Method for serum-free culture of cartilage cells and serum-free culture medium

A serum-free medium and chondrocyte technology, applied in the direction of cell culture active agent, bone/connective tissue cells, culture process, etc., can solve the problems of cell damage, chondrocytes that cannot be used for transplantation, and chondrocytes that cannot be well cultivated

Active Publication Date: 2017-02-22
REGENESIS SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Damage to cells if collagenase is overactive
Therefore, if serum-free culture is carried out in a state where collagenase is attached to cells, in addition to being unable to culture chondrocytes well, the resulting chondrocytes may not be applicable to transplantation etc.

Method used

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  • Method for serum-free culture of cartilage cells and serum-free culture medium
  • Method for serum-free culture of cartilage cells and serum-free culture medium
  • Method for serum-free culture of cartilage cells and serum-free culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Digestion of Tissue - Culture Preparation -

[0069] Primary human chondrocytes were isolated individually from articular cartilage biopsies and the samples were subdivided. Then, immerse in a 0.1% to 0.3% collagenase solution, and perform enzyme digestion at 37° C. for 4 to 5 hours. The obtained cells were collected by centrifugation at 1,200 rpm for 5 minutes.

[0070] FGF 100 ng / mL, SAG 0.5 μM, HC 400 ng / mL, IGF 5 ng / mL and insulin 5 μg / mL were added to DMEM medium and used as a medium. Cultured cells previously obtained at 1000 cells / cm 2 seeding density. At 37°C, 5% CO 2 cultured in an environment.

[0071] figure 1 It is a photograph in place of the drawing showing the cultured chondrocytes from the 0th day to the 16th day of culture in Example 1. It can be seen that the cartilage tissue increases slowly.

[0072] figure 2 It is a photograph in place of the drawing showing cultured chondrocytes from the 19th day to the 41st day of culture in Example 1. ...

Embodiment 2

[0087] FGF2 100 ng / mL, SAG 0.5 μM, HC 400 ng / mL, IGF 5 ng / mL, and insulin 5 μg / mL were added to DMEM medium to be used as a medium for subculture. The cultured cells in embodiment 1 were treated with 10000 cells / cm 2 seeding density. At 37°C, 5% CO 2 cultured in an environment.

[0088] Figure 4 It is a photograph in place of a drawing showing cultured chondrocytes from day 1 to day 13 in Example 2 (subculture). It was observed about 10 days from the start of the culture that stickiness was observed in the medium, and when the medium was removed with an aspirator, filaments were pulled out. From day 13, the viscosity of the medium increased.

[0089] Figure 5 It is a photograph in place of the drawing showing the cultured chondrocytes from the 18th day to the 28th day of culture in Example 2. On the 18th day of culture, 50 μg / mL of ascorbic acid was added to the medium. As a result of trial and error, it was found that the addition of ascorbic acid for such a period ...

Embodiment 3

[0098] In order to confirm that chondrocytes cultured in the serum-free medium of the present invention are cells suitable for transplantation, they were transplanted into nude mice to investigate whether cartilage was formed in vivo.

[0099] First, chondrocytes derived from human auricular cartilage were prepared according to the procedure described in Example 1, and cultured with the serum-free medium of the present invention. The cultured chondrocytes were impregnated in collagen cotton fabric, and transplanted together with the cotton fabric at two places on the back of nude mice. Transplantation surgery and management of nude mice were performed according to known methods. Six months after the transplantation, the back of the nude mouse was cut open, and it was confirmed whether the cells were fixed at the transplantation site and cartilage was formed.

[0100] Figure 8 This is a photograph showing the incision of the back of a nude mouse 6 months after transplantatio...

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Abstract

The invention provides a method for serum-free culture of human cartilage cells and a serum-free culture medium. The method for serum-free culture of cartilage cells includes an enzymatic treatment step for treating a human cartilage cell-containing tissue with a protease; an inhibitor-treatment step for, after the enzymatic treatment step, treating the tissue with an inhibitor for the aforesaid protease; and a culture step for, after the inhibitor-treatment step, culturing the tissue in a serum-free culture medium that contains kartogenin and / or SAG, ITS, FGF2 and hydrocortisone. A serum-free culture medium for culturing cartilage cells, the serum-free culture medium containing kartogenin and / or SAG, ITS, FGF2 and hydrocortisone.

Description

technical field [0001] The present invention relates to a method for culturing chondrocytes in which cells are treated with proteolytic enzymes before serum-free culture, and a serum-free medium suitable for the method. Background technique [0002] Japanese Unexamined Patent Publication No. 2003-125787 discloses a serum-free medium used in chondrocyte differentiation culture and a method for culturing chondrocytes using the serum-free medium. [0003] Japanese Patent No. 3638614 discloses a chondrocyte culture medium and a method for culturing chondrocytes using the culture medium. Furthermore, ITS is disclosed in this document. [0004] JP 2002-529071 A discloses a serum-free medium for chondrocyte-like cells. [0005] In Japanese Special Publication No. 2009-540826, a method for culturing chondrocytes is disclosed, which comprises: separately isolating primary human chondrocytes from articular cartilage biopsies, performing 1-hour enzymatic digestion with protease (Stre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N1/00
CPCC12N1/00C12N5/0655C12N2500/90C12N2500/25C12N2501/105C12N2501/115C12N2501/39C12N2500/36C12N2500/33C12N2500/38C12N2501/33C12N2501/734C12N2501/999
Inventor 矢永博子矢永克
Owner REGENESIS SCI CO LTD
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