A cell culture medium additive and use thereof

Abstract

The application provides a cell culture medium additive and application thereof, and belongs to the technical field of biological reagent development. The chlorogenic acid and ailanthus oil are compounded in the application, so that the problem of limited cell proliferation speed in the prior art can be overcome. The cell culture medium additive provided by the application is added to a cell culture medium, so that the cell proliferation rate can be significantly improved, a large number of cells can be obtained in a short time, and the demand of experiments in various fields for cell materials can be met. The application proves that 1%, 100 mg / L of a chlorogenic acid solution and 0.1%, 50 mg / L of an ailanthus oil solution together as a cell culture medium additive can significantly improve the cell proliferation rate, the conventional culture level of six days can be reached on the second day of culture, and the cell proliferation rate reaches 213.79% on the sixth day, so that a certain synergistic promotion effect exists between the two.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological reagent development, and particularly relates to a cell culture medium additive and application thereof. BACKGROUND

[0002] Cell culture is a culture technology that takes cells from in-vivo tissues, and makes them survive, grow, reproduce, and maintain their structure and function in an in-vitro environment that simulates the in-vivo environment. Through cell culture, the life process of cells, cell carcinogenesis, cell engineering, and other issues can be studied. The cultured living cells have the characteristics of large quantity, uniformity, and repeatability, can be regulated by various physical, chemical, biological, and other factors, and can be studied by various methods such as inversion, fluorescence, electron, laser confocal microscopy, flow cytometry, immunohistochemistry, in-situ hybridization, and isotope labeling, and has been widely used in different scientific research fields.

[0003] Most cells have limited proliferation speed, and a large number of required cells cannot be obtained in a short time. Expanding the production scale of cell proliferation will increase unnecessary costs. In the existing technology, the methods for improving the in-vitro expansion efficiency of cells mainly include feeder layer culture method and promoting cell proliferation by optimizing cell culture medium formula. However, the feeder layer culture method introduces impurity cells from other animal sources, and the purity of seed cells obtained by this method is poor, which limits its further application. The research on optimizing the culture medium formula to improve the cell proliferation speed mainly adds growth hormone and the like, which may affect the physiological condition of cells and is not suitable for industrial application. SUMMARY

[0004] The present application aims to provide a cell culture medium additive to solve the problem of limited cell proliferation speed in the prior art.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0006] The present application provides a cell culture medium additive, which comprises a chlorogenic acid solution and a atranol solution.

[0007] The concentration of the chlorogenic acid solution is 75-125 mg / L.

[0008] The concentration of the atranol solution is 40-60 mg / L.

[0009] Preferably, the volume ratio of the chlorogenic acid solution to the atranol solution is 0.8-1.2:0.08-0.15.

[0010] The present application also provides the application of the above-mentioned cell culture medium additive in ex-vivo cell culture.

[0011] Preferably, the cell culture medium additive is added to the cell culture medium for ex vivo cell culture.

[0012] The adding amount of the cell culture medium additive is 0.8-1.4% of the total volume of the cell culture medium.

[0013] Preferably, the ex vivo cell is an epithelial cell, a fibroblast cell or a neural cell.

[0014] Preferably, the epithelial cell is a CHO-K1 cell, a 293 cell, a vero cell, a hela cell, an MDCK cell, an Sf9 cell or an IEC-6 cell.

[0015] Preferably, the cell culture medium is DMEM complete medium, RPMI 1640 complete medium, MEM complete medium, DMEM / F12 complete medium, M199 complete medium, IMDM complete medium or L15 complete medium.

[0016] Technical effects and advantages of the present application:

[0017] The present application can significantly improve the proliferation rate of cells by compounding chlorogenic acid and ailanthone and adding them to the cell culture medium, and a large number of cells can be obtained in a short time to meet the demand for cell materials in various fields. The present application proves that 1%, 100 mg / L of chlorogenic acid solution and 0.1%, 50 mg / L of ailanthone solution together as cell culture medium additives can significantly improve the proliferation rate of cells, and the level of conventional culture for six days can be reached on the second day of culture, and the cell proliferation rate reaches 213.79% on the sixth day, and there is a certain synergistic promotion between the two. DETAILED DESCRIPTION

[0018] The present application provides a cell culture medium additive, which comprises chlorogenic acid and ailanthone; the chlorogenic acid and ailanthone are preferably used in the form of a solution, the solvent used in the chlorogenic acid solution and the ailanthone solution is preferably Dulbecco's phosphate buffer, the concentration of the chlorogenic acid solution is 75-125 mg / L, preferably 80-120 mg / L; the concentration of the ailanthone solution is 40-60 mg / L, preferably 45-55 mg / L, the volume ratio of the chlorogenic acid solution to the ailanthone solution is preferably 0.8-1.2:0.08-0.15, further preferably 0.9-1.1:0.09-0.12; the chlorogenic acid solution and the ailanthone solution of the present application can be packaged separately before use or mixed together.

[0019] The application further provides application of the cell culture medium additive in ex vivo cell culture, preferably the cell culture medium additive is added into a cell culture medium for ex vivo cell culture; the addition amount of the cell culture medium additive is preferably 0.8-1.4% of the total volume of the cell culture medium, and is further preferably 0.9-1.1%; the ex vivo cell is preferably an epithelial cell, a fibroblast cell or a neural cell; the epithelial cell is preferably a CHO-K1 cell, a 293 cell, a vero cell, a hela cell, an MDCK cell, an Sf9 cell or an IEC-6 cell; the cell culture medium is preferably a complete culture medium, which can meet the normal growth and proliferation of cells, and is further preferably a DMEM complete culture medium, an RPMI1640 complete culture medium, a MEM complete culture medium, a DMEM / F12 complete culture medium, an M199 complete culture medium, an IMDM complete culture medium or an L15 complete culture medium; the cell culture medium additive is preferably stored at 2-6°C in the dark after being added into the cell culture medium.

[0020] The technical solutions provided by the application will be described in detail below in combination with examples, but they should not be understood as limiting the protection scope of the application.

[0021] Chlorogenic acid (327-97-9), HPLC≥98%, purchased from Chengdu Zhongbiao Hua Pure Biotechnology Co., Ltd.;

[0022] Jujubosone (29913-86-8), HPLC≥98%, purchased from Sichuan Weikeqi Biotechnology Co., Ltd.;

[0023] The rat small intestinal epithelial cell IEC-6 cell strain (batch number: CRL-1592) is purchased from the American ATCC Company

[0024] DMEM / F12 complete culture medium (containing 10% FBS) is purchased from Wuhan Punsai Life Science and Technology Co., Ltd.

[0025] Example 1

[0026] Chlorogenic acid is prepared into a 100mg / L chlorogenic acid solution with D-PBS, and jujubosone is prepared into a 50mg / L jujubosone solution with D-PBS, which are added into DMEM / F12 complete culture medium, the addition amount of the chlorogenic acid solution is 1% of the DMEM / F12 complete culture medium, and the addition amount of the jujubosone solution is 0.1% of the DMEM / F12 complete culture medium, and the preparation is stored at 4°C in the dark after being completed.

[0027] Example 2

[0028] Chlorogenic acid was prepared into a 125 mg / L chlorogenic acid solution with D-PBS, and ailanto was prepared into a 60 mg / L ailanto solution with D-PBS, which were added into DMEM / F12 complete culture medium, the added amount of the chlorogenic acid solution was 1.2% of the DMEM / F12 complete culture medium, and the added amount of the ailanto solution was 0.15% of the DMEM / F12 complete culture medium, and the preparation was stored at 4°C in the dark after completion.

[0029] Example 3

[0030] Chlorogenic acid was prepared into a 75 mg / L chlorogenic acid solution with D-PBS, and ailanto was prepared into a 40 mg / L ailanto solution with D-PBS, which were added into DMEM / F12 complete culture medium, the added amount of the chlorogenic acid solution was 0.8% of the DMEM / F12 complete culture medium, and the added amount of the ailanto solution was 0.08% of the DMEM / F12 complete culture medium, and the preparation was stored at 4°C in the dark after completion.

[0031] Comparative Example 1

[0032] Chlorogenic acid was prepared into a 100 mg / L chlorogenic acid solution with D-PBS, which was added into DMEM / F12 complete culture medium, and the added amount was 1% of the DMEM / F12 complete culture medium, and the preparation was stored at 4°C in the dark after completion.

[0033] Comparative Example 2

[0034] Ailanto was prepared into a 50 mg / L ailanto solution with D-PBS, which was added into DMEM / F12 complete culture medium, and the added amount was 0.1% of the DMEM / F12 complete culture medium, and the preparation was stored at 4°C in the dark after completion.

[0035] Comparative Example 3

[0036] Chlorogenic acid was prepared into a 50 mg / L chlorogenic acid solution with D-PBS, and ailanto was prepared into a 50 mg / L ailanto solution with D-PBS, which were added into DMEM / F12 complete culture medium, the added amount of the chlorogenic acid solution was 0.5% of the DMEM / F12 complete culture medium, and the added amount of the ailanto solution was 0.1% of the DMEM / F12 complete culture medium, and the preparation was stored at 4°C in the dark after completion.

[0037] Comparative Example 4

[0038] Chlorogenic acid was prepared into a 150 mg / L chlorogenic acid solution with D-PBS, and ailanto was prepared into a 50 mg / L ailanto solution with D-PBS, which were added into DMEM / F12 complete culture medium, the added amount of the chlorogenic acid solution was 2% of the DMEM / F12 complete culture medium, and the added amount of the ailanto solution was 0.1% of the DMEM / F12 complete culture medium, and the preparation was placed in a 4°C dark storage.

[0039] Comparative Example 5

[0040] Chlorogenic acid was prepared into a 100 mg / L chlorogenic acid solution with D-PBS, and ailanto was prepared into a 20 mg / L ailanto solution with D-PBS, which were added into DMEM / F12 complete culture medium, the added amount of the chlorogenic acid solution was 1% of the DMEM / F12 complete culture medium, and the added amount of the ailanto solution was 0.05% of the DMEM / F12 complete culture medium, and the preparation was placed in a 4°C dark storage.

[0041] Comparative Example 6

[0042] Chlorogenic acid was prepared into a 100 mg / L chlorogenic acid solution with D-PBS, and ailanto was prepared into a 100 mg / L ailanto solution with D-PBS, which were added into DMEM / F12 complete culture medium, the added amount of the chlorogenic acid solution was 1% of the DMEM / F12 complete culture medium, and the added amount of the ailanto solution was 0.5% of the DMEM / F12 complete culture medium, and the preparation was placed in a 4°C dark storage.

[0043] Experimental Example 1

[0044] The IEC-6 cell strain was thawed and recovered, and then subcultured, and when it was subcultured to the 5th generation, it was divided into groups, which were experimental groups A, B, C, D, E, F, G, and H, corresponding to Experimental Example 1, Comparative Examples 1-6, and a control group (DMEM / F12 complete culture medium without treatment), respectively.

[0045] The culture medium of each group of cell strains was replaced with the culture medium of Experimental Example 1, Comparative Examples 1-6, and the control group, respectively, and the initial concentration of each group of cells was 10 4 OD 540 = 0.35), and it was placed in a 37°C, 5% CO2 constant-temperature incubator for culture, and the experiment was started.

[0046] MTT method was used to detect cell growth, and after 22h, 44h, 68h, 92h, 116h, 140h after the start of the culture experiment, one piece of cell culture plate was taken out and 0.5mg / ml of MTT was added, and it was placed back in a 37℃, 5% CO2 incubator for 4h, the supernatant was discarded, 100ul DMSO was added to each well, mixed, and the OD value was measured at 540nm, and the cell proliferation rate at each time point was calculated, the cell proliferation rate at the corresponding time point = [1-(control group OD value-experimental group OD value) / control group OD value] x 100%, each group was repeated three times, and the experimental results were recorded, as shown in Tables 1-2 below:

[0047] Table 1 OD value determination results

[0048] experimental group 1d 2d 3d 4d 5d 6d A 0.45±0.02 0.58±0.05 0.70±0.01 0.85±0.04 0.99±0.02 1.24±0.05 B 0.38±0.03 0.44±0.02 0.55±0.06 0.63±0.03 0.70±0.01 0.83±0.04 C 0.39±0.04 0.46±0.04 0.52±0.02 0.64±0.03 0.68±0.02 0.69±0.06 D 0.38±0.02 0.42±0.02 0.48±0.02 0.56±0.04 0.64±0.05 0.72±0.04 E 0.37±0.04 0.43±0.07 0.46±0.04 0.52±0.06 0.60±0.06 0.68±0.04 F 0.39±0.03 0.42±0.03 0.46±0.03 0.52±0.02 0.58±0.01 0.64±0.02 G 0.36±0.05 0.38±0.02 0.40±0.02 0.41±0.04 0.37±0.02 0.35±0.06 H 0.38±0.04 0.42±0.01 0.46±0.04 0.50±0.01 0.54±0.03 0.58±0.08

[0049] Table 2 cell proliferation rate statistical results

[0050]

[0051]

[0052] It can be known from the analysis of the experimental result data that the 1%, 100mg / L chlorogenic acid solution and the 0.1%, 50mg / L ailanthone solution provided by the present application together as cell culture medium additives can significantly improve the cell proliferation rate, and the cell proliferation rate can reach the level of conventional culture for six days on the second day of culture, and the cell proliferation rate reaches 213.79% on the sixth day, and there is a certain synergistic promotion effect between them. The promotion effect of the compound drug group on cell proliferation is significantly better than that of the drug monomer, and the drug dosage also determines the effect, wherein after 24h of experiment, the higher dosage of chlorogenic acid and ailanthone added makes the proliferation rate of IEC-6 cell strain lower, and the higher dosage of ailanthone added even presents the phenomenon of inhibiting cell strain proliferation in the later stage of the experiment, which may be that high-dose ailanthone can produce certain toxicity to cells.

[0053] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A cell culture medium additive, characterized in that, The chlorogenic acid solution and the ailanthus oil solution; The concentration of the chlorogenic acid solution is 75-125 mg / L; The concentration of the ailanthus oil solution is 40-60 mg / L.

2. The cell culture medium supplement of claim 1, wherein, The volume ratio of the chlorogenic acid solution to the ailanthus oil solution is 0.8-1.2:0.08-0.

15.

3. Use of the cell culture medium additive according to claim 1 or 2 in ex vivo cell culture.

4. Use of a cell culture medium additive according to claim 3 in ex vivo cell culturing, characterized in that, The cell culture medium additive is added to a cell culture medium for ex vivo cell culture. The addition volume of the cell culture medium additive is 0.8-1.4% of the total volume of the cell culture medium.

5. Use of a cell culture medium additive according to claim 4 in ex vivo cell culturing, characterized in that, The ex vivo cell is an epithelial cell, a fibroblast cell or a neural cell.

6. Use of a cell culture medium additive according to claim 5 in ex vivo cell culturing, characterized in that, The epithelial cell is a CHO-K1 cell, a 293 cell, a vero cell, a hela cell, an MDCK cell, an Sf9 cell or an IEC-6 cell.

7. Use of a cell culture medium additive according to claim 4 in ex vivo cell culturing, characterized in that, The cell culture medium is DMEM complete medium, RPMI 1640 complete medium, MEM complete medium, DMEM / F12 complete medium, M199 complete medium, IMDM complete medium or L15 complete medium.

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