CN103861147A - Method for culturing human melanocyte based on nano-fiber scaffold - Google Patents
Method for culturing human melanocyte based on nano-fiber scaffold Download PDFInfo
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Abstract
The invention discloses a method for culturing human melanocyte based on a nano-fiber scaffold. Melanocyte or melanocyte, fibroblast and keratinocyte can be (co-cultured) cultured and transferred by utilizing a nano-fiber scaffold which is constructed by utilizing a tissue engineering technology and has an excellent biocompatibility, and the method is applicable to depigmentation (such as leukoderma) and can be used for regulating skin color. The invention also relates to preparation of the nano-fiber scaffold and construction of a cell-nano-fiber scaffold composite material. Because the nano-fiber scaffold has a porous structure, cell culture and activity maintenance can be promoted.
Description
Technical field
The invention belongs to biomedical sector, relate in particular to a kind of melanocyte cultural method of the nano fiber scaffold based on biomaterial.
background of invention
Vitiligo is the de-property lost of a kind of acquired skin pigment disease, shows as local or general property depigmentation, and to form white macula as feature, histology and immunocytochemistry show that its skin lesion epidermal melanophore disappears.Vitiligo sickness rate in crowd reaches 0.5-2%, and for want of melanin of white macula place, is easily even caused canceration by sun burns, also can bring social worry to cause mental illness to patient simultaneously.Traditional treatment means comprises endo-medicine, ultraviolet radiation and surgical operation therapy etc.But the cure rate of Drug therapy and ultraviolet radiation is limited.Surgical operation therapy method mainly comprises Autologous epidermis transplanting, the transplanting of melanocyte suspension etc., these Therapeutic Method confirm it is effectively, but epidermic grafting needs large area bark fetching, and in cell suspension transplanting, cell suspension easily runs off, affect the treatment, and be not suitable for large area transplanting.Build biocompatible scaffold, melanocyte is cultivated and on support, formed cell skin graft and reach and cultivate and shift integrated, can effectively improve the weak point of suspension transplantation therapy.
Utilize tissue engineering technique, by cell loading and transfer, for treatment vitiligo new approach is provided.CN 1919352A discloses a kind of nano fibrous tissue recovery support of chitosan-containing, but the mechanical strength of support is low.CN 1952227A discloses with a kind of gelatine-chitosan fibrous framework for bionic extracellular matrix, and after support soaks, its structure is easily damaged.CN 101445971A discloses a kind of fibroin-chitosan nano fiber scaffold of imitative extracellular matrix, the Immunoreactivity of the introducing possibility trigger cell of fibroin albumen.CN 103194856A discloses a kind of chitosan-sericin nanofiber with antibacterial action, as wound dressing.Existing disclosed nano fiber scaffold patent is mainly used in the reparation of organizational project, there is no the open report of cultivating for human melanocyte.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, a kind of melanocytic cultural method based on nano fiber scaffold is provided.
The object of the invention is to be achieved through the following technical solutions: a kind of melanocytic cultural method based on nano fiber scaffold, it is characterized in that, comprise the following steps:
(1) prepare the nano fiber scaffold of biomaterial: after one or both biocompatible materialses are mixed by the part by weight of 1:50-50:1, be dissolved in solvent, be made into the solution that percentage by weight is 1-10%, pass through electrostatic spinning, prepare the nano fiber scaffold that thickness is 30-200 micron, under vacuum, 20-50oC, dry 2-48 hour; Then with cross-linking agent to nano fiber scaffold at 20-50oC crosslinking Treatment 0.5-24 hour, after crosslinking Treatment with purified rinse water Ex-all residual cross-linking agent; Then soak 24 hours with sodium hydroxide-aqueous sodium carbonate, be neutral with washed with de-ionized water to pH value of solution again, in described sodium hydroxide-aqueous sodium carbonate, the gross mass of sodium hydroxide and sodium carbonate is that 1-10:90-99 mixes composition with the quality proportioning of water, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1; Under air blast condition, under 30-120oC, dry 2-48 hour again.
(2) building cell-nano fiber scaffold composite: nano fiber scaffold is for before inoculating cell, is 75% with volumetric concentration alcohol-pickled 12 hours, subsequently again with the clean residual ethanol of PBS, and is placed under uviol lamp and irradiates 3-24 hour.Complete after sterilization, in Tissue Culture Plate, inject cell culture fluid, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, saturated humidity condition hatches.Melanocyte, fibroblast and the keratinocyte that number of cells ratio in 1-40:0-200:0-100 is cultivated altogether with
individual cell/cm
2density be seeded on the nano fiber scaffold of hatching, add the cell culture fluid of 1-10 milliliter simultaneously, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, cultivate under saturated humidity condition, within every two days, change a cell culture fluid, incubation time is 1-10 days, obtains the composite of cell-nano fiber scaffold.
Described solvent comprises formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide etc. and the mixed solvent with dichloromethane thereof, and the weight ratio of mixed solvent is 5:1-1:1.
Described electrostatic spinning condition: voltage is that 5-30KV, propelling speed are that 0.1-10ml/h, receiving range are that 5-30cm, temperature are 10-50 oC.
Described cell culture fluid is containing compositions such as 100 milliliters of F12 culture medium, 0.1-100 milliliter FBS, 10-50000 nanogram CT, 10-50000 microgram IBMX, 0.1-100 milliliter Glutamine and 100-500000 microgram Gentamicin.
The invention has the beneficial effects as follows: characterize by the biocompatibility nano fiber scaffold material (Fig. 1) to gained, there is loose structure (Fig. 2), be applicable to Growth of Cells.There is good mechanical performance simultaneously, at preparation, load, transfer and migration process, fragmentation and fracture do not occur, meet the requirement of shifting and transplanting.In the building process of cell-nano fiber scaffold composite, the form of cell on biocompatibility nano fiber scaffold material is good, the nanofibrous structures of porous benefits the renewal of culture fluid, thereby has promoted the propagation of cell, makes cell keep good activity (Fig. 3).
Brief description of the drawings
Fig. 1 nano fiber scaffold;
Fig. 2 nano fiber scaffold SEM stereoscan photograph;
Fig. 3 melanocyte is growthform figure on nano fiber scaffold.
Detailed description of the invention
The invention belongs to biomedical sector, utilize tissue engineering technique to build and have a good biocompatibility nano fiber scaffold material, (being total to) cultivation for melanocyte or its with fibroblast, keratinocyte, realizes In vitro culture and the transplanting of cell.Be specifically related to have the preparation of biocompatibility nano fiber scaffold material, structure and the application thereof of cell-nano fiber scaffold composite, meet the requirement that depigmentation disease (as vitiligo) and skin color regulate.
The present invention selects the material that biocompatibility is good, nano fiber scaffold material preparation condition gentleness, and the nanofibrous structures of porous benefits the renewal of culture fluid, thereby has promoted the propagation of cell, makes cell keep good active bio safety.Through the nano fiber scaffold material of crosslinking Treatment, there is superior mechanical performance, meet and can directly from Tissue Culture Plate, take out the requirement of shifting and transplanting for cell.In addition, the present invention is by regulating inoculum density, incubation time and the cultural method of cell, keep cytoactive, realize the regulation and control to Different Individual skin color, the expression that melanocytic effect is succeeded in transplanting, there is actual operability, for the adjusting of large area depigmentation disease and skin color provides a great convenience.
A kind of cell culture processes based on nano fiber scaffold of the present invention, comprises the following steps:
(1) prepare the nano fiber scaffold of biomaterial: after one or both biocompatible materialses are mixed by the part by weight of 1:50-50:1, be dissolved in solvent, be made into the solution that percentage by weight is 1-10%, pass through electrostatic spinning, prepare the nano fiber scaffold that thickness is 30-200 micron, under vacuum, 20-50oC, dry 2-48 hour; Then with cross-linking agent to nano fiber scaffold at 20-50oC crosslinking Treatment 0.5-24 hour, after crosslinking Treatment with purified rinse water Ex-all residual cross-linking agent; Then soak 24 hours with sodium hydroxide-aqueous sodium carbonate, be neutral with washed with de-ionized water to pH value of solution again, in described sodium hydroxide-aqueous sodium carbonate, the gross mass of sodium hydroxide and sodium carbonate is that 1-10:90-99 mixes composition with the quality proportioning of water, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1; Under air blast condition, under 30-120oC, dry 2-48 hour again.
(2) building cell-nano fiber scaffold composite: nano fiber scaffold is for before inoculating cell, is 75% with volumetric concentration alcohol-pickled 12 hours, subsequently again with the clean residual ethanol of PBS, and is placed under uviol lamp and irradiates 3-24 hour.Complete after sterilization, in Tissue Culture Plate, inject cell culture fluid, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, saturated humidity condition hatches.Melanocyte, fibroblast and the keratinocyte that number of cells ratio in 1-40:0-200:0-100 is cultivated altogether with
individual cell/cm
2density be seeded on the nano fiber scaffold of hatching, add the cell culture fluid of 1-10 milliliter simultaneously, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, cultivate under saturated humidity condition, within every two days, change a cell culture fluid, incubation time is 1-10 days, obtains the composite of cell-nano fiber scaffold.
Solvent comprises formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide etc. and the mixed solvent with dichloromethane thereof, and the weight ratio of mixed solvent is 5:1-1:1.
Electrostatic spinning condition: voltage is that 5-30KV, propelling speed are that 0.1-10ml/h, receiving range are that 5-30cm, temperature are 10-50 oC.
Cell culture fluid is containing compositions such as 100 milliliters of F12 culture medium, 0.1-100 milliliter FBS, 10-50000 nanogram CT, 10-50000 microgram IBMX, 0.1-100 milliliter Glutamine and 100-500000 microgram Gentamicin.
Describe the present invention in detail according to embodiment below, it is more obvious that object of the present invention and effect will become.
The preparation of embodiment 1. chitosan nano fiber timbering materials
A, chitosan is dissolved with trifluoroacetic acid/dichloromethane weight ratio 3:1 mixed solvent, be made into the chitosan solution that concentration is 5 %;
B, be that 15KV, propelling speed are that 1ml/h, receiving range are that 15cm, temperature are under 20oC condition, to carry out electrostatic spinning at voltage;
C, chitosan nano fiber support is dried 12 hours under vacuum, 50oC;
Under d, 20oC, chitosan nano fiber support is cross-linked 2 hours with sodium sulfate, uses the residual cross-linking agent of purified rinse water Ex-all after crosslinking Treatment;
E, the NaOH solution soaking chitosan nano fiber support that is 4% with mass fraction 24 hours, then rush to PH=7.2-7.4 with lot of pure water;
F, chitosan nano fiber is propped up and is placed in convection oven, under 50oC condition dry 24 hours;
G, obtain can be used for the chitosan nano fiber timbering material of cell culture.
The preparation of embodiment 2. chitosan-gelatin nano fiber scaffold materials
A, chitosan-gelatin (weight proportion 10:1) is dissolved with trifluoroethanol/dichloromethane (weight proportion 2:1) mixed solvent, be made into concentration and be 4% chitosan-gelatin solution;
B, be that 20KV, propelling speed are that 3ml/h, receiving range are that 18cm, temperature are under 30oC condition, to carry out electrostatic spinning at voltage;
C, chitosan-gelatin nano fiber scaffold is dried 24 hours under vacuum, 25oC;
Under d, 20oC, chitosan-gelatin nano fiber scaffold is cross-linked 12 hours with sodium citrate, uses the residual cross-linking agent of purified rinse water Ex-all after crosslinking Treatment;
The Na of e, use mass fraction 8%
2cO
3solution soaking chitosan-gelatin nano fiber scaffold 24 hours, then rush to PH=7.2-7.4 with lot of pure water;
F, chitosan-gelatin nano fiber scaffold is placed in to convection oven, under 50oC condition dry 24 hours;
G, obtain can be used for the chitosan-gelatin nano fiber scaffold material of cell culture.
The preparation of embodiment 3. chitosans-Polyethylene Glycol nano fiber scaffold material
A, by dmso solution for chitosan-Polyethylene Glycol (weight proportion 5:1), be made into concentration and be chitosan-polyglycol solution of 6%;
B, be that 25KV, propelling speed are that 3ml/h, receiving range are that 12cm, temperature are under 40oC condition, to carry out electrostatic spinning at voltage;
C, chitosan-Polyethylene Glycol nano fiber scaffold is dried 24 hours under vacuum, 25oC;
Under d, 50oC, chitosan-Polyethylene Glycol nano fiber scaffold is used glutaraldehyde cross-linking 12 hours, uses the residual cross-linking agent of purified rinse water Ex-all after crosslinking Treatment;
E, the NaOH/Na that is 6% with mass fraction
2cO
3(weight ratio 1:1) solution soaking chitosan-Polyethylene Glycol nano fiber scaffold 24 hours, then rush to PH=7.2-7.4 with lot of pure water;
F, chitosan-Polyethylene Glycol nano fiber scaffold is placed in to convection oven, under 50oC condition dry 24 hours;
G, obtain can be used for chitosan-Polyethylene Glycol nano fiber scaffold material of cell culture.
The structure of embodiment 4. melanocytes-chitosan nano fiber prop composite
A, the chitosan nano fiber described in example 1 is propped up to be placed on volume fraction be in 75% ethanol, to soak after 12 hours to rinse well with PBS;
B, the Tissue Culture Plate of cover housing polysaccharide nano fiber scaffold is placed under uviol lamp and is irradiated 12 hours;
C, in Tissue Culture Plate, inject 3 ml cells culture fluid, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, hatch 4 hours under saturated humidity;
D, by melanocyte with
individual cell/cm
2density be seeded on chitosan nano fiber support, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, cultivate under saturated humidity;
E, within every two days, change one time cell culture fluid, incubation time is 6 days;
F, obtain melanocyte-chitosan nano fiber prop composite, the transplanting that can be further used for.
The structure of embodiment 5. melanocytes/fibroblast-chitosan-gelatin nano-fiber composite material
A, the chitosan-gelatin nano fiber scaffold described in example 2 is placed in to volume fraction is that 75% ethanol soaks after 12 hours and rinses well with PBS;
B, the Tissue Culture Plate of cover housing polysaccharide-gelatine nano fiber support is placed under uviol lamp and is irradiated 12 hours;
C, in Tissue Culture Plate, inject 3 ml cells culture fluid, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, hatch 4 hours under saturated humidity;
D, by melanocyte and fibroblast taking ratio as 1:2, density is
individual cell/cm
2be seeded on chitosan-gelatin nano fiber scaffold, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, cultivate under saturated humidity;
E, within every two days, change one time cell culture fluid, incubation time is 1-10 days;
F, melanocyte and fibroblast-chitosan-gelatin nano-fiber composite material, the transplanting that can be further used for.
The structure of embodiment 6. melanocytes/fibroblast/keratinocyte-chitosan-Polyethylene Glycol nano fiber scaffold composite
A, the chitosan-Polyethylene Glycol nano fiber scaffold described in example 3 is placed in to volume fraction is that 75% ethanol soaks after 12 hours and rinses well with PBS;
B, the Tissue Culture Plate of cover housing polysaccharide-Polyethylene Glycol nano fiber scaffold is placed under uviol lamp and is irradiated 12 hours;
C, in Tissue Culture Plate, inject 3 ml cells culture fluid, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, hatch 4 hours under saturated humidity;
D, by melanocyte, fibroblast, keratinocyte ratio with 1:2:1, density is
individual cell/cm
2be seeded on chitosan-Polyethylene Glycol nano fiber scaffold, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, cultivate under saturated humidity;
E, within every two days, change one time cell culture fluid, incubation time is 6 days;
F, melanocyte/fibroblast/keratinocyte-chitosan-Polyethylene Glycol nano fiber scaffold composite, the transplanting that can be further used for.
The present invention utilizes tissue engineering technique to realize In vitro culture, load and the transplanting of melanocyte or itself and fibroblast and keratinocyte.By cell-nano fiber scaffold composite is transplanted to depigmenting skin wound place, make melanocyte or itself and fibroblast and keratinocyte move to skin wounds place by carrier material, for decolouring place skin provides melanocyte, thereby realize the adjusting of depigmentation disease (as vitiligo) and skin color.
Claims (6)
1. the melanocytic cultural method based on nano fiber scaffold, is characterized in that, comprises the following steps:
(1) prepare the nano fiber scaffold of biomaterial: after one or both biocompatible materialses are mixed by the part by weight of 1:50-50:1, be dissolved in solvent, be made into the solution that percentage by weight is 1-10%, pass through electrostatic spinning, prepare the nano fiber scaffold that thickness is 30-200 micron, under vacuum, 20-50oC, dry 2-48 hour; Then with cross-linking agent to nano fiber scaffold at 20-50oC crosslinking Treatment 0.5-24 hour, after crosslinking Treatment with purified rinse water Ex-all residual cross-linking agent; Then soak 24 hours with sodium hydroxide-aqueous sodium carbonate, be neutral with washed with de-ionized water to pH value of solution again, in described sodium hydroxide-aqueous sodium carbonate, the gross mass of sodium hydroxide and sodium carbonate is that 1-10:90-99 mixes composition with the quality proportioning of water, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1; Under air blast condition, under 30-120oC, dry 2-48 hour again;
(2) building cell-nano fiber scaffold composite: nano fiber scaffold is for before inoculating cell, is 75% with volumetric concentration alcohol-pickled 12 hours, subsequently again with the clean residual ethanol of PBS, and is placed under uviol lamp and irradiates 3-24 hour; Complete after sterilization, in Tissue Culture Plate, inject cell culture fluid, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, saturated humidity condition hatches; Melanocyte, fibroblast and the keratinocyte that number of cells ratio in 1-40:0-200:0-100 is cultivated altogether with
individual cell/cm
2density be seeded on the nano fiber scaffold of hatching, add the cell culture fluid of 1-10 milliliter simultaneously, be placed in the CO that cell culture incubator is 5% in 37oC, volume fraction
2, cultivate under saturated humidity condition, within every two days, change a cell culture fluid, incubation time is 1-10 days, obtains the composite of cell-nano fiber scaffold.
2. the human melanocyte cultural method based on nano fiber scaffold according to claim 1, it is characterized in that, described solvent is selected from formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide, or solvent be formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol or dimethyl sulfoxide with dichloromethane by weight ratio 5:1-1:1 mix the mixed solvent of composition.
3. the human melanocyte cultural method based on nano fiber scaffold according to claim 1, is characterized in that, described electrostatic spinning condition: voltage is that 5-30KV, propelling speed are that 0.1-10ml/h, receiving range are that 5-30cm, temperature are 10-50 oC.
4. the human melanocyte cultural method based on nano fiber scaffold according to claim 1, it is characterized in that, described cell culture fluid is mixed and is formed by compositions such as 100 milliliters of F12 culture medium, 0.1-100 milliliter FBS, 10-50000 nanogram CT, 10-50000 microgram IBMX, 0.1-100 milliliter Glutamine and 100-500000 microgram Gentamicin.
5. the human melanocyte cultural method based on nano fiber scaffold according to claim 1, it is characterized in that, described cross-linking agent comprises for the sodium sulfate of physical crosslinking, sodium citrate or sodium tripolyphosphate and for glutaraldehyde, Biformyl, salicylide or the vanillin etc. of chemical crosslinking.
6. the human melanocyte cultural method based on nano fiber scaffold according to claim 1, it is characterized in that, described biocompatible materials is selected from chitosan, gelatin, collagen protein, fibrin, hyaluronic acid, chondroitin sulfate, Polyethylene Glycol, polyvinyl alcohol etc.
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| CN105326863A (en) * | 2015-11-27 | 2016-02-17 | 广州市朴道联信生物科技有限公司 | Method for preparing composite membrane for treating leucoderma from autologous follicle melanocytes |
| CN112831459A (en) * | 2019-11-25 | 2021-05-25 | 杭州协合医疗用品有限公司 | Collagen melanocyte compound, preparation method and application |
| CN113215089A (en) * | 2020-10-11 | 2021-08-06 | 西北农林科技大学 | Manufacturing method of edible chitosan 3D gel scaffold for cell culture meat |
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| CN114438615A (en) * | 2022-02-28 | 2022-05-06 | 上海食未生物科技有限公司 | Large-scale production method of soybean protein fiber scaffold for cell culture meat |
| CN114622296A (en) * | 2022-02-28 | 2022-06-14 | 上海食未生物科技有限公司 | Method for continuously preparing edible fiber scaffold for cell culture meat |
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| CN114622296A (en) * | 2022-02-28 | 2022-06-14 | 上海食未生物科技有限公司 | Method for continuously preparing edible fiber scaffold for cell culture meat |
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