CN105154388A - Skin keratinocyte isolation and culture method - Google Patents
Skin keratinocyte isolation and culture method Download PDFInfo
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- CN105154388A CN105154388A CN201510456338.2A CN201510456338A CN105154388A CN 105154388 A CN105154388 A CN 105154388A CN 201510456338 A CN201510456338 A CN 201510456338A CN 105154388 A CN105154388 A CN 105154388A
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Abstract
In order to solve the problems of an existing keratinocyte isolation and culture method which is low in efficiency, causes a rejection reaction and the like, the invention provides a keratinocyte isolation and culture method; digestion is carried out in two steps by virtue of Trypsin/EDTA digestion liquid and I-shaped collagenase, and keratinocytes which are relatively strong in proliferative capacity are mainly extracted from a basal layer and extracted around hair follicle; relatively simplex keratinocytes are obtained; and the keratinocytes are high in activity, easy in adhesion to wall and easy in proliferation.
Description
Technical field
The present invention relates to a kind of separation preparation method of skin keratinocytes, particularly relate to a kind of for cultivate obtain tissue engineering epidermis skin keratinocytes be separated, cultural method.
Background technology
Skin keratinocytes culture has at scientific research, cosmetics and medical field to be applied extremely widely.That extracts from epidermis and some dermis layer has multiplication capacity keratinocyte, carries out amplification cultivation to it, the epidermic cell storehouse that vigor of can setting up is higher.
On medical field, during the skin injury diseases such as treatment vitiligo, burn, melanoma, scar and striae of pregnancy, traditional pharmacological agent or photochemotherapy there will be more unstable, need continued therapy.And the words of getting skin grafting dermepenthesis from self other position can cause and get skin zone's position skin injury, or there is the situation that skin source is in short supply.Therefore skin injury position can be treated by keratinocyte culture.
In addition, current cosmetics market is boundless, its security accordingly and both effectivenessly also become particularly important.Before putting on market, human body skin can be replaced to carry out susceptibility test and both effectiveness reflection by skin keratinocytes culture.So both for clinical trial provides conveniently, additionally reduce the hazardness of test failure, human keratinized cell disclosed in the people such as Lin Xiangmei evaluates the cytotoxicity (toxicology impurity, 2008,22 (1): 75-76) of makeup simultaneously.
Skin keratinocytes is separated has multiple method at present as Tr method, Tr-C method, Dispase method etc., but also often meet a lot of problem as: easily mix with other cell grow and can not long-term survival, vigor low, not easily attach, need special substrate, special culture medium etc.
The cultural method of skin keratinocytes has trophoderm culture method and non-trophoblast Serium-free Culture.The most frequently used is trophoderm culture method at present, normal adopt the fibroblast strain 3T3 cell of mouse embryoma through C060 irradiate or mitomycin process inactivation as substrate, it can promote that cell attachment can suppress again human fibroblasts to breed.But 3T3 may produce harmful meta-bolites, and with the product of turning out containing the nutrient solution of bovine serum for human body time, easily cause rejection.
Summary of the invention
Be separated for current keratinocyte and cultivate the efficiency existed low, there is the problems such as rejection, the invention provides a kind of keratinocyte and be separated and cultural method.
The present invention first aspect provides a kind of separation method of keratinocyte, comprising:
Viable skin tissue removes yellow fatty layer, retains epidermis and whole or at least part of skin corium;
Viable skin tissue is placed in Trypsin/EDTA Digestive system and carries out cold digestion;
Add pancreatin stop buffer, collect face tissue;
The face tissue of collection is placed in the digestion of I shape Collagenase heat;
Collecting cell suspension.
The present invention second aspect is to provide a kind of cultural method of keratinocyte, comprising:
Viable skin tissue removes yellow fatty layer, retains epidermis and whole or at least part of skin corium;
Viable skin tissue is placed in Trypsin/EDTA Digestive system and carries out cold digestion;
Add pancreatin stop buffer, collect face tissue;
The face tissue of collection is placed in the digestion of I shape Collagenase heat;
Collecting cell suspension, inoculation culture.
In method of the present invention, described skin histology can be the skin of organism any part, such as, any one or several position in chest, belly, back, buttocks, four limbs.
In method of the present invention, before described viable skin organizes cold digestion, can also fragmentation be carried out, form particle.
In method of the present invention, in described skin histology, the skin corium thickness of reservation is preferably 0.2-5mm, is more preferably 0.5-3mm, is more preferably 1-2mm.
In method of the present invention, described Trypsin/EDTA Digestive system concentration is preferably 0.01-0.2wt%, is more preferably 0.02-0.1wt%, is more preferably 0.03-0.08wt%, be more preferably 0.04-0.07wt%, be more preferably 0.05-0.06wt%.
In method of the present invention, described cold digestion temperature, within the scope of 2-8 DEG C, is more preferably within the scope of 3-6 DEG C, is more preferably within the scope of 4-5 DEG C.
In method of the present invention, described cold digestion time is preferably 6-24 hour, is more preferably 8-22 hour, is more preferably 10-20 hour, more preferably 12-18 hour, more preferably 14-16 hour.
In method of the present invention, I shape collagen protein enzyme concn is preferably 0.01%-1%, is more preferably 0.02%-0.8%, is more preferably 0.04%-0.7%, be more preferably 0.05%-0.5%.
In method of the present invention, described heat digestion temperature, within the scope of 35-38 DEG C, is more preferably 36-37.5 DEG C of scope, is more preferably 36.5-37 DEG C of scope.
In method of the present invention, described hot digestion time is preferably 1-10 hour, is more preferably 2-8 hour, is more preferably 3-6 hour.
Wherein, described heat digestion is preferably carried out under agitation.
In method of the present invention, the substratum of described inoculation culture can be conventional substratum, is preferably KGM-Gold or DKSFM substratum in the present invention.
Wherein, in described substratum, additional factor can also be added, as Transferrins,iron complexes, Regular Insulin, hormone, Urogastron, as any one or a few in hydrocortisone, progesterone, Transferrins,iron complexes, iron chelating agent.
Wherein, in KGM-Gold substratum, addible additional factor at least can comprise any one or a few in hydrocortisone (Hydrocortisone), Transferrins,iron complexes (Transferrin), suprarenin (Epinephrine), GA-1000, BPE, rhEGF, Regular Insulin.
Wherein, in DKSFM substratum, addible additional factor at least can comprise EGF, penicillin (Penicillin), Streptomycin sulphate (Streptomycin), any one or a few in amphotericin B (AmphotericinB).
In method of the present invention, in described seeded process, inoculating cell density is preferably 10
3-10
7cells/ml, is more preferably 10
4-10
6cells/ml, is more preferably (0.2-6) × 10
5cells/ml, is more preferably (0.5-3) × 10
5cells/ml, is more preferably (1-2) × 10
5cells/ml.
In method of the present invention, described cultivation is preferably carried out when non-trophoblast serum-free.
In a kind of preferred embodiment of method of the present invention, before described cold digestion, also comprise viable skin tissue pre-treatment step, described pre-treatment at least comprise cleaning, sterilization in any one or a few.
Wherein, described sterilization is preferably with any one or a few sterilization in the tincture of iodine, alcohol.
Wherein, described cleaning is preferably and at least adopts DPBS cleaning.
Wherein, described disinfecting time is preferably 10s-5min, is more preferably 20s-3min, is more preferably 30s-1min, is more preferably 35s-45s.
Third aspect of the present invention is to provide face tissue's culture that a kind of described cultural method obtains.
The present invention the 4th aspect is to provide the application of a kind of described cultural method or described face tissue culture.
Wherein, described application can be selected from:
The foundation in epidermic cell storehouse;
The material of susceptibility test is carried out in preparation to cosmetics;
Preparation skin regeneration material.
Application in described preparation skin regeneration material, as scar reparation, striae of pregnancy removal, skin injury treatment and treatment dermatosis in any one or a few skin regeneration material.
Wherein, described dermatosis comprises: pigment change and macle class, fungi infestation class, virus infection class dermatosis, such as vitiligo, freckle, albinism, tinea versicolor, chloasma, purpura, nevus cell nevus, nevus fuscoceruleus ophthalmomaxillaris, erythema caloricum, the tinea manuum, tinea pedis, ringworm of the body, bleb, wart etc.
Wherein, any one or a few in the dermal atrophy that skin injury comprises surgical wound surface, sunburn, burn, frostbite, scratch, incised wound, ulcer, wound cause.Described surgical wound surface is as the surface of a wound after the excision such as tumour, birthmark.Described ulcer can comprise metabolic disease as chronic ulcers such as chronic ulcer of skin, pressure ulcer, venous ulcers after diabetic foot ulcer, radiotherapy and/or chemotherapy.
Wherein, described skin regeneration material, for implanting the surface of a wound, substitutes or covers disease damage skin.
The present invention has following advantage:
1) the present invention adopts two step digestion methods, is mainly extracted stratum basale and the stronger keratinocyte of parafollicular multiplication capacity, can obtains more single keratinocyte, vigor is high, easily adherent, easily breed.
2) the inventive method is applicable to all people normal skin tissue, comprises band hair position.
3) the inventive method is without the need to trophoderm without the need to bovine serum, directly can be layered in pretreated culture dish or bottle and cultivate, be similar to the CMC model of physiological status, eliminate the impact of foreign protein.
4) substratum of the inventive method use is only for the cultivation of skin keratinocytes, avoids the pollution of other cell.
5) the skin keratinocytes culture that the inventive method obtains has at scientific research, cosmetics and medical field to be applied extremely widely.
Accompanying drawing explanation
Fig. 1 is that Tr method cultivates keratinocyte result (A is result after 3 days, and B is result after 5 days);
Fig. 2 is that the present invention two step digestion method cultivates keratinocyte result (A is result after 3 days, and B is result after 5 days);
Fig. 3 is that the present invention adopts skin of chest to cultivate keratinocyte result (A is result after 2 days, and B is result after 5 days);
Fig. 4 is that the present invention adopts skin of foreskin to cultivate keratinocyte result (A is result after 2 days, and B is result after 5 days).
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1: be successfully separated also enlarged culturing with adult normal skin of chest and go out human epidermal cell
(1) materials and methods
Sample: the adult normal skin of chest (comprising epidermis and corium) that hospital surgical is discarded, about 3cm
2.
Cell culture medium: KGM-Gold.
(2) isolation and culture of cell
A) get skin zone position, hair first need be removed in crinite position, and blocked up also need of stratum corneum is removed in advance, then carries out disinfection with 75% cotton ball soaked in alcohol, gathers tissue.
B) fresh skin sample is placed in HBSS/DPBS/DMEM, and 4 DEG C of preservations are transported to laboratory, survey area.
C) sample is placed in the tincture of iodine respectively and 75% alcohol is sterilized, then cleans with DPBS.
D) remove yellow fatty layer and major part white skin corium in tissue, retain epidermis and the thick skin corium of 0.2-2mm, skin graft is resolved into molecule.
E) skin particles is placed in 0.05%Trypsin+EDTA solution, under 4 DEG C of environment, places 12-18 hour.
F) pancreatin stop buffer is added.
G) with 100um cell filter collecting by filtration face tissue.
H) the I-type collagen enzyme that concentration is 0.05%-0.5% is added in face tissue, 37 DEG C of magnetic agitation 2-6 hour.
I) collecting by filtration cell suspension, then use DMEM (GIBCO) to clean cell bottle, collecting by filtration cell suspension.
J) centrifugal and washed cell precipitates, and counting, by 10
5the inoculum density of cells/mL is inoculated in culture vessel.
K) the Trypsin+EDTA digestion with 0.05% can be continued if any residual residue, carry out cell recovery.
When cellular segregation goes out, quantity is more, there is agglomerate (Fig. 3 A), second day starts adherent gradually, can see under microscope that keratinocyte is cloned, along with the increase of cultivated days, Keratiocyte growth is preponderated, squeeze away other contaminating cell, these clones constantly propagation connect in flakes gradually, in typical " paving stone shape ", as illustrated in figure 3b.Cell pollutes without inoblast substantially.Go down to posterity and frozen after still can keep good state and multiplication capacity.
Embodiment 2: be successfully separated also enlarged culturing with adult normal skin of chest and go out human epidermal cell
(1) materials and methods
Sample: the adult normal skin of chest (comprising epidermis and corium) that hospital surgical is discarded, about 3cm
2.
Cell culture medium: DKSFM.
(2) isolation and culture of cell
A) get skin zone position, hair first need be removed in crinite position, and blocked up also need of stratum corneum is removed in advance, then carries out disinfection with 75% cotton ball soaked in alcohol, gathers tissue.
B) fresh skin sample is placed in HBSS/DPBS/DMEM, and 4 DEG C of preservations are transported to laboratory, survey area.
C) sample is placed in the tincture of iodine respectively and 75% alcohol is sterilized, then cleans with DPBS.
D) remove yellow fatty layer and major part white skin corium in tissue, retain epidermis and the thick skin corium of 0.2-2mm, skin graft is resolved into molecule.
E) skin particles is placed in 0.05%Trypsin+EDTA solution, under 4 DEG C of environment, places 12-18 hour.
F) pancreatin stop buffer is added.
G) with 100um cell filter collecting by filtration face tissue.
H) the I-type collagen enzyme that concentration is 0.05%-0.5% is added in face tissue, 37 DEG C of magnetic agitation 2-6 hour.
I) collecting by filtration cell suspension, then use DMEM (GIBCO) to clean cell bottle, collecting by filtration cell suspension.
J) centrifugal and washed cell precipitates, and counting, by 10
5the inoculum density of cells/mL is inoculated in culture vessel.
When cellular segregation goes out, quantity is more, there is agglomerate, second day starts adherent gradually, can see under microscope that keratinocyte is cloned, along with the increase of cultivated days, Keratiocyte growth is preponderated, squeeze away other contaminating cell, these clones constantly propagation connect in flakes gradually, in typical " paving stone shape ", as shown in Figure 3.Cell pollutes without inoblast substantially.Go down to posterity and frozen after still can keep good state and multiplication capacity.
Example 2: with healthy male foreskin preparation table chrotoplast
(1) materials and methods
Sample: get people's healthy male and to peritomize postoperative skin of foreskin
(2) isolation and culture of cell: method is identical with example 1.
(3) result: the cell be separated to is easy to adherent, clones more, comparatively fast can be linked to be sheet, in " paving stone " shape, as shown in Figure 4.Cell is purer, go down to posterity and frozen after still can keep good state and multiplication capacity.
Comparative example
Dispase method mainly acts on stratum basale thus epidermal area is separated with skin corium, and the high keratinocyte of multiplication capacity is mainly present in stratum basale and perifollicolar, therefore, Dispase method can injure these keratinocytes, cannot obtain high reactivity keratinocyte efficiently.
Be separated same sample by Tr method with two step digestion methods respectively and carry out control experiment, result as shown in Figure 1, Tr method was the little clone of appearance only a few of the 3rd day, cloning efficiency about 5% (Figure 1A), cloning efficiency about 20% (Figure 1B) is there is in two step digestion methods the significantly clone that has of the 3rd day, and the 5th day time Tr method clonal expansion slowly (Fig. 2 A), the clonal expansion ability of two step digestion methods obviously strengthens, local is linked to be sheet (Fig. 2 B).
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (17)
1. a separation method for keratinocyte, is characterized in that, comprising:
Viable skin tissue removes yellow fatty layer, retains epidermis and whole or at least part of skin corium;
Viable skin tissue is placed in Trypsin/EDTA Digestive system and carries out cold digestion;
Add pancreatin stop buffer, collect face tissue;
The face tissue of collection is placed in the digestion of I shape Collagenase heat;
Collecting cell suspension.
2. method according to claim 1, is characterized in that, in described skin histology, the skin corium thickness of reservation is 0.2-5mm.
3. method according to claim 1, is characterized in that, described cold digestion temperature is within the scope of 2-8 DEG C, and described cold digestion time is 6-24 hour.
4. method according to claim 1, is characterized in that, described heat digestion temperature is within the scope of 35-38 DEG C, and described hot digestion time is 1-10 hour.
5. method according to claim 1, is characterized in that, before described viable skin organizes cold digestion, carries out fragmentation, forms particle.
6. method according to claim 1, is characterized in that, before described cold digestion, also comprises viable skin tissue pre-treatment step, described pre-treatment at least comprise cleaning, sterilization in any one or a few.
7. a cultural method for keratinocyte, is characterized in that, comprising:
According to the separation method of keratinocyte described in claim 1, collecting cell suspension, carries out inoculation culture.
8. method according to claim 7, is characterized in that, the substratum of described inoculation culture is selected from KGM-Gold or DKSFM substratum.
9. method according to claim 8, is characterized in that, also adds additional factor in described substratum, described additional factor at least comprise in Transferrins,iron complexes, Regular Insulin, hormone, Urogastron any one or a few.
10. method according to claim 8 or claim 9, it is characterized in that, the additional factor added in KGM-Gold substratum can at least comprise in hydrocortisone, Transferrins,iron complexes, suprarenin, GA-1000, BPE, rhEGF, Regular Insulin any one or a few.
11. methods according to claim 8 or claim 9, it is characterized in that, in DKSFM substratum, addible additional factor can at least comprise EGF, penicillin, Streptomycin sulphate, any one or a few in amphotericin B.
12. methods according to claim 7, is characterized in that, in described seeded process, inoculating cell density is 10
3-10
7cells/ml.
13. methods according to claim 7, is characterized in that, described cultivation is carried out when non-trophoblast serum-free.
Face tissue's culture that cultural method described in 14. 1 kinds of claims 7 obtains.
The application of face tissue's culture described in cultural method described in 15. 1 kinds of claims 7 or claim 14, it is characterized in that, described application is selected from:
The foundation in epidermic cell storehouse;
The material of susceptibility test is carried out in preparation to cosmetics;
Preparation skin regeneration material.
16. application according to claim 15, is characterized in that, preparation skin regeneration material be selected from: for scar reparation, striae of pregnancy removal, skin injury treatment and treatment dermatosis in any one or a few skin regeneration material.
17. application according to claim 16, is characterized in that, described dermatosis comprises: pigment change and macle class, fungi infestation class, virus infection class dermatosis; Any one or a few in the dermal atrophy that skin injury comprises surgical wound surface, sunburn, burn, frostbite, scratch, incised wound, ulcer, wound cause.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108498863A (en) * | 2017-07-11 | 2018-09-07 | 上海白衣缘生物工程有限公司 | A kind of oral cavity sticking patch preparation method of compound oral mucosa epithelial cell |
| CN110170072A (en) * | 2019-05-31 | 2019-08-27 | 北京添易医学研究院 | The preparation of cell fibronectin and the method for repairing dermal tissue |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1528253A (en) * | 2003-10-17 | 2004-09-15 | 上海第二医科大学附属第九人民医院 | Tissue engineered composite skin and preparing method thereof |
| CN102086451A (en) * | 2009-12-07 | 2011-06-08 | 韩春茂 | Method for amplifying seed cells of skin tissue engineering |
| WO2011137485A1 (en) * | 2010-05-05 | 2011-11-10 | Sydney Ivf Limited | Media and methods for cell culture |
| CN104099289A (en) * | 2013-04-08 | 2014-10-15 | 瑞田投资有限公司 | Epidermal tissue culturing method, and prepared epidermal tissue culture and application thereof |
-
2015
- 2015-07-29 CN CN201510456338.2A patent/CN105154388B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1528253A (en) * | 2003-10-17 | 2004-09-15 | 上海第二医科大学附属第九人民医院 | Tissue engineered composite skin and preparing method thereof |
| CN102086451A (en) * | 2009-12-07 | 2011-06-08 | 韩春茂 | Method for amplifying seed cells of skin tissue engineering |
| WO2011137485A1 (en) * | 2010-05-05 | 2011-11-10 | Sydney Ivf Limited | Media and methods for cell culture |
| CN104099289A (en) * | 2013-04-08 | 2014-10-15 | 瑞田投资有限公司 | Epidermal tissue culturing method, and prepared epidermal tissue culture and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108498863A (en) * | 2017-07-11 | 2018-09-07 | 上海白衣缘生物工程有限公司 | A kind of oral cavity sticking patch preparation method of compound oral mucosa epithelial cell |
| CN110170072A (en) * | 2019-05-31 | 2019-08-27 | 北京添易医学研究院 | The preparation of cell fibronectin and the method for repairing dermal tissue |
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Effective date of registration: 20191029 Address after: Room 605, B1 building, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, Suzhou, Jiangsu Applicant after: Suzhou nuopu Regenerative Medicine Co. Ltd. Address before: 214092, Jiangsu, Wuxi Province, Binhu District, Mashan Mei Lu Road, No. 130, -3 Applicant before: H&B HEALTH GROUP Applicant before: Liu Hong |
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