iPS cell as well as reprogramming method and application thereof

A reprogramming and cell technology, applied in the field of cell biology, can solve the problems of increasing the insecurity of the process, affecting the later application, etc., to achieve the effect of facilitating the later application, high electroporation efficiency, and improving the transformation efficiency.

Pending Publication Date: 2019-04-05
博尔赛(上海)细胞生物技术有限公司
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Problems solved by technology

However, this method includes a step of viral infection induction, which i

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  • iPS cell as well as reprogramming method and application thereof
  • iPS cell as well as reprogramming method and application thereof
  • iPS cell as well as reprogramming method and application thereof

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Embodiment 1

[0065] 1. Isolate UDCs: collect 30-50ml mid-section urine; centrifuge at room temperature 200×g for 10min, discard supernatant, add 10ml washing buffer to resuspend, centrifuge at 200×g, discard supernatant, add 2m basal medium to resuspend cells; inoculate on coated cultured overnight at 37°C in 5% CO2. Three days later, add basal medium for culture, and three days later, half-change medium for REMC culture until UDCs clones grow. When the clones become dense, they are subcultured.

[0066] 2. Electroporation: Discard the medium in the old culture dish, add DPBS to wash, digest with 0.25% trypsin, collect cells, and count. Mix cells and plasmids (pCXLE-hOCT3 / 4-shp53, pCXLE-hSK, pCXLE-hUL) evenly (5×10 4 -2×10 5 cells / 10μl add 1μg of each of the three plasmids), according to 5X10 4 -2X10 5 cells / 10μl electroporation system for electroporation. After electroporation, the mixture of cells and plasmids was added to the culture plate containing REMC, and cultured overnight at...

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Abstract

The invention provides an iPS cell as well as a reprogramming method and application thereof. The reprogramming method comprises the following steps that (1) urine is collected, centrifuging is carried out, a supernate is discarded, precipitates are washed, and cells are resuspended; (2) a coated culture dish is inoculated with the resuspended cells obtained in step (1), culture is carried out overnight, then liquid is changed for culture until UDCs (urine-derived cells) clones grow out, and subculture is carried out; and (3) a culture medium for subculture is sucked and discarded, trypsinization is carried out, the cells are collected and counted, plasmids are subjected to transfection by using an electric transfection method and are added into a resuscitation culture plate for culture overnight, and then liquid is changed for culture, and iPS clones are screened, wherein the plasmids comprise pCXLE-hOCT3/4-shp53, pCXLE-hSK and pCXLE-hUL. The method has the advantages that materials are convenient to obtain, trauma and ethical problems are avoided, the safety is remarkably improved, and meanwhile the whole reprogramming cost is lowered.

Description

technical field [0001] The invention belongs to the field of cell biology, and relates to an iPS cell and its reprogramming method and application, mainly to a urine-derived cell-induced iPS cell and its reprogramming method and application, in particular to a urine-derived cell-induced iPS cell iPS cell and its electrotransfection reprogramming method and application. Background technique [0002] iPS cells are similar to embryonic stem cells (ES) in morphology, karyotype, telomerase activity, and in vitro differentiation potential, and can also express the same surface marker molecules. ES cells and iPS cells have the same genes. The difference is that genes related to cell pluripotency in ES cells can be expressed, such as: oct4, Sox2, etc. These genes are not expressed in differentiated somatic cells. By introducing exogenous genes related to pluripotency to activate pluripotency genes in somatic cells, so that somatic cells can be reprogrammed from differentiated sta...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N5/0696C12N2501/603C12N2501/11C12N2501/115C12N2501/135C12N2506/25
Inventor 李辉付余丛伟立李超杨茜茜高洋
Owner 博尔赛(上海)细胞生物技术有限公司
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