Cell culture system and method

Abstract

The invention relates to a method and a device for culturing cells. Methods and apparatus for providing seed culture, including a chemostat, turbidistat, or stabiliser, as well as methods and apparatus for medium replacement using gravitational settling separation during culture are provided.

Description

Technical Field

[0001] This invention relates to systems and methods for cell culture. More particularly, this invention relates to systems and methods for increasing cell volume and yield within an optimal timeframe. Background Technology

[0002] Providing cells at optimal condition and density is time-consuming and labor-intensive. For cell culture to be commercially viable, the required cell density needs to be achieved within the shortest possible timeframe. Time-consuming human interventions during the process to achieve this can reduce the efficiency of cell culture.

[0003] One example is producing enough cells to serve as starter cultures for production-scale bioreactors. This is commonly referred to as seed culture (seed culture, seed amplification system, or seed train). Seed culture involves culturing a small number of cells until they reach a sufficient cell density and are in the exponential growth phase, making them optimal for use as starter cultures in production bioreactors. This is a time-consuming and labor-intensive process, and the cells are only in the exponential phase for a short window. Once the cells have been transferred from the seed culture to larger production vessels, the bioreactor used for the seed culture must be cleaned up before it can be reused. Therefore, there is a pressing need to optimize this process and more efficiently provide cells in the exponential phase that are readily available for inoculating larger production reactors.

[0004] Another problem with current cell culture methods and systems is that nutrients in the growth medium are depleted over time and need to be replaced to achieve high-density cell cultures. State-of-the-art techniques for cell clarification are typically based on centrifugation, perfusion, or filtration, and therefore require additional equipment. These techniques are thus laborious and time-consuming. Furthermore, mechanical intervention in the culture system can lead to cell damage and death, which can be counterproductive and increase culture time.

[0005] The applicant seeks to provide cell culture methods and systems that address at least these issues to create time-efficient cell culture methods and systems that require less human intervention during cell production. Summary of the Invention

[0006] According to a first aspect of the present invention, a method for providing seed culture is provided, the method comprising:

[0007] Culture mammalian cells in a chemostat or turbidistat.

[0008] Continuous culture is carried out using a chemostat or turbidistat.

[0009] In one implementation, the method may further include changing the dilution rate.

[0010] In one implementation, the dilution rate can be approximately 0.026 days. -1 In one implementation, the dilution rate is approximately 0.28 days. -1 From approximately 1.11 days -1 Optionally, the dilution rate is 0.026 hours. -1 .

[0011] In one implementation, cell density and / or nutrient concentration and / or metabolite levels can be kept constant over time.

[0012] In one implementation, the cells can be maintained in the exponential phase.

[0013] In one embodiment, the volume of the chemistat or turbidistat can be from about 0.05 liters to about 1 liter.

[0014] In one embodiment, the volume of the chemistat or turbidistat can be from about 0.1 liters to about 50 liters.

[0015] In one implementation, the volume of the chemistat or turbidistat can be from about 0.5 liters to about 25 liters.

[0016] In one embodiment, the volume of the chemistat or turbidistat can be from about 1 liter to about 10 liters.

[0017] In one embodiment, the chemostat or turbidistat may include an inflow line for continuously supplying fresh culture medium to the culture vessel and an outflow line for continuously removing fresh culture medium from the culture vessel.

[0018] In one embodiment, the method may further include the step of seeding the chemostat or turbidistat at about 50,000 to about 150,000 cells / mL.

[0019] In one embodiment, the method may further include the step of seeding the chemostat or turbidistat at about 50,000 to about 100,000 cells / mL.

[0020] According to a second aspect of the invention, a chemistat or turbidity stabilizer is provided for use in the invention.

[0021] According to a third aspect of the present invention, a method for replacing culture medium for cell culture is provided, the method comprising the following steps:

[0022] Culture cells in a container.

[0023] Gravity settling separation, and

[0024] Culture medium replacement.

[0025] In one embodiment, the container may include a stirrer, an internal dip-tube attached to an outflow line for removing culture medium from the culture container, and an inflow line for supplying fresh culture medium to the culture container.

[0026] In one embodiment, the method may further include the step of removing agitation by disabling the agitator.

[0027] In one embodiment, the method may further include the step of concentrating cells using gravity sedimentation separation to form concentrated cell aggregations.

[0028] In one implementation, the step of removing agitation by disabling the agitator can be controlled by a biomass detector and / or a timer and / or a metabolite detector.

[0029] In one embodiment, the culture medium replacement step includes extending the aspirator tube into the container.

[0030] In one implementation, the extraction tube can extend up to approximately 95% of the container height.

[0031] In one implementation, the extraction tube can extract the growth medium from the container after the gravity sedimentation separation step.

[0032] In one implementation, the container can be replenished with fresh culture medium after the culture medium extraction step.

[0033] In one embodiment, the method may further include steps of controlling heat and / or gas bubbling.

[0034] In one embodiment, the container has a volume of about 1 liter to about 50,000 liters.

[0035] In one embodiment, the container's volume can be from about 50 liters to about 10,000 liters.

[0036] In one embodiment, the container's volume can be from about 100 liters to about 1,000 liters.

[0037] In one implementation, the cells may have a high tendency to form aggregates.

[0038] In one implementation, the cells can be genetically modified.

[0039] In one implementation, the cell may be eukaryotic.

[0040] In one implementation, the cell may be mammalian.

[0041] In one implementation, gravity settling separation can take approximately 5 minutes to approximately 3 hours.

[0042] In one implementation, gravity sedimentation separation can be carried out for approximately 20 minutes to approximately 2 hours.

[0043] According to a fourth aspect of the invention, a bioreactor is provided, the bioreactor comprising an inflow line for supplying fresh culture medium to a culture vessel, an outflow line for removing fresh culture medium from the culture vessel, and a vessel, wherein the vessel includes a stirrer and an internal suction tube in fluid communication with the outflow line.

[0044] In one embodiment, the bioreactor also includes a biomass detector and / or a timer and / or a metabolite detector.

[0045] In one implementation, the extraction tube may be extendable.

[0046] In one implementation, the extraction tube may extend from about 70% to about 95% of the container height.

[0047] In one embodiment, the bioreactor may also include equipment (devices) for controlling heat and / or gas bubbling.

[0048] In one implementation, the device can be reversibly activated.

[0049] In one embodiment, the volume of the bioreactor can be from about 1 liter to about 50,000 liters.

[0050] In one embodiment, the volume of the bioreactor can be from about 50 liters to about 10,000 liters.

[0051] In one embodiment, the volume of the bioreactor can be from about 100 liters to about 1,000 liters.

[0052] In one implementation, the bioreactor can be used to culture cells that have a high tendency to form aggregates.

[0053] In one implementation, the cells can be genetically modified.

[0054] In one implementation, the cell may be eukaryotic.

[0055] In one implementation, the cell may be mammalian.

[0056] According to a fifth aspect of the present invention, a cell culture method is provided, the cell culture method comprising:

[0057] The present invention provides a method for seed cultivation, and

[0058] The culture medium replacement method according to the present invention.

[0059] In one embodiment of the cell culture method, the seed culture can be provided in a chemostat or turbidistat, and the culture medium replacement can be performed at a predetermined time point after the incubation of the seed cultured cells from the chemostat or turbidistat has been carried out in a separate additional bioreactor.

[0060] According to a fifth aspect of the invention, a cell culture system is provided, the cell culture system comprising a chemostat or turbidistat according to the invention and a bioreactor according to the invention. Attached Figure Description

[0061] The invention will now be described with reference to the accompanying non-limiting drawings.

[0062] Figure 1 Schematic diagram of unit operation and FTE requirements for a 600L final bioreactor, and FTE requirements for typical seed culture.

[0063] Figure 2 Cell density, viability, and cumulative doubling time in batch reactors R6 and R9.

[0064] Figure 3 Cell density during the 42-day chemostat period, showing two steady-state periods and key initiatives.

[0065] Figure 4 Cell viability and mean cell diameter during 42 days of chemostatinization.

[0066] Figure 5 Cell images at 4x, 10x and 20x magnification on day 14 of the chemistat.

[0067] Figure 6 A schematic diagram depicts cell transfer using a 3L chemostat and batch reactor during 600L seed culture.

[0068] Figure 7 Cell density, % viability, and cumulative doubling time in 20L batch reactors R11 (inoculated from both the chemostat and the batch reactor) and R14 (inoculated from separate chemostats).

[0069] Figure 8BP_TB_006 "Improved" cell growth distribution curve.

[0070] Figure 9 BP_TB_006 “Improved” waste metabolite and nutrient distribution curves.

[0071] Figure 10 BP_TB_005 “Typical” cell growth curve.

[0072] Figure 11 BP_TB_005 Waste metabolites and nutrient curves.

[0073] Figure 12 Other examples of LPH growth curves. Detailed Implementation

[0074] In one aspect of the invention, a method for providing seed culture is provided, the method comprising culturing mammalian cells in a chemostat, turbidostat, or auxostat, using a chemostat, turbidostat, or auxostat for continuous culture. A chemostat is a bioreactor into which fresh culture medium is continuously added while a culture medium containing residual nutrients, metabolic end products, and microorganisms is continuously removed at the same rate to maintain a constant culture volume. A turbidostat operates in a similar manner to a chemostat but maintains a constant culture turbidity. A auxostat is also similar to a chemostat but uses feedback from measurements taken at the growth chamber to control the culture medium flow rate, thereby maintaining the measurements at a constant value.

[0075] Traditionally, seed cultures are run in batches, with cells transferred to subsequent processes (typically by increasing container volume) once an acceptable density is reached. Typically, cells will only be in an acceptable optimal growth kinetic state for cell transfer for a short period before nutrients begin to deplete or metabolites accumulate and cell growth kinetics decline. Additionally, there is a risk that cells will not grow under acceptable kinetics and therefore cannot be used as a cell source.

[0076] Operating under a chemostat eliminates the risk of cells failing to grow under acceptable kinetics and maintains growth kinetics at their optimal levels. Nutrients are maintained at levels sufficient for cell growth, while metabolites are removed and kept at low levels that do not inhibit cell proliferation. Chemostat culture also operates as a closed system. Compared to traditional seed culture, it achieves these benefits with minimal intervention, reducing the labor-intensive time required to obtain a source of healthy cells. Furthermore, the lack of human intervention results in reduced microbial contamination of the culture medium (which can be introduced otherwise through routine treatments). Therefore, antibiotic-free media can be used.

[0077] Therefore, chemostats, turbidistats, or steady-state reactors can be used for efficient seed culture because fresh medium is added constantly and consumed medium is removed constantly, maintaining the cells in the exponential phase. This means that cells are always readily available as seed culture for use in larger-scale production bioreactors.

[0078] The chemostat requires a continuous feed of fresh culture medium at the same rate as the removal of the culture from the bioreactor. The rate at which the culture is replaced by fresh medium is called the dilution rate. Since live cells will be removed from the bioreactor while they are still proliferating, it is important to ensure that the dilution rate is not so high that the culture is removed faster than the cells proliferate, resulting in a washout where all cells eventually leave the system. In one embodiment, the dilution rate is approximately 0.026 days. -1 In one embodiment, 561 mL / day of fresh culture medium is added and 561 mL / day of culture is discharged from the system. In one embodiment, the dilution rate is approximately 0.026 hours. -1 In one implementation, the dilution rate is approximately 0.624 days. -1 In one embodiment, the method further includes changing the dilution rate. In one embodiment, the dilution rate is approximately 0.05 days. -1 From approximately 0.011 days -1 Approximately 0.049 days -1 Approximately 0.048 days -1 Approximately 0.047 days -1 Approximately 0.047 days -1 Approximately 0.046 days -1 Approximately 0.045 days -1 Approximately 0.044 days -1 Approximately 0.043 days -1 Approximately 0.042 days -1 Approximately 0.041 days -1 Approximately 0.04 days -1 Approximately 0.039 days -1Approximately 0.038 days -1 Approximately 0.037 days -1 Approximately 0.036 days -1 Approximately 0.035 days -1 Approximately 0.034 days -1 Approximately 0.033 days -1 Approximately 0.032 days -1 Approximately 0.031 days -1 Approximately 0.030 days -1 Approximately 0.030 days -1 0.029 days -1 0.028 days -1 0.027 days -1 0.026 days -1 Approximately 0.025 days -1 0.024 days -1 0.023 days -1 0.022 days -1 0.021 days -1 Approximately 0.020 days -1 Approximately 0.019 days -1 Approximately 0.018 days -1 Approximately 0.017 days -1 Approximately 0.016 days -1 Approximately 0.015 days -1 Approximately 0.014 days -1 Approximately 0.013 days -1 Approximately 0.012 days -1 Approximately 0.011 days -1 Approximately 0.01 days -1 In one implementation, the dilution rate is approximately 0.28 days. -1 From approximately 1.11 days -1 In one implementation, the dilution rate is approximately 0.10 days. -1 Approximately 0.12 days -1 Approximately 0.14 days -1 Approximately 0.16 days -1 Approximately 0.18 days -1 Approximately 0.20 days -1 Approximately 0.22 days -1 Approximately 0.24 days -1 Approximately 0.26 days -1 Approximately 0.28 days -1 Approximately 0.30 days -1 Approximately 0.32 days -1 Approximately 0.34 days -1 Approximately 0.36 days -1 Approximately 0.38 days -1 Approximately 0.40 days-1 Approximately 0.42 days -1 Approximately 0.44 days -1 Approximately 0.46 days -1 Approximately 0.48 days -1 Approximately 0.50 days -1 Approximately 0.52 days -1 Approximately 0.54 days -1 Approximately 0.56 days -1 Approximately 0.58 days -1 Approximately 0.60 days -1 Approximately 0.62 days -1 Approximately 0.64 days -1 Approximately 0.66 days -1 Approximately 0.68 days -1 Approximately 0.70 days -1 Approximately 0.72 days -1 Approximately 0.74 days -1 Approximately 0.76 days -1 Approximately 0.78 days -1 Approximately 0.80 days -1 Approximately 0.82 days -1 Approximately 0.84 days -1 Approximately 0.86 days -1 Approximately 0.88 days -1 Approximately 0.90 days -1 Approximately 0.92 days -1 Approximately 0.94 days -1 Approximately 0.96 days -1 Approximately 0.98 days -1 Approximately 1.00 days -1 Approximately 1.02 days -1 Approximately 1.04 days -1 Approximately 1.06 days -1 Approximately 1.08 days -1 Approximately 1.10 days -1 Approximately 1.12 days -1 Approximately 1.14 days -1 Approximately 1.16 days -1 Approximately 1.18 days -1 Approximately 1.20 days -1 Approximately 1.30 days -1 Approximately 1.40 days -1 Approximately 1.50 days -1 Approximately 1.60 days -1 Approximately 1.70 days -1 Approximately 1.80 days -1 Approximately 1.90 days -1 Approximately 2.00 days -1 In one implementation, the dilution rate is approximately 0.0115 hours.-1 From approximately 0.0463 hours -1 In one implementation, the dilution rate is approximately 0.005 hours. -1 Approximately 0.0075 hours -1 Approximately 0.01 hours -1 Approximately 0.0115 hours -1 Approximately 0.012 hours -1 Approximately 0.014 hours -1 Approximately 0.016 hours -1 Approximately 0.018 hours -1 Approximately 0.020 hours -1 Approximately 0.022 hours -1 Approximately 0.024 hours -1 Approximately 0.026 hours -1 Approximately 0.028 hours -1 Approximately 0.030 hours -1 Approximately 0.032 hours -1 Approximately 0.034 hours -1 Approximately 0.036 hours -1 Approximately 0.038 hours -1 Approximately 0.040 hours -1 Approximately 0.042 hours -1 Approximately 0.044 hours -1 Approximately 0.046 hours -1 Approximately 0.050 hours -1 Approximately 0.060 hours -1 Approximately 0.070 hours -1 Approximately 0.080 hours -1 Approximately 0.090 hours -1 Approximately 0.10 hours -1 .

[0079] In some embodiments, cells are seeded into the bioreactor, and the system operates dynamically, with cell density increasing over time. Eventually, a steady state is reached where cell density, as well as nutrient and metabolite levels, do not change over time. At any given time point, the only required intervention is to replenish the culture medium tank and harvest the effluent container. In one embodiment, cell density and / or nutrient concentration and / or metabolite levels remain constant over time. In one embodiment, the cell density is approximately 0.05 × 10⁻⁶. 6 Cells / ml to approximately 200 × 10⁻⁶ 6 cells / ml, approximately 1×10 6 cells / ml, approximately 2×10⁻⁶ 6 cells / ml, approximately 3 × 10⁻⁶ 6 cells / ml, approximately 4 × 10⁻⁶6 cells / ml, approximately 5 × 10⁻⁶ 6 cells / ml, approximately 10 × 10 6 cells / ml, approximately 20 × 10⁻⁶ 6 cells / ml, approximately 30 × 10⁻⁶ 6 cells / ml, approximately 40 × 10⁻⁶ 6 cells / ml, approximately 50 × 10⁻⁶ 6 cells / ml, approximately 60 × 10⁻⁶ 6 cells / ml, approximately 70 × 10⁻⁶ 6 cells / ml, approximately 80 × 10⁻⁶ 6 cells / ml, approximately 90 × 10⁻⁶ 6 cells / ml, approximately 100 × 10⁻⁶ 6 cells / ml, approximately 110 × 10⁻⁶ 6 cells / ml, approximately 120 × 10⁻⁶ 6 cells / ml, approximately 130 × 10⁻⁶ 6 cells / ml, approximately 140 × 10⁻⁶ 6 cells / ml, approximately 150 × 10⁻⁶ 6 cells / ml, approximately 160 × 10⁻⁶ 6 cells / ml, approximately 170 × 10⁻⁶ 6 cells / ml, approximately 180 × 10⁻⁶ 6 cells / ml, approximately 190 × 10⁻⁶ 6 cells / ml, approximately 200 × 10⁻⁶ 6 Cells / ml. In one embodiment, the chemostat, turbidistat, or stabilizer can operate for approximately 42 days. In one embodiment, the chemostat, turbidistat, or stabilizer can operate for an extended period of time, provided sterility is maintained. In one embodiment, the chemostat, turbidistat, or stabilizer can operate for approximately 1 day, approximately 5 days, approximately 10 days, approximately 15 days, approximately 20 days, approximately 25 days, approximately 30 days, approximately 35 days, approximately 40 days, approximately 45 days, approximately 50 days, approximately 55 days, approximately 60 days, approximately 3 months, approximately 3.5 months, approximately 4 months, approximately 4.5 months, approximately 5 months, approximately 5.5 months, or approximately 6 months.

[0080] To culture cells, various chemostats, turbidistats, or stabilizers of different sizes and designs can be used. For example, in some embodiments, the culture volume of the chemostat, turbidistat, or stabilizer is approximately 0.05 L to approximately 1 L, 0.075 L to approximately 1 L, 0.1 L to approximately 1 L, 0.2 L to approximately 1 L, 0.25 L to approximately 1 L, approximately 0.5 L, approximately 1 L, approximately 1.5 L, approximately 2 L, approximately 3 L, approximately 4 L, approximately 5 L, approximately 6 L, approximately 7 L, approximately 8 L, approximately 9 L, approximately 10 L, approximately 15 L, approximately 20 L, approximately 25 L, approximately 30 L, approximately 35 L, approximately 40 L, or approximately 45 L. Approximately 50 liters, approximately 55 liters, approximately 60 liters, approximately 65 liters, approximately 70 liters, approximately 75 liters, approximately 80 liters, approximately 85 liters, approximately 90 liters, approximately 95 liters, approximately 100 liters, approximately 150 liters, approximately 200 liters, approximately 250 liters, approximately 300 liters, approximately 350 liters, approximately 400 liters, approximately 450 liters, approximately 500 liters, approximately 550 liters, approximately 600 liters, approximately 650 liters, approximately 700 liters, 750 liters, approximately 800 liters, approximately 850 liters, approximately 900 liters, approximately 950 liters, or approximately 1,000 liters. In some embodiments, the culture volume of the container is approximately 0.1 liters to approximately 0.25 liters, approximately 0.1 liters to approximately 0.5 liters, approximately 0.1 liters to approximately 1 liter, approximately 0.1 liters to approximately 1.5 liters, approximately 0.1 liters to approximately 2 liters, approximately 0.1 liters to approximately 2.5 liters, approximately 0.1 liters to approximately 3 liters, approximately 0.1 liters to approximately 3.5 liters, approximately 0.1 liters to approximately 4 liters, approximately 0.1 liters to approximately 4.5 liters, approximately 0.1 liters to approximately 5 liters, approximately 0.1 liters to approximately 7.5 liters, approximately 0.1 liters to approximately 10 liters, approximately 0.1 liters to approximately 15 liters, approximately 0.1 liters to approximately 20 liters, approximately 0.1 liters to approximately 25 liters, approximately 0.1 liters to approximately 30 liters, approximately 0.1 liters to approximately 35 liters, approximately 0.1 liters to approximately 45 liters, and approximately 0.1 liters to approximately 45 liters. Up to about 50 liters, about 0.5 liters to about 1 liter, about 0.5 liters to about 1.5 liters, about 0.5 liters to about 2 liters, about 0.5 liters to about 2.5 liters, about 0.5 liters to about 3 liters, about 0.5 liters to about 3.5 liters, about 0.5 liters to about 4 liters, about 0.5 liters to about 4.5 liters, about 0.5 liters to about 5 liters, about 0.5 liters to about 7.5 liters, about 0.5 liters to about 10 liters, about 0.5 liters to about 15 liters, about 0.5 liters to about 20 liters, about 0.5 liters to about 25 liters, about 1 liter to about 2 liters, about 1 liter to about 3 liters, about 1 liter to about 4 liters, about 1 liter to about 5 liters, about 1 liter to about 6 liters, about 1 liter to about 7 liters, about 1 liter to about 8 liters and about 1 liter to about 9 liters or about 1 liter to about 10 liters.

[0081] In one embodiment, the chemostat, turbidity stabilizer, or constant includes an inflow line for continuously supplying fresh culture medium to the culture vessel and an outflow line for continuously removing fresh culture medium from the culture vessel.

[0082] In some implementations, gravity settling separation is carried out for about 5 minutes to about 3 hours, about 5 minutes to about 2 hours, about 5 minutes to about 1 hour, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 20 minutes to about 2 hours, about 20 minutes to about 1 hour, about 20 minutes to about 45 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 3 hours, about 30 minutes to about 2 hours, and about 30 minutes to about 1 hour.

[0083] In one embodiment, the method of providing seed culture further includes the step of inoculating a chemostat, turbidistat, or stabilizer at about 50,000 to about 150,000 cells / mL. Low concentrations of cells have been found to be advantageous as starter cultures for chemostats or turbidistats.

[0084] According to one aspect of the present invention, a chemostat, turbidity regulator or stabilizer is provided for the above-mentioned method of providing seed culture.

[0085] According to one aspect, the present invention may relate to a culture medium replacement method for cell culture, the culture medium replacement method comprising the steps of: culturing cells in a container, gravity sedimentation separation, and culture medium replacement. Such a method allows for “light partial harvesting” of cells cultured in the container and is advantageous because it does not exert as much stress on the cells as centrifugation-based cell clarification methods, and this method is hereinafter referred to as “light partial harvesting”.

[0086] Common techniques for cell clarification are typically based on centrifugation or filtration, which require additional equipment and can cause cell damage. "Small fraction harvesting" achieves high levels of cell retention by taking advantage of the tendency of cells to settle under gravity in a still liquid. The only additional equipment required is a straight or segmented internal aspirator tube for aspirating used culture medium from the reactor while preventing cell clumps from leaving the reactor.

[0087] In one embodiment, the cells used in the culture medium replacement method have a high tendency to form aggregates. For example, suspension-adapted cells have a high tendency to form aggregates and settle in the absence of dynamic input. Suspension-adapted cells were previously adhesive cells but adapted for suspension cell culture. They retain some characteristics of adhesive cells, such as increased aggregation. In one embodiment, the cells have increased amounts of factors involved in aggregation, such as integrins. In one embodiment, the cells are genetically modified. For example, the cells may be immortalized. In one embodiment, the cells are genetically edited. In one embodiment, the cells are eukaryotic. In one embodiment, the cells are mammalian. For example, suspension-adapted cultured meat cells can be used in the method of the present invention. Growing cultured meat cells in suspension culture can be advantageous (compared to growing them in adhesive culture) because the cells can grow in large quantities.

[0088] The "small-scale harvest" technique can be implemented at prescribed time intervals throughout the culture or in response to the accumulation of metabolites. Therefore, "small-scale harvest" allows cells to grow to densities (or concentrations) higher than those achievable otherwise through simple batch culture or fed-batch culture, and thus can be considered a bioprocess enhancement strategy. Surprisingly, the present invention performs as well as it has, because conventional wisdom holds that cell sedimentation would be time-consuming and would damage or kill cells due to lack of oxygenation (due to the agitator being shut off during sedimentation). However, this is not the case, and the inventors have demonstrated that cells are not adversely affected by the process. In fact, the process benefits cells by providing fresh culture medium without potential contamination. The absence of human intervention in the closed system of the bioreactor is highly beneficial for preventing bacterial contamination.

[0089] In addition, by enhancing the cell culture process, the load on downstream processes (DSP) is reduced, which is economically advantageous.

[0090] According to another aspect of the invention, a bioreactor is provided, the bioreactor comprising an inflow line for supplying fresh culture medium to a culture vessel, an outflow line for removing fresh culture medium from the culture vessel, and a vessel, wherein the vessel includes a stirrer and an internal suction tube in fluid communication with the outflow line. Figure 1 The bioreactor can be used in the aforementioned methods of the present invention as described above.

[0091] Therefore, the method and related bioreactor of the present invention are advantageous because they are highly efficient and effective (capable of achieving high levels of cell retention), scalable (in terms of operational, functional and economic feasibility), and non-damaging to cells.

[0092] In one embodiment, the bioreactor vessel includes a stirrer, an internal suction line attached to an effluent line for removing culture medium from the culture vessel, and an influent line for supplying fresh culture medium to the culture vessel. In some embodiments, stirring in the vessel can be removed by deactivating the stirrer. Deactivation of the stirrer causes cells in the vessel to begin to aggregate and settle to the bottom of the vessel due to gravity within a set time period. Therefore, the method includes the step of concentrating cells to form concentrated cell aggregates using gravity sedimentation separation.

[0093] The discontinuation of the stirrer can be controlled using a biomass detector and / or a timer and / or a metabolite detector. As culture time progresses, nutrients in the medium will be depleted, and eventually, the medium will be unable to support the continued growth of cells in the logarithmic growth phase. Therefore, a metabolite detector can be used to determine when nutrients are depleted and to deactivate the stirrer, allowing gravity sedimentation to occur and the container to be ready for removal of the nutrient-depleted medium and replenishment with fresh medium. Alternatively, a biomass detector or probe can be used to determine the precise amount of time required for sedimentation based on biomass measurements. Alternatively, this can be accomplished using a timer at set time points.

[0094] In one embodiment, the culture medium replacement step includes extending a suction tube into the container. In some embodiments, the suction tube is a specific geometry and length to remove used culture medium without aspirating cell aggregates into the effluent stream. In some embodiments, the suction tube extends to approximately 95% of the container height. In one embodiment, the suction tube extends from the headplate to 10% to 30% of the container height above the bottom of the bioreactor. The suction tube is used to remove used culture medium from the reactor. The internal suction tube can be sized according to the geometry of the container used. In some embodiments, the suction tube should extend from the headplate to some point along the length (height) of the container.

[0095] Following the gravity sedimentation separation step, the growth medium can be extracted from the container using a pick tube. After the medium extraction step using the pick tube, fresh medium can be replenished to the container.

[0096] The method of the present invention may further include steps of controlling heat and / or gas bubbling. During the "small fraction harvest," it is necessary to prevent any disturbance to the settling cells; therefore, in some embodiments, gas bubbling may be shut off. Furthermore, during the "small fraction harvest," in some embodiments, the temperature control loop supplying heat to the culture may be shut off. In some embodiments, under settling conditions, the reactor contents may not be mixed, and therefore, supplying heat during the "small fraction harvest" process may cause overheating in the reactor and the formation of "hot spots." Therefore, the bioreactor may include means for controlling heat and / or gas bubbling. In one embodiment, the bioreactor may include equipment for controlling heat and / or gas bubbling.

[0097] Different containers of varying sizes and designs can be used to culture cells. For example, in some embodiments, the culture volume of the containers is approximately 0.015 liters, approximately 0.02 liters, approximately 0.025 liters, approximately 0.03 liters, approximately 0.035 liters, approximately 0.040 liters, approximately 0.045 liters, approximately 0.5 liters, approximately 1 liter, approximately 1.5 liters, approximately 2 liters, approximately 3 liters, approximately 4 liters, approximately 5 liters, approximately 6 liters, approximately 7 liters, approximately 8 liters, approximately 9 liters, approximately 10 liters, and approximately 15 liters. Approximately 20 liters, approximately 25 liters, approximately 30 liters, approximately 35 liters, approximately 40 liters, approximately 45 liters, approximately 50 liters, approximately 55 liters, approximately 60 liters, approximately 65 liters, approximately 70 liters, approximately 75 liters, approximately 80 liters, approximately 85 liters, approximately 90 liters, approximately 95 liters, approximately 100 liters, approximately 150 liters, approximately 200 liters, approximately 250 liters, approximately 300 liters, approximately 350 liters, approximately 400 liters, approximately 450 liters, approximately 500 liters Approximately 550 liters, approximately 600 liters, approximately 650 liters, approximately 700 liters, 750 liters, approximately 800 liters, approximately 850 liters, approximately 900 liters, approximately 950 liters, approximately 1,000 liters, approximately 1,500 liters, 2,000 liters, approximately 2,500 liters, approximately 3,000 liters, approximately 3,500 liters, approximately 4,000 liters, approximately 5,000 liters, approximately 5,500 liters, approximately 6,000 liters. Approximately 7,000 liters, approximately 8,000 liters, approximately 9,000 liters, approximately 10,000 liters, approximately 15,000 liters, approximately 20,000 liters, approximately 25,000 liters, approximately 30,000 liters, approximately 40,000 liters, approximately 50,000 liters, approximately 60,000 liters, approximately 70,000 liters, approximately 80,000 liters, approximately 90,000 liters, or approximately 100,000 liters.In some embodiments, the culture volume of the container is about 1 liter to about 50 liters, about 1 liter to about 100 liters, about 1 liter to about 150 liters, about 1 liter to about 200 liters, about 1 liter to about 250 liters, about 1 liter to about 300 liters, about 1 liter to about 400 liters, about 1 liter to about 500 liters, about 1 liter to about 600 liters, about 1 liter to about 700 liters, about 1 liter to about 800 liters, about 1 liter to about 900 liters, about 1 liter to about 1,000 liters, about 1 liter to about 2,000 liters, or about 1 liter to about 300 liters. 0 liters, approximately 1 liter to approximately 4,000 liters, approximately 1 liter to approximately 5,000 liters, approximately 1 liter to approximately 10,000 liters, approximately 1 liter to approximately 20,000 liters, approximately 1 liter to approximately 30,000 liters, approximately 1 liter to approximately 40,000 liters, approximately 1 liter to approximately 50,000 liters, approximately 50 liters to approximately 100 liters, approximately 50 liters to approximately 150 liters, approximately 50 liters to approximately 200 liters, approximately 50 liters to approximately 250 liters, approximately 50 liters to approximately 300 liters, approximately 50 liters to approximately 400 liters, approximately 50 liters to approximately 500 liters, approximately 5 0 liters to approximately 600 liters, approximately 50 liters to approximately 700 liters, approximately 50 liters to approximately 800 liters, approximately 50 liters to approximately 900 liters, approximately 50 liters to approximately 1,000 liters, approximately 50 liters to approximately 2,000 liters, approximately 50 liters to approximately 3,000 liters, approximately 50 liters to approximately 4,000 liters, approximately 50 liters to approximately 5,000 liters, approximately 50 liters to approximately 10,000 liters, approximately 50 liters to approximately 20,000 liters, approximately 100 liters to approximately 150 liters, approximately 100 liters to approximately 200 liters, approximately 100 liters to approximately 250 liters Approximately 100 liters to approximately 300 liters, approximately 100 liters to approximately 400 liters, approximately 100 liters to approximately 500 liters, approximately 100 liters to approximately 600 liters, approximately 100 liters to approximately 700 liters, approximately 100 liters to approximately 800 liters, approximately 100 liters to approximately 900 liters, approximately 100 liters to approximately 1,000 liters, approximately 100 liters to approximately 2,000 liters, approximately 100 liters to approximately 3,000 liters, approximately 100 liters to approximately 4,000 liters, approximately 100 liters to approximately 5,000 liters, or approximately 100 liters to approximately 10,000 liters.

[0098] According to one aspect of the invention, a bioreactor is provided for culturing cells with a high tendency to form aggregates. For example, suspension-adapted cells have a high tendency to form aggregates and settle in the absence of power input. In one embodiment, the cells are genetically modified. In one embodiment, the cells are genetically edited. In one embodiment, the cells are eukaryotic. In one embodiment, the cells are mammalian. For example, suspension-adapted cultured meat cells can be used in the method of the invention. Growing cultured meat cells in suspension culture can be advantageous compared to growing them in adhesion culture because the cells can grow in large quantities.

[0099] The term "cultivated meat" is used herein to describe meat grown from animal cells in vitro, as opposed to meat from slaughtered animals. Additional terms that may be used in the art to describe meat grown from animal cells in vitro include cultured meat, cell-grown meat, clean meat, lab-grown meat, test tube meat, in vitro meat, tube steak, synthetic meat, cell-cultured meat, cell-grown meat, tissue-engineered meat, engineered meat, artificial meat, and manmade meat. The phrases “cell-based meat,” “slaughter-free cell-based meat,” “in vitro produced meat,” “in vitro cell-based meat,” “cultured meat,” “slaughter-free cultured meat,” “in vitro produced cultured meat,” “in vitro meat,” “invitro cultured meat,” and other similar phrases are used interchangeably herein and refer to meat produced in vitro, starting with cultured cells, and in a manner that does not involve the slaughter of animals to obtain meat directly from the animals for dietary consumption. The modified cells of this invention are suitable for human and / or non-human consumption. In some embodiments, cell-based meat is suitable for consumption by animals such as domesticated animals. Therefore, the cell biomass described herein supports the growth of “pet food” (e.g., dog food, cat food, etc.).

[0100] The terms “animal” and “non-human animal” are used interchangeably in reference to animals and cells derived therefrom, and refer only to cells of non-human animals. Cells used in this invention can be of any other animal origin. However, the cells are not human cells. Cells suitable for cell agriculture are preferably non-human animal cells that provide any source of dietary protein, fat, and / or carbohydrates.

[0101] In one embodiment of various aspects of the invention, the modified cell is a primary cell. In another embodiment of various aspects of the invention, the modified cell is a somatic cell. Any somatic cell suitable for cell agriculture (i.e., the production of animal-derived foods from cell culture) is within the scope of the invention. For example, the cell may be an adipocyte or a muscle cell. For example, the cell may be selected from one or more of the following cell types: myoblasts, fibroblasts, myofibroblasts, adipose-derived stem cells, epithelial cells, mesenchymal stem cells, satellite cells, or hepatocytes.

[0102] A cell is a cell of a non-human animal suitable for human and animal consumption. These include animals such as non-human mammals, birds, fish, crustaceans, mollusks, reptiles, amphibians, or insects. Exemplary non-human mammals include those from the following genera: bovinae, camel (Camelidae), canine (Canidae), goat (Caprae), deer (Cervidae), cat (Felidae), horse (Equidae), lagomorphs, kangaroo (Macropodidae), sheep (Oves), rodents (Rodents), or pig (Suidae). A cell can be a cell of any livestock or poultry. A cell can be from a pig, a cow (e.g., a cattle), a sheep, a goat, a bird (avine), or a fish. A cell can be from a shrimp, prawn, crab, crayfish, and / or lobster. In one implementation, the animal is a pig or a cow (e.g., domesticated cattle).

[0103] The animals used in various aspects of this invention can be animal species used in agriculture. The animal species used are domesticated animals. Such animals are listed above. In a preferred embodiment, they include pigs, cattle (e.g., domesticated cattle), poultry (e.g., chickens, turkeys, ducks, geese), sheep, goats, equines, fish, crustaceans, or mollusks.

[0104] The method of this aspect of the invention can be used in continuous cell culture systems or batch cell culture systems.

[0105] According to one aspect of the present invention, a cell culture method is provided, the cell culture method comprising a method for providing seed culture according to the methods mentioned above, and a culture medium replacement method according to the methods mentioned above.

[0106] In one embodiment, the method of providing seed culture is carried out in a chemostat, turbidistat, or stabilizer, and the culture medium replacement method is performed at a predetermined time point after the incubation of cells from the seed culture in the chemostat, turbidistat, or stabilizer has been carried out in a separate additional bioreactor. The combination of providing seed culture using a chemostat, turbidistat, or stabilizer with the “small-batch harvest” method described above creates an advantageous technique for cell culture that limits human intervention and makes the entire process less laborious and time-consuming. This combined technique also ensures limited cell damage during the process and allows for high cell densities to be achieved without the drawbacks of mechanical processes used for cell clarification.

[0107] According to another aspect of the invention, a cell culture system is provided, the cell culture system comprising a chemostat, turbidistat or stabilizer as described above, and a bioreactor as described above.

[0108] As used herein, “and / or” should be considered as specifically disclosing each of the two specified features or components (whether or not accompanied by the other feature or component). For example, “A and / or B” should be considered as specifically disclosing (i) A, (ii) B, and (iii) each of A and B, as if each were listed separately herein. Unless the context otherwise indicates, the description and definition of the features set forth above are not limited to any particular aspect or embodiment of the invention, but are equally applicable to all aspects and embodiments described.

[0109] Example

[0110] Continuous seed culture (chemostat)

[0111] Upstream bioprocess seed culture is a time-consuming and labor-intensive task. It requires operators to manage culture vessels of varying sizes, from Erlenmeyer flasks to large-scale bioreactors. Seed culture also presents points of increased risk during cross-scale material transfers (e.g., when cells are thawed in a biosafety cabinet and transferred to culture vessels).

[0112] At Ivy Farm, typical seed culture, ranging from Erlenmeyer flask scale to a 600L pilot-scale bioreactor, employs... Figure 1 The format is as follows. Six FTEs are required to manage the seed culture container / bioreactor.

[0113] A key factor in successful bioreactor seed culture is ensuring that cells maintain favorable properties, enabling them to proliferate at every stage of the process. To this end, we employ the following selection criteria when deciding whether to transfer the cell population to the next (larger-scale) stage of the bioreactor process.

[0114] Table 1: Cell population selection criteria for cell transfer in seed culture

[0115]

[0116] During batch culture, careful timing is necessary to ensure cells do not exit their exponential growth phase; this is why the cumulative doubling time is set to <30 hours. For example, Figure 2 Reactor R6 in the middle of the bioreactor had exited its exponential growth phase (from day 3 to day 4). The cumulative doubling time of the cell population no longer met the criteria for transfer within our seed culture. This resulted in a short operating window (less than one day) during which sufficient cells were proliferating at the required growth rate to inoculate the next bioreactor-scale in the seed culture.

[0117] To extend the operational window for transferring seed culture cells, a chemostat was constructed. The chemostat was set at a dilution rate of 0.55 L / day (for a 1 L working volume) and a frequency of 0.05 × 10⁻⁶. 6 Seeding was performed at 100 cells / ml. Although seeding the chemostat at such a low density is unusual, we hypothesize that the lower seeding density for the chemostat maximizes the percentage of cells exhibiting the lag phase. The dilution rate used in the chemostat ensures precise control over cell population doubling time. When using a dilution rate of 0.55 vvd (e.g., ... Figure 2 When the chemostat used (as shown) is employed, only cell populations capable of multiplying at a 30-hour doubling time are able to maintain a stationary phase during cell growth. If the cell density in the bioreactor continues to increase (regardless of this dilution rate), they are multiplying at a doubling time of less than 30 hours. This widens the operating window, thereby allowing for the acquisition of inoculum for transfer within seed culture.

[0118] Figure 3 The ability to maintain a chemostat was demonstrated, where cells were in either a quiescent or logarithmic growth phase, as is typical for most operating bioreactors. This was performed at a dilution rate of 0.55 vvd (equivalent to a doubling time of 30 hours) for a duration of >40 days. The chemostat was demonstrated in a seed culture reactor that provided cells meeting the growth kinetics standards for 600L seed culture during a 40-day window (rather than <1 day). Cell density during the chemostat period was >2 × 10⁻⁶ cells / day during the quiescent phase. 6 Cells / ml. The cell density within the chemostat will vary as it is used multiple times to inoculate a 20L reactor.

[0119] During chemostatinization, cells met the % viability (>95%) and cell size (14–16 μm) criteria for all periods during which cells were in the growth or quiescent phase. Days 16–19 represent the only days during which a decrease in cell density was observed (due to degradation of some culture medium components caused by deviations in storage conditions). Cell morphology was shown in… Figure 5 In the middle stage, although cell morphology is subjective, these cells will pass morphological criteria given the presence of live cells and low levels of cell debris. In addition to providing nutrients and removing waste metabolites (providing a favorable environment for cells), continuous removal of culture medium from the chemostat will also help reduce the amount of cell debris present.

[0120] Figure 6 An example of the application of chemostats in seed culture is given. Cells were transferred from chemostats (R5) and batch culture (R9) to two 20L reactors as part of seed culture into a 600L bioreactor.

[0121] Figure 7 Growth curves of seeds from two 20L reactors using a chemistat are shown. These 20L reactors were then used to inoculate a 600L reactor.

[0122] 2: Partial Harvest (LPH) Medium Replacement Method

[0123] Waste metabolites have always been a challenge in meat cell culture. Certain waste metabolites (such as lactate and ammonium) inhibit cell proliferation when reached at specific concentrations during culture. Waste metabolites are unavoidable in mammalian cell culture, and removing or reducing them can present an invasive and difficult challenge. Typical methods involve complete culture medium replacement, but this is highly invasive and carries a high risk of contamination and cell loss.

[0124] "Partial Low-Volume Harvest" (LPH) is an in-situ method that replaces a controlled volume of culture medium while preserving cells without invasive separation techniques (i.e., centrifugation). Cells are allowed to passively settle within the bioreactor for up to 2.5 hours, and a portion of the supernatant from the used medium is removed and replaced with fresh medium. This allows cultures to undergo complete medium replacement with minimal cell loss and low risk of contamination.

[0125] During the LPH process, all control loops within the bioreactor (pH control, DO control, stirrer, and temperature) are shut off, and the cells in the culture are allowed to settle. Cells will settle for at least 2.5 hours, and the settling progress will be closely monitored using a capacitive biomass probe (Aber Futura). As the cells settle, the biomass probe will fall back to its zero point, indicating that all cells have settled below the probe and therefore below the culture medium outlet tube used for replacement. The culture medium is then removed at 80% of the working volume length and subsequently replaced with fresh culture medium.

[0126] BP_TB_006 was the first attempt at LPH. Figure 8 The cell growth distribution curves are shown, demonstrating the extended growth period achievable through LPH. Figure 9 The reduction in waste metabolites caused by culture medium replacement is shown.

[0127] Figure 10 and Figure 11 Typical growth curves and nutrient / metabolite curves for typical cultures are shown.

[0128] Figure 12 Other examples of LPH growth curves are shown.

[0129] Figures 8 to 11 This demonstrates that LPH is a method to increase cell density while significantly reducing the risk of contamination and minimizing the time cells spend outside the culture vessel. Compared to typical culture in BP_TB_005 (where the peak cell count is only slightly above 1E6 cells / mL), the peak was successfully increased to 5E6 cells / mL using the LPH method. Furthermore, ammonia, one of the most hazardous waste metabolites, was reduced by 80%, allowing cells to continue growing without inhibition.

Claims

1. A method for providing seed culture, the method comprising: Culture mammalian cells in a chemostat, turbidistat, or stabilizer. Continuous culture is carried out using the aforementioned chemostat, turbidistat, or stabilizer.

2. The method according to claim 1, further comprising changing the dilution rate.

3. The method of claim 2, wherein the dilution rate is approximately 0.28 days. -1 From approximately 1.11 days -1 Optionally, the dilution rate is 0.026 hours. -1 .

4. The method according to any one of the preceding claims, wherein cell density and / or nutrient concentration and / or metabolite level remain constant over time.

5. The method according to any one of the preceding claims, wherein the cells are maintained in the exponential phase.

6. The method according to any one of the preceding claims, wherein the volume of the chemistat, turbidity stabilizer or constant is about 0.05 liters to about 1 liter.

7. The method according to any one of the preceding claims, wherein the volume of the chemistat, turbidity stabilizer or constant is from about 0.1 liters to about 50 liters.

8. The method according to any one of claims 1 to 6, wherein the volume of the chemistat, turbidity stabilizer or steady-state device is from about 0.5 liters to about 25 liters.

9. The method according to any one of claims 1 to 6, wherein the volume of the chemistat, turbidity regulator or stabilizer is about 1 liter to about 10 liters.

10. The method according to any one of the preceding claims, wherein the chemostat, turbidity stabilizer or constant includes an inflow line for continuously supplying fresh culture medium to the culture vessel and an outflow line for continuously removing fresh culture medium from the culture vessel.

11. The method according to any one of the preceding claims, further comprising the step of seeding the chemostat, turbidistat, or stabilizer at about 50,000 to about 150,000 cells / mL.

12. The method according to any one of claims 1 to 10, further comprising the step of seeding the chemostat, turbidistat, or stabilizer at about 50,000 to about 100,000 cells / mL.

13. A chemistat, turbidity regulator, or steady-state device for use in the method of claims 1 to 10.

14. A method for replacing a culture medium for cell culture, the method comprising the following steps: Culture cells in a container. Gravity settling separation, and Culture medium replacement.

15. The culture medium replacement method of claim 14, wherein the container includes a stirrer, an internal aspiration tube attached to an outflow line for removing culture medium from the culture container, and an inflow line for supplying fresh culture medium to the culture container.

16. The culture medium replacement method according to claim 15, further comprising the step of removing the agitator by disabling the agitator.

17. The culture medium replacement method according to claim 16, further comprising the step of concentrating the cells by gravity sedimentation separation to form concentrated cell aggregates.

18. The culture medium replacement method according to claim 16 or claim 17, wherein the step of removing the agitation by disabling the agitator is controlled by a biomass probe and / or a timer and / or a metabolite detector.

19. The culture medium replacement method according to any one of claims 14 to 18, wherein the culture medium replacement step comprises extending a suction tube into the container.

20. The culture medium replacement method according to claim 19, wherein the aspirator extends to about 95% of the container height.

21. The culture medium replacement method according to claim 19 or 20, wherein after the gravity sedimentation separation step, the aspirator extracts the growth medium from the container.

22. The culture medium replacement method according to claim 21, wherein fresh culture medium is added to the container after the culture medium extraction step.

23. The culture medium replacement method according to any one of claims 14 to 22, wherein the culture medium replacement method further comprises the steps of controlling heat and / or gas bubbling.

24. The culture medium replacement method according to any one of claims 14 to 23, wherein the volume of the container is about 1 liter to about 50,000 liters.

25. The culture medium replacement method according to any one of claims 14 to 23, wherein the volume of the container is about 50 liters to about 10,000 liters.

26. The culture medium replacement method according to any one of claims 14 to 23, wherein the volume of the container is about 100 liters to about 1,000 liters.

27. The culture medium replacement method according to any one of claims 14 to 26, wherein the cells have a high tendency to form aggregates.

28. The culture medium replacement method according to any one of claims 14 to 27, wherein the cells are genetically modified.

29. The culture medium replacement method according to any one of claims 14 to 28, wherein the cells are eukaryotic.

30. The culture medium replacement method according to any one of claims 14 to 29, wherein the cells are mammalian.

31. The culture medium replacement method according to any one of claims 14 to 30, wherein gravity sedimentation separation is carried out for about 5 minutes to about 3 hours.

32. The culture medium replacement method according to any one of claims 14 to 31, wherein gravity sedimentation separation is carried out for about 20 minutes to about 2 hours.

33. A bioreactor comprising an inflow line for supplying fresh culture medium to a culture vessel, an outflow line for removing fresh culture medium from the culture vessel, and a vessel, wherein the vessel includes a stirrer and an internal suction tube in fluid communication with the outflow line.

34. The bioreactor of claim 33, further comprising a biomass detector and / or a timer and / or a metabolite detector.

35. The bioreactor according to claim 33 or claim 34, wherein the extraction tube is extendable.

36. The bioreactor according to any one of claims 33 to 35, wherein the extractor tube extends from about 70% to about 95% of the container height.

37. The bioreactor according to any one of claims 33 to 36, wherein the bioreactor further comprises equipment for controlling heat and / or gas bubbling.

38. The bioreactor of claim 37, wherein the device is reversibly activated.

39. The bioreactor according to any one of claims 33 to 38, wherein the volume of the bioreactor is from about 1 liter to about 50,000 liters.

40. The bioreactor according to any one of claims 33 to 38, wherein the volume of the bioreactor is about 50 liters to about 10,000 liters.

41. The bioreactor according to any one of claims 33 to 38, wherein the volume of the bioreactor is from about 100 liters to about 1,000 liters.

42. The bioreactor according to any one of claims 33 to 41, wherein the bioreactor is used to culture cells having a high tendency to form aggregates.

43. The use according to claim 42, wherein the cell is genetically modified.

44. The use according to claim 43, wherein the cell is eukaryotic.

45. The use according to claim 44, wherein the cell is mammalian.

46. ​​A cell culture method, the cell culture method comprising: The method for providing seed culture according to claims 1 to 10; as well as The culture medium replacement method according to claims 14 to 32.

47. The cell culture method of claim 50, wherein the method of providing seed culture is carried out in a chemostat, turbidistat, or stabilizer, and the culture medium replacement method is carried out at a predetermined time point after the incubation of the seed cultured cells from the chemostat, turbidistat, or stabilizer has been carried out in a separate additional bioreactor.

48. A cell culture system comprising a chemostat, turbidistat, or stabilizer according to claim 13, and a bioreactor according to claims 33 to 42.

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