Cell culture methods

Abstract

This invention provides a cell culture method that can effectively restore the HGF production capacity of human-derived adipose-derived stem cells. [Solution] An example of a cell culture method to which the present invention is applied mainly comprises the following steps: (1) Initial culture step (2) Subculture step (3) Expanded culture step (4) Cell harvesting step In the cell culture method according to an embodiment of the present invention, human adipose-derived stem cells are cultured in a commonly used adipose-derived stem cell medium in (1) the initial culture step and (2) the subculture step, and then in (3) the expansion culture step, they are cultured in an expansion culture medium having the characteristics of the present invention. The expansion culture medium is based on the culture medium composition of a serum-free medium for human stem cells, but does not contain L-glutamine and has a culture medium composition that includes a reducing agent, L-alanyl-L-glutamine, D(+)-glucose, and serum components or human platelet-derived cell culture additives. The reducing agent is ascorbic acid 2-glucoside.

Description

Technical Field

[0001] The present invention relates to a cell culture method. More specifically, it relates to a cell culture method capable of favorably restoring the ability of human-derived adipose-derived stem cells to produce HGF.

Background Art

[0002] Hepatocyte growth factor (HGF) is the most potent growth factor for hepatocyte regeneration. It is also produced in many organs other than the liver, such as the kidney, spleen, and lung, and is known as a cell growth factor having an organ regeneration promoting effect.

[0003] In addition, hepatocyte growth factor activates signal transduction pathways such as the PI3K-Akt pathway that promotes cell proliferation, survival, growth, metabolism, angiogenesis, etc., and the MAPK pathway that regulates important cell functions such as cell proliferation, differentiation, survival, apoptosis (cell death), etc. It indirectly suppresses the activity of p53, which is one of the aging markers, and as a result, has a function of preventing apoptosis.

[0004] Regarding this hepatocyte growth factor, it has been reported that the expression of hepatocyte growth factor in human adipose-derived stem cells (ADSCs) decreases with aging. That is, it is said that a phenomenon occurs in which the ability to produce hepatocyte growth factor (hereinafter referred to as "HGF production ability") decreases with aging.

[0005] Although there are individual differences in the expression level of hepatocyte growth factor in human adipose-derived stem cells, for example, when comparing the young age group in their 20s to 30s and the elderly age group over 70 years old, it has been confirmed that in the elderly age group, the expression level of hepatocyte growth factor in adipose-derived stem cells significantly decreases.

[0006] In addition, it has also been reported that the HGF production ability in adipose-derived stem cells decreases in diabetic patients regardless of age.

[0007] This phenomenon of decreased HGF production capacity in cells not only leads to a decline in important functions performed by hepatocyte growth factors, including cell cycle acceleration, but also induces apoptosis and accelerates human aging. Therefore, it is desirable to take appropriate measures and countermeasures.

[0008] In this context, various approaches are being investigated to restore the HGF-producing capacity of cells with reduced HGF production through regenerative medicine. For example, techniques are being attempted to increase the expression of hepatocyte growth factor in stem cells by introducing the HGF gene (see, for example, Non-Patent Document 1).

[0009] The technology described in Non-Patent Document 1 involves introducing the HGF gene into dental pulp-derived stem cells, transplanting the gene-transformed cells into rats, and investigating the therapeutic effect on ulcerative colitis in rats. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Ning Li et al., "Dental pulp stem cells overexpressing hepatocyte growth factor facilitate the repair of DSS-induced ulcerative colitis", Stem Cell Res Ther, (US), 2021 Jan 7;12(1):30. doi: 10.1186 / s13287-020-02098-4. [Overview of the project] [Problems that the invention aims to solve]

[0011] However, technologies that utilize cells into which genes have been introduced, such as the technology described in Non-Patent Document 1, are classified as Type 1 regenerative medicine, which carries the highest risk among the types of regenerative medicine.

[0012] More specifically, technologies that utilize cells into which genes have been introduced carry risks such as tumor risk due to genomic mutations caused by the introduced genes, or cell damage during gene introduction. These risks pose a major concern for clinical application in humans.

[0013] Therefore, from the perspective of ensuring safety, the use of genetically modified cells is a technology that is not easily adopted.

[0014] Therefore, the inventors focused on a technology using the subject's own somatic stem cells as a safer method that is easier to adopt for human treatment, and diligently investigated a cell culture method to improve HGF production capacity using human-derived adipose-derived stem cells.

[0015] This invention was conceived in view of the above points, and aims to provide a cell culture method that can effectively restore the HGF production ability in human-derived adipose-derived stem cells. [Means for solving the problem]

[0016] To achieve the above objectives, the present invention provides a cell culture method for restoring the HGF production ability of human-derived adipose-derived stem cells, comprising a serum-free medium-based culture medium containing a reducing agent and serum components or human platelet-derived cell culture additives, and comprising an expansion culture step in which adipose-derived stem cells are cultured in an expansion culture medium that suppresses the amount of ammonia produced by cells.

[0017] In this process, culturing adipose-derived stem cells in a culture medium containing a reducing agent during the expansion culture stage can improve the HGF production capacity of the adipose-derived stem cells. Furthermore, it becomes possible to restore the HGF production capacity of adipose-derived stem cells whose HGF production capacity has decreased.

[0018] Furthermore, in the expansion culture process, culturing stem cells in an expansion culture medium containing serum components or human platelet-derived cell culture additives can promote the proliferation of adipose-derived stem cells and enhance the buffering capacity of the culture medium through the nutrients and bioactive substances necessary for cell proliferation and maintenance contained therein.

[0019] Furthermore, by culturing adipose-derived stem cells in a culture medium that suppresses ammonia production by cells during the expansion culture process, the cytotoxic effects of ammonia and the stress on cellular metabolism caused by a rapid increase in the pH of the culture medium (alkalization) can be reduced, thereby promoting the proliferation of adipose-derived stem cells.

[0020] Furthermore, using human-derived adipose-derived stem cells is advantageous because, among the various tissues that can serve as raw materials for stem cells, they are relatively easy to collect and can be harvested with minimal invasiveness. This makes it easier to prepare the stem cells for culture and reduces the burden on the patient. In addition, adipose-derived stem cells proliferate easily when cultured, making them suitable for use in multiple treatments.

[0021] Furthermore, when the reducing agent is ascorbic acid 2-glucoside, ascorbic acid 2-glucoside can improve the HGF production capacity of adipose-derived stem cells.

[0022] Furthermore, when the reducing agent is included in the expanded culture medium at a final concentration of 1 mM to 100 mM, it is possible to sufficiently improve the HGF production capacity of adipose-derived stem cells.

[0023] On the other hand, if the final concentration of the reducing agent in the expanded culture medium is less than 1 mM, the degree of improvement in HGF production capacity when adipose-derived stem cells are cultured in the expanded culture medium containing the reducing agent may be insufficient. Also, if the final concentration of the reducing agent in the expanded culture medium exceeds 100 mM, the effect of improving HGF production capacity when adipose-derived stem cells are cultured will plateau.

[0024] In addition, when the expansion culture medium contains L-alanyl-L-glutamine and D(+)-glucose but does not contain L-glutamine, during the expansion culture process, when culturing adipose-derived stem cells, the amount of ammonia production can be sufficiently suppressed, and the adipose-derived stem cells can be proliferated well. That is, by including D(+)-glucose in the expansion culture medium, the medium becomes a high-glucose condition, and the amount of ammonia produced by adipose-derived stem cells through culturing can be reduced. Also, since the expansion culture medium does not contain L-glutamine, it is possible to suppress L-glutamine from being decomposed into ammonia in the medium. Furthermore, by including L-alanyl-L-glutamine in the expansion culture medium, the L-glutamine molecule, which is the main nutrient source of cells in the medium, can be supplemented as L-alanyl-L-glutamine, and the adipose-derived stem cells can be proliferated well.

[0025] In addition, the expansion culture process is a culture process that is carried out after primary culture for culturing adipose tissue and at least one subculture carried out after the primary culture. The primary culture and the subculture are carried out in an adipose-derived stem cell medium. When the amount of HGF production by adipose-derived stem cells after the expansion culture process is at least 1.20 times or more the amount of HGF production by adipose-derived stem cells after comparative culture in which adipose-derived stem cells are cultured in the adipose-derived stem cell medium after the primary culture and the subculture, it is possible to sufficiently improve the HGF production ability of adipose-derived stem cells by the expansion culture process. Here, the adipose-derived stem cell medium referred to means an existing adipose-derived stem cell medium (MSC), an adipose-derived stem cell medium to which a reducing agent has not been added, or an adipose-derived stem cell medium containing a very small amount of a reducing agent (for example, containing 50 to 250 μM of ascorbic acid in the medium).

[0026] In addition, when the concentration of ammonia contained in the culture supernatant after the expansion culture process is 2.0 mM or less, the cytotoxic effect of ammonia and the stress on cell metabolism caused by the rapid increase (alkalization) of the pH of the medium can be further reduced, and the proliferation of adipose-derived stem cells can be sufficiently promoted.

[0027] Furthermore, if the adipose-derived stem cells are derived from humans aged 70 or older or from diabetic patients, the HGF-producing capacity of the stem cells obtained from subjects whose HGF-producing capacity has decreased due to aging or diabetes can be restored, and these cells can be used in regenerative medicine using the subject's own adipose-derived stem cells. [Effects of the Invention]

[0028] The cell culture method according to the present invention is capable of effectively restoring the HGF production ability in human-derived adipose-derived stem cells. [Brief explanation of the drawing]

[0029] [Figure 1] This graph shows the results of HGF production in Examples 1-8 and Comparative Examples 1-8. [Figure 2] This graph shows the results of HGF production in Examples 9-11 and Comparative Examples 9-11. [Figure 3] This graph shows the results for ammonia levels in Examples 1-8 and Comparative Examples 1-8. [Figure 4] This graph shows the results for ammonia levels in Examples 9-11 and Comparative Examples 9-11. [Modes for carrying out the invention]

[0030] The following describes a cell culture method that is an example of a cell culture method to which the present invention is applied.

[0031] The cell culture method described here mainly consists of the following steps. (1) Initial culture process (2) Subculture process (3) Expanded culture process (4) Cell harvesting process

[0032] In the cell culture method according to an embodiment of the present invention, human adipose-derived stem cells are cultured in a commonly used adipose-derived stem cell medium in (1) the initial culture step and (2) the subculture step, and then in (3) the expansion culture step, they are cultured in an expansion culture medium having the characteristics of the present invention.

[0033] Furthermore, the cell culture method according to the embodiment of the present invention is a cell culture method that can improve HGF production ability by culturing adipose-derived stem cells using an expanded culture medium having the characteristics of the present invention, instead of using a commonly used adipose-derived stem cell medium during the (3) expanded culture step.

[0034] The following describes the details of each step in the cell culture method, which is an embodiment of the present invention.

[0035] (1) Initial culture process (1) In the initial culture step, adipose tissue is collected from the subject and cultured in a general adipose-derived stem cell medium until the next step, (2) in the subculture step, the cells are transplanted into a new culture vessel.

[0036] (1) In the initial culture process, the collected adipose tissue is cultured for a certain period of time while changing the culture medium in the culture plate, for example, on a substrate on which the adipose tissue is placed, until a certain percentage of adipose-derived stem cells are observed migrating from the adipose tissue.

[0037] Furthermore, (2) the subculture process involves seeding the adipose-derived stem cells that were cultured and proliferated in (1) the initial culture process into adipose-derived stem cell culture medium in a new culture vessel and culturing them for a certain period of time.

[0038] In this (2) subculture process, adipose-derived stem cells are cultured under predetermined conditions until they reach 70-80% confluent. (2) The subculture process further proliferates the adipose-derived stem cells.

[0039] Here, known adipose-derived stem cell media can be used in (1) the initial culture step and (2) the subculture step. For example, a medium made by adding UltraGRO (Corefront Co., Ltd.) (human platelet-derived cell culture additive) to KBM ADSC-2 (manufactured by Kojin Bio Co., Ltd.) can be used. The amount of UltraGRO to add is 5% of the medium volume in (1) the initial culture step and 2% of the medium volume in (2) the subculture step. In addition, the known adipose-derived stem cell medium (KBM ADSC-2) used here does not contain the reducing agent (ascorbic acid 2-glucoside) that is included in the expansion culture medium described later.

[0040] In the cell culture method according to the embodiment of the present invention, the number of times the (2) subculture step is performed does not necessarily have to be once. For example, before performing the next step, (3) expansion culture step, the (2) subculture step may be performed two or more times to proliferate adipose-derived stem cells. In this case, cells are seeded into a new culture vessel and cultured each time a subculture is performed.

[0041] Furthermore, it is not necessary to use KBM ADSC-2 as the adipose-derived stem cell medium in (1) the initial culture step and (2) the subculturing step; other adipose-derived stem cell media can be used. For example, media such as BSCM-PL1 (Funakoshi Co., Ltd.) or BSCM-SP1 (Funakoshi Co., Ltd.) can be used with serum components or human platelet-derived cell culture additives added. Alternatively, media such as MesenPRO RS Medium (Thermo Fisher Scientific Co., Ltd.) containing 2% serum or ADSC Growth Medium BulletKit (Lonza Co., Ltd.) containing FBS (fetal bovine serum) can also be used.

[0042] Furthermore, (3) the expansion culture process involves seeding the adipose-derived stem cells that have been cultured and proliferated in (2) the subculture process into the expanded culture medium of a new culture vessel and culturing them for a certain period of time.

[0043] In this (3) expansion culture step, the expression of hepatocyte growth factor in the cells can be improved by culturing adipose-derived stem cells in an expansion culture medium containing characteristic components described later. In this step, the cells are cultured using the expansion culture medium under predetermined conditions until they reach 70-80% confluent.

[0044] Furthermore, (3) the expanded culture process makes it possible to restore the HGF-producing ability of adipose-derived stem cells from subjects whose HGF-producing ability has decreased due to aging or diabetes, and to use these cells in regenerative medicine for the subjects.

[0045] (3) An example of a culture medium used for cultivation in the expansion culture process is a culture medium with the following contents:

[0046] Here, the expanded culture medium is based on the culture medium composition of a serum-free medium for human stem cells, but does not contain L-glutamine. It has a culture medium composition that includes a reducing agent, L-alanyl-L-glutamine, D(+)-glucose, and serum components or human platelet-derived cell culture additives. The reducing agent is ascorbic acid 2-glucoside. For example, autologous serum can be used as the serum component, and UltraGRO can be used as the human platelet-derived cell culture additive.

[0047] Furthermore, the base serum-free medium is based on the composition of, for example, Ex-MSC XF Medium (manufactured by Myoridge Co., Ltd., product code: ME-09H00152), a medium for stem cells, but without L-glutamine. Alternatively, it can be based on the composition of Ex-MSC XF Medium but containing L-alanyl-L-glutamine and D(+)-glucose. This is referred to as a modified Ex-MSC XF Medium medium.

[0048] Then, the expanded culture medium is created by adding ascorbic acid 2-glucoside and UltraGRO to the modified Ex-MSC XF Medium.

[0049] Furthermore, the modified Ex-MSC XF Medium contains L-alanyl-L-glutamine at a concentration of 469.3 mg / L and D(+)-glucose at a concentration of 3680.1 mg / L.

[0050] Furthermore, the expanded culture medium contains ascorbic acid 2-glucoside at a final concentration of 20 mM.

[0051] Furthermore, ascorbic acid 2-glucoside, used as a reducing agent, is dissolved in powder form in the solvent IMDM medium (Iscove's Modified Dulbecco's Medium), and 5N NaOH is added to adjust the pH to 7-8 before adding it to the modified Ex-MSC XF Medium. This ensures that the pH of the medium does not become acidic when ascorbic acid 2-glucoside is added to the modified Ex-MSC XF Medium.

[0052] Furthermore, the expanded culture medium contains UltraGRO as a human platelet-derived cell culture additive at a concentration of 2%.

[0053] Here, ascorbic acid 2-glucoside, a reducing agent, is a characteristic component that enhances the expression of hepatocyte growth factor in adipose-derived stem cells.

[0054] In this embodiment, the expanded culture medium contains ascorbic acid 2-glucoside at a final concentration of 20 mM, but the concentration of ascorbic acid 2-glucoside is not limited to this. Furthermore, the final concentration of ascorbic acid 2-glucoside in the medium is preferably in the range of 1 mM to 100 mM, from the standpoint of improving the expression of hepatocyte growth factor in adipose-derived stem cells.

[0055] On the other hand, if the final concentration of ascorbic acid 2-glucoside in the expanded culture medium is less than 1 mM, the improvement in hepatocyte growth factor expression in adipose-derived stem cells when cultured in expanded culture medium containing a reducing agent may be insufficient. Furthermore, if the final concentration of ascorbic acid 2-glucoside in the expanded culture medium exceeds 100 mM, the effect of improving hepatocyte growth factor expression in cultured adipose-derived stem cells will plateau.

[0056] Furthermore, L-alanyl-L-glutamine is a characteristic component that complements L-glutamine molecules, which are the main nutrient source for cells in the culture medium.

[0057] In the modified Ex-MSC XF Medium, the composition does not include L-glutamine to reduce the amount of ammonia produced by cells, as ammonia negatively affects the healthy proliferation of adipose-derived stem cells. On the other hand, since L-glutamine molecules are the main nutrient source for cells, L-alanyl-L-glutamine is included in the culture medium as the main nutrient source instead of L-glutamine in the expanded culture medium.

[0058] If L-glutamine is present in the culture medium, it will be broken down during the culture of adipose-derived stem cells, producing ammonia. This increases the ammonia concentration in the medium, and the cytotoxic effects of ammonia, along with the stress on cellular metabolism caused by the rapid increase in the medium's pH (alkalization), will inhibit the healthy proliferation of adipose-derived stem cells.

[0059] Therefore, by including L-alanyl-L-glutamine as a characteristic component in the culture medium instead of L-glutamine, L-alanyl-L-glutamine can be used as the main nutrient source in the medium, and the amount of ammonia produced in the medium can be kept low.

[0060] Furthermore, D(+)-glucose is a characteristic component that creates high-glucose conditions in the modified Ex-MSC XF Medium culture medium. By creating high-glucose conditions in the medium, the amount of ammonia produced by adipose-derived stem cells during culture can be reduced.

[0061] Here, the concentrations of L-alanyl-L-glutamine and D(+)-glucose contained in the modified Ex-MSC XF Medium are not limited to 469.3 mg / L for L-alanyl-L-glutamine and 3680.1 mg / L for D(+)-glucose, but can be set as appropriate.

[0062] Furthermore, UltraGRO is a human platelet-derived cell culture additive that promotes the proliferation of adipose-derived stem cells and enhances the buffering capacity of the culture medium by providing nutrients and bioactive substances necessary for cell proliferation and maintenance.

[0063] In this embodiment, UltraGRO is included in the expanded culture medium at a concentration of 2% or 5%, but the concentration of UltraGRO is not limited to these values ​​and can be set as appropriate.

[0064] Furthermore, UltraGRO is not necessarily required; autologous serum collected from the subject can be used as the serum component instead.

[0065] Furthermore, the type of serum-free medium for human stem cells that serves as the base for the expanded culture medium is not necessarily limited to Ex-MSC XF Medium; known serum-free medium compositions can be used as a base. For example, it is possible to create expanded culture media by modifying the composition of serum-free mediums for stem cells such as MSC NutriStem® XF Medium (manufactured by Sartorius Stedim Japan Co., Ltd.), R:STEM Medium for hMSC High Growth (Ref. No. EM1-500, manufactured by Rohto Pharmaceutical Co., Ltd.), Tele Stem Medium HG-1 (Serum-Free / Xeno-Free) (manufactured by Telebio Co., Ltd.), and ADSC-4 (manufactured by Kojin Bio Co., Ltd.).

[0066] In this (3) expansion culture step, culturing adipose-derived stem cells using an expansion culture medium can improve the expression of hepatocyte growth factor in the cells.

[0067] As an effect of this embodiment, for example, in elderly individuals aged 70 or older or diabetic patients whose HGF production capacity of adipose-derived stem cells has decreased, the amount of HGF produced by cells after the (3) expansion culture step is 1.20 times or more the amount of HGF produced by cells cultured using a general adipose-derived stem cell medium (KBM ADSC-2) with 2% UltraGRO added instead of the expansion culture medium at the same timing as the (3) expansion culture step, as a comparison.

[0068] In other words, it demonstrates the effect of fully restoring the HGF production capacity of adipose-derived stem cells, which has decreased in aging or diabetic patients.

[0069] Furthermore, as an effect of this embodiment, the concentration of ammonia in the culture supernatant after the (3) expansion culture step can be reduced to 2.0 mM or less. If the ammonia concentration is 2.0 mM or less, the effect of inhibiting the proliferation of adipose-derived stem cells during culture can be suppressed.

[0070] Furthermore, in (4) the cell retrieval process, after culturing the adipose-derived stem cells in (3) the expansion culture process until they reach 70-80% confluent, the adipose-derived stem cells are retrieved from the culture medium and suspended in physiological saline.

[0071] In this embodiment, by providing the collected cells to the subject as live suspension cells without freezing them within a certain period of time (for example, within 30 hours), it is possible to use adipose-derived stem cells with high HGF production capacity for treatment of the subject while maintaining a high cell viability rate of 93-98%.

[0072] Furthermore, when providing the cells to subjects, the live suspension cells can be administered intravenously, making it a less labor-intensive and safer method for use in treatment.

[0073] Here, (3) in the expansion culture process, by harvesting adipose-derived stem cells at 70-80% confluent, a high cell viability can be maintained.

[0074] For example, when treating a target individual, 100 million to 200 million adipose-derived stem cells cultured in (3) the expanded culture process can be used for treatment.

[0075] Furthermore, a major advantage of the cell culture method of this embodiment is that, in the (3) expansion culture step, the doubling time when culturing adipose-derived stem cells in the expansion culture medium is almost the same as the doubling time when culturing using a general adipose-derived stem cell medium (KBM ADSC-2, etc.). This makes it possible to manage and carry out the culture in the (3) expansion culture step with the same culture schedule as the (1) initial culture step and the (2) subculture step.

[0076] As described above, the cell culture method to which the present invention is applied is a method that can effectively restore the HGF production ability in human-derived adipose-derived stem cells. [Examples]

[0077] The following describes embodiments of the present invention.

[0078] Human adipose-derived stem cells were cultured using the following procedure, and the amount of HGF produced after culturing was confirmed.

[0079] (1.Culture method) [Primary culture] 1. Adipose tissue was subdivided with scissors and placed on a substrate (manufactured by Bio Mirai Kobo). This substrate was then placed in a 6-well plate, and 5% UltraGRO (manufactured by Corefront Co., Ltd.) was added to KBM ADSC-2 (manufactured by Kojin Bio Co., Ltd.) (5 ml / well). The mixture was then incubated at 37°C with 25% CO2 for 7 days. 2. After the start of culture, the cultures were observed under a microscope on the 4th and 7th days, and on the 7th day, the culture medium was changed using the same medium as described above. 3. Four to five days after the culture medium change described in item 2 above, the culture medium was changed again using the same culture medium as described above. 4. Four to eight days after the culture medium change described in step 3 above, when it is confirmed that more than 20% of the adipose-derived stem cells have migrated from the adipose tissue into the substrate, the next subculturing process is carried out.

[0080] [Subculture] 1. After primary culture, cells were detached from the substrate using TrypLE Select (Themo Fisher), the reaction was stopped with IMDM, and only the cells were collected by centrifugation. The collected cells were suspended again in IMDM and the number of cells was counted. Then, centrifugation (1600 rpm, 5 minutes) was performed, and the supernatant was discarded. 2. Cells were seeded in a T225 flask containing KBM ADSC-2 (50 ml) + UltraGRO 2% to a concentration of 800,000 to 1,000,000 cells, and cultured in an incubator at 37°C and 25% CO2 for 3 to 7 days until the cells became 70-80% confluent.

[0081] [Expanded Culture] 1. After subculturing, cells were detached from the flask using TrypLE Select, the reaction was stopped with IMDM, and only the cells were centrifuged (1600 rpm, 5 minutes). The supernatant was discarded and the cells were collected. The collected cells were suspended in IMDM and the number of cells was counted. 2. Cells were seeded to a density of 800,000 to 1,000,000 in a T225 flask containing the above-mentioned expanded culture medium (modified Ex-MSC XF Medium + 20 mM ascorbic acid 2-glucoside + 2% UltraGRO), and cultured in an incubator at 37°C and 25% CO2 for 3 to 7 days until the cells reached 70-80% confluent. At the same time as the expansion culture, instead of the expansion culture medium described above, the cells were cultured in KBM ADSC-2 (50 ml) + UltraGRO 2% medium, and these were used as the samples for Comparative Examples 1 to 11 described later.

[0082] [collect] 1. The supernatant was sampled from the flask after expansion culture and immediately frozen at -80°C to be used as a sample for HGF and ammonia measurement. 2. After the expansion culture, cells were detached from the flask using TrypLE Select, the reaction was stopped with IMDM, and only the cells were centrifuged (1600 rpm, 5 minutes). The supernatant was discarded and the cells were collected. The collected cells were suspended in IMDM and the number of cells was counted.

[0083] (2. Measurement of HGF production) The collected samples were used to measure HGF production using the AuthentiKine Human HGF Measurement ELISA Kit (manufactured by Cosmo Bio Co., Ltd.). (Sandwich method immunoassay) 1. Dilute the sample fourfold with the standard and the diluted sample solution, add 100 μl to each well of the microplate in the kit, and let stand at 37°C for 120 minutes. 2. Discard the liquid by aspirating or decanting. Wash four times with washing buffer. 3. Add 100 μl of diluted detection antibody to each well and let stand at 37°C for 60 minutes. 4. Discard the liquid by aspirating or decanting. Wash four times with washing buffer. 5. Add 100 μl of HRP-labeled reagent to each well and let stand at 37°C for 40 minutes. 6. Discard the liquid by aspirating or decanting. Wash four times with washing buffer. 7. Add 100 μl of TMB substrate to each well and let stand at 37°C for 15-20 minutes. 8. Add 100 μl of the reaction stop solution to the well. 9. Microplate reader (Tristar3, manufactured by Berthol Japan Co., Ltd.) was used for measurement at 450 nm, with a correction reference wavelength of 630 nm. 10. Since the measured values ​​were obtained in ng / ml, the amount of HGF produced (pg / cell) was calculated from the sample volume and the number of cells counted after collection.

[0084] (3. Measurement of ammonia) The collected samples were analyzed using LabAssay Ammonia (Modified Fujii-Okuda Method) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to measure the ammonia production in the culture supernatant. In this measurement, the sample is deproteinized by adding a deproteinizing reagent to remove color-inhibiting components and simultaneously inactivating various enzymes in the sample. Phenol and sodium pentacyanonitrosylferrate(III) are added to this supernatant, and after making it alkaline, it is oxidized with sodium hypochlorite to produce indophenol, which exhibits a blue color. The ammonia nitrogen concentration in the sample can be determined by measuring the absorbance of this blue color. The sample will be stored frozen until measurement, and measurement will begin within one hour of thawing. 1. Dilute the deproteinized solution and supernatant (standard) in a ratio of 4:1 and mix. 2. Centrifuge at 5000 × g, 4°C, for 15 minutes. Perform all subsequent operations within the well. 3. Add 70 μl of supernatant, chromogenic reagent A, chromogenic reagent B, and chromogenic reagent C, mix well, and incubate at 37°C for 20 minutes. 4. Mix well and let stand at room temperature for 30 minutes. 5. Measure the absorbance of the sample and standard solution using a 630nm microplate reader. 6.1 The amount of ammonia contained in the liquid volume of the flask was calculated. Here, the amount of ammonia contained in the supernatant of the culture medium was calculated as a value (μg / ml) converted to the volume of the culture medium.

[0085] (4. Examples and Comparative Examples) In this example, adipose-derived stem cells were collected from four subjects aged 70 years or older (elderly) (E-1, E-2, E-3, E-4), four subjects in their 40s (Y-1, Y-2, Y-3, Y-4), and three subjects with diabetes (A, B, C), and each test was performed. Samples obtained by primary culture, subculture, and expansion culture of cells collected from each subject were designated as Examples 1 to 11. In addition, as described above, samples cultured in KBM ADSC-2 (50 ml) + UltraGRO 2% medium instead of expansion culture medium at the expansion culture stage were designated as Comparative Examples 1 to 11. The relationship between each subject and Examples 1 to 11 and Comparative Examples 1 to 11 is shown in Tables 1 to 3 below.

[0086] The following shows the results for HGF production and ammonia levels in the examples and comparative examples.

[0087] Table 1 shows the results for HGF production levels in four subjects aged 70 or older (elderly) (E-1, E-2, E-3, E-4). Table 2 shows the results for HGF production levels of four subjects in their 40s (Y-1, Y-2, Y-3, Y-4). Table 3 shows the results for HGF production levels in three diabetic patients (A, B, and C). Figure 1 is a graph of the results from Tables 1 and 2. Figure 2 is a graph of the results from Table 3. The vertical axis in Figures 1 and 2 represents the HGF production amount (pg / cell).

[0088] Table 4 shows the ammonia levels of four subjects (E-1, E-2, E-3, E-4) who were 70 years of age or older (elderly). Table 5 shows the ammonia levels for four subjects in their 40s (Y-1, Y-2, Y-3, Y-4). Table 6 shows the ammonia levels of three diabetic patients (A, B, and C). Figure 3 is a graph of the results from Tables 4 and 5. Figure 4 is a graph of the results from Table 6. The vertical axis in Figures 3 and 4 represents the ammonia content (μg / ml).

[0089] (Table 1) JPEG0007900861000002.jpg23109

[0090] (Table 2) JPEG0007900861000003.jpg22109

[0091] (Table 3) JPEG0007900861000004.jpg2194

[0092] (Table 4) JPEG0007900861000005.jpg21109

[0093] (Table 5) JPEG0007900861000006.jpg22112

[0094] (Table 6) JPEG0007900861000007.jpg2297

[0095] As shown in Tables 1 and 2 and Figure 1, in each subject aged 70 and over (elderly) and each subject in their 40s, HGF production in each example was improved compared to the HGF production of the corresponding comparative example. Furthermore, in each subject aged 70 and over (elderly), the rate of improvement in HGF production when comparing the comparative example to the example was 1.27 to 1.45 times, confirming a significant improvement in HGF production. When comparing each subject aged 70 and over (elderly) with each subject in their 40s, the rate of improvement in HGF production tended to be better in the elderly subjects aged 70 and over (elderly).

[0096] As shown in Table 3 and Figure 2, in each diabetic patient subject, HGF production in each example was improved compared to the HGF production in the corresponding comparative example. Furthermore, in each diabetic patient subject, the improvement in HGF production when comparing the comparative example to the example was 1.20 to 1.39 times, confirming a significant improvement in HGF production.

[0097] As shown in Tables 4 and 5 and Figure 3, in each subject aged 70 years or older (elderly) and each subject in their 40s, the amount of ammonia produced in each example was reduced compared to the amount of ammonia produced in the corresponding comparative example, indicating that the amount of ammonia was suppressed.

[0098] The results shown in Table 6 and Figure 4 indicate that in each subject with diabetes, the amount of ammonia produced in each example was reduced compared to the amount of ammonia produced in the corresponding comparative example, demonstrating that ammonia levels were suppressed.

[0099] Furthermore, as mentioned above, it should be considered that ammonia concentrations exceeding 2.0 mM in the culture medium inhibit the proliferation of adipose-derived stem cells during culture. Here, an ammonia concentration of 2.0 mM in the culture medium corresponds to 36.08 μg / ml.

[0100] As shown in Tables 4 to 6, the amount of ammonia generated in Examples 1 to 11 was 36.08 μg / ml or less in all cases. That is, the ammonia concentration in the culture supernatant of Examples 1 to 11 was 2.0 mM or less, indicating that the amount of ammonia was suppressed in Examples 1 to 11, resulting in good proliferation of adipose-derived stem cells.

Claims

1. A cell culture method for restoring the HGF production ability of human-derived adipose-derived stem cells, The culture medium is based on a serum-free medium and contains a reducing agent and serum components or human platelet-derived cell culture additives, and has an expansion culture step in which adipose-derived stem cells are cultured in an expansion culture medium that suppresses the amount of ammonia produced by cells. The reducing agent is ascorbic acid 2-glucoside. The aforementioned expansion culture medium is characterized by containing L-alanyl-L-glutamine and D(+)-glucose, but not containing L-glutamine. The aforementioned expansion culture step is a culture step performed after primary culture in which adipose tissue is cultured, and at least one subculture performed after the primary culture. The reducing agent is included in the expanded culture medium at a final concentration of 20 mM. The primary culture and subsequent subcultures are performed in adipose-derived stem cell culture medium. Cell culture method.

2. The amount of HGF produced by adipose-derived stem cells after the expansion culture step is at least 1.20 times the amount of HGF produced by adipose-derived stem cells after a comparative culture in which adipose-derived stem cells are cultured in the adipose-derived stem cell medium after the primary culture and the subculture. The cell culture method according to claim 1.

3. The concentration of ammonia contained in the culture supernatant after the expansion culture step is 2.0 mM or less. The cell culture method according to claim 1 or claim 2.

4. The adipose-derived stem cells are characterized by being derived from a human being 70 years of age or older or from a patient with diabetes. The cell culture method according to claim 1 or claim 2.

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