Preparation method of cell microsphere of anchorage-dependent cell

A technique for adherent cells and cells is applied in the field of preparation of cell microspheres, which can solve the problems of complex and low-efficiency in the preparation process of stem cell microspheres, and achieve the effect of a simple preparation method.

Active Publication Date: 2020-01-14
青岛奥克生物开发有限公司
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Problems solved by technology

However, the production process of stem cell mi

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  • Preparation method of cell microsphere of anchorage-dependent cell
  • Preparation method of cell microsphere of anchorage-dependent cell
  • Preparation method of cell microsphere of anchorage-dependent cell

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[0019] The present invention provides a method for preparing cell microspheres of adherent cells, comprising the following steps: (1) centrifuging the adherent cells after recovering, resuspending with stem cell culture medium, and inoculating them on a cell culture plate;

[0020] (2) Cultivate the inoculated cells at a temperature of 35-37°C, and continue culturing for 1-3 days when the cell confluence reaches 100%;

[0021] (3) Place the cultured cell culture plate on a pre-cooled stainless steel plate at 12-18°C, place it for 5 minutes, gently bump one side of the cell culture plate, and supplement the stem cell culture medium, The supplementary volume of the stem cell culture medium is half of the volume of the stem cell culture medium when the cells are resuspended in step (1);

[0022] (4) Place the cell culture plate supplemented with the stem cell culture medium at a temperature of 35-37° C. for 1 day to obtain cell microspheres.

[0023] In the present invention, wh...

Embodiment 1

[0029] 1. Take cryopreserved and revived umbilical cord mesenchymal cells or digested cells, and take samples for cell counting.

[0030] 2. Press 5×10 3 / cm 2 Inoculate into 96-well plate, the area of ​​each well is 0.32cm 2 , so each well requires 1.6×10 3 Cells, according to the bottom area of ​​a 96-well plate, 1.5×10 5 Centrifuge the cells at 1000rpm for 10min, discard the supernatant and resuspend the cells in 9.6mL medium, mix the cells by pipetting and inoculate 96-well plates, 100μL per well.

[0031] 3. Place the 96-well plate in a 37°C cell incubator for culture, and change the medium every three and a half days. When the cell confluence reaches 100%, continue to culture for 1 to 3 days.

[0032] 4. Take the 96-well plate out of the incubator, and quickly place it on a stainless steel plate at about 15°C to ensure that the bottom of the 96-well plate is in good contact with the stainless steel plate. After placing it for 5 minutes, gently bump the side of the 96...

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Abstract

The invention provides a preparation method of a cell microsphere of an anchorage-dependent cell, and relates to the technical field of cell microspheres. According to the preparation method providedby the invention, through temperature reduction, collision and fluid infusion, one side of a cell layer on the hole bottom can be curled, and a cell on the whole hole bottom is naturally curled to form a point so as to form the cell microsphere under the action of a tensile force of the cell layer. A metabolic capability difference of the cell microsphere and an equal-plane growing stem cell is tested through an MTT experiment, and the result shows that the cell microsphere and the plate culture cell have no obvious difference on metabolism. The preparation method provided by the invention issimple, a large number of cell microspheres can be prepared at one time, and in addition, the diameter sizes of the cell microspheres can be further adjusted according to the area of the hole bottom of a culture plate.

Description

technical field [0001] The invention belongs to the technical field of cell microspheres, and in particular relates to a method for preparing cell microspheres of adherent cells. Background technique [0002] Stem cells are a type of cells capable of self-renewal and differentiation in the human body. With the identification and understanding of stem cells, due to their wide source, multi-differentiation potential and various physiological effects, stem cells are gradually favored in clinical applications. Significant results have been achieved in the quality of life of patients. [0003] The main sources of mesenchymal stem cells are placenta, umbilical cord, bone marrow and fat. At present, the culture method of mesenchymal stem cells in clinical application is mainly cultured in cell culture flasks or petri dishes, which is a two-dimensional plane culture. In order to simulate the growth environment in the body, researchers have developed many three-dimensional culture ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/09C12N5/02
CPCC12N5/0665C12N5/0693
Inventor 贾在美黄玉香刘超姜磊
Owner 青岛奥克生物开发有限公司
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