Multilayered cell sheet of cardiac stem cells and method of preparing the same

Patent metadata

Field	Value
Publication number	US-2021196761-A1
Application number	US-201715779073-A
Country	US
Kind code	A1
Filing date	Dec 18, 2017
Priority date	Dec 20, 2016
Publication date	Jul 1, 2021
Grant date	—

Abstract

Disclosed are a multilayered cell sheet of cardiac stem cells (CSCs) and a method of manufacturing the same. In particular, the present disclosure provides a method of manufacturing a multilayered cell sheet according to a single step culture procedure by using, as a three-dimensional matrix, a biodegradable natural polymer hydrogel and embedding CSCs in the hydrogel. The multilayered cell sheet of the present disclosure does not require any special device for the manufacturing, is manageable with good physicomechanical property, increases a cell engraftment rate after transplantation based on sufficient accumulation of various growth and protective factors and extracellular matrix between cells, and is also self-assembled by the cell-mediated hydrogel compaction, making nutrients transfer easy. Therefore, the multilayered cell sheet of the CSCs is expected to be usefully applicable as a therapeutic agent for myocardium regeneration.

First claim

1 . A method of manufacturing a multilayered cell sheet of cardiac stem cells (CSCs), the method comprising steps of:
(1) isolating and culturing CSCs; (2) embedding the cultured CSCs in a hydrogel; (3) culturing the hydrogel comprising the CSCs embedded therein under stressed culture conditions in which a physical support is applied; and (4) culturing the resulting hydrogel of step (3) under non-stressed culture conditions in which a physical support is removed.

2 . The method of claim 1 , wherein the CSCs are isolated from cardiac tissue.

3 . The method of claim 1 , wherein step (2) comprises mixing the cultured CSCs with a hydrogel in a solution base and converting the solution phase to a gel phase so that the CSCs are uniformly distributed in a three-dimensional manner in the hydrogel.

4 . The method of claim 1 , wherein the hydrogel comprises a fibrin hydrogel.

5 . The method of claim 4 , wherein the fibrin hydrogel comprises fibrinogen at a final concentration in a range of about 0.1% to about 1%, thrombin at a final concentration in a range of about 0.2 U/ml to about 2 U/ml, and plasminogen activator inhibitor (PAI) at a final concentration in a range of about 10 μg/ml to about 500 μg/ml.

6 . The method of claim 5 , wherein the PAI comprises tranexamic acid, aminomethylbenzoic acid, aminocaproic acid, or aprotinin.

7 . The method of claim 1 , wherein the cultured CSCs of step (2) are added to a thrombin solution.

8 . The method of claim 1 , wherein the cultured CSCs of step (2) have a density in a range of about 1×10 6 /ml to about 1×10 8 /ml.

9 . The method of claim 1 , wherein the stressed culture conditions of step (3) in which the physical support is applied comprises casting the hydrogel comprising the CSCs embedded therein on a circular, rectangular, or square mold so that the CSCs are cultured under a condition in which a physical support is applied.

10 . The method of claim 1 , wherein the stressed culture conditions of step (3) induce cell-to-cell adhesion and cell-to-hydrogel polymer adhesion.

11 . The method of claim 1 , wherein the stressed culture conditions of step (3) comprise accumulating extracellular matrix (ECM), anti-inflammatory factors, protective factors for myocardial cells, cardiomyogenic factors, and proangiogenic factors, which are produced and secreted from the CSCs.

12 . The method of claim 11 , wherein the ECM comprises fibronectin, laminin, and collagen type IV.

13 . The method of claim 11 , wherein the anti-inflammatory factors comprise interleukin (IL), such as IL-6 or IL-10, or transforming growth factor-β (TGF-β).

14 . The method of claim 11 , wherein the protective factors for myocardial cells comprises IL-6, IL-11, cardiotrophin-1, hepatocyte growth factor (HGF), insulin-like growth factor (IGF)-1, or leukemia inhibitory factor (LIF).

15 . The method of claim 11 , wherein the cardiomyogenic factors comprise fibroblast growth factor (FGF), bone morphogenetic protein (BMP), or Sox-17.

16 . The method of claim 11 , wherein the proangiogenic factors are angiopoietin based or FGF based, or comprise hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF).

17 . The method of claim 1 , wherein the non-stressed culture conditions of step (4) induce cell-mediated hydrogel compaction so that water and culture media in the hydrogel are extruded.

18 . The method of claim 1 , wherein the multilayered cell sheet of the CSCs consists of about 10 layers to about 50 layers.

19 . A multilayered cell sheet of cardiac stem cells (CSCs), comprising:
a hydrogel in which the CSCs are embedded; and extracellular matrix (ECM), anti-inflammatory factors, protective factors for myocardial cells, cardiomyogenic factors, and proangiogenic factors, which are secreted from the CSCs and accumulated in the hydrogel.

20 . The multilayered cell sheet of claim 19 , wherein the hydrogel comprises a fibrin hydrogel.

21 . The multilayered cell sheet of claim 20 , wherein the fibrin hydrogel contains fibrinogen at a final concentration in a range of about 0.1% to about 1%, thrombin at a final concentration in a range of about 0.2 U/ml to about 2 U/ml, plasminogen activator inhibitor (PAI) at a final concentration in a range of about 10 μ/ml to about 500 μ/ml.

22 . The multilayered cell sheet of claim 19 , wherein the CSCs have a density in a range of about 1×10 6 /ml to about 1×10 8 /ml.

23 . The multilayered cell sheet of claim 19 , wherein the multilayered cell sheet of the CSCs consists of about 10 layers to about 50 layers.

24 . A composition for treating a heart disease, the composition comprising, as an active ingredient, the multilayered cell sheet of the CNCs of claim 19 or a culture of the multilayered cell sheet.

25 . The composition of claim 24 , wherein the composition reduces the degree of myocardium fibrosis that is induced by cardiac injury, promotes angiogenesis and myocardial regeneration, and promotes cardiac contraction function by increasing a thickness of myocardium.

26 . The composition of claim 24 , wherein the heart disease comprises ischaemic heart disease or primary (idiopathic) or secondary cardiomyopathy.

27 . The composition of claim 26 , wherein the primary (idiopathic) cardiomyopathy comprises dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), or restrictive cardiomyopathy.

Assignees

• Univ Inje Ind Acad Coop Found
• Univ Ajou Ind Academic Coop Found

Inventors

• Yang Young-Il
• Park Ki-Dong
• Lee Won-Jin
• Choi Min-Young
• Park Kyung-Min
• Lee Yun-Ki

Classifications

• C12N2501/105

  Insulin-like growth factors [IGF] · CPC title

• C12N2513/00

  3D culture · CPC title

• C12N2533/56

  Fibrin; Thrombin · CPC title

• C12N5/0657

  Cardiomyocytes; Heart cells · CPC title

• C12N2501/11

  Epidermal growth factor [EGF] · CPC title