Culture medium and culture method of infrapatellar fat pad stem cells

Abstract

The present invention generally relates to the field of cell culture technology, and specifically, to a culture medium and a culture method for infrapatellar fat pad stem cells. The infrapatellar fat pad stem cell culture medium comprises a basal culture medium, and working concentrations of 3v / v%-7v / v% platelet lysate, 10ng / mL-30ng / mL TGF-β1, 0.5μmol / L-1.5μmol / L dexamethasone, and 0.1μg / mL-1μg / mL non-cytokine proinflammatory factors. The culture medium can promote the growth of IPFP-ASCs and has a significant promoting effect on the in vitro cartilage differentiation ability of IPFP-ASCs.

Description

Technical Field

[0001] The present invention generally relates to the field of cell culture technology, and in particular to a culture medium and a culture method for infrapatellar fat pad stem cells. Background Art

[0002] Articular cartilage lacks the ability to regenerate and heal, making the repair of cartilage damage a major clinical challenge. However, the rapid development of regenerative medicine has brought hope for solving this problem. At present, some countries and regions have begun clinical trials of stem cell repair of articular cartilage, although these studies are still in the early stages of exploration and experimentation. In order to improve the effect of cartilage repair, many researchers have begun to focus on strategies to optimize and improve the therapeutic function of stem cells, which involves the selection of stem cell sources, pretreatment processes, and the development of auxiliary materials.

[0003] The infrapatellar fat pad (IPFP), also known as the Hoffa fat pad, is named after the famous surgeon Albert Hoffa who first described its anatomical structure in 1904. It is located between the femoral condyle, tibial plateau, patella and patellar tendon, and is mainly composed of fat cells. In the past, it was generally believed that IFP mainly played a role in cushioning and lubrication, but the latest research has revealed that IFP may participate in the occurrence and differentiation of articular cartilage through interaction with synovium, articular cartilage and subchondral bone.

[0004] Mesenchymal stem cells, with their excellent self-renewal ability, multidirectional differentiation potential, and cross-germ layer development plasticity, have shown great development potential in the field of tissue engineering construction and gene therapy. These unique characteristics have made mesenchymal stem cells the focus of attention in the medical community, and are expected to bring revolutionary breakthroughs in future disease treatment and tissue repair. It is well known that mesenchymal stem cells have abundant sources, and mesenchymal stem cells from different sources have different differentiation and functional characteristics. Infrapatellar Fat Pad derived stem cells (IPFP-ASCs) are important cells for realizing the function of IPFP. Compared with stem cells from other sources such as bone marrow, synovium, and subcutaneous fat, IPFP-ASCs are easier to obtain and have strong proliferation ability, which gives them significant advantages in the field of cell therapy and regenerative medicine. Compared with adipose-derived stem cells, IPFP-ASCs show superior performance in chondrogenic and osteogenic differentiation ability. This characteristic gives IPFP-ASCs great application potential in tissue engineering and repair of damaged tissues, providing more possibilities for future medical treatment. Different culture methods of IPFP-ASCs have a significant impact on their osteogenic potential, but compared with other mesenchymal stem cells, there are few studies on the culture and differentiation of IPFP-ASCs using existing technologies, and the relevant principles are not clear. Summary of the invention

[0005] The present invention provides the following technical solutions:

[0006] The first aspect of the present invention relates to an infrapatellar fat pad stem cell culture medium, which comprises a basal culture medium, and working concentrations of 3v / v%-7v / v% platelet lysate, 10ng / mL-30ng / mL TGF-β1, 0.5μmol / L-1.5μmol / L dexamethasone and 0.1μg / mL-1μg / mL non-cytokine proinflammatory factors.

[0007] A second aspect of the present invention relates to a method for culturing infrapatellar fat pad stem cells, comprising culturing infrapatellar fat pad stem cells using the infrapatellar fat pad stem cell culture medium as described above.

[0008] The third aspect of the present invention relates to a pharmaceutical composition, which comprises infrapatellar fat pad stem cells cultured according to the method described above.

[0009] A fourth aspect of the present invention relates to the use of infrapatellar fat pad stem cells cultured according to the above method in the preparation of a drug for treating cartilage injury-related diseases.

[0010] In the inventor's previous research, it was found that in the process of NK cell culture, the addition of non-cytokine proinflammatory factors can significantly improve the purity and proliferation ability of NK cells, and enhance the killing ability of NK cells. However, whether it can still have a similar effect on other cells, especially non-immune cells, is unknown. In the present invention, the inventors used non-cytokine proinflammatory factors in the culture of IPFP-ASCs and found that it can also promote the increase of IPFP-ASCs, and unexpectedly found that it has a significant promoting effect on the in vitro induced cartilage differentiation ability of IPFP-ASCs. This shows that non-cytokine proinflammatory factors may have effects similar to joint inflammation in vivo. In view of the fact that there are still very few in vitro cultures of IPFP-ASCs in the prior art, the present invention is of great significance for the in vitro culture of IPFP-ASCs and the promotion of their chondrogenic differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 The cell proliferation of IPFP-ASCs in different culture medium components; A is the cell proliferation curve; B is the light microscope photo of cells on the 10th day of culture (scale bar = 200 μm);

[0013] Figure 2 The changes of mRNA of genes related to chondrogenic differentiation under different culture conditions; *p<0.05, **p<0.01, ***p<0.001, vs Ctrl; #p<0.05, vs DT;

[0014] Figure 3 The expression changes of SOX-9 protein under different culture conditions; A is the electrophoresis band; B is the statistical result; *p<0.05, ***p<0.001, vs Ctrl; #p<0.05, vs DT. DETAILED DESCRIPTION

[0015] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0016] Unless otherwise indicated, the meaning of all terms (including technical and scientific terms) used to disclose the present invention is the same as that commonly understood by those of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0017] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0018] As used herein, the terms "comprising", "including" and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0019] Numerical ranges expressed as endpoints herein include all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0020] The present invention relates to concentration values, and its meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuations within the range of ±0.1%. For values ​​that are large or do not require too fine control, its meaning is also allowed to include greater fluctuations. For example, 100mM can allow fluctuations within the range of ±1%, ±2%, ±5%, etc. Involving molecular weight, its meaning is allowed to include fluctuations of ±10%.

[0021] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0022] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0023] In the present invention, "preferably", "better", "more preferably", and "suitably" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent of each other.

[0024] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0025] Infrapatellar fat pad stem cell culture medium, comprising a basal culture medium, and working concentrations of 3v / v%-7v / v% platelet lysate, 10ng / mL-30ng / mL TGF-β1, 0.5μmol / L-1.5μmol / L dexamethasone, and 0.1μg / mL-1μg / mL non-cytokine proinflammatory factors.

[0026] In the present invention, the infrapatellar fat pad stem cell culture medium can be packaged in the form of a solution, or in the form of a dry powder composition (such as a freeze-dried powder or a spray-dried powder), and then dissolved when used. When it is a solution, the ratio of the components in the composition or solution satisfies the predetermined concentration when formulated as a working concentration. It is usually packaged in the form of a working concentration or a mother liquor. In the present invention, "working concentration" is used to limit the proportion of the main active components of the culture medium composition when culturing infrapatellar fat pad stem cells. The concentration of the main active components in the infrapatellar fat pad stem cell culture medium can be a working concentration, or it can be a mother liquor that can be diluted to this concentration (for example, a 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times concentrated mother liquor).

[0027] It is understood that for ingredients such as dexamethasone, non-cytokine proinflammatory factors, etc., it can include variants that maintain their activity, typically as pharmaceutically acceptable salts. As used herein, the phrase "pharmaceutically acceptable salt" refers to a salt of an active compound that has the same pharmacological activity as the active compound and is neither biologically undesirable nor otherwise undesirable. Salts can be formed, for example, with organic acids or inorganic acids. Non-limiting examples of suitable acids include acetic acid, acetylsalicylic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzoic acid, benzenesulfonic acid, bisulfic acid, boric acid, butyric acid, camphoric acid, camphorsulfonic acid, carbonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfic acid, ethanesulfonic acid, formic acid, fumaric acid, glyceric acid, glycerophosphoric acid, glycine, glucoheptanoic acid, gluconic acid, glutamic acid, glutaric acid, glycolic acid, hemisulfic acid, heptanoic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, isethionic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthylanesulfonic acid, naphthylsulfonic acid, and naphthylsulfonic acid. acid), nicotinic acid, nitrous acid, oxalic acid, nonanoic acid, phosphoric acid, propionic acid, saccharin, salicylic acid, sorbic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanic acid, thioglycolic acid, thiosulfuric acid, toluenesulfonic acid (tosylic acid), undecylenic acid, naturally and synthetically derived amino acids.

[0028] In some embodiments, the infrapatellar fat pad stem cell culture medium contains working concentrations of 15 ng / mL-25 ng / mL TGF-β1, 0.7 μmol / L-1.3 μmol / L dexamethasone, and 0.3 μg / mL-0.7 μg / mL non-cytokine proinflammatory factors.

[0029] In some embodiments, it comprises working concentrations of 17 ng / mL-23 ng / mL TGF-β1, 0.8 μmol / L-1.2 μmol / L dexamethasone, and 0.4 μg / mL-0.6 μg / mL non-cytokine proinflammatory factors.

[0030] In some embodiments, it comprises working concentrations of 20 ng / mL TGF-β1, 1.0 μmol / L dexamethasone, and 0.5 μg / mL non-cytokine proinflammatory factors.

[0031] The present invention does not exclude the use of other culture media, or the addition of some conventional nutritional supplements, such as salts, antibiotics, vitamins and amino acids, etc., in the culture media of the present invention. Amino acid supplements can include any amino acids, including glycine, alanine, valine, leucine, isoleucine, arginine, lysine, aspartic acid, cysteine, cysteine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, asparagine, glutamine, histidine and serine. Antibiotics such as gentamicin, penicillin, streptomycin, ampicillin, kanamycin and the like.

[0032] Buffers may also be contained in the culture medium of the present application, and its type is not specifically limited and may be a type well known to those skilled in the art." buffer " is a solution that resists pH changes by the effect of its acid-base conjugate component. The various buffers that can be used according to the desired pH (and microbial growth and metabolic characteristics, microbial culture culture system, pH control and culture medium used) of, for example, buffers are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., edited by Calbiochem Corporation (1975). In one embodiment, the buffer has a pH in the range of about 2 to about 9, or about 3 to about 8, or about 4 to about 7, or about 5 to about 7. The non-limiting examples of the buffer of pH in the control range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate and ammonium buffer, and combinations of these. In some embodiments, the buffer is selected from 3-(N-morpholino)propanesulfonic acid (MOPS) free acid, 3-(N-morpholino)propanesulfonic acid (MOPS) Na, hydroxyethylpiperazineethanesulfonic acid (HEPES), and sodium bicarbonate.

[0033] In some embodiments, some formulation aids, such as defoaming agents, may also be added to the culture medium. In some embodiments, the defoaming agent of the culture medium formulation comprises an ionic or nonionic surfactant.

[0034] In some embodiments, the non-cytokine proinflammatory factor is serum amyloid A (SSA).

[0035] It is easy to understand that the basic culture medium is a culture medium that is conducive to the culture of infrapatellar fat pad stem cells. In some embodiments, the basic culture medium is selected from X VIVO-15, DMEM, RPMI1640, AIM-V or α-MEM culture medium.

[0036] In some embodiments, the DMEM is low-glucose DMEM.

[0037] In some embodiments, the infrapatellar fat pad stem cell culture medium further contains 80 U / mL-120 U / mL of penicillin and 80 U / mL-120 U / mL of streptomycin.

[0038] The concentration of platelet lysate is 3 v / v%-7 v / v%, specifically 4 v / v%, 5 v / v%, 6 v / v%.

[0039] According to another aspect of the present invention, a method for culturing infrapatellar fat pad stem cells is provided, comprising culturing infrapatellar fat pad stem cells using the infrapatellar fat pad stem cell culture medium as described above.

[0040] In the present invention, unless otherwise specified, the cell culture environment is about 5% CO 2 and about 37°C.

[0041] According to yet another aspect of the present invention, there is also a pharmaceutical composition, which specifically comprises infrapatellar fat pad stem cells cultured by the method described above.

[0042] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier or adjuvant.

[0043] According to another aspect of the present invention, it also relates to the use of infrapatellar fat pad stem cells cultured by the method as described above in the preparation of drugs for treating diseases related to cartilage damage.

[0044] Diseases associated with cartilage damage are typically degenerative conditions such as osteoarthritis, which can lead to disability, pain during joint movement, and deformity of bone joints.

[0045] The subjects to which the drug is applicable include all members of the animal kingdom that are prone to the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human.

[0046] As used herein, "treatment" includes any method for obtaining a beneficial or desired result (including clinical effect) in the subject's condition. Beneficial or desired clinical effects may include, but are not limited to, alleviating or improving one or more symptoms or conditions, reducing the extent of the disease, stabilizing the disease state (i.e., not worsening), delaying or slowing the progression of the disease, improving and reducing the recurrence of the disease. Treatment can prevent the disease from occurring; alleviate the symptoms of the disease, completely or partially remove the root cause of the disease, shorten the duration of the disease, or achieve a combination of the above. "Treatment" as used herein may also include preventive treatment. Treatment methods include administering a therapeutically effective amount of an active agent to the subject. The administration step may consist of a single administration, or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the drug used in the treatment, or a combination thereof. It should also be understood that the effective dose of the agent used for treatment or prevention may increase or decrease during a specific treatment or prevention regimen. By standard diagnostic assays known in the art, changes in dosage may occur and become apparent. In some cases, long-term administration may be required. For example, the drug is administered to the subject in an amount and duration sufficient to treat the patient.

[0047] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, and can also be based on other experimental methods known in the art, or according to the conditions recommended by the manufacturer.

[0048] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0049] In the following embodiments:

[0050] Platelet lysate was purchased from Beijing Novi Biotechnology Co., Ltd.

[0051] Human recombinant serum amyloid A (SSA) was purchased from Peprotech (Rocky Hill, NJ, USA).

[0052] Recombinant human TGF-β1 protein was purchased from abcam, USA.

[0053] Dexamethasone was purchased from Cyagen, USA.

[0054] Example 1 Isolation of infrapatellar fat pad-derived stem cells

[0055] This experiment selected healthy New Zealand male white rabbits as experimental subjects. These rabbits were all 4 months old and weighed between 3 and 3.5 kg. In order to ensure the smooth progress of the experiment, we first anesthetized the experimental rabbits with general anesthesia. Subsequently, through surgical operation, we were able to accurately cut the infrapatellar fat pad in the rabbit's knee joint. Next, using the enzymatic digestion method, we successfully isolated and extracted infrapatellar fat pad-derived stem cells (IPFP-SCs) from the infrapatellar fat pad.

[0056] 1) Obtaining adipose tissue

[0057] First, 5% chloral hydrate solution was used, with a dose of 0.7 ml per 100 g of body weight per rabbit, and injected slowly through the ear vein to ensure general anesthesia of the experimental rabbit. After the anesthesia took effect, the experimental rabbit was skinned and fixed on the animal experimental table and restrained with a bandage. Subsequently, the surgical area was routinely disinfected and a surgical drape was laid.

[0058] Next, the rabbit's knee was centered in front of the knee as the surgical incision, and the joint capsule was cut through the medial side of the patella. Then, a sharp incision was made along the medial side of the patellar tendon, and the patella was dislocated laterally to expose the infrapatellar fat pad. After careful separation, the infrapatellar fat pad was successfully removed.

[0059] To ensure the cleanliness and sterility of the fat pad tissue, it should be immediately placed in sterile saline and quickly sent to the laboratory for follow-up processing. After flushing the surgical area, the patella is repositioned and the surgical incision is sutured layer by layer.

[0060] Finally, in order to prevent infection, the experimental rabbits were injected intramuscularly with penicillin sodium (dose of 50,000 IU / Kg). After marking the experimental rabbits, they were put back into the rabbit cages.

[0061] During the entire process, the experimental operating procedures should be strictly followed to ensure the accuracy and reliability of the experimental results.

[0062] 2) Obtaining stromal vascular fraction (SVF)

[0063] To obtain stromal vascular fraction (SVF), the removed infrapatellar fat pad tissue was first sent to the clean bench for strict aseptic operation. The tissue was rinsed 3 to 6 times with PBS solution containing 1% penicillin-streptomycin (PS) to ensure cleanliness. The unnecessary components such as synovium and blood vessels on the surface of the tissue were carefully trimmed to remove.

[0064] Then, the processed fat pad tissue was cut into small particles, ensuring that the particle diameter was less than 1 mm. 3, in order to facilitate the subsequent digestion process. These adipose tissue fragments were transferred to a sterile centrifuge tube and digested with 0.1% type I collagenase (Collagenase I) solution. The volume ratio of adipose tissue to digestion solution was controlled between 1:3 and 1:4 to ensure the digestion effect.

[0065] The sample was placed in a constant temperature shaking box and tissue digestion was performed at 37°C and 150 rpm for 60 minutes. After the digestion was completed, 1 / 4 volume of low-glucose DMEM medium solution containing 10% fetal bovine serum (FBS) was added to stop the digestion process.

[0066] Next, the digested mixture was filtered through a 100-mesh cell sieve to remove larger tissue fragments. Subsequently, it was centrifuged at 280 g for 10 minutes. After centrifugation, we discarded the upper layer of digestion fluid, and the bottom of the tube was the desired infrapatellar fat pad stromal vascular fraction (SVF). This step ensured the purity and separation of SVF, providing high-quality samples for subsequent experiments.

[0067] Example 2 Cultivation and Identification of Infrapatellar Fat Pad-Derived Stem Cells

[0068] The SVF fraction obtained in Example 1 was resuspended in culture medium and the cell concentration was adjusted to 1×10 6 / mL, placed in 5% CO 2 The cells were cultured at 37°C for 3 days, and the medium was subsequently changed every 2-3 days.

[0069] When it is observed under a microscope that the cells have covered about 80% of the bottom of the culture flask, the cells are passaged. First, the original culture medium is aspirated, and then washed twice with PBS solution to remove residual culture medium and impurities. Next, the cells are digested with an appropriate amount of trypsin. When gaps begin to appear between cells and the cytoplasm begins to shrink under the microscope, culture medium is immediately added to terminate the digestion. Subsequently, the bottom of the culture flask is gently blown with a pipette to ensure that the cells can completely fall off the bottom of the flask. After that, we perform cell passage at a ratio of 1:2. The passaged cells are placed in a cell culture incubator for culture. The medium is changed every 2-3 days to maintain a stable growth environment for the cells. The morphology and proliferation of the cells are regularly observed under a microscope to ensure the healthy growth of the cells.

[0070] In order to compare the effects, the culture medium is divided into three groups, and the formulas are:

[0071] Control group (Ctrl): low-glucose DMEM + 5v / v% platelet lysate;

[0072] Dexamethasone-TGF-β1 group (DT): low-glucose DMEM + 5 v / v% platelet lysate + 20 ng / mL TGF-β1 + 1.0 μmol / L dexamethasone;

[0073] Proinflammatory factor group (SSA): low-glucose DMEM+5v / v% platelet lysate+20ng / mL TGF-β1+1.0μmol / L dexamethasone+0.5μg / mL SSA.

[0074] The third generation IPFP-ASCs were taken and cell surface specific markers were identified by flow cytometry. The results showed that CD90 and CD105 were highly expressed in each group of cell populations, while CD11b, CD34 and HLA-II were negatively expressed, and the negative expression rate was less than 2%, which was consistent with the MSCs CD phenotype characteristics defined by the International Society for Cellular Therapy (ISCT). This shows that the cultured IPFP-ASCs still meet the characteristics of MSCs.

[0075] Table 1 Flow cytometry detection of IPFP-ASCs phenotype

[0076]

[0077] Negative <2%; ++++80%~95%; +++++>95%.

[0078] Example 3 Effects of different culture medium components on cell proliferation rate

[0079] IPFP-ASCs growth curve drawing: The third generation of adipose-derived mesenchymal stem cells were taken and resuspended in culture medium. The cells were counted on a cell counting plate. 200 μL was used per well, and about 6×10 6 L -1 The cells were seeded in 96-well plates and placed in 5% CO 2 The cells were cultured in a cell culture incubator at 37°C and saturated humidity. Six wells were randomly selected at regular intervals every day and 20 μL / well of 5% MTT was added. After 4 hours of culture, the liquid in the culture plate was aspirated, 150 μL DMSO / well was added, and after 10 minutes of shaking, the absorbance (A) at 490 nm was measured. The average value of A of the six wells was taken, and the growth curve was drawn with A as the ordinate and time (d) as the abscissa.

[0080] The growth curves of the three groups are shown in Figure 1 As shown in Figure A. It can be seen that the fertility curves of the third generation cells in each group showed a typical "S" shape. The cell incubation period of the Ctrl and DT groups was about 3-4 days, and entered the plateau phase after about 8 days of culture; while the incubation period of the SSA group was shortened and entered the plateau phase around the 6th day. This shows that the addition of SSA does not cause cytotoxicity, on the contrary, it can promote the cell proliferation of IPFP-ASCs.

[0081] Light microscopic photos of cell growth in each group on day 10 are shown in Figure 1 As shown in Figure B, it can be seen that the cells basically cover the bottom of the culture plate, with the typical spiral growth morphology of IPFP-ASCs.

[0082] Example 4 Effects of different culture medium components on chondrogenic differentiation of IPFP-ASCs

[0083] 1. Detection of changes in gene expression

[0084] The present invention uses qRT-PCR to detect genes related to stemness and chondrogenic differentiation of IPFP-ASCs. The cell sample is the cells cultured on the 10th day of Example 3. The experimental steps are briefly described as follows:

[0085] 1.1 Use Trizol method to extract total cell RNA, take 1 μL RNA and measure the concentration OD260 / 280 ratio with spectrophotometer to determine whether the extraction quality is qualified.

[0086] 1.2 Reverse transcription:

[0087] 1) Preparation: Preparation of mixture on ice:

[0088] Total RNA 0.5 μg

[0089] Random primer (0.5μg / μL) 1μL

[0090] Add DEPC water to 11 μL

[0091] Mix well and centrifuge for 3 to 5 seconds.

[0092] 2) Incubate at 65°C for 5 min. Cool on ice and centrifuge briefly.

[0093] 3) Add in sequence:

[0094] 5×reaction buffer 4μL

[0095] RiboLock Ribonuclease Inhibitor(20u / μL)1μL

[0096] 10mM dNTPmix 2μL

[0097] Mix well and centrifuge briefly.

[0098] 4) Incubate at 37°C for 5 min

[0099] 5) Add 2 μL of M-MuLV reverse transcriptase (20u / μL) to a total volume of 20 μL.

[0100] 6) Room temperature for 5 minutes.

[0101] 7) Incubate at 42°C for 60 min.

[0102] 8) Heat at 70°C for 10 min to terminate the reaction. Cool on ice.

[0103] 9) The reverse transcribed DNA can be stored at -20℃.

[0104] 1.3 Real-time quantitative PCR

[0105] 1) Reaction system:

[0106]

[0107]

[0108] A total of 20 μL system, after adding the sample, centrifuge with a support <2000rbpm.

[0109] Table 2 Primer sequences

[0110]

[0111] Reaction conditions:

[0112] 95℃30s

[0113] 95℃0s

[0114] Annealing temperature 5s

[0115] 72℃10s

[0116] 40 cycles

[0117] 95℃0s

[0118] 65℃30s

[0119] 95℃0.1 / Continuous cooling

[0120] 40℃30s

[0121] 1.4 Test results Figure 2 As shown in the figure, the results of one-way analysis of variance (ANOVA) showed that compared with the Ctrl group, SOX-9, COL2A1, and ACAN in the DT and SSA groups were significantly increased, and the SSA group had a further significant increase compared with the DT group. SOX-9, COL2A1, and ACAN are all genes closely related to chondrogenic differentiation, and their increase suggests that the culture medium has a stronger ability to promote chondrogenic differentiation.

[0122] 1.5 In order to further verify the changes in mRNA, we selected SOX-9, the most representative gene related to cartilage differentiation, for protein level verification. The content of SOX-9 protein in the protein of cells cultured on the 10th day of Example 3 was detected by Western Blot method. The results are as follows: Figure 3 As shown in Figure 2, it can be seen that the chondrocyte synthesis marker SOX-9 in the SSA group is still significantly increased compared with the DT group, which indicates that the pro-inflammatory substance SSA has a significant promoting effect on the in vitro chondrogenic differentiation ability of PFP-ASCs.

[0123] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A culture medium for infrapatellar fat pad stem cells, characterized in that: Contains basal medium, and working concentrations of 3v / v%-7v / v% platelet lysate, 10ng / mL-30ng / mL TGF-β1, 0.5μmol / L-1.5μmol / L dexamethasone, and 0.1μg / mL-1μg / mL serum amyloid A; The basic culture medium is low-glucose DMEM.

2. The infrapatellar fat pad stem cell culture medium according to claim 1, characterized in that It contains working concentrations of 15 ng / mL-25 ng / mL TGF-β1, 0.7 μmol / L-1.3 μmol / L dexamethasone, and 0.3 μg / mL-0.7 μg / mL serum amyloid A.

3. The infrapatellar fat pad stem cell culture medium according to claim 2, characterized in that It contains working concentrations of 17 ng / mL-23 ng / mL TGF-β1, 0.8 μmol / L-1.2 μmol / L dexamethasone, and 0.4 μg / mL-0.6 μg / mL serum amyloid A.

4. The infrapatellar fat pad stem cell culture medium according to any one of claims 1 to 3, characterized in that It also contains penicillin 80U / mL-120U / mL and streptomycin 80U / mL-120U / mL.

5. A method for culturing infrapatellar fat pad stem cells, characterized in that: The method comprises culturing infrapatellar fat pad stem cells using the infrapatellar fat pad stem cell culture medium described in any one of claims 1 to 4.

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