Method for carrying out PDGF-BB biological activity detection by using serum and serum-free culture medium

Abstract

The invention belongs to the technical field of biology, and discloses a method for detecting PDGF-BB biological activity by using serum and a serum-free culture medium. According to the present invention, the Yicosai MSC serum-free basic culture medium is used, and Yicosai fetal calf serum is matched to detect the PDGF-BB sample, such that advantages of high sensitivity, good repeatability, high precision, convenient use and the like are provided.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for detecting the biological activity of PDGF-BB using serum and serum-free medium. Background Art

[0002] Platelet-Derived Growth Factor (PDGF) is an alkaline glycoprotein synthesized by bone marrow macrophages and stored in platelet α-granules. Its molecular weight is 28-35 kDa, and it forms homo- or hetero-dimers (such as PDGF-AA, BB, AB, etc.) by A and B chains linked by disulfide bonds. Among them, PDGF-BB is the core member of signal regulation because of its strongest binding ability to all receptor subtypes (PDGFR-αα, αβ, ββ). The biological functions of PDGF-BB cover a wide range of physiological processes from embryonic development to tissue repair. For example, as a core regulator of connective tissue hyperplasia, PDGF can activate tyrosine kinase receptors (PDGFR-α / β), trigger signal pathways such as Ras-MAPK and PI3K / Akt, and drive the proliferation, migration and differentiation of various mesenchymal-derived cells such as vascular smooth muscle cells, fibroblasts and glial cells. In addition, at the initial stage of injury, PDGF can also promote the release of growth factors such as TGF-β and bFGF by activating macrophages, forming a cascade amplification effect, and at the same time enhancing the chemotactic activity of neutrophils and monocytes to coordinate the dynamic balance between inflammation and repair.

[0003] Since PDGF-BB can promote the division and proliferation of various cells, it is widely used as an important mitogen in various cell culture media. To ensure the stability between batches of the medium, it is very important to detect the biological activity of PDGF-BB. According to the requirements of the pharmacopoeia, the bioactivity detection of PDGF-BB usually needs to be determined based on its functional characteristics. For example, the ability of PDGF-BB to promote the growth of specific cells (such as fibroblasts or smooth muscle cells) is evaluated through cell proliferation experiments. In cell proliferation experiments, in order to prevent different baseline proliferations caused by inconsistent cell states from interfering with the results, low-serum medium (such as 0.5-1% FBS) is often used 24 hours before the experiment to make the cells enter the quiescent phase (G0 / G1 phase). However, there are still PDGF-binding proteins in the serum in the low-serum medium, which will lead to poor starvation treatment effect. When PDGF-BB is added after starvation, the experimental sensitivity is low, and the experimental background is high, and the signal-to-noise ratio usually does not exceed 2. In addition, the requirements for serum in pharmacopoeia-compliant experiments are relatively high. If the quality of the serum is not uniform, the repeatability and reliability of cell proliferation experiments will be significantly reduced, directly affecting the accuracy of PDGF-BB bioactivity detection results. Therefore, the accurate detection of PDGF-BB biological activity is still an urgent problem to be solved. Summary of the Invention

[0004] To solve the technical problems mentioned in the background art, the present invention provides a method for detecting the biological activity of PDGF-BB, and the technical solution is as follows:

[0005] A method for detecting the biological activity of PDGF-BB using serum and serum-free medium. For cell culture and amplification, DMEM high-glucose complete medium containing 10% ExCell fetal bovine serum is used, and for cell starvation, ExCell MSC serum-free basal medium is used.

[0006] Preferably, the cells are NIH / 3T3 cells.

[0007] Preferably, it specifically includes the following steps:

[0008] S1. Culture and amplification of NIH / 3T3 cells: Use DMEM high-glucose complete medium containing 10% ExCell fetal bovine serum to amplify and culture NIH / 3T3 cells at 37 °C, 5% CO2, and saturated humidity. Inoculate at a density of 4×10 4 cells / cm 2 and passage every 2 days;

[0009] S2. Plating: After culturing NIH / 3T3 cells for 2 generations, discard the medium in the culture flask, digest and collect NIH / 3T3 cells, resuspend them with DMEM high-glucose complete medium containing 10% ExCell fetal bovine serum to form a cell suspension containing 4×10 4 cells per 1 mL, inoculate 0.1 mL per well into a 96-well cell culture plate, and place it in an incubator at 37 °C and 5% CO2 for overnight culture;

[0010] S3. Starve cells: Discard the medium in the 96-well plate, add 100 μL of ExCell MSC serum-free basal medium to each well, and starve for 24 h;

[0011] S4. Add PDGF-BB sample: Dilute the PDGF-BB sample with ExCell MSC serum-free basal medium to 1000 ng / mL, perform 5-fold serial dilution to obtain 9 concentrations. Discard the medium in the 96-well plate, add 100 μL of the diluted PDGF-BB sample to each well, set 2-3 replicates for each dilution concentration, and set another 100 μL of ExCell MSC serum-free basal medium culture medium control well. Incubate at 37 °C and 5% CO2 for 52 ± 4 h;

[0012] S5. Color development and calculation of the activity of the PDGF-BB sample: Add 100 μL of CCK-8 diluted 10-fold with EXCELL MSC serum-free basal medium to each well in the dark, place it in an incubator at 37°C and 5% CO2 for incubation for 1.5 - 2 hours. Measure the absorbance at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Process it using a computer program or four-parameter regression calculation method. Use the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB, and simultaneously calculate the signal-to-noise ratio (signal-to-noise ratio = maximum OD value of the sample well / OD value of the control well).

[0013] The beneficial effects of the present invention mainly lie in that during the process of detecting PDGF-BB, the applicant gradually established the method of this application through long-term experiments, extensive exploration, and repeated demonstration. By using the method mentioned in this patent and combining it with specific culture media and serum, the biological activity of PDGF-BB can be detected simply and accurately.

[0014] The key innovation point of the present invention is to culture cells using a culture medium containing 10% serum and then subsequently replace it with EXCELL MSC serum-free basal medium to detect the biological activity of the PDGF-BB sample. In the past, for the detection of such factors, cells were cultured using a culture medium containing 10% serum and then subsequently replaced with a low-concentration serum-containing culture medium to detect the biological activity of the relevant factors. Since this method contains serum throughout the process and the cell starvation stage still has serum providing nutrients, after starvation, when factors such as PDGF-BB are added, the sensitivity is low, the experimental background is high, and the signal-to-noise ratio usually does not exceed 2.

[0015] However, after culturing cells with serum and then adding EXCELL MSC serum-free medium, the formula of this medium is unique and simple. When cells are starved, while ensuring that the cells do not die, the addition of factors such as PDGF-BB can produce the maximum signal-to-noise ratio.

[0016] The detection of the PDGF-BB sample using EXCELL MSC serum-free basal medium combined with EXCELL fetal bovine serum has the advantages of high sensitivity, good repeatability, high precision, and convenient use. Description of the Drawings

[0017] Figure 1 It is: the results of detecting the biological activity of the PDGF-BB sample by two-fold dilution methods;

[0018] Figure 2 It is: the accuracy detection results of the method of the present invention;

[0019] Figure 3 It is: the precision detection results of the method of the present invention;

[0020] Figure 4are: the results of biological detection of PDGF-BB samples with different brand sera;

[0021] Figure 5 are: the results of biological detection of PDGF-BB samples with different serum-free basal media;

[0022] Figure 6 are: the results of biological activity detection of PDGF-BB samples by using the method of the present invention and the pharmacopoeia method. Detailed implementation manners

[0023] The technical solutions of the present invention will be further described below in conjunction with specific embodiments. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0024] Experimental materials: NIH / 3T3 cells (mouse embryonic fibroblasts, from ATCC, catalog number: CRL1658), EXCELL fetal bovine serum (from EXCELL, catalog number: FND500), DMEM high-glucose complete medium (from Sigma, catalog number: D5648), EXCELL MSC serum-free basal medium (from EXCELL, catalog number: BA0301), Gibco fetal bovine serum (from Gibco, catalog number: A5670701), Sijiqing fetal bovine serum (from Sijiqing, catalog number: 11011-8611), EXCELL 293 serum-free medium (from EXCELL, catalog number: HE000-N011), Youkang MSC serum-free basal medium (from Youkang, catalog number: NC0107), PDGF-BB international standard purchased from NIBSC (94 / 728), PDGF-BB (from R&D SYSTEMS, catalog number: 220-BB), PDGF-BB (from Dongkang, catalog number: Y03401).

[0025] Example 1. Determine the serial dilution method

[0026] 1. NIH / 3T3 cell culture and amplification: Expand and culture NIH / 3T3 cells in DMEM high-glucose complete medium containing 10% EXCELL fetal bovine serum at 37°C, 5% CO2, and saturated humidity. Inoculate at a density of 4×10 4 cells / cm 2 and passage every 2 days.

[0027] 2. Plating: After culturing NIH / 3T3 cells for 2 passages, discard the medium in the culture flask, digest and collect the NIH / 3T3 cells, and resuspend them in DMEM high-glucose complete medium containing 10% ExCell fetal bovine serum to form a cell suspension with 4×10 4 cells per 1 mL. Inoculate 0.1 mL per well into a 96-well cell culture plate and place it in an incubator at 37°C and 5% CO2 for overnight culture.

[0028] 3. Starving cells: Discard the medium in the 96-well plate, add 100 μL of ExCell MSC serum-free basal medium to each well, and starve for 24 h.

[0029] 4. Adding PDGF-BB samples: Dilute the PDGF-BB samples (from R&D SYSTEMS, catalog number: 220-BB) to 1000 ng / mL with ExCell MSC serum-free basal medium, perform 5-fold serial dilution to obtain 9 dilutions, and 10-fold dilution to obtain 7 dilutions (see Table 1). Discard the medium in the 96-well plate, add 100 μL of the PDGF-BB samples diluted by the two serial dilution methods to each well, and set 3 replicates for each dilution concentration. Additionally, set a culture medium control well (100 μL of ExCell MSC serum-free basal medium), and incubate at 37°C and 5% CO2 for 52 ± 4 h.

[0030] Table 1 Gradient dilution concentrations of PDGF-BB

[0031]

[0032] 5. Color development and calculating the activity of PDGF-BB samples: Add 100 μL of CCK-8 diluted 10-fold with ExCell MSC serum-free basal medium to each well in the dark, place it in an incubator at 37°C and 5% CO2 for incubation for 1.5 - 2 hours. Measure the absorbance value (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and perform data processing using a computer program or four-parameter regression calculation method. Use the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB, and simultaneously calculate the signal-to-noise ratio (signal-to-noise ratio = maximum OD value of the sample well / OD value of the control well).

[0033] 6. Result analysis: The detection results are as Figure 1 shown in [Figure] and Table 2. When the PDGF-BB samples are serially diluted using the 5-fold dilution method, the measured OD values are processed using a computer program or four-parameter regression calculation method, resulting in a higher signal-to-noise ratio, and the R 2 is greater than that of the 10-fold dilution method. Therefore, the 5-fold serial dilution method is superior to the 10-fold serial dilution method.

[0034] Table 2 Optimization of serial dilution method

[0035] Dilution ratio 5-fold dilution 10-fold dilution EC50 1.224 1.176 <![CDATA[R 2 > 0.9948 0.9764 Signal-to-noise ratio 9.660 6.940

[0036] Example 2, Accuracy Detection

[0037] 1. NIH / 3T3 cell culture and amplification: Expand and culture NIH / 3T3 cells in DMEM high-glucose complete medium containing 10% EKOSAI fetal bovine serum under the conditions of 37°C, 5% CO2, and saturated humidity. Inoculate at a density of 4×10 4 cells / cm 2 and passage once every 2 days.

[0038] 2. Plating: After culturing NIH / 3T3 cells for 2 passages, discard the medium in the culture flask, digest and collect NIH / 3T3 cells, and resuspend them in DMEM high-glucose complete medium containing 10% EKOSAI fetal bovine serum to form a cell suspension with 4×10 4 cells per 1 mL. Inoculate 0.1 mL per well into a 96-well cell culture plate and culture overnight in an incubator at 37°C and 5% CO2.

[0039] 3. Starve cells: Discard the medium in the 96-well plate and add 100 μL of EKOSAI MSC serum-free basal medium to each well for 24 h of starvation.

[0040] 4. Add PDGF-BB samples: After reconstituting the PDGF-BB international standard according to the instructions, dilute the PDGF-BB standard to 1000 ng / mL with EKOSAI MSC serum-free basal medium at 5 different potency levels (64%, 80%, 100%, 125%, 156%) (note: except for the 100% potency level, the actual concentrations of the other potency levels are 640 ng / mL, 800 ng / mL, 1250 ng / mL, and 1560 ng / mL respectively), and then perform 5-fold serial dilutions to obtain 9 concentrations. Discard the medium in the 96-well plate and add 100 μL of the diluted PDGF-BB samples to each well. Set 2 replicates for each dilution concentration. Additionally, set a culture medium control well (100 μL of EKOSAI MSC serum-free basal medium), and incubate at 37°C and 5% CO2 for 52 ± 4 h.

[0041] 5. Color development and calculation of the activity of the PDGF-BB sample: Add 100 μL of CCK-8 diluted 10-fold with EXCELL MSC serum-free basal medium to each well in the dark, and place it in an incubator at 37 °C and 5% CO₂ for incubation for 1.5 - 2 hours. Measure the absorbance value (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Repeat the experiment 3 times for the same sample according to the above steps. Use a computer program or four-parameter regression calculation method for processing. With the sample concentration as the abscissa and the OD value as the ordinate, calculate the half-maximal effective concentration (EC50) of PDGF-BB, and at the same time calculate the signal-to-noise ratio (signal-to-noise ratio = maximum OD value of the sample well / OD value of the control well).

[0042] 6. Result analysis: The detection results are shown in Figure 2 , and the recovery rates of 5 different titer levels range from 93.39% to 106.26%, and the CV values are between 3.09% and 8.34%, < 30%, within the normal requirements range. The regression equation is y = 1.0272x - 0.0011, and the slope of 1.0272 is within the range of 0.8 - 1.25. The consistency between the measured results and the expected results is good (R 2 = 0.9819). The above results indicate that the detection method of the present invention has high accuracy.

[0043] Example 3. Precision detection

[0044] 1. NIH / 3T3 cell culture and amplification: Expand and culture NIH / 3T3 cells using DMEM high-glucose complete medium containing 10% EXCELL fetal bovine serum under the conditions of 37 °C, 5% CO₂, and saturated humidity. Inoculate at a density of 4×10 4 cells / cm 2 and passage once every 2 days.

[0045] 2. Plating: After culturing NIH / 3T3 cells for 2 generations, discard the medium in the culture flask, digest and collect NIH / 3T3 cells, and resuspend them with DMEM high-glucose complete medium containing 10% EXCELL fetal bovine serum to form a cell suspension containing 4×10 4 cells per 1 mL. Inoculate 0.1 mL per well into a 96-well cell culture plate and place it in an incubator at 37 °C and 5% CO₂ for overnight culture.

[0046] 3. Starve the cells: Discard the medium in the 96-well plate, add 100 μL of EXCELL MSC serum-free basal medium to each well, and starve for 24 h.

[0047] 4. Add PDGF-BB samples: Dilute the PDGF-BB samples (from Dongkang, product number: Y03401) to 1000 ng / mL with EKSAI MSC serum-free basal medium and perform 5-fold serial dilutions to obtain 9 concentrations. Discard the medium in the 96-well plates, add 100 μL of the diluted PDGF-BB samples to each well, and set 3 replicates for each dilution concentration. Additionally, set a culture medium control well (100 μL of EKSAI MSC serum-free basal medium), and incubate at 37 °C with 5% CO2 for 52 ± 4 h.

[0048] 5. Color development and calculate the activity of PDGF-BB samples: Add 100 μL of CCK-8 diluted 10-fold with EKSAI MSC serum-free basal medium to each well in the dark, and place it in an incubator at 37 °C with 5% CO2 for 1.5 - 2 hours. Measure the absorbance value (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Repeat the detection of the same sample 3 times. At the same time, another experimenter detects the same batch of PDGF-BB samples according to the above steps and repeats the detection 3 times. Use a computer program or four-parameter regression calculation method for processing. Take the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB. Calculate the signal-to-noise ratio simultaneously (signal-to-noise ratio = maximum OD value of the sample well / OD value of the control well).

[0049] 6. Result analysis: The detection results are shown in Figure 3 , and two detectors each detect the same sample 3 times, and there is no significant statistical difference in the EC50 of the samples measured by the two. The results indicate that the detection method of the present invention has high precision.

[0050] Example 4. Repeatability detection

[0051] 1. NIH / 3T3 cell culture and amplification: Amplify and culture NIH / 3T3 cells using DMEM high-glucose complete medium containing 10% EKSAI fetal bovine serum under the conditions of 37 °C, 5% CO2, and saturated humidity. Inoculate at a density of 4×10 4 cells / cm 2 , and passage once every 2 days.

[0052] 2. Plating: After culturing NIH / 3T3 cells for 2 passages, discard the medium in the culture flask, digest and collect NIH / 3T3 cells, and resuspend them with DMEM high-glucose complete medium containing 10% EKSAI fetal bovine serum to form a cell suspension containing 4×10 4 cells per 1 mL. Inoculate 0.1 mL into each well of a 96-well cell culture plate and place it in an incubator at 37 °C with 5% CO2 to culture overnight.

[0053] 3. Starve the cells: Discard the medium in the 96-well plates, add 100 μL of EKSAI MSC serum-free basal medium to each well, and starve for 24 h.

[0054] 4. Addition of PDGF-BB sample: Dilute the PDGF-BB sample (from Dongkang, catalog number: Y03401) with EKSEI MSC serum-free basal medium to 1000 ng / mL, and perform 5-fold serial dilution to obtain 9 concentrations. Discard the medium in the 96-well plate, add 100 μL of the diluted PDGF-BB sample to each well, and set 3 replicates for each dilution concentration. Additionally, set a culture medium control well (100 μL of EKSEI MSC serum-free basal medium), and incubate at 37 °C and 5% CO2 for 52 ± 4 h.

[0055] 5. Color development and calculation of the activity of PDGF-BB sample: Add 100 μL of CCK-8 diluted 10-fold with EKSEI MSC serum-free basal medium to each well in the dark, and place it in an incubator at 37 °C and 5% CO2 for 1.5 - 2 hours. Measure the absorbance value (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Repeat the experiment 3 times for the same sample according to the above steps. Use a computer program or four-parameter regression calculation method for processing. Take the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB, and simultaneously calculate the signal-to-noise ratio (signal-to-noise ratio = maximum OD value of the sample well / OD value of the control well).

[0056] 6. Result analysis: The detection results are shown in Table 3. For the same sample, the signal-to-noise ratios in the 3 experimental results are all around 9 times, the average value of EC50 is 1.686 ng / mL, and the coefficient of variation CV (%) is 1.457 ± 0.0246, < 30%, within the conventional requirements. The above results indicate that the detection method of the present invention has good repeatability.

[0057] Table 3 Repeatability

[0058]

[0059] Example 5. Comparison of different brands of fetal bovine serum

[0060] 1. Culturing and amplifying NIH / 3T3 cells:

[0061] Use DMEM high-glucose complete medium containing Gibco fetal bovine serum, Sijiqing fetal bovine serum, and EKSEI fetal bovine serum to amplify and culture NIH / 3T3 cells respectively under the conditions of 37 °C, 5% CO2, and saturated humidity. Inoculate at a density of 4 × 10 4 cells / cm 2 and passage once every 2 days.

[0062] 2. Plating: After culturing NIH / 3T3 cells for 2 passages, discard the medium in the culture flask, digest and collect NIH / 3T3 cells cultured with 3 brands of fetal bovine serum, and resuspend the cells with complete culture medium prepared with 3 brands of serum respectively, with each 1 mL containing 4×10 4 cells. Inoculate into 3 96-well cell culture plates at 0.1 mL per well, and place them in an incubator at 37°C and 5% CO2 for overnight culture.

[0063] 3. Starving cells: Discard the medium in the 3 96-well plates respectively, and add 100 μL of EXCELL MSC serum-free basal medium to each well for starving for 24 h.

[0064] 4. Adding PDGF-BB samples: Dilute the PDGF-BB samples (from Dongkang, catalog number: Y03401) with EXCELL MSC serum-free basal medium to 1000 ng / mL, and perform 5-fold serial dilution to obtain 9 concentrations. Discard the medium in the 96-well plates, and add 100 μL of the diluted PDGF-BB samples to each well. Set 3 replicates for each dilution concentration. Additionally, set a culture medium control well (100 μL of EXCELL MSC serum-free basal medium), and incubate at 37°C and 5% CO2 for 52 ± 4 h.

[0065] 5. Color development and calculating the activity of PDGF-BB samples: Add 100 μL of CCK-8 diluted 10-fold with EXCELL MSC serum-free basal medium to each well in the dark, and place it in an incubator at 37°C and 5% CO2 for incubation for 1.5 - 2 h. Measure the absorbance value (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader, and perform processing using a computer program or four-parameter regression calculation method. Use the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB. At the same time, calculate the signal-to-noise ratio (signal-to-noise ratio = maximum OD value of the sample well / OD value of the control well).

[0066] 6. Result analysis: The detection results are shown in Table 4 and Figure 4 as follows. When detecting the activity of PDGF-BB samples using NIH / 3T3 cells cultured and amplified with Gibco fetal bovine serum and Sijiqing fetal bovine serum, due to the poor state of the amplified NIH / 3T3 cells, the obtained signal-to-noise ratio is lower than that of EXCELL fetal bovine serum, and the coefficient of variation of the signal-to-noise ratio between replicates is higher. Therefore, EXCELL fetal bovine serum is more suitable for the activity detection of PDGF-BB samples in the present invention.

[0067] Table 4 Comparison of different brands of fetal bovine serum

[0068]

[0069]

[0070] Example 6. Comparison of Different Serum-Free Basal Media

[0071] 1. Culturing and Amplifying NIH / 3T3 Cells: Amplify and culture NIH / 3T3 cells in DMEM high-glucose complete medium containing 10% ExCell fetal bovine serum under the conditions of 37 °C, 5% CO2, and saturated humidity. Seed the cells at a density of 4×10 4 cells / cm 2 and passage them every 2 days.

[0072] 2. Plating: After culturing NIH / 3T3 cells for 2 passages, discard the medium in the culture flask, digest and collect the NIH / 3T3 cells, resuspend the cells in DMEM high-glucose complete medium containing 10% ExCell fetal bovine serum, and seed the cells at a cell density of 4×10 3 per well into 3 96-well cell culture plates, and place them in an incubator at 37 °C and 5% CO2 for overnight culture.

[0073] 3. Starving Cells: Discard the medium in the 3 96-well plates, and add 100 μL of ExCell MSC serum-free basal medium, 293 serum-free medium, and Youkang MSC serum-free basal medium to each well of the cells in the 3 96-well plates respectively, and starve the cells for 24 h.

[0074] 4. Adding PDGF-BB Samples: Dilute the PDGF-BB samples (from Dongkang, catalog number: Y03401) to 1000 ng / mL with ExCell MSC serum-free basal medium, 293 serum-free medium, and Youkang MSC serum-free basal medium respectively, and perform 5-fold serial dilution with the corresponding serum-free basal medium to obtain 9 dilution concentrations. Discard the medium in the 96-well plates, add 100 μL of the diluted PDGF-BB samples to each well, and set 3 replicates for each dilution concentration. Additionally, set culture medium control wells (100 μL of ExCell MSC serum-free basal medium, 293 serum-free medium, and Youkang MSC serum-free basal medium), and incubate at 37 °C and 5% CO2 for 52 ± 4 h.

[0075] 5. Color Development and Calculating the Activity of PDGF-BB Samples: In the 3 96-well plates, add 100 μL of CCK-8 diluted 10-fold with ExCell MSC serum-free basal medium, 293 serum-free medium, and Youkang MSC serum-free basal medium to each well respectively in the dark, and place them in an incubator at 37 °C and 5% CO2 for incubation for 1.5 - 2 hours. Measure the absorbance (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader, and perform data processing using a computer program or four-parameter regression calculation method. Use the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB. At the same time, calculate the signal-to-noise ratio (signal-to-noise ratio = maximum OD value of the sample well / OD value of the control well).

[0076] 6. Result Analysis: The test results are shown in Table 5 and Figure 5 As shown, when using EXCELL MSC Serum-Free Basal Medium to detect the activity of PDGF-BB samples, since its formulation shows the best environment in the cell starvation step (ensuring that all inoculated cells do not die and are in a situation of relatively lacking nutrients), the signal-to-noise ratio obtained is higher than that of the other two serum-free basal media, and the coefficient of variation of the signal-to-noise ratio between replicate groups is lower, indicating that EXCELL MSC Serum-Free Basal Medium is more suitable for the activity detection of PDGF-BB samples.

[0077] Table 5 Comparison of Different Serum-Free Basal Media

[0078]

[0079] Example 7. Comparison with the Pharmacopoeia Method

[0080] 1. NIH / 3T3 Cell Culture and Amplification: Use DMEM high-glucose complete medium containing 10% EXCELL fetal bovine serum to amplify and culture NIH / 3T3 cells under the conditions of 37°C, 5% CO2, and saturated humidity. Inoculate at a density of 4×10 4 cells / cm 2 and passage once every 2 days.

[0081] 2. Plating: After culturing NIH / 3T3 cells for 2 generations, discard the medium in the culture flask, digest and collect NIH / 3T3 cells, and resuspend them in 2 centrifuge tubes with complete medium prepared with serum. One tube refers to the method for determining the biological activity of human epidermal growth factor (EGF) in the Chinese Pharmacopoeia (page 595, Volume III of the Chinese Pharmacopoeia) to adjust the cell concentration to 6×10 4 / mL, and the other tube adjusts the cell concentration to 4×10 4 / mL according to the method of the present invention. Then, inoculate 0.1 mL per well into 2 96-well cell culture plates and place them in an incubator at 37°C and 5% CO2 for overnight culture.

[0082] 3. Starving Cells: Discard the medium in the 2 96-well plates respectively. One 96-well plate refers to the pharmacopoeia method and add 100 μL of DMEM medium containing 0.4% EXCELL fetal bovine serum to each well, and the other 96-well plate add 100 μL of EXCELL MSC Serum-Free Basal Medium to each well for 24 hours of starvation.

[0083] 4. Add PDGF-BB samples: Dilute the PDGF-BB samples (from Dongkang, product number: Y03401) to 1000 ng / mL. One 96-well plate is diluted with DMEM medium containing 0.4% ExCell fetal bovine serum, and the other 96-well plate is diluted with ExCell MSC serum-free basal medium, and perform 5-fold serial dilution to obtain 9 concentrations. Discard the medium in the 96-well plate, and add 100 μL of PDGF-BB samples diluted with different media to each well. Each dilution concentration is set with 3 replicates. Additionally, set up culture medium control wells (100 μL of DMEM medium containing 0.4% ExCell fetal bovine serum and 100 μL of ExCell MSC serum-free basal medium). Among them, the well plates loaded according to the pharmacopoeia method are placed in an incubator at 37°C and 5% CO2 for 64 - 72 h, and the well plates loaded according to the method of the present invention are placed in an incubator at 37°C and 5% CO2 for 52 ± 4 h.

[0084] 5. Color development and calculate the activity of PDGF-BB samples: Add 100 μL of CCK-8 diluted 10-fold with DMEM medium containing 0.4% ExCell fetal bovine serum and 100 μL of ExCell MSC serum-free basal medium to each well in the dark, and place them in an incubator at 37°C and 5% CO2 for 1.5 - 2 hours. Measure the absorbance value (OD value) at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and perform processing using a computer program or four-parameter regression calculation method. Take the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB. At the same time, calculate the signal-to-noise ratio (signal-to-noise ratio = maximum OD value of sample wells / OD value of control wells).

[0085] 6. Result analysis: The detection results are shown in Table 6 and Figure 6 As shown, compared with the pharmacopoeia method, when using the medium and method of the present invention for the biological activity detection of PDGF-BB, a higher signal-to-noise ratio is obtained, and the R 2 is higher. Therefore, the medium and application method of the present invention are more suitable for the biological activity detection of PDGF-BB samples.

[0086] Table 6 Comparison with the pharmacopoeia method

[0087] Detection method The method of the present invention Pharmacopoeia method Signal-to-noise ratio 11.918 1.994 CV(%) 5.664 1.671 EC50(ng / mL) 1.939 1.876 <![CDATA[R 2 > 0.9831 0.9591

[0088] The above-described embodiments merely represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for detecting the biological activity of PDGF-BB using serum and serum-free medium, characterized in that, Cell culture expansion was carried out using DMEM high-glucose complete medium containing 10% ExCell Fetal Bovine Serum, and the cells were starved using ExCell MSC Serum-Free Basal Medium.

2. The method for detecting the biological activity of PDGF-BB by using serum and serum-free medium according to claim 1, wherein The cells described above were NIH / 3T3 cells.

3. A method for detecting the biological activity of PDGF-BB using serum and serum-free medium according to claim 2, characterized in that Specifically, it includes the following steps: S1. NIH / 3T3 cell culture and amplification: Amplify and culture NIH / 3T3 cells in high-glucose DMEM complete medium containing 10% ExCell fetal bovine serum under the conditions of 37 °C, 5% CO2, and saturated humidity. Inoculate at a density of 4×10 4 cells / cm 2 , and passage once every 2 days; S2. Plating: After culturing NIH / 3T3 cells for 2 passages, discard the medium in the culture flask, digest and collect the NIH / 3T3 cells, and resuspend them with DMEM high-glucose complete medium containing 10% EXCELL fetal bovine serum to form a cell suspension containing 4×10 4 cells per 1 mL. Inoculate 0.1 mL per well into a 96-well cell culture plate and culture overnight in an incubator at 37 °C and 5% CO2; S3. Starve the cells: Discard the medium in the 96-well plate, add 100 μL of ExCell MSC Serum-Free Basal Medium to each well, and starve for 24 h. S4. Add PDGF-BB samples: Dilute the PDGF-BB samples with ExCell MSC Serum-Free Basal Medium to 1000 ng / mL, perform 5-fold serial dilution to obtain 9 concentrations, discard the medium in the 96-well plate, add 100 μL of the diluted PDGF-BB samples to each well, set 2 - 3 replicates for each dilution concentration, and set another 100 μL of ExCell MSC Serum-Free Basal Medium culture medium control well. Incubate at 37 °C and 5% CO2 for 52 ± 4 h. S5. Color development and calculate the activity of PDGF-BB samples: Add 100 μL of CCK-8 diluted 10-fold with ExCell MSC Serum-Free Basal Medium to each well in the dark, place it in an incubator at 37 °C and 5% CO2 for incubation for 1.5 - 2 hours, measure the absorbance at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader, and perform processing using a computer program or four-parameter regression calculation method. Use the sample concentration as the abscissa and the OD value as the ordinate to calculate the half-maximal effective concentration (EC50) of PDGF-BB, and judge the biological activity of the PDGF-BB samples according to the EC50.

4. A method for detecting the biological activity of PDGF-BB using serum and serum-free medium according to claim 3, characterized in that, In the above S5, the relative biological activity of the PDGF-BB sample = standard product EC50 / test product EC50 × 100%, and the unit of the EC50 is ng / mL.

Images