A CN106676060 A CN 106676060A
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Abstract
The method provides a gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration. The method comprises the following steps: regenerating a hair follicle by culturing the outer root sheath of a vibrissa hair follicle, reestablishing the three-dimensional morphology of the hair follicle, simulating a body environment in a culture chamber, and culturing the outer root sheath of the hair follicle and embryo skin by utilizing the characteristics that the survival time of new hair follicles is relatively long, the neomorphosis is regular, and the hair follicles are not easy to collapse. Animal tissues (the hair follicles and fetal rat skin) are cultured on a gas-liquid interface by simulating the body environment, the cultured animal hair follicles and embryo skin have certain physiological functions, and the embryo skin can be healed without scars after being subjected to manual minimally invasive surgery; the gas-liquid interface three-dimensional culture method for skin and hair follicle regeneration is a method for culturing stem cells and rapidly establishing a medical regeneration model, and hair follicle regeneration and skin scarless healing mechanisms can be explored outside the body; the design is reasonable, the operation is simple, and an in vitro tissue regeneration and wound healing model can be easily established in short time.
Description
Technical field：
Skin of the present invention and hair follicle regeneration technical field, and in particular to the gas-liquid interface of a kind of skin and hair follicle regeneration is three-dimensional
Cultural method.
Background technology：
Perifollicolar has skin, and skin is wrapped in whole body, while being also interior environment as the maximum organ of body
First important protective barrier between natural environment, protects body from the invasion and attack of various factors in environment, maintains interior environment
Stable state.Wherein hair follicle has begun to development as the main accessory organ of skin in embryonic stage, and hair follicle is to be enclosed in hair
The bourse of root, internal layer is that epithelial tissue hair follicle is connected with epidermis, and outer layer is that connective tissue hair follicle is connected with corium.
Hair follicles maturity is made up of the external root sheath and inner root sheath of epithelium part and the hair papilla and dermal sheath of dermal partial.The development of hair follicle
The interaction of series of complex between epidermis and corium and formed, with self renewal and the characteristics of cyclical growth.
In addition the growth cycle of hair follicle can be divided into trophophase, phase of decline and rest period, and the morphological characteristic of hair follicle is different within three cycles.
The artificial skin containing corium and epidermis is successfully developed recently both at home and abroad, but these skins and has not contained
Hair follicle, the ability of its protective barrier effect is substantially reduced, it is well known that hair follicle plays a significant role in skin injury reparation,
Behind artificial skin without hair follicle transplanting affected part, in the event of secondary damage, it will the wound surface formation to patient is difficult to what is healed
Cicatrix.In addition, at present the domestic and international Mechanism Study to hair follicle cell biology and hair follicle stem cells wound repairing is concentrated mainly on
On skin of living body, while to the research of hair follicle regeneration mechanism aspect also mainly based on internal hair follicle growth situation.These grind
The source of material is studied carefully based on live body, and live body is affected by the various factors of environment, therefore final result of study and differ
Surely the biological property under hair follicle cell rest state can be reflected, while the mobility of live body is also brought for experimentation
Inconvenience.So, up to the present, the research to hair follicle regeneration mechanism and hair follicle stem cells wound repairing mechanism is not still very bright
.
Some researchers use hair follicle dermal papilla cell, raw coal bunker cell to carry out the mixing of different proportion, so
Hairless mouse back is injected afterwards, trial induces hair follicle tissue, although result can be found that the hair follicle sample tissue of some growths, but
The morphologic observation of hair follicle is simultaneously unintelligible, and observed hair follicle sample is organized as agglomerating sample, can not distinguish hair follicle structure, therefore
The model of research hair follicle regeneration mechanism cannot be become.Also there are some researchers to carry out consubstantiality using mice antenna hair follicle in addition
Research hair follicle biology is carried out with heteroplastic transplantation, although find that hair follicle cell has certain Immune privilege, but transplant
The research of its growth conditions of hair follicle afterwards and exemption mechanism is not deeply carried out, while and having researcher to mice antenna hair
Capsule carries out different cross section detachments, then carries out In vitro culture, observes growth conditions, but final result is unsatisfactory,
Hair follicle tissue's rate of growth of detachment is too low, while stick together with culture dish, and hair follicle tissue is invaded completely in culture medium, carefully
The harmful substance that born of the same parents' metabolism is discharged easily kills hair follicle cell, therefore the hair follicle tissue cultivated regenerates situation and is not so good as
Meaning, can not clearly observe hair follicle regeneration state.
In addition, in the research in terms of skin Scarless wound healing mechanism, up to the present, the mechanism of skin Scarless wound healing
Still without clear and definite, the phenomenon that wound surface heals without cicatrix from the point of view of current result of study, wound surface without cicatrix this phenomenon only
Occur in Embryo tissue prometaphase, to late embryogenesis the Scarless wound healing final result of wound surface is but unable to reach.And research embryo's wound
Pregnant Mus amount required for the Scarless wound healing mechanism of face is big, and tracks a large amount of mice oestrus, and becomes pregnant for mice, and records
Mice embryonic natural law, workload is big, and easily causes confusion.Simultaneously carrying out surgery to the pregnant Mus uterus of live body makes wound, but makes wound
Afterwards and suture uterus pregnant Mus later stage survival rate it is extremely low, operation technique is improper to cause pregnant Mus wound infection, cause pregnant Mus a large amount of
Death, therefore the experimental result for finally giving not is to be expected experimental result.
At present, hair follicle and skin are the focuses of the disciplinary study such as cytobiology and skin wound reparation and dermatological,
It is related to the positioning of hair follicle stem cells, the morphological analysis of hair follicle, hair follicle signal transduction, somatomedin, cytokine and corium
The biological function research of many aspects such as the interaction and epidermis between.And tentatively illustrated and positioned hair follicle now
The knuckle portion of the position of stem cell, i.e. raw coal bunker.Therefore, how hair follicle regeneration mechanism is studied using hair follicle stem cells
And research hair follicle stem cells have become the development trend of current research to the mechanism of wound repair.
It is not successfully established hair follicle regeneration model and embryo skin Scarless wound healing model in prior art in vitro；At present
Cultural method simulation body physiological environment, it is unstable, be easily affected by the external environment；Needed for can not quickly setting up in vitro again
Raw model and Scarless wound healing model, before the test in phase modeling, waste plenty of time and material resources.
From the foregoing, develop a kind of reconstruction in vitro hair follicle tissue form and set up skin Scarless wound healing model, and
Model method with certain physiological function, will have important existing to research hair follicle regeneration mechanism and wound surface Scarless wound healing mechanism
Sincere justice and application prospect.
The content of the invention：
In order to solve the above problems, the present invention provides the gas-liquid interface three-dimensional culture method of a kind of skin and hair follicle regeneration,
Methods described makes hair follicle regeneration, and the three-dimensional configuration of reconstructed hair follicles by cultivating antenna raw coal bunker, is deposited using hair follicles outgrowth
Live time is longer, and neomorphosis is regular, and hair follicle is difficult the characteristics of subsiding, and culture cell simulation body environment, culture has necessarily
Physiological function regeneration hair follicle or Scarless wound healing embryo skin；
Further, methods described includes：
S1：Make gas-liquid interface support
Using 40 μ L rat-tail I-type collagen equivalent volumes PBS, uniform application is straight in disposable 90mm under aseptic condition
On the culture dish inwall in footpath, Sterile ophthalmic tweezer takes Nuclepore polycarbonate membranes, is laid in the culture dish for filling Collagen type-I
In, ParafilmTM culture dish, ultraviolet irradiation 30min treats its rat-tail I-type collagen and Nuclepore polycarbonate membranes
After natural drying, 4 DEG C of refrigerators are put into standby；
S2：Prepare culture fluid
Using culture fluid based on low sugar MEM, hyclone FCS is added, is fabricated to the culture fluid of 5%FCS+MEM,
Mycillin 100mg is added in the 5%FCS+MEM culture fluid of 2mL, during culture raw coal bunker BFGF (fibroblasts are added
Basic fibroblast growth factor) 10ng/mL, add EGF (epidermal growth factor) 8ng/mL during culture embryo skin；
S3：Obtain antenna raw coal bunker and cultivate
Disconnected cervical approach puts to death the neonatal rat of new life 1-5d, and whole neonatal rat is put in 75% ethanol and sterilizes, and is then transferred into ultra-clean work
Make platform, bilateral antenna portion skin histology is cut along the neonatal rat corners of the mouth with eye scissorss under anatomic microscope, skin histology is put into nothing
In bacterium MEM culture fluid, ophthalmic tweezers are utilized under sterile liquid three-dimensional environment, isolate complete antenna hair follicle, it is aobvious using ophthalmology
Micro- surgery knife cuts off hair follicle hair bulb portion, extracts hair follicle inner root sheath, does not damage the middle and upper part of raw coal bunker, the form of acquisition
Complete raw coal bunker middle and upper part, the integrity in Preserve follicles knuckle portion；
S4：Obtain and cultivate embryo skin
The good culture fluid of 2mL proportionings is added in 30mm culture dishs, the Nuclepore polycarbonate membranes handled well are swum in
On culture fluid, raw coal bunker is transferred on the Nuclepore polycarbonate membranes of floating using Sterile ophthalmic tweezer, is placed on 37
DEG C, 5%CO2Cultivated in incubator；
Further, the material of gas-liquid interface described in S1 adopts Nuclepore Merlon microporous membranes, and tare weight is light, can
Floating, do not pollute culture fluid, described Nuclepore polycarbonate membranes are made up of high-quality polycarbonate film, per
Nuclepore polycarbonate membranes are circular, and specification is different, and aperture is different；
Further, support described in S1 is processed by Nuclepore Merlon microporous membrane through I types rat tail collagen protein
It is obtained；
Further, it is stem cells hyperplasia label EdU that additive is cultivated described in S2, hormone, antibiotic or cell because
Son；
Further, the culture medium in gas-liquid interface culture described in S2 is DMEM, MEM or RPMI-1640 culture fluid；
Further, import hyclone of the hyclone described in S2 from inactivation mycoplasma；
Further, the 1-5d for obtaining time selection mice just birth of the hair follicle of mice antenna described in S3, for obtaining
Mice antenna raw coal bunker, the mice antenna raw coal bunker culture access time is 1-6d；
Further, the skin of tire Corium Mus described in the S4 acquisition time is E14-E17d, the tire Mus Skins culture access time
It is 1-7d；
Beneficial effects of the present invention are as follows：
1) simulate body environment and animal tissue's (hair follicle, tire Corium Mus skin), the animal wool cultivated are cultivated on gas-liquid interface
Capsule, embryo skin have certain physiological function, and the artificial minimally invasive rear embryo skin of embryo skin can be realized being healed without cicatrix；
2) the gas-liquid interface three-dimensional culture method of skin and hair follicle regeneration is culturing stem cells and quickly sets up medical science regeneration
The method of model, can explore hair follicle regeneration and skin Scarless wound healing mechanism from external；
3) the inventive method is reasonable in design, simple to operate, and vitro tissue regeneration and wound healing are easily set up at short notice
Mould；
4) hair follicle regeneration model and embryo skin Scarless wound healing model are successfully established in vitro；
5) cultural method simulation body physiological environment, stable, it is not easy to be affected by the external environment；
6) required regenerating model and Scarless wound healing model can be quickly set up in vitro, before the test in phase modeling, saved
Plenty of time and material resources.
Description of the drawings：
Fig. 1 is the gas-liquid interface dimensional culture assembling pictorial diagram of skin and hair follicle regeneration；
When Fig. 2 is raw coal bunker culture 3d, frozen section H.E dyeing observation hair follicle structure；
When Fig. 3 is raw coal bunker culture 5d, frozen section H.E dyeing observation hair follicle structure；
Fig. 4 is Zeiss inverted fluorescence microscope under purple light passage, hair follicle regeneration structure, cell arrangement figure；
When Fig. 5 is raw coal bunker culture 5d, α-SMA Fluorescent Staining Observation hair follicle structure (green) figures；
When Fig. 6 is embryo skin artificial minimally invasive culture 3d, frozen section H.E dyeing observation embryo skins heal aspect graph；
When Fig. 7 is embryo skin artificial minimally invasive culture 7d, frozen section H.E dyeing observation embryo skins heal aspect graph；
Fig. 8 is the appropriate stem cell labeling thing EdU of addition, and raw coal bunker culture 12h, immunofluorescence dyeing observation hair follicle is new
Raw cell (green), core redyes (redness) aspect graph；
Fig. 9 is the appropriate stem cell labeling thing EdU of addition, embryo skin culture 12h, immunofluorescence dyeing observation skin revitalization
Cell (green), core redyes (redness) form.
Specific embodiment：
In order that the objects, technical solutions and advantages of the present invention become more apparent, it is right below in conjunction with drawings and Examples
The present invention is explained in further detail.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, and
It is not used in the restriction present invention.Conversely, the present invention cover it is any be defined by the claims the present invention spirit and scope on do
Replacement, modification, equivalent method and scheme.Further, in order that the public has a better understanding to the present invention, below to this
It is detailed to describe some specific detail sections in the detailed description of invention.It is thin without these for a person skilled in the art
The description of section part can also completely understand the present invention.
Below in conjunction with the accompanying drawings the invention will be further described with specific embodiment, but not as a limitation of the invention.
Enumerate most preferred embodiment for the present invention below：
As shown in Fig. 1-Fig. 9, it is an object of the invention to provide the support of a kind of embryo skin and vibrissa follicles in vitro
Method, simulates body physiological environment so that embryo skin and hair follicle activity activity in vitro is improved and extends growth time, from
And more easily study embryo skin and heal without cicatrix and hair follicle regeneration mechanism.
The gas-liquid interface three-dimensional culture method of skin follicle regeneration, it is comprised the following steps：
S1：The making of gas-liquid interface support
Nuclepore polycarbonate membranes, are made up of high-quality polycarbonate film, every Nuclepore polycarbonate membrane
Circular, specification is different, and aperture is different, using 40 μ L rat-tail I-type collagen equivalent volumes PBS, uniformly applies under aseptic condition
It is put on the culture dish inwall of disposable 90mm diameters, Sterile ophthalmic tweezer takes Nuclepore polycarbonate membranes, is laid in painting
In the culture dish of full Collagen type-I, ParafilmTM culture dish, ultraviolet irradiation 30min, treat its rat-tail I-type collagen and
After Nuclepore polycarbonate membranes are spontaneously dried, 4 DEG C of refrigerators are put into standby.
S2：The preparation of culture fluid
Culture fluid based on low sugar MEM used in test, adds hyclone (FCS), is fabricated to 5%FCS+MEM's
Culture fluid, adds mycillin 100mg in the 5%FCS+MEM culture fluid of 2mL, BFGF is added during culture raw coal bunker (into fibre
Dimension Cellular alkaline somatomedin) 10ng/mL, add EGF (epidermal growth factor) 8ng/mL during culture embryo skin.
S3:The acquisition and culture of antenna raw coal bunker
Disconnected cervical approach puts to death the neonatal rat of new life 1-5d, and whole neonatal rat is put in 75% ethanol and sterilizes, and is then transferred into ultra-clean work
Make platform, bilateral antenna portion skin histology is cut along the neonatal rat corners of the mouth with eye scissorss under anatomic microscope, skin histology is put into nothing
In bacterium MEM culture fluid, ophthalmic tweezers are utilized under sterile liquid three-dimensional environment, isolate complete antenna hair follicle, it is aobvious using ophthalmology
Micro- surgery knife cuts off hair follicle hair bulb portion, carefully extracts hair follicle inner root sheath, should not damage the middle and upper part of raw coal bunker, obtains
The complete raw coal bunker middle and upper part of form, the integrity in Preserve follicles knuckle portion.
The good culture fluid of 2mL proportionings is added in 30mm culture dishs, by the Nuclepore polycarbonate membranes handled well floating
On culture fluid, raw coal bunker is transferred on the Nuclepore polycarbonate membranes of floating using Sterile ophthalmic tweezer, is placed on
37 DEG C, 5%CO2Cultivated in incubator.
S4:The acquisition and culture of embryo skin
Pregnant age is taken for the pregnant Mus between 14-17d (E14-E17), cervical approach of breaking is put to death pregnant Mus, is soaked in 75% ethanol 10min,
Clean 3 times in aseptic PBS, in super-clean bench under aseptic condition, uterus is taken out, clean 3 times in aseptic PBS, remove surface residual
Stagnant blood liquid and mucus.After cutting off uterus, the embryo with fetal membrane is taken out, cleaned 3 times in aseptic PBS.Fetal membrane is torn with tweezers, is taken
Go out tire Mus.Tail and extremity are cut off with eye scissorss after tire Mus are rinsed in aseptic PBS, and is cut along back both sides with eye scissorss
Open, with tweezers tire Mus skin of back is carefully peeled off, using micro- ophthalmologic operation hilt embryo skin regular quadrilateral is cut into.It is used in combination
Card punch makes wound at tetragon skin center, measures and records and makes wound diameter.The good training of 2mL proportionings is added in 30mm culture dishs
Nutrient solution, the Nuclepore polycarbonate membranes handled well is swum on culture fluid, using Sterile ophthalmic tweezer raw coal bunker
It is transferred on the Nuclepore polycarbonate membranes of floating, is placed on 37 DEG C, 5%CO2Cultivated in incubator.
Embodiment 1：
(1) making of gas-liquid interface support：
Nuclepore polycarbonate membranes, are made up of high-quality polycarbonate film, every Nucle-pore polycarbonate membrane
Circular, specification is different, and aperture is different, using 40 μ L rat-tail I-type collagen equivalent volumes PBS, uniformly applies under aseptic condition
It is put on the culture dish inwall of disposable 90mm diameters, Sterile ophthalmic tweezer takes Nuclepore polycarbonate membranes, is laid in painting
In the culture dish of full Collagen type-I, ParafilmTM culture dish, ultraviolet irradiation 30min, treat its rat-tail I-type collagen and
After Nuclepore polycarbonate membranes are spontaneously dried, 4 DEG C of refrigerators are put into standby.
(2) preparation of culture fluid：
Culture fluid based on low sugar MEM used in experiment, adds hyclone (FCS), is fabricated to 5%FCS+MEM's
Culture fluid, in the 5%FCS+MEM culture fluid of 2mL add mycillin 50mg/mL, culture raw coal bunker when add BFGF (into
Fibrocyte basic fibroblast growth factor) 10ng/mL, add EGF (epidermal growth factor) 8ng/mL during culture embryo skin.
(3) acquisition and culture of antenna raw coal bunker
Disconnected cervical approach puts to death the neonatal rat of new life 1-5d, and whole neonatal rat is put in 75% ethanol and sterilizes, and is then transferred into ultra-clean work
Make platform, bilateral antenna portion skin histology is cut along the neonatal rat corners of the mouth with eye scissorss under anatomic microscope, skin histology is put into nothing
In bacterium MEM culture fluid, ophthalmic tweezers are utilized under sterile liquid three-dimensional environment, isolate complete antenna hair follicle, it is aobvious using ophthalmology
Micro- surgery knife cuts off hair follicle hair bulb portion, carefully extracts hair follicle inner root sheath, should not damage the middle and upper part of raw coal bunker, obtains
The complete raw coal bunker middle and upper part of form, the integrity in Preserve follicles knuckle portion.
The good culture fluid of 2mL proportionings is added in 35mm culture dishs, the Nuclepore polycarbonate membranes handled well are swum in
On culture fluid (Fig. 1), raw coal bunker is transferred on the Nuclepore polycarbonate membranes of floating using Sterile ophthalmic tweezer,
It is placed on 37 DEG C, 5%CO2Cultivated in incubator.
During raw coal bunker culture 3d, H.E dyeing, it can be seen that hair follicles outgrowth form (Fig. 2).During culture 5d, H.E dyes
Color, is clear that hair follicles outgrowth form more full (Fig. 3).Zeiss inverted fluorescence microscope is observed under purple light passage
Hair follicle regeneration structure, it is seen that hair follicles outgrowth cell ordered arrangement (Fig. 4).During raw coal bunker culture 5d, α-SMA fluorescence stainings are seen
Examine hair follicle new life framing structure (green) (Fig. 5).
Embodiment 2：
Pregnant age is taken for the pregnant Mus between 14-17d (E14-E17), cervical approach of breaking is put to death pregnant Mus, is soaked in 75% ethanol lOmin,
Clean 3 times in aseptic PBS, in super-clean bench under aseptic condition, uterus is taken out, clean 3 times in aseptic PBS, remove surface residual
Stagnant blood liquid and mucus.After cutting off uterus, the embryo with fetal membrane is taken out, cleaned 3 times in aseptic PBS.Fetal membrane is torn with tweezers, is taken
Go out tire Mus.Cut off along back both sides with eye scissorss after tire Mus are rinsed in aseptic PBS, with tweezers tire Mus back skin is carefully peeled off
Skin, using micro- ophthalmologic operation hilt embryo skin regular quadrilateral is cut into.And wound is made at tetragon skin center with card punch,
Measure and record and make wound diameter.
The good culture fluid of 2mL proportionings is added in 30mm culture dishs, by the Nucle-pore polycarbonate membranes handled well drift
Float on culture fluid, raw coal bunker is transferred on the Nuclepore polycarbonate membranes of floating using Sterile ophthalmic tweezer, put
In 37 DEG C, 5%CO2Cultivated in incubator.After embryo skin man wound, during culture 3d, H.E dyeing, observation wound surface is healed
Conjunction situation (Fig. 6), after embryo skin man wound, during culture 7d, H.E dyeing, observation wound healing situation (Fig. 7).
Embodiment 3：
On Nuclepore polycarbonate membranes, the μ L stem cells hyperplasia label EdU of Deca 2, Sterile ophthalmic tweezer obtains dissection
The raw coal bunker for obtaining is transferred on the Nuclepore polycarbonate membranes of floating, and raw coal bunker and stem cells hyperplasia mark
Note thing EdU is fully contacted, and is placed on 37 DEG C, 5%CO2Culture 12h, immunofluorescence dyeing observation hair follicle stem cells are carried out in incubator
Vegetative state (Fig. 8).
Embodiment 4：
On Nuclepore polycarbonate membranes, the μ L stem cells hyperplasia label EdU of Deca 2, Sterile ophthalmic tweezer obtains dissection
The embryo skin for obtaining is transferred on the Nuclepore polycarbonate membranes of floating, and embryo skin and stem cells hyperplasia label
EdU is fully contacted, and is placed on 37 DEG C, 5%CO2Culture 12h, immunofluorescence dyeing observation embryo skin stem cell are carried out in incubator
Vegetative state (Fig. 9).
In the present invention, Nuclepore polycarbonate membranes：It is made up of high-quality polycarbonate film, aperture is accurate, there is pole
Resistance to infiltration well and thermostability, film is smooth, pollution-free to sample.
Rat-tail I-type collagen：It is a kind of natural medium, with promotion cultured cell in vitro (particularly epithelial cell)
Adherent effect, while being also a kind of natural binder.
Embodiment described above, simply one kind of the present invention more preferably specific embodiment, those skilled in the art
The usual variations and alternatives that member is carried out in the range of technical solution of the present invention all should be comprising within the scope of the present invention.
Claims (9)
1. the gas-liquid interface three-dimensional culture method of a kind of skin and hair follicle regeneration, it is characterised in that：Methods described is touched by culture
Palpus raw coal bunker makes hair follicle regeneration, and the three-dimensional configuration of reconstructed hair follicles, neomorphosis longer using the hair follicles outgrowth time-to-live
Regular, hair follicle is difficult the characteristics of subsiding, culture cell simulation body environment, culture raw coal bunker and embryo skin.
2. method according to claim 1, it is characterised in that methods described includes：
S1：Make gas-liquid interface support
Using 40 μ L rat-tail I-type collagen equivalent volumes PBS, uniform application is in disposable 90mm diameters under aseptic condition
On culture dish inwall, Sterile ophthalmic tweezer takes Nuclepore polycarbonate membranes, in being laid in the culture dish for filling Collagen type-I,
ParafilmTM culture dish, ultraviolet irradiation 30min, treats its rat-tail I-type collagen and Nuclepore polycarbonate membrane natures
After drying, 4 DEG C of refrigerators are put into standby；
S2：Prepare culture fluid
Using culture fluid based on low sugar MEM, hyclone FCS is added, be fabricated to the culture fluid of 5%FCS+MEM, 2mL's
Mycillin 100mg is added in 5%FCS+MEM culture fluid, BFGF is added during culture raw coal bunker, and (fibroblast alkalescence is raw
The long factor) 10ng/mL, add EGF (epidermal growth factor) 8ng/mL during culture embryo skin；
S3：Obtain antenna raw coal bunker and cultivate
Disconnected cervical approach puts to death the neonatal rat of new life 1-5d, and whole neonatal rat is put in 75% ethanol and sterilizes, and is then transferred into superclean bench,
Bilateral antenna portion skin histology is cut along the neonatal rat corners of the mouth with eye scissorss under anatomic microscope, skin histology is put into aseptic MEM
In culture fluid, ophthalmic tweezers are utilized under sterile liquid three-dimensional environment, complete antenna hair follicle is isolated, using ophthalmic microsurgical
Knife cuts off hair follicle hair bulb portion, extracts hair follicle inner root sheath, does not damage the middle and upper part of raw coal bunker, and the form of acquisition is complete
Raw coal bunker middle and upper part, the integrity in Preserve follicles knuckle portion；
S4：Obtain and cultivate embryo skin
The good culture fluid of 2mL proportionings is added in 30mm culture dishs, the Nuclepore polycarbonate membranes handled well are swum in into training
On nutrient solution, raw coal bunker is transferred on the Nuclepore polycarbonate membranes of floating using Sterile ophthalmic tweezer, is placed on 37
DEG C, 5%CO2Cultivated in incubator.
3. method according to claim 2, it is characterised in that：The material of gas-liquid interface described in S1 is poly- using Nuclepore
Carbonic ester microporous membrane, tare weight is light, and culture fluid is not polluted in floatability, and described Nuclepore polycarbonate membranes are gathered by high-quality
Carbonic ester film is made, and every Nuclepore polycarbonate membrane is circular, and specification is different, and aperture is different.
4. method according to claim 2, it is characterised in that：Support described in S1 is by Nuclepore Merlon microporous membranes
It is obtained through the process of I types rat tail collagen protein.
5. method according to claim 2, it is characterised in that：It is stem cells hyperplasia label that additive is cultivated described in S2
EdU, hormone, antibiotic or cytokine.
6. method according to claim 2, it is characterised in that：Culture medium in gas-liquid interface culture described in S2 be DMEM,
MEM or RPMI-1640 culture fluid.
7. method according to claim 2, it is characterised in that：Import tire of the hyclone described in S2 from inactivation mycoplasma
Ox blood serum.
8. method according to claim 2, it is characterised in that：The acquisition time of the hair follicle of mice antenna described in S3 chooses mice
The 1-5d being just born, for obtaining mice antenna raw coal bunker, the mice antenna raw coal bunker culture access time is
1-6d。
9. method according to claim 2, it is characterised in that：The skin of tire Corium Mus described in the S4 acquisition time is E14-E17d, institute
It is 1-7d to state tire Mus Skins culture access time.
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| CN110755689A (en) * | 2019-11-08 | 2020-02-07 | 中国医学科学院整形外科医院 | Three-dimensional reconstruction method of hair follicle and hair follicle prepared by reconstruction method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110093306A (en) * | 2018-01-31 | 2019-08-06 | 中山大学 | Artificial hair follicle and its preparation method and application |
| CN110093306B (en) * | 2018-01-31 | 2022-12-02 | 中山大学 | Artificial hair follicle and preparation method and application thereof |
| WO2019215557A1 (en) * | 2018-05-07 | 2019-11-14 | Reelabs Pvt. Ltd. | The method of autologous primary hair follicles preparation in 3d culture |
| CN110157617A (en) * | 2019-04-29 | 2019-08-23 | 重庆市肿瘤研究所 | Cervical cancer cell three-dimensional cultivation device and its application |
| CN110157617B (en) * | 2019-04-29 | 2021-07-16 | 重庆大学附属肿瘤医院 | Cervical cancer cell three-dimensional culture device and application thereof |
| CN110408542A (en) * | 2019-08-22 | 2019-11-05 | 合肥学院 | A kind of skin chip of liquid phase culture |
| CN110755689A (en) * | 2019-11-08 | 2020-02-07 | 中国医学科学院整形外科医院 | Three-dimensional reconstruction method of hair follicle and hair follicle prepared by reconstruction method |
| CN110755689B (en) * | 2019-11-08 | 2022-01-25 | 中国医学科学院整形外科医院 | Three-dimensional reconstruction method of hair follicle and hair follicle prepared by reconstruction method |
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