Safe and efficient method for low-oxygen induction of adipogenic differentiation of cells and application

Abstract

The invention discloses a safe and efficient method for hypoxia induction of adipogenic differentiation of cells and application, and belongs to the technical field of cell culture. According to the method, cells are amplified to a certain number in a reactor in a suspension environment, and the cells are induced to be subjected to suspension differentiation into fat by reducing the dissolved oxygen level to low oxygen. According to the method, cell adipogenic differentiation is induced only through physical factor regulation, and compared with a traditional chemical inducer differentiation adipogenic method, an exogenous reagent is not introduced in the differentiation process, so that the food safety is guaranteed; by utilizing the method, adipogenic differentiation of cells can be completed within 4-6 days, amplification and adipogenic differentiation of the cells can be completed within about 10 days, and the differentiation time is shortened; and moreover, the fat can be differentiated in a large-scale reactor of 200 L and the like, and the method can be applied to large-scale preparation of cell culture meat.

Description

Technical Field

[0001] This application belongs to the technical field of cell culture, and specifically relates to a safe and efficient method for hypoxic induction of cell adipogenic differentiation and its application. Background Art

[0002] Cultured meat (also known as cell-cultured meat or cultivated meat) is a technology for producing edible meat by culturing animal cells in vitro. Compared with the production methods of traditional animal husbandry, the nutritional components of cultured meat can be adjusted according to needs to meet the health requirements of consumers, and it can also reduce environmental pollution, improve animal welfare, and reduce disease risks. The main components of traditional meat include skeletal muscle, fat, and connective tissue (mainly composed of collagen). During the production process of cultured meat, after the seed cells are amplified on a large scale, they can achieve functional differentiation through specific induction conditions. Among them, the differentiation of adipocytes plays a crucial role in the texture, flavor, and nutritional components of meat.

[0003] There have been many studies on methods for in vitro induction of cell adipogenic differentiation. Chemical induction is a common adipogenic induction method. Experimental studies have shown that by regulating the drug combination in the culture system, cells can be effectively induced to differentiate into the adipose lineage. Classic drugs include phosphodiesterase inhibitors (IBMX), dexamethasone, insulin, rosiglitazone, indomethacin, etc. These drugs activate transcription factors such as PPARγ to promote cell adipogenic differentiation. Although these chemical induction differentiation methods are effective, they involve food safety issues, and the time for adipogenic differentiation is usually more than 10 days, which is not conducive to industrial production. With the rapid development of the cultured meat industry, it has become particularly important to find a more natural, safe, and efficient adipogenic differentiation method.

[0004] In the current technical path, the seed cells of cultured meat can either use acellular plant scaffolds (such as sterilized tissues of onions and shiitake mushrooms) as biocompatible materials for adherent production of fat products, or generate suspended fat globules through self-organization technology. However, the above methods are all based on chemical inducers, and both the biocompatible materials and the self-organization technology have difficulties in large-scale production. Currently, there is no technology to optimize cell adipogenic differentiation only through physical factor regulation. Summary of the Invention

[0005] 1. Problems to be Solved In view of the conflict between the existing technology's reliance on chemical substances to induce adipogenic differentiation of cells and food safety supervision, this application provides a safe and efficient method for hypoxic induction of adipogenic differentiation of cells and its application. This method involves adjusting the dissolved oxygen concentration in the cell culture system. Specifically, after cell amplification, the dissolved oxygen in the culture system is reduced to induce adipogenic differentiation of cells, which can effectively improve the efficiency and maturity of cell adipogenic differentiation, overcome the limitations of traditional drug induction methods, and can be used for the preparation of cultured meat.

[0006] 2. Technical Solution To solve the above problems, the technical solutions adopted in this application are as follows: This application provides a safe and efficient method for hypoxic induction of adipogenic differentiation of cells, which includes the following steps: S1, culturing cells using a culture medium for cell amplification, and controlling the dissolved oxygen not to be lower than 35% during the culturing process; this stage is mainly the cell amplification stage, in which the number of cells increases to obtain a sufficient number of cells; S2, after cell amplification, controlling the dissolved oxygen not to be higher than 20% and continuing to culture to induce adipogenic differentiation of cells; in this stage, the cells do not stop amplifying, but mainly hypoxic induction of adipogenic differentiation of cells, and the lipid droplets in the cells increase in size and number.

[0007] Furthermore, the above cells are a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture. This porcine muscle stem cell line can be amplified in suspension and can complete adipogenic differentiation in the suspended state.

[0008] Furthermore, the porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture is named Young Pig Muscle Stem Cell Strain YP-S4-S-SC, which is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C2022372. The deposit date is December 7, 2022. For details, see the Chinese invention patent with the publication number CN116555171A.

[0009] Furthermore, the above culture medium is a serum-free culture medium.

[0010] Furthermore, in the above S1, the dissolved oxygen is controlled not to be lower than 36%.

[0011] Furthermore, in the above S1, the dissolved oxygen is controlled not to be lower than 37%.

[0012] Furthermore, in the above S1, the dissolved oxygen is controlled not to be lower than 38%.

[0013] Furthermore, in the above S1, the dissolved oxygen is controlled not to be lower than 39%.

[0014] Furthermore, in the above S1, the dissolved oxygen is controlled not to be lower than 40%.

[0015] Furthermore, in the above S1, the dissolved oxygen at the beginning of cultivation is controlled to be 90% - 100%.

[0016] Furthermore, in the above S2, the dissolved oxygen is controlled to be 1% - 20%.

[0017] Furthermore, in the above S2, the dissolved oxygen is controlled to be 9% - 20%.

[0018] Furthermore, in the above S2, the dissolved oxygen is controlled to be 9% - 15%.

[0019] Furthermore, in the above S1, the cultivation time is 3 - 10 days.

[0020] Furthermore, in the above S1, the cultivation time is 4 - 8 days.

[0021] Furthermore, in the above S1, the cultivation time is 5 - 6 days.

[0022] Furthermore, in the above S2, the cultivation time is 3 - 10 days.

[0023] Furthermore, in the above S2, the cultivation time is 4 - 8 days.

[0024] Furthermore, in the above S2, the cultivation time is 5 - 6 days.

[0025] Furthermore, in the above method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions, the cultivation process is a fed-batch cultivation process. Starting from the 3rd day of cultivation, a serum-free feed medium is supplemented, and then the feed medium is supplemented every 2 days.

[0026] Furthermore, in the above fed-batch cultivation process, the volume of the feed medium can be adjusted according to the viable cell density, cell viability, glucose, lactate, ammonium ion, and amino acid concentrations detected by sampling. As an example, in this application, a serum-free feed medium with a volume of 10% of the cultivation volume is supplemented.

[0027] Furthermore, the above S1 includes: The cells harvested by cell resuscitation are inoculated into a stirred bioreactor containing a serum-free medium at a density of (1 - 10)×10 5 cells / mL. The stirring speed is set to 80 - 240 rpm, the pH is set to (6.0 - 8.0) ± 0.2, the temperature is set to 34 - 40°C, the minimum dissolved oxygen is set, and a serum-free feed medium is supplemented starting from the 3rd day of cultivation, and then the serum-free feed medium is supplemented every 2 days.

[0028] Furthermore, the above S1 includes: The cells harvested by cell resuscitation are inoculated at a density of (2 - 5)×10 5Cells were inoculated into a bioreactor containing serum-free medium at a density of (1~10)×10

[0029] Furthermore, in the above method for safely and efficiently inducing adipogenic differentiation of cells under hypoxia, the culture process is a perfusion culture process. Perfusion culture starts on the 3rd day of culture at a rate of 0.5 VVD; on the 4th day, it is 1 VVD; and starting from the 5th day, it is 2 VVD.

[0030] Furthermore, the above S1 includes: The cells harvested by cell resuscitation were inoculated into a wave bioreactor containing serum-free medium at a density of (1~10)×10 5 cells / mL. The angle of the wave reactor was set to 6~9°, the swing speed was set to 15~30 rpm, the pH was set to (7.0~7.6)±0.2, the temperature was set to 34~40°C, and the minimum dissolved oxygen was set. Perfusion culture started on the 3rd day of culture.

[0031] Furthermore, the above S1 includes: The cells harvested by cell resuscitation were inoculated into a wave bioreactor at a density of (2~5)×10 5 cells / mL. The angle of the wave reactor was set to 7°, the swing speed was set to 20 rpm, the pH was set to 7.2±0.2, and the temperature was set to 37°C.

[0032] Furthermore, in the above method for safely and efficiently inducing adipogenic differentiation of cells under hypoxia, cell resuscitation was also included before S1, including: The cells were inoculated into serum-free medium at a density of (1~10)×10 5 cells / mL. The cells were centrifuged and collected for subculture every 2~6 days. After obtaining a sufficient number of cells by repeating the above steps, the cells were centrifuged and harvested.

[0033] Furthermore, in the above cell resuscitation, the cells were inoculated into serum-free medium at a density of (2~5)×10 5 cells / mL, and the cells were centrifuged and collected for subculture every 3 days.

[0034] The present application also provides the use of the above method for safely and efficiently inducing adipogenic differentiation of cells under hypoxia in the preparation of cultured meat from cells, specifically for inducing adipogenesis of cells.

[0035] 3. Beneficial effects Compared with the prior art, the beneficial effects of the present application are as follows: (1) A method and application for safely and efficiently inducing adipogenic differentiation of cells under hypoxia provided by the present application. In this method, cells are amplified to a certain number in a reactor with a suspension environment, and adipogenic differentiation of the cells is induced by reducing the dissolved oxygen level to hypoxia. The present application only regulates and induces adipogenic differentiation of cells through physical factors. Compared with the traditional method of differentiating into fat using chemical inducers, no exogenous reagents are introduced during the differentiation process, ensuring food safety; using the above method, cells can complete adipogenic differentiation in 4 - 6 days, and complete cell amplification and adipogenic differentiation within 10 days, shortening the differentiation time; and adipogenic differentiation can be completed in large-scale reactors such as 200 L, and it can be applied to the large-scale preparation of cultured meat.

[0036] (2) A method and application for safely and efficiently inducing adipogenic differentiation of cells under hypoxia provided by the present application, especially using a perfusion culture process for cell amplification and differentiation. During the cell differentiation process, a relatively high amplification rate is still maintained. Description of the Drawings

[0037] Figure 1 It shows the change of dissolved oxygen level in a 1 L stirred reactor in Example 1.

[0038] Figure 2 It shows the cell growth curve during the process of hypoxic induction of cell suspension differentiation into fat based on the fed-batch process in Example 1.

[0039] Figure 3 It shows the oil red staining photos of lipid droplets of hypoxic induction of cell suspension differentiation into fat (hypoxic differentiation) and non-hypoxic treatment (control). The unit size in the figure represents 50 μm.

[0040] Figure 4 It shows the change of dissolved oxygen level in a 1 L stirred reactor in Comparative Example 1.

[0041] Figure 5 It shows the change of dissolved oxygen level in a 200 L stirred reactor in Example 2.

[0042] Figure 6 It shows the cell growth curve during the process of hypoxic induction of cell suspension differentiation into fat based on the fed-batch process (200 L pilot scale) in Example 2.

[0043] Figure 7 It shows the oil red staining photo of lipid droplets of hypoxic induction of cell suspension differentiation into fat in Example 2. The unit size in the figure represents 50 μm.

[0044] Figure 8 It shows the change of dissolved oxygen level in a 50 L wave reactor in Example 3.

[0045] Figure 9It is the cell growth curve during the process of hypoxic induction of cell suspension differentiation into fat based on the perfusion culture process in Example 3.

[0046] Figure 10 It is a photo of Oil Red staining of lipid droplets in the process of hypoxic induction of cell suspension differentiation into fat in Example 3.

[0047] Figure 11 It is a photo of cell culture pork balls made from the cell raw materials of hypoxic induction differentiation into fat obtained in this application. Detailed implementation manners

[0048] The following further describes this application in combination with specific examples.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0050] For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0051] Concentrations, amounts, and other numerical data may be presented in range format herein. It should be understood that such range format is only used for convenience and brevity and should be interpreted flexibly as including not only the values explicitly recited as the range limits but also all individual values or sub-ranges subsumed within the stated range as if each value and sub-range were explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted as including not only the explicitly recited limit values of 1 to about 4.5 but also the individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that only recite one numerical value, such as "less than about 4.5", which should be interpreted as including all of the above values and ranges. In addition, this interpretation should apply regardless of the breadth of the range or feature described. Those skilled in the art know that "about" and "around" are numerical values that can achieve the same effect within a certain range of the relevant numerical value.

[0052] In this application, unless otherwise specified, the cells are porcine muscle stem cell lines adapted to carrier-free and serum-free suspension culture previously domesticated by the applicant, named porcine muscle stem cell strain YP-S4-S-SC, which is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, the deposit number being CCTCC NO: C2022372, and the deposit date being December 7, 2022. For details, see the Chinese invention patent with the publication number CN116555171A.

[0053] In this application, unless otherwise specified, the serum-free medium for culturing is a chemically defined medium for the in vitro proliferation of myogenic cells, including cell culture supplement factors and cell proliferation medium, without serum components. Specifically, it is the complete chemically defined cell proliferation medium of Group 14 in Example 1 of the Chinese invention patent with the publication number CN114574433A. The serum-free feeding medium is also a serum-free medium.

[0054] In this application, unless otherwise specified, dissolved oxygen, also known as dissolved oxygen, refers to the content of molecular oxygen dissolved in water, which is usually affected by temperature, salinity, and biological activities in water. The unit is usually mg / L. In this application, 100% dissolved oxygen is the saturated dissolved oxygen level of the medium (aqueous phase) at 37°C, approximately 6.6 mg / L.

[0055] As used in this application, the medium replacement rate (Volume to Volume Dilution, VVD) refers to the ratio of the volume of fresh medium added to the culture system per unit time to the total volume of the culture system, usually expressed as a volume ratio per hour, and the unit is h -1 。

[0056] In this application, unless otherwise specified, cell density refers to the number of cells contained in a unit volume (such as per milliliter) of cell suspension, which reflects the density of cells in the culture system.

[0057] In this application, unless otherwise specified, cell viability refers to the proportion of live cells in the total number of cells, usually expressed as a percentage, and is an important indicator to measure the health status of cells and the suitability of culture conditions.

[0058] In this application, unless otherwise specified, intracellular lipid droplets are observed and analyzed through oil red staining detection of lipid droplets. Oil red O is a lipid-soluble azo dye, and its chemical structure enables it to specifically bind to lipid substances. When it encounters intracellular lipid droplets, since lipid droplets are mainly composed of lipid components such as neutral fat (such as triglyceride), oil red O molecules can dissolve in the lipid droplets, thus staining the lipid droplets red. Based on the principle of similar solubility, oil red O preferentially interacts with the lipid components in intracellular lipid droplets and does not bind to other water-soluble components in the cell. This specificity enables clear differentiation of lipid droplets from other intracellular structures under a microscope, thereby localizing and observing lipid droplets. The oil red staining detection of lipid droplets includes the following steps: Take cell counting. According to the cell counting results, at 3×10 6Take the cell suspension of the cells into a 1.5 mL EP tube, place the EP tube in a centrifuge for centrifugation, centrifuge at 330×g for 5 min, and then remove the supernatant; resuspend the cells with 500 μL of 4% paraformaldehyde and fix them at room temperature for 30 min or overnight at 4°C; centrifuge the fixed cells at 330×g for 5 min, remove the supernatant, resuspend and wash them with 500 μL of PBS, and then centrifuge again under the same conditions to remove the supernatant; slowly add 200 μL of the detergent in the Oil Red O staining kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number: CO158M) along the inner wall of the centrifuge tube to cover the surface of the cell pellet. During the liquid addition process, prevent the cells from being blown up. After 20 s, aspirate the detergent, add 200 μL of Oil Red dye, disperse the cells, and stain them in the dark for 10 min; after the staining is completed, centrifuge at 330×g for 5 min, remove the supernatant, add the detergent to cover the surface of the cell pellet, aspirate the detergent after 20 s, add 200 μL of PBS to resuspend, aspirate 20 μL of the resuspended cells and add them to a 96-well plate; then turn on the inverted light microscope and the microscope software, place the cells under the microscope, and take pictures at 400×.

[0059] In this application, a 1 L stirred reactor was purchased from Guangzhou Aibotai Biotechnology Co., Ltd., and the model is my-Control. The stirred reactor keeps the dissolved oxygen in the reactor at about the lowest dissolved oxygen level by setting the parameter of the lowest dissolved oxygen.

[0060] In this application, a 200 L stirred reactor was purchased from Shanghai Duoning Biotechnology Co., Ltd., and the model is DuoBioX® Pro. The stirred reactor keeps the dissolved oxygen in the reactor at about the lowest dissolved oxygen level by setting the parameter of the lowest dissolved oxygen.

[0061] In this application, a 50 L wave reactor was purchased from Wuhan Saikecheng Technology Co., Ltd., and the model is SKC600. The wave reactor keeps the dissolved oxygen in the reactor at about the lowest dissolved oxygen level by setting the parameter of the lowest dissolved oxygen.

[0062] Example 1 This example provides a safe and efficient method for inducing adipogenic differentiation of cells under hypoxia, specifically a method for inducing adipogenic differentiation of cell suspension under hypoxia.

[0063] The method for inducing adipogenic differentiation of cell suspension under hypoxia includes: S1, cell flask amplification Resuscitate the stem cell line YP-S4-S-SC using serum-free medium, at 2×10 5Inoculate at a density of cells / mL into a 125 mL shake flask containing serum-free medium, set the temperature to 37 °C, the rotation speed to 120 rpm, and centrifuge at 500×g for 5 min on the 3rd day to collect the cells; S2, cell reactor amplification Inoculate the cells harvested in S1 at a density of 2×10 5 cells / mL into a 1 L stirred reactor containing serum-free medium; set the stirring speed of the stirred reactor to 120 rpm, the pH to 7.2 ± 0.2, the temperature to 37 °C, and set the minimum dissolved oxygen to 40%; Start supplementing 10% of the culture volume of serum-free feeding medium on the 3rd day of culture, and then supplement 10% of the culture volume of serum-free feeding medium every 2 days; S3, cell reactor differentiation After 5 days of cell reactor amplification, adjust the minimum dissolved oxygen of the stirred reactor to 10% to induce hypoxic-induced cell suspension differentiation into fat. Continuously supplement 10% of the culture volume of serum-free feeding medium every 2 days, and complete cell suspension differentiation after continuous culture for 5 days.

[0064] During the cell reactor amplification culture, track and detect the dissolved oxygen in the stirred reactor. The results are shown in Table 1 and Figure 1 as shown. The dissolved oxygen in the cell reactor gradually decreases from 98% with the amplification of the cells and drops to about 40%. Because the minimum dissolved oxygen is set to 40%, the cell reactor will supplement oxygen at this time and maintain it at about 40%; at D6, because the minimum dissolved oxygen is set to 10%, the dissolved oxygen in the cell reactor quickly drops to about 10%.

[0065] Table 1

[0066] During the cell reactor amplification culture, obtain the corresponding growth curve by counting every day. The growth curve is as Figure 2 shown, indicating that the cell growth condition is good and approaching the peak density at D5.

[0067] During the cell reactor differentiation culture, obtain the corresponding growth curve by counting every day and conduct oil red staining detection of lipid droplets. The growth curve is as Figure 2 shown. After approaching the peak density at D5, hypoxic differentiation starts. During the cell differentiation process, the cell number can still be maintained, and the viability always remains above 80%.

[0068] The oil red staining result of lipid droplets after hypoxic-induced cell differentiation is as Figure 3 shown in the hypoxic differentiation group in. A small amount of lipid droplets appear in the cells at D7, the lipid droplets in the cells increase and enlarge from D8 to D10, and a large number of cells form multilocular lipid droplets can be seen at D10.

[0069] Comparative Example 1 This comparative example provides a method for inducing the suspension differentiation of cells into fat.

[0070] Referring to Example 1, the difference is that after 5 days of cell amplification, the minimum dissolved oxygen setting is not adjusted, that is, the minimum dissolved oxygen setting is maintained at 40%, and 10% serum-free feeding medium is supplemented every two days. After continuous culture for 5 days, cell suspension differentiation is completed.

[0071] During the amplification culture of the cell reactor, the dissolved oxygen in the stirred reactor was tracked and detected. The results are shown in Table 2 and Figure 4 As shown, the dissolved oxygen in the cell reactor gradually decreased from 95% with the amplification of cells and dropped to about 40%. Since the minimum dissolved oxygen setting is 40%, the cell reactor will supplement oxygen at this time, so that the dissolved oxygen in the reactor is always maintained at about 40%.

[0072] Table 2

[0073] During the differentiation culture of the cell reactor, oil red staining detection of lipid droplets was performed every day. The oil red staining results of lipid droplets are as Figure 3 shown in the control group in. During the culture process from D5 to D10, there was no significant lipid droplet accumulation in the cells.

[0074] Example 2 This example provides a safe and efficient method for inducing adipogenic differentiation of cells under hypoxia, specifically a method for inducing suspension differentiation of cells into fat under hypoxia.

[0075] For the method of inducing suspension differentiation of cells into fat under hypoxia, referring to Example 1, in this example, the amplification and differentiation of the cell reactor are at the pilot scale.

[0076] Specifically include: S1, cell flask amplification Resuscitate the stem cell line YP-S4-S-SC using serum-free medium and inoculate it in a 125 mL flask containing serum-free medium at a density of 2×10 5 cells / mL. Set the temperature to 37°C and the rotation speed to 120 rpm. Centrifuge at 500 g for 5 min on the 3rd day to collect the cells; inoculate at a density of 2×10 5 cells / mL in a 1000 mL flask and centrifuge to collect the cells on the 3rd day; inoculate at a density of 2×10 5 cells / mL in a 5000 mL flask and centrifuge to collect the cells on the 3rd day; inoculate at a density of 2×10 5 cells / mL in a 50 L stirred reactor and centrifuge to collect the cells on the 3rd day; S2, Cell Reactor Amplification The cells harvested in S1 were inoculated into a 200 L stirred reactor containing serum-free medium at a density of 2×10 5 cells / mL; the stirring speed of the stirred reactor was set at 85 rpm, the pH was set at 7.2 ± 0.2, the temperature was set at 37°C, and the minimum dissolved oxygen was set at 40%; Starting from the 3rd day of culture, 10% of the culture volume of serum-free feeding medium was supplemented, and then 10% of the culture volume of serum-free feeding medium was supplemented every 2 days; S3, Cell Reactor Differentiation After 5 days of cell reactor amplification, the minimum dissolved oxygen of the stirred reactor was adjusted to 10% to induce hypoxic-induced cell suspension differentiation into fat. 10% of serum-free feeding medium was continuously supplemented every 2 days, and cell suspension differentiation was completed after continuous culture for 5 days.

[0077] During the amplification culture of the cell reactor, the dissolved oxygen in the stirred reactor was tracked and detected. The results are shown in Table 3 and Figure 5 as shown. The dissolved oxygen in the cell reactor gradually decreased from 95% with the amplification of cells and dropped to about 40%. Since the minimum dissolved oxygen was set at 40%, the cell reactor would supplement oxygen and maintain it at about 40%; at D6, since the minimum dissolved oxygen was set at 10%, the dissolved oxygen in the cell reactor rapidly dropped to about 10%.

[0078] Table 3

[0079] During the amplification culture of the cell reactor, the corresponding growth curve was obtained by counting every day. The growth curve is as Figure 6 shown. In the culture of a 200 L pilot-scale reactor, the cell growth condition was still good.

[0080] During the differentiation culture of the cell reactor, the corresponding growth curve was obtained by counting every day and the oil red staining of lipid droplets was detected at D5, D8, and D10. The growth curve is as Figure 6 shown. During the cell differentiation process, the cell number could still be maintained, and the viability was always maintained above 80%.

[0081] The oil red staining results of lipid droplets after hypoxic-induced cell differentiation are as Figure 7 shown. During the differentiation process of cells from D5 to D10, cells continuously accumulated lipid droplets, and a large number of cells formed multilocular lipid droplets were visible at D10.

[0082] Example 3 This example provides a safe and efficient method for hypoxic-induced cell adipogenic differentiation, specifically a method for hypoxic-induced cell suspension differentiation into fat.

[0083] Method for inducing hypoxia-induced cell suspension differentiation into fat, referring to Example 1, specifically including: S1, cell flask amplification Resuscitate the stem cell line YP-S4-S-SC using serum-free medium and inoculate it into a 125 mL flask containing serum-free medium at a density of 2×10 5 cells / mL. Set the temperature to 37°C and the rotation speed to 120 rpm. Centrifuge at 500 g for 5 min on the 3rd day to collect the cells; inoculate the cells into a 1000 mL flask at a density of 2×10 5 cells / mL and centrifuge to collect the cells on the 3rd day; S2, cell reactor amplification Inoculate the cells harvested in S1 into a 50 L wave reactor containing serum-free medium at a density of 2×10 5 cells / mL; Set the angle of the wave reactor to 7°, the swing speed to 20 rpm, the pH to 7.2±0.2, the temperature to 37°C, and set the minimum dissolved oxygen to 40%; Start perfusion culture on the 3rd day of culture at a rate of 0.5 medium replacement rate (Volume to Volume Dilution, VVD); perform perfusion culture at a rate of 1 VVD on the 4th day; perform perfusion culture at a rate of 2 VVD on the 5th day and subsequent days; S3, cell reactor differentiation After 5 days of cell reactor amplification, adjust the minimum dissolved oxygen in the reactor to 10% to induce hypoxia-induced cell suspension differentiation into fat. During this period, continue perfusion culture at 2 VVD and complete cell suspension differentiation after continuous culture for 5 days.

[0084] During the cell reactor amplification culture period, track and detect the dissolved oxygen in the reactor. The results are shown in Table 4 and Figure 8 as follows. The dissolved oxygen in the cell reactor first increases (the wave reactor culture setting requires continuous air supply, and the initial ventilation volume is greater than the cell oxygen consumption, so the dissolved oxygen level will increase) and then decreases as the cells expand. It drops to about 50% at D5; at D6, because the minimum dissolved oxygen is set to 10%, the dissolved oxygen in the cell reactor quickly drops to about 10%.

[0085] Table 4

[0086] During the cell reactor amplification culture period, count every day to obtain the corresponding growth curve. The growth curve is as Figure 9As shown, in the 50-L pilot-scale reactor culture, the cell growth condition was still good, and the cell density was slightly higher than the corresponding cell densities in Example 1 and Example 2.

[0087] During the differentiation culture of the cell reactor, the corresponding growth curves were obtained by counting every day, and the oil red staining of lipid droplets was detected at D5 - D10. The growth curves were as Figure 9 shown. During the cell differentiation process, the cell number could still be maintained, and the cell density was much higher than the corresponding cell densities in Example 1 and Example 2. It can be seen that the perfusion culture process can obtain a higher peak density than the fed-batch culture process, and the viability was always maintained above 80%.

[0088] The oil red staining results of lipid droplets after hypoxic induction of cell differentiation were as Figure 10 shown. During the differentiation process of cells at D5 - D10, cells continuously accumulated lipid droplets, and a large number of cells formed multilocular lipid droplets at D10.

[0089] Example 4 This example provides a safe and efficient method for hypoxic induction of cell adipogenic differentiation in the present application and the application of the hypoxic induction differentiated adipocytes prepared thereby in the preparation of cell culture meat.

[0090] In this example, its addition was mainly evaluated for the nutritional impact on plant meatballs, and the specific steps were as follows: (1) Preparation of cell culture meat: The powder and auxiliary materials such as ice water were stirred evenly, and shredded soy protein isolate and 20% of the cells after hypoxic induction of differentiation into fat (Example 2) were added. After kneading into balls, they were fried at 170°C for 1 min for shaping; through boiling, after the central temperature of the meatballs reached 72°C, they were taken out and cooled to room temperature in cold water, and the formed meatball products were frozen and stored at -20°C ( Figure 11 ).

[0091] Table 5 Nutritional composition of cell culture meatballs and plant meatballs

[0092] (2)Nutritional composition detection of cultured meat: Take the formed meatball products and thaw them. The protein content is detected by the Kjeldahl method, the fat content is detected by the Soxhlet extraction method, the moisture is detected by the drying method, the ash is obtained by incinerating and carbonizing at 500 °C and then cooling and weighing, the carbohydrate is obtained by the subtraction method, that is, the total weight minus the protein, fat, moisture and ash content, and the energy is calculated by measuring the energy that can be released by the main nutritional components in the food (such as protein, fat and carbohydrate). The results are shown in Table 5. For the cultured meatballs with 20% cell raw materials added, compared with the plant meatballs without addition, the protein and fat contents are significantly increased, indicating that the addition of cell raw materials effectively improves the nutritional value of plant meatballs and makes them closer to the nutritional composition of traditional meat products.

Claims

1. A safe and efficient method for inducing adipogenic differentiation of cells under hypoxia, characterized in that, The method includes the following steps: S1. Cultivate cells using a culture medium for cell expansion, and control the dissolved oxygen to be not less than 35% during the cultivation process; S2. After cell expansion, control the dissolved oxygen to be not higher than 20%, and continue cultivation to induce adipogenic differentiation of the cells.

2. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to claim 1, characterized in that, in S1, the dissolved oxygen at the beginning of cultivation is controlled to be 90% - 100%; in S2, the dissolved oxygen is controlled to be 1% - 20%.

3. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to claim 2, characterized in that, in S2, the dissolved oxygen is controlled to be 9% - 20%.

4. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to claim 2 or 3, characterized in that, in S1, the cultivation time is 3 - 10 days; and / or in S2, the cultivation time is 3 - 10 days.

5. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to claim 4, characterized in that, the cells are a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture; and / or the culture medium is a serum-free culture medium.

6. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to claim 5, characterized in that, the cultivation process is a fed-batch culture process; or the cultivation process is a perfusion culture process.

7. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to claim 6, characterized in that, the cultivation process is a fed-batch culture process, and S1 includes: The cells harvested by cell resuscitation were inoculated into a stirred bioreactor containing serum-free medium at a density of (1-10)×10 5 cells / mL. The stirring speed was set at 80-240 rpm, the pH was set at (6.0-8.0)±0.2, the temperature was set at 34-40°C, and the minimum dissolved oxygen was set. Starting from the 3rd day of culture, serum-free feeding medium was supplemented, and then the feeding medium was supplemented every 2 days.

8. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to claim 6, characterized in that, the cultivation process is a perfusion culture process, and S1 includes: The cells harvested by cell resuscitation were inoculated into a wave bioreactor containing serum-free medium at a density of (1-10)×10 5 cells / mL. The angle of the wave reactor was set at 6-9°, the swing speed was set at 15-30 rpm, the pH was set at (7.0-7.6)±0.2, the temperature was set at 34-40°C, and the minimum dissolved oxygen was set. Perfusion culture was started on the 3rd day of culture at a rate of 0.5 VVD; on the 4th day it was 1 VVD; and from the 5th day it was 2 VVD.

9. A method for safely and efficiently inducing adipogenic differentiation of cells under hypoxia according to claim 7 or 8, characterized in that The cell resuscitation includes: The cells were inoculated in a serum-free medium at a density of (1-10)×10 5 cells / mL, and the cells were centrifuged and collected for subculture every 2-6 days. After obtaining a sufficient number of cells by repeating the process, the cells were harvested by centrifugation.

10. Use of a method for safely and efficiently inducing adipogenic differentiation of cells under hypoxic conditions according to any one of claims 1 - 9 in the preparation of cultured meat.

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