High-stability PDGF-BB activity detection method

Abstract

The invention discloses a high-stability PDGF-BB activity detection method, ATDC5 cells are applied to PDGF-BB activity detection for the first time, the stability of the detection method is greatly improved, and on the basis, the brand-new high-stability PDGF-BB activity detection method is provided for the field. The technical problems that in the prior art, cells used for PDGF-BB activity detection are 3T3 cells, detection is unstable, and the failure probability is large are solved, a brand new thought and strategy are provided for PDGF-BB activity detection, and the application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a highly stable PDGF-BB activity detection method. Background Art

[0002] Recombinant human platelet-derived growth factor (PDGF) is a common peptide regulatory factor that can stimulate connective tissue proliferation. It is composed of two different polypeptide chains, which are connected into dimers by disulfide bonds. Different combinations of dimers form three isomers, namely PDGF-AA, PDGF-BB, and PDGF-AB. Among them, PDGF-AA is a homodimer composed of two A chains, which has the effect of promoting cell proliferation, survival and migration. PDGF-BB is a homodimer composed of two B chains, which plays a beneficial cardioprotective role in the heart ischemia-reperfusion injury model. PDGF-AB is a heterodimer composed of one A chain and one B chain, which has the ability to bind to and activate PDGFRα and PDGFRβ receptors.

[0003] PDGF-BB contributes to cell growth, angiogenesis and vascular development, can regulate blasts, osteoclasts and promote bone healing, highlighting its importance in the basic physiological processes required for coordinating tissue homeostasis. The activity detection of recombinant proteins is a key parameter for product quality detection. The activity and quality are directly related to whether the product can be released for sale, whether it can meet the use requirements of clinical applications or basic research. The quantitative activity detection methods of PDGF-BB currently provided on the market mostly use Balb / c 3T3 and NIH / 3T3 cells, and the cell selection is relatively single, and the detection method is extremely unstable, which greatly increases the difficulty of evaluating the batch consistency of the activity of the factor. Therefore, it is currently in urgent need of developing a new method of good stability, simple operation, and capable of simultaneously and rapidly performing multiple batches of PDGF-BB activity detection in this area.

[0004] At present, there are no studies or reports on the use of ATDC5 cells to evaluate the activity of PDGF-BB. Summary of the invention

[0005] In order to overcome the above-mentioned technical problems existing in the art, the purpose of the present invention is to provide a highly stable PDGF-BB activity detection method, which is a PDGF-BB activity detection method based on ATDC5 cells. The method has good stability and simple operation, and can quickly perform activity detection of multiple batches of PDGF-BB at the same time, overcoming the technical problems that the PDGF-BB quantitative activity detection methods in the prior art mostly use Balb / c 3T3 and NIH / 3T3 cells, the cell selection is relatively single, and the detection method is extremely unstable. The method provided by the present invention lays a foundation for the art to provide PDGF-BB proteins with guaranteed quality and stable batches.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides a highly stable method for detecting PDGF-BB activity.

[0008] Furthermore, the method comprises: using ATDC5 cells to detect the biological activity of PDGF-BB.

[0009] Further, the method comprises the following steps:

[0010] (1) Collect ATDC5 cells and perform cell starvation;

[0011] (2) diluting the PDGF-BB sample and reference product;

[0012] (3) adding the diluted PDGF-BB sample and reference product to the ATDC5 cells obtained in step (1) for culturing;

[0013] (4) adding a luminescent 3D cell viability detection reagent, shaking in the dark, and then incubating in the dark, performing chemiluminescence detection, and fitting a four-parameter curve based on the measured luminescence value to determine the biological activity of PDGF-BB.

[0014] Furthermore, the ATDC5 cells described in step (1) were plated at a density of 6×10 4 / mL;

[0015] Optionally, prepare ATDC5 cell suspension using complete culture medium;

[0016] Optionally, the volume of cell suspension added to each well was 100 μL;

[0017] Optionally, the culture conditions of the ATDC5 cells are: 37° C., CO 2 incubator for 17 h;

[0018] Optionally, the formula of the complete culture medium is: DMEM: Ham's F12 (1:1), FBS, PS (100×);

[0019] Optionally, the contents of the components in the complete culture medium are: 98% DMEM: Ham's F12 (1:1), 1% FBS, and 1% PS (100×).

[0020] Furthermore, the cell starvation in step (1) includes: after culturing ATDC5 cells for 17 hours, discarding the complete culture medium, adding 1×DPBS, discarding the 1×DPBS after a short stay, adding a maintenance solution, and performing cell starvation culture;

[0021] Optionally, the amount of 1×DPBS added is: 100 μL per well;

[0022] Optionally, the amount of the maintenance solution added is: 100 μL per well;

[0023] Optionally, the starvation culture conditions are: starvation for 24 h in a 37° C., CO 2 incubator;

[0024] Optionally, the maintenance solution has a formula of: DMEM: Ham's F12 (1:1), FBS, PS (100×);

[0025] Optionally, the contents of the components in the maintenance solution are: 98% DMEM: Ham's F12 (1:1), 1% FBS, 1% PS (100×).

[0026] Furthermore, the amount of the diluted PDGF-BB sample or reference product added in step (3) is: 100 μL per well;

[0027] Optionally, the ATDC5 cells are ATDC5 cells from which the maintenance solution has been discarded;

[0028] Optionally, the culture conditions are: 37° C., CO 2 incubator for 72 hours.

[0029] Furthermore, the luminescent 3D cell viability detection reagent described in step (4) is CellTiter-Lumi TM 3D cell viability detection reagent, CellTiter- 3D cell viability assay reagent or Luminescent 3D Cell Viability Assay Reagent;

[0030] Optionally, the amount of the luminescence 3D cell viability detection reagent added is: 100 μL per well;

[0031] Optionally, the conditions for the light-proof shaking are: shaking at 500 rpm for 5 min at room temperature and light-proof;

[0032] Optionally, the conditions for incubation in the dark are: incubation in the dark at room temperature for 5 min;

[0033] Optionally, chemiluminescence detection is performed using a multifunctional microplate reader.

[0034] In the present invention, the PDGF-BB refers to the abbreviation of platelet-derived growth factor-BB, which belongs to the PDGF family. PDGF-BB is a homodimer formed by two B chains connected by a disulfide bond. PDGF-BB is mainly derived from the α particles of platelets. When blood vessels are damaged, platelets are activated and aggregated at the site of injury, and then release a variety of growth factors including PDGF-BB. In addition, a variety of cells such as smooth muscle cells, activated macrophages, etc. can also synthesize and secrete PDGF-BB.

[0035] In the present invention, the ATDC5 cells are a chondrocyte cell line derived from mouse teratoma cells AT805. ATDC5 cells exhibit continuous phenotypic transformation in vitro, including various stages from mesenchymal condensation to calcification. Bone morphogenetic protein-2 (BMP-2) can stimulate early and late differentiation of ATDC5 cells, and activation of parathyroid hormone (PTH) / PTH-related peptide (PTHrP) receptors can inhibit this differentiation ability. Under insulin stimulation, ATDC5 cells can differentiate into chondrocytes, form cartilage nodules, and express type II collagen and type X collagen, and finally mineralize, which fully reflects the chondrogenesis process. Because ATDC5 cells have clear chondrogenesis characteristics, they are widely considered to be an ideal in vitro model for studying factors affecting cell behavior during cartilage development.

[0036] In some embodiments, the present invention has no particular limitation on the specific source of the ATDC5 cells, and any commercial ATDC5 cells or any self-constructed ATDC5 cells used in PDGF-BB activity detection will fall within the protection scope of the present invention.

[0037] In the present invention, the luminescent 3D cell viability detection is a technology that quantitatively detects the viability of 3D cultured cells based on the principle of bioluminescence, and has the characteristics of high sensitivity and high stability. The detection principle is as follows: Intracellular adenosine triphosphate (ATP) is the direct source of cellular energy, and its content is relatively stable in living cells. The luminescent 3D cell viability detection uses firefly luciferase to catalyze the conversion of the substrate luciferin. This process requires ATP to provide energy, and the reaction will emit photons. The intensity of the luminescent signal is proportional to the ATP content, and then proportional to the number and viability of the cells.

[0038] In some embodiments, the luminescent 3D cell viability detection reagent is not limited to the CellTiter-Lumi TM 3D Cell Viability Detection Reagents, CellTiter 3D cell viability assay reagent or Luminescent 3D cell viability detection reagent. Any reagent that can be used for luminescent 3D cell viability detection will fall within the scope of protection of the present invention.

[0039] In some embodiments, the method of determining the biological activity of PDGF-BB by fitting a four-parameter curve according to the measured luminescence value comprises fitting an S-shaped curve with four parameters to obtain ED 50 .

[0040] In some embodiments, the ED 50 It refers to the effective concentration of the recombinant protein that can cause 50% of the test system (such as cells, enzyme reaction system, etc.) to produce a specific biological activity reaction. For example, in a cell proliferation experiment, the recombinant protein is used as a factor to stimulate cell proliferation, and its ED 50 It is the protein concentration that causes 50% of the cells to produce the expected proliferation response.

[0041] In some embodiments, the ED 50 It is an important indicator to measure the quality of recombinant protein products. 50 , can ensure that the activity of each batch of recombinant protein produced can meet certain standards, ensuring the stability and consistency of product quality. For example, when producing PDGF-BB protein, the active ED 50 Testing can ensure that different batches of products have similar activity in stimulating cellular responses.

[0042] In some embodiments, the ED 50 It is a key reference data when developing recombinant protein-based therapeutic drugs, diagnostic reagents and other application products. For therapeutic drugs, appropriate ED 50 It can guide the dosage design of clinical applications and ensure that the drug can effectively play a role in the body. 50 It can help determine the optimal working concentration of the reagent and improve the accuracy and sensitivity of diagnosis.

[0043] In the present invention, the DMEM:Ham's F12 (1:1) refers to a composite culture medium obtained by mixing DMEM culture medium and Ham's F12 culture medium in a volume ratio of 1:1. This mixed culture medium combines the advantages of both DMEM and Ham's F12, including both the rich amino acids and high-concentration glucose nutrients in DMEM and the various trace elements and growth factors in Ham's F12. Such a combination can provide cells with a more comprehensive and balanced nutritional environment, which helps to meet the complex nutritional needs of different cell types.

[0044] Among them, DMEM (Dulbecco's Modified Eagle Medium) and Ham's F12 are two commonly used cell culture media. DMEM is a high-sugar culture medium rich in amino acids and vitamins, which can provide cells with rich nutrients and support the growth and proliferation of a variety of cells. It contains a high concentration of glucose (generally 4.5g / L) and is suitable for culturing cells with high energy requirements, such as mammalian fibroblasts and certain tumor cells. Ham's F12 medium is a culture medium with more complex nutrients and contains a variety of trace elements and growth factors. It is particularly suitable for culturing some difficult-to-culture cell types, such as some primary cells and hybridoma cells.

[0045] In the present invention, the FBS refers to fetal bovine serum (FBS), which is a nutritional supplement widely used in cell culture. It is a serum component separated from the blood of fetal bovines. The FBS contains protein components, growth factors, hormones and other nutrients. In cell culture, it can provide nutritional support, promote cell growth and proliferation, and maintain cell survival and function.

[0046] Among them, FBS contains a variety of proteins, such as albumin and globulin. Albumin can act as a carrier protein to bind and transport nutrients and metabolites such as fatty acids, hormones, and metal ions, ensuring that these substances can be effectively taken up and utilized by cells in the cell culture medium. Globulin plays a role in immune regulation and other aspects, helping to maintain the stability of the cell culture environment.

[0047] Among them, FBS is rich in a variety of growth factors, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), etc. These growth factors are essential for cell proliferation, differentiation and survival. For example, EGF can stimulate the proliferation of epithelial cells, and FGF can promote the growth of fibroblasts and vascular endothelial cells, playing a key role in the cell growth process of tissue repair and cell culture.

[0048] Among them, FBS contains multiple hormones such as insulin and thyroid hormone. Insulin can regulate the sugar metabolism of cells, promote the uptake and utilization of glucose by cells, and provide energy for cells. Thyroid hormone helps maintain the metabolic activity of cells and promotes cell growth and differentiation.

[0049] Among them, FBS also contains nutrients such as amino acids, glucose, and fatty acids, which provide the necessary energy and material basis for cell growth. At the same time, it also contains some trace elements, such as iron, zinc, and copper, which are involved in various enzymatic reactions and metabolic processes in cells.

[0050] In the present invention, the PS refers to Penicillin-Streptomycin (PS), which is a mixture of Penicillin and Streptomycin, and is widely used in fields such as cell culture for preventing and controlling bacterial contamination. Among them, penicillin is a natural antibiotic extracted from the culture fluid of Penicillium, belonging to the β-lactam antibiotics, which mainly inhibits the synthesis of bacterial cell walls. Streptomycin is an aminoglycoside antibiotic extracted from the culture fluid of Streptomyces griseus. It is an alkaline compound composed of streptavidin, streptose and N-methyl-L-glucosamine, which mainly acts on the bacterial ribosome 30S subunit and interferes with the synthesis of bacterial proteins.

[0051] In a specific embodiment of the present invention, the present invention first discovered through comparative experiments that the method provided by the present invention (using ATDC5 cells) is more effective than the conventional activity detection method in the prior art (using NIH / 3T3 cells conventionally used in the prior art) in terms of ED 50 The difference in values ​​is small, the linearity is excellent, and the experimental stability is high. It can facilitate the subsequent research and application of PDGF-BB protein, and is a highly stable PDGF-BB activity detection method. This comparison result is a technical effect that is unexpected by those skilled in the art based on the content disclosed in the prior art.

[0052] The second aspect of the present invention provides a highly stable PDGF-BB activity detection reagent.

[0053] Further, the reagents include ATDC5 cells, PDGF-BB reference products, and luminescence 3D cell viability detection reagents;

[0054] Optionally, the reagents further include complete culture medium and maintenance solution.

[0055] Furthermore, the complete culture medium is the complete culture medium described in the first aspect of the present invention.

[0056] Furthermore, the maintenance solution is the maintenance solution described in the first aspect of the present invention.

[0057] In some embodiments, the PDGF-BB reference refers to a PDGF-BB standard or a PDGF-BB reference substance, which is a PDGF-BB recombinant protein of known activity or concentration, and is used to establish a standard curve so that the activity or concentration of the PDGF-BB recombinant protein in the sample can be calculated by comparing the signal intensity of the unknown sample with the standard curve.

[0058] The third aspect of the present invention provides a highly stable PDGF-BB activity detection kit.

[0059] Furthermore, the kit comprises the reagents described in the second aspect of the present invention.

[0060] In some embodiments, the kit further comprises instructions or labels, which may include detailed steps on how to process the sample to be tested, how to detect the biological activity of the PDGF-BB protein in the sample to be tested, and other relevant instructions.

[0061] In some embodiments, the components contained in the kit are provided in one or more containers, or fixed on a matrix, and the container can be any conventional sterile container capable of containing the above components, such as a microcentrifuge tube, an ampoule or a bottle.

[0062] In some embodiments, the sterile container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a sachet, a blister pack, or other suitable container forms known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil or other materials.

[0063] The fourth aspect of the present invention provides the use of the method described in the first aspect of the present invention in controlling the quality of PDGF-BB protein.

[0064] The fifth aspect of the present invention provides the use of ATDC5 cells in detecting PDGF-BB activity with high stability;

[0065] Optionally, ATDC5 cells are used to detect PDGF-BB activity with high stability using the method described in the first aspect of the present invention.

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] The present invention applies ATDC5 cells to PDGF-BB activity detection for the first time, which greatly improves the stability of the detection method. Based on this, the present invention provides a new high-stability PDGF-BB activity detection method in the art, overcomes the technical problem that the cells used in the PDGF-BB activity detection in the current prior art are 3T3 cells, the detection is unstable, and the probability of failure is high, and provides a new idea and strategy for PDGF-BB activity detection, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : The result graph corresponding to the detection of PDGF-BB activity using the method provided by the present invention;

[0069] Figure 2 : The results of the ATDC5 cell group experiment 1 to detect PDGF-BB activity according to the method described in the first experiment;

[0070] Figure 3 : The results of the ATDC5 cell group experiment 2 for detecting PDGF-BB activity;

[0071] Figure 4 : The results of the ATDC5 cell group experiment 3 to detect the activity of PDGF-BB;

[0072] Figure 5 : The results of the method described in Experiment 1 of the NIH / 3T3 cell group for detecting PDGF-BB activity;

[0073] Figure 6 : The results of the NIH / 3T3 cell group experiment 2 for detecting PDGF-BB activity;

[0074] Figure 7 : The results of detecting PDGF-BB activity using the method described in Experiment 3 of the NIH / 3T3 cell group correspond to the results. DETAILED DESCRIPTION

[0075] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0076] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0077] Embodiment A highly stable PDGF-BB activity detection method

[0078] 1. Experimental Materials

[0079] ATDC5 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.; PDGF-BB protein was purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.; CellTiter-Lumi TM 3D cell viability detection reagents were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0080] 2. Experimental methods

[0081] (1) Cell preparation: ATDC5 cells were collected and plated at a density of 6 × 10 4 / mL, add 100 μL of cell suspension to each well and adjust the cell suspension using complete medium. The complete medium formula is: DMEM: Ham's F12 (1:1) (89%) + FBS (10%) + PS (100×) (1%).

[0082] (2) Cell plating: 100 μL of the cell suspension prepared in step (1) above was added to each well of a 96-well white-bottom plate and placed in a 37°C CO2 incubator for 17 h to allow the cells to adhere to the plate.

[0083] (3) Cell starvation: After culturing the cells for 17 hours, remove the 96-well white-bottom plate, discard the complete medium, add 1×DPBS (100 μL per well), and after a short stay, discard the 1×DPBS, add 100 μL of maintenance solution per well, and place in a 37°C CO2 incubator for starvation for 24 hours. The maintenance solution formula is: DMEM: Ham's F12 (1:1) (98%) + FBS (1%) + PS (100×) (1%).

[0084] (4) Add PDGF-BB protein: dilute PDGF-BB protein to a final concentration of 1 μg / mL, and then dilute it to 0.0153 ng / mL in 4-fold ratios, with the final concentration being 0. Take out the 96-well white-bottom plate that has been starved in step (3) above, and discard the maintenance solution. Add the diluted PDGF-BB protein to the 96-well white-bottom plate, 100 μL per well, and place it in a 37°C CO2 incubator for 72 hours.

[0085] (5) Add luminescent 3D cell viability detection reagent: After the cells are co-cultured with PDGF-BB protein for 72 hours, remove the 96-well white bottom plate and add CellTiter-Lumi TM 3D cell viability detection reagent, 100 μL per well. Oscillate at 500 rpm for 5 min at room temperature in the dark, then incubate at room temperature in the dark for 5 min. Perform chemiluminescence detection on a multifunctional microplate reader and read the chemiluminescence detection value every 5 min until the reading is stopped at 25 min.

[0086] (6) GraphPad software was used for data processing, with the x-axis representing the value of different PDGF-BB concentrations and the y-axis representing the luminescence value. A four-parameter S-shaped curve was fitted to obtain the ED 50 .

[0087] 3. Experimental results

[0088] The results are as follows Figure 1 As shown, the results show that the detection method described in this example can detect the activity of PDGF-BB protein with high stability.

[0089] Comparative Example Comparison of the effects of the ATDC5 cells used in the present invention and the NIH / 3T3 cells conventionally used in the current prior art

[0090] 1. Experimental Materials

[0091] ATDC5 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.; NIH / 3T3 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0092] 2. Experimental methods

[0093] For ATDC5 cells and NIH / 3T3 cells, the same detection method (ie, the detection method in the above-mentioned embodiment) was used to conduct three experiments for comparison.

[0094] Except for the different cell types between the two groups of experiments, other experimental conditions were exactly the same as in the above-mentioned embodiment.

[0095] 3. Experimental results

[0096] The results are shown in Table 1 and Figure 2-7 The results show that the ATDC5 cells used in the present invention are much more stable than the NIH / 3T3 cells conventionally used in the prior art: ATDC5 cells R 2 All > 0.9 and can fit a typical S-shaped curve, ED 50 The values ​​are not much different. However, NIH / 3T3 cells can occasionally fit a typical S-shaped curve, R 2 Occasionally >0.9, ED 50 The value fluctuates greatly and the experimental success rate is low. This comparison result is a technical effect that cannot be expected by those skilled in the art based on the content disclosed in the prior art.

[0097] Table 1 Comparative experimental results

[0098]

[0099] The above results show that the method provided by the present invention is more effective than conventional activity detection methods. 50 The value difference is small, the linearity is excellent, and the experimental stability is high. It can facilitate the subsequent research and application of PDGF-BB protein and is a highly stable PDGF-BB activity detection method.

Claims

1. A highly stable PDGF-BB activity detection method, characterized in that, The method comprises: using ATDC5 cells to detect the biological activity of PDGF-BB.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) Collect ATDC5 cells and perform cell starvation; (2) diluting the PDGF-BB sample and reference product; (3) adding the diluted PDGF-BB sample and reference product to the ATDC5 cells obtained in step (1) for culturing; (4) adding a luminescent 3D cell viability detection reagent, shaking in the dark, and then incubating in the dark, performing chemiluminescence detection, and fitting a four-parameter curve based on the measured luminescence value to determine the biological activity of PDGF-BB.

3. The method according to claim 1, characterized in that: The ATDC5 cells described in step (1) were plated at a density of 6×10 4 / mL; Optionally, prepare ATDC5 cell suspension using complete culture medium; Optionally, the volume of cell suspension added to each well is 100 μL; Optionally, the culture conditions of the ATDC5 cells are: 37° C., CO 2 incubator for 17 h; Optionally, the formula of the complete culture medium is: DMEM: Ham's F12 (1:1), FBS, PS (100×); Optionally, the contents of the components in the complete culture medium are: 98% DMEM: Ham's F12 (1:1), 1% FBS, 1% PS (100×).

4. The method according to claim 1, characterized in that The cell starvation in step (1) includes: after culturing ATDC5 cells for 17 hours, discarding the complete culture medium, adding 1×DPBS, discarding the 1×DPBS after a short stay, adding maintenance solution, and performing cell starvation culture; Optionally, the amount of 1×DPBS added is: 100 μL per well; Optionally, the amount of the maintenance solution added is: 100 μL per well; Optionally, the starvation culture conditions are: starvation for 24 h in a 37° C., CO 2 incubator; Optionally, the maintenance solution has a formula of: DMEM: Ham's F12 (1:1), FBS, PS (100×); Optionally, the contents of the components in the maintenance solution are: 98% DMEM: Ham's F12 (1:1), 1% FBS, 1% PS (100×).

5. The method according to claim 1, characterized in that The amount of the diluted PDGF-BB sample or reference product added in step (3) is: 100 μL per well; Optionally, the ATDC5 cells are ATDC5 cells from which the maintenance solution has been discarded; Optionally, the culture conditions are: 37° C., CO 2 incubator for 72 hours.

6. The method according to claim 1, characterized in that The luminescent 3D cell viability detection reagent described in step (4) is CellTiter-Lumi TM 3D cell viability detection reagents, 3D cell viability assay reagent or Luminescent 3D Cell Viability Assay Reagent; Optionally, the amount of the luminescence 3D cell viability detection reagent added is: 100 μL per well; Optionally, the conditions for the light-proof shaking are: shaking at 500 rpm for 5 min at room temperature and light-proof; Optionally, the conditions for incubation in the dark are: incubation in the dark at room temperature for 5 min; Optionally, chemiluminescence detection is performed using a multifunctional microplate reader.

7. A highly stable PDGF-BB activity detection reagent, characterized in that: The reagents include ATDC5 cells, PDGF-BB reference product, and luminescence 3D cell viability detection reagent; Optionally, the reagents further include complete culture medium and maintenance solution.

8. A highly stable PDGF-BB activity detection kit, characterized in that: The kit comprises the reagent according to claim 7.

9. Use of the method described in any one of claims 1-6 in controlling the quality of PDGF-BB protein.

10. Application of ATDC5 cells in the detection of PDGF-BB activity with high stability; Optionally, ATDC5 cells are used to detect PDGF-BB activity with high stability using the method described in any one of claims 1 to 6.

Images