Method for producing myocardial stem cell used for treatment and/or prevention of cardiac arrest

A manufacturing method and a technology for heart failure, which are applied in the field of manufacturing liposomes of cardiac stem cells, and cell preparations for treating and/or preventing heart failure, and can solve problems such as limited survival effect of transplanted cells and limited improvement effect of survival rate

Inactive Publication Date: 2019-08-02
卢卡科学株式会社
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As the problem of myocardial stem cell transplantation, for example, in the myocardial stem cell transplantation experiment in the pig ischemia-reperfusion model, the transplanted cell survival effect is limited (non-patent document 4); The effect of improving the survival rate in the transplantation experiment is limited (non-patent literature 5); etc.

Method used

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  • Method for producing myocardial stem cell used for treatment and/or prevention of cardiac arrest
  • Method for producing myocardial stem cell used for treatment and/or prevention of cardiac arrest
  • Method for producing myocardial stem cell used for treatment and/or prevention of cardiac arrest

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1. Preparation of CPC that delivers resveratrol to mitochondria

[0079] (1) Purification of mouse CPC

[0080] According to the method of Oh et al. (PNAS. 2003, 100, pp. 12313-12318), mouse CPC was isolated and purified as follows.

[0081] The heart was removed from 8-week-old c57BL6 / J male mice and subjected to collagenase treatment and Percoll density gradient treatment, thereby extracting a cell population containing CPC. After the obtained cell population was subjected to primary culture, the MACS system was used for sorting, and the selectively extracted Sca-1 positive CPCs were subcultured, thereby isolating mouse CPCs. For the isolated CPC, the quality of surface marker proteins was quantified by flow cytometry (FACS), and the gene expression levels of myocardial transcription factors and structural proteins were quantified by PCR. It was confirmed that these values ​​are consistent with the reported values ​​(not Display Data).

[0082] (2) Preparation of R...

Embodiment 2

[0089] Example 2. Cardiac myoblast protective effect of CPC delivering resveratrol to mitochondria (in vitro experiment)

[0090] (1) Import of RES-MITO-Porter to CPC

[0091] The CPC suspended in DMEM-F12 medium was divided into 1×10 6 Inoculate each well into a 6-well plate and incubate at 37°C for 24 hours. The RES-MITO-Porter prepared in (2) of Example 1 was added to each well and incubated for 2 hours, thereby introducing the RES-MITO-Porter into the CPC, and used it for subsequent experiments. This CPC into which RES-MITO-Porter was introduced is referred to as MA-cell.

[0092] (2) Cell survival rate under cell damage caused by doxorubicin

[0093] The rat cardiac myoblasts H9c2 cells (purchased from ATCC) and MA-cells prepared in (1) were mixed in DMEM-F12 medium to make H9c2 cells 3×10 4 Pcs / well, MA-cell is 1×10 4 Pieces / well, the mixture was incubated at 37°C for 24 hours. Add doxorubicin to the co-culture medium to a final concentration of 10μg / mL (low dosage) or 50μg / m...

Embodiment 3

[0099] Example 3. The protective effect of CPC delivering resveratrol to mitochondria on myocardial injury (in vivo experiment)

[0100] (1) Preparation of doxorubicin myocardial injury model mice and administration of each CPC

[0101] In the heart of normal mice (male C57 / BL6 mice aged 6 to 8 weeks) MA-cells (1×10 6 A) cell transplantation to prepare the MA-cell transplantation group (n=6). After 24 hours of transplantation, referring to the method of Zhang et al. (Nature Medicine 2012, 18, pp. 1639-1642), 200 μL of doxorubicin / PBS was administered into the abdominal cavity of mice at 25 mg / kg only once to induce myocardial damage. In the group without cell transplantation (untreated group) and the group in which CPC was transplanted instead of MA-cells (CPC transplantation group), myocardial injury was induced in the same manner as described above. In addition, a group without doxorubicin administration (normal group) was prepared for each group.

[0102] (2) Survival rate aft...

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Abstract

The present invention provides a novel myocardial stem cell for transplantation that enables maintaining of a therapeutic effect on and / or preventative effect against cardiac arrest for a prolonged period; a method for producing said myocardial stem cell; and a cell preparation containing said myocardial stem cell. The present invention pertains to: a production method for a myocardial stem cell to be used for the treatment and / or prevention of cardiac arrest, the method comprising a step for introducing into a myocardial stem cell a complex of a mitochondria-targeted carrier and a mitochondria activating agent; a cell produced by said production method and a cell preparation containing same; and a liposome used to produce said cell. The present invention is able to provide a myocardial stem cell that is capable of maintaining a therapeutic effect by cell transplantation and / or a preventative effect thereby for a prolonged period.

Description

Technical field [0001] The present invention relates to a method for producing a new cardiac stem cell for the treatment and / or prevention of heart failure, a cell population containing cardiac stem cells in which mitochondria are activated, and a cell population containing the cardiac stem cells or cell population for the treatment and / or prevention of heart failure Cell preparations and liposomes used to manufacture the cardiac stem cells. Background technique [0002] Heart failure is caused by heart-related diseases such as myocardial infarction, cardiomyopathy, or angina pectoris, or diseases other than the heart, such as high blood pressure, kidney disease, or side effects of chemotherapy for malignant tumors, and the function of the heart is reduced to the lungs and the whole body. Symptoms of supplying the necessary amount of blood. Heart failure is the leading cause of death after cancer in Japan. As its fundamental treatment, heart transplantation can be cited. Howeve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K9/127A61K31/05A61K35/34A61K47/24A61K47/42A61P9/04C12N5/074
CPCA61K9/127A61K31/05A61K35/34A61K45/00A61K47/24A61K47/42A61P9/04A61K9/0019C12N5/0662C12N2501/999
Inventor 原岛秀吉山田勇磨阿部二郎武田充人
Owner 卢卡科学株式会社
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