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Cartilage tissue cell viability assaying method

A cartilage tissue and detection method technology, applied in the field of biomedicine, can solve the problems of high cost and difficult implementation, and achieve the effects of reliable results, avoiding damage, and convenient reagent configuration

Active Publication Date: 2016-07-20
TAISHAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for detecting the survival rate of cartilage tissue cells, which aims to solve the problems of high cost and difficult implementation in the current detection method for the repair and treatment effect of articular cartilage damage

Method used

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  • Cartilage tissue cell viability assaying method
  • Cartilage tissue cell viability assaying method

Examples

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Effect test

Embodiment 1

[0048] Example 1: Cut the cartilage tissue into small pieces of 1mm×1mm×1mm; digest with 0.25% trypsin-EDTA2Na in a 37°C water bath for 80 minutes; centrifuge and then continue to digest with 0.2% type II collagenase in a 37°C water bath for 2 hours; Most of the cartilage is digested and turned into flocculent, add DMEM culture medium to stop the digestion, filter through a 200-mesh screen, take the filtrate and centrifuge at 1200rpm for 5min, discard the supernatant; add FDA (50mg / l) and EB (10mg / l) to the cell suspension l), incubate at 37°C in the dark for 20 minutes; after centrifugation, make the bottom cells into cell smears and detect them under 450nm wavelength laser excitation, use IPP image analysis software to count the green and red cells in the picture, and calculate the survival rate of cartilage tissue cells 68.37%. The cell survival rate was 83.04% detected by tissue section fluorescence method.

Embodiment 2

[0049] Example 2: Cut the cartilage tissue into small pieces of 1mm×1mm×1mm; digest with 0.25% trypsin-EDTA2Na in a 37°C water bath for 40 minutes; centrifuge and then continue to digest with 0.2% type II collagenase in a 37°C water bath for 4 hours; Most of the cartilage was digested and became flocculent. Add DMEM culture medium to stop the digestion and then filter through a 200-mesh screen. Take the filtrate and centrifuge at 1500rpm for 5min, discard the supernatant; add FDA (50mg / l) and EB (10mg / l) to the cell suspension. l), incubate at 37°C in the dark for 20 minutes; after centrifugation, make the bottom cells into cell smears and detect them under 450nm wavelength laser excitation, use IPP image analysis software to count the green and red cells in the picture, and calculate the survival rate of cartilage tissue cells 83.97%. The cell survival rate was 83.04% detected by tissue section fluorescence method.

Embodiment 3

[0050] Example 3: Cut the cartilage tissue into small pieces of 1mm×1mm×1mm; digest with 0.25% trypsin-EDTA2Na in a 37°C water bath for 20 minutes; centrifuge and then continue to digest with 0.2% type II collagenase in a 37°C water bath for 8 hours; Most of the cartilage is digested and turned into flocculent, add DMEM culture medium to stop digestion, filter with 200 mesh screen, take the filtrate and centrifuge at 1400rpm for 5min, discard the supernatant; add FDA (50mg / l) and EB (10mg / l) to the cell suspension l), incubate at 37°C in the dark for 20 minutes; after centrifugation, make the bottom cells into cell smears and detect them under 480nm wavelength laser excitation, use IPP image analysis software to count the green and red cells in the picture, and calculate the survival rate of cartilage tissue cells 76.59%. The cell survival rate was 83.04% detected by tissue section fluorescence method.

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Abstract

The invention discloses a cartilage tissue cell viability assaying method. The cartilage tissue cell viability assaying method includes: shearing cartilage tissues into small fragments and digesting the small fragments by 0.25% trypsin-EDTA2Na water bath; centrifuging and then continuing digesting by 0.2% type-II collagenase water bath; after the cartilage tissues become flocculent, adding a DMEM culture solution to suspend digestion and filtering by a 200-mesh screen, centrifuging filtrate and discarding liquid supernatant; adding FDA and EB into a cell suspension and incubating away from light; counting green and red cells in an image by IPP image analysis software and computing cartilage tissue cell viability according to a formula. The cartilage tissue cell viability assaying method is simpler to operate and beneficial to digesting and separating cartilage cells sufficiently, reduces damage to the cartilage cells during digesting and separating, is conducive to maintaining cell viability, avoids complexity caused by different excitation wavelengths of a double-fluorescent stain, and is used for screening articular cartilage injury treatment schemes and evaluating tissue bank cartilage preservation effects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to a method for detecting the survival rate of cartilage tissue cells. Background technique [0002] In recent years, with the improvement of people's living standards in our country and the comprehensive popularization of sports, the number of patients with articular cartilage injuries has increased significantly. At present, there is no ideal method for the repair and treatment of articular cartilage injury, and it is still a medical problem to be solved urgently in the world. Currently, clinical methods for the treatment of articular cartilage injuries include: microfracture, autologous chondrocyte transplantation, tissue engineered cartilage transplantation, autologous and allogeneic articular cartilage transplantation, etc. The effectiveness of the above methods in treating cartilage defects requires reliable detection indicators to evaluate, and the survival rate of carti...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
CPCG01N33/5005
Inventor 亓建洪宋洪强曹法民谢地胡遵杰周路
Owner TAISHAN MEDICAL UNIV
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