Culture method for inducing mesenchymal stem cell to differentiate for forming osteocyte under condition without CO2

A technology of osteoblasts and mesenchymal stem cells, which is applied in the direction of animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of being unable to carry and achieve the effect of saving equipment consumption and good differentiation effect

Inactive Publication Date: 2012-07-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitations of the payload, volume, device design and safety of the spacecraft, it is often impossible to carry CO 2 Storage tanks to provide the high concentration of CO required for space cell culture 2

Method used

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  • Culture method for inducing mesenchymal stem cell to differentiate for forming osteocyte under condition without CO2
  • Culture method for inducing mesenchymal stem cell to differentiate for forming osteocyte under condition without CO2
  • Culture method for inducing mesenchymal stem cell to differentiate for forming osteocyte under condition without CO2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: new basal medium preparation

[0027] Table 1: Detailed formulation of the novel basal medium of the present invention

[0028]

[0029] The concrete formula of novel basal medium of the present invention is referring to Table 1, and the preparation method of novel basal medium is as follows:

[0030] 1) After sterilizing with triple-distilled water, wash the utensils required for preparing the culture medium with sterilized triple-distilled water and dry them for later use.

[0031] 2) Take 1 pack of L-DMEM cell culture medium dry powder (Gibco company, article number 31600034, net weight 10.0g, containing 6.4g sodium chloride, excluding sodium bicarbonate and disodium hydrogen phosphate dodecahydrate), dissolve in 950ml and sterilize Triple distilled water, fully stirred with a magnetic stirrer to dissolve. Then take by weighing 0.41g sodium bicarbonate (Sinopharm Group Chemical Reagent Co., Ltd.), 1.5g disodium hydrogen phosphate dodecahydrate (Sin...

Embodiment 2

[0034] Example 2: Preparation of human bone marrow mesenchymal stem cells

[0035] The isolated adult bone marrow was taken for primary isolation and purification of human bone marrow mesenchymal stem cells, and the cells expanded to the third passage were used to induce differentiation into osteocytes. The specific method is as follows:

[0036] 1) Collect 3-4ml of isolated adult bone marrow, add 35-45ml of phosphate buffer solution (PBS), and centrifuge at 1800rpm for 5min;

[0037] 2) Suck off the supernatant and fat layer until 8ml of liquid remains in the centrifuge tube;

[0038]3) After the remaining 8ml of the liquid and the precipitate were repeatedly pipetted evenly, they were slowly added to the Ficoll separation solution with a density of 1.077g / ml (Pharmacia, Shanghai), and centrifuged at 2000rpm for 25min.

[0039] 4) Collect the mononuclear cell layer, add 35-45ml PBS, and centrifuge at 1800rpm for 5min.

[0040] 5) After discarding the supernatant, resuspend...

Embodiment 3

[0043] Example 3: Cell inoculation and experimental group design

[0044] 1) Collect the human bone marrow mesenchymal stem cells cultured to the third passage, adjust the cell density to 1×10 5 cells / ml to prepare cell suspension.

[0045] 2) Inoculate the above cell suspension into a 24-well plate at 0.5ml / well, that is, inoculate 5×10 4 cells. Place the 24-well plate in 5% CO 2 , cultivated overnight in a 37°C constant temperature incubator.

[0046] 3) When the cells are completely adhered to the wall and grow to about 80% confluence, replace the basal medium or induction medium according to the grouping conditions in Table 2 for culture or induction differentiation experiments, and change the medium once every 2 days.

[0047] The experimental group design is as follows:

[0048] Table 2: Treatment grouping and culture condition settings

[0049] treatment group

CO 2 condition

Medium type

1

none

Novel basal culture medium of th...

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Abstract

The invention provides a culture method for inducing mesenchymal stem cell to differentiate for forming osteocyte under the condition without CO2. The culture method comprises the following steps of: culturing the third-generation human mesenchymal stem cell under the conditions of 5 percent of CO2 and 37 DEG C and standing overnight; after the cell is completely attached to the wall and grows until 80 percents of cell is fused, replacing an inductive culture medium and culturing under the conditions of 37 DEG C without CO2 for 14-21 days to obtain osteoblast, wherein the inductive culture medium is formed by adding fetal calf serum, penicillin, streptomycin, hexadecadrol, beta-sodium glycerophosphate and vitamin C in a basal medium and is prepared from the following components calculatedby final concentration: 0.41g/L of sodium bicarbonate, 1.5g/L of disodium hydrogen phosphate dodecahydrate, 0.07g/L of monopotassium phosphate and 8.17g/L of sodium chloride; and solvent of the inductive culture medium is an L-DMEM cell culture medium free of sodium bicarbonate. According to the novel culture medium formula provided by the invention, the mesenchymal stem cell osteoblast can be normally induced to differentiate; the differentiation effect is favorable, so that the consumption of equipment is reduced; and the novel culture medium formula is suitable for cell culture and research on induced differentiation under the condition without CO2.

Description

(1) Technical field [0001] The present invention relates to a CO-free 2 A culture method for inducing mesenchymal stem cells to differentiate into osteoblasts under conditions. (2) Background technology [0002] The in vitro culture technology of cells has experienced about one hundred years of development history, and has developed rapidly since the late 1950s. As the most basic technical means of biological research, in vitro cell culture technology is widely used in various research fields. At present, the in vitro culture system of cells usually includes: conventional medium (containing various amino acids, carbohydrates, inorganic salts, vitamins, etc. required for cell growth), 10% serum, 20% O 2 , 5%CO 2 And 37 ℃ constant temperature and so on. CO 2 It is to provide the components needed for normal growth of cells, but it must be combined with high concentration sodium bicarbonate in conventional medium to form a buffer system to maintain pH stability. If there ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
Inventor 王金福陈键
Owner ZHEJIANG UNIV
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