CN112760279A - Mesenchymal stem cell culture medium - Google Patents

Mesenchymal stem cell culture medium Download PDF

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CN112760279A
CN112760279A CN202110031395.1A CN202110031395A CN112760279A CN 112760279 A CN112760279 A CN 112760279A CN 202110031395 A CN202110031395 A CN 202110031395A CN 112760279 A CN112760279 A CN 112760279A
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culture medium
mesenchymal stem
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杨晓晨
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Shengmei Gene Technology Beijing Co ltd
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Abstract

The invention relates to the technical field of cell culture proliferation, in particular to a mesenchymal stem cell culture medium which comprises a basic culture medium, wherein 0.1-0.2 wt% of enamel matrix protein derivatives are added into the basic culture medium, and the enamel matrix protein derivatives are derivatives of enamel matrix proteins extracted from young pig tooth embryos and comprise amelogenin, enamel mature protein, enamel protein, vulcanized enamel protein, protease and growth factor-like substances. The culture medium is scientific and reasonable in design, and compared with the traditional culture medium in which the enamel matrix protein derivative is added, the enamel matrix protein derivative plays an important role in inducing cell proliferation and differentiation and signal conduction; the enamel matrix protein derivative has good biocompatibility and biological safety, and can well promote the proliferation of mesenchymal stem cells.

Description

Mesenchymal stem cell culture medium
Technical Field
The invention relates to the technical field of cell culture proliferation, in particular to a mesenchymal stem cell culture medium.
Background
The culture medium is a solution for maintaining the survival and growth of cells in vitro, and is divided into a natural culture medium and a synthetic culture medium. Wherein the synthetic medium is artificially synthesized according to the species and amount of substances required for cell survival.
Mesenchymal stem cells are important members of the stem cell family, are primitive cells with self-replication and multidirectional differentiation potential, are the original cells of the body, and are the primitive cells forming various tissues and organs of the human body. Under certain conditions, the mesenchymal stem cells can be differentiated into various functional cells or tissues and organs, and are suitable for seed cells for repairing tissue and organ damage caused by aging and pathological changes. Therefore, the mesenchymal stem cells can be cultured in vitro to provide important medical materials.
The traditional culture medium is used for culturing the mesenchymal stem cells, the cell growth speed is low, and the growth characteristics and the differentiation potential of the mesenchymal stem cells can be influenced to a certain extent in the growth process, so that certain difficulty is caused in subsequent medical application. In addition, current research shows that the culture of mesenchymal stem cells by using 10% FBS culture medium or human platelet lysate culture medium can cause some immune response and influence the normal growth of cells.
Disclosure of Invention
The present invention is directed to overcoming the above problems in the conventional art and providing a mesenchymal stem cell culture medium.
In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:
a mesenchymal stem cell culture medium comprises a basic culture medium, wherein enamel matrix protein derivatives are added to the basic culture medium.
Further, in the mesenchymal stem cell culture medium, the addition amount of the enamel matrix protein derivative is 0.1-0.2 wt%.
Further, in the above mesenchymal stem cell culture medium, the enamel matrix protein derivative is a derivative of enamel matrix protein extracted from a young pig tooth embryo.
Further, in the mesenchymal stem cell culture medium, the enamel matrix protein derivative includes amelogenin, enamel mature protein, amelogenin, sulfurized enamel protein, protease and growth factor-like substances.
Further, in the mesenchymal stem cell culture medium, the enamel matrix protein derivative comprises the following components in parts by weight: 50-70 parts of amelogenin, 10-15 parts of amelogenin, 5-10 parts of glaze mature protein, 5-10 parts of glaze protein, 1-5 parts of vulcanized glaze protein, 1-3 parts of protease and 0.1-0.5 part of biological growth factor.
Further, in the above mesenchymal stem cell culture medium, the biological growth factor comprises a basic fibroblast growth factor and an epidermal growth factor.
Further, in the mesenchymal stem cell culture medium, the mass ratio of the basic fibroblast growth factor to the epidermal growth factor is 1: 1-2.
Further, in the mesenchymal stem cell culture medium, the components of the basic culture medium include: anhydrous calcium chloride, ferric nitrate, potassium chloride, anhydrous magnesium sulfate, sodium chloride, anhydrous sodium dihydrogen phosphate, succinic acid, sodium succinate, L-arginine hydrochloride, L-cystine hydrochloride, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, D-calcium pantothenate, choline tartrate, folic acid, inositol, nicotinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, glucose, sodium pyruvate, and phenol red.
Further, in the mesenchymal stem cell culture medium, the content of each component in the basic culture medium is as follows: 400mg/L of anhydrous calcium chloride 200-, 20-200mg/L, L-serine 20-200mg/L, L-threonine 20-200mg/L, L-tryptophan 5-50mg/L, L-tyrosine 20-200mg/L, L-valine 20-200mg/L, D-calcium pantothenate 1-10mg/L, choline tartrate 5-20mg/L, folic acid 1-10mg/L, inositol 5-10mg/L, nicotinamide 1-10mg/L, riboflavin 0.1-1mg/L, thiamine hydrochloride 1-10mg/L, pyridoxine hydrochloride 1-10mg/L, glucose 500 mg/L, sodium pyruvate 50-200mg/L, calcium pantothenate 1-10mg/L, and pharmaceutically acceptable salts thereof, 5-20mg/L of phenol red.
Further, in the mesenchymal stem cell culture medium, the content of each component in the basic culture medium is as follows: 265mg/L of anhydrous calcium chloride, 0.1mg/L of ferric nitrate, 400mg/L of potassium chloride, 97.67mg/L of anhydrous magnesium sulfate, 6400mg/L of sodium chloride, 109mg/L of anhydrous sodium dihydrogen phosphate, 75mg/L of succinic acid, 100mg/L, L of sodium succinate, 84mg/L, L of arginine hydrochloride, 63mg/L of cystine hydrochloride, 30mg/L, L of glycine, 42mg/L, L of histidine hydrochloride, 105mg/L, L of isoleucine, 105mg/L, L of leucine hydrochloride, 146mg/L, L of methionine, 30mg/L, L of phenylalanine, 66mg/L, L of serine, 42mg/L, L of threonine, 95mg/L, L of tryptophan, 16mg/L of serine, 72mg/L, L mg of L-tyrosine, 94mg/L, D mg/L of calcium pantothenate, 7.2mg/L of choline tartrate, 4mg/L of folic acid, 7.2mg/L of inositol, 4mg/L of nicotinamide, 0.4mg/L of riboflavin, 4mg/L of ammonium sulfate hydrochloride, 4mg/L of pyridoxine hydrochloride, 1000mg/L of glucose, 110mg/L of sodium pyruvate and 9.3mg/L of phenol red.
The invention has the beneficial effects that:
the invention adds enamel matrix protein derivatives in a basic culture medium, the enamel matrix protein derivatives are extracted from the young pig tooth germ, and the enamel matrix protein derivatives are mainly a mixture formed by amelogenin, mature enamel protein, sulfide enamel protein, protease, growth factor-like substances and the like, and play an important role in inducing cell proliferation and differentiation and signal conduction; the enamel matrix protein derivative has good biocompatibility and biological safety, and can well promote the proliferation of mesenchymal stem cells.
Of course, it is not necessary for any one product that embodies the invention to achieve all of the above advantages simultaneously.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a graph of growth of mesenchymal stem cells;
FIG. 2 is an observation view of mesenchymal stem cells obtained by culture medium culture;
FIG. 3 is a graph showing the results of flow cytometry;
fig. 4 is a graph showing the results of the measurement of osteogenic differentiation and adipogenic differentiation performance of the obtained mesenchymal stem cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A mesenchymal stem cell culture medium comprises a basal culture medium, wherein the basal culture medium is added with enamel matrix protein derivatives. The addition amount of enamel matrix protein derivative is 0.15 wt%. Wherein, the components of the basic culture medium are shown in the following table 1:
TABLE 1 composition of basal Medium
Figure BDA0002892317930000041
Figure BDA0002892317930000051
In this example, the enamel matrix protein derivative is a derivative of enamel matrix protein extracted from a young pig tooth germ. The composition of the enamel matrix protein derivative comprises: 60 parts of amelogenin, 12 parts of amelogenin, 8 parts of mature enamel protein, 7 parts of enamel protein, 3 parts of vulcanized enamel protein, 2 parts of protease and 0.35 part of biological growth-like factor.
In this embodiment, the biological-like growth factors include basic fibroblast growth factor and epidermal growth factor. The mass ratio of the basic fibroblast growth factor to the epidermal growth factor is 1: 2.
in this example, mesenchymal stem cells were cultured in a basal medium supplemented with enamel matrix protein derivatives and a basal medium not supplemented with enamel matrix protein derivatives, respectively, and cell growth curves were plotted according to the following steps:
s1: digesting the mesenchymal stem cells with good growth state;
s2: 12 orifice plates at 5X 105cells/hole are paved;
s3: cell counts were performed every 24h digestion 2 wells;
s4: the results were recorded and growth curves were plotted.
The result is shown in fig. 1, compared with the basal medium without the enamel matrix protein derivative, the basal medium with the enamel matrix protein derivative has the advantages that the mesenchymal stem cells grow faster under the former condition, and an obvious S-shaped growth curve is obtained, which indicates that the mesenchymal stem cell culture medium has a certain promotion effect on the growth of the mesenchymal stem cells.
The mesenchymal stem cells obtained by culturing the mesenchymal stem cell culture medium of the embodiment are shown in figure 2.
Example 2
Immunophenotypic testing
Mesenchymal stem cells cultured using the mesenchymal stem cell culture medium modified in example 1 (i.e., basal medium supplemented with enamel matrix protein derivative) were collected and adjusted to a cell concentration of 1X 106Antibodies were added per ml and incubated at room temperature for 30min in the dark. After completion of the incubation, the mixture was centrifuged at 1000rpm for 5min, and the supernatant was discarded. Adding 2ml PBS containing 1% fetal calf serum into each tube, mixing by vortex, centrifuging at 1000rpm for 5min, discarding supernatant, adding 0.5ml PBS into each tube, mixing by vortex, and detecting on machine with 1h internal flow cytometer.
The detection result of the flow cytometry is shown in fig. 3, the mesenchymal stem cells cultured by the improved mesenchymal stem cell culture medium express CD73 (96.6%), CD90 (99.2%), CD105 (97.7%), do not express CD45, CD34, CD11b, CD19 and HLA-DR, accord with the phenotype characteristics of the mesenchymal stem cells, and have no statistical difference in surface antigen expression (P > 0.05).
Osteogenic differentiation Performance test
Digesting the cells cultured in the improved mesenchymal stem cell culture medium, and then performing cell culture on the cells according to the ratio of 1 × 103Inoculating the inoculum size of cells/cm2 in a six-hole plate, adding an improved culture medium for 24h, adding an osteogenesis/chondrogenesis induction culture medium, continuously culturing for 2-4 weeks, changing the culture solution every 2-3 days, judging the formation of osteoblasts by an alizarin red staining method after the culture is finished, and identifying the formation of cartilage by an Alisin blue staining method.
After 1 week of culture, the cells are changed from spindle shape to polygon shape, and filamentous protrusions appear around the cells and can extend to the periphery, and the shape is obviously changed. Over 2 weeks of culture, the plaques are calcified, mineralized progressively in the cell matrix and begin to form a multi-layered nodule structure. After osteogenesis induction, red-stained calcium nodules appear after alizarin red staining; after chondrogenesis induction, the proteoglycan synthesized by blue chondrocytes appears after Alisin blue staining. The mesenchymal stem cells grown by the improved culture medium can still maintain good osteogenic differentiation potential.
Experiment on adipogenic differentiation Performance
Digesting the cells cultured in the improved mesenchymal stem cell culture medium, and then performing cell culture on the cells according to the ratio of 1 × 103cells/cm2The inoculation amount of the strain is inoculated in a six-hole plate, an improved mesenchymal stem cell culture medium is added for 24 hours, then a fat forming induction culture medium is added for continuous culture for 2-4 weeks, the liquid is changed every 2-3 days, and the fat drop formation is identified by oil red staining after the culture is finished.
Culturing for 3 days, gradually shrinking and shortening the cells from a spindle shape, wherein more than 90% of the cells become cubic or polygonal, and the shape is obviously changed; continuously culturing for 1 week, wherein the tiny fat drops appear in the cells, and the fat drops gradually expand and fuse with the prolonging of the culture time; after 2 weeks of culture, the whole cells were filled with droplets of fat that fused into clumps. Oil red staining revealed that intracellular fat was specifically stained red, as shown in fig. 4. The mesenchymal stem cells grown by the improved culture medium can still maintain good adipogenic differentiation potential.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims (10)

1. A mesenchymal stem cell culture medium comprises a basic culture medium, and is characterized in that: the basal medium is added with enamel matrix protein derivatives.
2. The mesenchymal stem cell culture medium of claim 1, wherein: the addition amount of the enamel matrix protein derivative is 0.1-0.2 wt%.
3. The mesenchymal stem cell culture medium of claim 1, wherein: the enamel matrix protein derivative is extracted from the teeth germ of the piglet.
4. The mesenchymal stem cell culture medium of claim 3, wherein: the enamel matrix protein derivative comprises amelogenin, mature enamel protein, sulfide enamel protein, protease and growth factor-like substances.
5. The mesenchymal stem cell culture medium of claim 4, wherein: the enamel matrix protein derivative comprises the following components in parts by weight: 50-70 parts of amelogenin, 10-15 parts of amelogenin, 5-10 parts of glaze mature protein, 5-10 parts of glaze protein, 1-5 parts of vulcanized glaze protein, 1-3 parts of protease and 0.1-0.5 part of biological growth factor.
6. The mesenchymal stem cell culture medium of claim 4, wherein: the biology-like growth factor comprises basic fibroblast growth factor and epidermoid growth factor.
7. The mesenchymal stem cell culture medium of claim 4, wherein: the mass ratio of the basic fibroblast growth factor to the epidermal growth factor is 1: 1-2.
8. The mesenchymal stem cell culture medium of claim 1, wherein: the basic culture medium comprises the following components: anhydrous calcium chloride, ferric nitrate, potassium chloride, anhydrous magnesium sulfate, sodium chloride, anhydrous sodium dihydrogen phosphate, succinic acid, sodium succinate, L-arginine hydrochloride, L-cystine hydrochloride, glycine, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, D-calcium pantothenate, choline tartrate, folic acid, inositol, nicotinamide, riboflavin, thiamine hydrochloride, pyridoxine hydrochloride, glucose, sodium pyruvate, and phenol red.
9. The mesenchymal stem cell culture medium of claim 8, wherein: the basic culture medium comprises the following components in percentage by weight: 400mg/L of anhydrous calcium chloride 200-, 20-200mg/L, L-serine 20-200mg/L, L-threonine 20-200mg/L, L-tryptophan 5-50mg/L, L-tyrosine 20-200mg/L, L-valine 20-200mg/L, D-calcium pantothenate 1-10mg/L, choline tartrate 5-20mg/L, folic acid 1-10mg/L, inositol 5-10mg/L, nicotinamide 1-10mg/L, riboflavin 0.1-1mg/L, thiamine hydrochloride 1-10mg/L, pyridoxine hydrochloride 1-10mg/L, glucose 500 mg/L, sodium pyruvate 50-200mg/L, calcium pantothenate 1-10mg/L, and pharmaceutically acceptable salts thereof, 5-20mg/L of phenol red.
10. The mesenchymal stem cell culture medium of claim 9, wherein: the basic culture medium comprises the following components in percentage by weight: 265mg/L of anhydrous calcium chloride, 0.1mg/L of ferric nitrate, 400mg/L of potassium chloride, 97.67mg/L of anhydrous magnesium sulfate, 6400mg/L of sodium chloride, 109mg/L of anhydrous sodium dihydrogen phosphate, 75mg/L of succinic acid, 100mg/L, L of sodium succinate, 84mg/L, L of arginine hydrochloride, 63mg/L of cystine hydrochloride, 30mg/L, L of glycine, 42mg/L, L of histidine hydrochloride, 105mg/L, L of isoleucine, 105mg/L, L of leucine hydrochloride, 146mg/L, L of methionine, 30mg/L, L of phenylalanine, 66mg/L, L of serine, 42mg/L, L of threonine, 95mg/L, L of tryptophan, 16mg/L of serine, 72mg/L, L mg of L-tyrosine, 94mg/L, D mg/L of calcium pantothenate, 7.2mg/L of choline tartrate, 4mg/L of folic acid, 7.2mg/L of inositol, 4mg/L of nicotinamide, 0.4mg/L of riboflavin, 4mg/L of ammonium sulfate hydrochloride, 4mg/L of pyridoxine hydrochloride, 1000mg/L of glucose, 110mg/L of sodium pyruvate and 9.3mg/L of phenol red.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2162531A1 (en) * 2007-05-04 2010-03-17 Ascendia AB Method and means for culturing osteoblastic cells
JP2016192962A (en) * 2015-04-01 2016-11-17 学校法人慶應義塾 METHOD FOR PRODUCING HUMAN ODONTOGENIC EPITHELIAL CELL AND HUMAN ODONTOGENIC MESENCHYMAL CELL FROM HUMAN iPS CELL

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2162531A1 (en) * 2007-05-04 2010-03-17 Ascendia AB Method and means for culturing osteoblastic cells
JP2016192962A (en) * 2015-04-01 2016-11-17 学校法人慶應義塾 METHOD FOR PRODUCING HUMAN ODONTOGENIC EPITHELIAL CELL AND HUMAN ODONTOGENIC MESENCHYMAL CELL FROM HUMAN iPS CELL

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JUE ET AL.: "The effects of enamel matrix derivative on the proliferation and differentiation of human mesenchymal stem cells", 《CLINICAL ORAL IMPLANTS RESEARCH》 *
孟茂花等: "釉基质衍生物诱导骨髓间充质干细胞成骨分化的效应及机制", 《中国组织工程研究》 *
陈宗道等: "《食品生物技术概论》", 31 January 2008 *

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