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Method for generating autologous melanocytes based on 3D suspension iPS (induced pluripotent stem cells) and application

A technology for the generation of melanocytes and cells, applied in the biological field, can solve the problems of uneven size and shape of embryoid bodies, unable to obtain mature melanocytes, low differentiation efficiency, etc., and achieve the effect of excellent performance advantages.

Active Publication Date: 2018-11-13
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the technical problems that the size and shape of embryoid bodies are not uniform in the existing melanocyte induction and differentiation schemes, the differentiation efficiency is low, and a large number of epithelioid cells are generated during differentiation, and mature melanocytes are often not obtained after several passages. The purpose of the present invention is to overcome the defects in the prior art and provide a new method for generating autologous melanocytes. This method adopts a method based on 3D suspension to induce iPS cells to generate autologous melanocytes.

Method used

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  • Method for generating autologous melanocytes based on 3D suspension iPS (induced pluripotent stem cells) and application
  • Method for generating autologous melanocytes based on 3D suspension iPS (induced pluripotent stem cells) and application
  • Method for generating autologous melanocytes based on 3D suspension iPS (induced pluripotent stem cells) and application

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Experimental program
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Effect test

Embodiment 1

[0037] Traditional method (2D differentiation):

[0038] Such as figure 1 As shown, the size and shape of embryoid bodies produced by traditional methods (such as mechanical methods) are highly heterogeneous ( figure 1 A); These embryoid bodies will be fully spread out when cultured on a 2D plane, generating a large number of epithelioid cells, while the proportion of dendritic cells is very small ( figure 1 B) Single cell digestion was carried out at 21 days of differentiation, and it can also be observed that most cells in the adherent single cells maintain polygonal epithelioid cell morphology, and the proportion of dendritic cells is very small ( figure 1 C); due to the low proportion of melanocytes and slow proliferation, while the high proportion of epithelioid cells and rapid proliferation, mature melanocytes are often not obtained after several passages ( figure 1 D).

Embodiment 2

[0040] a. Production of embryoid bodies by single-cell method

[0041] When the iPS cell clone grows to a moderate size, add the iPS single-cell digestive enzyme ACCUTASE TM (InnovativeCell Technologie), put it at room temperature for 5-7 minutes, add mTeSR (Stemcell Technologies) medium and blow it into iPS single cell suspension, discard the supernatant after centrifugation, add mTeSR medium to resuspend and count, and inoculate iPS single cells into Elplasia TM Three-dimensional culture plate (Kuraray) inside ( figure 2 A) The seeding density is taken as an example in a 24-well plate, and 5×10 5 Cells were added with ROCK inhibitor Y27632 (Wako) at a final concentration of 10 μM, cultured at 37 degrees, and after 24 hours, cultured embryoid bodies with uniform shape and size were formed ( figure 2 B) Gently pipet and aspirate the embryoid body, transfer it to a low-adhesion plate (Corning) to continue culturing, and change the medium every day.

[0042] b. 3D Suspe...

Embodiment 3

[0053] Choose embryoid bodies of different culture days and sizes for 3D suspension induced differentiation:

[0054] In the process of inducing differentiation, the present invention selects embryoid bodies with different culture days and sizes to induce differentiation, and compares the effects of culture days and embryoid body sizes on induced differentiation. Such as image 3 As shown, the embryoid bodies with a loose structure and a diameter of less than 200 μm were cultured for less than 3 days ( image 3 A), in the early stage of differentiation (differentiation day 14), there will be a large number of vacuolar-like structures ( image 3 B), after attachment (differentiation day 21), these structures showed a flat and wide shape, without the ability to proliferate ( image 3 C). However, embryoid bodies with a diameter greater than 700 μm ( image 3 D), after differentiation (15th day of differentiation), most of the cells in the central dark area could not adhere t...

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Abstract

The invention relates to a method for generating cells, in particular to a method for generating autologous melanocytes based on 3D suspension iPS (induced pluripotent stem cells) and an application,and belongs to the field of biotechnology. According to the method, iPS are digested into single cells, embryoid bodies with uniform shape and size are generated by a three-dimensional culture plate,2D plane adherence is changed to 3D suspension culture in the early induction process 14 days before differentiation, so that the proportion of epithelioid cells in the differentiation process is reduced, differentiation efficiency of the melanocytes is improved, selection of the embryoid bodies before differentiation, the digestion time point of the single cells and culture medium components areoptimized, and the proliferation state of the melanocytes is improved; the obtained melanocytes have the characteristics highly close to those of normal melanocytes in vitro, and have characteristicssignificantly superior to those of normal melanocytes in the in-vivo transplantation process.

Description

technical field [0001] The invention relates to a method for generating cells, in particular to a method and application for inducing iPS cells to generate autologous melanocytes by 3D suspension, and belongs to the field of biotechnology. Background technique [0002] Melanocytes are the only cell type that produce melanin to protect skin cells from UV rays. Human melanocytes can be directly isolated from the epidermis. However, their number in the epidermis is extremely limited, and their ability to proliferate in vitro is weak, which greatly limits the application of melanocytes in autologous cell transplantation therapy and drug screening. . [0003] In addition to the epidermis, melanocytes can also be differentiated through other pathways, such as melanin stem cells and melanin precursor cells, dermal stem cells, hair follicle stem cells, hair follicle outer root sheath stem cells, embryonic neural crest stem cells, and embryonic stem cells. However, these methods ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61K35/36A61P17/00
CPCA61K35/36A61P17/00C12N5/0626C12N2500/34C12N2500/38C12N2501/115C12N2501/125C12N2509/00C12N2513/00A61K35/545C12N2501/33C12N2501/365C12N2501/39C12N2501/415C12N2501/998C12N2506/45C12N2533/52C12N5/0062
Inventor 刘莉萍李遇梅郑允文郭宁宁
Owner JIANGSU UNIV
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