Quantitative detection method for evaluating cartilage cell proliferation promoting capability of mesenchymal stem cells

Abstract

The invention discloses a quantitative detection method for evaluating the chondrocyte proliferation promoting capacity of mesenchymal stem cells, and the chondrocyte proliferation promoting capacity of the mesenchymal stem cells is evaluated by detecting the secretion amount of PDGF-AA in mesenchymal stem cell culture supernatant. According to the method, the chondrocyte proliferation promoting capacity of the mesenchymal stem cells can be subjected to potency judgment, the chondrocyte proliferation promoting capacity of the mesenchymal stem cells can be evaluated by applying the method, and the qualified mesenchymal stem cells obtained by applying the method can be applied to preparation of osteoarthritis treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, and particularly relates to a quantitative detection method for evaluating the ability of mesenchymal stem cells to promote the proliferation of chondrocytes. BACKGROUND

[0002] PDGF-AA is an important cell growth regulator and belongs to the platelet-derived growth factor (PDGF) family. The PDGF family is composed of four polypeptide chains, namely PDGF-A, PDGF-B, PDGF-C and PDGF-D. Among them, PDGF-AA is a homodimer formed by two A chains through disulfide bonds.

[0003] PDGF-AA is an effective mitogen and chemotactic factor, which plays a key physiological role in the body. It is mainly derived from platelet alpha granules, but also expressed in various cell types, especially one of the important secretory factors of MSCs. PDGF-AA exerts its effects by binding to its cell surface receptor PDGFRα. The receptor dimerizes upon ligand binding and cross-phosphorylates intracellular tyrosine residues. These phosphorylated residues serve as binding sites for downstream signaling components and activate phospholipase PLCγ, phosphatidylinositol 3-kinase (PI3K) and Ras-MAPK signaling.

[0004] In vivo and in vitro experiments have shown that mesenchymal stem cells (MSCs) have the ability to promote the proliferation and repair of chondrocytes. They can participate in cartilage neogenesis by directly differentiating into chondroblasts, and they can also secrete various cytokines to act on surrounding chondrocytes or chondrocyte precursors to promote their proliferation and migration and inhibit their apoptosis, thereby initiating the endogenous repair process. Therefore, accurately evaluating the ability of MSCs to promote the proliferation of chondrocytes is crucial for quality control, efficacy prediction and individualized treatment plan development of MSCs as a therapeutic product.

[0005] The current method for evaluating the ability of MSCs to promote the proliferation of chondrocytes has significant shortcomings: Direct co-culture method: MSCs are directly mixed and cultured with chondrocytes, and the number of chondrocytes is counted to evaluate the proliferation effect. This method is complicated to operate, cannot exclude the interference of MSCs proliferation, has low accuracy, and cannot be standardized.

[0006] Indirect method of conditioned medium: collect the culture supernatant of MSCs (conditioned medium) and use it to culture chondrocytes, and then detect the proliferation of chondrocytes by cell counting, CCK-8 or MTT method, etc. Although this method avoids the interference of direct cell contact, the whole process is time-consuming (usually 5-7 days are needed), the steps are complicated, the repeatability is easily affected by operation, and the operator has high technical requirements.

[0007] Since chondrocytes are not immortal cell lines, new chondrocytes need to be isolated after they cannot be passaged, and different chondrocyte strains or different generations have batch differences, which makes it impossible to compare the promotion of chondrocyte proliferation function of different batches of MSCs.

[0008] At present, the method based on cytology can only provide a "yes or no" or "strong or weak" overall function conclusion, and cannot carry out precise quantitative evaluation. Without reference standard, batch comparison of MSCs cannot be carried out, and reliable basis for standardization of cell products cannot be provided. SUMMARY

[0009] In view of the problems in the prior art, the present application provides a novel quantitative detection method for evaluating the chondrocyte proliferation promoting ability of mesenchymal stem cells, and a method for effectively detecting and evaluating the chondrocyte proliferation promoting ability of MSCs.

[0010] The present application is realized in the following way: a quantitative detection method for evaluating the chondrocyte proliferation promoting ability of mesenchymal stem cells, detecting the secretion amount of PDGF-AA in the culture supernatant of mesenchymal stem cells, and determining the chondrocyte proliferation promoting ability of mesenchymal stem cells according to the secretion amount of PDGF-AA.

[0011] Specifically includes the following steps: Step one, mesenchymal stem cells are inoculated in a hole plate or a culture bottle at a seeding density of 2×10 4 / cm 2 , complete culture medium is added for culture for 24 hours, the original culture medium is discarded, DPBS is used for washing, and serum-free culture medium is added for continuous culture for 24 hours; the culture medium is added in an amount of 0.2mL / cm 2 ; Step two, collect the conditioned medium, and detect the content of PDGF-AA therein by a PDGF-AA ELISA kit; Step three, calculate the PDGF-AA level in each mL of the conditioned medium according to the standard curve; Step four, when the expression amount of PDGF-AA is >100pg / mL, it is determined that the MSCs have the ability to promote chondrocyte proliferation.

[0012] Further, the mesenchymal stem cells are placenta, umbilical cord, bone marrow, fat, gum-derived mesenchymal stem cells.

[0013] Further, the species are human, dog, cat, horse, rat or mouse.

[0014] Further, the complete medium is DMEM / F12 medium or IMDM medium containing 5-20% FBS, and the percentage is the volume fraction.

[0015] Further, the serum-free medium is DMEM / F12 medium or IMDM medium.

[0016] Further, the well plate is a 6-well plate, a 24-well plate or a 96-well plate, and the culture bottle is a T25 culture bottle.

[0017] The present application has the advantages and technical effects: 1. The present application finds a new use of PDGF-AA for evaluating the ability of MSCs to promote chondrocyte proliferation.

[0018] 2. The present application is a quantitative method for evaluating the ability of MSCs to promote chondrocyte proliferation, which uses ELISA quantitative detection method, draws a standard curve through standard samples, and performs accurate quantification. The results are objective, accurate, high in sensitivity and good in repeatability, and can compare the efficacy of different batches of MSCs and perform quality control grading.

[0019] 3. The present application can perform simultaneous detection of multiple samples. The present application can be performed in a T25 bottle, a 6-well plate, a 24-well plate or a 96-well plate, can set multiple duplicate wells and control groups, is suitable for medium-throughput sample screening, and significantly reduces the detection cost and time.

[0020] 4. The present application has wide application. The present application can not only be used for evaluating the quality of MSCs, but also can be used for screening inducing agents capable of enhancing the function of MSCs to promote chondrocyte proliferation. For example, after MSCs are induced by a specific inducing agent, whether the secretion amount of PDGF-AA in the culture supernatant of MSCs is up-regulated is detected, so as to determine whether the function of MSCs to promote chondrocyte proliferation is enhanced. By analogy, the present application can also screen culture conditions or gene modification schemes for enhancing the function of MSCs to promote chondrocyte proliferation, and provide a powerful tool for optimizing MSCs treatment strategy. Furthermore, the present application can be used for detecting qualified MSCs for the treatment of osteoarthritis, including: degenerative arthritis, rheumatoid arthritis, arthritis caused by hip dysplasia, traumatic arthritis, gouty arthritis, reactive arthritis and ankylosing arthritis. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1Figures showing the morphology of chondrocytes cultured alone and in co-culture with MSCs (a: canine chondrocytes, b: canine chondrocytes after co-culture with MSCs).

[0022] Figure 2 Figures showing the chondrocyte proliferation-promoting rate of different MSC samples.

[0023] Figure 3 Figures showing the PDGF-AA secretion amount of different MSC samples.

[0024] Figure 4 Figures showing the correlation between the chondrocyte proliferation-promoting rate and the PDGF-AA secretion amount of different MSC samples.

[0025] Figure 5 Figures showing the chondrocyte proliferation-promoting rate of MSCs after addition of a PDGFRa inhibitor.

[0026] Figure 6 Figures showing the determination curve of chondrocyte proliferation-promoting ability.

[0027] Figure 7 Figures showing the detection of chondrocyte proliferation-promoting ability of MSCs.

[0028] Figure 8 Figures showing the chondrocyte repair-promoting effect of MSCs on a rabbit osteoarthritis model. DETAILED DESCRIPTION

[0029] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be given below with reference to the drawings. It should be understood that the specific embodiments described herein are only used to explain the present application and should not be used to limit the present application.

[0030] The quantitative detection method for evaluating the chondrocyte proliferation-promoting ability of mesenchymal stem cells of the present application detects the secretion amount of PDGF-AA in the culture supernatant of mesenchymal stem cells, and determines the chondrocyte proliferation-promoting ability of mesenchymal stem cells according to the secretion amount of PDGF-AA.

[0031] Specifically includes the following steps: Step 1: Mesenchymal stem cells are inoculated in a well plate or a culture flask at a seeding density of 2 x 10 4 / cm 2 After 24 hours of culture in complete medium, the original culture medium is discarded, the well plate or culture flask is washed with DPBS, and serum-free medium is added for further culture for 24 hours. The amount of culture medium added is 0.2 mL / cm 2 . Step 2: The conditioned medium is collected, and the content of PDGF-AA therein is detected by a PDGF-AA ELISA kit. Step three, according to the standard curve, calculate the PDGF-AA level in each mL of conditional medium; Step four, when the expression of PDGF-AA is >100 pg / mL, determine that the MSCs have the ability to promote chondrocyte proliferation.

[0032] Further, the mesenchymal stem cells are placenta, umbilical cord, bone marrow, fat, gum-derived mesenchymal stem cells.

[0033] Further, the species are human, dog, cat, horse, rat or mouse.

[0034] Further, the complete culture medium is DMEM / F12 culture medium or IMDM culture medium containing 5-20% FBS, and the percentage is the volume fraction.

[0035] Further, the serum-free culture medium is DMEM / F12 culture medium or IMDM culture medium.

[0036] Further, the well plate is a 6-well plate, a 24-well plate or a 96-well plate, and the culture bottle is a T25 culture bottle.

[0037] The present application can be used for the detection of the chondrocyte proliferation-promoting ability of MSCs, the preclinical efficacy evaluation of MSCs for treating osteoarthritis (OA), and the quality control of MSC preparations.

[0038] The following will be described in combination with specific examples as follows.

[0039] Material source: the mesenchymal stem cells are umbilical cord, placenta, fat, bone marrow, gum-derived mesenchymal stem cells, and the species source is human, dog, cat, horse, rat or mouse. The following examples take canine placenta-derived mesenchymal stem cells as an example. The canine placenta-derived mesenchymal stem cells are derived from Tianjin Bai'en Biotechnology Co., Ltd.

[0040] The DMEM / F12 culture medium is purchased from Thermo Fisher Scientific.

[0041] The FBS is purchased from Yikosai Biological Company.

[0042] The transwell is purchased from Thermo Fisher Scientific.

[0043] The CP-673451 is purchased from Cayman Chemical.

[0044] The PDGF-AA ELISA kit is purchased from Abeam Company.

[0045] The PDGF-AA factor is purchased from MCE Company.

[0046] The canine chondrocytes are purchased from Hefei Hesheng Biological Technology Co., Ltd.

[0047] Example 1: Correlation between MSCs promoting chondrocyte proliferation rate and PDGF-AA secreted by MSCs Step 1, Effect of MSCs cells on chondrocyte proliferation; 4x10 4 chondrocytes were seeded in the lower chamber of transwell co-culture system, and 2x10 4 MSCs cells were seeded in the upper chamber. After co-culturing MSCs and chondrocytes for 24 hours, chondrocytes in the lower chamber were digested, and the number of chondrocytes was detected by trypan blue counting method. The proliferation rate of chondrocytes promoted by MSCs was obtained by comparing with the number of chondrocytes cultured alone. Nine different individual canine MSCs cells were selected for the experiment, and the cells for detection were P4 generation. The morphology of chondrocytes co-cultured with MSCs and chondrocytes cultured alone showed no difference (Fig. 1). Figure 1 The number of chondrocytes after co-culture was more than that of chondrocytes cultured alone, and the ability of different MSCs cells to promote chondrocyte proliferation was different, with individual differences (Fig. 2). Figure 2 The specific data are shown in Table 1.

[0048] Table 1 Proliferation rate of different MSCs cells promoting chondrocytes

[0049] Step 2, Detection of PDGF-AA secretion in MSCs culture supernatant; MSCs were seeded in 6-well plates at a seeding density of 2x10 4 / cm 2 , and cultured in a cell incubator with 5% CO2, 37°C, and saturated humidity of 95%. The culture medium was DMEM / F12 medium containing 10% fetal bovine serum, and the medium was added at a volume of 0.2 mL / cm 2 . After 24 hours of culture, the original culture medium was discarded, and the cells were washed with DPBS, and the serum-free DMEM / F12 medium was replaced, and the medium was added at a volume of 0.2 mL / cm 2 . After 24 hours of culture, the conditioned medium was collected, and the PDGF-AA secretion in the culture supernatant was determined by PDGF-AA ELISA kit. The PDGF-AA secretion of different MSCs cells was different, with individual differences (Fig. 3). Figure 3 The specific data are shown in Table 2.

[0050] Table 2 PDGF-AA secretion of different MSCs cells

[0051] Step 3, correlation analysis between PDGF-AA secretion amount of MSCs and its ability to promote chondrocyte proliferation.

[0052] Correlation analysis between PDGF-AA secretion amount of MSCs in step 2 and the proliferation rate of chondrocytes promoted by the corresponding MSCs in step 1 was performed. Statistical analysis showed that the Pearson correlation coefficient = 0.9514, p < 0.001, and there was a very significant positive correlation between them. Figure 4

[0053] Example 2: PDGF-AA secretion amount determines the ability of MSCs to promote chondrocyte proliferation 5 x 10 4 chondrocytes were planted in the lower chamber of the transwell co-culture system, 2.5 x 10 4 MSCs were planted in the upper chamber, and PDGFRα selective inhibitor CP-673451 (0.5 μM) was added to the co-culture system. After co-culturing for 24 hours, the chondrocytes in the lower chamber were digested, and the proliferation number of chondrocytes was detected by trypan blue counting method. The experimental results showed that after completely inhibiting PDGFRα, the promoting effect of MSCs supernatant on chondrocyte proliferation almost disappeared Figure 5 , which indicated that the promoting effect of MSCs on chondrocyte proliferation was mainly mediated by PDGF-AA and PDGFRα signaling pathway. Therefore, the PDGF-AA secretion amount of MSCs can be used to detect the ability of MSCs to promote chondrocyte proliferation, and further PDGF-AA can be used to evaluate the ability of MSCs to promote cartilage repair.

[0054] Example 3: determination of the ability of MSCs to promote chondrocyte proliferation Chondrocytes were inoculated in 6-well plates at a seeding density of 2 x 10 4 / cm 2 , and were cultured in a cell incubator with 5% CO2, 37°C, and a saturated humidity of 95%. The culture medium was DMEM / F12 medium containing 10% fetal bovine serum, and the medium addition amount was 0.2 mL / cm 2 . After 24 hours of culture, the original culture medium was discarded, the cells were washed with DPBS, and the serum-free DMEM / F12 medium was replaced, and the medium addition amount was 0.2 mL / cm 2 ​, PDGF-AA factor was added, and the level of PDGF-AA factor was 0, 25, 50, 75, 100, 150, 200, and 250 pg / mL, respectively. After 24 hours of culture, the chondrocytes were digested, and the number of chondrocyte proliferation was detected by trypan blue counting method. The experimental results showed that low concentration of PDGF-AA had no obvious effect on the proliferation of chondrocytes. When the level of PDGF-AA was greater than 100 pg / mL, the growth rate of chondrocytes was greater than 70%, thus determining that it had the ability to promote chondrocyte proliferation. Figure 6 ).

[0055] Example 4: Detection of the ability of MSCs to promote chondrocyte proliferation Three strains of canine placenta MSCs (No. 202510, 202513, and 202528) were inoculated in a 6-well plate at a seeding density of 2×10 4 / cm 2 . After 24 hours of culture in complete medium, the original culture medium was discarded, and DPBS was used for washing, and then serum-free medium was added for continuous culture for 24 hours. The amount of culture medium added was 0.2 mL / cm 2 . The conditioned medium was collected, and the content of PDGF-AA was determined by PDGF-AA ELISA kit. The detection result of No. 202510 was 135.26±1.08 pg / mL, which determined that it had the ability to promote chondrocyte proliferation. The detection result of No. 202513 was 110.42±3.25 pg / mL, which determined that it had the ability to promote chondrocyte proliferation. The detection result of No. 202528 was 85.50±2.13 pg / mL, which determined that it had no ability to promote chondrocyte proliferation. Figure 7 ).

[0056] Example 5: In vivo experiment to verify the ability of MSCs to promote chondrocyte proliferation First, a rabbit osteoarthritis model was established by chemical burn method. After the modeling was successful, the animals were divided into four groups: sham operation group (4), model control group (6), treatment group 1 (6), and treatment group 2 (6). The treatment group was injected with MSCs for cell treatment, and the model control group was injected with normal saline. Treatment group 1 applied canine placenta MSCs (No. 202510) in Example 4 for cell treatment, and treatment group 2 applied canine placenta MSCs (No. 202528) in Example 4 for cell treatment. The cell dose was 1×10 6 / joint, and the drug was given twice at an interval of 1 week. After 4 weeks of continuous observation, the tissue was taken for gross dissection evaluation and histopathological examination (HE staining). Figure 8It can be seen that the cartilage injury of the cell treatment group 1 is repaired and improved to a certain extent, and there is a statistical difference compared with the model control group, while the cartilage repair effect of the cell treatment group 2 is limited, and no obvious difference is found compared with the model control group.

[0057] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A quantitative detection method for evaluating the ability of mesenchymal stem cells to promote the proliferation of chondrocytes, characterized by, The secretion amount of PDGF-AA in the mesenchymal stem cell culture supernatant is detected, and when the expression amount of PDGF-AA is greater than 100 pg / mL, it is determined that the mesenchymal stem cell has the ability to promote chondrocyte proliferation.

2. The method according to claim 1, wherein the quantitative evaluation of the ability of mesenchymal stem cells to promote the proliferation of chondrocytes is performed by measuring the expression of the following genes: collagen type II alpha 1 (Col2al), aggrecan (Acan), and / or fibroblast growth factor 9 (Fgf9). The method comprises the following steps: Step one, the mesenchymal stem cells are inoculated in a hole plate or a culture bottle at an inoculation density of 2×104 / cm2, complete culture medium is added and cultured for 24 hours, then the original culture medium is discarded, DPBS is used for washing, and serum-free culture medium is added for continuous culture for 24 hours; the culture medium is added in an amount of 0.2 mL / cm2; Step two, the conditioned medium is collected, and the content of PDGF-AA therein is detected through a PDGF-AA ELISA kit; Step three, according to a standard curve, the PDGF-AA level in each mL of the conditioned medium is calculated; Step four, when the expression amount of PDGF-AA is greater than 100 pg / mL, it is determined that the MSCs have the ability to promote chondrocyte proliferation.

3. The method according to claim 1 or 2, wherein the method is used for quantitatively evaluating the ability of mesenchymal stem cells to promote the proliferation of chondrocytes. The mesenchymal stem cells are placenta, umbilical cord, bone marrow, fat, gum-derived mesenchymal stem cells.

4. The method according to claim 3, wherein the quantitative evaluation of the ability of mesenchymal stem cells to promote chondrocyte proliferation is performed. The species are human, dog, cat, horse, rat or mouse.

5. The method of claim 2, wherein the method is used for quantitatively evaluating the ability of mesenchymal stem cells to promote chondrocyte proliferation. The complete culture medium is DMEM / F12 culture medium or IMDM culture medium containing 5% to 20% FBS in volume fraction.

6. The method according to claim 2, wherein the ability of mesenchymal stem cells to promote chondrocyte proliferation is quantitatively evaluated. The serum-free culture medium is DMEM / F12 culture medium or IMDM culture medium.

7. The method according to claim 2, wherein the ability of mesenchymal stem cells to promote chondrocyte proliferation is quantitatively evaluated. The hole plate is a 6-hole plate, a 24-hole plate or a 96-hole plate, and the culture bottle is a T25 culture bottle.

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