Mesenchymal stem cell conditioned medium and preparation method thereof

Abstract

The invention discloses a mesenchymal stem cell conditioned medium and a preparation method thereof. The preparation method comprises the following steps: culturing immortalized stem cells in an antibiotic-free serum-free medium under a starvation condition for a preset time; and filtering to obtain filtrate, and adding HPL to obtain the mesenchymal stem cell conditioned medium. According to the present invention, the mesenchymal stem cell conditioned medium and the preparation method thereof can be provided, and the mesenchymal stem cell conditioned medium without the animal source additive can be used for stem cell culture so as to enhance the cell proliferation ability and well maintain the multi-directional differentiation ability of stem cells.

Description

Technical Field

[0001] The present disclosure generally relates to the field of bio-industry technology, and specifically relates to a mesenchymal stem cell conditioned medium and a preparation method thereof. Background Art

[0002] Stem cell therapy has received widespread attention due to its potential in treating a variety of diseases. Its application ranges from tissue repair to organ regeneration to the treatment of degenerative diseases. For example, umbilical cord mesenchymal stem cells are an important type of stem cell. They have the characteristics of differentiation potential, immune regulation, hematopoietic support, and self-replication. They are highly valued in the medical field and are used in regenerative medicine, developmental research, tissue engineering, and medical aesthetics. Since the number of stem cells in the body is limited, in order to meet the needs of clinical treatment and research, researchers often need to expand and proliferate stem cells through in vitro culture.

[0003] At present, commonly used mesenchymal stem cell culture media contain a certain concentration of animal-derived proteins, such as fetal bovine serum (FBS), to provide necessary growth factors and nutrients. Although these animal-derived components can promote the proliferation of stem cells, they have potential risks in clinical applications, such as the possibility of spreading viruses and inducing immune responses. In order to overcome these problems, some researchers have proposed the use of culture media without xenogeneic components.

[0004] However, when used for stem cell culture, culture media without xenogeneic components usually have a weak effect on the proliferation ability, immunomodulatory function and multidirectional differentiation potential of mesenchymal stem cells. Therefore, it is urgent to provide a mesenchymal stem cell culture medium with stable quality and without xenogeneic components. Summary of the invention

[0005] The present disclosure is proposed in view of the above-mentioned state of the prior art, and its purpose is to provide a mesenchymal stem cell conditioned medium and a preparation method thereof. The mesenchymal stem cell conditioned medium without animal-derived additives can be used for stem cell culture to enhance cell proliferation ability and better maintain the multidirectional differentiation ability of stem cells.

[0006] To this end, the first aspect of the present disclosure provides a method for preparing a mesenchymal stem cell conditioned medium, comprising the following steps: culturing immortalized stem cells in a serum-free medium without antibiotics and under starvation conditions for a predetermined time; filtering to obtain a filtrate, adding HPL to obtain a mesenchymal stem cell conditioned medium.

[0007] In the first aspect of the present disclosure, the immortalized stem cells used have a high degree of self-renewal or replication ability, and the quality is stable after immortalization induction; the cell viability is stronger, the self-renewal and proliferation ability is high, and the cytokine secretion ability is strong, and further, it can be stimulated to secrete a large amount of cytokines by serum-free starvation culture for a period of time; and the use of antibiotic-free culture medium can help eliminate antibiotic residues. Compared with the existing method of adding various reagents such as high-concentration glucose to stimulate, the preparation method of the present invention is simple, easy to operate, does not introduce exogenous substances, and can reduce side effects such as allergies; and the test results of the present invention show that the obtained conditioned medium has a stronger proliferation ability. Thus, according to the present invention, a mesenchymal stem cell conditioned medium without animal-derived additives can be prepared, which can be used for stem cell culture to enhance cell proliferation ability and better maintain the ability of stem cells to differentiate in multiple directions.

[0008] In the preparation method involved in the first aspect of the present disclosure, optionally, the immortalized stem cells are immortalized adipose-derived stem cells from human fat. In this case, the immortalized adipose-derived stem cells from human fat are selected because they are stable and do not introduce animal-derived additives. The cytokines contained in the culture medium finally prepared can help enhance cell proliferation ability during stem cell culture and better maintain the ability of stem cells to differentiate in multiple directions.

[0009] In the preparation method involved in the first aspect of the present disclosure, optionally, the preparation method of the immortalized adipose stem cells is: obtaining primary adipose stem cells from fat; and infecting the primary adipose stem cells with SV40 lentivirus to obtain the immortalized adipose stem cells. This can help to obtain stable immortalized adipose stem cells.

[0010] In the preparation method involved in the first aspect of the present disclosure, optionally, after obtaining the immortalized adipose-derived stem cells, the immortalized adipose-derived stem cells are subjected to puromycin screening, flow immunophenotyping detection, osteogenic adipogenic cartilage induction differentiation detection and / or proliferation ability detection, so as to select the immortalized adipose-derived stem cells. In this case, it can be helpful to obtain target cells, such as obtaining stable immortalized adipose-derived stem cells, or obtaining immortalized adipose-derived stem cells with stronger proliferation and differentiation effects, so as to prepare a conditioned medium with better effects.

[0011] In the preparation method involved in the first aspect of the present disclosure, optionally, in the flow immunophenotyping test, the selected immortalized adipose stem cells meet the immunophenotype CD29, CD44, CD73, CD90, CD105 are positive, and the positive rate is not less than 95%; CD31, CD34, CD45, HLA-DR are negative, and the negative rate is not higher than 2%; in the proliferation ability test, the selected immortalized adipose stem cells meet the doubling time of less than 18 hours. Thus, it can help to obtain immortalized adipose stem cells with stronger proliferation and differentiation effects, thereby preparing a conditioned medium with better effects.

[0012] In the preparation method involved in the first aspect of the present disclosure, optionally, the immortalized stem cells are first cultured in a medium without antibiotics and passaged to the p5 generation or above; then immortalized stem cells in the logarithmic growth phase are selected and cultured in a serum-free medium without antibiotics and under starvation conditions. In this case, it is possible to obtain a filtrate containing a large amount of cytokines secreted by the immortalized stem cells.

[0013] In the preparation method involved in the first aspect of the present disclosure, optionally, the concentration of HPL is 0.1% to 10%, thereby helping to improve the ability of the prepared conditioned medium to promote the proliferation and differentiation of mesenchymal stem cells.

[0014] In the preparation method involved in the first aspect of the present disclosure, optionally, the subject of the mesenchymal stem cell conditioned medium is umbilical cord mesenchymal stem cells. When the conditioned medium of the present disclosure is used to culture umbilical cord mesenchymal stem cells, the cell proliferation ability of umbilical cord mesenchymal stem cells can be better enhanced, and the multidirectional differentiation ability of umbilical cord mesenchymal stem cells can be better maintained.

[0015] In the preparation method of the first aspect of the present disclosure, optionally, the serum-free medium without antibiotics is MEM alpha medium containing 3% HPL. In this case, it can help provide a basic growth environment for immortalized stem cells and construct a culture environment with starvation conditions.

[0016] In a second aspect of the present disclosure, a mesenchymal stem cell conditioned medium is provided, comprising a filtrate of immortalized stem cells and HPL, wherein the filtrate of the immortalized stem cells is obtained by filtering the immortalized stem cells after culturing them for a predetermined time in a serum-free medium without antibiotics and under starvation conditions. In this case, the conditioned medium contains a large amount of cytokines secreted by the immortalized stem cells, and there is no need to introduce additional animal-derived additives. When used for stem cell culture, only the conditioned medium provided by the present disclosure can enhance the cell proliferation ability and better maintain the multidirectional differentiation ability of the stem cells.

[0017] According to the present disclosure, a mesenchymal stem cell conditioned medium free of animal-derived additives and a preparation method thereof can be provided. The mesenchymal stem cell conditioned medium used for stem cell culture can enhance cell proliferation ability and better maintain the multidirectional differentiation ability of stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present disclosure will now be explained in further detail by way of example only with reference to the accompanying drawings, in which: Figure 1 Schematic diagram showing the immunophenotypic detection results of immortalized adipose stem cells involved in the examples of the present disclosure.

[0019] Figure 2 Schematic diagram showing the morphology of immortalized adipose stem cells involved in the examples of the present disclosure.

[0020] Figure 3 Schematic diagram showing the osteogenic induction results of the osteogenic, adipogenic and chondrogenic differentiation detection of immortalized adipose stem cells involved in the examples of the present disclosure.

[0021] Figure 4 Schematic diagram showing the adipogenic induction results of the osteogenic, adipogenic, and chondrogenic differentiation detection of immortalized adipose stem cells involved in the examples of the present disclosure.

[0022] Figure 5 Schematic diagram showing the morphology of umbilical cord mesenchymal stem cells cultured using conditioned medium according to an example of the present disclosure.

[0023] Figure 6 Schematic diagram showing the results of immunophenotypic detection of umbilical cord mesenchymal stem cells cultured with conditioned medium according to an example of the present disclosure.

[0024] Figure 7 Schematic diagram showing the osteoinduction results of the third generation umbilical cord mesenchymal stem cells cultured with conditioned medium according to the examples of the present disclosure.

[0025] Figure 8 Schematic diagram showing the results of adipogenesis induction of third-generation umbilical cord mesenchymal stem cells cultured with conditioned medium according to an example of the present disclosure. DETAILED DESCRIPTION

[0026] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings.

[0027] It should be noted that the terms "including" and "having" and any variations thereof in the present disclosure, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0028] In addition, the subheadings and the like involved in the following description of the present disclosure are not intended to limit the content or scope of the present disclosure, but are merely used as a reminder for reading. Such subheadings should neither be understood as being used to divide the content of the article, nor should the content under the subheading be limited to the scope of the subheading.

[0029] The present disclosure relates to a method for preparing a mesenchymal stem cell conditioned medium, and the prepared mesenchymal stem cell conditioned medium can enhance cell proliferation ability and better maintain the multidirectional differentiation ability of stem cells when used for stem cell culture. In the present disclosure, the method for preparing the mesenchymal stem cell conditioned medium can be referred to as "preparation method" or "method for preparing conditioned medium".

[0030] The present disclosure also relates to a mesenchymal stem cell conditioned medium, which can enhance cell proliferation ability and better maintain the ability of stem cells to differentiate in multiple directions. In the present disclosure, the mesenchymal stem cell conditioned medium can be referred to as "conditioned medium" or "stem cell conditioned medium".

[0031] Hereinafter, the mesenchymal stem cell conditioned medium and the preparation method thereof involved in the present disclosure will be described in conjunction with the accompanying drawings.

[0032] In some examples, the expansion method may include the following steps: culturing the immortalized stem cells in a serum-free medium without antibiotics for a predetermined time (step S100); filtering to obtain a filtrate to obtain a mesenchymal stem cell conditioned medium (step S200). In the present disclosure, the immortalized stem cells used have a high self-renewal ability or replication ability, and the quality is stable after immortalization induction; their cell viability is stronger, the self-renewal and proliferation ability is high, and they have a strong cytokine secretion ability; and the use of antibiotic-free medium for culturing can help eliminate antibiotic residues. Compared with the existing method of adding various reagents such as high-concentration glucose for stimulation, the preparation method of the present invention is simple, easy to operate, does not introduce exogenous substances, and can reduce side effects such as allergies.

[0033] In the present disclosure, the filtrate containing a large amount of cytokines secreted by immortalized stem cells can also be referred to as a nutrient supplement or promoter. In other words, the present disclosure can also provide a nutrient supplement or promoter that can be used for cell culture.

[0034] In some examples, in step S100, the immortalized stem cells can be cultured in a serum-free medium without antibiotics and under starvation conditions for a predetermined time. In this case, the immortalized stem cells can be stimulated to secrete a large amount of cytokines by serum-free starvation culture for a period of time, which helps to enhance the cell proliferation ability when the formed conditioned medium is used for stem cell culture, and better maintain the multidirectional differentiation ability of the stem cells.

[0035] In some examples, starvation conditions may refer to conditions that place immortalized stem cells in an environment lacking nutrients. For example, starvation conditions may be created by limiting or eliminating nutrients such as growth factors and serum in the cell culture medium. In some examples, immortalized stem cells may also be stimulated by adjusting other culture conditions in the surrounding environment (such as oxygen concentration, pH value, temperature, etc.).

[0036] In some examples, in step S100, preferably, the serum-free culture medium without antibiotics can be MEM alpha culture medium containing 3% HPL. In this case, it can help provide a basic growth environment for immortalized stem cells and construct a culture environment with starvation conditions.

[0037] In some examples, the predetermined time can be 24h-48h. For example, the predetermined time can be 24h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, or 48h. In this case, the immortalized stem cells can be stimulated to secrete a large amount of cytokines as much as possible.

[0038] In some examples, in step S100, the immortalized stem cells may be immortalized stem cells derived from the umbilical cord, bone marrow, fat, muscle, or dental pulp. In some examples, preferably, in step S100, the immortalized stem cells may be immortalized adipose stem cells derived from human fat. That is, the immortalized stem cells may be obtained from human fat. In this case, the immortalized adipose stem cells derived from human fat are selected for their stable source and no animal-derived additives are introduced. The cytokines contained in the culture medium finally prepared can help enhance the cell proliferation ability during stem cell culture and better maintain the multidirectional differentiation ability of stem cells. Subsequently, taking the immortalized stem cells as immortalized adipose stem cells as an example, the preparation method involved in the present disclosure is further described in detail.

[0039] In some examples, the cytokines secreted by the immortalized adipose stem cells may be cytokines that help promote the proliferation and differentiation of mesenchymal stem cells. For example, the cytokines may include platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor (TGF), and epidermal growth factor (EGF).

[0040] In some examples, the method for preparing immortalized stem cells can be: obtaining stem cells from a target tissue; and immortalizing the stem cells to obtain immortalized stem cells. In some examples, stem cells can be immortalized by gene editing technology, viral vector-mediated gene transfer, cell fusion, or epigenetic regulation.

[0041] In some examples, the method for preparing immortalized adipose-derived stem cells may include: obtaining primary adipose-derived stem cells from fat; and infecting the primary adipose-derived stem cells with SV40 (Simian Virus 40) lentivirus to obtain the immortalized adipose-derived stem cells. This can help obtain stable immortalized adipose-derived stem cells.

[0042] In some examples, after obtaining the immortalized adipose-derived stem cells, an immortalization test may be performed on the immortalized adipose-derived stem cells, thereby facilitating obtaining stable immortalized adipose-derived stem cells.

[0043] In some examples, after obtaining the immortalized adipose stem cells, the immortalized adipose stem cells can be selected. In some examples, the immortalized adipose stem cells can be subjected to puromycin screening, flow immunophenotyping detection, osteogenic adipogenic cartilage induction differentiation detection and / or proliferation ability detection. In this case, it can help to obtain target cells, such as obtaining stable immortalized adipose stem cells, or obtaining immortalized adipose stem cells with stronger proliferation and differentiation effects, thereby preparing a conditioned medium with better effects.

[0044] In some examples, in flow cytometry immunophenotyping, the selected immortalized adipose stem cells meet the immunophenotypes of CD29, CD44, CD73, CD90, and CD105, which are positive, and the positive rate is not less than 95%; CD31, CD34, CD45, and HLA-DR are negative, and the negative rate is not higher than 2%. This can help obtain immortalized adipose stem cells with stronger proliferation and differentiation effects, thereby preparing a more effective conditioned medium.

[0045] In some examples, in the proliferation capacity test, the immortalized adipose-derived stem cells selected satisfy a doubling time of less than 18 hours, thereby being helpful in obtaining immortalized adipose-derived stem cells with stronger proliferation and differentiation effects, thereby preparing a conditioned medium with better effects.

[0046] In some examples, the immortalized stem cells can be first cultured in a medium without antibiotics and passaged to the p5 generation or above; then the immortalized stem cells in the logarithmic growth phase are selected and cultured in a serum-free medium without antibiotics and under starvation conditions. Among them, the logarithmic growth phase refers to the most active stage of proliferation of immortalized stem cells in the life cycle, at which time the cell proliferation rate is high, the morphology is stable, the function is active and the genetics is stable. In this case, it can be helpful to obtain a filtrate containing a large amount of cytokines secreted by immortalized stem cells. In some examples, the predetermined time for culturing immortalized stem cells in a serum-free medium without antibiotics can be 24h-48h.

[0047] In some examples, immortalized adipose-derived stem cells can be cultured in an antibiotic-free medium at least at P2 and later, thereby helping to eliminate the possibility of antibiotic residues in the culture medium.

[0048] In some examples, in step S200, HPL (human platelet lysate) can be added to the filtrate to obtain a mesenchymal stem cell conditioned medium. HPL contains a variety of growth factors, proteins, and other bioactive molecules that contribute to the proliferation and differentiation of mesenchymal stem cells, and can be used as a serum substitute in cell culture. In this case, the addition of HPL can help improve the ability of the prepared conditioned medium to promote the proliferation and differentiation of mesenchymal stem cells.

[0049] In some examples, the concentration of HPL can be 0.1% to 10%. For example, the concentration of HPL can be 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. Thus, it can help to improve the ability of the prepared conditioned medium to promote the proliferation and differentiation of mesenchymal stem cells. In some examples, preferably, the concentration of HPL can be 3%.

[0050] In some examples, the target of the mesenchymal stem cell conditioned medium may include umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, placental mesenchymal stem cells, amniotic fluid-derived mesenchymal stem cells, synovial mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, liver-derived mesenchymal stem cells, and corneal-derived mesenchymal stem cells. In some examples, preferably, the target of the mesenchymal stem cell conditioned medium may include umbilical cord mesenchymal stem cells. When the conditioned medium of the present disclosure is used to culture umbilical cord mesenchymal stem cells, the cell proliferation ability of umbilical cord mesenchymal stem cells can be better enhanced, and the ability of umbilical cord mesenchymal stem cells to differentiate in multiple directions can be better maintained.

[0051] When the target of the conditioned medium is limited to specific stem cells, the conditioned medium of the present disclosure may also be referred to as specific stem cells + conditioned medium. For example, when the target of the conditioned medium is umbilical cord mesenchymal stem cells, the conditioned medium of the present disclosure may also be referred to as umbilical cord mesenchymal stem cell conditioned medium.

[0052] In some examples, preferably, the preparation method disclosed herein may include: obtaining primary adipose stem cells from fat; infecting primary adipose stem cells with SV40 lentivirus to obtain immortalized adipose stem cells; culturing immortalized stem cells in a medium without antibiotics and passage to p5 or above; selecting immortalized stem cells in the logarithmic growth phase and culturing them in a serum-free medium without antibiotics and under starvation conditions for a predetermined time; filtering to obtain a filtrate, adding HPL, and obtaining a mesenchymal stem cell conditioned medium. In this case, the immortalized stem cells used have a high self-renewal ability or replication ability, and the quality is stable after immortalization induction; their cell viability is stronger, the self-renewal and proliferation ability is high, and they have a strong cytokine secretion ability, and further by serum-free starvation culture for a period of time can stimulate them to secrete a large amount of cytokines; and the use of antibiotic-free culture medium can help eliminate antibiotic residues. Compared with the existing method of adding various reagents such as high-concentration glucose to stimulate, the preparation method of the present invention is simple, easy to operate, does not introduce exogenous substances, and can reduce side effects such as allergies; and the test results of the present invention show that the obtained conditioned medium has a strong proliferation ability.

[0053] In summary, the preparation method according to the present invention can prepare a mesenchymal stem cell conditioned medium without animal-derived additives, which can be used for stem cell culture to enhance cell proliferation ability and better maintain the multidirectional differentiation ability of stem cells.

[0054] As mentioned above, the present disclosure can also provide a mesenchymal stem cell conditioned medium. Among them, the mesenchymal stem cell conditioned medium can be prepared by the aforementioned preparation method, and the parts already described in the previous preparation method are not repeated here. In some examples, the conditioned medium of the present disclosure can also be prepared by other methods.

[0055] In some examples, the conditioned medium may include a filtrate of immortalized stem cells, wherein the filtrate of immortalized stem cells may be obtained by filtering the immortalized stem cells after culturing them in a serum-free medium without antibiotics for a predetermined period of time. In this case, the conditioned medium contains a large amount of cytokines secreted by the immortalized stem cells, and no animal-derived additives need to be introduced. When used for stem cell culture, the cell proliferation ability can be enhanced and the multidirectional differentiation ability of the stem cells can be better maintained by using only the conditioned medium provided by the present disclosure.

[0056] In some examples, the filtrate of the immortalized stem cells can be obtained by filtering the immortalized stem cells after culturing them for a predetermined time in a serum-free medium without antibiotics and under starvation conditions. In some examples, the conditioned medium can include HPL. Thus, it can help to improve the ability of the prepared conditioned medium to promote the proliferation and differentiation of mesenchymal stem cells. In some examples, the concentration of HPL in the conditioned medium can be 0.1% to 10%. In some examples, preferably, the concentration of HPL can be 3%.

[0057] In some examples, the conditioned medium may include a basal medium (MEM medium). In some examples, preferably, the basal medium may be MEM alpha medium. Thus, it is possible to provide basic conditions for the proliferation and differentiation of stem cells. In some examples, preferably, the conditioned medium may be composed of a filtrate of immortalized stem cells, HPL, and a basal medium.

[0058] It should be noted that, although the present disclosure aims to provide a conditioned medium without animal-derived additives, in clinical applications, animal-derived additives may also be added for use according to actual needs.

[0059] In summary, according to the present disclosure, a mesenchymal stem cell conditioned medium free of animal-derived additives can be provided, which can be used for stem cell culture to enhance cell proliferation ability and better maintain the multidirectional differentiation ability of stem cells.

[0060] The present disclosure is described in detail below through examples and comparative examples. However, the following examples or comparative examples are only provided as examples to specifically illustrate the present disclosure, and do not define or restrict the scope of the invention disclosed in this application.

[0061] Unless otherwise specified, the reagents, materials, and instruments used in the examples and comparative examples of the present disclosure are all commercially available.

[0062] 1. Preparation of Conditioned Medium 1. Bluntly peel off the fat tissue, clean and remove the fascia tissue and small blood vessels. Cut the fat pad into pieces and transfer it to a 15mL centrifuge tube.

[0063] 2. Add 3-4 times the volume of 0.1% type I collagenase solution and shake in a 37°C water bath for about 20 minutes until the cell mixture becomes a milky and thick liquid. During this period, the fat tissue can be blown to assist digestion.

[0064] 3. Centrifuge at 250×g, 4°C for 5 min. Note that the centrifuge is precooled to 4°C.

[0065] 4. Carefully remove the upper layer of fat, taking care not to damage the gelatinous precipitate below. Add 5 mL of complete culture medium, resuspend the precipitate, transfer to a new centrifuge tube, and centrifuge again for 5 minutes.

[0066] 5. Repeat the washing step and remove the supernatant.

[0067] 6. Add 8 mL of complete culture medium to resuspend the pellet, transfer to a T25 culture flask, and place at 37°C, 5% CO2.

[0068] 7. After the cells have been attached to the wall for 48 hours, observe and change the medium for the first time.

[0069] 8. When changing the medium, discard the culture medium to remove the non-adherent cells.

[0070] 9. The culture medium was replaced every two days. When the cell confluence reached 80%-90%, primary adipose-derived stem cells were infected with SV40 lentivirus.

[0071] 10. The virus-infected cells were subjected to puromycin screening to select stable immortalized adipose stem cells.

[0072] 11. The immortalized adipose-derived stem cells screened were subjected to morphological observation, flow cytometry, and osteogenic and adipogenic induction, and it was concluded that the immortalized adipose-derived stem cells have stem cell characteristics. The results are shown in Figure 1-Figure 4 . Figure 1 Schematic diagram showing the immunophenotypic detection results of immortalized adipose stem cells involved in the examples of the present disclosure. Figure 2 Schematic diagram showing the morphology of immortalized adipose stem cells involved in the examples of the present disclosure. Figure 3 Schematic diagram showing the osteogenic induction results of the osteogenic, adipogenic and chondrogenic differentiation detection of immortalized adipose stem cells involved in the examples of the present disclosure. Figure 4 Schematic diagram showing the adipogenic induction results of the osteogenic, adipogenic, and chondrogenic differentiation detection of immortalized adipose stem cells involved in the examples of the present disclosure.

[0073] 12. Cultivate immortalized adipose-derived stem cells in the logarithmic growth phase in MEM alpha medium containing 3% HPL and activate them under serum-free starvation conditions for 24-48 hours; 13. Take the supernatant and filter to remove the cells, obtain the filtrate, add 3% HPL to obtain the conditioned medium.

[0074] 2. Functional verification of conditioned medium 1. Cell test Cell Counting Kit-8 (CCK-8 for short) reagent can be used for simple and accurate cell proliferation and toxicity analysis. Its basic principle is: the reagent contains WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonate)-2H-tetrazole monosodium salt), which is reduced to a highly water-soluble yellow formazan product (Formazan dye) by dehydrogenase in cells under the action of electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells. Therefore, this characteristic can be used to directly perform cell proliferation and toxicity analysis.

[0075] Umbilical cord mesenchymal stem cells were seeded in 96-well plates at 5000 cells / well and divided into experimental groups and positive and negative control groups, with 6 replicates in each group. After overnight adherence, the experimental group was replaced with the conditioned medium, the control group 1 used MEM alpha medium containing 3% HPL, and the control group 2 used MEM alpha medium containing 10% FBS. After continued culture for 24 hours, the supernatant was discarded, and 100 μl of MEM alpha basal medium containing 10% CCK8 detection solution was added to each well. After incubation for 4 hours, the absorbance values ​​at 450nm and 630nm were detected with a microplate reader.

[0076] Test results Table 1

[0077] Figure 5 Schematic diagram showing the morphology of umbilical cord mesenchymal stem cells cultured using conditioned medium according to an example of the present disclosure.

[0078] From the above results, it can be seen that the conditioned medium is superior to the medium simply adding HPL or FBS, and can greatly improve the cell proliferation ability.

[0079] 2. Flow cytometry Umbilical cord mesenchymal stem cells were continuously cultured with the conditioned medium to the 10th generation and the purity was analyzed.

[0080] When the cells were confluent to 80%, Try PLE was used to digest the cells, centrifuged and the cell pellet was collected. The cells were washed with PBS, counted, and evenly divided into 7 tubes, with 1*10^5 cells collected in each tube. The cells were centrifuged again, resuspended in 100μL PBS, and the corresponding antibodies were added to each tube for incubation in the dark for 30 minutes.

[0081] Table 2

[0082] After incubation, add 1 mL of PBS to each tube and centrifuge, discard the supernatant, harvest the labeled cells, resuspend each tube with 200 μL of PBS, filter into a flow cytometer, and test on the flow cytometer. Figure 6 Schematic diagram showing the results of immunophenotypic detection of umbilical cord mesenchymal stem cells cultured using conditioned medium according to the disclosed example. It can be seen that the positive markers CD73, CD44, CD90, and CD105 of umbilical cord mesenchymal stem cells cultured using the conditioned medium are expressed at more than 98%, and the negative markers CD34, CD45, and HLA-DR are expressed at less than 2%. The results show that the purity of human umbilical cord mesenchymal stem cells obtained by continuous culture using the conditioned medium is high.

[0083] 3. Osteogenesis and Adipogenesis Induction The umbilical cord mesenchymal stem cells cultured to the third generation with the conditioned medium were subjected to osteogenic and adipogenic induction experiments.

[0084] 1. Osteogenic Induction When the umbilical cord mesenchymal stem cells reach 80-90% confluence, digest them. Inoculate the umbilical cord mesenchymal stem cells in a six-well plate, with about 3x10 ^5 2 ml of the culture medium of the present invention was added to each well. The cells were placed in an incubator at 37°C and 5% CO2 for culture. After 24 hours, the old culture medium was removed and 2 ml of osteogenic induction medium (Cyagen, HUXUC-90021) was added. The medium was changed every three days and the induction lasted for 2 weeks. After 2 weeks, calcium nodules were formed and alizarin red staining was performed. The staining results were observed under a microscope. Figure 7 Schematic diagram showing the results of osteoinduction of the third generation of umbilical cord mesenchymal stem cells cultured with conditioned medium according to the examples of the present disclosure. Figure 7 It can be seen that under the culture conditions of the human umbilical cord mesenchymal stem cell culture medium disclosed in the present invention, the human umbilical cord mesenchymal stem cells can maintain their differentiation potential and differentiate into osteocytes.

[0085] 2. Adipogenesis Induction When the umbilical cord mesenchymal stem cells reach 80-90% confluence, digest them. Inoculate the cells in a six-well plate, with about 3x10 cells per well. ^5For each cell, add 2 ml / well of this conditioned medium. Culture in a 37°C, 5% CO2 incubator. Change the medium every three days until the cells reach 100% fusion. When the cells reach 100% fusion, remove the old medium and add 2 ml / well of Adipogenic Induction Medium A (Cyagen, HUXUB-90031) to start induction. After three days, replace with Adipogenic Induction Medium B (Cyagen, HUXUB-90031) for maintenance. After 24 hours, replace with Adipogenic Induction Medium A for induction, and repeat this for 3 cycles. When there are more lipid droplets but they are relatively small, use Adipogenic Induction Medium B for maintenance for 3-5 days to increase the size of the lipid droplets. Stain with Oil Red O. Observe the staining results under a microscope. Figure 8 Schematic diagram showing the results of adipogenesis induction of the third generation of umbilical cord mesenchymal stem cells cultured with conditioned medium according to the examples of the present disclosure. Figure 8 It can be seen that under the culture conditions of the human umbilical cord mesenchymal stem cell culture medium disclosed in the present invention, the human umbilical cord mesenchymal stem cells can maintain their differentiation potential and can differentiate into adipocytes.

[0086] In summary, the mesenchymal stem cell conditioned medium disclosed herein, which does not contain animal-derived additives, can enhance cell proliferation ability and better maintain the multidirectional differentiation ability of stem cells when used for stem cell culture.

[0087] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. A method for preparing a mesenchymal stem cell conditioned medium, characterized in that: The following steps are involved: culturing the immortalized stem cells in a serum-free medium without antibiotics and under starvation conditions for a predetermined time; The filtrate was obtained by filtration, and HPL was added to obtain a mesenchymal stem cell conditioned medium.

2. The preparation method according to claim 1, characterized in that: The immortalized stem cells are immortalized adipose stem cells derived from human adipose.

3. The preparation method according to claim 2, characterized in that: The method for preparing the immortalized adipose stem cells comprises: obtaining primary adipose stem cells from fat; and infecting the primary adipose stem cells with SV40 lentivirus to obtain the immortalized adipose stem cells.

4. The preparation method according to claim 3, characterized in that: After obtaining the immortalized adipose stem cells, the immortalized adipose stem cells are subjected to puromycin screening, flow cytometry immunophenotyping detection, osteogenic, adipogenic, and chondrogenic differentiation detection, and / or proliferation ability detection, so as to select the immortalized adipose stem cells.

5. The preparation method according to claim 4, characterized in that: In the flow immunophenotyping test, the selected immortalized adipose stem cells meet the immunophenotype CD29, CD44, CD73, CD90, CD105 are positive, and the positive rate is not less than 95%; CD31, CD34, CD45, HLA-DR are negative, and the negative rate is not higher than 2%; In the proliferation capacity detection, the selected immortalized adipose stem cells satisfy a doubling time of less than 18 hours.

6. The preparation method according to claim 1, characterized in that: First, the immortalized stem cells are cultured in a medium without antibiotics and passaged to the p5 generation or above; then, immortalized stem cells in the logarithmic growth phase are selected and cultured in a serum-free medium without antibiotics under starvation conditions.

7. The preparation method according to claim 1, characterized in that: The concentration of the HPL is 0.1% to 10%.

8. The preparation method according to claim 1, characterized in that: The target of the mesenchymal stem cell conditioned medium is umbilical cord mesenchymal stem cells.

9. The preparation method according to claim 1, characterized in that: The serum-free medium without antibiotics is MEM alpha medium containing 3% HPL.

10. A mesenchymal stem cell conditioned medium, characterized in that: The invention comprises a filtrate of immortalized stem cells and HPL, wherein the filtrate of immortalized stem cells is obtained by filtering the immortalized stem cells after culturing them in a serum-free medium without antibiotics and under starvation conditions for a predetermined time.

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