CN111748522A - Stem cell culture medium and application thereof - Google Patents

Stem cell culture medium and application thereof Download PDF

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CN111748522A
CN111748522A CN202010760134.9A CN202010760134A CN111748522A CN 111748522 A CN111748522 A CN 111748522A CN 202010760134 A CN202010760134 A CN 202010760134A CN 111748522 A CN111748522 A CN 111748522A
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culture medium
cell culture
stem cell
vitamin
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刘洋
李艳菊
王飞清
何志旭
舒丽萍
朱晓东
刘广洋
梁惠玲
黄美
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Affiliated Hospital of Guizhou Medical University
First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine
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First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine
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Abstract

The invention discloses a stem cell culture medium, which belongs to the technical field of stem cell culture and comprises the following substances: serum-free culture medium, platelet lysate, glutamine, non-essential amino acids, compound vitamins, lipids and plant extraction additive. The cell culture medium is suitable for culturing stem cells separated from tissues or organs with stem cells such as bone marrow, umbilical cords, adipose tissues and the like, can improve the amplification speed of the cells by 4-6 times, does not influence the differentiation potential of the cells, and obviously improves the purity and proliferation capacity of stem cell culture.

Description

Stem cell culture medium and application thereof
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a stem cell culture medium and application thereof.
Background
Many researches show that the stem cells can not only self-renew, but also differentiate into other functional cells under proper conditions, so that the stem cells are expected to become an effective means for treating human intractable diseases. However, stem cells are present in a very small amount in normal adult tissues, and how to rapidly expand and culture stem cells in vitro is an important technique for studying the mechanism of action of stem cells and exploring therapeutic methods for treating human diseases.
Stem cells are present in minor amounts, but are widely distributed in various tissues and organs of mammals, including, but not limited to, bone marrow, umbilical cord, adipose tissue, brain tissue, retina, heart, liver, lung, and skin. Numerous studies have shown that these low stem cells can be further expanded in vitro by appropriate methods, one of the most common stem cell culture media being medium supplemented with 10% fetal bovine serum. However, stem cells cultured in this medium not only grow relatively slowly, but also have a greatly reduced potential to differentiate into functional cells after more than 5 passages.
The application numbers are: 201810430129.4 discloses a stem cell culture medium, its application and stem cell culture method, which ensures the expansion stability of stem cells and shortens the culture time. However, the culture effect of stem cells of different tissues is unstable, and further improvement and enhancement are still needed.
Disclosure of Invention
The invention aims to provide a stem cell culture medium and application thereof.
The technical purpose of the invention is realized by the following technical scheme:
a stem cell culture medium comprises the following substances in parts by weight:
85-90 parts of a serum-free culture medium, 7-10 parts of a platelet lysate, 2-3 parts of glutamine, 3-5 parts of non-essential amino acid, 2-4 parts of vitamin complex, 1-3 parts of lipid and 0.5-1 part of a plant extraction additive.
Preferably, the composition comprises the following substances in parts by weight:
86-88 parts of serum-free culture medium, 8-9 parts of platelet lysate, 2.3-2.6 parts of glutamine, 3.5-4.5 parts of non-essential amino acid, 2.5-3.5 parts of vitamin complex, 1.5-2.5 parts of lipid and 0.6-0.8 part of plant extraction additive.
More preferably, the following substances are included in corresponding parts by weight:
87 parts of serum-free culture medium, 8.5 parts of platelet lysate, 2.5 parts of glutamine, 4 parts of nonessential amino acid, 3 parts of composite vitamin, 2 parts of lipid and 0.7 part of plant extraction additive.
Further, the compound vitamin comprises the following substances in parts by weight: 10-14 parts of choline chloride, 3-6 parts of folic acid, 2-5 parts of inositol, 4-8 parts of nicotinamide, 6-12 parts of riboflavin, 7-11 parts of vitamin B12, 20-25 parts of vitamin C and 15-20 parts of vitamin E.
Preferably, the compound vitamin comprises the following substances in parts by weight: 12 parts of choline chloride, 5 parts of folic acid, 4 parts of inositol, 6 parts of nicotinamide, 9 parts of riboflavin, 9 parts of vitamin B12, 24 parts of vitamin C and 18 parts of vitamin E.
Furthermore, the lipid is composed of the following substances in parts by weight: 20-25 parts of oleic acid, 5-10 parts of cholesterol, 4-8 parts of linoleic acid, 3-6 parts of lipoic acid and 1-5 parts of lipid.
Furthermore, the lipid is composed of the following substances in parts by weight: 24 parts of oleic acid, 8 parts of cholesterol, 6 parts of linoleic acid, 5 parts of lipoic acid and 3 parts of lipid.
Further, the preparation method of the plant extraction additive comprises the following steps:
(1) cleaning Morinda citrifolia, juicing, and filtering to obtain fruit pulp for later use;
(2) adding a complex enzyme accounting for 0.3-0.5% of the total mass of the fruit pulp into the fruit pulp, controlling the enzymolysis temperature to be 38-43 ℃, and performing enzymolysis treatment for 2-2.5 hours to obtain an enzymolysis liquid for later use; the compound enzyme is prepared by correspondingly mixing cellulase, hemicellulase, beta-glucanase and pectinase according to a weight ratio of 5-7: 4-6: 1-3: 2-4;
(3) centrifuging the enzymolysis solution, collecting the centrifugate, and standing to obtain supernatant of the centrifugate for later use;
(4) and filtering the supernatant to obtain the plant extract additive.
Further, the aperture of the filter membrane is controlled to be 0.7-5 μm during the filtration treatment in the step (4).
The application of a stem cell culture medium is used for culturing stem cells.
Compared with the prior art, the invention has the following advantages:
the invention changes the raw material components and the proportion ratio of the stem cell culture medium, wherein the serum-free culture medium is favorable for improving the amplification purity of stem cells, so that vascular endothelial cells, fiber cells and the like can not grow, the added platelet lysate contains factors required by the proliferation and division of the stem cells, the added glutamine and non-essential amino acid provide the required nutrient supply for the stem cells, the compound vitamins and lipids also protect the rapid stability of the growth of the stem cells, the added plant extraction additive is extracted from noni fruits serving as a raw material and is rich in an extracting agent of active ingredients such as silonin and the like, and the substance can promote the growth of the cells, activate and improve the activity of tyrosinase, eliminate free radicals and enhance the service life and the amplification capacity of the cells. Under the synergistic effect of the raw material components, the cell culture medium is suitable for culturing stem cells separated from tissues or organs with stem cells such as bone marrow, umbilical cord, adipose tissue and the like, can improve the amplification speed of the cells by 4-6 times, does not influence the differentiation potential of the cells, obviously improves the purity and the proliferation capacity of stem cell culture, and has great popularization and application values.
Detailed Description
The following will clearly and completely describe the technical solutions of 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 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 stem cell culture medium comprises the following substances in parts by weight:
components Parts by weight
Serum-free medium 85 portions of
Platelet lysate 7 portions of
Glutamine 2 portions of
Non-essential amino acids 3 portions of
Compound vitamin 2 portions of
Lipids 1 part of
Plant extract additive 0.5 portion
The compound vitamin comprises the following substances in parts by weight: 10 parts of choline chloride, 3 parts of folic acid, 2 parts of inositol, 4 parts of nicotinamide, 6 parts of riboflavin, 7 parts of vitamin B12, 20 parts of vitamin C and 15 parts of vitamin E.
The lipid comprises the following substances in parts by weight: 20 parts of oleic acid, 5 parts of cholesterol, 4 parts of linoleic acid, 3 parts of lipoic acid and 1 part of lipid.
The preparation method of the plant extraction additive comprises the following steps:
(1) cleaning Morinda citrifolia, juicing, and filtering to obtain fruit pulp for later use;
(2) adding complex enzyme with the total mass of 0.3% into the fruit pulp, controlling the enzymolysis temperature to be 38 ℃, and performing enzymolysis treatment for 2 hours to obtain enzymolysis liquid for later use; the complex enzyme is prepared by correspondingly mixing cellulase, hemicellulase, beta-glucanase and pectinase according to the weight ratio of 5:4:1: 2;
(3) centrifuging the enzymolysis solution, collecting the centrifugate, and standing to obtain supernatant of the centrifugate for later use;
(4) filtering the supernatant to obtain filter membrane with pore diameter of 0.7 μm, and collecting the plant extract additive.
Example 2
A stem cell culture medium comprises the following substances in parts by weight:
components Parts by weight
Serum-free medium 86 portions of
Platelet lysate 8 portions of
Glutamine 2.3 parts of
Non-essential amino acids 3.5 parts of
Compound vitamin 2.5 parts of
Lipids 1.5 parts of
Plant extract additive 0.6 part
The compound vitamin comprises the following substances in parts by weight: 10 parts of choline chloride, 5 parts of folic acid, 2 parts of inositol, 4 parts of nicotinamide, 6 parts of riboflavin, 9 parts of vitamin B12, 20 parts of vitamin C and 15 parts of vitamin E.
The lipid comprises the following substances in parts by weight: 20 parts of oleic acid, 8 parts of cholesterol, 6 parts of linoleic acid, 3 parts of lipoic acid and 1 part of lipid.
The preparation method of the plant extraction additive comprises the following steps:
(1) cleaning Morinda citrifolia, juicing, and filtering to obtain fruit pulp for later use;
(2) adding complex enzyme with the total mass of 0.3% into the fruit pulp, controlling the enzymolysis temperature to be 40 ℃, and performing enzymolysis treatment for 2 hours to obtain enzymolysis liquid for later use; the compound enzyme is prepared by mixing cellulase, hemicellulase, beta-glucanase and pectinase according to the weight ratio of 5:4:1: 3, mixing;
(3) centrifuging the enzymolysis solution, collecting the centrifugate, and standing to obtain supernatant of the centrifugate for later use;
(4) filtering the supernatant to obtain filter membrane with pore diameter of 1 μm, and collecting the plant extract additive.
Example 3
A stem cell culture medium comprises the following substances in parts by weight:
components Parts by weight
Serum-free medium 87 portions
Platelet lysate 8.5 parts of
Glutamine 2.5 parts of
Non-essential amino acids 4 portions of
Compound vitamin 3 portions of
Lipids 2 portions of
Plant extract additive 0.7 portion of
The compound vitamin comprises the following substances in parts by weight: 12 parts of choline chloride, 5 parts of folic acid, 4 parts of inositol, 6 parts of nicotinamide, 9 parts of riboflavin, 9 parts of vitamin B12, 24 parts of vitamin C and 18 parts of vitamin E.
The lipid comprises the following substances in parts by weight: 24 parts of oleic acid, 8 parts of cholesterol, 6 parts of linoleic acid, 5 parts of lipoic acid and 3 parts of lipid.
The preparation method of the plant extraction additive comprises the following steps:
(1) cleaning Morinda citrifolia, juicing, and filtering to obtain fruit pulp for later use;
(2) adding complex enzyme with the total mass of 0.4% into the fruit pulp, controlling the enzymolysis temperature at 40 ℃, and performing enzymolysis treatment for 2.3h to obtain enzymolysis liquid for later use; the complex enzyme is prepared by correspondingly mixing cellulase, hemicellulase, beta-glucanase and pectinase according to the weight ratio of 6:5:2: 3;
(3) centrifuging the enzymolysis solution, collecting the centrifugate, and standing to obtain supernatant of the centrifugate for later use;
(4) filtering the supernatant to obtain filter membrane with pore diameter of 2 μm, and collecting the plant extract additive.
Example 4
A stem cell culture medium comprises the following substances in parts by weight:
components Parts by weight
Serum-free medium 88 portions of
Platelet lysate 9 portions of
Glutamine 2.6 parts of
Non-essential amino acids 4.5 parts of
Compound vitamin 3.5 parts of
Lipids 2.5 parts of
Plant extract additive 0.8 portion of
The compound vitamin comprises the following substances in parts by weight: 12 parts of choline chloride, 6 parts of folic acid, 5 parts of inositol, 8 parts of nicotinamide, 10 parts of riboflavin, 9 parts of vitamin B12, 25 parts of vitamin C and 20 parts of vitamin E.
The lipid comprises the following substances in parts by weight: 25 parts of oleic acid, 8 parts of cholesterol, 8 parts of linoleic acid, 5 parts of lipoic acid and 4 parts of lipid.
The preparation method of the plant extraction additive comprises the following steps:
(1) cleaning Morinda citrifolia, juicing, and filtering to obtain fruit pulp for later use;
(2) adding complex enzyme with the total mass of 0.5% into the fruit pulp, controlling the enzymolysis temperature at 40 ℃, and performing enzymolysis for 2.3h to obtain enzymolysis liquid for later use; the complex enzyme is prepared by correspondingly mixing cellulase, hemicellulase, beta-glucanase and pectinase according to the weight ratio of 7:6:3: 3;
(3) centrifuging the enzymolysis solution, collecting the centrifugate, and standing to obtain supernatant of the centrifugate for later use;
(4) filtering the supernatant to obtain filter membrane with pore diameter of 4 μm, and collecting the plant extract additive.
Example 5
A stem cell culture medium comprises the following substances in parts by weight:
Figure BDA0002612839950000071
Figure BDA0002612839950000081
the compound vitamin comprises the following substances in parts by weight: 14 parts of choline chloride, 6 parts of folic acid, 5 parts of inositol, 8 parts of nicotinamide, 12 parts of riboflavin, 11 parts of vitamin B12, 25 parts of vitamin C and 20 parts of vitamin E.
The lipid comprises the following substances in parts by weight: 25 parts of oleic acid, 10 parts of cholesterol, 8 parts of linoleic acid, 6 parts of lipoic acid and 5 parts of lipid.
The preparation method of the plant extraction additive comprises the following steps:
(1) cleaning Morinda citrifolia, juicing, and filtering to obtain fruit pulp for later use;
(2) adding complex enzyme with the total mass of 0.5% into the fruit pulp, controlling the enzymolysis temperature at 43 ℃, and performing enzymolysis treatment for 2.5h to obtain enzymolysis liquid for later use; the complex enzyme is prepared by correspondingly mixing cellulase, hemicellulase, beta-glucanase and pectinase according to the weight ratio of 7:6:3: 4;
(3) centrifuging the enzymolysis solution, collecting the centrifugate, and standing to obtain supernatant of the centrifugate for later use;
(4) filtering the supernatant to obtain filter membrane with pore diameter of 5 μm, and collecting the plant extract additive.
Comparative example 1
A stem cell culture medium comprises the following substances in parts by weight:
Figure BDA0002612839950000082
Figure BDA0002612839950000091
this comparative example 1 is compared to example 3 with the only difference that the plant extract additive components are omitted except that the process steps are the same.
The serum-free medium used in examples 1 to 5 and comparative example 1 was supplied by CellGenix corporation, Cat. 24803-; platelet lysate is supplied by the company commpass biological, cat #: PLS 2; glutamine is glutamine, Gln; non-essential amino acids are NEAA, non-essentialamic acid.
Control group
The application numbers are: 201810430129.4 discloses a stem cell culture medium.
In order to compare the effects of the invention, experiments on the proliferation acceleration effect of stem cells of different tissues are carried out, and the experiments specifically comprise:
1. test medium: stem cell culture media corresponding to example 2, comparative example 1, and control;
2. culturing a stem cell source: human stem cells (ADSC, BMSC, UMBSC) derived from three tissues and organs, namely fat, marrow and umbilical cord;
3. in vitro cell culture experiments: the cell inoculation density is controlled to be 10-20%, and after inoculation, a culture medium is added for culture (37 ℃, 5% CO)2)；
The cell density is measured every 8 hours by an MTT method, and the specific experimental result data are shown in the following tables 1-3:
TABLE 1
Figure BDA0002612839950000092
Figure BDA0002612839950000101
TABLE 2
Figure BDA0002612839950000102
TABLE 3
Figure BDA0002612839950000103
As can be seen from the above tables 1 to 3, the culture medium corresponding to the embodiment 2 of the present invention can rapidly realize proliferation of stem cells, and has a good and stable proliferation promoting effect on different types of stem cells.
In order to further compare the effects of the invention, an experiment for analyzing the influence of the potency of the adipogenic cells is carried out, which specifically comprises the following steps:
1. test medium: stem cell culture media corresponding to example 2, comparative example 1, and control;
2. culturing a stem cell source: human stem cells (ADSC, BMSC, UMBSC) derived from three tissues and organs, namely fat, marrow and umbilical cord;
3. in vitro cell culture experiments: in order to further examine the influence of the stem cells on the differentiation potential after 10 generations of expansion by the culture medium described in the present invention, the differentiation experiments of adipogenic cells after the expansion of human stem cells (ADSC, BMSC, UMBSC) derived from three tissues and organs, fat, marrow and umbilical cord, were determined according to the Methods described in the literature (Methods in Molecular Biology,2011, Volume 702, Part 3, 193-; the specific method is that the stem cells are induced by replacing a adipogenic culture medium when the density of the stem cells is about 80 percent, and the adipogenic culture medium has the formula: DMEM/F12 medium with 1% FBS, 1mM dexamethasone, 200mM indomethacin, 0.5nM 3-isobutyl-1-methylxanthine (IBMX), 10mg/ml insulin. The solution was changed once every two days, and oil red O staining was performed two weeks after induction. The oil red O staining solution is prepared from oil red O, and the oil red O storage solution: weighing 0.5g of oil red O, adding into 100ml of isopropanol, and filtering for 0.22 mm; oil red O working fluid: and (4) sucking 6ml of oil red O storage liquid, adding into 4ml of distilled water, and uniformly mixing. The specific method is that after the adipogenic induction is successful, the culture medium is sucked out and washed for 2 times by PBS; adding newly-prepared oil red O staining solution for dyeing for 1 hour; the staining solution was removed, washed 3 times with PBS for 2min each time, and observed with a microscope. After the fat cells are stained, the number of positive cells is counted, and the data of the specific experiment result are shown in the following table 4:
Figure BDA0002612839950000111
as can be seen from the above Table 4, the culture medium of the present invention can well ensure the differentiation potential of stem cells, and even after 10 generations of expansion, the culture medium still has a cell differentiation rate of 90%, and compared with the existing matrix, the differentiation potential is significantly improved.
In conclusion, the stem cell culture medium can well ensure the differentiation potential of stem cells, obviously improve the purity and proliferation capacity of stem cell culture, and has great popularization and application values.

Claims (10)

1. A stem cell culture medium is characterized by comprising the following substances in parts by weight:
85-90 parts of a serum-free culture medium, 7-10 parts of a platelet lysate, 2-3 parts of glutamine, 3-5 parts of non-essential amino acid, 2-4 parts of vitamin complex, 1-3 parts of lipid and 0.5-1 part of a plant extraction additive.
2. The stem cell culture medium according to claim 1, comprising the following substances in parts by weight:
86-88 parts of serum-free culture medium, 8-9 parts of platelet lysate, 2.3-2.6 parts of glutamine, 3.5-4.5 parts of non-essential amino acid, 2.5-3.5 parts of vitamin complex, 1.5-2.5 parts of lipid and 0.6-0.8 part of plant extraction additive.
3. The stem cell culture medium according to claim 2, comprising the following substances in parts by weight:
87 parts of serum-free culture medium, 8.5 parts of platelet lysate, 2.5 parts of glutamine, 4 parts of nonessential amino acid, 3 parts of composite vitamin, 2 parts of lipid and 0.7 part of plant extraction additive.
4. The stem cell culture medium according to any one of claims 1 to 3, wherein the vitamin complex comprises the following substances in parts by weight: 10-14 parts of choline chloride, 3-6 parts of folic acid, 2-5 parts of inositol, 4-8 parts of nicotinamide, 6-12 parts of riboflavin, 7-11 parts of vitamin B12, 20-25 parts of vitamin C and 15-20 parts of vitamin E.
5. The stem cell culture medium according to claim 4, wherein the vitamin complex comprises the following substances in parts by weight: 12 parts of choline chloride, 5 parts of folic acid, 4 parts of inositol, 6 parts of nicotinamide, 9 parts of riboflavin, 9 parts of vitamin B12, 24 parts of vitamin C and 18 parts of vitamin E.
6. The stem cell culture medium according to any one of claims 1 to 3, wherein the lipid is composed of the following substances in parts by weight: 20-25 parts of oleic acid, 5-10 parts of cholesterol, 4-8 parts of linoleic acid, 3-6 parts of lipoic acid and 1-5 parts of lipid.
7. The stem cell culture medium of claim 6, wherein the lipid is comprised of, in parts by weight: 24 parts of oleic acid, 8 parts of cholesterol, 6 parts of linoleic acid, 5 parts of lipoic acid and 3 parts of lipid.
8. The stem cell culture medium according to any one of claims 1 to 3, wherein the preparation method of the plant extract additive comprises the following steps:
(1) cleaning Morinda citrifolia, juicing, and filtering to obtain fruit pulp for later use;
(2) adding a complex enzyme accounting for 0.3-0.5% of the total mass of the fruit pulp into the fruit pulp, controlling the enzymolysis temperature to be 38-43 ℃, and performing enzymolysis treatment for 2-2.5 hours to obtain an enzymolysis liquid for later use; the compound enzyme is prepared by correspondingly mixing cellulase, hemicellulase, beta-glucanase and pectinase according to a weight ratio of 5-7: 4-6: 1-3: 2-4;
(3) centrifuging the enzymolysis solution, collecting the centrifugate, and standing to obtain supernatant of the centrifugate for later use;
(4) and filtering the supernatant to obtain the plant extract additive.
9. The stem cell culture medium according to claim 8, wherein the pore size of the filter membrane in the filtration treatment in the step (4) is controlled to be 0.7 to 5 μm.
10. Use of a stem cell culture medium according to any one of claims 1 to 9 for culturing stem cells.
CN202010760134.9A 2020-07-31 2020-07-31 Stem cell culture medium and application thereof Pending CN111748522A (en)

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