CN105112353A - Mixed cultivation method of keratinocyte and melanocyte and application - Google Patents
Mixed cultivation method of keratinocyte and melanocyte and application Download PDFInfo
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Abstract
The invention provides a mixed cultivation method of keratinocyte and melanocyte comprising: breaking the skin sample reserving epidermal layer and at least part of corium layer, cold digesting by pancreatin; collecting deposition organization; heat digesting by I type collagenase, collecting cells; primary inoculating, culturing in KGM-Gold medium; subcultring to the cells merge, culturing in KGM-Gold medium to the cells differentiate and stratify; collecting the formed skin sheet. The mixed cultivation method of keratinocyte and melanocyte uses the medium only for skin keratinocyte and melanocyte with which can avoid pollution of the other cells, and can get skin sheet including functional melanocyte and distributing uniformly. The mixed cultivation method of keratinocyte and melanocyte can get large area of skin sheet from quite small area of autologous sample, solve the problem of shortage of the skin source at skin grafting for the patient with large-area skin diseases, and apply in skin diseases of pigment deficiency such as leucoderma and so on.
Description
Technical field
The present invention relates to a kind of cell culture processes, particularly relate to method and the application thereof of a kind of keratinocyte and melanophore (melanocyte) mixed culture.
Background technology
The difference of the colour of skin depends primarily on quantity, size, the Type and distribution situation of melanocyte in skin.Melanocyte is a kind of protein derivatives, is produced by the melanophore in epidermis.
Partial skin patient parts of body occurs that hickie is due to melanocyte in skin disappearance, destroyed or nonfunctional, and then affects skin pigment manufacturing system and cause.If vitiligo is that a kind of common acquired character limit office property or general property skin pigment depigmentation are sick, be that other similar diseases also comprise the pigment disorders such as black mole, chloasma, freckle, nevus fuscoceruleus ophthalmomaxillaris because the melanophore disappearance of skin causes.This disease to patient's physiology and can cause serious harm psychologically.Current methods for the treatment of comprises local and uses reflunomide, NB-UVB/PUVA, excimer laser etc., but curative effect is low, and with certain side effect.
The method of other the above-mentioned disease for the treatment of also comprises: 1) transplantation surgeries technology, as negative pressure suction method, razor graft transplanting, small grafts, but there is skin source problem in short supply concerning big area patient; 2) melanophore transfer methods, disclosed in CN102266586B, melanophore transplants melanophore cultural method disclosed in coating carrier, CN101892286B, and melanocyte suspension formulation disclosed in CN100354001C, all can be used for melanophore to transplant, but there is the poor problem of melanoma coincidence risk, uniformity coefficient in the method, and its maximum technological difficulties are that melanocyte growth is poor, cannot increase in a large number in a short time.
On the other hand, be separated melanophore without other ad hoc approach except substratum, the melanophore quantity be directly separated ex vivo is few, and has limited homogeneity and multiplication capacity, and therefore having no efficiency can say.Further, have report to claim, when using in vitro just from epidermal area during isolated melanophore, the stimulation of described cell to UVB etc. has no reaction, again or the expression of the gene relevant to B16 cell have decline.
Summary of the invention
For the problem existing for current melanophore vitro culture, the invention provides a kind of method of keratinocyte and melanophore co-cultivation.
The present invention first aspect is to provide a kind of serum free medium for keratinocyte and melanocyte, and described substratum comprises: KGM-Gold substratum and KGM substratum.
Wherein, in the substratum described in first, described KGM-Gold substratum and the separate existence of KGM substratum.
The present invention second aspect is to provide a kind of method of keratinocyte and melanophore co-cultivation, and step comprises:
The dermatological specimens of reservation table cortex and at least part of skin corium, after fragmentation, carries out the cold digestion of pancreatin;
Collecting precipitation tissue, carries out heat digestion with type i collagen enzyme, collecting cell;
Primary inoculation, carries out in KGM-Gold substratum;
Secondary Culture, to cell confluency, cultivates in KGM substratum.
Third aspect of the present invention is to provide a kind of method preparing artificial epidermis, and step comprises:
The dermatological specimens of reservation table cortex and at least part of skin corium, after fragmentation, carries out the cold digestion of pancreatin;
Collecting precipitation tissue, carries out heat digestion with type i collagen enzyme, collecting cell;
Primary inoculation, cultivates in KGM-Gold substratum;
Secondary Culture, to cell confluency, carries out being cultured to cytodifferentiation layering in KGM substratum;
Collect the skin graft formed.
In an advantageous embodiment, be added with additional factor in described KGM-Gold substratum, described additional factor can be selected from hydrocortisone (hydrocortisone), source of iron, suprarenin (epinephrine), ox pituitary gland extract (BPE), Urogastron, Regular Insulin, microbiotic any one or a few.
In an advantageous embodiment, be added with additional factor in described KGM substratum, described additional factor can be selected from KSR (Knockout serum substitute), keratinocyte growth factor, hydrocortisone, Urogastron, microbiotic, source of iron any one or a few.
Wherein, described source of iron can be can be accepted by cell TfR and obtain the material of ferro element, as Transferrins,iron complexes (transferrin) and can play in the Transferrins,iron complexes surrogate of phase same-action any one or a few.
Wherein, Transferrins,iron complexes can be any one or a few in recombinant transferrin, people source Transferrins,iron complexes, animal source Transferrins,iron complexes (as ox source Transferrins,iron complexes).
Wherein, described Transferrins,iron complexes surrogate can be any one or a few in ironic citrate, ferric cacodylate, Gluconate Ferrecex.
Wherein, described Regular Insulin can be Recombulin, people source Regular Insulin, animal source Regular Insulin (as ox source Regular Insulin) and insulin-like growth factor.
Wherein, described microbiotic can be selected from penicillin (Penicillin), Streptomycin sulphate (streptomycin), gentamicin (Gentamicin), GA-1000, amphotericin B (amphotericinB), Pen/Strep (Penicillin-streptomycin) any one or a few.
Wherein, described keratinocyte growth factor is as any one or a few in human keratinocyte Growth Factor (HKGS), KGF-2, rhKGF-2, KGF-1, rhKGF-1.
Wherein, described Urogastron is as any one or a few in recombinant human epidermal growth factor (rhEGF), EGF.
Described keratinocyte and melanocyte can be come from same organism or different organism in an advantageous embodiment, also can be the different sites or the same area that come from same organism.
Described dermatological specimens can be allosome and/or autologous skin sample, and is preferably autologous skin sample.Described dermatological specimens can be the skin being derived from any one or a few positions such as chest, belly, back, buttocks, four limbs.
In an advantageous embodiment, in described viable skin sample, skin corium accounts for the volume ratio of skin corium in initial skin sample, is preferably 15-50%, is more preferably 20-45%, is more preferably 25-40%, be more preferably 30-35%.
In an advantageous embodiment, epidermal keratinocyte and melanocyte quantitative proportion are preferably 100:(0.001-80), be preferably 100:(0.01-60), be preferably 100:(0.1-50), be preferably 100:(0.5-40), be preferably 100:(1-30), be preferably 100:(5-20), be preferably 100:(10-15).
In an advantageous embodiment, in described dermatological specimens, skin corium thickness is preferably 0.2-4mm, is more preferably 0.3-3.5mm, is more preferably 0.5-3.0mm, is more preferably 0.8-2.5mm, is more preferably 1.0-2.2mm, is more preferably 1.2-2.0mm.
In an advantageous embodiment, described Secondary Culture is preferably original cuiture 2-8 days, is preferably 3-6 days, is more preferably 4-5 days and carries out.
In an advantageous embodiment, the time of the cold digestion of described pancreatin is preferably 8-24 hour, is more preferably 10-22 hour, is more preferably 11-20 hour, be more preferably 12-18 hour, be more preferably 14-16 hour.
In an advantageous embodiment, in the cold digestive process of described pancreatin, pancreatin mass concentration is preferably 0.01-0.1%, is more preferably 0.02-0.08%, is more preferably 0.04-0.07%, is more preferably 0.05-0.06%.
In an advantageous embodiment, the time of described I type pancreatin heat digestion is preferably 1-10 hour, is more preferably 1.5-8 hour, is more preferably 2-6 hour, be more preferably 3-5 hour.
In an advantageous embodiment, carry out in the process of cultivating in KGM-Gold substratum, substratum is changed interval and is preferably at least 12 hours, is more preferably 24 hours, is more preferably 36 hours.
In an advantageous embodiment, cultivate 1-10 days in KGM substratum after, collect skin graft, after being more preferably 2-9 days, be more preferably 3-8 days after, be more preferably 4-6 days after collect skin graft.
Beneficial effect of the present invention is as follows:
(1) the inventive method is applicable to proprietary normal skin tissue, comprises band hair position.
(2) 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.
(3) the inventive method needs epidermis when separation and Extraction and carries corium, because melanophore is mainly present in basal layer of epidermis, and can extract the strong keratinocyte of more multiplication capacities.
(4) substratum of the inventive method use is only for skin keratinocytes and melanocytic cultivation, avoids the pollution of other cell.
(5) present approach provides a kind of method of epidermic cell and melanocyte Dual culture, and can obtain and comprise functional melanophore and the epidermis diaphragm be evenly distributed, can be applied on the dermatosis of the pigment loss such as vitiligo.
(6) the inventive method can obtain large-area epidermis diaphragm by very small area from sample body, skin source problem in short supply when solving large area skin patient grafts.
In sum, the method for keratinocyte provided by the invention and melanophore Dual culture, can be trained the epidermis of melanin-containing cell for transplanting.The method carries out amplification cultivation in vitro, connects in flakes and divide to multilayered structure, solving Pi Yuan problem in short supply.And the keratinocyte of epidermis also participates in formation and the metabolism (melanophore and keratinocyte are collectively referred to as epidermal melanin unit) of the black pigment of skin.
Accompanying drawing explanation
Fig. 1 is that the embodiment of the present invention 1 method cultivates the epidermis diaphragm cell state obtained;
Fig. 2 is that the embodiment of the present invention 2 method cultivates the epidermis diaphragm cell state obtained;
Fig. 3 is that the embodiment of the present invention 2 method cultivates the epidermis diaphragm treatment vitiligo design sketch obtained, wherein,
Before Fig. 3 A-Fig. 3 C is followed successively by treatment, treat 1 month, the photo for the treatment of after 6 months.
Embodiment
Example 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.
Skins culture base: KGM-Gold and KGM
(2) cellular segregation and skin graft are cultivated
A) dermatological specimens in DMEM nutrient solution refrigerated shipment to laboratory, survey area.
B) sample is cleaned with PBS; Repeated washing sample again after the tincture of iodine, alcohol disinfecting, removes yellow fatty layer and major part white skin corium in tissue, retains epidermis and the thick skin corium of 0.2-3mm, skin graft is resolved into molecule.
C) 0.05% pancreatin cold digestion sample 12-18 hour; Add pancreatin stop buffer; Collecting by filtration face tissue.
D) add type i collagen enzyme magnetic agitation heat digestion 2-6 hour, after filtering, collected after centrifugation epidermic cell is seeded in culture dish and cultivates.
E) change a KGM-Gold substratum every other day, in KGM-Gold substratum, be added with hydrocortisone, Transferrins,iron complexes, GA-1000, BPE, rhEGF, Regular Insulin, suprarenin, AmphotericinB etc.
F) go down to posterity during 3-6 days.
G) change keratinocyte substratum KGM after cell confluency, in KGM substratum, be added with KSR, KGS, hydrocortisone, rhEGF, Pen/Strep, Transferrins,iron complexes, EGF etc.
H) cell confluency is cultivated after 4-8 days again and is carried out skin graft stripping.
I) the epidermis diaphragm under stripping is done to the distribution of haematoxylin dyeing and dopa stain inspection stratum basale melanocyte and keratinocyte.
(3) result
Epidermis diaphragm cell state is good more even.Haematoxylin dyeing and dopa stain result show melanocyte and are dyed to black and are evenly distributed at stratum basale, and account for 10% of keratinocyte sum, cell is dendron shape, (as Fig. 1) in good condition.
Example 2: get people's healthy male peritomize postoperative skin of foreskin be successfully separated and enlarged culturing go out human epidermal cell
(1) materials and methods
Sample: the skin of foreskin (comprising epidermis and corium) that the healthy male that hospital surgical is discarded is peritomized postoperative, about 4cm
2.
Skins culture base: KGM-Gold and KGM
(2) cellular segregation and skin graft are cultivated
A) dermatological specimens in DMEM nutrient solution refrigerated shipment to laboratory, survey area.
B) sample is cleaned with PBS; Repeated washing sample again after the tincture of iodine, alcohol disinfecting, removes yellow fatty layer and major part white skin corium in tissue, retains epidermis and the thick skin corium of 1-3mm, skin graft is resolved into molecule.
C) the cold digestion sample of 0.05% pancreatin 15 hours; Add pancreatin stop buffer; Collecting by filtration face tissue.
D) add type i collagen enzyme magnetic agitation heat digestion 5 hours, after filtering, collected after centrifugation epidermic cell is seeded in culture dish and cultivates.
E) change a KGM-Gold substratum every other day, in KGM-Gold substratum, be added with hydrocortisone, Transferrins,iron complexes, GA-1000, BPE, rhEGF, Regular Insulin, suprarenin, AmphotericinB etc.
F) the 5th day time go down to posterity.
G) change keratinocyte substratum KGM after cell confluency, in KGM substratum, be added with KSR, KGS, hydrocortisone, rhEGF, Pen/Strep, Transferrins,iron complexes, EGF etc.
H) cell confluency is cultivated after 6 days again and is carried out skin graft stripping.
I) the epidermis diaphragm under stripping is done to the distribution of haematoxylin dyeing and dopa stain inspection stratum basale melanocyte and keratinocyte.
(3) result
Epidermis diaphragm is more smooth, cell is purer, substantially be all keratinocyte and melanophore, haematoxylin dyeing and dopa stain result show melanocyte and are dyed to black in stratum basale distribution uniform, account for 20% of keratinocyte sum, cell is dendron shape, (as Fig. 2) in good condition.
Fig. 3 is that patients with vitiligo autologous keratinocytes and melanophore Dual culture form epidermis and carry out transplantation treatment result.Fig. 3 A is the state (in figure, the black line institute region of arrow indication is the region of determining to carry out transplantation treatment) before treatment, Fig. 3 B is the state for the treatment of after 1 month, can find out, compared with other non-transplantation sites, the inventive method gained skin graft has obvious result for the treatment of, and Fig. 3 C is the state of transplanting latter 6 months, can find out, transplant region pigment calmness completely, with surrounding normal skin color closely.
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 (12)
1., for a serum free medium for keratinocyte and melanocyte, it is characterized in that, described substratum comprises: KGM-Gold substratum and KGM substratum.
2. substratum according to claim 1, is characterized in that, described KGM-Gold substratum and the separate existence of KGM substratum.
3. substratum according to claim 1, it is characterized in that, be added with additional factor in described KGM-Gold substratum, described additional factor can be selected from hydrocortisone, source of iron, suprarenin, ox pituitary gland extract (BPE), Urogastron, Regular Insulin, microbiotic any one or a few.
4. substratum according to claim 1, it is characterized in that, be added with additional factor in described KGM substratum, described additional factor can be selected from KSR, keratinocyte growth factor, hydrocortisone, Urogastron, microbiotic, source of iron any one or a few.
5. the substratum according to claim 3 or 4, is characterized in that, described microbiotic be selected from penicillin, Streptomycin sulphate, gentamicin, GA-1000, amphotericin B, Pen/Strep any one or a few.
6. the substratum according to claim 3 or 4, is characterized in that, described Urogastron be selected from recombinant human epidermal growth factor, EGF any one or a few.
7. substratum according to claim 4, is characterized in that, described keratinocyte growth factor be selected from HKGS, KGF-2, rhKGF-2, KGF-1, rhKGF-1 any one or a few.
8. a method for keratinocyte and melanophore co-cultivation, is characterized in that, step comprises:
The dermatological specimens of reservation table cortex and at least part of skin corium, after fragmentation, carries out the cold digestion of pancreatin;
Collecting precipitation tissue, carries out heat digestion with type i collagen enzyme, collecting cell;
Primary inoculation, carries out in KGM-Gold substratum described in claim 1;
Secondary Culture, to cell confluency, cultivates in KGM substratum described in claim 1.
9. method according to claim 8, is characterized in that, the volume ratio that skin corium accounts for skin corium in initial skin sample is 15-50%.
10. method according to claim 8 or claim 9, it is characterized in that, in described dermatological specimens, skin corium thickness is 0.2-4mm.
11. methods according to claim 8, is characterized in that, epidermal keratinocyte and melanocyte quantitative proportion are 100:(0.001-80).
12. 1 kinds of methods preparing artificial epidermis, it is characterized in that, step comprises:
The dermatological specimens of reservation table cortex and at least part of skin corium, after fragmentation, carries out the cold digestion of pancreatin;
Collecting precipitation tissue, carries out heat digestion with type i collagen enzyme, collecting cell;
Primary inoculation, carries out in KGM-Gold substratum described in claim 1;
Secondary Culture, to cell confluency, carries out in KGM substratum described in claim 1 being cultured to cytodifferentiation layering;
Collect the skin graft formed.
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| CN106520674A (en) * | 2016-12-24 | 2017-03-22 | 严志海 | Serum-free medium for in-vitro culture of epidermal melanocytes |
| CN107151648A (en) * | 2017-06-18 | 2017-09-12 | 广东博溪生物科技有限公司 | A kind of melanocyte and keratinocyte co-cultivation culture medium |
| CN107287151A (en) * | 2017-06-18 | 2017-10-24 | 广东博溪生物科技有限公司 | A kind of construction method of the vitro skin test model containing melanocyte |
| CN107326004A (en) * | 2017-06-18 | 2017-11-07 | 广东博溪生物科技有限公司 | Build the melanocyte and keratinocyte gas-liquid face culture medium of restructuring skin model |
| CN107338216A (en) * | 2016-11-14 | 2017-11-10 | 上海睿泰生物科技股份有限公司 | Artificial epidermal graft, its preparation method and application |
| CN108203705A (en) * | 2018-02-11 | 2018-06-26 | 山西农业大学 | The co-culture method of mouse melanin cell and keratinocyte |
| CN108567996A (en) * | 2017-03-07 | 2018-09-25 | 武汉北度生物科技有限公司 | A kind of preparation and its application of 3D multilayer structures cell patch |
| CN111117945A (en) * | 2019-12-31 | 2020-05-08 | 广东博溪生物科技有限公司 | Melanin-containing epidermal skin model and construction method and application thereof |
| CN112481194A (en) * | 2020-12-02 | 2021-03-12 | 深圳清华大学研究院 | Method for preparing cell suspension with melanocyte activity |
| 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 |
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| CN107338216A (en) * | 2016-11-14 | 2017-11-10 | 上海睿泰生物科技股份有限公司 | Artificial epidermal graft, its preparation method and application |
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| CN111117945A (en) * | 2019-12-31 | 2020-05-08 | 广东博溪生物科技有限公司 | Melanin-containing epidermal skin model and construction method and application thereof |
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| CN112481194A (en) * | 2020-12-02 | 2021-03-12 | 深圳清华大学研究院 | Method for preparing cell suspension with melanocyte activity |
| CN112481194B (en) * | 2020-12-02 | 2023-03-21 | 深圳清华大学研究院 | Method for preparing cell suspension with melanocyte activity |
| 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 |
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Application publication date: 20151202 |