Method of isolation of culture of vascular endothelial cells, medium for maintaining characteristics of vascular endothelial cells, and culture method including same
Inventors
LEE, Shin Jeong • Yoon, Young Sup
Assignees
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Publication Number
US-12447181-B2
Publication Date
2025-10-21
Expiration Date
Abstract
The present specification provides: a method of isolation of a pure culture of vascular endothelial cells, the method capable of isolating homogeneous endothelial cells adhered to a matrix for a specific time in a cell line of an endothelial cell lineage differentiated from human pluripotent stem cells; a medium for maintaining characteristics of vascular endothelial cells, comprising high-purity vascular endothelial cells isolated through the method, 4 ng/ml to 6 ng/ml of FGF2, 5 ng/ml to 10 ng/ml of EGF, 10 ng/ml to 30 ng/ml of VEGF-A, 20 ng/ml to 50 ng/ml of ascorbic acid, and DMEM/F-12 as active ingredients; and a culture method comprising same.
Core Innovation
The invention provides a method of separating vascular endothelial cells by obtaining a cell line of an endothelial cell lineage differentiated from human pluripotent stem cells, filtering the obtained cell line using a filter, and culturing the filtered cell line on a matrix for a period of 4 to 20 hours. The method then separates homogenous endothelial cells attached to the matrix from the cultured cell line.
In the described workflow, the separation relies on timed matrix adhesion following filtering of a pluripotent-stem-derived endothelial lineage cell line, producing a separated fraction enriched in endothelial markers. The approach is directed to obtaining homogenous endothelial cells attached to the matrix that represent the endothelial component of the cultured cell line.
The method is further defined by selecting filter pore spacing, matrix composition, and endothelial maintenance conditions, including DMEM/F-12 supplemented with defined growth factors and ascorbic acid. The resulting separated endothelial cells show marker enrichment, including CDH5 and VWF, with CDH5 maintained through passage in the described maintenance method.
Claims Coverage
The document includes one independent claim. The independent claim covers a multi-step separation workflow based on filtration and timed matrix adhesion, followed by separation of matrix-attached homogenous endothelial cells, with dependent claims adding quantitative and composition constraints and measurable marker-enrichment parameters.
Separating vascular endothelial cells via filter and timed matrix adhesion
obtaining a cell line of an endothelial cell lineage differentiated from human pluripotent stem cells from a differentiation medium; filtering the obtained cell line using a filter; culturing the filtered cell line on a matrix for a period of 4 to 20 hours; and separating homogenous endothelial cells attached to the matrix from the cultured cell line.
Filtering with a defined pore-spacing range
using a filter whose pore spacing is between 20 μm and 40 μm.
Matrix composed of collagen at a defined concentration
using a matrix made of collagen at a concentration of 0.1 mg/ml.
Endothelial maintenance using DMEM/F-12 with defined supplements
culturing the filtered cell line in DMEM/F-12 medium supplemented with cell growth factors and ascorbic acid.
Using specified endothelial growth factor supplements
using cell growth factors that include at least one specified growth factor such as FGF family members, EGF, KGF, HGF, TGF-α/TGF-β2, angiopoietins, erythropoietin, neuropilin, IGF-1, osteopoline, pleiotrophin, activin, endothelin 01, and VEGF-A.
CDH5 gene expression increases after separation
the gene expression level of CDH5 increases to 12 times higher after the separation.
Overall, the claim set centers on separating homogenous vascular endothelial cells by filtering a human pluripotent-stem-derived endothelial lineage cell line, applying timed matrix adhesion, and separating matrix-attached cells, while dependent claims specify filter pore spacing, collagen matrix composition, endothelial maintenance medium components, defined growth-factor supplements, and quantified CDH5 upregulation after separation.
Stated Advantages
Simpler and more economical purification compared with conventional purification.
Avoids animal-derived serum/feeder components.
Supports mass production.
Provides angiogenic/vascular regeneration capability for cardiovascular disease treatment.
Documented Applications
Treatment of cardiovascular diseases including ischemic heart disease, myocardial infarction, and stroke, using a cell therapeutic composition comprising the separated cells.
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