Serum-free medium for culturing vascular endothelial cells and use thereof

Abstract

The application discloses a serum-free culture medium for vascular endothelial cells and application thereof, and belongs to the technical field of cell culture. The serum-free culture medium for vascular endothelial cells comprises a basic culture medium, and further comprises human serum albumin, endothelial cell growth factor, epidermal growth factor, follicular inhibin-like recombinant protein 1, fibroblast growth factor, insulin-like growth factor, heparin sodium, hydrocortisone, L-glutamine and ascorbic acid. The serum-free culture medium has the advantages of clear components, stable quality and low cost, can promote the division and proliferation of vascular endothelial cells, can better maintain the state of endothelial cells, has high scientific research and medical application values, and can make the cultured vascular endothelial cells have the advantages of high proliferation speed, short doubling time, good cell state, high survival rate and high activity. In addition, the serum-free culture medium does not contain xenoantigen components, will not cause an immune rejection reaction with the human body, and is suitable for industrialized production of vascular endothelial cell related products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a serum-free culture medium for vascular endothelial cells and application thereof. BACKGROUND

[0002] In recent years, vascular endothelial cells have attracted extensive attention from scholars. Vascular endothelial cells refer to a monolayer of flat epithelium lining the inner surface of the cardiovascular system, blood vessels and lymphatic vessels. They form the inner wall of blood vessels, can phagocytose foreign bodies, bacteria, necrotic and aging tissues, and are also involved in many physiological activities, including vasoconstriction and vasodilation to control blood pressure; permeability; blood clotting (involved in thrombus formation and fibrinolysis); improving arteriosclerosis; angiogenesis; inflammation and swelling (such as edema); immunity and pathological development, such as atherosclerosis and malignant tumors.

[0003] Vascular endothelial cells have significant therapeutic effects and broad application prospects in stem cell medicine, and are used to treat various refractory diseases, including atherosclerosis, myocardial infarction, diabetic complications, hypertension, new coronavirus disease, malignant tumors, etc. On the one hand, vascular endothelial cells and their extracellular vesicles can be directly used for the treatment of these diseases. On the other hand, vascular endothelial cells can be used as a therapeutic target for certain diseases.

[0004] Therefore, it is particularly important to develop a special culture medium suitable for the growth and reproduction of vascular endothelial cells, to maintain their high cell activity and cell morphology, to achieve the large-scale and rapid reproduction of vascular endothelial cells, and to meet the market demand for vascular endothelial cells.

[0005] However, most of the culture media on the market contain animal serum such as fetal bovine serum. Although animal serum such as fetal bovine serum is rich in nutrients and has a significant effect on promoting cell proliferation and normal growth, the serum is expensive and its chemical composition is uncertain, and the quality of batches also has great instability, which can easily lead to deviations in the culture results and is not conducive to the industrialized production of cultured cells. In addition, the components of fetal bovine serum added to the culture medium may become an infection source of blood-borne pathogenic bacteria and a xenogeneic antigen, increasing the risk of disease in patients or causing immune rejection reactions in patients.

[0006] Therefore, it is particularly necessary to develop a serum-free, chemically defined special culture medium for vascular endothelial cells for the clinical application of vascular endothelial cells and the marketing of related products. SUMMARY

[0007] The purpose of the present application is to provide a serum-free culture medium for vascular endothelial cells and application thereof, to solve the problem that there is no serum-free, chemically stable and good culture effect special culture medium for vascular endothelial cells on the market at present, to fill the market gap and help the rapid marketing of vascular endothelial cell related products.

[0008] To achieve the above object, the application provides a serum-free medium for vascular endothelial cells, which comprises a basic medium and further comprises human serum albumin, endothelial cell growth factor, epidermal growth factor, follistatin-like recombinant protein 1, fibroblast growth factor, insulin-like growth factor, heparin sodium, hydrocortisone, L-glutamine and ascorbic acid.

[0009] Preferably, the basic medium comprises but is not limited to M199 basic medium, DMEM / F12 basic medium, IMDM basic medium and DMEM basic medium.

[0010] Preferably, the basic medium further comprises 0.1-5% human serum albumin, 1-50 ng / mL endothelial cell growth factor, 1-50 ng / mL epidermal growth factor, 1-20 ng / mL follistatin-like recombinant protein 1, 1-20 ng / mL fibroblast growth factor, 5-50 ng / mL insulin-like growth factor, 10-200 μg / mL heparin sodium, 0.01-10 μg / mL hydrocortisone, 1-20 mM L-glutamine and 100-200 μg / mL ascorbic acid.

[0011] Preferably, the basic medium further comprises 0.5-3% human serum albumin, 2-15 ng / mL endothelial cell growth factor, 2-15 ng / mL epidermal growth factor, 1-9 ng / mL follistatin-like recombinant protein 1, 1-9 ng / mL fibroblast growth factor, 10-20 ng / mL insulin-like growth factor, 80-130 μg / mL heparin sodium, 0.1-5 μg / mL hydrocortisone, 1-9 mM L-glutamine and 110-170 μg / mL ascorbic acid.

[0012] Preferably, the basic medium further comprises 2% human serum albumin, 8 ng / mL endothelial cell growth factor, 10 ng / mL epidermal growth factor, 4 ng / mL follistatin-like recombinant protein 1, 5 ng / mL fibroblast growth factor, 15 ng / mL insulin-like growth factor, 100 μg / mL heparin sodium, 1 μg / mL hydrocortisone, 5 mM L-glutamine and 150 μg / mL ascorbic acid.

[0013] Preferably, the basic medium further comprises 0.5% human serum albumin, 2 ng / mL endothelial cell growth factor, 3 ng / mL epidermal growth factor, 2 ng / mL follicular suppressin-like recombinant protein 1 recombinant protein, 1 ng / mL fibroblast growth factor, 10 ng / mL insulin-like growth factor, 90 ug / mL sodium heparin sulfate, 0.5 ug / mL hydrocortisone, 2 mM L-glutamine, 110 ug / mL ascorbic acid.

[0014] Preferably, the basic medium further comprises 3% human serum albumin, 14 ng / mL endothelial cell growth factor, 15 ng / mL epidermal growth factor, 8 ng / mL follicular suppressin-like recombinant protein 1 recombinant protein, 9 ng / mL fibroblast growth factor, 18 ng / mL insulin-like growth factor, 130 ug / mL sodium heparin sulfate, 4 ug / mL hydrocortisone, 9 mM L-glutamine, 170 ug / mL ascorbic acid.

[0015] The application of a serum-free medium for vascular endothelial cells in culturing vascular endothelial cells.

[0016] Therefore, the serum-free medium for vascular endothelial cells provided by the application has the following technical effects:

[0017] 1. The medium composition is clear and stable, and the production cost is greatly reduced by replacing acetylheparin sulfate with low-cost sodium heparin sulfate, which is suitable for industrial production.

[0018] 2. The medium has a very good promoting effect on the division and proliferation of vascular endothelial cells, and can better maintain the stem cell state, which has high scientific research and medical application value.

[0019] 3. The cultured vascular endothelial cells have a fast proliferation speed, a short doubling time, a good cell state, a high survival rate, and strong activity.

[0020] 4. The medium does not contain animal serum and does not contain antigen components, and will not produce an immune rejection reaction with the human body, and has good safety.

[0021] The technical solutions of the application will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the description of the embodiments of the application will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0023] Figure 1is the state of each group of cells when grown to 24h;

[0024] Figure 2 is the state of each group of cells when grown to 48h;

[0025] Figure 3 is the identification result of the vascular endothelial cells cultured by the application by CD31 flow cytometry. DETAILED DESCRIPTION

[0026] The technical solutions of the application are further described below by means of the drawings and examples.

[0027] In order to make the purpose, technical solutions and advantages of the application more clear, thorough and complete, the technical solutions in the two embodiments of the application are described clearly and completely below by means of the drawings and examples.

[0028] The reagent information used in the examples is shown in Table 1.

[0029] Table 1 reagent information used in the examples

[0030]

[0031] Example 1

[0032] Pre-isolated and frozen P3 generation umbilical vein vascular endothelial cells were selected, and after resuscitation, they were subcultured to P4 generation, the cells were digested, centrifuged, and the supernatant was removed. The cells were resuspended in M199 complete culture medium containing 10% FBS, 100 μg / mL HSS and 5 mM L-Gln, and the cell density was adjusted to 1×10 5 / mL. The cells were inoculated into a 6-well plate, 2 mL per well, 3 replicate wells per group, and placed in a 5% CO2, 100% humidity, 37°C cell culture incubator for overnight culture.

[0033] After the cells were completely attached, the cells were transferred to new culture medium for continuous culture for 48h. The composition of the culture medium was: 2% HBS, 8 ng / mL ECGS, 10 ng / mL EGF, 4 ng / mL rFSTL1, 5 ng / mL bFGF, 15 ng / mL IGF-1, 100 μg / mL HSS, 1 μg / mL H-cort, 5 mM L-Gln, 150 μg / mL AA, and the volume was supplemented to 50 mL with M199 culture medium.

[0034] Example 2

[0035] The cell resuscitation, subculture method and example one are completely same, after the cell completely adheres, the new culture medium component that turns to is: 0.5% HBS, 2ng / mL ECGS, 3ng / mL EGF, 2ng / mL rFSTL1, 1ng / mL bFGF, 10ng / mL IGF-1, 90 μg / mL HSS, 0.5 μg / mL H-cort, 2mM L-Gln, 110 μg / mL AA, with M199 culture medium supplement volume to 50 mL.

[0036] Example three

[0037] The cell resuscitation, subculture method and example one are completely same, after the cell completely adheres, the new culture medium component that turns to is: 0.5% HBS, 2ng / mL ECGS, 3ng / mL EGF, 2ng / mL rFSTL1, 1ng / mL bFGF, 10ng / mL IGF-1, 90 μg / mL HSS, 0.5 μg / mL H-cort, 2mM L-Gln, 110 μg / mL AA, with M199 culture medium supplement volume to 50 mL.

[0038] Comparative experiment

[0039] The cell resuscitation, subculture method and example one are completely same, after the cell completely adheres, the new culture medium component that turns to is: 0.5% HBS, 2ng / mL ECGS, 3ng / mL EGF, 2ng / mL rFSTL1, 1ng / mL bFGF, 10ng / mL IGF-1, 90 μg / mL HSS, 0.5 μg / mL H-cort, 2mM L-Gln, 110 μg / mL AA, with M199 culture medium supplement volume to 50 mL.

[0040] Comparative example 1: 10% FBS, 10ng / mL ECGS, 100 μg / mL HSS, 5mM L-Gln, with M199 culture medium supplement volume to 50 mL.

[0041] Comparative example 2: 2% FBS, 10ng / mL EGF, 5ng / mL VEGF, 5ng / mL bFGF, 15ng / mL IGF-1, 0.75U / mL HS, 1 μg / mL H-cort, 10mM L-Gln, 150 μg / mL AA, with M199 culture medium supplement volume to 50 mL.

[0042] Example one and comparative example are set up 3 biological repeats respectively. At the time of cell turning new culture medium and cultivating to 24h and 48h, the cell state in example one, comparative example 1 and comparative example 2 is observed and photographed respectively. Then respectively digest and collect cell, calculate cell total number, live cell number, cell viability and cell proliferation multiple.

[0043] Result analysis

[0044] The results of Comparative Example 1 and Comparative Example 2 were compared with those of Example 1. The experimental results were statistically analyzed using GraphPad 8.0.2 software. Unpaired t-tests were used for pairwise comparisons between groups. The significance level was set at α = 0.05. When P ≤ 0.05, the difference was significant; when P < 0.01, the difference was extremely significant; and when P > 0.05, the difference was not significant.

[0045] The state of the three groups of cells after 24 hours of growth is as follows: Figure 1 As shown in the figures, Figure a represents cells from Comparative Example 1; Figure b represents cells from Comparative Example 2; Figure c represents cells from the present invention group; and Figures d and f are magnified views of Figures a and c, respectively. It can be seen from the figures that the cell confluence in Comparative Example 1 (a) reached approximately 80%; the cell confluence in Comparative Example 2 (b) was approximately 70%, and the cell morphology showed a tendency to differentiate into fibroblasts; the cell confluence in the present invention group (c) was approximately 85%, and the cells generally exhibited a typical oval epithelial-like morphology and were in good condition (f).

[0046] The state of the three groups of cells after 48 hours of growth is as follows: Figure 2 As shown in the figures, Figure a represents cells from Comparative Example 1; Figure b represents cells from Comparative Example 2; Figure c represents cells from the present invention group; and Figures df are magnified views of Figures a and c, respectively. It can be seen from the figures that the cell confluence in Comparative Example 1 (a) reached approximately 90%; the cell confluence in Comparative Example 2 (b) was approximately 85%, with a significant number of cells differentiating towards fibroblasts; and the cell confluence in the present invention group (c) was approximately 95%.

[0047] Figure 3 The results of CD31 flow cytometry staining of vascular endothelial cells from this invention group show a cell purity greater than 99.9%. Figure 3 and Figure 2 (f) It can be seen that the cells cultured in this invention are basically in a typical oval epithelial-like morphology, with high cell transparency and excellent condition.

[0048] The above results indicate that the culture medium formulation of the present invention can better maintain cell morphology.

[0049] Table 2 Comparison of total cell count in each group

[0050] Group Total cell number t value P value Comparative Example 1 (6.06 ± 0.46) x 10 5 ]] 3.051 0.0380 Comparative Example 2 (3.86 ± 0.22) x 10 5 ]] 4.667 0.0095 The Invention (6.52 ± 0.37) x 10 5 ]] - -

[0051] The total number of cells in each group after 48 hours of growth is shown in Table 2. The total number of cells in the group of this invention was (6.52±0.37)×10⁻⁶. 5 The total cell count in control group 1 was (6.06±0.46)×10⁻⁶. 5 Compared with the control group of the present invention, t = 3.051, P = 0.0380, the difference was significant. The total number of cells in the two control groups was (3.86 ± 0.22) × 10⁻⁶. 5The difference is extremely significant compared with the group of the application, t = 4.667, P = 0.0095.

[0052] Comparison of the number of living cells in each group is shown in Table 3.

[0053] Group Viable cell number t value P value Comparative Example 1 (5.83 ± 0.44) x 10 5 ]] 3.053 0.0379 Comparative Example 2 (3.57 ± 0.39) x 10 5 ]] 4.930 0.0079 The Invention (6.39 ± 0.27) x 10 5 ]] - -

[0054] The number of living cells in each group is shown in Table 3. The total number of living cells in the group of the application is (6.39 ± 0.27) x 10 5 The total number of living cells in the group of Comparative Example 1 is (5.83 ± 0.44) x 10 5 The difference is significant compared with the group of the application, t = 3.053, P = 0.0379. The total number of living cells in the group of Comparative Example 2 is (3.57 ± 0.39) x 10 5 The difference is extremely significant compared with the group of the application, t = 4.930, P = 0.0079.

[0055] Comparison of the cell viability in each group is shown in Table 4.

[0056] Group Cell viability (%) t value P value Comparative Example 1 95.93±0.05 62.680 <0.0001 Comparative Example 2 92.06±0.04 207.200 <0.0001 The Invention 98.04±0.03 - -

[0057] The cell viability in each group is shown in Table 4. The cell viability in the group of the application is (98.04 ± 0.03) %, the cell viability in the group of Comparative Example 1 is (95.93 ± 0.05) %, the difference is extremely significant compared with the group of the application, t = 62.680, P < 0.0001. The cell viability in the group of Comparative Example 2 is (92.06 ± 0.04) %, the difference is extremely significant compared with the group of the application, t = 207.200, P < 0.0001.

[0058] Comparison of the cell proliferation fold in each group is shown in Table 5.

[0059] Group Cell proliferation fold t value P value Comparative Example 1 2.03±0.15 4.996 0.0075 Comparative Example 2 0.93±0.04 9.327 0.0007 The Invention 2.26±0.10 - -

[0060] The cell proliferation fold in each group is shown in Table 5. The cell proliferation fold in the group of the application is (2.26 ± 0.10) fold, the cell proliferation fold in the group of Comparative Example 1 is (2.03 ± 0.15) fold, the difference is extremely significant compared with the group of the application, t = 4.996, P = 0.0075. The cell proliferation fold in the group of Comparative Example 2 is (0.93 ± 0.04) fold, the difference is extremely significant compared with the group of the application, t = 9.327, P = 0.0007.

[0061] The above results show that the formula of the application has very good proliferation and division promoting effect on vascular endothelial cells, can maintain higher cell viability, shorten cell doubling time, and is very beneficial to the industrialization of cell related products.

[0062] Therefore, the serum-free culture medium for vascular endothelial cells has the advantages of clear components, stable quality, low cost, good promotion effect on the division and proliferation of vascular endothelial cells, better maintenance of endothelial cell state, high scientific research and medical application value, fast proliferation speed of cultured vascular endothelial cells, short doubling time, good cell state, high survival rate, high activity, no animal serum in the culture medium, no xenogeneic antigen components, no immune rejection reaction with the human body, and suitability for industrialized production of vascular endothelial cell related products.

[0063] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A serum-free medium for vascular endothelial cells, characterized by comprising: The serum-free culture medium for vascular endothelial cells is composed of a basic medium, 0.1-5% human serum albumin, 1-50 ng / mL endothelial cell growth factor ECGS, 1-50 ng / mL epidermal growth factor, 1-20 ng / mL follicular suppressin-like protein 1 recombinant protein, 1-20 ng / mL fibroblast growth factor, 5-50 ng / mL insulin-like growth factor, 10-200 μg / mL sodium heparin sulfate, 0.01-10 μg / mL hydrocortisone, 1-20 mM L-glutamine and 100-200 μg / mL ascorbic acid; the basic medium comprises M199 basic medium. ​ 2. The serum-free medium for vascular endothelial cells according to claim 1, characterized by: The serum-free culture medium for vascular endothelial cells is composed of a basic medium, 0.5-3% human serum albumin, 2-15 ng / mL endothelial cell growth factor ECGS, 2-15 ng / mL epidermal growth factor, 1-9 ng / mL follicular suppressin-like protein 1 recombinant protein, 1-9 ng / mL fibroblast growth factor, 10-20 ng / mL insulin-like growth factor, 80-130 μg / mL sodium heparin sulfate, 0.1-5 μg / mL hydrocortisone, 1-9 mM L-glutamine and 110-170 μg / mL ascorbic acid.

3. The serum-free medium for vascular endothelial cells according to claim 1, wherein: the serum-free medium for vascular endothelial cells is a medium for culturing vascular endothelial cells in which the concentration of the serum is 0.5% or less. The serum-free culture medium for vascular endothelial cells is composed of a basic medium, 0.5-3% human serum albumin, 2-15 ng / mL endothelial cell growth factor ECGS, 2-15 ng / mL epidermal growth factor, 1-9 ng / mL follicular suppressin-like protein 1 recombinant protein, 1-9 ng / mL fibroblast growth factor, 10-20 ng / mL insulin-like growth factor, 80-130 μg / mL sodium heparin sulfate, 0.1-5 μg / mL hydrocortisone, 1-9 mM L-glutamine and 110-170 μg / mL ascorbic acid.

4. The serum-free culture medium for vascular endothelial cells according to claim 1, characterized in that: The serum-free culture medium for vascular endothelial cells is composed of a basic medium, 0.5-3% human serum albumin, 2-15 ng / mL endothelial cell growth factor ECGS, 2-15 ng / mL epidermal growth factor, 1-9 ng / mL follicular suppressin-like protein 1 recombinant protein, 1-9 ng / mL fibroblast growth factor, 10-20 ng / mL insulin-like growth factor, 80-130 μg / mL sodium heparin sulfate, 0.1-5 μg / mL hydrocortisone, 1-9 mM L-glutamine and 110-170 μg / mL ascorbic acid.

5. The serum-free culture medium for vascular endothelial cells according to claim 1, characterized in that: The serum-free culture medium for vascular endothelial cells is composed of a basic medium, 0.5-3% human serum albumin, 2-15 ng / mL endothelial cell growth factor ECGS, 2-15 ng / mL epidermal growth factor, 1-9 ng / mL follicular suppressin-like protein 1 recombinant protein, 1-9 ng / mL fibroblast growth factor, 10-20 ng / mL insulin-like growth factor, 80-130 μg / mL sodium heparin sulfate, 0.1-5 μg / mL hydrocortisone, 1-9 mM L-glutamine and 110-170 μg / mL ascorbic acid.

6. Use of the serum-free culture medium for vascular endothelial cells according to any one of claims 1-5 in culturing vascular endothelial cells.

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