A fast and efficient method for isolating chondrocytes

A high-efficiency technology for chondrocytes, applied in the field of rapid and efficient separation of chondrocytes, can solve the problems of inability to digest and separate chondrocytes, denature type II collagenase, and reduce digestion, and achieve good protection, fast and efficient separation, and high activity Effect

Active Publication Date: 2019-10-25
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most common method for isolating chondrocytes is to obtain them through type II collagenase digestion, but this method has many shortcomings, mainly including: (1) Although type II collagenase is a mild digestive enzyme, it still exists The damage to cells, especially when the digestion is excessive, the damage to cells is very obvious; (2) Type Ⅱ collagenase is susceptible to denaturation due to the influence of temperature, pH, other proteases, etc., so type Ⅱ collagenase is used In the process of chondrocytes, it is very easy to lose activity, reduce the digestion effect, and seriously lead to the inability to realize the digestion and separation of chondrocytes

Method used

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  • A fast and efficient method for isolating chondrocytes
  • A fast and efficient method for isolating chondrocytes
  • A fast and efficient method for isolating chondrocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A method for rapidly and efficiently separating chondrocytes, comprising the steps of:

[0029] Obtain 10g of cartilage tissue sample, add 1mL type II collagenase to it, wash with PBS with double antibody, and cut it into pieces to 1mm 3 , then add 5 times the volume of cartilage tissue sample type II collagenase, place on a shaker at 37°C, add 0.5% human serum albumin at the same time, and digest at 100rpm for 30min;

[0030] Then add 3% amifostine solution, and continue to digest at 100 rpm for 4 hours; after digestion, filter through a 200-mesh filter, centrifuge to precipitate, and collect chondrocytes for counting.

Embodiment 2

[0032] A method for rapidly and efficiently separating chondrocytes, comprising the steps of:

[0033] Obtain 10g of cartilage tissue sample, add 1.5mL type II collagenase to it, wash with PBS with double antibody, and cut it into pieces to 1mm 3 , then add 8 times the volume of type II collagenase of the cartilage tissue sample, place it on a shaker at 37°C, add 2% human serum albumin at the same time, and digest at 150rpm for 100min;

[0034] Then add 2% amifostine solution, and continue to digest at 100 rpm for 2 hours; after digestion, filter through a 200-mesh filter, centrifuge to precipitate, and collect chondrocytes for counting.

Embodiment 3

[0036] A method for rapidly and efficiently separating chondrocytes, comprising the steps of:

[0037] Obtain 10g of cartilage tissue sample, add 2mL type II collagenase to it, wash with PBS with double antibody, and cut it into pieces to 1mm 3 , and then add 10 times the volume of type II collagenase of the cartilage tissue sample, place it on a shaker at 37°C, add 1% human serum albumin at the same time, and digest at 200rpm for 80min;

[0038] Then add 5% amifostine solution, and continue to digest at 100 rpm for 3 hours; after digestion, filter through a 200-mesh filter, centrifuge to precipitate, and collect chondrocytes for counting.

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Abstract

The invention relates to the technical field of in-vitro tissue cell separation, and particularly relates to a method for quickly and efficiently separating cartilage cells. A human serum albumin is added in the early digestion stage to buffer the decomposition effect of other extrinsic protease on type II collagenase, thus ensuring that the type II collagenase is not damaged and has high activity; and an amifostine solution is further added in the late digestion stage to favorably protect cartilage cells and effectively prevent the cartilage cells from being damaged in the digestion process, thereby being beneficial to obtaining more primary cartilage cells with higher activity.

Description

technical field [0001] The invention relates to the technical field of in vitro tissue cell separation, in particular to a method for rapidly and efficiently separating chondrocytes. Background technique [0002] Cartilage is an avascular tissue composed of chondrocytes and extracellular matrix. Chondrocytes are the only cell type in articular cartilage. Function of articular cartilage. Under normal physiological conditions, the synthesis and catabolism of chondrocytes maintain a dynamic balance; while chondrocytes mainly rely on joint movement and extrusion to absorb nutrients, so the self-repair ability of cartilage damage is relatively weak, and its repair after damage has always been It is one of the problems in the medical field. In recent years, with the emergence of tissue engineering technology and new biomaterials, autologous chondrocyte transplantation has been used in the treatment of cartilage injuries at home and abroad. Clinically, cartilage tissue materials...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2509/00
Inventor 陈海佳葛啸虎王一飞应杰张维敏
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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