CN104491931A - Sebaceous gland-containing skin tissue as well as formation method and application thereof - Google Patents
Sebaceous gland-containing skin tissue as well as formation method and application thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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Abstract
The invention discloses a sebaceous gland-containing skin tissue as well as a formation method and an application thereof. A method for forming sebaceous gland comprises the step of co-culturing hypodermal cells and epidermal stem cells in a gel. By virtue of the method, the skin tissue with sebaceous gland can be formed.
Description
Technical field
The present invention relates to cytobiology and regenerative medicine field, particularly, the method and application thereof that form sebaceous gland is related to, more specifically, relate to the method forming sebaceous gland, the purposes of method in skin regeneration of the skin histology containing sebaceous gland and formation sebaceous gland.
Background technology
Skin, as the maximum tissue of human body, is the barrier contacted with external environment, when the factor such as environmental damage or disease causes skin injury, usually causes damage and the infection of skin.China is populous, and annual burn reaches 1,500 ten thousand people with ulcers, and wherein need to carry out dermatoplastic case 3,500,000 people, skin demand is more than 400,000,000 square centimeters.Therefore, find a kind of desirable skin substitute products is a urgent need to solve the problem clinically always.Along with the develop rapidly of cytobiology, materialogy, biochemistry, biotechnology, implantology, people have had more deep understanding to the biological characteristics of epidermal keratinocytes, the processing of artificial material and synthesis etc., and making to build organization engineering skin in vitro becomes possibility.
Sebaceous gland safeguards the most important accessory organ of skin texture function-stable, because sebaceous gland and perspiration and the epithelial cell etc. come off form one deck sebum perspiration latex film at skin surface, its thickness 7-10 μm, it is the three-dimensional lipid conformation of skin surface, can promote the integrity of skin barrier.If lack this layer of three-dimensional cortex structure, pachylosis will be there will be and hair is withered.Because the partial skin of palm, the sufficient sole of the foot and finger, toes is not because have sebaceous gland, so often there is chapped skin phenomenon.And although present organization engineering skin has the corium similar with normal skin and epidermal area; but shortage sebaceous gland; moist, the antibacterial antioxidative protection of nutrition and water tariff collection cannot be provided for skin; after skin transplantation; occur that contraction of skin distortion is serious, the appearance easily problem such as dry and cracked and susceptible, the performance of organization engineering skin is starkly lower than natural normal skin simultaneously.
But existing organization engineering skin still haves much room for improvement.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is to propose a kind of method forming sebaceous gland.
It should be noted that, the present invention completes based on the following work of inventor:
Inventor finds, derive from the epidermal stem cells of epiderm skin and derive from the hypodermal cell of dermis of skin, all there are the potentiality of a large amount of amplification, and in vitro after separation and Culture, the feature of epidermal stem cells is high expressed integrin CD29, CD49f and K15 (keratoprotein 15), and in follow-up going down to posterity, the characteristic scalar memory developed by molecule of two kinds of stem cell is stablized.Above-mentioned two kinds of cells increase by inventor respectively in different cultivating systems, then two kinds of cells are transplanted to nude mice skin in proportion, through histologic analysis, find that there is the new sebaceous gland structure formed.
Thus, according to an aspect of the present invention, the invention provides a kind of method forming sebaceous gland.According to embodiments of the invention, the method comprises: in gel, carry out Dual culture to hypodermal cell and epidermal stem cells.The discovery that inventor is surprised, the method can form the skin histology with sebaceous gland.According to embodiments of the invention, the application is formed has the method for the skin histology of sebaceous gland, simple, conveniently fast, easily to control, is easy to accomplish scale production.
According to embodiments of the invention, described Dual culture carries out the subcutaneous of animal.Thus, promote the regeneration of skin histology of sebaceous gland, and the structure of the skin histology of regeneration and normal skin similar.
In addition, the method for one-tenth sebaceous gland according to the above embodiment of the present invention, can also have following additional technical characteristic:
According to embodiments of the invention, described hypodermal cell comprise corium stem cell and fibroblastic one of at least.
According to embodiments of the invention, described hypodermal cell is fibroblast, and containing insulin, dexamethasone, lattice row ketone and XAV939 (3 in described gel, 5,7,8-tetrahydrochysene-2-[4-(trifluoromethyl) phenyl]-4H-thiapyran also [4,3-d] pyrimidin-4-one).Thus, chemical reagent induction epidermal stem cells is divided into free sebaceous gland, and then is formed and the similar sebaceous gland structure of normal skin.
The final concentration of described insulin is 50-150 μ g/mL, and the final concentration of described dexamethasone is 10
-4-10
-6m, the final concentration of described class lattice row ketone is 150-250 μM, and the final concentration of described XAV939 is 5-15 μM.Thus, chemical reagent induction epidermal stem cells and fibroblast are divided into the effective of free sebaceous gland.
According to the embodiment of the present invention, the final concentration of described insulin is 100 μ g/mL, and the final concentration of described dexamethasone is 10
-5m, the final concentration of described class lattice row ketone is 200 μMs, and the final concentration of described XAV939 is 10 μMs.Thus, chemical reagent induction epidermal stem cells and fibroblast to be divided into the effect of free sebaceous gland better.
Wherein, it should be noted that, after the term " final concentration " used in the application refers to and various chemical reagent is added the gel containing cell mixing, the concentration of this chemical reagent in gel.
According to embodiments of the invention, the number ratio of described hypodermal cell and the mixing of described epidermal stem cells is 10:1 ~ 1:10.
According to embodiments of the invention, for every 3mm
2skin, the quantity of cell mixing is no less than 10
4individual.Thus, the quantity of cell mixing meets the demand of sebaceous gland skin tissue regeneration.
According to embodiments of the invention, described gel is at least one being selected from Matrigel, collagen or hydrogel.
According to embodiments of the invention, described hypodermal cell and described epidermal stem cells are the cell deriving from autologous, allogeneic or xenogenesis.Wherein it should be noted that, described in derive from xenogenesis cell regenerate for the sebaceous gland of immunodeficient animals.Thus, the sebaceous gland skin histology rejection effect of regeneration is little.
According to embodiments of the invention, described hypodermal cell and described epidermal stem cells separately in advance in liquid medium within by adhere-wall culture 0 ~ 30 generation, containing cell active factor and extracellular matrix molecule in described fluid medium, described cell active factor is at least one being selected from bFGF, EGF, and described extracellular matrix molecule is at least one being selected from hyaluronic acid, fibronectin splicing variants.Thus, the activity of hypodermal cell and epidermal stem cells is high, and cytometaplasia is little.
According to embodiments of the invention, the method comprises further: utilize the fat secretion containing the skin tissue surface of regenerated bark adipose gland described in original position Mass Spectrometric Identification.Thereby, it is possible to the fat secretion situation of the described skin tissue surface containing regenerated bark adipose gland of more accurate qualification.
According to a further aspect in the invention, present invention also offers a kind of skin histology containing sebaceous gland.According to embodiments of the invention, described sebaceous gland is formed by the method described in the aforesaid embodiment of the present invention.Inventor is surprised to find, and this skin histology has the structure of the sebaceous gland similar with normal skin tissue.
In accordance with a further aspect of the present invention, the present invention also puies forward the purposes of aforesaid method much of that in skin regeneration.According to the embodiment of the present invention, described method is used for regenerated bark adipose gland, or the skin histology containing sebaceous gland.Inventor is surprised to find, in skin regeneration, apply the method, can obtain and have the sebaceous gland similar with normal skin.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows the picture of the expression of results of the cell surface marker molecule CD49f of epidermal stem cells according to an embodiment of the invention;
Fig. 2 shows the picture of the expression of results of the cell surface marker Nestin of corium stem cell according to an embodiment of the invention;
Fig. 3 shows the picture of the confocal microscopy of normal skin tissue's section according to an embodiment of the invention;
Fig. 4 shows according to an embodiment of the invention by the picture of the confocal microscopy of the Skin slice formed after epidermal stem cells and corium stem cell co-transplantation;
The picture of the confocal microscopy of the Skin slice that Fig. 5 is formed after inducing co-transplantation with chemical reagent after showing and being mixed by epidermal stem cells and fibroblast according to an embodiment of the invention;
Fig. 6 shows the picture of the smegma result of the skin histology formed after MALDI Mass Spectrometer Method is according to an embodiment of the invention transplanted.
Detailed description of the invention
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings.Being exemplary below by the embodiment be described with reference to the drawings, only for explaining the present invention, and can not limitation of the present invention being interpreted as.
Unreceipted concrete technology or condition in the embodiment of the application, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Be described below in detail embodiments of the invention, if no special instructions, in following embodiment, isolated skin stem cell is separated from the skin of C57 mice or the scalp tissue of people and cultivates; Corium stem cell media consists of DMEM/F12 (3:1) (buying in Invitrogen company), containing 2%B27 (buying in Invitrogen company), 40ng/ μ L bFGF (Peprotech) and 20ng/ μ L EGF (Peprotech); Fibroblastic culture medium is that DMEM contains 10% hyclone; Accutase is purchased from sigma; 3M film is Tegaderm
tMfilm; Suspension cell culture ware, purchased from Guangzhou Ztel's biofiltration Products Co., Ltd, article No. is MCD-000-090.Adherent cell culture dish, purchased from Corning company.Matrigel is purchased from BD company.Laboratory animal is all purchased from Guangdong Medical Lab Animal Center.
Embodiment 1
The separation of epidermal stem cells and cultivation and qualification process specific as follows:
(1) get tire Mus full thickness skin, add mass concentration 0.25%dispase II enzyme 4 DEG C and spend the night.
(2) peel off the epiderm skin of tire Mus, add mass concentration 0.25% collagenase after being shredded by epidermis, cessation reaction after 37 DEG C of digestion 30min, obtains the primary cell of postdigestive epidermal stem cells.
(3) primary cell of acquisition is crossed 80 eye mesh screens, then the centrifugal 10min of 300 × g, with CnT-07 (being produced by CELLnTEC company) culture medium suspension cell after abandoning supernatant.
(4) primary cell that CnT-07 suspends is inoculated in and is covered with on the 10CM Tissue Culture Plate of mice type i collagen, after hatching 60min, with 0.01mol/L HBSS washed cell 2 times, then add CnT-07 and cultivate.
(5) when primary cell grows to 60% ~ 80% fusion, use Accutase enzymic digestion, then go down to posterity in 1: 2 ratio.
(6) qualification of epidermal stem cells: the i.e. qualification of cell surface marker, method is as follows: get the 1st, 3,5 generation epidermal stem cells do cell climbing sheet, it is carried out to the immunofluorescence dyeing of integrin CD29 and CD49f and K15, the coloring case of observation of cell.Observe and find, the equal high expressed of the integrin CD29 of epidermal stem cells, CD49f and K15, concrete outcome as shown in Figure 1.
Embodiment 2
The separation of corium stem cell and cultivation and qualification process specific as follows:
(1) get tire Mus full thickness skin, after skin is shredded, add mass concentration 0.25%dispase enzyme 4 DEG C and spend the night.
(2) peel off the epidermis of tire Mus, be separated corium, and shredded by corium, add mass concentration 0.25% trypsin, cessation reaction after 37 DEG C of digestion 20 ~ 40min, obtains the primary cell of postdigestive corium stem cell.
(3) primary cell of corium stem cell is crossed 200 eye mesh screens, then, the centrifugal 10min of 300 × g, after abandoning supernatant, with corium stem cell media suspension primary cell.
(4) corium stem cell media suspension primary cell is inoculated in not in adhere-wall culture ware.
(5) after there is more cell ball in not adhere-wall culture ware, by corium stem cell in 1: 2 ratio go down to posterity.
(6) qualification of corium stem cell: the i.e. qualification of cell surface marker, concrete grammar is as follows: get the 1st, 3,5 generation corium stem cell do cell rejection tablet, immunofluorescence dyeing is carried out to Nestin and Fibronectin of corium stem cell, the coloring case of observation of cell.Immunofluorescence analysis finds, the equal high expressed of Nestin and Fibronectin of corium stem cell, wherein, the result of the Nestin positive as shown in Figure 2.
Embodiment 3
Utilize corium stem cell to induce epidermal stem cells to form sebaceous gland structure, detailed process is as follows:
(1) cultivate the epidermal stem cells obtained in embodiment 1, by the 3rd generation epidermal stem cells with after Accutase enzymic digestion, prepare the epidermal stem cells suspension of total cell number 1,000,000 in PBS.
(2) cultivate the corium stem cell obtained in embodiment 2, preparation the 3rd generation corium stem cell total cell number 1,000,000 cell suspension is in PBS.
(3) by two kinds of cell suspension mixing in (1) and (2), the centrifugal 5min of 300g.
(4) get centrifugal after cell, abandon supernatant, in cell precipitation, add 10 microlitre Matrigel, mixing, stand-by.
(5) by the pentobarbital sodium anesthesia of 1% of BALB/c nude mice, on its skin of back, manufacture area with card punch and be about 7mm
2wound.
(6) Matrigel being mixed with skin progenitor cell prepared in step (4) is transplanted to nude mice skin wound surface.
(7) cover nude mice wound surface with 3M film, then with binder by nude mice wound dressing.
(8) normally raise nude mice, strip the bandage off after 3 weeks and remove 3M film, cut the skin histology of cell transplantation, and after fixing dyeing, observe sebaceous gland structure, find that there is a large amount of sebaceous gland structure and formed.
Embodiment 4
Utilize chemical reagent to induce epidermal stem cells to form sebaceous gland structure, detailed process is as follows:
(1) cultivate the epidermal stem cells obtained in embodiment 1, by the 3rd generation epidermal stem cells with after Accutase enzymic digestion, the cell suspension of preparation 2,000,000 is in PBS.
(2) by fibroblast with after Accutase enzymic digestion, preparation 2,000,000 cell suspension in PBS.
(3) by the two kinds of cell suspension mixing in (1) and (2), and be divided into 2 parts, a as experimental group, another part as a control group, 300g, centrifugal 5min.
(4) by above-mentioned 2 parts centrifugal after cell abandon supernatant, in cell precipitation, add 10 microlitre Matrigel respectively, mixing.
(5) in the Matrixgel of experimental group, appropriate insulin is added, dexamethasone, class lattice row ketone and XAV939, its ultimate density is made to be: insulin 100 μ g/mL, dexamethasone 10-5M, class lattice row ketone 200 μMs, XAV93910 μM, by abundant for above-mentioned chemical reagent pressure-vaccum mixing.Matched group does not add above-mentioned chemical reagent.
(6) by the pentobarbital sodium anesthesia of 1% of BALB/c nude mice, on its skin of back, manufacture area with card punch and be about 7mm
2wound.
(7) Matrigel that 2 groups that prepare in step (5) are mixed with skin progenitor cell is transplanted to nude mice skin wound surface respectively.
(8) cover nude mice wound surface with 3M film, then with binder by nude mice wound dressing.
(9) normally raise nude mice, strip the bandage off after 3 weeks and remove 3M film, cut the skin histology of cell transplantation, and after fixing dyeing, observe sebaceous gland structure.Discovery experimental group has the formation of a large amount of sebaceous gland structure and matched group only has the sebaceous gland structure of seldom amount to be formed.
Embodiment 5
Tectology qualification is carried out to the skin histology of the stem cell transplantation of experimental group and matched group in embodiment 3 and embodiment 4, specific as follows:
1, epidermis stem cell regenerating sebaceous gland structure organization section is prepared:
(1) cut the skin tissue area of the stem cell transplantation of experimental group and matched group in embodiment 3 and embodiment 4 respectively, 4% paraformaldehyde fixedly spends the night, and PBS soaks 3 hours, and 30% sucrose dewaters 8 hours.
(2) OCT investing tissue, frozen section, gets the tissue slice of 10 μm of thickness.
(3) tissue slice is dried naturally.
(4) tissue slice PBS is washed three times, each 5 minutes.
(5) with 0.2-0.5%triton X-100 (PBS preparation), permeable membrane process is done 10 minutes to tissue slice.
(6) the tissue slice PBS after permeable membrane process is washed three times, each 5 minutes.
(7) tissue slice 2%BSA is closed 30 minutes, then wash twice with PBS.
(8) tissue slice after closing is added 1 of antibiotin Cy3 labelling and resists, incubated at room 3 hours.
(9) tissue slice PBS anti-for connection 1 is washed three times, each 5 minutes.
(10) 0.5 μ g/ml DAPI (PBS preparation) dyeing 10 minutes is added.
(11) stained tissue section PBS is washed three times, remove unnecessary DAPI.
(12) tissue slice removing unnecessary DAPI is added 20 μ l mountant mountings.
(13) the tissue staining sample that mounting is good is placed in 4 degree of refrigerators, can deposit more than 2 weeks.
2, the tectology qualification of tissue slice:
Adopt confocal microscopy tissue slice, observe and find, the sebaceous gland of normal skin, immunofluorescence dyeing shows it has large number of biological element (Biotin) to synthesize, and cell is rendered as sky balloon-shaped structure; Have the formation of sebaceous gland structure in the regenerated bark skin tissue of embodiment 3, the sebaceous gland in its configuration and normal skin is similar; The tissue (i.e. experimental group) having chemical reagent to induce in embodiment 4 has the sebaceous gland structure of large number of biological element synthesis, and matched group does not almost have similar structure to be formed, and concrete outcome as in Figure 3-5.
Embodiment 6
Utilize the method for the fat secretion ability of the sebaceous gland of the skin histology after transplanting in original position Mass Spectrometric Identification embodiment 4 specific as follows:
(1) by dermatoplastic for stem cell in embodiment 4 BALB/c nude mice with 1% pentobarbital sodium anesthesia, its skin of back cleaned by the ethanol with 100%, and the oil substances of its skin surface is all washed off.
(2) skin histology after the cell transplantation of experimental group and matched group is cut respectively, the skin histology of 2mm diameter is cut with skin biopsy apparatus, comprising normal skin and stem cell transplantation skin, the incubator this tissue being placed in 37 DEG C hatches 2 hours, and its in-house sebum is secreted to skin surface.
(3) before utilizing the secretion of the above-mentioned skin tissue surface oils and fats of original position Mass Spectrometric Identification, pretreatment need be carried out to tissue slice, its process is the CHCA matrix solution becoming 15g/L with the 50%ACN solution containing 0.1%TFA, promote 100 μ l matrix solutions with peristaltic pump with 5 μ l/s to be evenly sprayed on tissue surface, when tissue surface solution will be done, again spray matrix solution, circulate 2 times.
(4) tissue slice having sprayed substrate is put at room temperature, after solvent volatilization crystallization, put into the dry 2h of exsiccator.
(5) dried tissue slice conductive tape is attached on MALDI rustless steel target plate, sends into spectrometer analysis.
(6) Mass Spectrometry Conditions gets every sheet tissue slice 3 points at random, and cumulative 1000 scan-datas of each point obtain mass spectrum, obtain total mass spectrum after 3 points are cumulative.Detect by cation reflective mode, mass scan range m/z 100-1000; Cation linear model detects, mass scan range m/z 100-2000.
As shown in Figure 6, wherein red line represents the oils and fats of normal smegma to result, and the oils and fats of green line representative regeneration smegma, mass spectral results shows, regenerated bark adipose gland has the fat secretion ability similar with normal sebaceous gland.
In the description of this description, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. form a method for sebaceous gland, it is characterized in that, comprising:
In gel, Dual culture is carried out to hypodermal cell and epidermal stem cells.
2. method according to claim 1, is characterized in that, described Dual culture carries out the subcutaneous of animal.
3. method according to claim 1, is characterized in that, described hypodermal cell comprise corium stem cell and fibroblastic one of at least,
Optionally, described hypodermal cell is fibroblast, and containing insulin, dexamethasone, lattice row ketone and XAV939 in described gel,
Optionally, the final concentration of described insulin is 50-150 μ g/mL, and the final concentration of described dexamethasone is 10
-4-10
-6m, the final concentration of described class lattice row ketone is 150-250 μM, and the final concentration of described XAV939 is 5-15 μM,
Optionally, the final concentration of described insulin is 100 μ g/mL, and the final concentration of described dexamethasone is 10
-5m, the final concentration of described class lattice row ketone is 200 μMs, and the final concentration of described XAV939 is 10 μMs.
4. method according to claim 1, is characterized in that, the number ratio of described hypodermal cell and the mixing of described epidermal stem cells is 10:1 ~ 1:10,
Optionally, for every 3mm
2skin, the quantity of cell mixing is no less than 10
4individual.
5. method according to claim 1, is characterized in that, described gel is at least one being selected from Matrigel, collagen or hydrogel.
6. method according to claim 1, is characterized in that, described hypodermal cell and described epidermal stem cells are the cell deriving from autologous, allogeneic or xenogenesis.
7. method according to claim 1, it is characterized in that, described hypodermal cell and described epidermal stem cells separately cultivated for 0 ~ 30 generation in liquid medium within advance, wherein, corium stem cell is suspension culture, cell culture medium consists of DMEM/F12 (3:1), 2%B27,40ng/ μ L bFGF and 20ng/ μ LEGF; Epidermal stem cells is adhere-wall culture, and cell culture medium is CnT-07 culture medium; Wherein, further containing cell active factor and extracellular matrix molecule in described corium stem cell fluid medium, wherein said cell active factor is at least one being selected from bFGF, EGF, and described extracellular matrix molecule is at least one being selected from hyaluronic acid, fibronectin splicing variants.
8. method according to claim 1, is characterized in that, comprises further: utilize the fat secretion containing the skin tissue surface of regenerated bark adipose gland described in original position Mass Spectrometric Identification.
9. the skin histology containing sebaceous gland, is characterized in that, described sebaceous gland is formed by the method described in any one of claim 1-8.
10. the purposes of the method described in any one of claim 1-8 in skin regeneration.
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| CN201410723513.5A CN104491931B (en) | 2014-12-02 | 2014-12-02 | Sebaceous gland-containing skin tissue as well as formation method and application thereof |
| PCT/CN2015/071777 WO2016086527A1 (en) | 2014-12-02 | 2015-01-28 | Sebaceous gland-containing skin tissue, formation method and usage thereof |
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| CN107802891A (en) * | 2017-11-09 | 2018-03-16 | 清华大学深圳研究生院 | Organization engineering skin and preparation method thereof |
| CN109943520A (en) * | 2019-03-08 | 2019-06-28 | 北京达博威迎医药技术有限公司 | The separation and culture of sweat gland cells obtain the method and its application of sweat gland organoid |
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| CN113310963B (en) * | 2021-05-31 | 2023-12-01 | 四川大学华西医院 | Improved neutrophil NETs immunofluorescence detection method |
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| CN107802891A (en) * | 2017-11-09 | 2018-03-16 | 清华大学深圳研究生院 | Organization engineering skin and preparation method thereof |
| CN109943520A (en) * | 2019-03-08 | 2019-06-28 | 北京达博威迎医药技术有限公司 | The separation and culture of sweat gland cells obtain the method and its application of sweat gland organoid |
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| CN104491931B (en) | 2017-04-12 |
| WO2016086527A1 (en) | 2016-06-09 |
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