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Cell preparation method

A cell and somatic cell technology, applied in biochemical equipment and methods, animal cells, cell culture medium, etc., can solve the problems of safety cost and time, cell tumorization, etc.

Active Publication Date: 2018-12-21
KYOTO PREFECTURAL PUBLIC UNIV CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the method of introducing a gene, there is a possibility that the cells may become tumorous due to the influence of the introduced gene or carrier
In addition, there are also issues such as the cost and time required to prove safety

Method used

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preparation example Construction

[0250] Preparation of target somatic cells can be confirmed by known methods such as detection of markers.

[0251] Examples thereof are described below, but they are not limited to one marker, and are preferably confirmed by combining the detection of several markers.

[0252] The evaluation of the preparation of the target somatic cells can also be carried out by analyzing the summary of mRNA expression such as transcriptional analysis of the prepared somatic cells, the analysis of the summary of protein expression such as proteome analysis, etc., and the analysis of the target somatic cells derived from a living body. The results are compared.

[0253] For example, the acquisition of osteoblasts can be confirmed by real-time PCR-based measurement of ALP (alkaline phosphatase) gene, osteocalcin (Osteocalcin, OC) gene, osteopontin (Osteopontin) gene, Runx2 gene mRNA , Alizarin Red S-based staining (production of mineralized (mineralized) bone matrix), etc. It should be note...

Embodiment 1

[0318] Example 1 ( figure 1 )

[0319] Fibroblasts (human dermal fibroblasts; HDFs) derived from normal human skin were suspended in ordinary medium (Dulbecco's modified minimum essential medium supplemented with 10% FBS; DMEM). Put it in 5×10 3 The concentration of cells / well was seeded in a 24-well plate (day 0) in 5% CO 2 / 95% humidified air at 37°C to start culturing. On the next day, the culture supernatant was aspirated, and 500 μL / well of a normal medium, a mineralization-inducing medium, or a mineralization-inducing medium supplemented with each compound was added as described in the figure.

[0320] The mineralization induction medium was prepared by adding 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone to 10% FBS DMEM. Once every 3 to 4 days, replace the culture medium with fresh culture medium, and cultivate until the 24th day.

[0321] On day 24, the culture medium was aspirated from each well, washed with PBS(-), and then fixed w...

Embodiment 2

[0328] Example 2 ( figure 2 )

[0329]Fibroblasts (human dermal fibroblasts; HDF) derived from normal human skin were suspended in ordinary medium (Dulbecco's modified minimum essential medium supplemented with 10% FBS; DMEM). Put it in 5×10 3 The concentration of cells / well was seeded in a 24-well plate (day 0) in 5% CO 2 / 95% humidified air at 37°C to start culturing. The next day, the culture supernatant was aspirated, and a mineralization-inducing medium or a mineralization-inducing medium to which each compound was added was added at 500 μL / well as described in the figure.

[0330] The mineralization induction medium was prepared by adding 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone to 10% FBS DMEM. Once every 3 to 4 days, replace the culture medium with fresh culture medium, and cultivate until the 24th day.

[0331] On day 24, the culture medium was aspirated from each well, washed with PBS(-), and then fixed with 10% formalin. Af...

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Abstract

Provided is a method for preparing a somatic cell characterized by comprising culturing, in the presence of a TGF-beta pathway inhibitor, a differentiated somatic cell of a mammal in a medium for inducing the differentiation of a somatic cell other than the differentiated somatic cell described above and thus converting the differentiated somatic cell into the other somatic cell.

Description

technical field [0001] Cross References to Related Applications [0002] This application claims priority based on Japanese Patent Application No. 2015-207529 for which it applied on October 21, 2015, and takes in the whole indication of this in this specification by reference. [0003] The present invention mainly relates to the preparation method of cells. Specifically, it relates to a method for preparing cells based on direct reprogramming. The present invention also relates to inducers for converting differentiated somatic cells into other somatic cells. Background technique [0004] In recent years, attention has been paid to regenerative medical technology, which treats diseases by supplementing functional and morphological abnormalities by transplanting cells to patients. [0005] For example, for osteoblasts, if osteoblasts are transplanted into the lesion for the purpose of repairing bone defects caused by bone tumors, trauma, osteomyelitis, etc., and bone defec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/077C12N5/0775C12N5/078C12N5/079
CPCC12N5/0662C12N5/0658C12N5/0654C12N5/0655C12N2506/1307C12N2506/1384C12N2501/727C12N2501/39C12N2501/395C12N2501/115C12N2501/11C12N2501/33C12N2501/01C12N2501/415C12N5/0018C12N5/0622C12N5/0652C12N5/0653C12N5/0684C12N2501/385C12N5/0656C12N2501/15C12N5/0663
Inventor 山本健太岸田纲郎素轮善弘山本俊郎松田修
Owner KYOTO PREFECTURAL PUBLIC UNIV CORP
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