A method for preparing induced pluripotent stem cell chromosomes

A technology of pluripotent stem cells and chromosomes, which is applied in the field of preparation of induced pluripotent stem cell chromosomes, can solve the problems of optimal final concentration and optimal blocking time interval without reference data, chromosomes affecting karyotype analysis, high-resolution chromosome length, etc. , to achieve the effect of high success rate, large quantity and high resolution

Inactive Publication Date: 2020-05-29
杭州市妇产科医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, since there are no domestic reports on harvesting induced pluripotent stem cell chromosomes using cell cycle synchronization, due to the inconsistency of cell types and cell cycles, the optimal final concentration and retardation of the synchronization reagent added when harvesting induced pluripotent stem cells There are no reference materials for the optimal time interval between release, harvest, and need to be re-explored; due to the long high-resolution chromosomes, the dispersion conditions also need to be re-optimized during film production, so as to avoid excessive crossover of chromosomes from affecting karyotype analysis

Method used

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  • A method for preparing induced pluripotent stem cell chromosomes

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Embodiment 1

[0043] A method for preparing induced pluripotent stem cell chromosomes, comprising the steps of:

[0044] (1) Cultivation of stem cells: Human stem cells were placed at 37°C with 5% CO 2 Open culture in the incubator, the medium (commercially available) was changed daily;

[0045] (2) Synchronization of the cell cycle: replace the fresh medium at 8:00 a.m. the day before stem cell harvesting, add 100 microliters of fluorouracil deoxynucleoside (concentration 1 μg / ml) to the culture flask at 10:00 a.m., and continue to add Cultivate in an incubator; add 200µl thymine (concentration 2.4mg / ml) to the culture flask at 8:00 the next day, and continue culturing, add 100µl colchicine (concentration 20µg / ml) to the culture flask at 11:00 am, and mix well After placing in the incubator for 15 minutes, take out the culture bottle for harvesting stem cells;

[0046] (3) Stem cell harvesting:

[0047] A. Aspirate the culture medium in the culture bottle into the centrifuge tube, add 2...

Embodiment 2

[0064] A method for preparing induced pluripotent stem cell chromosomes, comprising the steps of:

[0065] (1) Stem cell culture: Stem cells were placed at 37°C, 5% CO 2 Open culture in the incubator, the medium (commercially available) was changed daily;

[0066] (2) Synchronization of the cell cycle: replace the fresh medium at 8:00 a.m. the day before stem cell harvesting, add 100 microliters of fluorouracil deoxynucleoside (concentration 1 μg / ml) to the culture flask at 10:00 a.m., and continue to add Cultivate in an incubator; add 200µl thymine (concentration 2.4mg / ml) to the culture flask at 8:00 the next day, and continue culturing, add 100µl colchicine (concentration 20µg / ml) to the culture flask at 11:00 am, and mix well After placing in the incubator for 15 minutes, take out the culture bottle for harvesting stem cells;

[0067] (3) Stem cell harvesting:

[0068] A. Aspirate the culture medium in the culture bottle into the centrifuge tube, add 2ml of normal salin...

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Abstract

The invention discloses a preparation method of induced pluripotent stem cells chromosome, comprising the following steps: (1) culture of stem cells; (2) cell cycle synchronization; (3) acquisition ofstem cells: extracting stem cells from a culture bottle to obtain a cell suspension; (4) dripping; and (5) staining. The invention has advantages of high resolution and large quantity of chromosomesduring karyotype identification of stem cells. Thus, success rate of karyotype analysis is high.

Description

technical field [0001] The invention relates to a method for preparing chromosomes, in particular to a method for preparing induced pluripotent stem cell chromosomes. Background technique [0002] Induced pluripotent stem cells (Induced Pluripotent Stem Cells, iPSCs) refer to the reprogramming of somatic cells into pluripotent stem cells with the characteristics and functions of embryonic stem cells. The research on induced pluripotent stem cells began in 2006. Japan's Takahashi et al. first induced induced pluripotent stem cells based on adult fibroblasts, and confirmed that the obtained cells had the characteristics and functions of embryonic stem cells, setting off an upsurge in induced stem cell research. It has been confirmed that induced pluripotent stem cells, similar to embryonic stem cells, can self-renew and differentiate into various types of somatic cells. They are not only of great value in replacement therapy, but also can be used to establish in vitro disease ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N1/30
Inventor 王昊吴凌峰梁平
Owner 杭州市妇产科医院
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