Top > Search of International Patents > TISSUE FRAGMENT

TISSUE FRAGMENT

Foreign code F180009641
File No. 4601
Posted date Nov 21, 2018
Country WIPO
International application number 2015JP079364
International publication number WO 2016060260
Date of international filing Oct 16, 2015
Date of international publication Apr 21, 2016
Priority data
  • P2014-212008 (Oct 16, 2014) JP
  • P2014-212010 (Oct 16, 2014) JP
  • P2014-212014 (Oct 16, 2014) JP
Title TISSUE FRAGMENT
Abstract The present invention provides a method for producing a tissue fragment, particularly a myocardial tissue fragment, which contains cultured cells having an oriented configuration, said method comprising culturing cells on an oriented fiber sheet. The present invention also provides a tissue fragment produced by the method according to the present invention. The present invention further provides: a device for evaluating an electrophysiological function of a cell; and a method for evaluating an electrophysiological function of a cell. The present invention still further provides a sheet for culturing cells.
Outline of related art and contending technology BACKGROUND ART
Severe heart failure including myocardial infarction patient is about 80 million people in Japan of the disease and, about 18 million people die each year. At present the only effective treatment for heart transplantation but, in Japan and extremely serious shortage of donors, such as immune rejection also suffers. Pluripotent stem cells (for example, embryonic pluripotent stem cells (ES) or induced pluripotent stem cell (iPS) ) established by, as a therapeutic alternative to heart transplantation, myocardial cell regeneration treatment and can be formed to restore myocardial function expected. Is for use in therapy in clinical transplantation, to imitate the structure having the same sequence as in vivo, the mature high safe myocardial tissue piece is required to be obtained.
A method for inducing human ES/iPS cells into cardiac muscle cells have been proposed various. Used to induce a current of heart muscle cells is the most general method, cytokine of embryoid bodies (for example, DKK1, bFGF, activin A (ActivinA), and BMP4) and the suspension culture, or mouse END2 (the proximal endoderm-like cells (visceralendoderm) ) co-culture is the removal of the (non-patent document 1-4).
The inventors of the present invention, novel low-molecular-weight compound is used as an efficient stable myocardial differentiation induction method (-98%) clinical grade proposed (non-patent document 5, patent document 1). Myocardial cells induced by such a method, human differentiated cell is an important maturation marker of heart muscle cells is the amount of β-MHC gene expression is less than the amount of expression of the adult human heart (-10%), sequence does not have the myocardial tissue structure, sufficient mature electrophysiological there occurs a problem cannot be obtained.
Myocardial cell transplantation method has been proposed as the method can be divided roughly into the following 2 types: (1) a disease of heart failure and cardiac cells by direct injection of the cells in a sheet form of the method (2), to a method for implanting a disease.
Derived from human ES cells by direct injection of heart muscle cells, improve myocardial infarction in guinea pig has succeeded (non-patent document 6) report. However direct injection method, into a cell solution which flows out from the myocardial tissue, which are applied to the systemic blood flow such as this happens, the low efficiency of cell transplantation is pointed out. The number of the cell engraftment % or less, is not sufficient therapeutic effect is considered to be.
In addition, the temperature-responsive culture dish as a cell sheet technique of the cells with a technology in which a sheet has been proposed (non-patent document 7). And using such a technique myocardial sheet which, in clinical trials to transplant it has been started. However, this constructed using the techniques of the myocardial cell is a single-layered sheet, orientation is not randomly distributed, which mimic the structure of the myocardium within the living body cannot be. Therefore, myocardial cell contraction force is weak, the degree of maturity is low, a problem in that the handling is difficult to lower strength still remains. In addition, remains formed on a support sheet for the production of implantable cardiac muscle cells has not been reported yet.
More effective for use in transplantation and assays, in biological cells which mimic the structure and the fabrication of a piece of tissue structure has been desired, micro-engineering technique in biological cells which mimic the structure studies have been attracting attention. For example in vivo, and a cardiac muscle cells are an array structure, the microline or, punch or micro, fiber or other technique, attempts are sequenced cardiomyocyte culture have been reported (non-patent document 8-14).
The evaluation of the development of a drug safety in conventional, animal-derived primary culture cells or an animal has been used, and the functions in cells of the tissue human bio species difference, various problems are caused by a lot of the differences in the are. In addition, at present, all areas of the electrocardiogram QT extension for a medicament for cardiotoxicity in non-clinical experimental practice guideline is obtained. Such as iPS cells induced pluripotent stem cells into cardiac muscle cells, myocardial tissue obtained and cultured to evaluate the toxicity of the drug piece applied in the evaluation with respect to the behavior or a high demand of the industry.
Is the electrophysiological evaluation of cells, intracellular recording the field potential can be patch-clamp method is have been frequently used. Patch-clamp method is however, an infested cells by patch electrodes, a long-time measurement difficult. In addition, the procedure is not suitable for screening difficult.
Owing to the development of multi-electrode technique, the human ES/iPS cell-derived multi-electrode on the cultured myocardial cells, their electrophysiology function and it is possible to evaluate the (non-patent document 15 and 16). This technique for measuring activity extracellular potential, without damaging the cells, while the function observed for a long period of cell culture, can be measured. Essential to this technology in drug discovery as well as evaluate the toxicity of the drug, such as human ES cells and iPS cells from pluripotent stem cell into a cardiomyocyte differentiation induction and maturity can be possible to monitor the processes respectively.
Myocardial differentiation induction obtained in conventional myocardial cell tissue of heart muscle cells are aligned in a direction not scattered muscle contraction force is weak, and obtaining an electrocardiogram QT extension experiment or the like to arrhythmia occurs frequently, weak adhesion between the electrodes, the long-term culture cannot be set, for use in drug discovery applications, in the evaluation system is provided as a stable but still has a problem. In the first place, the cardiomyocytes random, or lumps had grown to electrical signals from myocardial tissue, myocardial tissue in vivo behavior difficult to say that sufficiently mimic.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 配向性ファイバーシートと、該配向性ファイバー上の培養心筋細胞とを含む、心筋組織片。

[請求項2]
 配向性ファイバーシートが培養心筋細胞層の間に介在している、心筋組織片。

[請求項3]
 該心筋細胞が多能性幹細胞から誘導された心筋細胞である請求項1または2に記載の心筋組織片。

[請求項4]
 配向性ファイバーシートにおけるファイバーの直径が0.1μm~5μm、シートの幅1mm当たりのファイバーの本数30~15000本、シートの厚みが0.1μm~20μmである、請求項1~3いずれかに記載の心筋組織片。

[請求項5]
 心筋細胞層の数が10層以上である、請求項1~4いずれかに記載の心筋組織片。

[請求項6]
 配向性ファイバーシートが生体内分解性の材料で構成されている、請求項1~5いずれかに記載の心筋組織片。

[請求項7]
 配向性ファイバーシートが分解されにくい素材で構成されている、請求項1~5いずれかに記載の心筋組織片。

[請求項8]
 配向性ファイバーシートが、周囲にフレーム部を有する、請求項1~7何れかに記載の心筋組織片。

[請求項9]
 フレーム部が配向性ファイバーシートと同一素材で構成されている、請求項8記載の心筋組織片。

[請求項10]
 フレーム部が配向性ファイバーシートと別素材で構成されている、請求項8記載の心筋組織片。

[請求項11]
 複数の配向性ファイバーシートと、該複数の配向性ファイバーシート上で培養された複数の心筋細胞層を含み、配向性ファイバーシートが同一配向方向にて心筋細胞層間に介在している、請求項1~10いずれかに記載の心筋組織片。

[請求項12]
 さらに培養用チャンバー部分を有し、該心筋細胞が、該配向性ファイバーシート上の培養用チャンバー中にある、請求項1~4、6および7いずれかに記載の心筋組織片。

[請求項13]
 該心筋細胞のβ-MHCの発現量が成人の正常心筋細胞の10%以上である、請求項1~12いずれかに記載の心筋組織片。

[請求項14]
 細胞の機能評価に用いるための、請求項1~13いずれかに記載の心筋組織片。

[請求項15]
 移植に用いるための、請求項6、8~11および13いずれかに記載の心筋組織片。

[請求項16]
 複数の心筋組織片を配向性ファイバーシートの配向が同一となるよう重ねて培養する工程を含む、請求項11に記載の心筋組織片の製造方法。

[請求項17]
 得られた心筋組織片の電気信号を、該心筋組織片と接触させた多電極アレイにより検出して心筋細胞の機能を評価する工程をさらに含む、請求項16に記載の方法。

[請求項18]
 請求項14に記載の心筋組織片を多電極アレイに接触させて電気信号を検出することを含む、心筋細胞の機能評価方法。

[請求項19]
 細胞機能を指標に医薬候補物質の有効性の評価を行う、請求項18に記載の方法。

[請求項20]
 細胞機能を指標に物質の安全性の評価を行う、請求項18に記載の方法。

[請求項21]
 滅菌された包装材および、その中に封入されている請求項1~15いずれかに記載の心筋組織片を含む製品。

[請求項22]
 多電極アレイおよび、該多電極アレイ上の配向性ファイバーシートを含む、心筋細胞機能評価用デバイス。

[請求項23]
 該配向性ファイバーシート上に培養用チャンバー部分をさらに有する、請求項22に記載の心筋細胞機能評価用デバイス。

[請求項24]
 該培養用チャンバー部分の中に培養心筋細胞をさらに含む、請求項23に記載の心筋細胞機能評価用デバイス。

[請求項25]
 請求項24記載のデバイスの培養心筋細胞の電気信号を多電極アレイにより検出する工程を含む、心筋細胞機能評価方法。

[請求項26]
 シート状細胞培養部と該細胞培養部の周囲にフレームを含む、細胞培養用シート。

[請求項27]
 該シート状細胞培養部がファイバーシートである、請求項26に記載の細胞培養用シート。

[請求項28]
 シート状細胞培養部とフレームが同一素材で構成されている、請求項26または27に記載の細胞培養用シート。

[請求項29]
 該シート状細胞培養部とフレームが異なる素材で構成されている、請求項26または27に記載の細胞培養用シート。

[請求項30]
 該ファイバーシートにおいて、ファイバーがランダムな構造を有する、請求項27~29いずれかに記載の細胞培養用シート。

[請求項31]
 該ファイバーシートにおいて、ファイバーが配向性の構造を有する、請求項27~29いずれかに記載の細胞培養用シート。

[請求項32]
 該ファイバーシートが生体内分解性の素材で構成されている、請求項27~31いずれかに記載の細胞培養用シート。

[請求項33]
 該ファイバーシートが分解されにくい素材で構成されている、請求項27~31いずれかに記載の細胞培養用シート。

[請求項34]
 フレーム上に、スペーサーをさらに備える、請求項26~33いずれかに記載の細胞培養用シート。

[請求項35]
 請求項26~34何れかに記載の細胞培養用シートと、該細胞培養用シート上の培養細胞を含む、組織片。

[請求項36]
 複数の細胞培養用シートが培養細胞層間に介在している、請求項35記載の組織片。

[請求項37]
 滅菌された包装材および、その中に封入されている請求項35または36に記載の組織片を含む製品。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KYOTO UNIVERSITY
  • Inventor
  • LIU, Li
  • LI, Junjun
  • MINAMI, Itsunari
  • CHEN, Yong
  • NAKATSUJI, Norio
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
Please contact us by e-mail or facsimile if you have any interests on this patent. Thanks.

PAGE TOP

close
close
close
close
close
close