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New undifferentiated stem cell removal and myocardial purification and refinement culture medium

A technology for cell culture and cardiomyocytes, applied in the direction of cell culture medium, cell culture active agent, cell culture support/coating, etc.

Active Publication Date: 2017-08-18
HEARTSEED INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Also, in times of rapid energy demand, energy is obtained by maximizing anaerobic respiration of the glycolytic system, so that pyruvate production exceeds its consumption, leading to activation of the lactic acid fermentation pathway

Method used

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  • New undifferentiated stem cell removal and myocardial purification and refinement culture medium
  • New undifferentiated stem cell removal and myocardial purification and refinement culture medium
  • New undifferentiated stem cell removal and myocardial purification and refinement culture medium

Examples

Experimental program
Comparison scheme
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Embodiment approach

[0084] Based on the above-mentioned first embodiment of the present invention, the inventors of the present invention cultured cells in a cell culture medium that did not contain glutamine in the amino acid composition; Serine and glycine-free cell culture medium; or by culturing in a cell culture medium that does not contain arginine in the amino acid composition, cell death can be induced to undifferentiated stem cells, and therefore, as a result, it is possible to provide A method for inducing cell death in undifferentiated stem cells.

[0085] In the first embodiment of the present invention, in order to induce cell death of undifferentiated stem cells, the cell culture is continued for 12 hours to 360 hours, preferably 24 to 240 hours, more preferably 48 to 120 hours.

[0086] As described above, when the various cardiomyocyte culture conditions listed in Patent Document 2 are applied to human cells, there may be very few remaining non-cardiomyocytes or undifferentiated ...

Embodiment 1

[0125] Example 1: Culture of Cells

[0126] In this example, culture of undifferentiated stem cells (embryonic stem cells and induced pluripotent stem cells), culture of differentiated cells, and culture of cardiomyocytes differentiated from undifferentiated stem cells were performed.

[0127] Human embryonic stem cells (hESCs) were obtained from Professor Norio Nakatsuji of the Stem Cell Medical Research Center affiliated with the Institute of Regenerative Medicine, Kyoto University, and human induced pluripotent stem cells were obtained from Professor Shinya Yamanaka, National University Corporation, Kyoto University iPS Cell Research Institute . The human embryonic stem cells and human induced pluripotent stem cells were maintained in undifferentiated culture using Matrigel (BD Bioscience, Cat No. 354277). As a culture medium, mTeSR1 (STEMCELL Technologies Inc., Cat No. 11875-119) was used. As a culture medium for maintaining undifferentiation, besides mTeSR1, there are E...

Embodiment 2

[0136] Example 2: Amino Acid Requirements of Cultured Undifferentiated Stem Cells Under Culture Conditions

[0137] In this example, using human embryonic stem cells or human induced pluripotent stem cells as undifferentiated stem cells, it was investigated which amino acid is required for undifferentiated stem cells under culture conditions.

[0138] To study the depletion of non-essential and semi-essential amino acids that are particularly active in human embryonic stem cells, the amino acid concentrations in the culture medium were determined. Specifically, in a 3.5 cm culture dish, 1.5 × 10 6 Cells were cultured, and then the composition of the culture medium before the initiation of cell culture and the composition of the culture medium after cell culture were analyzed.

[0139] Amino acid analysis was carried out according to the system of Xinbao et al. (Shimbo, K., Rapid Commun. Mass Spectrom., 2009, 23, 1483-1492). The supernatant after cell culture was transferred ...

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Abstract

The present invention addresses the problem of finding new conditions that make it possible to more completely induce cell death in non-myocardial cells or in undifferentiated stem cells and to select only myocardial cells. In order to solve such a problem, the present application: provides a cell culture fluid that is used to cause cell death in undifferentiated stem cells, wherein glutamine is not contained in the amino acid composition; and further provides is a method for inducing cell death in non-myocardial cells by performing culturing in the cell culture fluid. The present application further provides a cell culture fluid that is used to selectively select myocardial cells, in which lactic acid, pyruvic acid, or fatty acid is added, sugar is not contained, and glutamine is not contained in the amino acid composition. Further provided is a method to selectively select myocardial cells by culturing a mixture of myocardial cells and non-myocardial cells in the cell culture fluid.

Description

technical field [0001] The present invention is concerned with providing a medium that can be used to remove undifferentiated stem cells and purify refined cardiomyocytes. Background technique [0002] Since the cardiomyocytes in the adult human body lose their proliferative activity, they have to continue to rely on heart transplantation for severe myocardial infarction, cardiomyopathy and other diseases. However, in recent years, research on pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has progressed, and it is possible to differentiate / induce these pluripotent stem cells to produce As cardiomyocytes, the cardiomyocytes thus induced are used in transplantation medicine. [0003] However, cardiomyocytes under non-physiological conditions (i.e., in vitro conditions) differentiate in the same manner as physiological development, first forming undifferentiated mesoderm cells, some of which undergo presumptive cardiomyoc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2500/32C12N2500/38C12N2501/998C12N2501/999C12N2506/02C12N2506/45C12N2533/52C12N2533/90C12N5/00C12N5/0018C12N5/0068C12N5/0087C12N5/0607C12N5/0623C12Q1/045C12N2500/30C12N2500/34
Inventor 福田惠一藤田淳远山周吾
Owner HEARTSEED INC