CN116751747B - Serum-free induction medium and induction method for promoting mesenchymal stem cells to differentiate into neurons - Google Patents
Serum-free induction medium and induction method for promoting mesenchymal stem cells to differentiate into neurons Download PDFInfo
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Abstract
The invention discloses a serum-free induction medium and an induction method for promoting mesenchymal stem cells to differentiate into neurons, and belongs to the technical field of cell culture. The culture medium contains nutritional factors and human growth promoting small molecules required by cells, has no heterologous protein, and can provide a good environment for maintaining the growth and proliferation of cells. By adding the piperlongin, the gentione, the troxerutin and the kaempferol, the invention can obviously improve the adherence of mesenchymal stem cells, promote the proliferation rate of cells, maintain the cell surface phenotype and the multiple differentiation potential of cells, and can better protect the stem cells. In addition, the components added in the invention are clear, the effect is stable, no batch-to-batch difference exists, and the invention is suitable for standardized process production.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a serum-free induction medium and an induction method for promoting mesenchymal stem cells to differentiate into neurons.
Background
Mesenchymal Stem Cells (MSCs) are found to have excellent neural differentiation potential, and are applied to nerve regeneration tissue engineering and treatment of neurological diseases such as alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic lateral sclerosis and the like. Differentiation of MSCs into neurons or neurons in vitro helps to improve the efficacy of stem cell therapies. The efficient, low-cost, stable and rapid neural differentiation scheme is helpful for improving the differentiation efficiency of MSCs and promoting the preclinical and clinical application thereof.
However, the market has high price, a plurality of expensive factors are added to promote cell differentiation, the cost is high, the period of inducing into neurons is long, the time of operators is long, frequent periodic operation is needed, and great manpower and material resources are consumed. Secondly, the types of the induced products in the market are complex, the ingredients are uncertain, and the batch difference is obvious.
Disclosure of Invention
The invention aims to provide a serum-free induction culture medium for promoting mesenchymal stem cells to differentiate into neurons, which contains nutritional factors and human growth-promoting small molecules required by the cells, does not contain heterologous proteins and can provide a good environment for maintaining the growth and proliferation of the cells. By adding the piperlongin, the gentione, the troxerutin and the kaempferol, the adherence of mesenchymal stem cells can be obviously improved, the proliferation rate of the cells can be promoted, the cell surface phenotype can be maintained, the multiple differentiation potential of the cells can be maintained, and the stem cells can be well protected. In addition, the components added in the invention are clear, the effect is stable, no batch-to-batch difference exists, and the invention is suitable for standardized process production.
The invention is realized by the following technical scheme:
a serum-free induction medium that promotes differentiation of mesenchymal stem cells into neurons, comprising:
recombinant human fibronectin, human epidermal growth factor EGF, human basic fibroblast growth factor bFGF, piperlongin, gentione, troxerutin and kaempferol are mixed in DMEM high-sugar basal medium.
A preparation method of a serum-free induction medium for promoting mesenchymal stem cells to differentiate into neurons comprises the following steps:
the concentrated solutions of recombinant human fibronectin, human epidermal growth factor EGF, human basic fibroblast growth factor bFGF, piperlongin, gentione, troxerutin and kaempferol are sequentially mixed according to the following ratio of 1:1000, adding the mixture into a DMEM high-sugar basal medium, uniformly mixing, and then filtering and sterilizing by using a filter membrane to finish.
Preferably, the concentration of the recombinant human fibronectin is 1-20 mg/L, the concentration of the human EGF is 1-40 mu g/L, the concentration of the bFGF is 1-40 mu g/L, the concentration of the Litsea cubeba is 10-50 mu mol/L, the concentration of the gentian is 10-100 mu mol/L, the concentration of the troxerutin is 1-20 mu mol/L and the concentration of the kaempferol is 5-50 mu mol/L.
Preferably, the concentration of the recombinant human fibronectin is 5-15 mg/L, the concentration of the human EGF is 10-30 mug/L, the concentration of the bFGF is 10-30 mug/L, the concentration of the Litsea cubeba is 20-45 mug/L, the concentration of the gentian is 30-80 mug/L, the concentration of the troxerutin is 5-15 mug/L and the concentration of the kaempferol is 15-40 mug/L.
Preferably, the recombinant human fibronectin has a concentration of 10 mg/L, human EGF has a concentration of 20 μg/L, bFGF has a concentration of 20 μg/L, piperlongin has a concentration of 40 μmol/L, gentian has a concentration of 50 μmol/L, troxerutin has a concentration of 10 μmol/L and kaempferol has a concentration of 30 μmol/L.
Preferably, the DMEM high-sugar basal medium is stored at 0-8deg.C, and the concentrated solution is stored below-20deg.C.
Use of a serum-free induction medium for promoting differentiation of mesenchymal stem cells into neurons in a medium for promoting differentiation of mesenchymal stem cells into neurons.
An induction method for promoting mesenchymal stem cells to differentiate into neurons, comprising the following steps:
s1, after cell resuscitating culture or subculture, adding a common DMEM complete medium to continue culture after inoculation;
s2, after the cells grow fully, replacing the cells with the serum-free induction culture medium for promoting the mesenchymal stem cells to differentiate into neurons according to claim 1, and continuing to culture;
s3, changing the liquid once every 2-3 days, and culturing for 2-4 weeks to observe the cell morphology and observe the neural differentiation condition by staining after passage.
Compared with the prior art, the invention has at least the following technical effects:
the invention provides a serum-free induction culture medium for promoting mesenchymal stem cells to differentiate into neurons, which contains nutritional factors and human-derived growth-promoting small molecules required by the cells, has no heterologous protein, and can provide a good environment for maintaining the growth and proliferation of the cells. By adding the piperlongin, the gentione, the troxerutin and the kaempferol, the invention can obviously improve the adherence of mesenchymal stem cells, promote the proliferation rate of cells, maintain the cell surface phenotype and the multiple differentiation potential of cells, and can better protect the stem cells. In addition, the components added in the invention are clear, the effect is stable, no batch-to-batch difference exists, and the invention is suitable for standardized process production.
Drawings
FIG. 1 is a schematic diagram showing the cell morphology of test example one;
FIG. 2 is a schematic representation of cell expression Nestin staining in a three immunofluorescence assay of test examples;
FIG. 3 is a schematic representation of the staining of cells expressing TUJ1 in a three immunofluorescence assay of test example.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
The components and reagents involved in the culture medium are all conventional commercial products or can be obtained by conventional technical means in the field. DMEM high sugar medium was sourced from Biological industries (israel).
Example 1:
a culture medium that promotes differentiation of mesenchymal stem cells into neurons: the components comprise:
recombinant human fibronectin: 1 mg/L, human epidermal growth factor EGF: 1. Mu.g/L, human basic fibroblast growth factor bFGF:1 μg/L, fructus Litseae: 10 mu mol/L, gentianone: 10 mu mol/L, troxerutin: 1 mu mol/L, kaempferol: 5. Mu. Mol/L.
A method for preparing a culture medium for promoting mesenchymal stem cells to differentiate into neurons, comprising the following steps:
s1, preparing a concentrated solution with the concentration of the additive combination: recombinant human fibronectin: 1 mg/mL, human epidermal growth factor EGF: 1. Mu.g/mL, human basic fibroblast growth factor bFGF:21 μg/mL, fructus Litseae: 10mmol/L, gentianone: 10mmol/L, troxerutin: 1 mmol/L, kaempferol: 5 mmol/L.
S2, taking a DMEM high-sugar culture medium, and sequentially taking the concentrated solution prepared in the step S1 according to the following steps of 1:1000, adding recombinant human fibronectin, human EGF, human bFGF, fructus Litseae, gentian, troxerutin, and kaempferol into DMEM basic culture medium, and mixing.
And S3, filtering and sterilizing the culture medium by using a filter membrane to obtain the mesenchymal stem cell culture medium.
An induction method for promoting mesenchymal stem cells to differentiate into neurons comprises the following steps:
s1, after the mesenchymal stem cells are subjected to cell resuscitating culture, adding a common DMEM complete medium for continuous culture after inoculation;
s2, after the cells grow fully, changing into a culture medium for promoting the mesenchymal stem cells to differentiate into neurons, and continuing culturing;
s3, changing the liquid once every 2-3 days, and culturing for 2-4 weeks to observe the cell morphology and observe the neural differentiation condition by staining after passage.
Example 2:
a composition of a medium for promoting differentiation of mesenchymal stem cells into neurons comprising: recombinant human fibronectin: 10 mg/L, human epidermal growth factor EGF: 20. Mu.g/L, human basic fibroblast growth factor bFGF:20 mug/L, fructus Litseae: 40 mu mol/L, gentianone: 50 mu mol/L, troxerutin: 10 mu mol/L, kaempferol: 30. Mu. Mol/L.
A method for preparing a culture medium for promoting mesenchymal stem cells to differentiate into neurons, comprising the following steps:
s1, preparing a concentrated solution with the concentration of the additive combination: recombinant human fibronectin: 10 mg/mL, human epidermal growth factor EGF: 20. Mu.g/mL, human basic fibroblast growth factor bFGF:20 μg/mL, fructus Litseae: 40mmol/L, gentianone: 50mmol/L, troxerutin: 10mmol/L, kaempferol: 30mmol/L.
S2, taking a DMEM high-sugar culture medium, and sequentially taking the concentrated solution prepared in the step S1 according to the following steps of 1:1000, adding recombinant human fibronectin, human EGF, human bFGF, fructus Litseae, gentian, troxerutin, and kaempferol into DMEM basic culture medium, and mixing.
And S3, filtering and sterilizing the culture medium by using a filter membrane to obtain the mesenchymal stem cell culture medium.
An induction method for promoting mesenchymal stem cells to differentiate into neurons comprises the following steps:
s1, after the mesenchymal stem cells are subjected to cell resuscitating culture, adding a common DMEM complete medium for continuous culture after inoculation;
s2, after the cells grow fully, changing into a culture medium for promoting the mesenchymal stem cells to differentiate into neurons, and continuing culturing;
s3, changing the liquid once every 2-3 days, and culturing for 2-4 weeks to observe the cell morphology and observe the neural differentiation condition by staining after passage.
Example 3:
a composition of a medium for promoting differentiation of mesenchymal stem cells into neurons comprising: recombinant human fibronectin: 20 mg/L, human epidermal growth factor EGF: 40. Mu.g/L, human basic fibroblast growth factor bFGF: 40. Mu.g/L, fructus Litseae: 50 mu mol/L, gentianone: 100 mu mol/L troxerutin: 20 mu mol/L, kaempferol: 50. Mu. Mol/L.
A method for preparing a culture medium for promoting mesenchymal stem cells to differentiate into neurons, comprising the following steps:
s1, preparing a concentrated solution with the concentration of the additive combination: recombinant human fibronectin: 20 mg/mL, human epidermal growth factor EGF: 40. Mu.g/mL, human basic fibroblast growth factor bFGF: 40. Mu.g/mL, fructus Litseae: 50mmol/L, gentianone: 100mmol/L, troxerutin: 20 mmol/L, kaempferol: 50 mmol/L.
S2, taking a DMEM high-sugar culture medium, and sequentially taking the concentrated solution prepared in the step S1 according to the following steps of 1:1000, adding recombinant human fibronectin, human EGF, human bFGF, fructus Litseae, gentian, troxerutin, and kaempferol into DMEM basic culture medium, and mixing.
And S3, filtering and sterilizing the culture medium by using a filter membrane to obtain the mesenchymal stem cell culture medium.
A preparation method and an induction method of a culture medium for promoting mesenchymal stem cells to differentiate into neurons are as follows:
s1, after the mesenchymal stem cells are subjected to cell resuscitating culture, adding a common DMEM complete medium for continuous culture after inoculation;
s2, after the cells grow fully, changing into a culture medium for promoting the mesenchymal stem cells to differentiate into neurons, and continuing culturing;
s3, changing the liquid once every 2-3 days, and culturing for 2-4 weeks to observe the cell morphology and observe the neural differentiation condition by staining after passage.
Comparative example 1: the steps and parameters of the other steps and parameters are the same as those of the example 2, except that no piperlongin, gentione, troxerutin and kaempferol are added.
Comparative example 2: the steps and parameters were the same as in example 2 without addition of cubelin.
Comparative example 3: no gentian was added and the remaining steps and parameters were the same as in example 2.
Comparative example 4: troxerutin was not added, and the rest steps and parameters were the same as in example 2.
Comparative example 5: the steps and parameters were the same as in example 2 without adding kaempferol.
Experimental example one, wall-attached detection of mesenchymal Stem cells
Umbilical cord mesenchymal stem cells are resuscitated or passaged, the frozen stock solution is removed after centrifugation, the stem cells are resuspended and inoculated and then covered in a DMEM complete medium, the culture medium which promotes the differentiation of the mesenchymal stem cells into neurons is carried out for 24 hours after the culture, and the culture medium is inoculated in a culture flask and placed in a culture box at 37 ℃ for culture. After 24h incubation, the stem cells were stained with 0.4% trypan blue, observed and counted for attachment. Culturing was continued for 2-3 days and cell morphology was observed by inverted light microscopy.
As can be seen from the results in Table 1, the culture medium for promoting the differentiation of mesenchymal stem cells into neurons of the present invention can obtain a very high adherence rate, and examples 1 to 3 all exceed 90% adherence rate, which indicates that the present invention can promote fine adherence and increase the survival rate of cells.
FIG. 1 is a schematic diagram showing morphological observation of test example one.
As shown in FIG. 1, the cells in the culture medium in all examples exhibited classical fibroblastic shape, indicating that all the culture media of the invention are suitable for the culture of mesenchymal stem cells.
The result of comparative example 1, which is not added with any small molecule of traditional Chinese medicine, shows that the adherence rate is lower.
Compared with the culture medium without adding any small Chinese medicinal molecules, the wall attachment rate of the comparative examples 2-5 is obviously improved, but the wall attachment rate of cells can be influenced by reducing the addition of small Chinese medicinal molecules.
Test example two, cell proliferation assay of mesenchymal Stem cells
Umbilical cord mesenchymal stem cells 10 4 The concentration of individual cells/mL was inoculated in 96-well plates, and after cell attachment, the culture medium of examples 1 to 3 and comparative examples 1 to 5, which promoted differentiation of mesenchymal stem cells into neurons, was replaced. After 24h of incubation, CCK-8 was used to bind to living stem cells and form a detectable colored pellet.
10. Mu.L of CCK-8 solution was added at working concentration and 6 biological replicates were performed. The microplate reader measured absorbance at a wavelength of 450nm and calculated the relative proliferation rates relative to all other examples and comparative examples of comparative example 1.
As can be seen from the results of table 2, the culture medium of examples 1 to 3, which promotes differentiation of mesenchymal stem cells into neurons, can significantly improve the cell proliferation rate compared to the culture medium of comparative example 1, to which no small molecule of chinese medicine was added.
Compared with the culture medium without any small traditional Chinese medicine molecules, the cell proliferation rate of comparative examples 2-5 is obviously improved, but the cell attachment rate can be affected by reducing the addition of one small traditional Chinese medicine molecule.
Test example III, neural differentiation induced by mesenchymal Stem cells
Umbilical cord mesenchymal stem cells were passaged and inoculated into 6-well plates, and were first cultured with DMEM complete medium until the cells were grown completely adherent and observed to be confluent to the bottom 80% or more, and replaced with the medium of example 2 that promoted differentiation of mesenchymal stem cells into neurons and the medium of comparative example 1. After 2-3 weeks of culture, the cell morphology was observed and immunofluorescent staining was performed to observe the neuronal differentiation status.
DAPI:4', 6-diamidino-2-phenylindole; nestin: a nestin; TUJ1: class III β -tubulin; merge is the composite, DAPI blue, nestin and TUJ1 green, superimposed.
FIG. 2 is a schematic representation of the staining results of Nestin-expressing cells of example 2 and comparative example 1.
As shown in FIG. 3, the staining results of the comparison cells of example 2 and comparative example 1 for TUJ1 expression are schematically shown.
As shown in fig. 2 and 3, the culture medium for promoting the differentiation of the mesenchymal stem cells into neurons can effectively promote the induction and differentiation of the mesenchymal stem cells into neurons, and express neuron markers Nestin and TUJ1. However, when the piperlongumin, the gentian, the troxerutin and the kaempferol in the culture medium are removed, the stem cells do not carry out nerve differentiation and do not express neuron markers.
Finally, it should be noted that: the foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A serum-free induction medium for promoting differentiation of mesenchymal stem cells into neurons, comprising:
mixing recombinant human fibronectin, human epidermal growth factor EGF, human basic fibroblast growth factor bFGF, piperlongin, gentione, troxerutin and kaempferol in a DMEM high-sugar basal medium;
the concentration of the recombinant human fibronectin is 10 mg/L, the concentration of the human epidermal growth factor EGF is 20 mu g/L, the concentration of the human basic fibroblast growth factor bFGF is 20 mu g/L, the concentration of the cubin is 40 mu mol/L, the concentration of the gentione is 50 mu mol/L, the concentration of the troxerutin is 10 mu mol/L and the concentration of the kaempferol is 30 mu mol/L.
2. The method for preparing a serum-free induction medium for promoting differentiation of mesenchymal stem cells into neurons according to claim 1, comprising the steps of:
the concentrated solutions of recombinant human fibronectin, human epidermal growth factor EGF, human basic fibroblast growth factor bFGF, piperlongin, gentione, troxerutin and kaempferol are sequentially mixed according to the following ratio of 1:1000, adding the mixture into a DMEM high-sugar basal medium, uniformly mixing, and then filtering and sterilizing by using a filter membrane to finish.
3. The method for preparing a serum-free induction medium for promoting differentiation of mesenchymal stem cells into neurons according to claim 2, wherein the DMEM-high sugar basal medium is stored at 0-8 ℃ and the concentrated solution is stored below-20 ℃.
4. Use of the serum-free induction medium for promoting differentiation of mesenchymal stem cells into neurons according to claim 1 in a medium for promoting differentiation of mesenchymal stem cells into neurons.
5. An induction method for promoting differentiation of mesenchymal stem cells into neurons, comprising the steps of:
s1, after cell resuscitating culture or subculture, adding a common DMEM complete medium to continue culture after inoculation;
s2, after the cells grow fully, replacing the cells with the serum-free induction culture medium for promoting the mesenchymal stem cells to differentiate into neurons according to claim 1, and continuing to culture;
s3, changing the liquid once every 2-3 days, and culturing for 2-4 weeks to observe the cell morphology and observe the neural differentiation condition by staining after passage.
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