Method for improving osteogenic differentiation of umbilical cord mesenchymal stem cells under GMP (good manufacturing practice) system

Abstract

The invention relates to a method for improving osteogenic differentiation of umbilical cord mesenchymal stem cells under a GMP (good manufacturing practice) system, belongs to the technical field of stem cells, and solves the problem of poor osteogenic induction effect of the umbilical cord mesenchymal stem cells under the GMP system in the existing method. A method for improving osteogenic differentiation of umbilical cord mesenchymal stem cells under a GMP (good manufacturing practice) system comprises the following steps: in an osteogenic induction process of the umbilical cord mesenchymal stem cells, designing reasonable supplement frequency and volume according to an induction period, and supplementing an MSC (mesenchymal stem cells) osteogenic induction complete medium. According to the method disclosed by the invention, the osteogenic capability of the human umbilical cord mesenchymal stem cells is improved by using a conventional MSC osteogenic induction complete medium and by virtue of an operation method that the MSC osteogenic induction complete medium is supplemented at the initial stage of induction through reasonable replacement frequency and volume setting.

Description

Technical Field

[0001] The invention relates to the technical field of stem cells, and in particular to a method for improving the osteogenic differentiation of umbilical cord mesenchymal stem cells under a GMP system. Background Art

[0002] Mesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multidirectional differentiation potentials. They are mainly found in connective tissue and organ stroma, such as bone marrow, blood, umbilical cord, cord blood, placenta, fat, etc. They have the characteristics of regulating inflammation, anti-apoptosis and promoting angiogenesis. They are excellent tools for regenerative medicine, inflammatory diseases, high-throughput screening and toxicology. They can differentiate into osteoblasts under appropriate induction conditions. Human umbilical cord mesenchymal stem cells (UC-MSCs) can be harvested non-invasively, have osteogenic differentiation and good proliferation ability, low immunogenicity and strong immunosuppression, which makes them have broad prospects in clinical medicine and bone tissue engineering.

[0003] Osteogenic differentiation of MSCs is an important evidence for identifying the osteogenic ability of MSCs and is the basis of bone tissue engineering. In the process of osteogenic differentiation, osteoblasts first synthesize extracellular matrices such as type 1 collagen, osteocalcin and osteopontin, and release calcium ions and enzymes such as alkaline phosphatase through vesicles. Calcium ions are deposited on collagen fibers under the action of alkaline phosphatase, completing the mineralization process of the extracellular matrix and finally forming bone tissue. Mineralized nodules, also called bone nodules, are a sign of bone maturity and a morphological feature of bone tissue function; the formation of calcium nodules is identified by Alizarin red staining.

[0004] With the development of science and technology, it has become a trend to study and produce stem cells as a kind of cell biological preparation. Cell biological preparations must comply with the Good Manufacturing Practice of Medical Products (GMP). The production cells must use serum-free culture medium that does not contain animal-derived substances, and add cell additives with clear components to promote growth. Cells cultured in this culture medium have stronger proliferation ability after expansion than those cultured in serum culture medium. Previously, most of the cells used for inducing osteogenic differentiation of MSCs were cultured in serum culture medium. Compared with serum-free culture medium under GMP system, MSCs cultured in serum system have weaker proliferation activity and are easy to differentiate. Studies have shown that the existing induction methods have poor osteogenic induction effects on human umbilical cord mesenchymal stem cells (UC-MSCs) produced by serum-free culture under GMP system. Therefore, the existing induction methods need to be optimized. Summary of the invention

[0005] In view of the above analysis, the embodiments of the present invention aim to provide a method for improving the osteogenic differentiation of umbilical cord mesenchymal stem cells derived from a GMP system, so as to solve the problem that the existing induction methods have a very poor osteogenic induction effect on human umbilical cord mesenchymal stem cells (UC-MSCs) produced by serum-free culture under a GMP system.

[0006] The purpose of the present invention is mainly achieved through the following solutions:

[0007] The present invention provides a method for improving the osteogenic differentiation of umbilical cord mesenchymal stem cells under a GMP system, comprising the following steps:

[0008] S1: resuscitated cells;

[0009] S2: When cells reach the first confluence, use TrypLE TM Select enzyme to digest the cells, and then terminate the digestion reaction with phosphate buffer;

[0010] S3: Transfer the digested cell suspension into a conical sterile centrifuge tube and centrifuge;

[0011] S4: discard the supernatant, resuspend the cells in umbilical cord mesenchymal stem cell serum-free medium and count them;

[0012] S5: Coating the cell culture dish;

[0013] S6: Inoculate the cells into the coated multi-well plate, add umbilical cord mesenchymal stem cell serum-free culture medium to each well, and place the plate in an incubator to culture the cells;

[0014] S7: When the cells reach the second degree of confluence, the serum-free medium of umbilical cord mesenchymal stem cells is discarded, and MSC osteogenic induction complete medium is added to each well, and the cells are continued to be cultured in the incubator;

[0015] S8: During the first induction period, MSC osteogenic induction complete medium was added and the cells were cultured in the incubator continuously;

[0016] S9: The second induction period, add MSC osteogenic induction complete medium, and continue to culture the cells in the incubator;

[0017] S10: The third induction period, observing the calcium deposition in the culture medium and on the cell surface and the formation of bone nodules, and judging whether to stop the induction culture according to the evaluation rules;

[0018] S11: After the induction culture was completed, the culture supernatant was discarded, the cells were washed twice with phosphate buffer, and a neutral paraformaldehyde solution was added to each well to fix the cells at room temperature;

[0019] S12: discard the fixative, wash the cells twice with phosphate buffer, and add alizarin red dye to each well at room temperature to stain the cells;

[0020] S13: discard the alizarin red dye solution, wash the cells three times with phosphate buffer, add fresh PBS, observe the bone nodules and calcium deposition on the cell surface under a microscope, and perform quantitative analysis on the bone nodules and calcium deposition on the cell surface.

[0021] Furthermore, in step S2, the first cell fusion degree is 90-95%.

[0022] Furthermore, in step S7, the second cell fusion degree is 60-70%.

[0023] Further, in step S8, the first induction period is 5-7 days after the start of induction;

[0024] Furthermore, in step S8, the amount of the added MSC osteogenic induction complete culture medium is 300-600 μL / well.

[0025] Furthermore, in step S9, the second induction period is the 10th to 14th day from the start of induction.

[0026] Furthermore, in step S9, the amount of the added MSC osteogenic induction complete culture medium is 300-600 μL / well.

[0027] Furthermore, in step S10, the third induction period is the 15th to 21st day from the start of induction.

[0028] Furthermore, in step S10, the evaluation rules include:

[0029] If there are <5 calcium deposition spots on the cell surface and >50 bone nodules in the culture medium, 500-1000 μL of culture medium is aspirated from the upper layer of the culture medium;

[0030] If there are less than 5 calcium deposition points on the cell surface and less than 50 bone nodules in the culture medium, remove 500-1000 μL of culture medium from the upper layer of the culture medium, and replenish each well with MSC osteogenic induction complete culture medium and continue to observe for 5-10 days;

[0031] When the number of calcium deposition points on the cell surface is greater than 50, the cell induction culture is stopped.

[0032] Furthermore, the amount of the supplemented MSC osteogenic induction complete culture medium is 300-600 μL / well.

[0033] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0034] 1. Compared with the traditional method of frequently replacing MSC osteogenic induction complete culture medium for osteogenic induction, the present invention supplements the MSC osteogenic induction complete culture medium at the early stage of induction according to the induction cycle. Through reasonable replacement frequency and volume setting, it can meet the needs of cell nutrition without making the nutrition in the culture medium too rich, which is beneficial to the osteogenic differentiation of human umbilical cord mesenchymal stem cells; at the same time, human umbilical cord mesenchymal stem cells will secrete cytokines that promote osteogenic differentiation during the osteogenic differentiation process. Frequent replacement of MSC osteogenic induction complete culture medium is likely to lose these cytokines that promote osteogenic differentiation. The present invention supplements the MSC osteogenic induction complete culture medium at the early stage of induction, so that the cytokines that are beneficial to differentiation secreted during the osteogenic differentiation of cells can accumulate, further promote the osteogenic differentiation of cells, and improve the efficiency and effect of osteogenic differentiation of human umbilical cord mesenchymal stem cells.

[0035] 2. The present invention improves the osteogenic ability of human umbilical cord mesenchymal stem cells by using conventional MSC osteogenic induction complete culture medium, by reasonably changing the frequency and volume setting, and by supplementing the MSC osteogenic induction complete culture medium at the early stage of induction.

[0036] 3. The method of osteogenic differentiation of human umbilical cord mesenchymal stem cells of the present invention is also applicable to the induction of osteogenic differentiation of mesenchymal stem cells from other sources under the GMP system, including but not limited to adipose mesenchymal stem cells, placental mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells, blood mesenchymal stem cells, etc. The method of the present invention can be used for the identification of in vitro osteogenic differentiation of mesenchymal stem cells and the exploration of in vitro induction of bone tissue in bone tissue engineering based on mesenchymal stem cells.

[0037] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference symbols denote like components throughout the drawings.

[0039] Figure 1 is a cell surface micrograph of Example 1;

[0040] Figure 2 It is the cell surface micrograph of Comparative Example 1;

[0041] Figure 3This is a result diagram of measuring the absorbance value at 490 nm after dissolving Alizarin Red with glacial acetic acid-ethanol solution in Example 1 and Comparative Example 1;

[0042] Figure 4 is a cell surface micrograph of Example 2;

[0043] Figure 5 It is the cell surface micrograph of Comparative Example 2;

[0044] Figure 6 This is a graph showing the absorbance values ​​measured at 490 nm after dissolving Alizarin Red in glacial acetic acid-ethanol solution in Example 2 and Comparative Example 2. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

[0046] The present invention provides a method for improving the osteogenic differentiation of umbilical cord mesenchymal stem cells derived from a GMP system, comprising the following steps:

[0047] A method for improving osteogenic differentiation of umbilical cord mesenchymal stem cells under a GMP system, characterized by comprising the following steps:

[0048] S1: Resuscitation cells

[0049] Take out the umbilical cord mesenchymal stem cells to be tested from the liquid nitrogen tank, quickly thaw the cells in a 37°C water bath, wipe the surface of the cryopreservation tube with alcohol, and move it into a biosafety cabinet. Quickly transfer the cell suspension to 4 mL of mesenchymal stem cell serum-free medium and centrifuge at 1000 rpm for 3 minutes; discard the supernatant and transfer the mesenchymal stem cells to a 10 cm cell culture dish, add 10 mL of umbilical cord mesenchymal stem cell serum-free medium, and culture for 2-3 days;

[0050] The umbilical cord mesenchymal stem cell serum-free culture medium is a conventional umbilical cord mesenchymal stem cell serum-free culture medium, preferably 3D Mesenchymal stem cell serum-free medium, GMP-grade MSC serum-free medium from Youkang Bio.

[0051] S2: When cells reach the first confluence, use TrypLE TM Select enzyme to digest the cells, and then terminate the digestion reaction with phosphate buffered saline (PBS);

[0052] When the first confluency is 90-95%, use 1 mL 1X TrypLE TMSelect enzyme digested the cells for 2 min, and the digestion reaction was terminated with phosphate buffered saline (PBS);

[0053] It should be noted that when the cells reach a confluence of 90-95%, it can ensure that more cells are obtained and the cells are in good condition. If the cell confluence is too high, the cell growth state after passaging will be poor. If the cell confluence is low, sufficient cells cannot be obtained after passaging for subsequent experimental steps.

[0054] Specifically, before terminating the digestion reaction with phosphate buffered saline (PBS), PBS should be preheated to 37°C to ensure that TrypLE TM The activity of the Select enzyme can be quickly neutralized; when cells begin to detach from the serum-free medium, gently tap the cell culture dish to promote cell detachment, then aspirate the TrypLE TM Select enzyme solution; then add 4 mL of preheated PBS to the cell culture dish to terminate the digestion reaction process; use a pipette to gently blow the cells in the cell culture dish to completely detach them from the culture medium and form a cell suspension.

[0055] S3: Transfer the digested cell suspension into a conical sterile centrifuge tube and centrifuge;

[0056] Specifically, the digested cell suspension was transferred into a 15 mL conical-bottom sterile centrifuge tube and centrifuged at 1000 rpm for 3 min;

[0057] S4: discard the supernatant, resuspend the cells in umbilical cord mesenchymal stem cell serum-free medium and count them;

[0058] Specifically, the amount of the umbilical cord mesenchymal stem cell serum-free culture medium is 1 mL.

[0059] S5: Coating of cell culture dishes

[0060] Add 1 mL of 0.1% sterile gelatin solution to the 6-well plate, incubate at 37°C for 30 minutes, aspirate the coating solution and dry it for later use.

[0061] S6: Inoculate the cells into the coated multi-well plate, add umbilical cord mesenchymal stem cell serum-free culture medium to each well, and place the plate in an incubator to culture the cells;

[0062] Specifically, according to 2×104cells / cm 2 The cells were seeded into the coated multi-well plate at a density of 1.5 μg / mL, 2 mL of umbilical cord mesenchymal stem cell serum-free medium was added to each well, and the plate was cultured in a 37°C, 5% CO2 incubator;

[0063] S7: When the cells reach the second degree of confluence, the serum-free medium of umbilical cord mesenchymal stem cells is discarded, and MSC osteogenic induction complete medium is added to each well, and the cells are continued to be cultured in the incubator;

[0064] Specifically, the second confluence is 60-70%, the umbilical cord mesenchymal stem cell serum-free culture medium is discarded, 2 mL of MSC osteogenic induction complete culture medium is added to each well, and the cells are placed in a 37° C., 5% CO2 incubator for culture;

[0065] The MSC osteogenic induction complete medium is a conventional MSC osteogenic induction complete medium, preferably Saiye Biotech human umbilical cord osteogenic induction complete medium or Fuyuan Biotech human umbilical cord osteogenic induction complete medium;

[0066] It should be noted that when the cell confluence reaches 60-70%, the serum-free medium of umbilical cord mesenchymal stem cells is discarded and replaced with MSC osteogenic induction complete medium. This is because within 2-3 days of changing from the serum-free medium of umbilical cord mesenchymal stem cells to the complete medium of MSC osteogenic induction, the umbilical cord mesenchymal stem cells will rapidly proliferate and differentiate into osteogenic precursor cells. The cell confluence of 60-70% can ensure that the cells have enough suitable proliferation space to rapidly proliferate and differentiate. Finally, the cell confluence basically reaches 100%, and the secretion of inorganic salts and extracellular matrix is ​​more, which makes it easier to form bone nodules. If the cell confluence before induction is too high, contact inhibition will occur when the cells are not fully proliferated and differentiated. If the cell confluence before induction is too low, the cell confluence cannot reach 100% after the umbilical cord mesenchymal stem cells proliferate and differentiate into osteogenic precursor cells, which will cause poor cell adhesion and finally reduce the ability of the cells to form bone nodules.

[0067] It should be noted that cells are easy to fall off after osteogenic induction. Therefore, before replacing the umbilical cord mesenchymal stem cell serum-free medium with MSC osteogenic induction complete medium, the MSC osteogenic induction complete medium must be rewarmed. The MSC osteogenic induction complete medium needed for the day can be taken into a 15mL cryopreservation tube and incubated in a 37°C water bath for 5 minutes; when adding MSC osteogenic induction complete medium, the action should be gentle and slowly added along the side wall of the culture dish to avoid blowing up the cells; the time and effect of osteogenic induction are closely related to the cell state. The cell state is observed under a light microscope. Under normal conditions, the cells are spindle-shaped under the light microscope, and the cells in poor condition are round. If the cell state is not good at this time, the cells should be re-inoculated for the experiment.

[0068] S8: During the first induction period, MSC osteogenic induction complete medium was added and the cells were cultured in the incubator continuously;

[0069] The first induction period is 5-7 days after the induction begins, and the amount of MSC osteogenic induction complete medium added is 300-600 μL / well, and the cells continue to be cultured in a 37° C., 5% CO 2 incubator.

[0070] S9: The second induction period, add MSC osteogenic induction complete medium, and continue to culture the cells in the incubator;

[0071] The second induction period is from the 10th to the 14th day after the induction, and the amount of MSC osteogenic induction complete medium added is 300-600 μL / well, and the cells are cultured in a 37° C., 5% CO 2 incubator.

[0072] It should be noted that in steps S8 and S9, the amount of MSC osteogenic induction complete medium added is 500 μL / well, which can not only dilute the harmful substances produced in the MSC osteogenic induction complete medium during the culture process, but also replenish the volume of the MSC osteogenic induction complete medium evaporated during the culture process; if the amount added is too large, it is easy to cause the subsequent calcium nodules to be difficult to deposit on the cell surface after formation and float in the culture medium; if the amount added is too small, it is easy to cause the total volume of the culture medium in the culture dish to become smaller and smaller and unable to cover the cells, ultimately affecting the cell state and even causing death.

[0073] S10: The third induction period, observing the calcium deposition in the culture medium and on the cell surface and the formation of bone nodules, and judging whether to stop the induction culture according to the evaluation rules;

[0074] The third induction period is 15-21 days after the start of induction, and the formation of calcium deposits and calcium nodules in the culture medium and on the cell surface is observed under a light microscope; the evaluation rules include: if the number of calcium deposition points on the cell surface is less than 5, and the number of bone nodules in the culture medium is greater than 50, 500-1000 μL of culture medium can be carefully aspirated from the upper layer of the culture medium to promote bone nodule deposition; if the number of calcium deposition points on the cell surface is less than 5, and the number of bone nodules in the culture medium is less than 50, 500-1000 μL of culture medium can be aspirated from the upper layer of the culture medium, and 300-600 μL / well MSC osteogenic induction complete culture medium is supplemented and observation continues for 5-10 days; if the number of calcium deposition points on the cell surface is greater than 50, the induction culture is stopped.

[0075] S11: After the induction culture, the culture supernatant was discarded, the cells were washed twice with phosphate buffered saline (PBS), and a neutral paraformaldehyde solution was added to each well to fix the cells at room temperature;

[0076] Specifically, after the induction culture was completed, the culture supernatant was discarded, the cells were washed twice with phosphate buffered saline (PBS), 1 mL of 4% neutral paraformaldehyde solution was added to each well, and the cells were fixed at room temperature for 20 minutes.

[0077] S12: discard the fixative, wash the cells twice with phosphate buffered saline (PBS), and add alizarin red dye to each well at room temperature to stain the cells;

[0078] Specifically, the fixative was discarded, the cells were washed twice with phosphate buffered saline (PBS), 1 mL of 0.2% (w / v) alizarin red dye solution was added to each well, and the cells were stained at room temperature for 15 min;

[0079] S13: discard the Alizarin Red dye solution, wash the cells three times with phosphate buffered saline (PBS), add fresh PBS, observe the bone nodules and calcium deposition on the cell surface under a microscope, and perform quantitative analysis on the bone nodules and calcium deposition on the cell surface.

[0080] Specifically, the alizarin red dye solution was discarded, the cells were washed three times with phosphate buffered saline (PBS), and 1 mL of fresh PBS was added; the bone nodules and calcium deposits on the cell surface were observed under a microscope and photographed; then the PBS was aspirated, 1 mL of glacial acetic acid-ethanol solution was added, wherein the mass ratio of 10% glacial acetic acid to anhydrous ethanol in the glacial acetic acid-ethanol solution was 8:2, and the solution was placed on a horizontal shaker at a speed of 60-90 rpm / min, and incubated at room temperature for 25-45 min to fully dissolve the alizarin red; 200 μL of the dissolved solution was taken into a 96-well plate, and the absorbance at 490 nm was measured on an ELISA reader to quantitatively analyze the bone nodules and calcium deposits on the cell surface.

[0081] It should be noted that existing studies have shown that although human umbilical cord mesenchymal stem cells cultured in serum systems and GMP systems have the ability to induce differentiation into adipogenesis, osteoblastogenesis, and chondrogenesis, with the increase of cell generations, human umbilical cord mesenchymal stem cells cultured in the GMP system show a relatively uniform vortex-like growth, while human umbilical cord mesenchymal stem cells cultured in the serum system gradually show cell differentiation or aging phenomena, that is, human umbilical cord mesenchymal stem cells cultured in the serum system are easier to differentiate than those in the GMP system, and osteogenic induction is essentially a differentiation process, that is, human umbilical cord mesenchymal stem cells cultured in the GMP system are more difficult to osteogenesis than those cultured in the serum system, that is, the existing osteogenic induction methods for human umbilical cord mesenchymal stem cells cultured in the serum system are less effective in inducing osteogenic changes in human umbilical cord mesenchymal stem cells cultured in the GMP system. The present invention performs osteogenic induction on human umbilical cord mesenchymal stem cells by supplementing MSC osteogenic induction complete culture medium at the initial stage of induction instead of replacing the MSC osteogenic induction complete culture medium. Compared with the traditional method of frequently replacing the MSC osteogenic induction complete culture medium for osteogenic induction, the present invention performs an operation method of supplementing the MSC osteogenic induction complete culture medium at the initial stage of induction according to the induction cycle. Through reasonable replacement frequency and volume setting, the demand for cell nutrition can be met without making the nutrition in the culture medium too rich, which is beneficial to the osteogenic differentiation of human umbilical cord mesenchymal stem cells. At the same time, human umbilical cord mesenchymal stem cells secrete cytokines that promote osteogenic differentiation during the osteogenic differentiation process. Frequent replacement of the MSC osteogenic induction complete culture medium is likely to lose these cytokines that promote osteogenic differentiation. The present invention performs an operation method of supplementing the MSC osteogenic induction complete culture medium at the initial stage of induction, which can accumulate cytokines that are beneficial to differentiation and further promote cell osteogenic differentiation, thereby improving the efficiency and effect of osteogenic differentiation of human umbilical cord mesenchymal stem cells.

[0082] The method of osteogenic differentiation of human umbilical cord mesenchymal stem cells of the present invention is also applicable to the induction of osteogenic differentiation of mesenchymal stem cells from other sources under the GMP system, including but not limited to adipose mesenchymal stem cells, placental mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells, blood mesenchymal stem cells, etc. The method of the present invention can be used for the identification of in vitro osteogenic differentiation of mesenchymal stem cells and the exploration of in vitro induction of bone tissue in bone tissue engineering based on mesenchymal stem cells.

[0083] Example 1

[0084] In this example, the human umbilical cord osteogenic kit (Cat. No.: HUXUC-90021) of Saiye Company was used to induce osteogenic growth of umbilical cord mesenchymal stem cells derived from the GMP system (referred to as the Modified method group), including the following steps:

[0085] S1: Resuscitation cells

[0086] Take out the umbilical cord mesenchymal stem cells to be tested from the liquid nitrogen tank, quickly thaw the cells in a 37°C water bath, wipe the surface of the cryopreservation tube with alcohol, and move it into a biosafety cabinet. Quickly transfer the cell suspension to 4 mL of mesenchymal stem cell serum-free medium and centrifuge at 1000 rpm for 3 min. After discarding the supernatant, transfer the mesenchymal stem cells to a 10 cm cell culture dish and add 10 mL of CytoNiche (Huakan Bio) 3D Mesenchymal stem cells were cultured in serum-free medium for 3 days;

[0087] S2: When cells reach 90% confluency, add 1 mL 1X TrypLE TM Select enzyme digested the cells for 2 min, and the digestion reaction was terminated with phosphate buffered saline (PBS);

[0088] Specifically, before terminating the digestion reaction with phosphate buffered saline (PBS), PBS was preheated to 37°C to ensure that TrypLE TM The activity of the Select enzyme can be quickly neutralized; when cells begin to detach from the serum-free medium, gently tap the cell culture dish to promote cell detachment, then aspirate the TrypLE TM Select enzyme solution; then add 4 mL of preheated PBS to the cell culture dish to terminate the digestion reaction process; use a pipette to gently blow the cells in the cell culture dish to completely detach them from the culture medium and form a cell suspension.

[0089] S3: Transfer the digested cell suspension into a 15 mL conical sterile centrifuge tube and centrifuge at 1000 rpm for 3 min.

[0090] S4: Discard the supernatant and use 1 mL CytoNiche (HuaKan Biotechnology) 3D Resuspend the cells in serum-free medium for mesenchymal stem cells and count them;

[0091] S5: Coating of cell culture dishes

[0092] Add 1 mL of 0.1% sterile gelatin solution to the 6-well plate, incubate at 37°C for 30 min, aspirate the coating solution and dry it for later use.

[0093] S6: 20,000 pieces / cm 2 The cells were seeded into a coated 6-well plate at a density of 1.5 μg / mL, 2 mL of umbilical cord mesenchymal stem cell serum-free medium was added to each well, and the plates were cultured in a 37°C, 5% CO2 incubator.

[0094] S7: When the cell confluence reaches 65%, discard the CytoNiche 3D Serum-free medium for mesenchymal stem cells: 2 mL of Saiye Biotech human umbilical cord osteogenic induction complete medium was added to each well and cultured in a 37°C, 5% CO2 incubator.

[0095] It should be noted that when using CytoNiche 3D Before replacing the serum-free medium of mesenchymal stem cells with the complete medium of human umbilical cord osteogenic induction of Saiye Bio, the complete medium of human umbilical cord osteogenic induction of Saiye Bio must be thawed. Take the complete medium of human umbilical cord osteogenic induction of Saiye Bio into a 15mL cryopreservation tube and incubate it in a 37℃ water bath for 5min; observe the cell status under a light microscope, and the cell status is good.

[0096] S8: From the beginning of induction to the sixth day, each well was supplemented with 500 μL of Saiye Biotech human umbilical cord osteogenic induction complete medium and cultured in a 37°C, 5% CO2 incubator.

[0097] S9: From the beginning of induction to the 13th day, each well was supplemented with 500 μL of Saiye Biotech human umbilical cord osteogenic induction complete medium and cultured in a 37°C, 5% CO2 incubator.

[0098] S10: From the start of induction to the 19th day, the calcium deposition in the culture medium and on the cell surface and the formation of bone nodules were observed under a light microscope. It was found that almost no calcium deposition points were seen on the cell surface at this time, and the number of bone nodules in the culture medium was only 36 (less than 50). Then 500 μL of culture medium was aspirated from the upper layer of the culture medium, and 500 μL / hole of Seychem Biotechnology human umbilical cord osteogenic induction complete culture medium was added and continued observation was continued. From the start of induction to the 28th day, about 80 (>50) calcium deposition points were observed on the cell surface, and the induction culture was stopped.

[0099] S11: After the induction culture was completed, the culture supernatant was discarded, the cells were washed twice with phosphate buffered saline (PBS), 1 mL of 4% neutral paraformaldehyde solution was added to each well, and the cells were fixed at room temperature for 20 min;

[0100] S12: discard the fixative, wash the cells twice with phosphate buffered saline (PBS), add 1 mL of 0.2% (w / v) alizarin red dye to each well, and stain for 15 min at room temperature;

[0101] S13: discard the alizarin red dye, wash the cells three times with phosphate buffered saline (PBS), and add 1 mL of fresh PBS;

[0102] Observe the bone nodules and calcium deposits on the cell surface under a microscope. Black granular substances can be seen on the cell surface, which are bone nodules and calcium deposits. Take pictures. Figure 1As indicated by the arrow;

[0103] Aspirate PBS, add 1 mL of glacial acetic acid-ethanol solution, wherein the mass ratio of 10% glacial acetic acid to anhydrous ethanol in the glacial acetic acid-ethanol solution is 8:2, place on a horizontal shaker at a speed of 60-90 rpm / min, and incubate at room temperature for 25-45 min to fully dissolve Alizarin Red; take 200 μL of the dissolved solution into a 96-well plate, measure the absorbance at 490 nm on an ELISA reader, and perform quantitative analysis of bone nodules and calcium deposition on the cell surface. The measurement shows that the absorbance at 490 nm is 0.82.

[0104] Comparative Example 1

[0105] In this comparative example, the human umbilical cord osteogenic kit (Cat. No.: HUXUC-90021) of CytoNiche was used to induce osteogenic growth of umbilical cord mesenchymal stem cells derived from the GMP system (referred to as the Control group). The difference from Example 1 is the treatment method of human umbilical cord osteogenic induction complete medium in steps S8-S10. The remaining steps are the same as Example 1. In this comparative example, CytoNiche 3D The serum-free medium of mesenchymal stem cells was replaced with the complete medium of human umbilical cord osteogenic induction of Saiye Bio. After the induction culture of human umbilical cord mesenchymal stem cells began, the complete medium of human umbilical cord osteogenic induction of Saiye Bio was replaced every 3 days in each well, and the number of calcium deposition points on the cell surface was observed by light microscopy at the same time. On the 21st day of induction, sporadic calcium deposition points were observed on the cell surface. After that, half of the medium was replaced in each well every 3 days, that is, 1 mL of medium was aspirated from the upper layer of the medium in each well every 3 days, and 1 mL of fresh complete medium of human umbilical cord osteogenic induction of Saiye Bio was added. Alizarin red staining and quantitative analysis were performed on the 28th day of induction. The bone nodules and calcium deposition on the cell surface were observed under a microscope. Black granular substances on the cell surface were bone nodules and calcium deposition, and photos were taken, such as Figure 2 As indicated by the arrow;

[0106] The absorbance at 490 nm was measured on an ELISA instrument, and the bone nodules and calcium deposition on the cell surface were quantitatively analyzed. The absorbance at 490 nm was found to be 0.078.

[0107] Example 2

[0108] In this example, human umbilical cord osteogenic kit (Cat. No.: FY200006) of Fuyuan Company was used to induce osteogenic growth of umbilical cord mesenchymal stem cells derived from the GMP system (referred to as the Modified method group), including the following steps:

[0109] S1: Resuscitation cells

[0110] Take out the umbilical cord mesenchymal stem cells to be tested from the liquid nitrogen tank, quickly thaw the cells in a 37°C water bath, wipe the surface of the cryopreservation tube with alcohol, and move it into a biosafety cabinet. Quickly transfer the cell suspension to 4 mL of mesenchymal stem cell serum-free medium and centrifuge at 1000 rpm for 3 minutes; discard the supernatant and transfer the mesenchymal stem cells to a 10 cm cell culture dish, add 10 mL of Yokom Bio's GMP-grade MSC serum-free medium, and culture for 3 days;

[0111] S2: When cells reach 95% confluency, add 1 mL 1X TrypLE TM Select enzyme digested the cells for 2 min, and the digestion reaction was terminated with phosphate buffered saline (PBS);

[0112] Specifically, before terminating the digestion reaction with phosphate buffered saline (PBS), PBS was preheated to 37°C to ensure that TrypLE TM The activity of the Select enzyme can be quickly neutralized; when cells begin to detach from the serum-free medium, gently tap the cell culture dish to promote cell detachment, then aspirate the TrypLE TM Select enzyme solution; then add 4 mL of preheated PBS to the cell culture dish to terminate the digestion reaction process; use a pipette to gently blow the cells in the cell culture dish to completely detach them from the culture medium and form a cell suspension.

[0113] S3: Transfer the digested cell suspension into a 15 mL conical sterile centrifuge tube and centrifuge at 1000 rpm for 3 min.

[0114] S4: Discard the supernatant, resuspend the cells in 1 mL of Yokom Bio GMP-grade MSC serum-free medium and count them;

[0115] S5: Coating of cell culture dishes

[0116] Add 1 mL of 0.1% sterile gelatin solution to the 6-well plate, incubate at 37°C for 30 min, aspirate the coating solution and dry it for later use.

[0117] S6: 20,000 pieces / cm 2 The cells were seeded into a coated 6-well plate at a density of 1.5 μg / mL, 2 mL of umbilical cord mesenchymal stem cell serum-free medium was added to each well, and the plates were cultured in a 37°C, 5% CO2 incubator.

[0118] S7: When the cell confluence reaches 70%, discard the GMP-grade MSC serum-free medium of Youkang Bio, add 2 mL of human umbilical cord osteogenic induction complete medium of Fuyuan Bio to each well, and culture in a 37°C, 5% CO2 incubator;

[0119] It should be noted that before replacing the GMP-grade MSC serum-free culture medium of Youkang Bio with the human umbilical cord osteogenic induction complete culture medium of Fuyuan Bio, the human umbilical cord osteogenic induction complete culture medium of Fuyuan Bio must be thawed. Take the human umbilical cord osteogenic induction complete culture medium of Saiye Bio into a 15mL cryopreservation tube and incubate it in a 37°C water bath for 5 minutes; observe the cell status under a light microscope and the cell status is good.

[0120] S8: From the beginning of induction to the sixth day, each well was supplemented with 500 μL of Saiye Biotech human umbilical cord osteogenic induction complete medium and cultured in a 37°C, 5% CO2 incubator.

[0121] S9: From the beginning of induction to the 13th day, each well was supplemented with 500 μL of Saiye Biotech human umbilical cord osteogenic induction complete medium and cultured in a 37°C, 5% CO2 incubator.

[0122] S10: From the start of induction to the 18th day, the calcium deposition in the culture medium and on the cell surface and the formation of bone nodules were observed under a light microscope. It was found that the number of calcium deposition points on the cell surface was about 70 (>50) at this time, and the induction culture was stopped.

[0123] S11: After the induction culture was completed, the culture supernatant was discarded, the cells were washed twice with phosphate buffered saline (PBS), 1 mL of 4% neutral paraformaldehyde solution was added to each well, and the cells were fixed at room temperature for 20 min;

[0124] S12: discard the fixative, wash the cells twice with phosphate buffered saline (PBS), add 1 mL of 0.2% (w / v) alizarin red dye to each well, and stain for 15 min at room temperature;

[0125] S13: discard the alizarin red dye, wash the cells three times with phosphate buffered saline (PBS), and add 1 mL of fresh PBS;

[0126] Observe the bone nodules and calcium deposits on the cell surface under a microscope. Black granular substances can be seen on the cell surface, which are bone nodules and calcium deposits. Take pictures. Figure 4 As indicated by the arrow;

[0127] Aspirate PBS, add 1 mL of glacial acetic acid-ethanol solution, wherein the mass ratio of 10% glacial acetic acid to anhydrous ethanol in the glacial acetic acid-ethanol solution is 8:2, place on a horizontal shaker at a speed of 60-90 rpm / min, and incubate at room temperature for 25-45 min to fully dissolve Alizarin Red; take 200 μL of the dissolved solution into a 96-well plate, measure the absorbance at 490 nm on an ELISA reader, and perform quantitative analysis of bone nodules and calcium deposition on the cell surface. The measurement shows that the absorbance at 490 nm is 0.98.

[0128] Comparative Example 2

[0129] In this comparative example, human umbilical cord osteogenic kit (Cat. No.: FY200006) of Fuyuan Company was used to induce osteogenic growth of umbilical cord mesenchymal stem cells derived from the GMP system (referred to as the Control group). The difference from Example 1 lies in the method for treating the complete human umbilical cord osteogenic induction medium in steps S8-S10, and the remaining steps are the same as Example 1. In this comparative example, after replacing the GMP-grade MSC serum-free medium of Youkang Bio with the complete human umbilical cord osteogenic induction medium of Fuyuan Bio, and inducing and culturing human umbilical cord mesenchymal stem cells, the complete human umbilical cord osteogenic induction medium of Fuyuan Bio was replaced in each well every 3 days, and the number of calcium deposition points on the cell surface was observed by light microscopy at the same time. Alizarin red staining and quantitative analysis were performed on the 18th day of induction, and bone nodules and calcium deposition on the cell surface were observed under a microscope. A very small amount of sporadic black granular substances, i.e., bone nodules and calcium deposition, were observed on the cell surface, and photos were taken. Figure 5 As indicated by the arrow;

[0130] The absorbance at 490 nm was measured on an ELISA instrument, and the bone nodules and calcium deposition on the cell surface were quantitatively analyzed. The absorbance at 490 nm was found to be 0.04.

[0131] By comparing Example 1 and Comparative Example 1, it can be seen that there are a small amount of bone nodules and calcium deposition on the cell surface of the Control group, and a large amount of bone nodules and calcium deposition on the cell surface of the Modified method group. The absorbance value of the solution at 490nm (scale is 100μm) was measured to indirectly quantify the calcium deposition. The absorbance value of the Control group at 490nm was 0.078, and the absorbance value of the Modifiedmethod group at 490nm was 0.82, which was 10.5 times higher than that of the Control group. In Example 1, in which the method of the present invention was used to induce osteogenic induction of human umbilical cord mesenchymal stem cells, the osteogenic induction effect was better. Figure 3 This is a graph showing the absorbance values ​​measured at 490 nm after dissolving Alizarin Red in glacial acetic acid-ethanol solution in Example 1 and Comparative Example 1.

[0132] Comparison of Example 2 and Comparative Example 2 shows that there are a small amount of bone nodules and calcium deposits on the cell surface of the Control group, and a large amount of bone nodules and calcium deposits on the cell surface of the Modified method group. The absorbance value of the solution at 490nm (scale is 100μm) was measured to indirectly quantify the calcium deposition. The absorbance value of the Control group at 490nm was 0.04, and the absorbance value of the Modifiedmethod group at 490nm was 0.98, which was 24.5 times higher than that of the Control group; Example 1 in which the method of the present invention was used to induce osteogenic induction of human umbilical cord mesenchymal stem cells, the effect of osteogenic induction was better. Figure 6This is a graph showing the absorbance values ​​measured at 490 nm after dissolving Alizarin Red in glacial acetic acid-ethanol solution in Example 2 and Comparative Example 2.

[0133] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for improving osteogenic differentiation of umbilical cord mesenchymal stem cells under a GMP system, characterized in that: The following steps are involved: S1: resuscitated cells; S2: When cells reach the first confluence, use TrypLE TM Select enzyme to digest the cells, and then terminate the digestion reaction with phosphate buffer; S3: Transfer the digested cell suspension into a conical sterile centrifuge tube and centrifuge; S4: discard the supernatant, resuspend the cells in umbilical cord mesenchymal stem cell serum-free medium and count them; S5: Coating the cell culture dish; S6: Inoculate the cells into the coated multi-well plate, add umbilical cord mesenchymal stem cell serum-free culture medium to each well, and place the plate in an incubator to culture the cells; S7: When the cells reach the second degree of confluence, the serum-free medium of umbilical cord mesenchymal stem cells is discarded, and MSC osteogenic induction complete medium is added to each well, and the cells are continued to be cultured in the incubator; S8: During the first induction period, MSC osteogenic induction complete medium was added and the cells were cultured in the incubator continuously; S9: The second induction period, add MSC osteogenic induction complete medium, and continue to culture the cells in the incubator; S10: The third induction period, observing the calcium deposition in the culture medium and on the cell surface and the formation of bone nodules, and judging whether to stop the induction culture according to the evaluation rules; S11: After the induction culture was completed, the culture supernatant was discarded, the cells were washed twice with phosphate buffer, and a neutral paraformaldehyde solution was added to each well to fix the cells at room temperature; S12: discard the fixative, wash the cells twice with phosphate buffer, and add alizarin red dye to each well at room temperature to stain the cells; S13: discard the alizarin red dye solution, wash the cells three times with phosphate buffer, add fresh PBS, observe the bone nodules and calcium deposition on the cell surface under a microscope, and perform quantitative analysis on the bone nodules and calcium deposition on the cell surface.

2. The method according to claim 1, characterized in that In step S2, the first cell confluence degree is 90-95%.

3. The method according to claim 1, characterized in that In step S7, the second cell confluence degree is 60-70%.

4. The method according to claim 1, characterized in that In step S8, the first induction period is 5-7 days after the start of induction.

5. The method according to claim 4, characterized in that In step S8, the amount of the added MSC osteogenic induction complete culture medium is 300-600 μL / well.

6. The method according to claim 5, characterized in that In step S9, the second induction period is the 10th to 14th day from the start of induction.

7. The method according to claim 6, characterized in that In step S9, the amount of the added MSC osteogenic induction complete culture medium is 300-600 μL / well.

8. The method according to claim 1, characterized in that In step S10, the third induction period is 15-21 days from the start of induction.

9. The method according to claim 8, characterized in that In step S10, the evaluation rules include: If there are <5 calcium deposition spots on the cell surface and >50 bone nodules in the culture medium, 500-1000 μL of culture medium is aspirated from the upper layer of the culture medium; If there are less than 5 calcium deposition points on the cell surface and less than 50 bone nodules in the culture medium, remove 500-1000 μL of culture medium from the upper layer of the culture medium, and replenish each well with MSC osteogenic induction complete culture medium and continue to observe for 5-10 days; When the number of calcium deposition points on the cell surface is greater than 50, the cell induction culture is stopped.

10. The method according to claim 9, characterized in that The amount of the supplemented MSC osteogenic induction complete medium is 300-600 μL / well.

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