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Method for directional differentiation from autologous adipose-derived mesenchymal stem cells to melanocytes

A technology of melanocytes and mesenchymal stem cells, which is applied in the field of directed differentiation of autologous adipose-derived mesenchymal stem cells into melanocytes, which can solve the problems of low yield and difficult cultivation, and achieve the effects of wide sources, increased differentiation rate, and accelerated maturation

Inactive Publication Date: 2017-07-25
安徽安龙基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the defects of low yield and difficult culture in the prior art, and provide a method for directional differentiation of autologous adipose-derived mesenchymal stem cells into melanocytes to solve the above problems

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0053] 2. Preparation of adipose-derived mesenchymal stem cells:

[0054] (5) Obtain adipose tissue through surgery;

[0055] (6) Use surgical scissors to cut the adipose tissue into 1mm pieces 3 small pieces;

[0056] (7) Digest with 10ml of high-sugar DMEM medium containing 0.01% (m / v) type Ⅰ collagenase and 0.05% (m / v) neutral protease at 37°C for 55-65min;

[0057] (8) Pipette the above cell suspension 4-5 times, and centrifuge at 860×g for 10 minutes at room temperature;

[0058] (9) Discard the upper layer of suspended fat particles and mature adipocytes, obtain the bottom cell mass, resuspend the cells in PBS solution, and filter with a 100 μm pore size filter;

[0059] (10) Count the cell filtrate, add excess 1×BD IMag™ Buffer, centrifuge at 1500rpm for 5min, and discard the supernatant;

[0060] (11) Thoroughly vortex the CD31 magnetic beads evenly, and then every 1×10 7 Add 50 μL CD31 magnetic beads to each cell, mix well, and incubate at 30°C for 25-35 minutes;...

Embodiment 1

[0082] The invention discloses a method for directional differentiation of autologous adipose-derived mesenchymal stem cells into melanocytes. The specific steps are as follows:

[0083] 1. Preparation of melanocyte conditioned medium:

[0084] (1) The fifth-generation human epidermal melanocytes were inoculated into T75 cell culture flasks containing 30ml of complete melanocyte medium at a density of 5×10 3 / ml;

[0085] (2) Observe the growth state of the cells. After entering the rapid growth period, give low-dose narrow-band ultraviolet radiation (NB-UVB) with an intensity of 0.5J / cm 2 , the time is 30s;

[0086] (3) After 24 hours, collect the supernatant and centrifuge for 5 minutes;

[0087] (4) Filter and sterilize the supernatant with a 0.22 μm pore size filter membrane to prepare the melanocyte conditioned medium, store it in the dark at 4°C, and set aside;

[0088] 2. Preparation of adipose-derived mesenchymal stem cells:

[0089] (5) Obtain adipose tissue thro...

Embodiment 2

[0117] The invention discloses a method for directional differentiation of autologous adipose-derived mesenchymal stem cells into melanocytes. The specific steps are as follows:

[0118] 1. Preparation of melanocyte conditioned medium:

[0119] (1) The fifth-generation human epidermal melanocytes were inoculated into T75 cell culture flasks containing 30ml of complete melanocyte medium at a density of 5×10 3 / ml;

[0120] (2) Observe the growth state of the cells. After entering the rapid growth period, give low-dose narrow-band ultraviolet radiation (NB-UVB) with an intensity of 0.5J / cm 2 , the time is 32 s;

[0121] (3) After 24 hours, collect the supernatant and centrifuge for 5 minutes;

[0122] (4) Filter and sterilize the supernatant with a 0.22 μm pore size filter membrane to prepare the melanocyte conditioned medium, store it in the dark at 4°C, and set aside;

[0123] 2. Preparation of adipose-derived mesenchymal stem cells:

[0124] (5) Obtain adipose tissue thr...

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Abstract

The invention discloses a method for directional differentiation from autologous adipose-derived mesenchymal stem cells to melanocytes. The method comprises the following concrete steps: a melanocyte conditioned medium is prepared; adipose mesenchymal stem cells are prepared; directional differentiation of melanocytes is carried out, human epidermal melanocyte cell strains and a melanocyte complete medium are both purchased from Biovol Technologies, volume of adipose tissue is 20cm<3>, and a DMEM contains 10%(v / v)FBS and 40U / ml gentamicin sulphate. The method has the advantages of high differentiation efficiency, easy acquisition, and the like.

Description

technical field [0001] The invention belongs to the field of tissue engineering, and in particular relates to a method for directional differentiation of autologous adipose mesenchymal stem cells into melanocytes. Background technique [0002] Vitiligo is a common primary localized or generalized skin depigmentation disease, which is caused by the loss of function of skin melanocytes. The mechanism is still unclear. Its incidence rate is about 1%. Vitiligo patients may be discriminated against in society, which brings huge psychological pressure to patients and seriously affects the normal life of patients. [0003] At present, there are various treatments for vitiligo, such as drug therapy, photochemical therapy, ultraviolet light therapy, laser therapy, surgical skin transplantation therapy, cell transplantation therapy and so on. But above-mentioned method is better to the curative effect of local small-area symptom patient, but then curative effect is unsatisfactory fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0775
CPCC12N5/0626C12N5/0667C12N2500/25C12N2500/38C12N2500/84C12N2501/01C12N2501/115C12N2501/125C12N2501/365C12N2501/415C12N2506/1384C12N2509/00C12N2529/10
Inventor 韦玉军陆宝石李航吴远航苏军
Owner 安徽安龙基因科技有限公司
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