CN113621567A - Composition and application thereof, stem cell culture medium and stem cell culture method - Google Patents
Composition and application thereof, stem cell culture medium and stem cell culture method Download PDFInfo
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- CN113621567A CN113621567A CN202110780032.8A CN202110780032A CN113621567A CN 113621567 A CN113621567 A CN 113621567A CN 202110780032 A CN202110780032 A CN 202110780032A CN 113621567 A CN113621567 A CN 113621567A
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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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Abstract
The invention relates to a composition, application thereof, a stem cell culture medium and a stem cell culture method, wherein the composition comprises serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor, and the composition does not contain serum. The composition has high safety when used in stem cell culture medium, and can delay aging of stem cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a composition and application thereof, a stem cell culture medium and a stem cell culture method.
Background
Stem cells (stem cells) are a type of pluripotent cells with self-replicating capacity (self-rejuvenating). Under certain conditions, stem cells can differentiate into a variety of functional cells. Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, originating from the mesoderm and ectoderm in early developmental stages. MSCs have a commonality of stem cells, i.e., the ability to self-renew, differentiate and home. Under a specific external differentiation environment, MSCs can differentiate into various histiocytes such as nerves, heart, bone, cartilage, fat, epithelium and the like, and are considered as one of the most promising source cells for cell therapy technology.
Currently, in vitro expansion of MSCs is performed in medium supplemented with Fetal Bovine Serum (FBS), human autologous serum, or serum analogues. However, from the perspective of potential therapeutic applications of MSC cultures, the presence of animal or human serum in MSC cultures has the following drawbacks: first, bovine serum, human serum or other animal sera may contain blood-borne pathogens such as viruses and mad cow disease prions, Bovine Spongiform Encephalopathy (BSE), etc. Second, animal serum elicits the production of antibodies against xenobiotic proteins, which elicit the immune response of the recipient patient. Third, animal sera showed lot-to-lot variation, possibly leading to inconsistent performance.
In view of this, a large number of serum-free medium products should be produced. It is widely used in the culture of mammalian and invertebrate cells to produce monoclonal antibodies, viral antigens, recombinant proteins, and the like. Most of serum-free media currently contain transferrin, which transports ions into cells, and insulin, albumin, fibrin, fetuin, and other proteins that regulate the uptake of glucose, and proteins added to serum-free media perform various functions in cell culture, such as providing a substrate required for cell adhesion, resisting shear stress of bioreactor, and serving as carriers for lipids and other growth differentiation factors. However, current serum-free media tend to cause rapid senescence of stem cells during culture expansion.
Disclosure of Invention
Based on this, there is a need for a composition that is highly safe and can slow down the aging of stem cells.
A composition comprising serum albumin, basic fibroblast growth factor, transforming growth factor β 1, and epidermal growth factor.
The composition can slow down the aging speed of stem cells in a serum-free culture medium after being added into the serum-free stem cell culture medium through the mutual matching of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor; the composition does not contain serum, and has high safety; in addition, when the serum-free culture medium added with the composition is used, the culture container does not need to be coated with fibronectin, the process of culturing the stem cells is simplified, and the cost is saved.
In one embodiment, the mass ratio of the serum albumin, the basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor is (0.5-50 μ g): (10 ng-30 ng): (0.3 ng-1 ng): (5 ng-15 ng), the composition does not contain serum.
In one embodiment, the mass ratio of serum albumin to basic fibroblast growth factor is (1 μ g to 10 μ g): (15 ng-20 ng);
And/or the mass ratio of the serum albumin to the transforming growth factor beta 1 is (1-10 mug): (0.5 ng-0.75 ng);
and/or the mass ratio of the serum albumin to the epidermal growth factor is (1-10 mug): (7.5 ng-10 ng).
In one embodiment, the composition further comprises at least one of an antioxidant, an antibiotic, transferrin, dexamethasone, and a lipid material.
In one embodiment, the antioxidant is selected from at least one of ascorbic acid 2-phosphate, superoxide dismutase, catalase, and glutathione peroxidase.
In one embodiment, the antibiotic is selected from at least one of penicillin and streptomycin.
In one embodiment, the composition comprises an antioxidant, and the ratio of the molar amount of the antioxidant to the mass of the serum albumin is (150nmol to 250 nmol): (0.5. mu.g-50. mu.g);
and/or the composition comprises an antibiotic, and the ratio of the titer of the antibiotic to the mass of the serum albumin (100U-2100U): (0.5. mu.g-50. mu.g);
and/or, the composition comprises transferrin, the volume of the transferrin to the mass of the serum albumin ratio is 0.01 mL: (0.5. mu.g-50. mu.g);
And/or the composition comprises dexamethasone, wherein the ratio of the molar amount of the dexamethasone to the mass of the serum albumin (50-150 pmol): (0.5. mu.g-50. mu.g);
and/or the composition comprises lipid substances, and the mass ratio of the lipid substances to the serum albumin is (30-50 pg): (0.5. mu.g-50. mu.g).
The composition is applied to stem cell culture media.
A stem cell culture medium, which comprises the composition and a serum-free culture medium;
in one embodiment, the serum-free medium is alpha-MEM.
In one embodiment, the stem cell culture medium can increase proliferation and/or delay senescence of stem cells.
In one embodiment, the final concentration of serum albumin is 0.5-50 μ g/mL, the final concentration of basic fibroblast growth factor is 10-30 ng/mL, the final concentration of transforming growth factor beta 1 is 0.3-1 ng/mL, and the final concentration of epidermal growth factor is 5-15 ng/mL.
In one embodiment, the serum-free medium is serum-free mesenchymal stem cell medium.
In one embodiment, the serum-free medium is DMEM/F12, alpha-MEM or DMEM.
A method for culturing stem cells, comprising the steps of:
and (3) inoculating the mesenchymal stem cells into the stem cell culture medium for culture.
In one embodiment, the stem cell is a mesenchymal stem cell.
Drawings
FIG. 1 is a mesenchymal stem cell in the stem cell culture medium of examples 1-5;
FIG. 2 is a mesenchymal stem cell in the stem cell culture medium of examples 6-8;
FIG. 3 is a mesenchymal stem cell in the stem cell culture medium of examples 9 to 11 and comparative example 1;
FIG. 4 is an image of mesenchymal stem cells in the stem cell culture medium of examples 1-8 and comparative example 1 after staining with β -galactosidase.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
One embodiment of the present invention provides a composition comprising, as components: serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor.
When used alone, the three growth factors, namely the basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor, have bidirectional effect and time effect on cells, and particularly can promote the rapid proliferation of the cells and accelerate the aging of the cells. The composition can slow down the aging speed of stem cells after being added into a serum-free stem cell culture medium by the mutual matching of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor, and the composition does not contain serum and has high safety. The composition is proved to be capable of slowing down the aging of the mesenchymal stem cells when being added into a serum-free stem cell culture medium.
Optionally, the mass ratio of the serum albumin, the basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor is (0.5-50 μ g): (10 ng-30 ng): (0.3 ng-1 ng): (5 ng-15 ng), the composition is serum-free.
In this embodiment, the serum albumin is recombinant human serum albumin; the basic fibroblast growth factor is recombinant human basic fibroblast growth factor (bFGF); the recombinant human serum albumin, the recombinant human basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor can be recombinant proteins produced by adopting a fermentation technology.
In one embodiment, the mass ratio of serum albumin to basic fibroblast growth factor is (1-20 μ g): (10 ng-20 ng). Further, the mass ratio of the serum albumin to the basic fibroblast growth factor is (1 to 10 μ g): (15 ng-20 ng). Furthermore, the mass ratio of the serum albumin to the basic fibroblast growth factor is (1-5 mug): (15 ng-20 ng).
In one embodiment, the mass ratio of serum albumin to transforming growth factor beta 1 is (1 μ g-10 μ g): (0.5 ng-1 ng). Further, the mass ratio of the serum albumin to the transforming growth factor beta 1 is (1-10 mug): (0.5 ng-0.75 ng). Furthermore, the mass ratio of the serum albumin to the transforming growth factor beta 1 is (1 mug-5 mug): (0.5 ng-1 ng).
In one embodiment, the mass ratio of serum albumin to epidermal growth factor is (1 μ g-10 μ g): (5 ng-10 ng). Further, the mass ratio of the serum albumin to the epidermal growth factor is (1 to 10 μ g): (7.5 ng-10 ng). Furthermore, the mass ratio of the serum albumin to the epidermal growth factor is (1-5 mug): (5 ng-10 ng).
In one embodiment, the mass ratio of the serum albumin, the basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor is (1-10 μ g): (10 ng-20 ng): (0.5 ng-1 ng): (5 ng-10 ng). Furthermore, the mass ratio of the serum albumin, the basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor is (1-5 mug): (15 ng-20 ng): (0.5 ng-1 ng): (5 ng-10 ng).
In one embodiment, the composition does not contain fibronectin (Fn).
In one embodiment, the composition consists of: serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor.
In some embodiments, the composition further comprises at least one of an antioxidant, an antibiotic, transferrin, dexamethasone, and a lipid material.
In particular, antioxidants are used to further reduce the damage of oxygen radicals to stem cells, slowing stem cell aging. Optionally, the antioxidant is selected from at least one of ascorbic acid 2-phosphate, superoxide dismutase, catalase, and glutathione peroxidase. Of course, in other embodiments, the antioxidant is not limited to the above-mentioned substances, and may be other substances having an antioxidant effect.
Optionally, the ratio of the molar amount of antioxidant to the mass of serum albumin is (150nmol to 250 nmol): (0.5. mu.g-50. mu.g). Further, the ratio of the molar amount of the antioxidant to the mass of the serum albumin is (150nmol to 200 nmol): (0.5 to 20. mu.g), further, the ratio of the molar amount of the antioxidant to the mass of the serum albumin is (150 to 200 nmol): (0.5. mu.g-10. mu.g).
Specifically, transferrin is used to avoid free radical production, protect cell growth, and maintain cell growth and proliferation. In this embodiment, the transferrin is recombinant transferrin.
In one embodiment, the ratio of the volume of transferrin to the mass of serum albumin is 0.01 mL: (0.5. mu.g-50. mu.g). In one particular example, the Transferrin is Insulin-Transferrin-Selenium (ITS).
Specifically, dexamethasone is an anti-inflammatory glucocorticoid, and plays a large number of physiological roles in cell survival, cell signal transduction and gene expression, and can induce production of phospholipase A2 inhibitor protein (lipocortin) and inhibit nitric oxide synthase-inducing activity.
In one embodiment, the ratio of the molar amount of dexamethasone to the mass of serum albumin (50 to 150 pmol): (0.5. mu.g-50. mu.g). Further, the ratio of the molar amount of dexamethasone to the mass of serum albumin (50 to 100 pmol): (0.5. mu.g-10. mu.g). Further, the ratio of the molar amount of dexamethasone to the mass of serum albumin (50 to 90 pmol): (0.5. mu.g-5. mu.g).
In particular, antibiotics are used to inhibit the growth of bacteria in a culture medium. Optionally, the antibiotic is selected from at least one of penicillin and streptomycin. Further, the antibiotic is a mixture of penicillin and streptomycin, and the ratio of the titer of the penicillin to the mass of the serum albumin (100U-200U): (0.5-50. mu.g), the ratio of the potency of streptomycin to the mass of serum albumin (100-2000U): (0.5. mu.g-50. mu.g). Of course, in other embodiments, the antibiotic is not limited to the above, and may be other antibiotics.
In one embodiment, the ratio of the potency of the antibiotic to the mass of serum albumin (100 to 2000U): (0.5. mu.g-50. mu.g). Further, the ratio of the potency of the antibiotic to the mass of the serum albumin (100 to 500U): (0.5. mu.g-20. mu.g). Further, the ratio of the potency of the antibiotic to the mass of the serum albumin (100 to 200U): (0.5. mu.g-10. mu.g).
Optionally, the lipid material is selected from at least one of linoleic acid, cholesterol and VE. It is understood that in other embodiments, the lipid material is not limited to the above, but may be other materials.
In one embodiment, the mass ratio of lipid material to serum albumin (30pg to 50 pg): (0.5. mu.g-50. mu.g). Further, the mass ratio of the lipid substance to the serum albumin (35pg to 45 pg): (0.5. mu.g-20. mu.g). Further, the mass ratio of lipid substance to serum albumin (35pg to 45 pg): (0.5. mu.g-10. mu.g).
In one embodiment, the composition consists of: serum albumin, basic fibroblast growth factor, transforming growth factor beta 1. Epidermal growth factor, antioxidant, antibiotic, transferrin, dexamethasone and lipid substances. The composition has the advantages of few component types, small batch-to-batch difference and high stability.
The composition can slow down the aging speed of cells when being added into a stem cell culture medium, so the invention also provides an application of the composition in the stem cell culture medium. In particular to the application of the composition in preparing stem cell culture medium. In one example, the stem cell is a mesenchymal stem cell. More specifically, the above composition is added to a stem cell culture medium to prepare a stem cell culture medium.
In addition, an embodiment of the present invention provides a method for preparing the above composition, comprising the steps of: the above mixture is prepared by uniformly mixing the components of the above composition.
The preparation method of the composition is simple, convenient and easy to operate, and is easy for large-scale production.
In addition, the invention also provides a stem cell culture medium, which comprises the composition and a serum-free culture medium.
Specifically, the serum-free culture medium is a serum-free mesenchymal stem cell culture medium. Alternatively, the serum-free medium is DMEM/F12, alpha-MEM or DMEM. Of course, in other embodiments, the serum-free medium is not limited to the above, and may be other serum-free media.
In one embodiment, the final concentration of serum albumin in the stem cell culture medium is 0.5-50. mu.g/mL, the final concentration of basic fibroblast growth factor is 10-30 ng/mL, the final concentration of transforming growth factor beta 1 is 0.3-1 ng/mL, and the final concentration of epidermal growth factor is 5-15 ng/mL. Optionally, in the stem cell culture medium, the final concentration of serum albumin is 1 to 10 μ g/mL, the final concentration of basic fibroblast growth factor is 15 to 20ng/mL, the final concentration of transforming growth factor beta 1 is 0.5 to 0.75ng/mL, and the final concentration of epidermal growth factor is 7.5 to 10 ng/mL. Optionally, in the stem cell culture medium, the final concentration of serum albumin is 1 to 5 μ g/mL, the final concentration of basic fibroblast growth factor is 10 to 20ng/mL, the final concentration of transforming growth factor beta 1 is 0.5 to 1ng/mL, and the final concentration of epidermal growth factor is 5 to 10 ng/mL.
In one embodiment, the composition comprises an antioxidant, and in this case, the antioxidant is present in the stem cell culture medium at a final concentration of 150nmol/mL to 250 nmol/mL. Further, the final concentration of the antioxidant in the stem cell culture medium is 150nmol/mL to 200 nmol/mL.
In one embodiment, the composition comprises transferrin, wherein the transferrin is present in the stem cell culture medium at a final concentration of 1% by volume.
In one embodiment, the composition comprises dexamethasone, wherein the final concentration of dexamethasone is 50 to 150pmol/mL in the stem cell culture medium. Further, the final concentration of dexamethasone in the stem cell culture medium is 50 to 100 pmol/mL. Further, the final concentration of dexamethasone in the stem cell culture medium is 50 to 90 pmol/mL.
In one embodiment, the composition comprises an antibiotic, wherein the potency of the antibiotic in the stem cell culture medium is between 100U/mL and 2100U/mL. Further, the titer of the antibiotic in the stem cell culture medium is 100U/mL-500U/mL. Furthermore, the titer of the antibiotic in the stem cell culture medium is 100U/mL-200U/mL.
In one embodiment, the composition comprises a lipid material, and the final concentration of the lipid material in the stem cell culture medium is between 30pg/mL and 50 pg/mL. Further, the final concentration of the lipid substance in the stem cell culture medium is 35pg/mL to 45 pg/mL.
In one embodiment, the stem cell culture medium does not contain fibronectin (Fn).
In one embodiment, the stem cell culture medium is composed of the composition and a serum-free medium.
The stem cell culture medium does not contain serum, comprises the composition, and can slow down cell aging and has high safety when used for culturing cells. In addition, when the above-mentioned stem cell culture medium is used, it is not necessary to pre-treat the culture vessel with fibronectin so that the culture vessel is coated with fibronectin, and the culture can be carried out directly with an empty culture vessel, which is time-saving and cost-saving. In addition, the stem cell culture medium has clear components, few component types, small batch-to-batch difference and high stability.
In addition, an embodiment of the present invention provides a method for preparing the stem cell culture medium, including the steps of: the above composition is added to a serum-free medium to prepare the above stem cell medium. The amount of the above composition added may be adjusted according to the final concentration of each component in the stem cell culture medium to be prepared.
The preparation method of the stem cell culture medium is simple, convenient and easy, and easy for industrial production, and the prepared stem cell culture medium can be used for stem cell culture and can slow down the aging of stem cells.
In addition, an embodiment of the present invention provides a method for culturing stem cells, including the steps of: the stem cells are inoculated into the above-mentioned stem cell culture medium for culture, wherein the culture vessel for culturing the stem cells does not have to be precoated with fibronectin.
Optionally, the stem cell is a mesenchymal stem cell. Further, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell. Of course, the mesenchymal stem cell is not limited to the umbilical cord mesenchymal stem cell, but may be other mesenchymal stem cells.
Optionally, the amount of mesenchymal stem cells inoculated is 0.6 × 104Per cm2～1×104Per cm2。
In one embodiment, the frozen stem cells are thawed and then cultured in a culture vessel containing the above-described stem cell culture medium, wherein the culture vessel is not pretreated with (i.e., the culture vessel is not coated with) fibronectin.
The culture method of the stem cells is simple and convenient, and the stem cells cultured according to the culture method of the stem cells are not easy to age.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. Unless otherwise specified, the recombinant human serum albumin in the following examples and comparative examples is TL-108 from Warcarb), the recombinant human basic fibroblast is 100-18B from Peprotech, the transforming growth factor beta 1 is 100-36E from Peprotech, the epidermal growth factor is GF316 from Sigma-Aldrich, the lipid substance is Gibco TMThe product of chemical ly Defined Lipid Concentrate, cat # 11905031, Insulin-Transferrin-Selenium (ITS) is PB180431 of Procell.
Examples 1 to 11
The stem cell culture media of each example consisted of the composition and α - Μ medium, the components of the composition of each example and the final concentrations in the stem cell culture media are shown in table 1. Wherein, the compositions in the stem cell culture media of the embodiments 1 to 10 are all composed of recombinant human serum albumin, recombinant human basic fibroblast, transforming growth factor beta 1, epidermal growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and ITS; the stem cell culture medium of example 11 consists of recombinant human serum albumin, recombinant human basic fibroblast, transforming growth factor β 1, epidermal growth factor and α - Μ Ε culture medium.
The preparation method of the stem cell culture medium of each embodiment includes but is not limited to the following steps:
the components of the composition were weighed at the final concentrations shown in table 1 and mixed with the α - Μ Ε Μ culture medium, respectively, to prepare the stem cell culture media of the examples.
Comparative example 1
The stem cell culture medium of comparative example 1 consists of a composition and a culture medium of α - Μ Ε Μ, the specific composition and content are shown in table 1.
The preparation method of the stem cell culture medium of comparative example 1 includes, but is not limited to, the following steps:
the components were weighed at the final concentrations shown in table 1 and mixed with the culture medium of α - Μ Ε Μ, the stem cell culture medium of comparative example 1 was prepared.
Comparative example 2
The composition of the stem cell culture medium of this comparative example was approximately the same as that of example 11, except that this comparative example replaced the epidermal growth factor of example 11 with the same amount of DGEA peptide. In this comparative example, the composition of the stem cell culture medium was: the culture medium comprises recombinant serum albumin, recombinant basic fibroblast growth factors, transforming growth factors beta 1, DGEA peptides and an alpha-MFLA culture medium, wherein the final concentration of the recombinant serum albumin is 2 mug/mL, the final concentration of the recombinant basic fibroblast growth factors is 20ng/mL, the final concentration of the transforming growth factors beta 1 is 0.5ng/mL, and the final concentration of the DGEA peptides is 5 ng/mL.
The preparation method of the stem cell culture medium of comparative example 2 includes, but is not limited to, the following steps:
the components were weighed at final concentrations and mixed with the culture medium of α - Μ Ε Μ, the stem cell culture medium of the present comparative example was prepared.
Comparative example 3
The composition of the stem cell culture medium of this comparative example was approximately the same as that of example 1, except that recombinant serum albumin was omitted from this comparative example. In this comparative example, the stem cell culture medium consisted of recombinant human basic fibroblast growth factor, transforming growth factor β 1, epidermal growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid material and ITS, the contents of each component being detailed in table 1.
Comparative example 4
The composition of the stem cell culture medium of this comparative example was approximately the same as that of example 1, except that the recombinant human basic fibroblast growth factor was omitted from this comparative example. In this comparative example, the stem cell culture medium consisted of recombinant human serum albumin, transforming growth factor β 1, epidermal growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipids and ITS, the contents of each component being specified in table 1.
Comparative example 5
The composition of the stem cell culture medium of this comparative example was approximately the same as that of example 1, except that the transforming growth factor beta 1 was omitted. In this comparative example, the stem cell culture medium consisted of recombinant human serum albumin, recombinant human basic fibroblast growth factor, epidermal growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipids and ITS, the contents of each component being specified in table 1.
Comparative example 6
The composition of the stem cell culture medium of this comparative example was approximately the same as that of example 1, except that epidermal growth factor was omitted. In this comparative example, the stem cell culture medium consisted of recombinant human serum albumin, recombinant human basic fibroblast growth factor, transforming growth factor β 1, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipids and ITS, the contents of each component being detailed in table 1.
TABLE 1
Testing
(1) And taking out the frozen umbilical cord mesenchymal stem cells of the 3 rd generation, and unfreezing the umbilical cord mesenchymal stem cells in a water bath at 37 ℃ to form a cell suspension.
(2) Slowly adding the cell suspension obtained in the step (1) into a prepared centrifuge tube (10 mL of DPBS is added in advance) in a super clean bench; sampling and counting after uniform resuspension.
(3) Centrifuging the cell suspension subjected to the heavy suspension in the step (2) at 1200rpm for 5min, and removing the supernatant; then adding a proper amount of DPBS (platelet-rich plasma-enhanced Raman Spectroscopy) to resuspend cells, centrifuging at 1200rpm for 5min, and removing supernatant to remove residual FBS;
(4) according to the counting results, corresponding cell suspensions were prepared using the media of each example and comparative example, respectively, each having a cell concentration of 1.0X 106Individual cells/mL.
(5) By 1.0X 104Individual cell/cm 2The wells were uniformly seeded in 6-well plates (uncoated with fibronectin) with 3 parallel wells set.
(6) Standing at 37 deg.C and 5% CO2Culturing in a cell culture box, and changing the culture solution every day on the 2 nd day. The following evaluation tests were carried out:
(I) cell morphology observation
The operation method comprises the following steps: the cell morphology of each group was observed and the image was collected under a microscope by inverting the cells cultured until day 1, day 2 and day 3, respectively, and the results are shown in FIGS. 1 to 3.
As is apparent from fig. 1 to 3, the stem cell culture media of examples 1 to 11 can be used for culturing the recovered mesenchymal stem cells, and the number of mesenchymal stem cells is significantly increased in the stem cell culture media of examples 3, 5, 7 and 8.
(II) cell expansion fold determination
The operation method comprises the following steps: after culturing for 3 days, the cells were collected from each group, and the amount of the collected cells was counted using a hemocytometer to calculate the fold expansion of the cells, wherein the ratio of the amount of the collected cells to the amount of the inoculated cells was the fold expansion of the cells, and the results are shown in table 2.
TABLE 2
As is apparent from table 2, the stem cell culture media of examples 1 to 11 can be used for culturing the restored mesenchymal stem cells, and among them, the mesenchymal stem cells in the stem cell culture media of example 5 have the highest expansion factor, and the stem cell culture media of example 3 are the next.
(III) cellular senescence assay
The operation method comprises the following steps: after 3 days of culture, each group was stained using a beta galactosidase assay, the degree of cell senescence was determined, and the cell surface antigen CD146 was detected using a flow cytometer. The results are shown in FIG. 4 and Table 3.
TABLE 3
In FIG. 4, the black part (blue in the color chart) is senescent cells (stained by beta-galactosidase), and it can be seen from FIG. 4 that the number of senescent cells in the stem cell culture medium of example 5 is minimal, and that of examples 3 and 7 times.
CD146 is a surface antigen marker associated with cellular senescence, with lower numbers indicating more rapid cellular senescence. As can be seen from Table 3, the CD146 ratios in examples 1-11 were high, with the CD146 ratio in example 5 being the highest.
As described above, it was found that the mesenchymal stem cells cultured in the stem cell culture media of examples 1 to 11 had a high expansion ratio and a slow aging rate, and it was not necessary to coat fibronectin in a culture vessel in advance.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A composition comprising serum albumin, basic fibroblast growth factor, transforming growth factor β 1, and epidermal growth factor;
preferably, the mass ratio of the serum albumin, the basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor is (0.5-50 μ g): (10 ng-30 ng): (0.3 ng-1 ng): (5 ng-15 ng), the composition does not contain serum.
2. The composition according to claim 1, wherein the mass ratio of serum albumin to basic fibroblast growth factor is (1-10 μ g): (15 ng-20 ng);
and/or the mass ratio of the serum albumin to the transforming growth factor beta 1 is (1-10 mug): (0.5 ng-0.75 ng);
And/or the mass ratio of the serum albumin to the epidermal growth factor is (1-10 mug): (7.5 ng-10 ng).
3. The composition of claim 1 or 2, further comprising at least one of an antioxidant, an antibiotic, transferrin, dexamethasone, and a lipid material;
optionally, the antioxidant is selected from at least one of ascorbic acid 2-phosphate, superoxide dismutase, catalase, and glutathione peroxidase;
optionally, the antibiotic is selected from at least one of penicillin and streptomycin.
4. The composition according to claim 1 or 2, characterized in that it comprises an antioxidant in a molar amount to the mass of serum albumin ratio (150-250 nmol): (0.5. mu.g-50. mu.g);
and/or the composition comprises an antibiotic, and the ratio of the titer of the antibiotic to the mass of the serum albumin (100U-2100U): (0.5. mu.g-50. mu.g);
and/or, the composition comprises transferrin, the volume of the transferrin to the mass of the serum albumin ratio is 0.01 mL: (0.5. mu.g-50. mu.g);
and/or the composition comprises dexamethasone, wherein the ratio of the molar amount of the dexamethasone to the mass of the serum albumin (50-150 pmol): (0.5. mu.g-50. mu.g);
And/or the composition comprises lipid substances, and the mass ratio of the lipid substances to the serum albumin is (30-50 pg): (0.5. mu.g-50. mu.g).
5. Use of a composition according to any one of claims 1 to 4 in a stem cell culture medium;
preferably, the stem cell is a mesenchymal stem cell.
6. A stem cell culture medium comprising the composition of any one of claims 1 to 4 and a serum-free medium;
preferably, the stem cell culture medium can increase proliferation and/or delay senescence of stem cells.
7. The stem cell culture medium of claim 6, wherein the final concentration of serum albumin is 0.5-50 μ g/mL, the final concentration of basic fibroblast growth factor is 10-30 ng/mL, the final concentration of transforming growth factor β 1 is 0.3-1 ng/mL, and the final concentration of epidermal growth factor is 5-15 ng/mL.
8. The stem cell culture medium according to claim 6 or 7, wherein the serum-free medium is a serum-free mesenchymal stem cell culture medium.
9. The stem cell culture medium of claim 8, wherein the serum-free medium is DMEM/F12, alpha-MEM, or DMEM;
Preferably, the serum-free medium is alpha-MEM.
10. A method for culturing stem cells, comprising the steps of:
inoculating the stem cells into the stem cell culture medium according to any one of claims 6 to 9 for culture;
preferably, the stem cell is a mesenchymal stem cell.
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