A safe and efficient method for inducing adipogenic differentiation of cells under hypoxia and application thereof

Abstract

The application discloses a safe and efficient method for inducing cell adipogenic differentiation under low oxygen and application thereof, and belongs to the technical field of cell culture. The method is to expand cells to a certain number in a reactor in a suspension environment, to induce the cell suspension differentiation into fat by reducing the dissolved oxygen level to low oxygen. The method is to induce cell adipogenic differentiation only by physical factors, compared with the traditional chemical inducer differentiation into fat method, no exogenous reagent is introduced in the differentiation process, and the food safety is ensured. The cell can complete the adipogenic differentiation in 4-6 days by using the method, the cell expansion and adipogenic differentiation are completed in about 10 days, the differentiation time is accelerated, and the differentiation into fat can be completed in a 200 L large-scale reactor, so that the method can be applied to the large-scale preparation of cell culture meat.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell culture, and particularly relates to a safe and efficient method for hypoxia-induced cell adipogenic differentiation and application thereof. BACKGROUND

[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 mode of traditional livestock farming, the nutritional components of cell cultured meat can be adjusted according to requirements to meet the health requirements of consumers, and can also reduce environmental pollution, improve animal welfare, reduce disease risk, etc. The main components of traditional meat include skeletal muscle, fat and connective tissue (main component is collagen). In the production process of cultured meat, after the seed cells are scaled up, functional differentiation can be achieved through specific induction conditions, and 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 inducing cells to differentiate into adipocytes in vitro. Chemical induction is a common method for adipogenic induction. Experimental studies have shown that by regulating the drug combination in the culture system, cells can be effectively induced to differentiate into the adipogenic lineage. The classic drugs include phosphodiesterase inhibitors (IBMX), dexamethasone, insulin, rosiglitazone and indomethacin, etc. By activating transcription factors such as PPARγ through drug combination, cell adipogenic differentiation is promoted. Although these chemical induction differentiation methods are effective, they involve food safety issues, and the adipogenic differentiation time is usually more than 10 days, which is not conducive to industrial production. With the rapid development of cell cultured meat industry, it is 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 decellularized plant scaffolds (such as sterilized tissues of onions and shiitake mushrooms) as biocompatible materials for adherent production of fat products, or generate suspended fat spheroids through self-organization technology. However, the above methods are all based on chemical inducers, and both biocompatible materials and self-organization technology have difficulties in scaled production. At present, there is no technology for optimizing cell adipogenic differentiation by adjusting only physical factors. SUMMARY

[0005] 1. Problem to be solved

[0006] The present application aims at the problem of conflict between chemical substance-induced cell adipogenic differentiation and food safety supervision in the prior art, and provides a safe and efficient method for low-oxygen-induced cell adipogenic differentiation and application. The method is to adjust the dissolved oxygen concentration in the cell culture system, specifically to reduce the dissolved oxygen in the culture system after cell expansion to induce cell adipogenic differentiation, which can effectively improve the cell adipogenic differentiation efficiency and maturity, overcome the limitations of traditional drug induction methods, and be used for the preparation of cell culture meat.

[0007] 2. Technical solution

[0008] To solve the above problems, the technical solution adopted by the present application is as follows:

[0009] The present application provides a safe and efficient method for low-oxygen-induced cell adipogenic differentiation, which comprises the following steps:

[0010] S1, using a culture medium to culture cells for cell expansion, and controlling the dissolved oxygen not to be lower than 35% during the culture process; this stage is mainly a cell expansion stage, in which the number of cells increases to obtain a sufficient number of cells;

[0011] S2, after cell expansion, the dissolved oxygen is controlled to be not higher than 20%, and the cells are cultured to induce cell adipogenic differentiation; in this stage, the cells have not stopped expanding, but mainly low-oxygen-induced cell adipogenic differentiation, and the lipid droplets in the cells increase.

[0012] Further, the above-mentioned cells are a pig muscle stem cell line adapted to serum-free and vector-free suspension culture, which can be expanded in suspension and can complete differentiation into adipose tissue in a suspended state.

[0013] Further, the above-mentioned pig muscle stem cell line adapted to serum-free and vector-free suspension culture is named piglet muscle stem cell strain YP-S4-S-SC, preserved in the China Center for Type Culture Collection, located in Wuhan University, Wuhan, China, with the preservation number CCTCC NO: C2022372, and the preservation date is December 7, 2022. For details, see Chinese invention patent CN116555171A.

[0014] Further, the above-mentioned culture medium is a serum-free culture medium.

[0015] Further, in S1, the dissolved oxygen is controlled to be not lower than 36%.

[0016] Further, in S1, the dissolved oxygen is controlled to be not lower than 37%.

[0017] Further, in S1, the dissolved oxygen is controlled to be not lower than 38%.

[0018] Further, in S1, the dissolved oxygen is controlled to be not lower than 39%.

[0019] Further, in the above S1, the dissolved oxygen is controlled to be not less than 40%.

[0020] Further, in the above S1, the dissolved oxygen at the beginning of the culture is controlled to be 90% to 100%.

[0021] Further, in the above S2, the dissolved oxygen is controlled to be 1% to 20%.

[0022] Further, in the above S2, the dissolved oxygen is controlled to be 9% to 20%.

[0023] Further, in the above S2, the dissolved oxygen is controlled to be 9% to 15%.

[0024] Further, in the above S1, the culture time is 3 to 10 days.

[0025] Further, in the above S1, the culture time is 4 to 8 days.

[0026] Further, in the above S1, the culture time is 5 to 6 days.

[0027] Further, in the above S2, the culture time is 3 to 10 days.

[0028] Further, in the above S2, the culture time is 4 to 8 days.

[0029] Further, in the above S2, the culture time is 5 to 6 days.

[0030] Further, in the above method for safely and efficiently inducing adipogenic differentiation of cells under low oxygen, the culture process is a fed-batch culture process, and serum-free feed medium is supplemented from the third day of culture, and then the feed medium is supplemented every 2 days.

[0031] Further, in the above fed-batch culture process, the volume of the feed medium can be adjusted according to the concentrations of the viable cell density, cell viability, glucose, lactic acid, ammonium ion, and amino acid detected by sampling, for example, 10% of the culture volume of serum-free feed medium is supplemented.

[0032] Further, the above S1 comprises:

[0033] The recovered cells are inoculated into a stirred bioreactor containing serum-free medium at a density of (1-10) x 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 serum-free feed medium is supplemented from the third day of culture, and then serum-free feed medium is supplemented every 2 days.

[0034] Further, the above S1 comprises:

[0035] The harvested cells are inoculated into a bioreactor containing serum-free medium at a density of (2-5)×10 5 cells / mL, the stirring speed is set to 120 rpm, the pH is set to 7.2±0.2, and the temperature is set to 37℃.

[0036] Further, the above-mentioned safe and efficient low-oxygen-induced cell adipogenic differentiation method, the culture process is perfusion culture process, from the third day of culture, the perfusion culture rate is 0.5 VVD; the fourth day is 1 VVD; the fifth day is 2 VVD.

[0037] Further, the above-mentioned S1 includes:

[0038] The harvested cells are inoculated into a wave bioreactor containing serum-free medium at a density of (1-10)×10 5 cells / mL, the wave reactor angle is set to 6-9°, the swing speed is set to 15-30 rpm, the pH is set to (7.0-7.6)±0.2, the temperature is set to 34-40℃, the minimum dissolved oxygen is set, and perfusion culture is started on the third day of culture.

[0039] Further, the above-mentioned S1 includes:

[0040] The harvested cells are inoculated into a wave bioreactor containing serum-free medium at a density of (2-5)×10 5 cells / mL, the wave reactor angle is set to 7°, the swing speed is set to 20 rpm, the pH is set to 7.2±0.2, and the temperature is set to 37℃.

[0041] Further, the above-mentioned safe and efficient low-oxygen-induced cell adipogenic differentiation method further includes cell resuscitation before S1, which includes:

[0042] The cells are inoculated in serum-free medium at a density of (1-10)×10 5 cells / mL, and the cells are centrifuged and passaged every 2-6 days, and after repeating to obtain sufficient number of cells, the cells are centrifuged and harvested.

[0043] Further, the above-mentioned cell resuscitation is inoculated in serum-free medium at a density of (2-5)×10 5 cells / mL, and the cells are centrifuged and passaged every 3 days.

[0044] The application also provides the use of the above-mentioned safe and efficient low-oxygen-induced cell adipogenic differentiation method in the preparation of cell culture meat, specifically to induce cell adipogenesis.

[0045] 3. Beneficial effects

[0046] The present application has the beneficial effects compared with the prior art, which are:

[0047] (1) The present application provides a safe and efficient method for low-oxygen-induced cell adipogenic differentiation and application. The method is to expand cells to a certain number in a suspension reactor, and induce cell suspension differentiation into adipogenic by reducing the dissolved oxygen level to low oxygen. The present application only adjusts the induction of cell adipogenic differentiation by physical factors, compared with the traditional chemical inducer differentiation method, no exogenous reagent is introduced in the differentiation process, which ensures the food safety; using the above method, the cell can complete the adipogenic differentiation in 4-6 days, and the cell expansion and adipogenic differentiation can be completed within 10 days, which shortens the differentiation time; and the differentiation and adipogenic can be completed in a 200 L large-scale reactor, which can be applied to the large-scale preparation of cell culture meat.

[0048] (2) The present application provides a safe and efficient method for low-oxygen-induced cell adipogenic differentiation and application, especially using perfusion culture process for cell expansion and differentiation. In the cell differentiation process, a high expansion rate is still maintained. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is the change of dissolved oxygen level in a 1 L stirred reactor in Example 1.

[0050] Figure 2 is the cell growth curve in the process of low-oxygen-induced cell suspension differentiation and adipogenic based on fed-batch process in Example 1.

[0051] Figure 3 is the oil red staining photograph of lipid droplets in the process of low-oxygen-induced cell suspension differentiation and adipogenic (low-oxygen differentiation) and without low-oxygen treatment (control), and the unit size in the figure represents 50 μm.

[0052] Figure 4 is the change of dissolved oxygen level in a 1 L stirred reactor in Comparative Example 1.

[0053] Figure 5 is the change of dissolved oxygen level in a 200 L stirred reactor in Example 2.

[0054] Figure 6 is the cell growth curve in the process of low-oxygen-induced cell suspension differentiation and adipogenic based on fed-batch process (200 L pilot test) in Example 2.

[0055] Figure 7 is the oil red staining photograph of lipid droplets in the process of low-oxygen-induced cell suspension differentiation and adipogenic in Example 2, and the unit size in the figure represents 50 μm.

[0056] Figure 8 is the change of dissolved oxygen level in a 50 L wave reactor in Example 3.

[0057] Figure 9 Figure 2 is a cell growth curve in a process of inducing cell suspension differentiation into fat by low oxygen based on perfusion culture process in Example 3.

[0058] Figure 10 Figure 3 is a photograph of oil red staining of lipid droplets in a process of inducing cell suspension differentiation into fat by low oxygen in Example 3.

[0059] Figure 11 Figure 4 is a photograph of cell culture pork balls made of cell raw material induced to differentiate into fat by low oxygen in the present application. DETAILED DESCRIPTION

[0060] The present application will be further described below with reference to specific examples.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs; the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0062] Unless otherwise indicated, conventional methods and techniques of molecular biology, microbiology, cell biology, and recombinant DNA techniques, as well as the specific conditions for carrying out the present application, are described in the literature in the field, such as Molecular Cloning: A Laboratory Manual, 3rd Ed., Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; Short Protocols in Molecular Biology: A Compendium of Techniques, 4th Ed., Ausubel et al., John Wiley & Sons, Inc., New York, 1999; and the like.

[0063] Concentrations, amounts, and other numerical data can be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be construed as having been followed to the rightmost significant figure of the number when it has been followed, even though some of the significant figures within the range might not be legible, to indicate the precision of the values. Only those individual values that fall within the range of values are to be considered as the preferred values in the range, unless the context clearly indicates otherwise. That is, all stated values and / or measurements can be understood as being preceded by the word "about". This means that the recited value or measurement can vary from the stated meaning by up to 1%, 2%, 5%, 10%, 15%, or even 20%, still falling within the meaning of the term used, unless otherwise indicated. For example, the range of "from about 1 to about 4.5" is to be interpreted as including only the recited values of 1 to 4.5, and not including individual values such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges recited with only one numerical value, such as "less than about 4.5", which is to be interpreted as including all the values and ranges described above. In addition, this interpretation should apply regardless of the breadth of the range or the characteristic being described. Those skilled in the art will appreciate that "about" "around" are values within a certain range of the relevant numerical value that can achieve the same effect.

[0064] In the present application, unless otherwise specified, the cells are the porcine muscle stem cell line YP-S4-S-SC previously domesticated by the applicant for adaptation to vector-free and serum-free suspension culture, which is named as porcine muscle stem cell line YP-S4-S-SC, deposited in the China Center for Type Culture Collection, located in Wuhan University, Wuhan, China, with the accession number CCTCC NO: C2022372, and the deposit date is December 7, 2022. For details, see the Chinese patent for invention with the publication number CN116555171A.

[0065] In the present application, unless otherwise specified, the serum-free culture medium for culture is a chemically defined cell proliferation culture medium for muscle-derived cells in vitro, which includes cell culture supplements and cell proliferation culture medium, and does not contain serum components. Specifically, it is the complete chemically defined cell proliferation culture medium of Group 14 in Example 1 of the Chinese patent for invention with the publication number CN114574433A. The serum-free feed medium is also a serum-free culture medium.

[0066] In the present 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 activity in water. The unit is usually mg / L. In the present application, 100% dissolved oxygen is the saturation level of dissolved oxygen of the culture medium (aqueous phase) at 37℃, which is about 6.6 mg / L.

[0067] As used herein, the volume to volume dilution (VVD) refers to the ratio of the volume of fresh culture medium added to the culture system per unit time to the total volume of the culture system, which is usually expressed in volume per hour, with the unit of h -1 .

[0068] In the present application, unless otherwise specified, the cell density refers to the number of cells contained in a unit volume (such as per milliliter) of cell suspension, which reflects the degree of cell concentration in the culture system.

[0069] In the present application, unless otherwise specified, the cell viability refers to the proportion of viable cells in the total number of cells, which is usually expressed in percentage, and is an important indicator for measuring the health status of cells and the suitability of culture conditions.

[0070] In the present application, unless otherwise specified, the lipid droplets in the cells are observed and analyzed by oil red staining of the lipid droplets. Oil red O is a liposoluble azo dye, and its chemical structure enables it to specifically bind to lipid substances. When encountering lipid droplets in cells, since the lipid droplets are mainly composed of neutral fats (such as triglycerides) and other lipid components, the oil red O molecules can dissolve in the lipid droplets, so that the lipid droplets are dyed red. Based on the principle of similar solubility, oil red O preferentially interacts with the lipid components in the lipid droplets in the cells, and does not bind to other water-soluble components in the cells. This specificity enables clear differentiation between lipid droplets and other structures in the cells under a microscope, so that the lipid droplets can be located and observed. The oil red staining detection of the lipid droplets includes the following steps:

[0071] Take the cell count, and according to the cell count result, take 3 x 10 6 cells to a 1.5 mL EP tube, and place the EP tube in a centrifuge. After centrifugation at 330 x g for 5 min, remove the supernatant. Resuspend the cells with 500 μL of 4% paraformaldehyde, and fix at room temperature for 30 min or at 4°C overnight. After centrifugation of the fixed cells at 330 x g for 5 min, remove the supernatant, resuspend and wash with 500 μL of PBS, and then centrifuge again under the same conditions to remove the supernatant. Slowly add 200 μL of the washing agent in the oil red O staining kit (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., product number: CO158M) along the inner wall of the centrifuge tube to cover the surface of the cell precipitate. Prevent the cells from being blown up during the liquid addition process. After 20 s, aspirate the washing agent, add 200 μL of oil red dye, blow the cells apart, and avoid light for 10 min of staining. After the staining is completed, centrifuge at 330 x g for 5 min to remove the supernatant, add the washing agent to cover the surface of the cell precipitate, aspirate the washing agent after 20 s, resuspend with 200 μL of PBS, and aspirate 20 μL of the resuspended cells to add to a 96-well plate. Then turn on the inverted light microscope and microscope software, place the cells under the microscope, and take a photo at 400 x.

[0072] In the present application, the 1 L stirred reactor is purchased from Guangzhou Aibeta Biotechnology Co., Ltd., and the model number is my-Control. The stirred reactor is set to the minimum dissolved oxygen parameter, so that the dissolved oxygen in the reactor is maintained at about the minimum dissolved oxygen level.

[0073] In the present application, the 200 L stirred reactor is purchased from Shanghai Doning Biotechnology Co., Ltd., and the model number is DuoBioX® Pro. The stirred reactor is set to the minimum dissolved oxygen parameter, so that the dissolved oxygen in the reactor is maintained at about the minimum dissolved oxygen level.

[0074] In the present application, the 50 L wave reactor is purchased from Wuhan Saikesheng Technology Co., Ltd., model number SKC600. The wave reactor is set to the minimum dissolved oxygen parameter, so that the dissolved oxygen in the reactor is maintained at about the minimum dissolved oxygen level.

[0075] Example 1

[0076] The present embodiment provides a safe and efficient method for inducing cell adipogenic differentiation under hypoxia, specifically a method for inducing cell suspension differentiation into adipocytes under hypoxia.

[0077] The method for inducing cell suspension differentiation into adipocytes under hypoxia comprises:

[0078] S1, cell flask expansion

[0079] The stem cell strain YP-S4-S-SC is resuscitated using serum-free medium, and inoculated in a 125 mL flask containing serum-free medium at a density of 2x10 5 cells / mL, with a temperature setting of 37°C and a rotation speed setting of 120 rpm. The cells are collected by centrifugation at 500xg for 5 min on the 3rd day;

[0080] S2, cell reactor expansion

[0081] The cells harvested in S1 are inoculated into a 1 L stirred reactor containing serum-free medium at a density of 2x10 5 cells / mL; the stirring speed of the stirred reactor is set to 120 rpm, the pH is set to 7.2±0.2, the temperature is set to 37°C, and the minimum dissolved oxygen is set to 40%;

[0082] Starting from the 3rd day of culture, 10% of the culture volume of serum-free feed medium is supplemented, and then every 2 days, 10% of the culture volume of serum-free feed medium is supplemented;

[0083] S3, cell reactor differentiation

[0084] After 5 days of cell reactor expansion, the minimum dissolved oxygen of the stirred reactor is adjusted to 10% for hypoxic induction of cell suspension differentiation into adipocytes, and 10% of the culture volume of serum-free feed medium is continuously supplemented every 2 days. After 5 days of continuous culture, the cell suspension differentiation is completed.

[0085] During the cell reactor expansion culture, the dissolved oxygen in the stirred reactor is tracked and detected. The results are shown in Table 1 and Figure 1 As the cells expand, the dissolved oxygen in the cell reactor gradually decreases from 98% to about 40%, because the minimum dissolved oxygen is set to 40%, at this time the cell reactor will supplement oxygen to maintain about 40%; at D6, because the minimum dissolved oxygen is set to 10%, the dissolved oxygen in the cell reactor rapidly decreases to about 10%.

[0086] Table 1

[0087]

[0088] During the expansion culture in the cell reactor, the cells were counted every day to obtain the corresponding growth curve. The growth curve is shown in Figure 2 , which indicates that the cells grew well and approached the peak density on D5.

[0089] During the differentiation culture in the cell reactor, the cells were counted every day to obtain the corresponding growth curve, and the oil red staining of lipid droplets was detected. The growth curve is shown in Figure 2 , which indicates that the cells started to differentiate under low oxygen after approaching the peak density on D5, and the cell number was maintained during the cell differentiation process, and the viability was always maintained above 80%.

[0090] The results of oil red staining of lipid droplets after low oxygen-induced cell differentiation are shown in Figure 3 , which indicates that a small amount of lipid droplets appeared in the cells on D7, the lipid droplets in the cells increased on D8-D10, and a large number of cells formed multilocular lipid droplets on D10.

[0091] Comparative Example 1

[0092] This comparative example provides a method for inducing cells to differentiate into lipids in suspension.

[0093] Reference Example 1, which is different in that after the cells are expanded for 5 days, the minimum dissolved oxygen setting is not adjusted, i.e., the minimum dissolved oxygen setting is maintained at 40%, and the 10% serum-free feed medium is supplemented every two days, and the cell suspension differentiation is completed after continuous culture for 5 days.

[0094] During the expansion culture in the cell reactor, the dissolved oxygen in the stirred reactor was tracked and detected, and the results are shown in Table 2 and Figure 4 , which indicates that the dissolved oxygen in the cell reactor gradually decreased from 95% to about 40% as the cells expanded, because the minimum dissolved oxygen setting was 40%, at this time the cell reactor would supplement oxygen, so that the dissolved oxygen in the reactor was always maintained at about 40%.

[0095] Table 2

[0096]

[0097] During the differentiation culture in the cell reactor, the oil red staining of lipid droplets was detected every day. The results of oil red staining of lipid droplets are shown in Figure 3 , which indicates that the cells did not have significant lipid droplet accumulation during the culture process on D5-D10.

[0098] Example 2

[0099] The embodiment provides a safe and efficient method for low-oxygen-induced cell adipogenic differentiation, in particular, a method for low-oxygen-induced cell suspension differentiation into adipocytes.

[0100] The method for low-oxygen-induced cell suspension differentiation into adipocytes is described in reference example 1, and in this embodiment, the cell reactor is expanded and differentiated to a pilot scale.

[0101] Specifically, it comprises the following steps:

[0102] S1, cell flask expansion

[0103] The stem cell strain YP-S4-S-SC is resuscitated using a serum-free medium, and is inoculated in a 125 mL flask containing the serum-free medium at a density of 2×10 5 cells / mL, and the temperature is set to 37°C and the rotation speed is set to 120 rpm; the cells are collected by centrifugation at 500 g for 5 min on the third day; the cells are inoculated in a 1000 mL flask at a density of 2×10 5 cells / mL, and the cells are collected by centrifugation on the third day; the cells are inoculated in a 5000 mL flask at a density of 2×10 5 cells / mL, and the cells are collected by centrifugation on the third day; the cells are inoculated in a 50 L stirred reactor at a density of 2×10 5 cells / mL, and the cells are collected by centrifugation on the third day.

[0104] S2, cell reactor expansion

[0105] The cells collected in S1 are inoculated in a 200 L stirred reactor containing a serum-free medium at a density of 2×10 5 cells / mL; the stirring speed of the stirred reactor is set to 85 rpm, the pH is set to 7.2±0.2, the temperature is set to 37°C, and the minimum dissolved oxygen is set to 40%;

[0106] The serum-free feed medium is supplemented at 10% of the culture volume on the third day of culture, and then the serum-free feed medium is supplemented at 10% of the culture volume every 2 days.

[0107] S3, cell reactor differentiation

[0108] After the cell reactor is expanded for 5 days, the minimum dissolved oxygen of the stirred reactor is adjusted to 10% to induce low-oxygen-induced cell suspension differentiation into adipocytes, and the serum-free feed medium is supplemented at 10% every 2 days; after continuous culture for 5 days, the cell suspension differentiation is completed.

[0109] During the cell reactor expansion culture, the dissolved oxygen in the stirred reactor is tracked and detected, and the results are shown in Tables 3 and Figure 5As shown, the dissolved oxygen in the cell reactor gradually decreased from 95% to about 40% as the cells were expanded, and the cell reactor maintained the dissolved oxygen at about 40% as the minimum dissolved oxygen was set to 40%. At D6, the dissolved oxygen in the cell reactor rapidly decreased to about 10% as the minimum dissolved oxygen was set to 10%.

[0110] Table 3

[0111]

[0112] During the expansion culture of the cell reactor, the corresponding growth curve was obtained by counting every day. The growth curve is as shown in Figure 6 As shown, the cell growth condition was still good in the 200 L pilot-scale reactor culture.

[0113] During the differentiation culture of the cell reactor, the corresponding growth curve was obtained by counting every day, and oil red staining of lipid droplets was detected at D5, D8 and D10. The growth curve is as shown in Figure 6 As shown, the cell number was maintained during the cell differentiation process, and the viability was always maintained above 80%.

[0114] The oil red staining results of the lipid droplets after low-oxygen-induced cell differentiation are as shown in Figure 7 As shown, the cells continuously accumulated lipid droplets during the differentiation process from D5 to D10, and a large number of cells formed multi-locular lipid droplets at D10.

[0115] Example 3

[0116] The present embodiment provides a safe and efficient method for low-oxygen-induced cell adipogenic differentiation, specifically a method for low-oxygen-induced cell suspension differentiation into adipocytes.

[0117] The method for low-oxygen-induced cell suspension differentiation into adipocytes is described in Reference Example 1, and specifically comprises:

[0118] S1, cell shake flask expansion

[0119] The stem cell strain YP-S4-S-SC was resuscitated using serum-free medium, and inoculated in a 125 mL shake flask containing serum-free medium at a density of 2x10 5 cells / mL, and the temperature was set to 37°C and the rotation speed was set to 120 rpm. The cells were collected by centrifugation at 500 g for 5 min on the 3rd day; the cells were inoculated in a 1000 mL shake flask at a density of 2x10 5 cells / mL, and the cells were collected by centrifugation on the 3rd day;

[0120] S2, cell reactor expansion

[0121] The cells harvested in S1 were inoculated in a cell reactor at a density of 2x10 5The cells were inoculated into a 50 L wave reactor containing serum-free medium at a density of 1.5 x 106cells / mL; the wave reactor angle was set to 7°, the swing speed was set to 20 rpm, the pH was set to 7.2±0.2, the temperature was set to 37℃, and the minimum dissolved oxygen was set to 40%;

[0122] From the third day of culture, perfusion culture was started at a rate of 0.5 volume to volume dilution (VVD); on the fourth day, the perfusion rate was 1 VVD; and on the fifth day and subsequently, the perfusion rate was 2 VVD.

[0123] S3, cell reactor differentiation

[0124] After the cell reactor was expanded for 5 days, the minimum dissolved oxygen in the reactor was adjusted to 10% for low-oxygen-induced cell suspension differentiation into fat, and perfusion culture was continuously performed at 2 VVD. After 5 days of continuous culture, cell suspension differentiation was completed.

[0125] During the expansion culture of the cell reactor, the dissolved oxygen in the reactor was tracked and detected. As shown in Table 4 and Figure 8 , the dissolved oxygen in the cell reactor first increased (the wave reactor culture setting needs to be constantly aerated, and the early aeration amount is greater than the cell oxygen consumption, so the dissolved oxygen level increases) and then decreases to about 50% on D5 as the cells expand. On D6, because the minimum dissolved oxygen was set to 10%, the dissolved oxygen in the cell reactor rapidly decreased to about 10%.

[0126] Table 4

[0127]

[0128] During the expansion culture of the cell reactor, the cells were counted every day to obtain the corresponding growth curve. As shown in Figure 9 , 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 density in Example 1 and Example 2.

[0129] During the differentiation culture of the cell reactor, the cells were counted every day to obtain the corresponding growth curve and oil red staining detection of lipid droplets on D5-D10. As shown in Figure 9 , the cell number can still be maintained during cell differentiation, and the cell density is much higher than the corresponding cell density in Example 1 and Example 2. It can be seen that the perfusion culture process can obtain a higher peak density than the batch feeding culture process, and the viability is always maintained above 80%.

[0130] The oil red staining results of the lipid droplets after low-oxygen-induced cell differentiation are as follows Figure 10As shown, cells continuously accumulated lipid droplets during the differentiation process from D5 to D10, and a large number of cells formed multilocular lipid droplets at D10.

[0131] Example 4

[0132] The present application provides a safe and efficient method for inducing cell adipogenic differentiation under hypoxia and the application of the prepared hypoxia-induced adipogenic cells in the preparation of cell culture meat.

[0133] In this example, the nutritional impact of the addition of the cells on the plant meatballs is evaluated, including the following steps:

[0134] (1) Preparation of cell culture meat: The powder and ice water and other auxiliary materials are stirred uniformly, and the large soybean protein and 20% hypoxia-induced adipogenic cells (Example 2) are added. After kneading into balls, they are fried at 170°C for 1 min to shape; through water boiling, the center temperature of the meatballs reaches 72°C, then they are taken out and cooled to room temperature in cold water. The shaped meatball product is frozen at -20°C for storage. Figure 11 ).

[0135] Table 5 Nutritional composition of cell culture meatballs and plant meatballs

[0136]

[0137] (2) Nutritional composition detection of cell culture meat: The shaped meatball product is thawed, and the protein content is detected by Kjeldahl nitrogen determination, the fat content is detected by Soxhlet extraction, the moisture content is detected by drying method, the ash content is obtained by high-temperature 500°C carbonization and cooling, and the carbohydrate content is obtained by difference method, i.e. total weight minus protein, fat, moisture and ash content, and the energy is calculated by measuring the energy released by the main nutritional components (such as protein, fat and carbohydrate) in the food. The results are shown in Table 5. The protein and fat content of the cell culture meatballs with 20% cell raw materials is significantly improved compared with the plant meatballs without addition, indicating that the addition of cell raw materials effectively improves the nutritional value of plant meatballs, making it closer to the nutritional composition of traditional meat products.

Claims

1. A safe and efficient method for inducing adipogenic differentiation of cells by hypoxia, characterized in that: The method comprises the following steps: S1, using culture medium to culture cells for cell expansion, controlling the dissolved oxygen at the beginning of the culture to 90%-100%, and setting the minimum dissolved oxygen to 40% during the culture process; S2, after cell expansion, the minimum dissolved oxygen was adjusted to 10%, and the cells were cultured to induce adipogenic differentiation; The cells are a porcine muscle stem cell line adapted to carrier-free and serum-free suspension culture, named young porcine muscle stem cell line YP-S4-S-SC, deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: C2022372 and a deposit date of December 7, 2022; The culture medium is a serum-free culture medium, specifically a complete cell proliferation culture medium with clear chemical composition of Group 14 in Example 1 of the Chinese invention patent publication number CN114574433A.

2. A safe and efficient method for inducing adipogenic differentiation of cells by hypoxia according to claim 1, characterized in that: In said S1, the culture time is 3 to 10 days; and / or In the S2, the culture time is 3 to 10 days.

3. A safe and efficient method for inducing adipogenic differentiation of cells by hypoxia according to claim 2, characterized in that: The culture process is a fed-batch culture process; or The culture process is perfusion culture process.

4. A safe and efficient method for inducing adipogenic differentiation of cells by hypoxia according to claim 3, characterized in that: The culture process is a fed-batch culture process, and S1 comprises: The cells harvested after cell recovery were divided into (1~10)×10 5 Cells / mL were inoculated into a stirred bioreactor containing serum-free medium. The stirring speed was set to 80-240 rpm, the pH was set to (6.0-8.0) ± 0.2, the temperature was set to 34-40°C, and the minimum dissolved oxygen was set. Serum-free feed medium was supplemented starting from the third day of culture and every two days thereafter.

5. A safe and efficient method for inducing adipogenic differentiation of cells by hypoxia according to claim 3, characterized in that: The culture process is a perfusion culture process, and S1 includes: The cells harvested after cell recovery were divided into (1~10)×10 5 The cells / mL density was inoculated into a wave bioreactor containing serum-free medium. The wave reactor angle 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℃, the minimum dissolved oxygen was set, and perfusion culture was started from the third day of culture at a rate of 0.5 VVD; 1 VVD on the fourth day; and 2 VVD starting from the fifth day.

6. A safe and efficient method for inducing adipogenic differentiation of cells by hypoxia according to claim 4 or 5, characterized in that: The cell recovery includes: The cells were divided into (1~10)×10 5 Cells were seeded at a density of 10 cells / mL in serum-free medium and collected by centrifugation every 2 to 6 days for passage. After repeated attempts to obtain a sufficient number of cells, the cells were harvested by centrifugation.

7. Use of the safe and efficient method for inducing adipogenic differentiation of cells under hypoxia according to any one of claims 1 to 6 in the preparation of cell-cultured meat.

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