CN116970559B - Induction medium for preparing adipose-derived mesenchymal stem cell factor and application thereof - Google Patents
Induction medium for preparing adipose-derived mesenchymal stem cell factor and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0602—Vertebrate cells
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Abstract
The invention discloses an induction culture medium for preparing adipose-derived mesenchymal stem cell factor, which comprises a basal culture medium and the following components added in the culture medium: linaclotide, sesamol, fructose, transferrin, glutathione. The linaclotide and sesamol are added into the culture medium to obviously stimulate the adipose-derived mesenchymal stem cells to secrete cytokines, so that the capacity of secreting cytokines by the adipose-derived mesenchymal stem cells is improved, the content of the cytokines in the culture medium is improved, and convenience is provided for the concentration application of the cytokines.
Description
Technical Field
The invention relates to the field of stem cells, in particular to an induction medium for preparing adipose-derived mesenchymal stem cell factors and application thereof.
Background
Mesenchymal stem cells are multipotent stem cells that share all the common properties of stem cells, namely self-renewal and multipotent differentiation. The application of the recombinant strain is the most in clinical application, and the recombinant strain can be combined with hematopoietic stem cells, so that the success rate of transplantation can be improved, and hematopoietic reconstitution can be accelerated. When the patient receives the large-dose chemotherapy, the mesenchymal stem cells and the hematopoietic stem cells are infused together, so that the recovery time of the blood cells of the patient can be obviously accelerated, and the method is safe and has no adverse reaction. Mesenchymal stem cells exist in various tissues (such as bone marrow, umbilical cord blood and umbilical cord tissue, placenta tissue, adipose tissue, etc.), and have a multidirectional differentiation potential, and are adult stem cells of non-hematopoietic stem cells. The stem cells have the potential of differentiating into various mesenchymal series cells (such as osteoblasts, chondroblasts, adipoblasts and the like) or non-mesenchymal series cells, and have unique cytokine secretion functions.
Adipose-derived stem cells refer to mesenchymal stem cells existing in adipose tissues, and studies have shown that adipose-derived stem cells can secrete various cytokines such as EGF epidermal growth factor, TGF-beta 1 transforming growth factor beta 1, FGF fibroblast growth factor, VEGF vascular endothelial growth factor, PDGF platelet-derived growth factor, etc. The adipose-derived mesenchymal stem cells secrete active factors and have the functions of improving inflammatory environment, regulating organism immune state, inhibiting apoptosis, promoting cell proliferation, promoting blood vessel regeneration, promoting wound healing and the like.
However, there are problems in the present adipose-derived mesenchymal stem cells, such as low content, unstable property and poor activity of the cytokines although the adipose-derived mesenchymal stem cells contain more cytokines, which greatly limit the wide clinical application of the cytokines of the adipose-derived mesenchymal stem cells.
Disclosure of Invention
In order to solve the problems, the invention provides an induction medium for preparing adipose-derived mesenchymal stem cell factors and application thereof, which can promote the adipose-derived mesenchymal stem cells to secrete the cell factors and can improve the content of the cell factors in cell fluid.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
an induction medium for preparing adipose-derived mesenchymal stem cell factor, comprising a basal medium and the following components added in the medium: linaclotide, sesamol, fructose, transferrin, glutathione.
Further, the basic culture medium is a DMEM/F12 culture medium, and the contents of each component in the culture medium are as follows: linaclotide 0.5-3.5 μg/mL, sesamol 1.2-4.8 μg/mL, transferrin 2.4-6.5 μg/mL, fructose 5.5-10ng/mL, glutathione 10-20ng/mL.
Preferably, the contents of each component in the culture medium are as follows: linaclotide 2.5 μg/mL, sesamol 3.7 μg/mL, transferrin 5.5 μg/mL, fructose 7ng/mL, glutathione 15ng/mL.
The application of the induction culture medium for preparing the adipose-derived mesenchymal stem cell factor comprises the following steps:
(1) Isolating adipose mesenchymal stem cells;
(2) Subculturing the adipose-derived mesenchymal stem cells until the cell confluence is 70-80%, and discarding the culture medium;
(3) And (3) inoculating the adipose-derived mesenchymal stem cells in the step (2) into the induction culture medium for induction culture.
Preferably, step (2) is subculturing to the adipose mesenchymal stem cells of the P2-P5 generation.
Preferably, the induction culture of step (3) is at 37℃with 5% CO 2 Is carried out under the condition of (2).
Preferably, the cell density in the induction medium in step (3) is 1-5X10 5 And each mL.
Preferably, the induction culture time in step (3) is 3-5d.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an induction culture medium for preparing adipose-derived mesenchymal stem cell factors and application thereof, and linaclotide and sesamol are added in the culture medium to obviously stimulate the adipose-derived mesenchymal stem cells to secrete the cell factors, so that the capacity of secreting the cell factors by the adipose-derived mesenchymal stem cells is improved, the content of the cell factors in a culture solution is improved, and convenience is provided for the concentration application of the cell factors.
Drawings
FIG. 1 is the EGF (epidermal growth factor) content in the cell culture solutions of comparative examples 1 to 6 according to example 1 of the present invention;
FIG. 2 shows the TGF-. Beta.1 (transforming growth factor-. Beta.1) content of the cell culture broth of comparative examples 1 to 6 according to example 1 of the present invention;
FIG. 3 shows the FGF (fibroblast growth factor) content of the cell culture solutions of comparative examples 1 to 6 according to example 1 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.
Example 1, an induction medium for the preparation of adipose-derived mesenchymal stem cell factor, comprising a basal medium and the following components added to the medium: linaclotide, sesamol, fructose, transferrin, glutathione; the basic culture medium is a DMEM/F12 culture medium, and the contents of each component in the culture medium are as follows: linaclotide 2.5 μg/mL, sesamol 3.7 μg/mL, transferrin 5.5 μg/mL, fructose 7ng/mL, glutathione 15ng/mL;
the application of the induction culture medium for preparing the adipose-derived mesenchymal stem cell factor comprises the following steps:
(1) Isolation of adipose-derived mesenchymal stem cells: adding normal saline into adipose tissue for cleaning, packaging adipose tissue into centrifuge tube, adding 0.5% type I collagenase, digesting at normal temperature for 30min, centrifuging, collecting bottom layer precipitated adipose mesenchymal stem cells, adding complete medium (DMEM/F12 medium+10% FBS), and resuspending to obtain a cell density of 1×10 4 Individual/mL, at 37 ℃,5% CO 2 Culturing under the condition;
(2) After the confluence of the adipose-derived mesenchymal stem cells in the step (1) reaches 80%, carrying out subculture, wherein the subculture ratio is 1:3, and discarding the culture medium after the confluence of the cells of the P3 generation is 70%;
(3) Resuspending the adipose-derived mesenchymal stem cells of step (2) with the above-mentioned induction medium to a cell density of 1×10 5 Inoculating 1mL of culture medium in 24-well plate at 37deg.C and 5% CO 2 Is induced to culture for 4d.
Example 2, an induction medium for the preparation of adipose-derived mesenchymal stem cell factor, comprising a basal medium and the following components added to the medium: linaclotide, sesamol, fructose, transferrin, glutathione; the basic culture medium is a DMEM/F12 culture medium, and the contents of each component in the culture medium are as follows: linaclotide 0.5 μg/mL, sesamol 1.2 μg/mL, transferrin 2.4 μg/mL, fructose 5.5ng/mL, glutathione 10ng/mL;
the application of the induction culture medium for preparing the adipose-derived mesenchymal stem cell factor comprises the following steps:
(1) Isolation of adipose-derived mesenchymal stem cells: adding normal saline into adipose tissue for cleaning, packaging adipose tissue into centrifuge tube, adding 0.5% type I collagenase, digesting at normal temperature for 30min, centrifuging, collecting bottom layer precipitated adipose mesenchymal stem cells, adding complete medium (DMEM/F12 medium+10% FBS), and resuspending to obtain a cell density of 1×10 4 Individual/mL, at 37 ℃,5% CO 2 Culturing under the condition;
(2) After the confluence of the adipose-derived mesenchymal stem cells in the step (1) reaches 80%, carrying out subculture, wherein the subculture ratio is 1:3, and discarding the culture medium after the confluence of the cells of the P2 generation is 75%;
(3) Resuspending the adipose-derived mesenchymal stem cells of step (2) with the above-mentioned induction medium to a cell density of 3×10 5 Inoculating 1mL of culture medium in 24-well plate at 37deg.C and 5% CO 2 Is induced to culture for 3d.
Example 3, an induction medium for the preparation of adipose-derived mesenchymal stem cell factor, comprising a basal medium and the following components added to the medium: linaclotide, sesamol, fructose, transferrin, glutathione; the basic culture medium is a DMEM/F12 culture medium, and the contents of each component in the culture medium are as follows: linaclotide 3.5 μg/mL, sesamol 4.8 μg/mL, transferrin 6.5 μg/mL, fructose 10ng/mL, glutathione 20ng/mL;
the application of the induction culture medium for preparing the adipose-derived mesenchymal stem cell factor comprises the following steps:
(1) Isolation of adipose-derived mesenchymal stem cells: adding normal saline into adipose tissue, washing, packaging into centrifuge tube, adding 0.5% type I collagenase, digestion at room temperature for 30min, centrifuging, and collecting the bottom precipitated fatThe mesenchymal stem cells were resuspended in complete medium (DMEM/F12 medium+10% FBS) at a cell density of 1×10 4 Individual/mL, at 37 ℃,5% CO 2 Culturing under the condition;
(2) After the confluence of the adipose-derived mesenchymal stem cells in the step (1) reaches 80%, carrying out subculture, wherein the subculture ratio is 1:3, and discarding the culture medium after the confluence of the cells of the P5 generation is 80%;
(3) Resuspending the adipose-derived mesenchymal stem cells of step (2) with the above-mentioned induction medium to a cell density of 5×10 5 Inoculating 1mL of culture medium in 24-well plate at 37deg.C and 5% CO 2 Is induced to culture for 5d.
Comparative example 1 is an induction medium for preparing adipose-derived mesenchymal stem cell factor and application thereof, and the difference from example 1 is that: linaclotide 0 μg/mL, otherwise the same as in example 1.
Comparative example 2 is an induction medium for preparing adipose-derived mesenchymal stem cell factor and its application, and the difference from example 1 is: linaclotide 5 μg/mL, the same as in example 1.
Comparative example 3 is an induction medium for preparing adipose-derived mesenchymal stem cell factor and its application, and the difference from example 1 is: sesamol 0. Mu.g/mL, and the same as in example 1.
Comparative example 4 is an induction medium for preparing adipose-derived mesenchymal stem cell factor and its application, and the difference from example 1 is that: the amount of sesamol was adjusted to 6.2. Mu.g/mL without addition of linaclotide.
Comparative example 5 is an induction medium for preparing adipose-derived mesenchymal stem cell factor and its application, and the difference from example 1 is that: the amount of linaclotide was adjusted to 6.2. Mu.g/mL without adding sesamol.
Comparative example 6 is an induction medium for preparing adipose-derived mesenchymal stem cell factor and application thereof, and the difference from example 1 is that: linaclotide and sesamol were not added.
The adipose-derived mesenchymal stem cells of example 1, comparative examples 1 to 6 were subjected to cell counting by trypan blue staining after completion of induction culture, respectively, and the cell viability was counted, and the results are shown in table 1.
TABLE 1
| Group of | Cell number (. Times.10) 5 Personal computer | Cell viability (%) |
| Example 1 | 1.63 | 98.29% |
| Comparative example 1 | 1.59 | 98.06% |
| Comparative example 2 | 1.61 | 97.83% |
| Comparative example 3 | 1.46 | 97.29% |
| Comparative example 4 | 1.57 | 98.15% |
| Comparative example 5 | 1.42 | 97.72% |
| Comparative example 6 | 1.39 | 97.24% |
It can be seen from Table 1 that the cell viability is not greatly different in example 1 and comparative examples 1 to 6, and that example 1 is superior to comparative examples 1 to 6 in terms of cell number.
The cell culture solutions of example 1 and comparative examples 1 to 6 were collected after centrifugation, and the contents of EGF (epidermal growth factor), TGF-. Beta.1 (transforming growth factor-. Beta.1) and FGF (fibroblast growth factor) were measured using ELISA kit, and the results are shown in FIGS. 1 to 3.
As can be seen from fig. 1 to 3, the contents of EGF (epidermal growth factor), TGF- β1 (transforming growth factor β1) and FGF (fibroblast growth factor) in example 1 are higher than those in comparative examples 1 to 6, which shows that the induction medium of the present invention effectively improves the capacity of the adipose-derived mesenchymal stem cells to secrete cytokines by adding linaclotide and sesamol, and the increase of the cytokines content in the culture solution is higher, especially the increase of the EGF (epidermal growth factor) content is most obvious.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (2)
1. An induction medium for preparing adipose-derived mesenchymal stem cell factor, which is characterized by comprising a basal medium and the following components added in the medium: linaclotide, sesamol, fructose, transferrin, glutathione; the basic culture medium is a DMEM/F12 culture medium, and the contents of each component in the culture medium are as follows: linaclotide 2.5 μg/mL, sesamol 3.7 μg/mL, transferrin 5.5 μg/mL, fructose 7ng/mL, glutathione 15ng/mL; the cytokines are: epidermal growth factor, transforming growth factor beta 1, fibroblast growth factor.
2. Use of an induction medium for the preparation of adipose-derived mesenchymal stem cell factor according to claim 1, comprising the steps of:
(1) Isolating adipose mesenchymal stem cells;
(2) Performing subculture on the adipose-derived mesenchymal stem cells, taking the adipose-derived mesenchymal stem cells subcultured to P2-P5 generation, culturing until the cell confluence is 70-80%, and discarding the culture medium;
(3) Inoculating the adipose-derived mesenchymal stem cells of the step (2) into the induction medium of claim 1 for induction culture, wherein the cell density in the induction medium is 1-5X10 5 The induction culture was performed at 37℃and 5% CO per mL 2 Is carried out under the condition of 3-5d induction culture time.
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