CN107338216A

CN107338216A - Artificial epidermal graft, its preparation method and application

Info

Publication number: CN107338216A
Application number: CN201611026605.3A
Authority: CN
Inventors: 仁科博道; 吴复跃; 何振东
Original assignee: Shanghai Rui Tai Biological Polytron Technologies Inc
Current assignee: Shanghai Rui Tai Biological Polytron Technologies Inc
Priority date: 2016-11-14
Filing date: 2016-11-14
Publication date: 2017-11-10

Abstract

The present invention relates to artificial epidermal graft, its preparation method and application.Specifically, the present invention provides a kind of artificial epidermal graft, and it is included：Melanocyte living forms cell and epidermal cell, wherein, the artificial epidermal graft does not include animal derived serum and protein and nutritional support cell.The present invention also provides the method for the artificial epidermal graft for preparing, by artificial application of the epidermal graft in treating skin disease and/or beauty or screening substances made from the above method.

Description

Artificial epidermal graft, its preparation method and application

Technical field

The present invention relates to field of biological product；In particular it relates to co-incubation epidermal cell forms cell with shape with melanocyte Into the method for artificial epidermal graft, and application of the above-mentioned artificial epidermal graft in medical treatment, beauty and/or screening substances.

Background technology

Skin refers to tissue of the body surface bag outside muscle, is the maximum organ of human body, mainly carry protection body, Perspire, feel the functions such as cold and hot and pressure.Skin covers whole body, and it makes various in vivo organize with organ from physical, mechanical Property, the chemically invasion and attack with pathogenic microorganism.Due to outside cause and/or organism self reason, skin may occur Various damages and/or lesion, some of which damage or lesion are directed not only to skin epidermal cells, and the melanocyte further related in skin is thin The missing of born of the same parents or damage (such as leucoderma).

Common leucoderma is a kind of illness caused by melanocyte in skin lacks.Its Etiological be melanocyte from Ruin, so as to be considered as a kind of autoimmune disease.The affected part of leucoderma can form hickie because melanin lacks, so that and body Other positions produce obvious aberration.

At present, it can typically be used for leucoderma and smear the medicines such as steroid ointment, active form vitamin ointment Therapeutic modality, it can also be treated sometimes using immunodepressant such as tacrolimus.In addition, existing therapy also includes：PUVA Therapy, short wavelength UV gamma therapy, and the therapy of the steroid for oral administration used during symptom development to whole body.On however, State therapy and affected part the problems such as still progressively expanding after curative effect deficiency, treatment be present.

However, when body starts to drug resistant, above-mentioned therapy is unable to reach Expected Results, and symptom will be by Step expands.In the case, another therapy can be used, i.e., obtains skin from patient's normal portions, and transplanted.This treatment The shortcomings that method, is, can not carry out the transplanting of large area¹。

Also adoptable another therapy is：Beyond patient's affected part, unconspicuous normal body position collection one is small Block skin, keratinocyte culture is carried out using the skin, and the epidermis grafts obtained will be cultivated to affected part.Research knot Fruit shows that this therapy is effective for segmental vitiligo.Why effective it is, is mainly based upon the pathology of leucoderma：Attack The immunocyte intrusion epidermis of itself melanocyte is hit, this not only causes the death of melanocyte, and even the cutin on periphery is formed carefully Born of the same parents are also together killed².Many reports show that in leucoderma, the keratinocyte of affected part also sustains damage^3-7。

According to research reports, under normal circumstances, keratinocyte can discharge many kinds of substance under UVB irradiation, these Material can promote propagation and the differentiation of hair follicle portion melanocyte formation cell^8,9.However, impaired keratinocyte then lack it is above-mentioned Ability, this is also the reason for most of UVB therapies fail.As long as on the contrary, the horn cell of affected part is substituted for normal epidermis Horn cell, it becomes possible to treat leucoderma.One of the reason for can curing leucoderma here it is transplanting normal epidermis skin graft.

However, the validity of above-mentioned this therapy is only limitted to the treatment for segmental vitiligo, it is for appearing in two The whole body type leucoderma of side is invalid.

According to such as preceding document report, the melanocyte of leucoderma affected part can be because of the attack by immunocyte and dead.No Cross, may still suffer from some in hair follicle root area does not have by the melanocyte formation cell of the antigen of immunocyte identification, because It is not affected by and attacks and existence.Thus, most UVB therapies are also exactly to utilize the principle, the epidermal cell at the position is lived Change, secrete the movement that cell is formed to melanocyte, differentiation and/or ripe favourable cell factor, melanocyte is formed cell generation black Pigment.But if the melanocyte at the position forms cell and also come to harm, not only UVB therapies can fail, and transplant normal angle Matter, which forms cell and remained on, does not reach therapeutic effect.And in the case of only melanocyte is transplanted, if immunocyte activity frequency Numerous, the survival rate of melanocyte can also be influenceed and low by immune cells attack¹⁰。

At present, although having reported several serum-frees, the skin keratin formation cell culture processes without feeder cells, It is to make cell differentiation in the state of serum-free, and the cultural method for the skin graft for being derived from being transplanted is so far without report Road.Also, also never attempt and develop in such skin graft of sening as an envoy to form cell containing melanocyte in this area, and make itself and table The cultural method that chrotoplast is bred, broken up jointly.

Therefore, this area stills need to treat the high security novel product of leucoderma and technological means.Also, except Outside leucoderma, this area need also remain for being used for treating or beautify other be related to epiderm skin and melanocyte formed cell by The high security novel product and technological means of the skin symptom and/or disease (such as skin burn, burn, scar etc.) of damage.

The content of the invention

It is an object of the present invention to provide a kind of artificial epidermal graft, the culture of the artificial epidermal graft is pacified with preparation process Quan Xinggao.Also, obtained artificial epidermal graft also forms cell comprising melanocyte, and this is for needing dermatoplasty and/or skin It is extremely advantageous for the disease (such as leucoderma) and/or situation (as beauty is conditions associated) of repairing.

The first aspect of the present invention provides a kind of artificial epidermal graft, and it is included：Melanocyte living forms cell and epidermal cell, Wherein, the artificial epidermal graft does not include animal derived serum and protein and nutritional support cell.

In some embodiments, the artificial epidermal graft does not include the allergy original of brain derived material and animal and plant source Matter；In another embodiment, the artificial epidermal graft, which also includes, is used to support the melanocyte formation cell and epidermal cell Support material, such as fibrin gel.

The second aspect of the present invention provides a kind of method for preparing artificial epidermal graft, and it comprises the following steps：

(i) skin histology pre-processes：Handled with enzyme solutions to decompose skin histology, obtain the prepared product for including living cells；

(ii) cell propagation and differentiation：Make the cell propagation in gained prepared product, and be divided into melanocyte and form cell and table Chrotoplast, further make the cell multiple stratification to form the artificial epidermal graft containing melanocyte formation cell；

Wherein, methods described does not use animal derived serum, protein and nutritional support cell.

In some embodiments, methods described also includes：After skin histology is decomposed with enzyme solutions, using enzyme level Agent completely inhibits tryptic activity, then carries out cell propagation and differentiation.

In other embodiments, methods described is additionally included in cell propagation with carrying out adaptation step, institute after differentiation State adaptation step and the embedding of the cell skin graft of the cell of differentiation or fragmentation is entered into suitable carrier, obtain the load containing noble cells Body.

In some embodiments, the carrier is fibrin.

In other embodiments, the enzyme solutions include proteolytic enzyme, and the proteolytic enzyme includes neutral egg One or more in white enzyme (for example, dispase), trypsase, clostridiopetidase A.

In other embodiments, in the atomization, progressively by differentiation nutrient solution relative to Multiplying culture liquid Accounting improves.

In some embodiments, the volume of the differentiation nutrient solution and Multiplying culture liquid that are added in cell differentiation procedure Than from 1:2 are gradually transition to all differentiation nutrient solutions.For example, from 1:1 to 2:1, then to 3:1, it is finally reached all differentiation Nutrient solution.

In some embodiments, the differentiation nutrient solution also includes on the basis of basic culture solution：Insulin, Corticotropin, Holo-transferrin and vitamin D.

In some embodiments, during the cell propagation, the passage to the cell was no more than for 5 generations.

In some embodiments, the basis for the Multiplying culture liquid that the Multiplying culture liquid used after passage uses before passage Upper another addition stem cell medium exchange SCF and Wnt-3a.

The third aspect of the present invention provides the artificial epidermal graft prepared using the inventive method.

The fourth aspect of the present invention provide the present invention artificial epidermal graft prepare be directed to using melanocyte in skin missing as Application in the disease (for example, leucoderma) or the product of situation of feature.

The artificial epidermal graft that the fifth aspect of the present invention provides the present invention is used for disease of skin or the medicine of situation in screening Or the application in cosmetics.

In the method for the invention, both without using the nutrient solution containing risk factor, for example, containing it is animal derived (for example, Humanized) serum, brain derived material, the protein of ox or pig source property, or the nutrient solution of the allergenic agent of animal and plant source, Again without using nutritional support cell (for example, support cell of small mouse).Methods described is safe, also, obtained Artificial epidermal graft also forms cell comprising melanocyte, and this is for needing by artificial skin the disease or shape treating and/or beautify Condition, especially it is for the treatment and/or beautification of (such as leucoderma) disease or situation characterized by melanocyte in skin lacks Extremely advantageous.

Those skilled in the art can be combined without departing from this hair to foregoing technical scheme and technical characteristic Bright inventive concept and protection domain.The other side of the present invention is due to this disclosure, to those skilled in the art For be obvious.

Brief description of the drawings

The invention will be further described below in conjunction with the accompanying drawings, and wherein these are shown only for illustrating the reality of the present invention Scheme is applied, rather than in order to limit to the scope of the present invention.

Fig. 1：Membrane surface culture VD₃Concentration influence (embodiment 5).(A)：1α,25(OH)₂VD₃:10^-11M；(B)：1α,25 (OH)₂VD₃:10^-9M。

Fig. 2：By the fibrin gel containing cell fromTake out what is be placed on the back of the hand and shoot in culture dish Photo (embodiment 9).

Fig. 3：The research 1 of differentiation method：Multistage differentiation, VD₃Concentration and passage number form cell number for melanin Influence (embodiment 11).

Fig. 4：The research 2 of differentiation method：Cell factor and VD₃Influence (the embodiment of cell maturation is formed for melanocyte 12).(A)：Control；(B)：Add cell factor；(C)：Add cell factor+VD₃。

Fig. 5：The research 3 of differentiation method：Cell factor and VD₃Influence (the embodiment of cell quantity is formed for melanocyte 13).(A)：Control；(B)：Add cell factor；(C)：Add VD₃。

Fig. 6：The research 4 of differentiation method：Maintenance effects (embodiment 14) of the ACTH for keratinocyte.Tieed up during differentiation Hold basal cell：(A) ACTH (+), (B) ACTH (-)；Upper figure below enlargement ratio is identical.

Fig. 7：The research 5 of differentiation method：The influence (embodiment 15) of insulin, Holo-transferrin for differentiation.Differentiation Add insulin and transferrins (i.e. IT), multiple stratification result and gained knot during application serum of acquisition on the basis of Shi Tianjia ACTH again Fruit is identical.

Fig. 8：The research 6 of differentiation method：Influence (embodiment 16) of the cell factor for tyrosinase activity. (A)：Wnt +SCF；(B)：SCF+ Endothelins.

Fig. 9：Different collection positions, form cell for melanocyte and the difference confirmation of quantity occur.(A) skin of chest；(B) Skin of foreskin.

Embodiment

By long-term and in-depth study, inventor is found that a kind of new method of cell culture.With it, energy It is enough serum-free, non-trophoblast environment in turn out skin epidermal cells, and form it into artificial epidermal graft.It is meanwhile described The melanocyte bred jointly with epidermal cell is also included in artificial epidermal graft and forms cell.Pass through the side of safety of the invention and low cost The artificial epidermal graft that method obtains can be used in a variety of applications, for example, the disease characterized by for being lacked by melanocyte in skin is (such as Leucoderma) treatment, the research and development that are carried out for the related medicine of disease of skin or cosmetics screen etc..The present invention is exactly herein Completed on the basis of it was found that.

Specifically, method of the invention be related to from the skin samples of collection separation (such as by protease at Reason) cell mass comprising epidermal cell and melanocyte formation cell, cultivating the cell mass makes it breed and/or pass on to obtain The cell quantity needed so that the culture cell differentiation formed containing epidermal cell and melanocyte formed cell artificial epidermal graft or So that noble cells is combined with suitable carrier material to form the skin graft material suitable for transplanting.

The difference of the method and common method in the prior art of the present invention essentially consists in：In the method for the invention, Both without using the nutrient solution containing risk factor, for example, containing animal derived (for example, humanized) serum, brain derived material, moving The protein of material resource (for example, ox or pig source property), or the nutrient solution of the allergenic agent of animal and plant source, and without using battalion Support sertoli cell (for example, the support cell of small mouse, feeder cells etc.).Methods described is safe, also, obtained Artificial epidermal graft also form cell comprising melanocyte, this is for needing the disease such as (leucoderma that dermatoplasty and/or skin are repaired Wind) and/or symptom (such as beauty related symptoms) for be extremely advantageous.

Term explanation

Term " leucoderma " used herein refers to a kind of common pigmented dermatosis, with skin, mucous membrane and/or hair Depigmentation be characterized.It shows as skin, size on mucous membrane and/or hair caused by melanocyte function lacks With different decolourising property hickie, surrounding skin color is normal or has pigment increase.The skin lesion of leucoderma may alternatively appear in whole body Each position, it is common in and refers to the back of the body, wrist, forearm, face, neck and surrounding genital etc..

Term " segmental vitiligo " is a kind of hypotype of leucoderma, is distributed along nervus cutaneus section.With it is other types of white Purplish or white patches on the skin wind facies ratio, it clinically has the characteristics of morbidity is early, skin lesion is asymmetric, progress is rapid.

Term " generalized vitiligo " is a kind of hypotype of leucoderma, and its skin lesion is the hickie of clear border, rounded, ovum Circular or not shaping.Any position of body, no conscious sympton can be betided, hickie exceedes more than the 50% of the body surface gross area, more Developed by prolonged illness.

Term " melanocyte " used herein namely " melanocyte ", it is be predominantly located at skin and mucous membrane special thin Born of the same parents, it can be synthesized and secrete melanin, and melanin is passed to the keratinocyte of surrounding, and light spoke is resisted to produce The protective effect penetrated." melanocyte stem cell " is primarily present in Hair Follicle Bulge (bulge) part, and it has a variety of differentiation potentials and height Multiplication capacity is spent, melanoblast can be broken up in suitable environmental conditions.Herein, " melanocyte stem cell " and " melanocyte is thin Born of the same parents " are referred to as " melanocyte formation cell ".

Term " epidermal cell " used herein refers mainly to the epithelial cell of animal skin, and it is except with general Outside the feature of chrotoplast, cutin can be also produced, there is very strong keratinization feature, plays a part of protecting skin.

Term " keratinocyte " is a kind of epidermal cell that can synthesize keratoprotein, and it is the main body of epidermis, by Epidermis deep layer starts gradually propagation, differentiation.Known in the art, keratinocyte finally produces keratoprotein, also, its to During horn cell develops, four layers, i.e. basalis, spinous layer, stratum granulosum and cuticula, this shape can be typically roughly divided into Process into two or more layers is herein referred to as " multiple stratification ".In the horn cell of keratinization, nucleus and organelle It is wholly absent, cell also loses physiological function.

Term " skin graft " used herein refers to a kind of flaky material for being used to repair the Shallow tissue defect of body surface organization, and it is logical Often be free of subcutaneus adipose tissue.In general, skin graft can be through transplanting or the skin for needing to repair, fill up being put in the form of dressing At skin, mucous membrane.

Term " nutritional support cell " used herein or " feeder cells " (feeder cells) have referred to this area Cell knowing, cultivating in vitro, it is usually used in aiding in or promoted the growth of target cell and propagation.Conventional nutritional support The example of cell has：The fetus fiber sprout cell of small mouse, 3T3 cells, skin fiber sprout cell, Chinese hamster ovary celI, COS-7 are thin Born of the same parents, Vero cells, MDBK cells, STO cells, BRL cells, SL-10 cells etc..

As used herein, " containing ", " having " or " comprising " include "comprising", " mainly by ... form ", " substantially By ... form " and " by ... form "；" mainly by ... form ", " substantially by ... form " and " by ... form " Belong to the subordinate concept of " containing ", " having " or " comprising ".

Artificial epidermal graft

The present invention provides a kind of artificial epidermal graft, and it is included：Melanocyte living forms cell and epidermal cell.Wherein, it is described Artificial epidermal graft does not include animal derived serum and protein and nutritional support cell.

In the artificial epidermal graft of the present invention, the allergenic agent not comprising brain derived material and animal and plant source.

In an example of the present invention, the artificial epidermal graft, which also includes, to be used to support the melanocyte to form cell and table The support material of chrotoplast.In an instantiation, the support material is fibrin gel.

The manufacture method of artificial epidermal graft

The present invention provides a kind of method of manufacture of intraocular epidermal graft.

In the method for the above-mentioned manufacture of intraocular epidermal graft of the present invention, including skin histology pretreatment：Handled with enzyme solutions To decompose skin histology, to obtain the prepared product for including living cells.

In the method for the invention, the skin histology pretreatment includes enzyme solutions impregnation steps.

In the method for the invention, enzyme solutions dipping is related to：The tissue obtained by tissue sampling is immersed and contains albumen The enzyme solutions of hydrolase.

In the illustrative methods of the present invention, enzyme solutions processing is carried out under 0 DEG C~37 DEG C of environment temperature.One In individual example, enzyme solutions processing is carried out under 4 DEG C~37 DEG C of environment temperature.

In the method for the invention, enzyme solutions processing does not use animal derived proteolytic enzyme.

In the illustrative methods of the present invention, enzyme solutions impregnation uses the proteolytic enzyme of Genetic Recombination. In one example, available proteolytic enzyme includes but is not limited to：Trypsase, TrypLE enzymes, neutral proteinase are (for example, divide Dissipate enzyme), clostridiopetidase A, release enzyme,Enzyme, Gelatinase B, metalloelastase, thermolysin etc., or its Meaning combination.

In the illustrative methods of the present invention, enzyme solutions impregnation can also use commercially available enzyme solutions.The city The enzyme solutions sold are such as, but not limited to：TrypZean (Sigma companies), TrypLE Select (GIBCO companies), TrypLE Express (GIBCO companies), Dispase dispases (Sanko Junyaku Co., Ltd.), clostridiopetidase A I, II, III, IV, V or VI type (and Guang Chun medicines Co., Ltd.) etc., or its any combination.

In the illustrative methods of the present invention, two or more enzyme solutions can be used, sequentially or simultaneously impregnate skin Skin tissue.In an example, disperse enzyme solutions to use before TrypLE solution.In another example, using TrypLE The enzyme solutions dipping of solution does not use scattered enzyme solutions.In another example, first using dispase and TrypLE successively or together When handle tissue, finally add clostridiopetidase A carry out digestion process.

In the illustrative methods of the present invention, proteolysis enzyme concentration in enzyme solutions with used enzyme species It is different and change.In general, concentration of the used proteolytic enzyme in cushioning liquid (such as PBS) be 0.001%~ 0.7%W/v).In an example, the concentration of the proteolytic enzyme can be：0.005%th, 0.01%, 0.05%, 0.1%th, 0.3%, 0.5%, 0.7%, or more state any two numerical value be end points formed scope.In an instantiation In, if using TrypLE in enzyme solutions, TrypLE can dilute certain multiple on the basis of original mother liquid concentration as needed (such as 1~10 times) uses afterwards.

In the illustrative methods of the present invention, in order to avoid cell death, added optionally in enzyme solutions anti- The only reagent of cell death, such as ROCK inhibitor.In an example, this kind of inhibitor includes but is not limited to：Y-27632、 Thiazovivin, Fasudil, GSK429286A, RKI-1447 etc..In a preferred embodiment, 1 μM~20 μM concentration are added The Y-27632 or ASUDIL of scope.

In the illustrative methods of the present invention, enzyme solutions handle the time span for continuing 0.1 hour~24 hours. In one example, enzyme solutions handle the time span for continuing 1 hour~16 hours.

In the illustrative methods of the present invention, enzyme solutions processing step also includes：To the thing by enzyme solutions processing Material is filtered, to remove indigested bulk tissue residue.In an example, filtering is residual using indigested bulk tissue The screen pack for the size that slag can not pass through is carried out.In another example, filtering is carried out using 40~100 μm of filter screen.

In the illustrative methods of the present invention, enzyme solutions processing step also includes：It is separated and recovered from from gained filtrate Cell and tissue fractionlet.In an example, the separation is carried out by centrifugation.

If necessary, undigested residue is reclaimed and with residue enzyme solutions to the processing of its repeated impregnations.The residue is used The proteolytic enzyme that enzyme solutions include is identical with the proteolytic enzyme in enzyme solutions previously used in enzyme solutions processing step It is or different.Then, gained cell and/or tissue fractionlet can be merged with gained cell before and/or tissue fractionlet, are used for Subsequent treatment.

In the method for the invention, the skin histology pretreatment also optionally includes tissue sampling step.The tissue Acquisition step is carried out before enzyme solutions processing step.

In the method for the invention, tissue sampling is related to：Contain the tissue of cell from dermal harvest.

In the illustrative methods of the present invention, the tissue sampling is autologous tissue's collection.

It is not particularly limited for the region of tissue sampling, it can be selected from any position of object whole skin.

In the illustrative methods of the present invention, selecting object neck area above, such as face, the skin of incidence Tissue.Because the content of the stem cell in skin histology according to the literature, more than neck is higher, and stem cell have be divided into The potential of cell needed for various skins.For example, may be selected head hair position skin as tissue sampling region because the area Contain hair follicle stem cells in domain.

The present invention an illustrative methods in, the inguinal skin of selecting object as tissue sampling region because Cell is formed containing a large amount of melanocytes in the region, this is equally beneficial for the artificial epidermal graft to form the present invention.

In the illustrative methods of the present invention, the tissue of collection is skin histology holostrome.In an example, adopt Epidermis and skin corium of the tissue of collection including at least skin.

Be not particularly limited for the age of the object of tissue sampling, preferred acquisition it is tissue-derived autologous in object.

In the method for the invention, the skin histology pretreatment also optionally includes inhibitor solution processing step.

In the method for the invention, inhibitor solution processing is related to：By the mixture by enzyme solutions processing and contain egg The inhibitor solution contact of white hydrolase inhibitor.Its main purpose is：Suppress the albumen used in previous enzyme solutions processing step Easily cause cell death present in hydrolysis enzyme solutions and/or cause the egg having a negative impact to follow-up cultivation and/or differentiation White enzyme (predominantly trypsase) activity.

In the illustrative methods of the present invention, the proteinase inhibitor can be, for example, trypsase presses down Preparation (soybean source, butter bean source property etc.), AKOLINE (Aprotinin) etc..In an example, commercially available egg can also be used White hydrolase inhibitor, such as, but not limited to, TRYPSIN INHIBITOR (soybean) (Wako companies), TRYPSIN INHIBITOR (butter bean) (Sigma companies), restructuring Aprotinin (tobacco) (Sigma companies) etc..

In the method for the invention, the used in amounts of proteinase inhibitor will consider following aspect.On the one hand, it is of the invention Inhibitor solution processing step and cell culture step in do not use serum, hence for the egg with tryptic activity White hydrolase, its complete deactivation need to be made as early as possible.Therefore, the suppression solution containing a large amount of proteinase inhibitors is necessary. On the other hand, if the dosage of proteinase inhibitor is excessive, can have a negative impact (example to follow-up cell propagation Such as, follow-up cell is hindered to breed).

In the illustrative methods of the present invention, in order to meet that above-mentioned both sides requirement (that is, makes trypsase simultaneously Active complete deactivation, while the suppression for subsequent cell propagation is not caused), it is normal that inhibitor solution dosage need to only reach this area The dosage of complete inhibition tryptic activity disclosed in rule technology and general knowledge.For example, inhibitor solution processing step In used proteinase inhibitor dosage be trypsase used in enzyme solutions processing step 2 times~10 times (volumes Than).In an example, the dosage of proteinase inhibitor used in inhibitor solution processing step is enzyme solutions processing 2 times of trypsase used in step~5 times.In an example, proteolysis used in inhibitor solution processing step The dosage of enzyme inhibitor is 2 times~3 times of trypsase used in enzyme solutions processing step.

In the illustrative methods of the present invention, inhibitor solution processing step is within the temperature range of 0 DEG C~37 DEG C Carry out.In an example, inhibitor solution processing step is carried out within the temperature range of 4 DEG C~25 DEG C.

In the illustrative methods of the present invention, inhibitor solution processing continues the time length of 10 minutes~6 hours Degree.In an example, the time span that inhibitor solution processing continues 10 minutes~2 hours.

In the illustrative methods of the present invention, inhibitor solution processing also includes：Impregnated from by inhibitor solution The material of processing removes inhibitor.In an example, the inhibitor is removed by centrifuging.

, can be by indigested tissue after inhibitor solution processing in the illustrative methods of the present invention Residue is recovered and is handled with residue with enzyme solutions, and the residue is walked with the proteolytic enzyme that enzyme solutions include and enzyme solutions processing Proteolytic enzyme used difference in rapid enzyme solutions, then as the cell obtained by the processing of residue enzyme solutions and suppression will be passed through The cell that agent solution handles to obtain is provided commonly for cell culture step.

In the method for the above-mentioned manufacture of intraocular epidermal graft of the present invention, including cell propagation and differentiation：Make gained prepared product In cell propagation, and be divided into melanocyte and form cell and epidermal cell, further make the cell multiple stratification with formed containing Melanocyte forms the artificial epidermal graft of cell.

In the method for the invention, cell propagation with atomization in without using the nutrient solution containing risk factor.It is described Risk factor include non-clinical level allosome or xenogenesis source property, follow-up cell culture may be polluted or influenceed, or Person may cause the component of undesirable immune response (such as allergy, repulsion etc.) in follow-up migration process, but should note Meaning, does not include apyrogenic recombinant protein herein.Exemplary risk factor includes：Animal derived serum, brain derived material, move The protein of material resource, or the allergenic agent of animal and plant source.

Specifically, the animal derived serum generally includes (but not limited to)：Human serum, monkey serum, ape serum, tire Cow's serum, cow's serum, Swine serum, horse serum, donkey serum, chicken serum, Serum of Quail, sheep serum, lowlenthal serum, dog serum, Cat serum, rabbit anteserum, mice serum, guinea pig serum and mouse serum etc..

The brain derived material generally includes (but not limited to)：Pituitary gland extract, brain extract and brain source property carry Take lipid etc..

The animal-based protein matter (such as ox or pig) refers to any protein from animal (such as ox or pig), its Particular example includes but is not limited to animal (such as ox or pig) source：Albumin, siderophillin, collagen, gelatin, enzyme Deng.

The allergenic agent of the animal and plant source generally includes (but not limited to)：Peanut, flour, shell, wheat, ox Material derived from breast and egg etc., and any other material that can be caused allergic reaction.It should be noted that do not include herein without heat The recombinant protein in source.

In the method for the invention, cell culture step is without using nutritional support cell.

In the conventional cell culture of field of tissue engineering technology, majority can use nutritional support cell (for example, feeder cells) To support the growth of target cell and propagation.Nutritional support cell type commonly used in the art includes but is not limited to：Mouse source The fetus fiber sprout cell of property, 3T3 cells, skin fiber sprout cell, Chinese hamster ovary celI, COS-7 cells, Vero cells, MDBK are thin Born of the same parents, STO cells, BRL cells, SL-10 cells, and other general culture strain cells.But in the methods of the invention, do not make With any similar external nutritional support cell.Therefore, nutritional support cell, Ke Yibao are not mixed into the cell finally obtained Demonstrate,prove its security.Likewise it is preferred that it is autogenous cell used by culture, to ensure that the cell of culture has high implantation rate.

In the method for the invention, suitable component can be separately added into be formed respectively in the basic culture solution of the present invention Multiplying culture liquid, Secondary Culture liquid and/or the differentiation nutrient solution of the present invention., can according to the different cultivation stage demands of the present invention Using different basic culture solutions, and appropriate component is added on this basis.Had no particular limits for basic culture solution, only Vitamin, mineral matter, amino acid necessary to the nutrient solution for needing to select contains cells survival, and do not contain any risk factor .

In an example, available basic culture solution is such as, but not limited to：MCDB151 nutrient solutions (Sigma companies), MCDB153 nutrient solutions (Sigma companies), MCDB154 nutrient solutions (Sigma companies), keratinocyte basic culture solution (Keratinocyte Basal Medium, Stemline companies), the keratinocyte serum-free medium of determinant (Defined Keratinocyte-SFM, Invitrogen companies), DMEM/F-12 nutrient solutions (Invitrogen companies) or it It is appropriately combined etc..

In the method for the invention, cell propagation Multiplying culture liquid used into selected basic culture solution by adding It is adapted to and/or promotes the additive of cell culture and propagation to prepare.It is adapted to the additive of cell culture to include, for example, cell Enhancer of proliferation, the protective agent (such as lipid additive, free radical scavenger) for preventing or reducing cell death, and its any group Close.

In an example, there is cell growth promoter in Multiplying culture liquid.In an instantiation, the cell The one or more that enhancer of proliferation includes but is not limited in following material：KGF (keratinocyte growth factor, such as FGF7), insulin, people's Holo-transferrin, selenite, monoethanolamine, lipid, vitamin C, and vitamin C inducer.On State in material, KGF addition concentration is preferably in the range of 0.1~100ng/L, and other additives in addition to KGF add Add concentration preferably in the range of 1~2000nM/L.

In an example, there is lipid additive in Multiplying culture liquid.In an instantiation, the lipid addition The one or more that agent includes but is not limited in following material：Arachidonic acid, cholesterol, DL- α-D-α-tocopherol acetate, sub- oil Acid, leukotrienes, myristic acid, oleic acid, palmitoleic acid, Retinol Palmitate, stearic acid, Tween-80, Pluronic F68 (can Purchased from Sigma companies, Invitrogen companies etc.), and its other lipid additives commonly used in the art.The lipid additive Concentration preferably in 1/10000~1/1000 (volume ratio, based on nutrient solution final volume).

In an example, there is free radical scavenger in Multiplying culture liquid.In an instantiation, the free radical The one or more that scavenger includes but is not limited in following material：Reduced glutathione, vitamin E, vitamin E induction Thing, catalase, superoxide dismutase (SOD) etc..Wherein, the addition concentration of the free radical scavenger preferably 1~ In the range of 1000nM/L.

In example of the present invention, the Multiplying culture liquid used in Multiplying culture is on the basis of basic culture solution Another addition, for example, insulin, people's Holo-transferrin, CD lipids concentrate, vitamin, keratinocyte growth factor, epidermis are thin The intracellular growth factor, fibroblast growth factor, ROCK inhibitor, corticotropin, antibiotic etc..

In an example, the one or more in following material can be also added into Multiplying culture liquid：Epidermal growth factor Sub (EGF), fibroblast growth factor (FGF2), HGF (HGF), insulin-like growth factor-i (IGF- 1), stem cell factor (SCF), Wnt-1, Wnt-3a, corticotropin (ACTH), endothelin -1 (ET-1), active form Vitamine D3, prostaglandin E2 (PGE2), the method as protectant Rho associated kinases (ROCK) inhibitor of cell death relax ground (Fasudi), Y-27632 etc..Above-mentioned additive is according to the addition scope use of routine, preferably 1nM/L~0.1mM/ L。

In the illustrative methods of the present invention, when cell starts culture, by cell with appropriate density, such as 1 × 10^2-6Individual cell/cm²Or 1 × 10^2-6Individual cell/mL density, it is inoculated in the Multiplying culture liquid in cell culture container, so Be placed on afterwards 37 DEG C CO2gas incubator or automatic culture apparatus in cultivated.

In the illustrative methods of the present invention, the cell culture container includes but is not limited to, culture dish, culture Bottle, microplate etc..In an example, in order to improve the tack between cell and culture vessel, collagen, gel, L-type can be used It is poly-D-lysine, D types poly-D-lysine, laminin, fibronectin, how poly- L-Orn, fibrin, transparent One or more cell supports in matter acid etc. are coated pretreatment to culture vessel with base material.In another example, Using the special culture vessel of suitable serum-free medium.

In the illustrative methods of the present invention, cell culture of the invention is using domestication training method.It should be understood that often The cell culture of rule typically just needed the whole nutrient solution of replacing every several days, and so-called " domestication culture " is then to change training Partly or entirely to retain when nutrient solution can make the composition of self growth of cell in original fluid.Specifically, domestication culture Represent when changing nutrient solution, be not that old nutrient solution is all removed and is replaced with new nutrient solution, but member-retaining portion or All old nutrient solutions, and new nutrient solution is added on this basis.It is right if there is the old nutrient solution in part to be discarded in the process The part old nutrient solution to be discarded extract.Centrifuged for example, old nutrient solution to be discarded is put into centrifuge tube, in removing Clear liquid, into centrifuge tube, pellet (including residual cell) adds new nutrient solution, makes its resuspension, is then added back again In culture vessel originally.In an example, the old nutrient solution of culture material volume 5~50% is retained when changing nutrient solution. In another example, the old nutrient solution of culture material volume 10~30% is retained.

In the illustrative methods of the present invention, cell of the invention propagation includes primary Multiplying culture.In a reality In example, the primary Multiplying culture is carried out using Multiplying culture liquid.

In example of the present invention, the base of Multiplying culture liquid used in primary Multiplying culture in basic culture solution Separately added on plinth, for example, insulin, people's Holo-transferrin, CD lipids concentrate, vitamin, keratinocyte growth factor, table Skin cell growth factor, fibroblast growth factor, ROCK inhibitor, corticotropin, antibiotic etc..

In an example, since cell culture, last the time progress of about 3~10 days and cultivate fluid exchange for the first time. In another example, since cell culture, last the time progress of about 3~8 days and cultivate fluid exchange for the first time.At another In example, since cell culture, last the time progress of about 3~6 days and cultivate fluid exchange for the first time.In an instantiation In, the first time culture fluid exchange does not remove old nutrient solution, but only adds fresh medium.

In an example, primary Multiplying culture of the invention continues 10~16 days.In another example, it is of the invention Primary Multiplying culture continues 10~14 days.In another example, primary Multiplying culture of the invention continues 10~12 days.

In the illustrative methods of the present invention, cell of the invention propagation also includes passage, and the cell passes In generation, can be carried out when the cell of primary Multiplying culture reaches fusion.In an instantiation, to the passage of Multiplying culture Less than 5 generations.In an instantiation, is less than to the passage of Multiplying culture 4 generations.In an instantiation, to propagation The passage of culture was less than for 3 generations.In a specific example, is less than to the passage of Multiplying culture 2 generations.It is specific at one In example, is no more than to the passage of Multiplying culture 1 generation.

In the illustrative methods of the present invention, cell of the invention propagation also includes Multiplying culture after passage.One In individual example, Multiplying culture uses Secondary Culture liquid after passage.In an instantiation, the Secondary Culture liquid is in foregoing increasing Grow another addition stem cell medium exchange SCF and Wnt-3a on the basis of nutrient solution.

In an example, Multiplying culture continues 3~10 days after the passage.In another example, after the passage Multiplying culture continues 5~7 days.

In the illustrative methods of the present invention, cell differentiation of the invention is carried out using differentiation nutrient solution.At one In example, differentiation nutrient solution configures on the basis of basic culture solution.

In an example, insulin is included in the differentiation nutrient solution.In an example, the pancreas broken up in nutrient solution Island cellulose content is 1~10mg/L.In another example, the insulin content broken up in nutrient solution is 3~8mg/L.Another In individual example, the insulin content broken up in nutrient solution is 5~7mg/L.

In an example, corticotropin (ACTH) is included in the differentiation nutrient solution.In an example, It is 0.5~3 μM to break up ACTH contents in nutrient solution.In another example, it is 0.8~2 μM to break up ACTH contents in nutrient solution. In another example, the ACTH contents broken up in nutrient solution are 1~1.5 μM.

In an example, Holo-transferrin is included in the differentiation nutrient solution.In an example, nutrient solution is broken up In Holo-transferrin content be 1~10mg/L.In another example, the Holo-transferrin content broken up in nutrient solution For 3~8mg/L.In another example, it is 5~7mg/L to break up Holo-transferrin content in nutrient solution.

In an example, vitamin D is included in the differentiation nutrient solution₃.In an example, break up in nutrient solution Vitamin D₃Content is 5 × 10^-12M~5 × 10^-11M.In another example, the vitamin D broken up in nutrient solution₃Content is 7 ×10^-12M~3 × 10^-11M.The vitamin D broken up in nutrient solution₃Content is 9 × 10^-12M~2 × 10^-11M.In an example In, vitamin D used₃It is 1 α, 25- dihydroxyvitamin Ds₃。

In an example, in nutrient solution used in cell differentiation procedure without keratinocyte growth factor (KGF) and Epithelical cell growth factor (EGF).

In the illustrative methods of the present invention, the calcium content in the differentiation nutrient solution is higher than the Multiplying culture liquid In calcium content.In an instantiation, the calcium content in Multiplying culture liquid is 0.05~0.15mM.It is specific real at another In example, the calcium content in Multiplying culture liquid is 0.08~0.12mM.In another instantiation, the calcium in Multiplying culture liquid contains Measure as 0.09~0.1mM.In an instantiation, the calcium content broken up in nutrient solution is 1~2mM.It is specific real at another In example, the calcium content broken up in nutrient solution is 1.5~1.8mM.

In an example, the cell differentiation is carried out using multistage differentiation mode.Specifically, described " multistage differentiation side Formula " refers to, and in atomization, will progressively break up nutrient solution and be improved relative to the accounting of Multiplying culture liquid, with thin needed for realization Born of the same parents break up.

In an example, the volume ratio of the differentiation nutrient solution and the Multiplying culture liquid that are added in cell differentiation procedure is from 1:2 It is gradually transition to all differentiation nutrient solutions.In another example, the differentiation nutrient solution added in cell differentiation procedure is with increasing The volume ratio of nutrient solution is grown from 1:1.5 are gradually transition to all differentiation nutrient solutions.In another example, cell differentiation procedure The volume ratio of differentiation nutrient solution and the Multiplying culture liquid of middle addition is from 1:1 is gradually transition to all differentiation nutrient solutions.

In an example, differentiation nutrient solution steps up relative to the volume score four-stage of Multiplying culture liquid. In one instantiation, the volume ratio of differentiation nutrient solution and Multiplying culture liquid with：0.8:1~1.2:1、 1.5:1~2.5:1、 2.8:1~4:1st, all differentiation nutrient solutions, divide four-stage to step up.In another instantiation, break up nutrient solution With the volume ratio of Multiplying culture liquid with：1:1、2:1、3:1st, all differentiation nutrient solutions, divide four-stage to step up.In this hair In a bright illustrative methods, above-mentioned each stage continues 2~4 days.

In the method for the invention, cell propagation also includes making the cell multiple stratification be formed containing melanocyte shape with differentiation Into the artificial epidermal graft of cell.In an example, cell mass can form laminated structure, preferably multilayer chip structure, wherein wrapping Cell is formed containing epidermal cell and melanocyte.The tablet is collected, is the artificial epidermal graft of the present invention.Alternatively, by the people Work epidermal graft is transferred on medical carrier, in case using.

In the above-mentioned method for preparing artificial epidermal graft of the present invention, for the further consideration for improving implantation rate, may be used also Including adaptation step.In the adaptation step, can make to obtain in cell culture step cell or artificial epidermal graft fragment embed into Enter in suitable carrier (such as fibrin), obtain artificial epidermal graft (such as the fibrin gel skin containing noble cells Piece).

In the illustrative examples of the present invention, the artificial epidermal graft that will be obtained by the inventive method culture, again Peeled off, and be embedded into fibrin by ferment treatment, fibrocyte skin graft is made.

In the illustrative examples of the present invention, the artificial epidermal graft that will be obtained by the inventive method culture passes through Ferment treatment is peeled off, and is transferred on appropriate holder to support the skin graft in preservation and/or transport.

The application of artificial epidermal graft

The invention further relates to the application that obtains artificial epidermal graft is prepared by the method for the present invention.

In the illustrative examples of the present invention, it is related to the artificial epidermal graft prepared by the inventive method and is preparing For treating the application using external member of the disease characterized by melanocyte in skin lacks.In an example, it is described with skin The disease that skin melanocyte missing is characterized is leucoderma.In an instantiation, the leucoderma is segmental vitiligo. In another instantiation, the leucoderma is whole body type leucoderma.

In an example, it is described to be applied in the following way using external member：First the affected part epidermis of patient is polished, Then this artificial epidermal graft is transplanted to the affected part.

In another illustrative examples of the present invention, it is related to the artificial epidermal graft prepared by the inventive method in pin Application in the research and development screening carried out to the related medicine of disease of skin or cosmetics.

In an example, the test compound or composition of the medicine or cosmetics related for disease of skin are applied To the artificial epidermal graft prepared by the inventive method, and by judging that can the compound or composition promote in skin graft Epidermal cell or melanocyte form the propagation (or reduction) of cell, to carry out research and development screening to the test compound or composition.

All number ranges provided herein be intended to clearly include falling all numerical value between endpoints of ranges and it Between number range.The feature that the feature or embodiment that can be mentioned to the present invention are mentioned is combined.This specification is taken off All features shown can be used in combination with any combinations thing form, each feature disclosed in specification, can provide phase with any The alternative characteristics substitution of same, impartial or similar purpose.Therefore except there is special instruction, disclosed feature is only impartial or similar The general example of feature.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.Those skilled in the art can make appropriate modification to the present invention, change, these modifications It is within the scope of the present invention with variation.

Unless otherwise indicated, otherwise percentage and number are calculated by weight.Unless otherwise defined, it is all used in text Specialty is identical with meaning known to one skilled in the art with scientific words.It is in addition, any similar or equal to described content Deng method and material all can be applied in the inventive method.Preferable implementation described in text only presents a demonstration it with material With.

The tissue sampling of embodiment 1.

Operate and carried out in the state of integral asepsis below.

Tissue-derived is 62 years old women.When obtaining its plastic aesthetic surgery from the discarded skin histology of face's excision Area about 2.0cm²Holostrome, be immersed in iodine disinfectant and sterilize 30 seconds, then (train biotechnology stock in Shanghai source with PBS Part Co., Ltd) solution cleans 3 times.It is positioned over after wash clean using aperture<In 70% alcoholic solution of 1 μm of filter screen filtration, Continue 30 seconds, sterilize again.Then using PBS solution (pH 7.4) separately cleaning 3 times.Tissue sterilizing operating scissors after wash clean Cut with tweezers, only retain the block containing hair follicle, further remove subcutaneous and dermal connective tissue, then shredded into operating scissors About 1mm²The fragment of size.

The enzyme solutions impregnation of embodiment 2.

(i) enzyme solutions dipping engineering 1

Prepare comprising 10 μM ROCK inhibitor (Y-27632, Wako Pure Chemical Industries, Ltd.) and 22U/ml disperse The PBS solution of enzyme (Diapase, GIBCO), the enzyme solutions of engineering 1 are made.It will as described above gather and pretreated tissue immerses It is previously cooled in 4 DEG C of 5mL enzyme solutions of engineering 1 produced above, is stood overnight at 4 DEG C, be acquired the ferment treatment of tissue.

(ii) enzyme solutions dipping engineering 2

By TrypLE Express enzyme solutions (1 ×) (being purchased from Thermo Fisher Scientific companies) with PBS5 times Dilution, and Y-27632 is added with 10 μM of concentration, the enzyme solutions of engineering 2 are made.By the ferment treatment blend that engineering 1 obtains from The heart, abandoning supernatant, immediately adding the enzyme solutions of 5mL engineerings 2 is resuspended tissue precipitation, and 37 DEG C are placed 20 minutes.

(iii) enzyme solutions dipping engineering 3

Type i collagen enzyme (Collagense Type I, GIBCO) solution (1400U/mL) is prepared in DMEM nutrient solutions, and Add antiseptic 1 μ g/ml Erythromycin E rythromycinAbbot companies) and 10U/ml Gentasin (the glad medicine company of occasion), make Obtain 3 antiseptics of engineering-enzyme solutions.The ferment treatment blend that above-mentioned enzyme solutions dipping engineering 2 obtains is centrifuged, abandoning supernatant, Immediately add 20mL 3 antiseptics of engineering-enzyme solutions (37 DEG C warm), and with magnetic stirring apparatus (500rpm) 37 DEG C stir 1.5 hours, obtain homogeneous blend.

The inhibitor solution impregnation of embodiment 3.

Inhibitor solution：In PBS with 2mg/mL allotment trypsin inhibitor (Trypsin Inhibitor (soybean), SIGMA) solution.

Homogeneous blend obtained by enzyme solutions impregnation is filtered through using 100 μm of nylon leaching net, retains filtrate, clearly Except non-slaking residue.Ferment treatment container previously used is rinsed with 40mL PBS, and further rinses screen pack, gained is rinsed Liquid and above-mentioned filtrate merge, and centrifugation, obtain cell precipitation.

Gained cell precipitation is further rinsed with 40mL PBS, then centrifugation.Then obtained cell precipitation is immersed In the inhibitor solution that 5mL is prepared in advance, it is resuspended, and room temperature 10 minutes.

The cell culture of embodiment 4.

(i) primary Multiplying culture

It is the specific formula of Multiplying culture liquid below：By the MCDB153 graininess nutrient solution of basic culture, (Sigma is public Department) insert in 1L distilled water for injection and dissolve, carry out filtration sterilization after adding 1.8g sodium acid carbonate.Then, 33.3mL is added Liquid medium DMEM (Dulbecco ' s Modified Eagle Medium, Sigma companies).Afterwards, addition is by going out The following material of bacterium processing is to shown concentration：

Rh- insulin (Version16 companies) (5mg/L), people's Holo-transferrin (Holo-human Transferrin, Sigma company) (5mg/L), CD lipids concentrate (GIBCO companies) (1mL/L), L-AA -2- phosphorus Acid esters (Sigma companies) (50mg/L), rh KGF/FGF7 (PROSPEC companies) (10 μ g/L), rh EGF (R＆D system house) (20 μ g/L), rh FGF2 (PeproTech companies) (10 μ g/L), Y-27632 (Wako companies) (5mg/L), B- 27Supplement XenoFree CTS (GIBCO companies) (10mL/L), ACTH (1-24) (people) (Wako companies) (1nM), 1 α, 25- dihydroxy vitamin d3 (Sigma companies) (10^-11M), erythromycin (Erythromycin, Wako company) (0.5mg/L), Gentamicin sulphate (Gentamicin Sulfate, 10,000U/L, the glad medicine company of occasion), nystatin (Nystatin, 1 μ g/L, LKT-Lab)。

6 orifice plates (COSTAR 3335 is used during Initial culture：Corning CeLLBIND surfaces).Centrifugation removes above-mentioned suppression Formulation soln, 12mL nutrient solutions are added to precipitation, cell is resuspended.The nutrient solution contains 10 μM of Y-27632.Then, with Cell is seeded in the hole of 6 orifice plates by 2mL cell suspensions/hole.After inoculation 2 days, per hole, addition includes 10 μM of Y-27632 2ml Nutrient solution.Cultivate the 4th day, add the 2mL nutrient solution for including 10 μM of Y-27632 to every hole again.Cultivate the 6th day, each hole is protected 1mL nutrient solutions are stayed, remaining old nutrient solution is suctioned out and centrifuged, after supernatant discarding, adding 18mL fresh medium makes cell weight It is outstanding.Gained re-suspension liquid is inoculated with 3mL/ holes.Liquid is changed in 3 days afterwards according to method same as described above.When cultivating by the 12nd day Merge, carry out following Secondary Culture engineering.

(ii) passage

With final concentration, 10 μM are added to Y-27632 in the nutrient solution for the cell for having reached fusion, and 37 DEG C stand 30 minutes. Then by broth out, 2%EDTA/PBS solution is added with 2mL/ holes, 37 DEG C stand 10 minutes.Then, added with 0.5mL/ holes Add TrypLE Express enzyme solutions (1 ×), 37 DEG C stand 5 minutes.Confirm that cell departs from from bottom hole portion by just putting microscope Afterwards, cell is gathered using pipettor.The cell of collection is collected with centrifuge tube, with 20mL PBS 2 times.After cleaning, it will obtain Cell immerse 5mL inhibitor solution (trypsin inhibitor (soybean):2mg/ml PBS) in, room temperature 10 minutes with On.

(iii) Multiplying culture after passing on

After centrifugation removes inhibitor solution, cell is resuspended in after being passed on added to 40mL in Multiplying culture liquid.Biography used It is another following two components of addition on the basis of primary Multiplying culture liquid for rear Multiplying culture liquid：Rh SCF (GIBCO companies) (2.5 μ g/L), rh Wnt-3a (StemRD companies) (2.5 μ g/L).Then ten are inoculated into the volume of 4mL/ wares Tissue Culture Dish (the Tissue Culture Dish of Corning 3295：60mm×15mm；CellBIND surfaces) in.37 DEG C are placed 3 days, are retained 1mL/ holes nutrient solution, old nutrient solution remove supernatant through centrifugation, cell are resuspended

In the fresh Secondary Culture liquid in 4mL/ holes, re-suspension liquid is inoculated, is further cultured for.Cultivate to reach for the 7th day and melt Close, carry out membrane surface culture (specifically described in embodiment 5).Retain two culture dishes and carry out differentiation culture.

(iv) culturing engineering 1 is broken up

Differentiation culture formula of liquid：1 μM of addition ACTH (Wako companies), insulin in DMEM nutrient solutions (Sigma companies) (Version16 companies) (5mg/L), people's Holo-transferrin (Sigma companies) (5mg/L), 1 α, 25- dihydroxy vitamin d3s (Sigma companies) (10^-11M), modulation forms.

Multiplying culture liquid (-)：According to being prepared above for identical mode described in primary Multiplying culture liquid, difference It is without KGF and EGF (i.e. without rh KGF/FGF7 (PROSPEC companies) (10 μ g/L), rh EGF (R＆D previously used System house) (20 μ g/L)).

The old nutrient solution in the Tissue Culture Dish for reaching fusion is removed, nutrient solution will be broken up:Multiplying culture liquid (-) is according to 1: 1 volume ratio is added in culture dish with always adding volume 4mL/ wares, starts differentiation culture.It was observed that the 1st day cell of differentiation goes out Existing gap, the gap for breaking up the 2nd day proliferative cell disappear.

(v) culturing engineering 2 is broken up

Break up the 3rd day, confirm that ware bottom space between cells disappears.After removing old nutrient solution, nutrient solution will be broken up:Multiplying culture Liquid (-) is according to 2:1 volume ratio is added in culture dish with always adding volume 4mL/ wares, is broken up.

(vi) culturing engineering 3 is broken up

Break up the 5th day, remove old nutrient solution.Nutrient solution will be broken up:Multiplying culture liquid (-) is according to 3:1 volume ratio is with total Add volume 4mL/ wares to be added in culture dish, then broken up.

(vii) culturing engineering 4 is broken up

Break up the 7th day, remove old nutrient solution.Nutrient solution will be broken up:Multiplying culture liquid (-) is according to all differentiation nutrient solutions Volume ratio always to add volume 4mL/ wares added in culture dish, finally broken up.Break up the 8th day and terminate differentiation.

1 α when the membrane surface culture engineering of embodiment 5. and differentiation, the research of 25- dihydroxy vitamin d3 concentration

During Secondary Culture, using the Secondary Culture engineering identical method with art heretofore taught from two culture dishes merged Cell is obtained, is seeded on two membrane surface culture filter screens.The differentiation culturing engineering being described above.Wherein, differentiation culture work Journey 1 is cultivated under interface；Cultivated since being broken up culturing engineering 2 at interface；When breaking up the culture of culturing engineering 4 Between extended and (be extended for from-the 8 day the 7th day-the 11 day the 7th day), carried out the differentiation culturing engineering of 11 days altogether, entered Row comparative studies.

As a result show, when membrane surface culture breaks up, by 1 α, 25- dihydroxy vitamin d3 concentration values are set in 10^-9M also may be used Skin graft is turned out, but 10^-11In the case of M, ideal thicker artificial epidermal graft can be turned out (referring to Fig. 1, Fig. 1 Skin graft section is shown, the figure multiplication factor of left and right two is identical).

6. artificial epidermal graft of embodiment is taken

The nutrient solution for finally breaking up the cell to finish is removed, remaining cell skin graft material is then immersed into dispase (Dispase) in solution (4mL/ wares, GIBCO), 37 DEG C are placed 1 hour.When cell skin graft material is in peelable state, make Skin shape thing is peeled off with sterilized tweezers and nylon membrane.Then, transfer them on hospital gauze, you can as transplanting table Skin is transplanted.

The collection of noble cells is resuspended in embodiment 7.

To remaining in the ware (2mL/ wares) of noble cells addition Y-27632 up to 10 μM of concentration, 37 DEG C stand 30 minutes with On.Old nutrient solution is removed, then adds TrypLE Express enzyme solutions (1 ×) (0.5mL/ holes), 37 DEG C stand 10 minutes.So After continue add TrypLE Express enzyme solutions (1 ×) (0.5mL/ holes), 37 DEG C stand 10 minutes.Then, it is aobvious by just putting After micro mirror confirms that cell can depart from from bottom, cell is gathered using pipettor.The cell collected is collected into centrifuge tube, used 20mL PBS 2 times.After cleaning, by (trypsin inhibitor is (big in the cell immersion 5mL of acquisition inhibitor solution Beans):2mg/ml PBS), room temperature more than 10 minutes.

Then, cell can be obtained by centrifuging, and the cell of acquisition is resuspended in PBS solution, as noble cells Suspension is standby.

The fibrin gel preformulation of embodiment 8.

Using medical Fibringluraas-Fibrin Sealant (people) kit (Shanghai Lay scholar), following two are prepared Kind liquid.

Fibrinogen and water for injection are placed in into 37 DEG C to warm 15 minutes, then, 2mL water for injection is added to fibre In fibrillarin original, make its dissolving.37 DEG C stand more than 20 minutes, molten as fibrinogen untill confirming to be completely dissolved Liquid is standby.

By factor and CaCl₂Solution is placed 15 minutes at 37 DEG C, then, 2mL water for injection is added into blood coagulation In proenzyme, and blown and beaten with pipettor is repeatedly soft until dissolving.Then, a part for the liquid is taken, 500 times is diluted with PBS, makees It is standby for diluted prothrombin solution.

It is prepared by fibrin gel of the embodiment 9. containing cell

By the noble cells suspension cell of acquisition described above and collection, (cell for being resuspended in PBS as described in Example 7 hangs Liquid) it is inoculated in(1 × 10 in culture dish³Individual cell/cm²), to eachCell in ware adds 1920 μ L's Diluted prothrombin solution (preparing as described in Example 8), makes itself and mixing with cells in ware.Then 120 μ L fibers are added Proteinogen is made 2mL solution (preparing as described in Example 8) and is gently added in all wares.Ware is placed on 4 DEG C of refrigerated overnights. Second day, the fibrin gel containing cell is peeled off from ware using tweezers and nylon membrane, it is subsequently used in transplanting.Fig. 2 Photo in show be placed in the back of the hand photographs fromCulture dish in the fibrin containing cell that takes out coagulate Glue.

Embodiment 10. forms cell to the melanocyte in cell skin graft and carries out DOPA dyeing confirmations

The melanocyte formation cell confirmed in cell skin graft (such as prepared by embodiment 6) is dyed by DOPA to whether there is.

First, by 3.3g NaH₂PO₄·2H₂O and 28.7g Na₂HPO₄·12H₂O is added in 1L distilled water, molten DOPA buffer solutions are made after solution.Then, 0.1g DOPA (3,4- dihydroxy-L-phenylalanine, Sigma companies) is added to In 100mL DOPA buffer solutions, and stirring in 30 minutes is carried out until being completely dissolved using agitator.The cell obtained will be peeled off Skin graft sample is immersed in the DOPA solution, and 37 DEG C stand 2 hours.Next using DOPA buffer solution for cleaning samples 3 times, then Sample is immersed in DOPA buffer solutions again, 50 DEG C stand 2 hours.After the completion of, confirm that melanocyte is formed using micro- sem observation The situation of cell.

The research 1 of the differentiation method of embodiment 11.

Two groups will be divided into by the cell through amplification cultivation being described above：First group is multistage differentiation group (G), and it is according to reality Apply the differentiation culturing engineering 1-4 of example 4 order culture；Second group is single-order differentiation group (S), and it is only with differentiation culturing engineering 4 In nutrient solution culture.Then, by observing the appearance number of DOPA staining positive cells come the above-mentioned training method pair of multilevel iudge Whether have an impact in the formation that melanocyte forms cell.

As a result reference can be made to Fig. 3.Fig. 3 shows that multistage differentiation, VD concentration and passage number form cell number for melanin Influence.Wherein, the DOPA positive cells in multistage differentiation group are pressed far more than the DOPA positive cells in single-order differentiation group, explanation The melanocyte quantity that the order culture of multistage differentiation (differentiation culturing engineering 1-4) described in embodiment 4 is obtained substantially occupies It is more.Also, when breaking up, either 10^-11M or 10^-91 α of M concentration, 25 (OH)₂VD₃Melanocyte quantity is not produced aobvious Writing influences.Multiple stratification occurs for the keratinocyte of only multistage differentiation.And P1 is more than P2 generations for the melanocyte quantity of culture Culture.

In addition, further acknowledge in multistage differentiation group, the keratinocyte multiple stratification observed in the cell of passage 1 generation (P1) Significantly more than pass on 2 generations (P2) cell.And do not occur keratinocyte multiple stratification in single-order differentiation group.

The research 2 of the differentiation method of embodiment 12.

Use the cell by the culture being described above to (i) the step of completing embodiment 4.With only with as described above Subculture 1 generation cell is used as " control sample "；Using addition rh SCF (GIBCO companies) (2.5 μ g/L) and rh in culture The cell of Wnt-3a (StemRD companies) (2.5 μ g/L) both cell factors is used as " cell factor addition sample "；To add Another addition 10 outside above two cell factor is added^-11The cell of M vitamine D3 (VD3) is used as " cell factor+vitamin D (VD) sample is added ".During cell fusion, differentiation culture is carried out using with multistage differentiation identical condition described above.Differentiation training After supporting, the maturity for being formed cell to the melanocyte of each sample using DOPA dyeing and HE dyeing is compared.

As a result such as Fig. 4 is shown, compared to control sample (A), melanocyte forms the maturation of cell in cell factor addition sample (B) Du Genggao, and the result in cell factor+VD addition samples (C) is more preferable than cell factor addition sample.In addition, display control sample Prematurity melanocyte in product forms orbicule, it is possible thereby to judge, when forming ripe melanocyte, to passage 1 The cell addition cell factor and VD in generation are effectively.

The research 3 of the differentiation method of embodiment 13.

Broken up using by cell of the culture being described above to (i) the step of completing embodiment 4.With only with culture The passage 1 generation cell of liquid culture is used as " control sample "；To add rh SCF (GIBCO companies) (2.5 μ g/L) and rh in culture The cell of Wnt-3a (StemRD companies) (2.5 μ g/L) both cell factors is used as " cell factor addition sample "；To add Add 10^-11M VD3 cell is as " VD adds sample ".During cell fusion, using with multistage differentiation identical described above Condition carries out differentiation culture.After differentiation culture, the melanocyte of each sample is dyed using DOPA, under stereomicroscope Take pictures, and observe the frequency of occurrences of dyed melanocyte.

As a result as shown in figure 5, being ordered as according to the number of melanocyte quantity：Cell factor addition sample ＞ VD addition samples Product ＞ control samples, this shows to add cell factor and the VD3 frequency of occurrences for being advantageous to improve melanocyte.

The research 4 (ACTH effect) of the differentiation method of embodiment 14.

Using by the cell that culture is described above.During differentiation, cell is divided into 1 μM of ACTH addition sample and no added sample Product (control sample), the cell in the differentiation stage of engineering 1 is observed using inverted microscope.

As a result showing, the cell size of ACTH addition samples is smaller than the cell of no added sample by more than 1/4 (referring to Fig. 6), This shows that ACTH maintains the basal cell state of keratinocyte.

The research 5 (influence of insulin, Holo-transferrin to differentiation) of the differentiation method of embodiment 15.

Use the cell by the culture being described above to (i) the step of completing embodiment 4.During differentiation, in 1 μM of addition It is common to add 5mg/L insulin (Version16 companies) and 5mg/L people's Holo-transferrin (Sigma public affairs on the basis of ACTH Department) into differentiation nutrient solution.

Test result indicates that the result (Fig. 7) that the addition of above-mentioned substance obtains (does not show with the result obtained by application serum Show) it is identical.Therefore, it can be realized using the cell culture method of the present invention and keratinocyte multilayer is formed by free serum culture The effect of change.

The research 6 (influence of the cell factor to tyrosinase activity) of the differentiation method of embodiment 16.

Skin obtained from people's chest and foreskin region is handled according to method as described above, separates and obtains melanocyte and formed carefully Born of the same parents.Research is from following these three cell factors rh SCF (GIBCO companies) (10 μ g/L), rh Wnt-3a (StemRD companies) The influence of (10 μ g/L), endothelin -1 (Wako companies) (10 μ g/L) to tyrosinase activity.

As a result such as Fig. 8 ((A)：Wnt+SCF；(B)：SCF+ Endothelins) shown in, show to add the better of Wnt+SCF. In addition, DOPA Coloration experiments are shown, the excellent effect degree of cell factor addition is ordered as：Wnt+SCF>Individually addition SCF, interior Skin element or Wnt>Wnt+ Endothelins>SCF+ Endothelins.

Embodiment 17. compares the quantity of the melanocyte of the skin histology of different parts collection

The skin from people's foreskin or chest is handled according to the method recorded above, cultivates, break up.Pass through DOPA The quantity variance of Study on dyeing melanocyte.

As a result show, the melanocyte from skin of foreskin is significantly more than the melanocyte from skin of chest.Therefore, may be used To judge that from the result that the skin of body different parts collection is cultivated be discrepant.

All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Bibliography (non-patent literature)：

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[2]Jasper G.van den Boorn1,4,Debby Konijnenberg1,4,Trees A.M.Dellemijn2,J.P.Wietze van der Veen1,Jan D.Bos1,Cornelis J.M.Melief3, Florry A.Vyth-Dreese2,5and Rosalie M.Luiten1,5；Autoimmune Destruction of Skin Melanocytes by Perilesional T Cells from Vitiligo Patients.J Invest Dermatol. (2009)129,2220–2232

[3]Bondanza S1,Maurelli R,Paterna P,Migliore E,Giacomo FD,Primavera G,Paionni E,Dellambra E,Guerra L.；Keratinocyte cultures from involved skin in vitiligo patients show an impaired in vitro behaviour.Pigment Cell Res.2007Aug；20(4):288-300

[4]Moellmann G,Klein-Angerer S,Scollay DA,Nordlund JJ,Lerner AB Extracellular granular material and degeneration of keratinocytes in the normally pigmented epidermis of patients with vitiligo.J Invest Dermatol (1982)79:321–30

[5]Bhawan J,Bhutani LK(1983)Keratinocyte damage in vitiligo.J Cutan Pathol 10:207–12

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Claims

1. a kind of artificial epidermal graft, it is included：Melanocyte living forms cell and epidermal cell, wherein, the artificial epidermal graft is not Include animal derived serum and protein and nutritional support cell.

2. artificial epidermal graft as claimed in claim 1, it is characterised in that the artificial epidermal graft do not include brain derived material and The allergenic agent of animal and plant source；In another embodiment, the artificial epidermal graft, which also includes, is used to support the melanocyte Form the support material of cell and epidermal cell, such as fibrin gel.

3. a kind of method for preparing artificial epidermal graft, it comprises the following steps：

(ii) cell propagation and differentiation：Make gained prepared product in cell propagation, and be divided into melanocyte formed cell and epidermis it is thin Born of the same parents, further make the cell multiple stratification to form the artificial epidermal graft containing melanocyte formation cell；

4. method as claimed in claim 3, it is characterised in that methods described also includes：Skin histology is being decomposed with enzyme solutions Afterwards, tryptic activity is completely inhibited using enzyme inhibitor, then carries out cell propagation and differentiation；In another embodiment In, methods described is additionally included in cell propagation and carries out adaptation step after differentiation, the adaptation step by the cell of differentiation or The cell skin graft embedding of fragmentation enters in suitable carrier, such as fibrin, obtains the carrier containing noble cells.

5. method as claimed in claim 3, it is characterised in that the enzyme solutions include proteolytic enzyme, the proteolysis Enzyme includes the one or more in neutral proteinase (for example, dispase), trypsase, clostridiopetidase A.

6. method as claimed in claim 3, it is characterised in that in the atomization, progressively will differentiation nutrient solution relative to The accounting of Multiplying culture liquid improves, for example, the volume ratio of the differentiation nutrient solution and Multiplying culture liquid added in cell differentiation procedure From 1:2 are gradually transition to all differentiation nutrient solutions, such as from 1:1 to 2:1, then to 3:1, it is finally reached all differentiation cultures Liquid；Preferably, the differentiation nutrient solution also includes on the basis of basic culture solution：It is insulin, corticotropin, complete Iron transferrins and vitamin D.

7. method as claimed in claim 3, it is characterised in that during the cell propagation, the passage to the cell No more than 5 generations；Wherein, another addition is dry on the basis of the Multiplying culture liquid that the Multiplying culture liquid used after passage uses before passage Cell culture factor S CF and Wnt-3a.

8. the artificial epidermal graft that the method as any one of claim 3-7 prepares.

9. artificial epidermal graft as claimed in claim 1 or artificial epidermal graft as claimed in claim 8 are directed to skin in preparation Application in disease (for example, leucoderma) or the product of situation that skin melanocyte missing is characterized.

10. artificial epidermal graft as claimed in claim 1 or artificial epidermal graft as claimed in claim 8 are used for skin in screening Application in the medicine or cosmetics of disease or situation.