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CN103861147B - A kind of cultural method of the human melanocyte based on nano fiber scaffold - Google Patents

A kind of cultural method of the human melanocyte based on nano fiber scaffold Download PDF

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CN103861147B
CN103861147B CN201410117947.0A CN201410117947A CN103861147B CN 103861147 B CN103861147 B CN 103861147B CN 201410117947 A CN201410117947 A CN 201410117947A CN 103861147 B CN103861147 B CN 103861147B
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nano fiber
fiber scaffold
cell
cultural method
cell culture
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CN103861147A (en
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许爱娥
王文俊
蒋森阳
尉晓冬
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No3 People's Hospital Hangzhou City
Zhejiang University ZJU
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No3 People's Hospital Hangzhou City
Zhejiang University ZJU
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Abstract

The invention discloses a kind of cultural method of the human melanocyte based on nano fiber scaffold, the present invention utilizes tissue engineering technique to build the nano fiber scaffold with good biocompatibility, carry out melanophore or itself and inoblast, (being total to) of keratinocyte cultivated and shifted, be applicable to the adjustment of depigmentation disease (as vitiligo) and skin color.The present invention relates to the structure of the preparation of nano fiber scaffold, cell-nano fiber scaffold matrix material.Owing to there is vesicular structure in nano fiber scaffold, facilitate the maintenance of cell cultures and activity.

Description

A kind of cultural method of the human melanocyte based on nano fiber scaffold
Technical field
The invention belongs to biomedical sector, particularly relate to a kind of melanophore cultural method of the nano fiber scaffold based on biomaterial.
background of invention
Vitiligo is a kind of acquired skin pigment depigmentation disease, shows as local or general property depigmentation, and to form hickie for feature, histology and immunocytochemistry show its skin and damage epidermal melanophore disappearance.Vitiligo sickness rate in crowd reaches 0.5-2%, and hickie place for want of melanochrome, is easily even caused canceration by sun burns, also social worry can be brought to cause mental illness to patient simultaneously.Traditional treatment means comprises endo-medicine, uviolizing and surgical operation therapy etc.But the curative ratio of pharmacological agent and uviolizing is limited.Surgical operation therapy method mainly comprises AUTOEPIDERMIC GRAFTING, the transplanting of melanophore suspension etc., it is effective that these methods for the treatment of confirm, but epidermic grafting needs big area to get skin, and in cell suspension transplanting, cell suspension easily runs off, affect the treatment, and be not suitable for big area transplanting.Build biocompatible scaffold, melanophore cultivation is formed on support cell skin graft and reach cultivation and transfer integration, effectively can improve the weak point of suspension transplantation therapy.
Utilize tissue engineering technique, by cell loading and transfer, for treatment vitiligo provides new approach.CN 1919352A discloses a kind of nano fibrous tissue recovery support of chitosan-containing, but the physical 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 silk fibroin.CN 103194856A discloses a kind of chitosan-silk gum nanofiber with anti-microbial effect, as wound dressings.Existing disclosed nano fiber scaffold patent is mainly used in the reparation of organizational project, there is no the open report cultivated 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, is characterized in that, comprise the following steps:
(1) nano fiber scaffold of biomaterial is prepared: by one or both biocompatible materialses by being dissolved in solvent after the part by weight mixing of 1:50-50:1, be made into the solution that weight percent 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 linking agent to nano fiber scaffold at 20-50oC crosslinking Treatment 0.5-24 hour, with the linking agent that purified rinse water Ex-all is residual after crosslinking Treatment; Then 24 hours are soaked 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 total mass of sodium hydroxide and sodium carbonate and the quality proportioning of water are that 1-10:90-99 mixes and forms, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1; Under 30-120oC, 2-48 hour is dried again under air blast condition.
(2) build cell-nano fiber scaffold matrix material: nano fiber scaffold is alcohol-pickled 12 hours of 75% by volumetric concentration before being used for inoculating cell, clean residual alcohol with PBS more subsequently, and irradiate 3-24 hour under being placed in ultraviolet lamp.After completing sterilization, in Tissue Culture Plate, inject cell culture fluid, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, saturated humidity condition hatches.By the melanophore of the number of cells ratio Dual culture in 1-40:0-200:0-100, inoblast and keratinocyte 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 cell culture incubator in 37oC, volume fraction be the CO of 5% 2, cultivate under saturated humidity condition, within every two days, change a cell culture fluid, incubation time is 1-10 days, namely obtains the matrix material of cell-nano fiber scaffold.
Described solvent comprises formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide (DMSO) etc. and the mixed solvent with methylene dichloride thereof, and the weight ratio of mixed solvent is 5:1-1:1.
Described electrospinning conditions: voltage is 5-30KV, propelling speed is 0.1-10ml/h, receiving range is 5-30cm, temperature is 10-50 oC.
Described cell culture fluid is containing 100 milliliters of compositions such as F12 substratum, 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: by characterizing the biocompatibility nano fiber scaffold material (Fig. 1) of gained, there is vesicular structure (Fig. 2), being applicable to Growth of Cells.There is good mechanical property simultaneously, at preparation, load, transfer and migration process, fragmentation and fracture do not occur, the requirement meeting transfer and transplant.In the building process of cell-nano fiber scaffold matrix material, the form of cell on biocompatibility nano fiber scaffold material is good, the nanofibrous structures of porous benefits the renewal of nutrient solution, thus facilitates the propagation of cell, the activity (Fig. 3) making cell keep good.
Accompanying drawing explanation
Fig. 1 nano fiber scaffold;
Fig. 2 nano fiber scaffold SEM stereoscan photograph;
Fig. 3 melanophore is growthhabit figure on nano fiber scaffold.
Embodiment
The invention belongs to biomedical sector, utilize tissue engineering technique to build and there is good biocompatibility nano fiber scaffold material, for melanophore or itself and inoblast, keratinocyte (being total to) cultivate, realize vitro culture and the transplanting of cell.Be specifically related to that there is the preparation of biocompatibility nano fiber scaffold material, the structure of cell-nano fiber scaffold matrix material and application thereof, meet the requirement of depigmentation disease (as vitiligo) and skin color adjustment.
The material that the present invention selects biocompatibility good, nano fiber scaffold material preparation condition is gentle, and the nanofibrous structures of porous benefits the renewal of nutrient solution, thus facilitates the propagation of cell, makes the active bio safety that cell keeps good.Through the nano fiber scaffold material of crosslinking Treatment, there is superior mechanical property, meet and directly can take out the requirement being used for cell transfer and transplanting from Tissue Culture Plate.In addition, the present invention is by regulating the inoculum density of cell, incubation time and cultural method, keep cytoactive, realize the regulation and control to Different Individual skin color, make melanocytic both effectiveness expression of succeeding in transplanting, there is actual operability, for the adjustment of big 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) nano fiber scaffold of biomaterial is prepared: by one or both biocompatible materialses by being dissolved in solvent after the part by weight mixing of 1:50-50:1, be made into the solution that weight percent 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 linking agent to nano fiber scaffold at 20-50oC crosslinking Treatment 0.5-24 hour, with the linking agent that purified rinse water Ex-all is residual after crosslinking Treatment; Then 24 hours are soaked 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 total mass of sodium hydroxide and sodium carbonate and the quality proportioning of water are that 1-10:90-99 mixes and forms, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1; Under 30-120oC, 2-48 hour is dried again under air blast condition.
(2) build cell-nano fiber scaffold matrix material: nano fiber scaffold is alcohol-pickled 12 hours of 75% by volumetric concentration before being used for inoculating cell, clean residual alcohol with PBS more subsequently, and irradiate 3-24 hour under being placed in ultraviolet lamp.After completing sterilization, in Tissue Culture Plate, inject cell culture fluid, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, saturated humidity condition hatches.By the melanophore of the number of cells ratio Dual culture in 1-40:0-200:0-100, inoblast and keratinocyte 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 cell culture incubator in 37oC, volume fraction be the CO of 5% 2, cultivate under saturated humidity condition, within every two days, change a cell culture fluid, incubation time is 1-10 days, namely obtains the matrix material of cell-nano fiber scaffold.
Solvent comprises formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide (DMSO) etc. and the mixed solvent with methylene dichloride thereof, and the weight ratio of mixed solvent is 5:1-1:1.
Electrospinning conditions: voltage is 5-30KV, propelling speed is 0.1-10ml/h, receiving range is 5-30cm, temperature is 10-50 oC.
Cell culture fluid is containing 100 milliliters of compositions such as F12 substratum, 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, object of the present invention and effect will become more obvious.
The preparation of embodiment 1. chitosan nano fiber timbering material
A, by chitosan with trifluoroacetic acid/dichloromethane weight ratio 3:1 mixed solvent dissolve, be made into the chitosan solution that concentration is 5 %;
B, voltage be 15KV, propelling speed is 1ml/h, receiving range is 15cm, temperature carries out electrostatic spinning under being 20oC condition;
C, chitosan nano fiber support to be dried 12 hours under vacuum, 50oC;
Under d, 20oC, chitosan nano fiber support sodium sulfate is cross-linked 2 hours, with the linking agent that purified rinse water Ex-all is residual after crosslinking Treatment;
E, with massfraction be 4% NaOH solution soak chitosan nano fiber support 24 hours, then to rush to PH=7.2-7.4 with lot of pure water;
F, chitosan nano fiber propped up be placed in convection oven, under 50oC condition dry 24 hours;
G, obtain the chitosan nano fiber timbering material that can be used for cell cultures.
The preparation of embodiment 2. chitosan-gelatin nano fiber scaffold material
A, by chitosan-gelatin (weight proportion 10:1) with trifluoroethanol/methylene dichloride (weight proportion 2:1) mixed solvent dissolve, be made into the chitosan-gelatin solution that concentration is 4%;
B, voltage be 20KV, propelling speed is 3ml/h, receiving range is 18cm, temperature carries out electrostatic spinning under being 30oC condition;
C, chitosan-gelatin nano fiber scaffold to be dried 24 hours under vacuum, 25oC;
Under d, 20oC, chitosan-gelatin nano fiber scaffold Trisodium Citrate is cross-linked 12 hours, with the linking agent that purified rinse water Ex-all is residual after crosslinking Treatment;
The Na of e, use massfraction 8% 2cO 3solution soaking chitosan-gelatin nano fiber scaffold 24 hours, then with the punching of lot of pure water to PH=7.2-7.4;
F, chitosan-gelatin nano fiber scaffold is placed in convection oven, under 50oC condition dry 24 hours;
G, obtain the chitosan-gelatin nano fiber scaffold material that can be used for cell cultures.
The preparation of embodiment 3. chitosans-polyoxyethylene glycol nano fiber scaffold material
A, by chitosan-polyoxyethylene glycol (weight proportion 5:1) dmso solution, be made into chitosan-polyglycol solution that concentration is 6%;
B, voltage be 25KV, propelling speed is 3ml/h, receiving range is 12cm, temperature carries out electrostatic spinning under being 40oC condition;
C, chitosan-polyoxyethylene glycol nano fiber scaffold to be dried 24 hours under vacuum, 25oC;
Under d, 50oC, chitosan-polyoxyethylene glycol nano fiber scaffold glutaraldehyde cross-linking 12 hours, with the linking agent that purified rinse water Ex-all is residual after crosslinking Treatment;
E, be the NaOH/Na of 6% with massfraction 2cO 3(weight ratio 1:1) solution soaking chitosan-polyoxyethylene glycol nano fiber scaffold 24 hours, then with the punching of lot of pure water to PH=7.2-7.4;
F, chitosan-polyoxyethylene glycol nano fiber scaffold is placed in convection oven, under 50oC condition dry 24 hours;
G, obtain the chitosan-polyoxyethylene glycol nano fiber scaffold material that can be used for cell cultures.
The structure of embodiment 4. melanophores-chitosan nano fiber prop composite
A, the chitosan nano fiber described in example 1 propped up to be placed on volume fraction be soak in the alcohol of 75% after 12 hours to rinse well with PBS;
B, the Tissue Culture Plate of cover housing glycan nano fiber scaffold is placed in ultraviolet lamp under irradiate 12 hours;
C, in Tissue Culture Plate, inject 3 ml cells nutrient solutions, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, hatching 4 hours under saturated humidity;
D, by melanophore with individual cell/cm 2density be seeded on chitosan nano fiber support, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, cultivate under saturated humidity;
E, within every two days, change a cell culture fluid, incubation time is 6 days;
F, obtain melanophore-chitosan nano fiber prop composite, the transplanting that can be further used for.
The structure of embodiment 5. melanophores/inoblast-chitosan-gelatin nano-fiber composite material
A, the chitosan-gelatin nano fiber scaffold described in example 2 is placed in volume fraction be 75% alcohol soak after 12 hours and rinse well with PBS;
B, the Tissue Culture Plate of cover housing glycan-gelatine nano fiber support is placed in ultraviolet lamp under irradiate 12 hours;
C, in Tissue Culture Plate, inject 3 ml cells nutrient solutions, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, hatching 4 hours under saturated humidity;
D, be 1:2 by melanophore and inoblast with ratio, density is individual cell/cm 2be seeded on chitosan-gelatin nano fiber scaffold, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, cultivate under saturated humidity;
E, within every two days, change a cell culture fluid, incubation time is 1-10 days;
F, melanophore and inoblast-chitosan-gelatin nano-fiber composite material, the transplanting that can be further used for.
The structure of embodiment 6. melanophores/inoblast/keratinocyte-chitosan-polyoxyethylene glycol nano fiber scaffold matrix material
A, the chitosan described in example 3-polyoxyethylene glycol nano fiber scaffold is placed in volume fraction be 75% alcohol soak after 12 hours and rinse well with PBS;
B, the Tissue Culture Plate of cover housing glycan-polyoxyethylene glycol nano fiber scaffold is placed in ultraviolet lamp under irradiate 12 hours;
C, in Tissue Culture Plate, inject 3 ml cells nutrient solutions, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, hatching 4 hours under saturated humidity;
D, by melanophore, inoblast, keratinocyte ratio with 1:2:1, density is individual cell/cm 2be seeded on chitosan-polyoxyethylene glycol nano fiber scaffold, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, cultivate under saturated humidity;
E, within every two days, change a cell culture fluid, incubation time is 6 days;
F, melanophore/inoblast/keratinocyte-chitosan-polyoxyethylene glycol nano fiber scaffold matrix material, the transplanting that can be further used for.
The present invention utilizes tissue engineering technique to realize the vitro culture of melanophore or itself and inoblast and keratinocyte, load and transplanting.By by cell-nano fiber scaffold composite implantation to depigmenting skin wound place, melanophore or itself and inoblast and keratinocyte is made to move to skin wounds by solid support material, for decolouring place skin provides melanophore, thus realize the adjustment of depigmentation disease (as vitiligo) and skin color.

Claims (6)

1., based on a melanocytic cultural method for nano fiber scaffold, it is characterized in that, comprise the following steps:
(1) nano fiber scaffold of biomaterial is prepared: by one or both biocompatible materialses by being dissolved in solvent after the part by weight mixing of 1:50-50:1, be made into the solution that weight percent 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 linking agent to nano fiber scaffold at 20-50oC crosslinking Treatment 0.5-24 hour, with the linking agent that purified rinse water Ex-all is residual after crosslinking Treatment; Then 24 hours are soaked 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 total mass of sodium hydroxide and sodium carbonate and the quality proportioning of water are that 1-10:90-99 mixes and forms, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1; Under 30-120oC, 2-48 hour is dried again under air blast condition;
(2) build cell-nano fiber scaffold matrix material: nano fiber scaffold is alcohol-pickled 12 hours of 75% by volumetric concentration before being used for inoculating cell, clean residual alcohol with PBS more subsequently, and irradiate 3-24 hour under being placed in ultraviolet lamp; After completing sterilization, in Tissue Culture Plate, inject cell culture fluid, be placed in cell culture incubator in 37oC, volume fraction be the CO of 5% 2, saturated humidity condition hatches; By the melanophore of the number of cells ratio Dual culture in 1-40:0-200:0-100, inoblast and keratinocyte 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 cell culture incubator in 37oC, volume fraction be the CO of 5% 2, cultivate under saturated humidity condition, within every two days, change a cell culture fluid, incubation time is 1-10 days, namely obtains the matrix material 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 (DMSO), or solvent be formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol or dimethyl sulfoxide (DMSO) with methylene dichloride by weight ratio 5:1-1:1 mix the mixed solvent formed.
3. the human melanocyte cultural method based on nano fiber scaffold according to claim 1, is characterized in that, described electrospinning conditions: voltage is 5-30KV, propelling speed is 0.1-10ml/h, receiving range is 5-30cm, temperature is 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 by 100 milliliters of F12 substratum, 0.1-100 milliliter FBS, 10-50000 nanogram CT, 10-50000 microgram IBMX, 0.1-100 milliliter Glutamine and 100-500000 microgram Gentamicin and forms.
5. the human melanocyte cultural method based on nano fiber scaffold according to claim 1, it is characterized in that, described linking agent comprise for the sodium sulfate of physical crosslinking, Trisodium Citrate or tripoly phosphate sodium STPP and for the glutaraldehyde of chemically crosslinked, oxalic dialdehyde, salicylic aldehyde or Vanillin.
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, scleroproein, hyaluronic acid, chondroitin sulfate, polyoxyethylene glycol, polyvinyl alcohol.
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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
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