A high-density culture method of porcine myoblasts based on bioreactor

Abstract

The present invention belongs to the technical field of bioreactors, and in particular relates to a high-density culture method for porcine myoblasts based on a bioreactor. Through the steps of resuscitating porcine myoblasts, cell-microcarrier attachment treatment, bioreactor stirring mode conversion, counting and centrifugal cell collection, the porcine myoblasts are difficult to reach a high density in a bioreactor, and a harvest density of 10^6 cells / mL is achieved in the bioreactor, meeting the requirements of cell culture meat production on cell yield; at the same time, the culture time is shortened by more than 20%, greatly saving energy consumption cost and time cost.

Description

Technical Field

[0001] The invention belongs to the technical field of bioreactors, and in particular relates to a high-density culture method of porcine myoblasts based on a bioreactor. Background Art

[0002] Cultured meat is an emerging food science and technology that produces edible meat based on in vitro animal cell culture. Cultured meat is considered to be the most likely food to solve the problems of meat production and meat consumption in the future. Its emergence can effectively reduce environmental pollution, improve resource utilization, and avoid animal slaughter.

[0003] The production process of cell-cultured meat mainly includes seed cell acquisition, cell proliferation in vitro, cell differentiation, collection and processing. Among them, the cell culture process involving high-density proliferation of seed cells in vitro is the core technology that promotes the industrial production of cell-cultured meat. The in vitro proliferation of cell-cultured meat seed cells depends on the bioreactor to provide them with stable three-dimensional culture environment conditions, such as temperature, pH, dissolved oxygen, etc. The inevitable problem of using bioreactors is that the shear force generated by the reactor's paddle stirring will greatly affect the vitality of the cells, making it difficult for the cells to reach high density.

[0004] At present, except for CHO, Vero, HEK293 and other mature cells whose culture density in bioreactors can exceed 10^6 cells / mL, most cells cannot be cultured at a density of more than 10^6 cells / mL, and this problem is also a common problem faced by the food and biopharmaceutical fields. There are still major defects in the cell culture process used for cell culture meat production, which are as follows:

[0005] (1) During the cell attachment process, the attachment efficiency is low, and the 24-hour attachment efficiency is generally only about 50%;

[0006] (2) The cell harvest density is low, and the density in the bioreactor is less than 10^6 cells / mL;

[0007] (3) Due to the influence of shear force, the cell culture cycle is long, and it takes more than 10 days to multiply 10 times in a bioreactor.

[0008] The specification of Chinese invention patent ZL202111679237.3 records a method for multiple amplification of 2BS cells in a bioreactor, including: S01, preparation of seed cells: culturing 2BS seed cells in a rotating bottle, digesting and making a cell suspension after growing into a dense monolayer, inoculating into a primary bioreactor for adherent culture, and obtaining 2BS seed cells; S02, online multiple amplification: 2BS seed cells grow into a dense monolayer on the microcarriers of the bioreactor, washing the microcarriers with sterilized PBS, washing the carriers thoroughly, and removing the culture fluid and serum; adjusting the pH value and temperature, adding trypsin to the microcarriers and mixing them thoroughly, adding serum-containing culture medium after digestion to terminate digestion, inoculating into a secondary bioreactor, and adding culture fluid for cell culture to amplify 2BS cells. The invention uses the above-mentioned culture method to make the 2BS adhesion rate as high as 95% or more, breaking through the technical threshold that the 2BS cells on the Cytodex1 microcarriers have low adhesion rate and are easy to fall off.

[0009] However, the continuous replenishment of oxygen, carbon dioxide and air for the cells mentioned in the patent will destroy the dissolved oxygen value, pH balance and the bubbles of the replenished gas will greatly reduce the cell-microcarrier attachment efficiency; the perfusion culture mode used therein will consume a lot of culture medium, such as in the production of cell cultured meat, which will increase the production cost dramatically. In addition, the continuous replenishment of oxygen, carbon dioxide and air is used during the culture process, and the bursting of bubbles produced by the replenishment will affect the cell state.

[0010] Chinese invention patent ZL200410017588.8 describes a method for three-dimensionally culturing and inducing bone marrow mesenchymal stem cells into chondrocytes, which includes the following steps: 1. Suspending and culturing bone marrow mesenchymal stem cells in a suspension culture device pre-installed with culture medium and microcarriers, and amplifying bone marrow mesenchymal stem cells under stirring; 2. Then adding chondrogenic induction culture medium to a culture device containing harvested Cytodex 3 microcarriers and bone marrow mesenchymal stem cells grown thereon, and inducing bone marrow mesenchymal stem cells to aggregate into chondrogenic cells. The method of the invention can obtain a large number of chondrocytes at one time in a short period of time, reducing the probability of contamination, reducing cell damage, and facilitating the transfer of nutrients between cells.

[0011] However, the patent did not achieve a high harvest density, and the culture environment of 37°C, 5% CO2, and saturated humidity mentioned therein was not sufficient to achieve high-density proliferation of porcine myoblasts in a bioreactor. Summary of the invention

[0012] In order to solve the above technical problems, the present invention provides a high-density culture method of porcine myoblasts based on a bioreactor. In view of the difficulty that porcine myoblasts are difficult to reach high density in a bioreactor, a harvest density of 10^6 cells / mL is achieved in the bioreactor, meeting the requirements of cell culture meat production for cell yield; at the same time, the culture time is shortened by more than 20%, greatly saving energy consumption and time costs.

[0013] A high-density culture method of porcine myoblasts based on a bioreactor to solve the above technical problems comprises the following steps:

[0014] (1) Recovery of porcine myoblasts: The porcine myoblasts stored in liquid nitrogen were quickly recovered by placing them in a water bath, centrifuged to remove the freezing solution, resuspended in culture medium A, and inoculated into two-dimensional culture containers such as cell culture dishes / culture bottles / cell factories for 2 to 3 generations of subculture;

[0015] (2) Cell-microcarrier attachment treatment: When the number of two-dimensionally cultured porcine myoblasts reaches a certain number, the culture medium is removed and the cells are washed with PBS. The digested porcine myoblasts are added to the cell suspension, and the digested porcine myoblasts are collected by centrifugation and inoculated into the bioreactor in proportion to the microcarriers. The culture medium B is added to the upper limit of the reaction system, and the cell-microcarrier attachment treatment is carried out in the bioreactor interval stirring mode. The culture is carried out for 22 to 26 hours, and 24 hours in the optimized scheme; wherein the cell to microcarrier inoculation ratio is 5 to 20:1; the cell to microcarrier inoculation ratio in the optimized scheme is 20:1. When the ratio is not within this range, the attachment efficiency will decrease, and a high inoculation ratio can shorten the culture time;

[0016] Cells and microcarriers require certain static conditions to adhere. If they are continuously stirred, the adhesion efficiency will be greatly reduced. Therefore, an interval stirring mode is required during the adhesion process, in which stirring allows cells and microcarriers to combine evenly, and interval static conditions promote cell adhesion to the microcarriers.

[0017] (3) Agitation conversion and counting: The bioreactor's intermittent agitation mode is switched to a continuous agitation mode, and counting is performed once every 1 to 2 days until the expected harvest density is reached;

[0018] In this step, the cell-microcarrier complex needs to be fully stirred and suspended to achieve better proliferation. The cells have already attached to the microcarriers, so there is no need to keep them still to allow them to attach better. Continuous stirring can make the cells evenly distributed in the culture environment, which is conducive to better nutrient absorption. At the same time, stirring can make the gases required by the cells better dissolved in the culture medium;

[0019] (4) After 8 days of culture, the cells were collected by centrifugation.

[0020] In the optimized scheme, the water bath condition for the porcine myoblasts in step (1) is 37-41°C; the water bath condition in the optimized scheme is 37°C.

[0021] The culture medium A in step (1) is a DMEM culture medium containing 2% to 15% fetal bovine serum.

[0022] The washing in step (2) is PBS washing, and the number of times of washing cells is 1 to 5 times.

[0023] The cells are digested by trypsin in step (2), the digestion time is 5 to 30 min, and the digestion temperature is 35 to 38°C.

[0024] The trypsin digestion temperature in step (2) is 37°C and the digestion time is 5 min.

[0025] In the further optimized scheme, the microcarrier in step (2) is the Huakan 3D TableTrix microcarrier.

[0026] The cell seeding density in step (2) can be 1*10^5 cells / mL. Here, the cell seeding density refers to the amount of cells and microcarriers inoculated in the bioreactor in proportion, and the seeding amount can be adjusted according to the volume of the bioreactor.

[0027] In a further optimized solution, the culture medium B in step (2) is a DMEM culture medium containing 2% to 15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine.

[0028] Adding glutamine to the cell culture medium promotes the attachment efficiency of porcine myoblasts; adding ascorbic acid diphosphate promotes the vitality of seed cells and the tolerance of porcine myoblasts to the shear force of the bioreactor. Adding 1% ascorbic acid diphosphate and 1% glutamine can shorten the time to reach a high cell density and reduce time costs.

[0029] In the optimized scheme, the stirring speed of the bioreactor in step (2) is 40-300 rpm, the interval stirring mode conditions are an interval time of 5-120 min, and the stirring time is 1-60 min; in the further optimized scheme, the stirring speed of the bioreactor is 150 rpm, the interval stirring mode conditions are an interval time of 60 min, and the stirring time is 10 min.

[0030] The rotation speed of the bioreactor in the continuous stirring mode in step (3) is 40 to 300 rpm; the rotation speed of the bioreactor in the continuous stirring mode in the optimization scheme is 150 rpm.

[0031] When the stirring parameters are within the appropriate range, cells can reach a high harvest density.

[0032] In further optimizing this case, the pH value in the bioreactor in step (3) is controlled at 7.2-7.4, and the dissolved oxygen value is controlled at 40%-80%.

[0033] The temperature in the bioreactor in steps (2) and (3) is 37°C to 41°C; in the optimized solution, the temperature in the bioreactor is 37°C.

[0034] The centrifugal cell collection conditions in steps (1), (2) and (4) are a centrifugal speed of 1000 to 2000 rpm and a centrifugal time of 5 to 15 min; in the optimized scheme, the centrifugal speed is 1000 rpm and the centrifugal time is 5 min.

[0035] Among the above reaction parameters, if they are not within this parameter range, the cells will proliferate slowly or fail to proliferate in the bioreactor, and the effects will vary greatly.

[0036] The method of the present invention solves the following technologies and achieves corresponding effects:

[0037] (1) In order to address the defect that the attachment efficiency is low and the 24-hour attachment efficiency is generally only about 50%, the present invention adds additives to the cell culture medium to promote the attachment of porcine myoblasts without affecting the state of the cells, and can stabilize the 24-hour attachment efficiency of the cells at more than 70%;

[0038] (2) In view of the disadvantage that the seed cell harvest density is low and the density in the bioreactor is lower than 10^6 cells / mL, this technical solution adopts the optimal bioreactor process parameters and cell-microcarrier ratio, and the porcine myoblast harvest density is stabilized at more than 1*10^6 cells / mL;

[0039] (3) In order to address the shortcoming that the cell culture cycle is long due to the influence of shear force, and it takes about 10 days to proliferate 10 times in a bioreactor, the technical solution adds additives to the cell culture medium to promote the vitality of seed cells, thereby promoting the tolerance of porcine myoblasts to the shear force of the bioreactor. The 10-fold proliferation time of porcine myoblasts in the bioreactor is stabilized within 9 days;

[0040] The method of the present invention is widely applicable and can be applied to small and medium-sized reactors, and can achieve good results. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a process flow chart of pig myoblast culture based on a bioreactor in the present invention;

[0042] Figure 2 This is a growth curve of porcine myoblasts in Example 5 of the present invention;

[0043] Figure 3This is a growth curve of porcine myoblasts in Example 2 of the present invention;

[0044] Figure 4 This is a comparison diagram of the pig myoblast attachment efficiency of Example 3 of the present invention;

[0045] Figure 5 This is a comparison chart of cell growth efficiency in Example 4 of the present invention;

[0046] Figure 6 A comparison diagram of the effects of different ratios of cell number to microcarriers in the present invention;

[0047] Figure 7 This is a comparison chart of the effects of different additives contained in the culture medium of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with specific embodiments:

[0049] As attached Figure 1 As shown in the figure, the bioreactor-based porcine myoblast culture method is divided into the following four steps:

[0050] (1) Resuscitating porcine myoblasts and inoculating the cells into two-dimensional culture vessels such as cell culture dishes / culture flasks / cell factories for culture;

[0051] (2) When the number of two-dimensionally cultured porcine myoblasts reaches a certain level, the cells are digested into a cell suspension. The digested porcine myoblasts are inoculated into a bioreactor together with microcarriers, and the cell-microcarrier attachment process is performed using the bioreactor interval stirring mode;

[0052] (3) Switch the interval stirring mode of the bioreactor to the continuous stirring mode, and count once every one or two days until the expected harvest density is reached. In the present invention, the expected harvest density can be about 2*10^6, and the expected harvest density can also be set according to one's own needs;

[0053] (4) Obtain cells by centrifugation.

[0054] The specific plan is as follows: Figure 1 As shown, porcine myoblasts (from Beijing black pigs) were selected as seed cells, and the cells were taken out of the liquid nitrogen tank and placed in a 37-41°C water bath for rapid recovery; the porcine myoblasts were then inoculated into culture dishes / culture bottles / cell factories and subcultured for 2 to 3 generations; the cells were then digested with trypsin into a cell suspension and inoculated into a bioreactor together with a certain proportion of microcarriers, and ascorbic acid diphosphate and glutamine were added to the culture medium; the bioreactor interval stirring mode was then turned on, and after 22 to 26 hours, it was switched to continuous stirring mode, and the cells were harvested after 8 days of culture.

[0055] The following is described in detail with different embodiments:

[0056] Example 1 Take out the porcine myoblasts (Beijing black pig) from the liquid nitrogen tank and place them in a 39°C water bath for rapid recovery. After centrifugation at 1500rpm for 10min, discard the freezing solution, add medium A to resuspend the cells and inoculate them in a cell culture dish for 2 to 3 generations of subculture. When the cells grow to about 2*10^7 (the initial inoculation cell amount is different when the culture volume is different), discard the supernatant of medium A, wash the cells once with PBS, add 8 mL of trypsin to digest at 35°C for 30 min, collect the cells in a 15 mL centrifuge tube and centrifuge at 1500 rpm for 10min, discard the supernatant and add 5 mL of medium B to resuspend the cells, and count them. Add about 1*10^7 cells and 1*10^6 microcarriers to a 500 mL bioreactor, and fill medium B to 200mL. Turn on the interval stirring mode of the bioreactor, with a stirring speed of 40 rpm, a stirring time of 60 min, an interval time of 100 min, a culture temperature of 41°C, and calculate the cell attachment rate after culturing for 26 h. Then, the continuous stirring mode was turned on with a stirring speed of 40 rpm, the dissolved oxygen value was maintained at 80%, and the pH was controlled at 7.4. After 8 days of culture, the cells were harvested by centrifugation at 1500 rpm for 10 min.

[0057] Culture medium A is DMEM culture medium containing 2% to 15% fetal bovine serum, and culture medium B is DMEM culture medium containing 2% to 15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine.

[0058] The cell attachment rate was 62.0% on the first day, and the cell density reached 0.70*10^6 / mL on the ninth day of culture. Porcine myoblasts showed a good growth trend.

[0059] Example 2 Porcine myoblasts (Beijing black pigs) were taken out of the liquid nitrogen tank and placed in a 38°C water bath for rapid recovery. After centrifugation at 2000 rpm for 8 min, the cryopreservation solution was discarded, medium A was added to resuspend the cells and inoculated into a cell culture dish for culture. When the cells grew to 2*10^7

[0060] When the number of cells reached about 100, the supernatant of medium A was discarded, the cells were washed 4 times with PBS, 6 mL of trypsin was added to digest at 38°C for 10 min, the cells were collected in a 15 mL centrifuge tube and centrifuged at 1200 rpm for 15 min, the supernatant was discarded and 5 mL of medium B was added to resuspend the cells and counted. About 5*10^6 cells and 1*10^6 microcarriers were added to a 500 mL bioreactor, and medium B was filled to 200 mL. The bioreactor was turned on in interval stirring mode, with a stirring speed of 40 rpm, a stirring time of 50 min, an interval time of 80 min, and a culture temperature of 38°C. The cell attachment rate was calculated after 22 h. Subsequently, the continuous stirring mode was turned on, with a stirring speed of 300 rpm, the dissolved oxygen value was maintained at 60%, and the pH was controlled at 7.3. After 8 days of culture, the cells were harvested by centrifugation at 1200 rpm for 15 min.

[0061] Culture medium A is DMEM culture medium containing 2% to 15% fetal bovine serum, and culture medium B is DMEM culture medium containing 2% to 15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine.

[0062] The cell attachment rate was 62.0% on the first day, and the cell density reached 0.65*10^6 / mL on the ninth day of culture. Porcine myoblasts showed a good growth trend.

[0063] Example 3 Take out the porcine myoblasts (Beijing black pig) from the liquid nitrogen tank and place them in a 40°C water bath for rapid recovery. After centrifugation at 1200rpm for 15min, discard the freezing solution, add medium A to resuspend the cells and inoculate them in a cell culture dish for 2 to 3 generations of subculture. When the cells grow to about 2*10^7, discard the supernatant of medium A, wash the cells three times with PBS, add 6 mL of trypsin to digest at 39°C for 5 min, collect the cells in a 15 mL centrifuge tube and centrifuge at 1800 rpm for 6min, discard the supernatant and add 5 mL of medium B to resuspend the cells, and count them. Add about 2*10^7 cells and 1*10^6 microcarriers to a 500 mL bioreactor, and fill medium B to 200 mL. Turn on the interval stirring mode of the bioreactor, with a stirring speed of 300 rpm, a stirring time of 1 min, an interval time of 5 min, a culture temperature of 38°C, and calculate the cell attachment rate after 25 h. Then, the continuous stirring mode was turned on with a stirring speed of 200 rpm, the dissolved oxygen value was maintained at 50%, and the pH was controlled at 7.2. After 8 days of culture, the cells were harvested by centrifugation at 1800 rpm for 6 minutes.

[0064] Culture medium A is DMEM culture medium containing 2% to 15% fetal bovine serum, and culture medium B is DMEM culture medium containing 2% to 15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine.

[0065] The cell attachment rate was 39% on the first day, and the cell density reached 0.61*10^6 cells / mL on the ninth day of culture. Porcine myoblasts showed a good growth trend.

[0066] In this example, the rotation speed of 300 rpm will affect cell attachment and the attachment efficiency is low. Therefore, the faster the stirring speed during the attachment process, the better the effect. During the culture stage, the rotation speed of 200 rpm makes the culture environment relatively mild, thereby achieving a higher density. After the attachment process, the cell tolerance to the rotation speed is better than the tolerance during the attachment process.

[0067] Example 4 Take out the porcine myoblasts (Beijing black pig) from the liquid nitrogen tank and place them in a 41°C water bath for rapid recovery. After centrifugation at 1800rpm for 12min, discard the cryopreservation solution, add medium A to resuspend the cells and inoculate them in a cell culture dish for 2 to 3 generations of subculture. When the cells grow to about 2*10^7, discard the supernatant of medium A, wash the cells 5 times with PBS, add 5 mL of trypsin to digest at 37°C for 5 min, collect the cells in a 15 mL centrifuge tube and centrifuge at 2000 rpm for 5min, discard the supernatant and add 5 mL of medium B to resuspend the cells, and count them. Add about 1.5*10^7 cells and 1*10^6 microcarriers to a 500 mL bioreactor, and fill medium B to 200 mL. Turn on the interval stirring mode of the bioreactor, with a stirring speed of 100 rpm, a stirring time of 30 min, an interval time of 120 min, a culture temperature of 40°C, and calculate the cell attachment rate after 23 h. Then, the continuous stirring mode was turned on, the stirring speed was 100 rpm, the dissolved oxygen value was maintained at 70%, and the pH was controlled at 7.2. After 8 days of culture, the cells were harvested by centrifugation at 2000 rpm for 5 minutes.

[0068] Culture medium A is DMEM culture medium containing 2% to 15% fetal bovine serum, and culture medium B is DMEM culture medium containing 2% to 15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine.

[0069] The cell attachment rate was 64% on the first day, and the cell density reached 0.912*10^6 / mL on the ninth day of culture. Porcine myoblasts showed a good growth trend.

[0070] Example 5 Take out the porcine myoblasts (Beijing black pig) from the liquid nitrogen tank and place them in a 37°C water bath for rapid recovery. After centrifugation at 1000rpm for 5min, discard the freezing solution, add medium A to resuspend the cells and inoculate them in a cell culture dish for 2 to 3 generations of subculture. When the cells grow to about 2*10^7, discard the supernatant of medium A, wash the cells three times with PBS, add 5 mL of trypsin to digest at 37°C for 5 min, collect the cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5min, discard the supernatant and add 5 mL of medium B to resuspend the cells, and count them. Add about 2*10^7 cells and 1*10^6 microcarriers to a 500 mL bioreactor, and fill medium B to 200 mL. Turn on the interval stirring mode of the bioreactor, with a stirring speed of 150 rpm, a stirring time of 10 min, an interval time of 60 min, a culture temperature of 37°C, and calculate the cell attachment rate after 24 h. Then the continuous stirring mode was turned on with a stirring speed of 150 rpm, the dissolved oxygen value was maintained at 40%, and the pH was controlled at 7.2. After 8 days of culture, the cells were harvested by centrifugation.

[0071] Culture medium A is DMEM culture medium containing 2% to 15% fetal bovine serum, and culture medium B is DMEM culture medium containing 2% to 15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine.

[0072] As attached Figure 2 As shown, when porcine myoblasts were cultured using the method of this embodiment, the cell attachment rate was 76% on the first day, and the cell density reached 1.25*10^6 cells / mL on the ninth day of culture, and the porcine myoblasts showed a good growth trend.

[0073] The stirring speed is optimized during the cell attachment process to achieve high attachment efficiency, which will give the cell culture a better starting point, thereby achieving a higher cell density. The attachment method and the culture method in the present invention are integrated to harvest high-density cells.

[0074] Example 6 Take out the porcine myoblasts (Beijing black pig) from the liquid nitrogen tank and place them in a 38°C water bath for rapid recovery. After centrifugation at 1000rpm for 5min, discard the freezing solution, add medium A to resuspend the cells and inoculate them in the cell factory for 2 to 3 generations of subculture. When the cells grow to about 3*10^8, discard the supernatant of medium A, wash the cells three times with PBS, add 5mL trypsin to digest at 37.5℃ for 5min, collect the cells in a 50mL centrifuge tube and centrifuge at 1000rpm for 5min, discard the supernatant and add 20mL medium to resuspend the cells, and count. Add about 2*10^8 cells and 1*10^7 microcarriers to a 5L bioreactor, and fill medium B to 3L. Turn on the interval stirring mode of the bioreactor, with a stirring speed of 155rpm, a stirring time of 9min, an interval time of 58min, a culture temperature of 36.5℃, and calculate the cell attachment rate after 24h. Then, the continuous stirring mode was turned on with a stirring speed of 155 rpm, the dissolved oxygen value was maintained at 40%, and the pH was controlled at 7.2. The cells were harvested after 8 days of culture.

[0075] Culture medium A is DMEM culture medium containing 2% to 15% fetal bovine serum, and culture medium B is DMEM culture medium containing 2% to 15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine.

[0076] As attached Figure 3 As shown, the porcine myoblasts were cultured using the process of this embodiment. The cell attachment rate was 72% on the first day, and the cell density reached 1.09*10^6 cells / mL on the ninth day of culture. The porcine myoblasts showed a good growth trend.

[0077] Example 7: Porcine myoblasts (Beijing black pigs) were taken out from the liquid nitrogen tank and placed in a 37°C water bath for rapid recovery. After centrifugation at 1000 rpm for 5 min, the freezing solution was discarded, culture medium A was added to resuspend the cells, and the cells were inoculated in a cell culture dish and subcultured for 2 to 3 generations. When the cells grew to about 4*10^7, the supernatant of culture medium A was discarded, the cells were washed three times with PBS, 5 mL of trypsin was added to digest at 37°C for 5 min, the cells were collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 5 mL of culture medium was added to resuspend the cells, and the cells were counted.

[0078] In this embodiment, two reaction groups were set up, and about 2*10^7 cells and 1*10^6 microcarriers were added to a 500 mL bioreactor. The first group was supplemented with complete medium A without addition to 200 mL, and the second group was supplemented with 1% glutamine on the basis of the first group of medium A. Subsequently, the bioreactor interval stirring mode was turned on, with a stirring speed of 150 rpm, a stirring time of 10 min, an interval time of 60 min, and a culture temperature of 37°C. After 24 h, the cell attachment rate was calculated and compared. Medium A is DMEM medium containing 2% to 15% fetal bovine serum.

[0079] As attached Figure 4 As shown, the left figure is a comparison of the attachment efficiency in this embodiment, and the attachment efficiency of the group containing additives is significantly higher than that of the group without additives; the right figure is a fluorescence image of cell attachment in this embodiment, and the cells are fixed and stained with DAPI. It can be seen from the figure that the attachment efficiency of the group containing additives is significantly higher than that of the group without additives.

[0080] Example 8: Porcine myoblasts (Beijing black pig) were taken out from the liquid nitrogen tank and placed in a 37°C water bath for rapid recovery. After centrifugation at 1000 rpm for 5 min, the freezing solution was discarded, culture medium was added to resuspend the cells and inoculated into a cell culture dish for culture. When the cells grew to about 4*10^7, the supernatant of the culture medium was discarded, the cells were washed three times with PBS, 5 mL of trypsin was added to digest at 37°C for 5 min, the cells were collected in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 5 mL of culture medium was added to resuspend the cells, and the cells were counted.

[0081] In this embodiment, two reaction groups were set up, and each group added about 2*10^7 cells and 1*10^6 microcarriers to a 500mL bioreactor. The first group was supplemented with complete medium A (i.e., DMEM medium containing 2% to 15% fetal bovine serum) to 200 mL, and the second group was supplemented with 1% ascorbic acid diphosphate and 1% glutamine, i.e., medium B, on the basis of the first group of culture medium. The bioreactor interval stirring mode was turned on, the stirring speed was 150 rpm, the stirring time was 10 min, the interval time was 60 min, the culture temperature was 37°C, and the cell attachment rate was calculated after 24 h. Subsequently, the continuous stirring mode was turned on, the stirring speed was 150 rpm, the dissolved oxygen value was maintained at 40%, the pH was controlled at 7.2, and the cell technology was harvested after 8 days of culture and compared.

[0082] As attached Figure 5 As shown, the culture efficiency of the group containing additives was higher than that of the group without additives. The cell harvest density of the porcine myoblasts in the group without additives was only 0.914*10^6 cells / mL after 9 days of culture. The cell harvest density of the porcine myoblasts in the group containing additives was 1.23*10^6 cells / mL after 9 days of culture, which was 30% higher than that of the group without additives.

[0083] Experiment 1 Experimental groups 1-6 were set up, and the other contents were the same as those in Example 5 (using the attachment conditions described in Example 5, and the culture medium used was culture medium A), wherein the cell-microcarrier inoculation ratios in experimental groups 1-6 were 1:1, 5:1, 10:1, 20:1, 50:1 and 100:1, respectively. After 24 hours, the cells were collected and the attachment efficiency was calculated: the number of cells counted per mL / the number of microcarriers per mL. The effect comparison experiment when the ratio of cell number to microcarrier was different is shown in the figure. Figure 6 shown.

[0084] like Figure 6 It can be seen that as the cell-microcarrier inoculation ratio increases, the cell attachment efficiency also increases, but after a certain ratio, the cell attachment efficiency becomes smaller. Therefore, a suitable cell-microcarrier inoculation ratio is important. In the present invention, the cell attachment efficiency is very good when the cell-microcarrier inoculation ratio is 5:1, 10:1 and 20:1.

[0085] from Figure 6 It can be seen that the attachment efficiency of 5:1, 10:1, and 20:1 is not much different, and there is no significant difference through statistical analysis. Statistically speaking, the effects of these three groups of ratios are roughly the same. Among the several ratios, 20:1 can be used because it can save the amount of microcarriers and material costs, which is more in line with the production needs of cell cultured meat.

[0086] Experiment 2 set up experimental groups 1-8, and the other contents were the same as those in Example 5, wherein both culture medium A and culture medium B were DMEM culture medium containing 2%-15% fetal bovine serum, and the continuous stirring speed, pH and DO value were different during the cell culture stage. The cells were harvested after 9 days of culture, as shown in Table 1 below for details.

[0087] Table 1 Comparison of cells harvested after 9-day culture at different stirring speeds, pH and DO

[0088] ;

[0089] The results in the above table show that the best effect is achieved when the continuous stirring speed of the bioreactor is 150 rpm, and the pH between 7.2 and 7.4 and the DO between 40 and 80 have no significant effect on the culture results.

[0090] Experiment 3 set up experimental groups 1-4, and the other contents adopted the contents described in Example 5 of the present invention, wherein both culture medium A and culture medium B were DMEM culture medium containing 2% to 15% fetal bovine serum, and the culture temperature was used as a variable. After 9 days of culture, the cells were harvested and counted to obtain the cell density, as shown in Table 2 below.

[0091] Table 2 Cells harvested and counted after 9 days of culture with variable culture temperature

[0092] ;

[0093] From Table 2 above, it can be concluded that the best culture effect is achieved when the culture temperature is 37°C.

[0094] Experiment 4 set up experimental groups 1-8 and control groups 1-2, and the other contents in experimental groups 1-8 adopted the contents described in Example 5 of the present invention, wherein both culture medium A and culture medium B were DMEM culture medium containing 2% to 15% fetal bovine serum, and the stirring speed during the interval stirring process of the cell attachment process was a constant, and the interval time and stirring time were variables. The cell attachment process of control group 1 was not stirred, and the cell attachment process of control group 2 was continuously stirred. After 24 hours, the cells were collected and the attachment efficiency was calculated: the number of cells counted per mL / the number of microcarriers per mL.

[0095] Table 3 Comparative experiment on the effects of continuous stirring mode and interval stirring mode in bioreactor

[0096] ;

[0097] From Table 3 above, it can be concluded that the best adhesion effect is achieved when the stirring speed is 150 rpm, the stirring time is 10 min, and the interval time is 60 min.

[0098] The continuous stirring mode and the intermittent stirring mode need to be compared only during the cell attachment process. During the culture process after attachment, the culture mode must be continuous stirring. Therefore, there is no need to compare the continuous stirring mode and the intermittent stirring mode during the culture process.

[0099] Experiment 5 set up experimental groups 1-4, and the other contents were the contents described in Example 5 of the present invention, wherein medium B was a variable, medium B in experimental group 1 had no additives, medium B in experimental group 2 had 1% glutamine additives, medium B in experimental group 3 had 1% ascorbic acid diphosphate additives, and medium B in experimental group 4 had 1% ascorbic acid diphosphate and 1% glutamine additives. The cell density of each group was measured, see the attached Figure 7 .

[0100] From the attached Figure 7 It can be concluded that 1% glutamine significantly improved the attachment efficiency of porcine myoblasts, but the effect of promoting cell growth was weaker than 1% ascorbyl diphosphate. However, 1% ascorbyl diphosphate had no significant effect on the attachment efficiency of porcine myoblasts. The combined use of 1% glutamine and 1% ascorbyl diphosphate achieved a better effect in promoting the growth of porcine myoblasts.

[0101] Reaction principle in the present invention:

[0102] (1) Cell attachment

[0103] In order to solve the problem of low attachment efficiency, which is generally only about 50% in 24 hours, the technology of the present invention carries out the design idea of ​​DOE test design-test result fitting-attachment process optimization, and obtains the best pig myoblast-microcarrier attachment process based on bioreactor. At the same time, high-throughput additive screening is carried out to screen glutamine, an edible additive that promotes the attachment efficiency of pig myoblasts, which greatly improves the attachment efficiency of pig myoblasts.

[0104] (2) Cell culture process

[0105] In view of the shortcomings of low seed cell harvest density, the density in the bioreactor is lower than 10^6 cells / mL, and the long cell culture cycle due to the influence of shear force, and 10-fold proliferation in the bioreactor requires about 10 days. The technology in the present invention first carries out the design concept of DOE experimental design-experimental result fitting-culture process optimization, and obtains the optimal porcine myoblast culture process based on bioreactor. At the same time, high-throughput additive screening is carried out to screen ascorbic acid diphosphate, an edible additive that promotes the activity of porcine myoblasts. The combination of culture process and additives can make porcine myoblasts grow from 10^5 cells / mL to 10^6 cells / mL in a bioreactor in just 8 days;

[0106] (3) The cell harvest density of porcine myoblasts (Beijing black pigs) in the scheme of the present invention is above 1*10^6 cells / mL, which can basically meet the density requirements for cell culture meat production. And by optimizing the porcine myoblast-microcarrier attachment process, the attachment efficiency is increased by 20%, cell loss is reduced, and production costs are saved. By optimizing the porcine myoblast culture process, the cell culture efficiency is improved and time costs are saved.

[0107] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A high-density culture method of porcine myoblasts based on a bioreactor, characterized in that: The following steps are involved: (1) Resuscitation of porcine myoblasts: Beijing black pig myoblasts stored in liquid nitrogen were placed in a water bath for resuscitation, centrifuged and resuspended in culture medium A and inoculated in a two-dimensional culture container for 2-3 generations; the culture medium A is DMEM culture medium containing 2%-15% fetal bovine serum; (2) Cell-microcarrier attachment treatment: remove the culture medium, wash and digest the cells into a cell suspension, then inoculate the centrifuged cells and microcarriers in a certain ratio in a bioreactor, add culture medium B and use the bioreactor interval stirring mode for cell-microcarrier attachment treatment, and culture for 22-26 hours; the cell-microcarrier inoculation ratio is 5:1, 10:1 or 20:1; the microcarrier is Huakan 3D TableTrix microcarrier; The culture medium B is a DMEM culture medium containing 2%-15% fetal bovine serum and supplemented with 1% ascorbic acid diphosphate and 1% glutamine; the stirring speed of the bioreactor is 150 rpm, the interval stirring mode conditions are an interval time of 60 min, and a stirring time of 10 min; the culture temperature in the bioreactor is 37° C.-41° C., the pH value in the bioreactor is 7.2-7.4, and the dissolved oxygen value is 40% - 80%; (3) Agitation conversion and counting: the bioreactor's interval agitation mode is switched to a continuous agitation mode, and counting is performed once every 1-2 days until the expected harvest density is reached; the rotation speed of the bioreactor in the continuous agitation mode is 150 rpm; (4) Collect cells by centrifugation; In the above steps (1), (2) and (4), the conditions for collecting cells by centrifugation are a centrifugal speed of 1000-2000 rpm and a centrifugal time of 5-15 min.

2. The method for high-density culture of porcine myoblasts based on a bioreactor according to claim 1, characterized in that: The cells in step (2) are digested by using trypsin, the digestion time is 5-30 min, and the digestion temperature is 35-38°C.

3. The method for high-density culture of porcine myoblasts based on a bioreactor according to claim 1, characterized in that: The culture temperature in step (2) is 37°C.

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