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Method for preparing pellets of chondrocytes from human induced pluripotent stem cells, and use thereof

A technology of pluripotent stem cells and chondrocytes, applied in the field of preparation of chondrocyte particles, can solve the problems of low differentiation speed, increased time, differentiation or deformation, etc.

Pending Publication Date: 2021-01-12
伊普塞尔有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has the following disadvantages: the differentiation rate is low and the phenotype is unstable, so unnecessary differentiation or deformation is likely to occur during the differentiation process, and it is necessary to extract a large amount of
And there are also problems such as apoptosis and calcification of chondrocytes due to blood vessel penetration due to the expression of genes related to cell hypertrophy after implantation in the body
[0028] Various methods for preparing chondrocytes from human induced pluripotent stem cells have been reported, but these methods have limitations in that the time required to differentiate from human induced pluripotent stem cells into chondrocytes is increased, or chondrocytes occur in cultured pellets The loosening of granules, or the acquisition of not fully mature chondrocytes, or differentiation into chondrocytes or non-chondrocytes, the degree of differentiation of each chondrocyte is different

Method used

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  • Method for preparing pellets of chondrocytes from human induced pluripotent stem cells, and use thereof
  • Method for preparing pellets of chondrocytes from human induced pluripotent stem cells, and use thereof
  • Method for preparing pellets of chondrocytes from human induced pluripotent stem cells, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Preparation of Human Induced Pluripotent Stem Cells

[0127] Human induced pluripotent stem cells (hiPSC) were prepared from cord blood mononuclear cells (CBMC). CBMCs obtained from the cord blood bank of St. Mary's Hospital in Seoul, Korea were used. Cord blood was diluted with phosphate buffered saline (PBS), and centrifuged through a Ficoll gradient at 850×g for 30 minutes to collect CBMCs, washed, frozen and stored until use. After the CBMC were thawed before use, they were resuspended in StemSpan medium (STEMCELL Technological, Vancouver, British Columbia, Canada) to which CC110 cytokine cocktail (Cytokine cocktail; STEMCELL) had been added, and incubated at 37°C in 5% CO 2 Cultured in the incubator for 5 days.

[0128] The cultured CBMC were divided into 3×10 5 was inoculated into 24-well plates, and CBMC-derived hiPSCs were obtained by inducing reprogramming using the CytoTune™-iPS20 Sendai reprogramming kit (sendai reprogram kit; A16518, Life Technologies) ac...

Embodiment 2

[0131] Formation of embryoid bodies from hiPSCs

[0132] The CBMC-derived hiPSCs prepared in the above Example 1 were resuspended in Aggrewell medium (STEMCELL) at 2×10 6 Concentrations of cells / well were seeded into 100-mm culture plates. The inoculated hiPSCs were cultured in a 37 °C incubator for 24 hours, and the next day the medium was replaced with TeSR-E8 medium (containing 543 μl / ml sodium bicarbonate (NaHCO 3 ), 64μg / ml of L-ascorbic acid 2-phosphate magnesium (L-Ascorbic acid2-phosphate magnesium), 14ng / ml of sodium selenite (Sodium selenite), 10 7 μg / ml of transferrin (Transferrin), 20 μg / ml of insulin, 100ng / ml of fibroblast growth factor-2 (FGF-2) and 2ng / ml of transforming growth factor beta 1 (transforming growth factor beta 1, TGF -β1) After adding glutamine (glutamine) and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) to the DMEM / F12 medium, an additional 6 days of adherent culture was carried out to form and obtain pseudo embryoid body.

Embodiment 3

[0134] Formation and isolation of growing cells from embryoid bodies

[0135] The embryoid body (embryoid body, EB) formed and obtained in the above-mentioned Example 2 was suspended in DMEM medium (Thermo Fisher Scientific) containing 20% ​​fetal bovine serum (Fetal Bovine Serum) and 10% penicillin / streptomycin, on gelatin-coated plates at 37°C in 5% CO 2 The formation of outgrowth cells (OG) was induced by culturing for 7 days. For this, culture plates were used which were completely dry after 30 minutes of coating the bottom with 0.1% gelatin.

[0136] Formed OG cells were isolated from gelatin-coated plates and passed through a 40 μm-sized cell strainer (cellstrainer; Thermo Fisher Scientific) to remove EB clumps, thereby isolating and obtaining only single-cell units of OG cells.

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Abstract

The present application claims priority for Korean patent application No. 10-2018-0072875 filed on June 25, 2018, the entire description of which is a reference for the present application. The present invention relates to a method for preparing chondrocyte pellets, comprising the steps of: (a) culturing human induced pluripotent stem cells so as to form and obtain embryoid bodies; (b) inducing the embryonic bodies obtained in step (a) to become outgrowth cells and isolating same; and (c) culturing the outgrowth cells isolated in step (b) in a pellet form. In addition, the present invention relates to: a pharmaceutical composition to be used in the treatment of arthritis, comprising the chondrocyte pellets prepared by the preparation method; or a method for preventing or treating arthritis, comprising a step of administering the chondrocyte pellets prepared by the method to an arthritis patient. Chondrocyte pellets of the present invention have a remarkably high rate of differentiationinto chondrocytes, have a uniform size, and have a homogeneous differentiation degree, and a pharmaceutical composition for treating arthritis, comprising same, has an excellent cartilage regeneration effect when administered to the site of cartilage damage, and is in an injectable form, which does not require surgery, such that through a simple procedure, the pain of a patient is relieved and acontinuous arthritis treatment effect can be provided even after the procedure.

Description

technical field [0001] This application claims the priority of Korean Patent Application No. 10-2018-0072875 filed on June 25, 2018, and the entire contents of the above specification are references of this application. [0002] The present invention relates to a method for preparing chondrocyte granules, comprising: step (a), forming and obtaining an embryoid body (embryoid body) by culturing human induced pluripotent stem cells; step (b), inducing The embryoid bodies obtained in the above step (a) become outgrowth cells and are separated; and step (c), culturing the outgrowth cells isolated in the above step (b) in the form of pellets. [0003] The present invention also relates to a pharmaceutical composition for treating arthritis, which comprises the chondrocyte particles prepared by the above preparation method. [0004] The present invention also relates to a method for preventing or treating arthritis, which includes the step of administering the chondrocyte particles...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/074A61K35/32A61P19/02A61P29/00A61K31/728
CPCA61P19/02A61K35/32C12N5/0655C12N2533/50C12N2533/54C12N2513/00C12N2506/45C12N2500/25C12N2501/115C12N2501/15C12N2501/39A61K31/728A61K2300/00C12N2501/155C12N2500/99
Inventor 朱址贤南有俊林芮利
Owner 伊普塞尔有限公司
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