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Method for evaluating state of cells

A cell state and cell technology, applied in the direction of material inspection products, instruments, analysis materials, etc., can solve the problems of poor detection sensitivity and achieve the effect of system improvement

Inactive Publication Date: 2011-10-19
HOKKAIDO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when detecting specific proteins, the detection sensitivity may deteriorate

Method used

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  • Method for evaluating state of cells
  • Method for evaluating state of cells
  • Method for evaluating state of cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Cells P19CL6 derived from mouse fetal carcinoma cells were induced to differentiate into cardiomyocytes, and glycomics of undifferentiated and differentiated cells was performed.

[0163] will be 3.7×10 5 Cells / 6cm dish (dish) from mouse fetal carcinoma cell P19CL6 were inoculated into DMEM medium containing 10% fetal bovine serum, and for the differentiation induction group, added 1% dimethyl sulfoxide (DMSO) For culture, as for the control group, culture was directly carried out without adding any substance. On the 16th day after the start of the culture, after confirming all beating (beating) in the differentiation induction group, the cells of the two groups were recovered, and all N-bonded sugar chains in the cells were captured and purified by the above-mentioned glycoblotting method. The total cellular protein equivalent to 200 μg was analyzed, and a quantitative expression profile was obtained by MALDI-TOF / MS. The expression amount of each sugar chain shown in...

Embodiment 2

[0177] The mouse embryonal tumor cell P19C6 was induced to differentiate into neural cells, and the following three steps were performed, namely undifferentiated, differentiated intermediates, and glycomics of differentiated cells.

[0178] Step 1: Undifferentiated Cells

[0179] The P19C6 cells were cultured in a cell culture dish (cell culture dish) using 15% FBS / DMEM as a culture medium for several days (subsequent culture). When reaching 80-90% confluency, recover the cells for glycomics test, or proceed to step 2 for inducing differentiation.

[0180] Step 2: Differentiate intermediate cells

[0181] For the cells that reached 80-90% confluence in step 1, a cell suspension was prepared by trypsin treatment, and a bacterial-grade Petri dish was used for hanging drop culture. At this point, 10 μL of cell suspension (10 5cells / mL) 100spots for suspension culture to form cell aggregates. As a medium, 10% FBS / αMEM supplemented with 1 μM retinoic acid was used for diffe...

Embodiment 3

[0198] Mouse ES cells were induced to differentiate with retinoic acid, and glycomics experiments were performed on undifferentiated and differentiated cells. The differentiation induction method and the quantitative analysis of sugar chain expression levels were carried out in the same manner as in Example 2. The quantitative analysis results of sugar chain expression levels are shown in Figure 3-1 and Figure 3-2 (the vertical axis of Figure 3-1 is enlarged). Further, based on this result, subclassification was performed with sugar chains of types thought to have a structure bisecting GlcNAc and types not having such a structure, and the results are shown in Figs. 3-3. This result indicates that cell differentiation can be quantitatively monitored by this method.

[0199] In addition, sugar chains of a type having bisected GlcNAc, sugar chains of a type not having bisected GlcNAc, and sugar chains of a high mannose type are as follows, respectively.

[0200] The sugar chain...

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Abstract

Provided are a novel marker of the state of cells and the differentiation level of the same which is usable as a substitute for a gene or a protein; a means for quantitatively understanding this marker; and a means for understanding the state of specific cells and the differentiation level of the same using this marker. A method for determining a sugar chain category to be used for evaluating the state of cells. The quantitative profile of N-linked sugar chains of cells before a change in the state thereof is obtained. Next, the quantitative profile of N-linked sugar chains contained in the cells after the change in the state thereof is obtained. Sugar chains showing variations in the contents thereof between these two quantitative profiles are extracted and the individual sugar chains contained in the extracted sugar chains are classified into the following categories based on sugar chain type: either the high-mannose type or the non-high-mannose type; (1) the presence / absence of a bisect; (2) the number fucose residues(0, 1, or 2 or more); (3) the presence / absence of sialic acid; and (4) the degree of branching (2 or 3 or more). From these five categories, a sugar chain category appropriate for evaluating the state of cells is determined. A method for evaluating the state of cells and the differentiation of cells using the thus determined sugar chain category.

Description

[0001] Cross-references to related applications [0002] This application claims the priority of Japanese Patent Application No. 2008-298819 for which it applied on November 21, 2008, and the whole description content is especially taken in here as an indication. technical field [0003] The present invention relates to a method for evaluating the state of cells, for example, a method for evaluating the differentiation level of stem cells or precursor cells induced to differentiate, particularly, the differentiation level of stem cells or precursor cells induced to differentiate into cardiomyocytes or nerve cells method of evaluation. Background technique [0004] With the development of cell engineering and regenerative medicine, methods for quantitatively and rapidly evaluating the state of cells are required. Conventionally, methods for evaluating the state of cells based on the expression levels of specific genes and proteins have been widely used. [0005] An example ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48G01N27/62G01N33/00
CPCG01N33/5023G01N2400/00
Inventor 西村绅一郎天野麻穗武川泰启
Owner HOKKAIDO UNIVERSITY