Low-serum suspension domestication culture method for DF-1 cells and application of low-serum suspension domestication culture method

Abstract

The invention discloses a low-serum suspension domestication culture method of DF-1 cells and application of the low-serum suspension domestication culture method, and belongs to the technical field of cell culture. The culture method comprises the following steps: after subculturing DF-1 cells, adding the DF-1 cells into a basic culture medium for primary suspension domestication; after one week of preliminary suspension domestication, adding a 10% FBS basal culture medium, and carrying out large-scale suspension domestication by adopting a rotating speed climbing mode; and after large-scale suspension domestication for 5-6 days, carrying out first passage on the DF-1 cells again, and resuspending the DF-1 cells by using a complete culture medium containing 5% of FBS. According to the low-serum suspension domestication culture method of the DF-1 cells, provided by the invention, the DF-1 cells can realize gradual adaptation in a relatively low mechanical stress and stable metabolism environment through a combined mode of two-dimensional culture, preliminary suspension domestication and large-scale culture and a stirring climbing and gradient serum reduction strategy; the continuity and the stability of the cells in the amplification and suspension transformation process are ensured, the cell agglomeration phenomenon is remarkably reduced, and the uniformity and the stability of the suspension state are improved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to a method for low-serum suspension culture of DF-1 cells and its application. Background Technology

[0002] Cell-cultured meat technology, a cutting-edge field integrating cell biology, tissue engineering, and bioreactor engineering, aims to produce edible muscle tissue through the in vitro culture of animal cells. Compared with traditional animal husbandry, this technology has advantages such as lower resource consumption, less environmental burden, significantly improved animal welfare, and higher food safety, and is considered an important development path for future sustainable protein supply. Current research mainly focuses on two aspects: first, cell source and domestication, constructing myoblast or fibroblast cell lines with high proliferative capacity and stability; second, optimization of culture systems, including the development of low-serum or serum-free culture media and the establishment of high-density cell culture processes suitable for large-scale production.

[0003] However, the industrialization of cell-cultured meat still faces significant bottlenecks, with the difficulty of large-scale production being the core issue. Existing suspension acclimation methods generally suffer from long cycles and low success rates. During the acclimation process, cells are prone to aggregation, shear damage, and the generation of numerous cell fragments, leading to uneven cell morphology, decreased proliferation efficiency, and difficulty in achieving stable, high-density continuous culture in bioreactors.

[0004] Among various avian cell lines, DF-1 cells are more suitable for developing suspension culture systems due to their stable origin, low dependence on adhesion, and rapid acclimatization. This cell line exhibits good proliferation capacity and growth consistency, maintaining stable expansion even under low serum conditions, which facilitates a smooth transition from adherent to suspension states. Furthermore, DF-1 cells have weak intercellular adhesion, high tolerance to mechanical forces generated by stirring and aeration, and stable overall growth, making them an ideal model cell for constructing large-scale suspension culture processes suitable for bioreactors.

[0005] Despite the numerous advantages of DF-1 cells, achieving stable suspension proliferation in bioreactors remains challenging due to environmental complexity. Agitation and aeration within bioreactors generate strong shear forces, which can easily cause cell damage or affect proliferation stability. While traditional protective agents such as Pluronic F-68 can alleviate shear stress to some extent, their ability to inhibit cell aggregation and maintain growth under low serum conditions is limited. To address this, current technologies attempt to introduce multi-component additives such as ITS, antioxidants, and reducing agents to improve the reactor microenvironment. However, their synergistic mechanisms, optimal concentrations, and suitable acclimatization rhythms are not yet systematically clarified. Furthermore, the lack of a controllable dynamic regulation strategy makes it difficult to simultaneously achieve shear stress adaptation and maintain viability under low serum conditions, resulting in insufficient process repeatability and scale-up stability. Summary of the Invention

[0006] The purpose of this invention is to provide a method for low-serum suspension acclimatization culture of DF-1 cells and its application, solving the problem of limitations in large-scale production of low-serum suspension acclimatization culture of DF-1 cells. This culture method uses a complete culture medium with shear protection, anti-aggregation, metabolic support, and serum replacement functions. It combines a gradual "rotation speed increase" and "gradient serum reduction" strategy to ensure a smooth transition of cells from adherent to suspension and from a high-serum to a low-serum environment. This culture method can significantly reduce serum usage, increase cell density and viability, and ensure controllable key process parameters, providing a feasible and scalable technical path for large-scale, low-cost production of cell-cultured meat.

[0007] This invention is achieved through the following technical solution: This invention provides a method for low-serum suspension culture of DF-1 cells, comprising: After thawing the DF-1 cell suspension, centrifuge the suspension, remove the supernatant, and resuspend the DF-1 cells in basal culture medium. After passage, DF-1 cells were added to basal culture medium for initial suspension acclimatization. After one week of initial suspension acclimatization, 10% FBS basal medium was added, and large-scale suspension acclimatization was carried out using a rotation speed ramp method. After 5-6 days of large-scale suspension acclimatization, the DF-1 cells were passaged for the first time and resuspended in complete medium containing 5% FBS to collect the DF-1 cells.

[0008] Further specifying, in the low serum suspension acclimation culture method for DF-1 cells, the DF-1 cells are passaged to a quantity of 5 × 10⁶ cells / year. 6 -1×10 7 indivual.

[0009] More specifically, in the low-serum suspension acclimatization culture method for DF-1 cells, the initial suspension acclimatization is carried out using a shake-flask culture method.

[0010] More specifically, in the low-serum suspension acclimatization culture method for DF-1 cells, the large-scale suspension acclimatization adopts a 3D culture method.

[0011] Further specifying, in the low-serum suspension acclimatization culture method for DF-1 cells, the reaction conditions for the large-scale suspension acclimatization include: (1) The temperature is 37~41℃; (2) The pH value is 7.2~7.4; (3) Dissolved oxygen level is 60%~80%; (4) The culture volume shall not be less than 100 mL.

[0012] Further specifying, in the low serum suspension acclimatization culture method for DF-1 cells, the rotation speed ramping method includes: The initial speed was increased from 50-60 rpm to 75-85 rpm at an increase rate of 8-12 rpm / d over a period of 2-3 days. It increases to 95-105 rpm at a rate of 16-24 rpm / d over 2-3 days.

[0013] Further specifying, in the low serum suspension acclimation culture method for DF-1 cells, the complete culture medium comprises: Pluronic F-68 at a concentration of 0.05%–0.2%, where the concentration can be 0.05%, 0.1%, 0.15%, 0.2%, etc., but not limited to the listed values; other unlisted values ​​within this range are also applicable; Tween 20 at a concentration of 0.01%–0.05%, where the concentration can be 0.01%, 0.02%, 0.03%, 0.05%, etc., but not limited to the listed values; other unlisted values ​​within this range are also applicable; ITS at an addition amount of 0.5×–5×, where the addition amount can be 0.5×, 1×, 5×, 3×, 4×, 5×, etc., but not limited to the listed values; other unlisted values ​​within this range are also applicable; and β-mercaptoethanol (β-ME) at a final concentration of 50–500 μM. The concentrations can be 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, etc., but are not limited to the listed values; other unlisted values ​​within this range are also applicable. The concentration of L-ascorbic acid-2-phosphate is 50–500 μM, and the concentrations can be 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, etc., but are not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0014] More specifically, in the low-serum suspension acclimatization culture method for DF-1 cells, after resuspension in the complete culture medium and after the proliferation of DF-1 cells has stabilized, the serum content in the complete culture medium is 2-3%.

[0015] More specifically, in the low serum suspension acclimation culture method for DF-1 cells, the basal culture medium is DMEM / F-12 medium.

[0016] The present invention also provides the application of the low serum suspension acclimatization culture method of DF-1 cells in cell engineering, vaccine preparation, and cell cultured meat.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: The low-serum suspension acclimatization culture method for DF-1 cells provided by this invention combines two-dimensional culture, preliminary suspension acclimatization, and large-scale culture. Through stirring and gradient serum reduction strategies, DF-1 cells can gradually adapt to a low mechanical stress and stable metabolic environment, ensuring the continuity and stability of cells during expansion and suspension transition. This significantly reduces cell aggregation, improves the uniformity and stability of the suspension state, enhances the consistency of suspension adaptation among different batches of cells, significantly improves suspension growth efficiency, avoids large-scale cell death caused by direct loading into the culture vessel, and increases the culture success rate.

[0018] This invention provides a low-serum suspension culture method for DF-1 cells, constructing a controllable process system that can stably achieve DF-1 cell suspension proliferation. The aim is to enable DF-1 cells to achieve good survival, proliferation, and metabolic capacity under low-serum conditions by designing a complete culture medium containing multiple additives and employing strategies such as increasing rotation speed and decreasing serum concentration. Simultaneously, by controlling reaction parameters, the consistency and reproducibility of suspension culture are improved, achieving stable and uniform suspension proliferation of cells in large-scale bioreactors. This enhances the controllability, reproducibility, and industrial scalability of the entire culture process, laying a technical foundation for the industrial-scale culture of DF-1 cells. With the continuous expansion of applications in cell engineering, vaccine preparation, and cultured meat, traditional high-serum, adhesion-dependent DF-1 culture methods can no longer meet the demands for low-cost, scalable, and continuous production. This method effectively solves the problems of poor suspension adaptability, long acclimatization period, and insufficient stability in large-scale culture caused by its natural adhesion characteristics. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a flowchart of the existing DF-1 cell suspension culture process; Figure 2 A flowchart of the low serum suspension acclimatization culture method for DF-1 cells provided by the present invention; Figure 3 This is a graph showing the proliferation of DF-1 cells in Examples 1-3 of Test 1 of this invention; Figure 4 This refers to the survival rate of DF-1 cells after culture in Examples 1-3 of Test 2 of this invention; Figure 5 These are microscopic images of DF-1 cells after culture in Examples 1-3 of Test 2 of this invention; Figure 6 This is a graph showing the proliferation of DF-1 cells in Examples 3-5 of Test 1 of this invention; Figure 7 This refers to the survival rate of DF-1 cells after culture in Examples 3-5 of Test 2 of this invention; Figure 8 These are microscopic images of DF-1 cells after culture in Examples 3-5 of Test 2 of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The illustrative embodiments and descriptions of this invention are for explanation only and are not intended to limit the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0021] Reference Figure 1 The DF-1 cell suspension culture technique mainly consists of the following three steps: Step 1: After thawing the frozen DF-1 cells, seed them into small-scale culture flasks or dishes using a standard adherent culture system (such as DMEM / F-12 medium), supplemented with 10% fetal bovine serum (FBS) and necessary growth factors to maintain cell viability and proliferation. Through continuous passage, stabilize the cell growth state and gradually expand to obtain a sufficient quantity of healthy cells, providing a seed cell source for subsequent culture in culture tanks.

[0022] Step Two: Once the cell number reaches the predetermined scale, transfer them to a bioreactor for large-scale culture. Based on the cell growth requirements, set the key operating parameters of the reactor, including temperature, pH, dissolved oxygen, and rotation speed, to ensure sufficient transfer of nutrients and oxygen within the system. During the culture process, regularly change the culture medium to avoid the accumulation of metabolic inhibitors, ensuring continuous cell proliferation and maintaining high viability.

[0023] Step 3: Once the target cell density is reached, stop stirring and collect the cells from the bioreactor using methods such as centrifugation. The resulting cells can be used for subsequent passage culture, functional experiments, or downstream product development.

[0024] The existing DF-1 cell suspension culture technology has the following main drawbacks, which can be divided into the following three parts: 1. Poor suspension adaptability and long acclimatization period. Currently, DF-1 cells mostly grow in an adherent manner, with tight cell adhesion and relying on the matrix for adhesion signals. Therefore, they are prone to aggregation in suspension systems, resulting in limited nutrient and oxygen transfer. Most published methods require a long-term, multi-generation suspension acclimation process, which is cumbersome, time-consuming, and has significant differences in suspension adaptability between different batches, leading to insufficient process stability.

[0025] 2. Uncontrollable cultivation conditions make it difficult to achieve stable growth in bioreactors. Existing technologies can achieve a certain degree of DF-1 suspension proliferation in shake flasks or small-scale reaction systems, but when scaled up to bioreactors, the following problems often arise: cell damage caused by stirring and shearing; difficulty in simultaneously maintaining pH and dissolved oxygen within suitable ranges; and uneven cell settling or floating, resulting in large fluctuations in cell growth curves and poor reproducibility. Furthermore, the lack of reactor parameter setting methods specifically tailored to the characteristics of DF-1 cells makes it difficult to obtain a stable process suitable for industrial scale-up.

[0026] 3. Existing culture media or additive systems do not adequately support suspension proliferation. Existing technologies mostly employ basal media such as DMEM, commonly used for adherent culture. However, the nutrient composition, adhesion factors, and serum dependence of these media are not suitable for the suspension growth requirements of DF-1 cells. In low-serum or even serum-free systems, cell adhesion decreases, the risk of aggregation increases, and the proliferation rate decreases significantly. Furthermore, the lack of a clear strategy for matching functional additives to promote suspension adhesion and improve cell viability makes it difficult to maintain metabolic homeostasis and proliferative capacity of DF-1 cells in suspension.

[0027] To address this, the present invention provides the following solution: A method for low-serum suspension culture of DF-1 cells, comprising: After thawing the frozen DF-1 cells, the suspension was centrifuged, the supernatant was removed, and the DF-1 cells were resuspended in basal culture medium. After passage, DF-1 cells were added to basal culture medium for initial suspension acclimatization. After one week of initial suspension acclimatization, 10% FBS basal medium was added, and large-scale suspension acclimatization was carried out using a rotation speed ramp method. After 5-6 days of large-scale suspension acclimatization, the DF-1 cells were passaged for the first time and resuspended in complete medium containing 5% FBS to collect the DF-1 cells.

[0028] Reference Figure 2As shown, the low-serum suspension acclimatization culture method for DF-1 cells provided by this invention, through a combination of two-dimensional culture, preliminary suspension acclimatization, and large-scale culture, and by employing a stirring and gradient serum reduction strategy, allows DF-1 cells to gradually adapt to a lower mechanical stress and stable metabolic environment. This ensures the continuity and stability of cells during expansion and suspension transition, significantly reduces cell aggregation, improves the uniformity and stability of the suspension state, enhances the consistency of suspension adaptation among different batches of cells, significantly improves suspension growth efficiency, avoids large-scale cell death caused by direct loading into the culture vessel, and increases the culture success rate.

[0029] This invention provides a low-serum suspension culture method for DF-1 cells, constructing a controllable process system that can stably achieve DF-1 cell suspension proliferation. The aim is to design a complete culture medium containing multiple additives, combined with a rotation speed increase and serum gradient decrease strategy, to enable DF-1 cells to achieve good survival and metabolic capacity under low-serum conditions. Simultaneously, by combining a controlled reaction parameter system, the consistency and reproducibility of suspension culture are improved, achieving stable and uniform suspension proliferation of cells in large-scale bioreactors. This enhances the controllability, reproducibility, and industrial scalability of the entire culture process, laying a technical foundation for the industrial-scale culture of DF-1 cells. With the continuous expansion of applications in cell engineering, vaccine preparation, and cultured meat, traditional high-serum, adhesion-dependent DF-1 culture methods can no longer meet the demands for low-cost, scalable, and continuous production. This method effectively solves the problems of poor suspension adaptability, long acclimatization period, and insufficient stability in large-scale culture caused by its natural adhesion characteristics.

[0030] This invention provides a low-serum suspension culture method for DF-1 cells, which promotes suspension adaptation through a serum gradient descent strategy. Since DF-1 cells naturally adhere to the culture surface and are highly dependent on high serum concentrations, this invention gradually reduces the serum concentration during continuous passages (initially 10% → intermediate 5% → final 2-3%), allowing cells to gradually adapt to the low-serum environment, reducing their need for adhesion signals, decreasing cell aggregation, and thus achieving stable suspension proliferation. This invention employs a serum gradient descent strategy to smoothly regulate cellular stress responses, enabling cells to maintain high proliferative capacity under low-serum conditions, reducing serum dependence and lowering production costs. Simultaneously, it maintains metabolic stability and avoids growth inhibition caused by mutational conditions.

[0031] The low-serum suspension culture method for DF-1 cells provided by this invention employs a complete culture medium containing a combination of various additives to enhance suspension growth. Adding substances such as Tween 20, Pluronic F-68, ITS, β-mercaptoethanol, and L-ascorbic acid-2-phosphate to the complete culture medium can promote DF-1 cell suspension growth in multiple ways: Tween 20 and F-68 reduce cell clumping and mitigate agitation and shear damage; ITS provides crucial nutritional support; β-mercaptoethanol enhances the reducing environment; and L-ascorbic acid-2-phosphate maintains cellular antioxidant capacity and metabolic homeostasis, thereby improving survival rate and proliferation efficiency.

[0032] The low-serum suspension acclimation culture method for DF-1 cells provided by this invention, through increasing the stirring speed and using a complete culture medium containing multiple additives, can establish a stable suspension growth environment in the reactor during large-scale suspension acclimation, reducing cell aggregation and cell debris, and significantly improving oxygen transfer and nutrient diffusion efficiency. This strategy has good scale-up consistency, is applicable to multi-stage bioreactor scale-up, and is convenient for industrial application.

[0033] The low-serum suspension culture method for DF-1 cells provided by this invention achieves scalable and stable culture through control of reaction parameters. Precise control of key parameters such as pH, dissolved oxygen, temperature, and stirring speed maintains uniform cell distribution, preventing uneven sedimentation or floating. Combined with the effects of serum gradients and functional additives, this method enables DF-1 cells to achieve a reproducible and stable suspension proliferation process under low-serum conditions, demonstrating feasibility for industrial-scale production.

[0034] More specifically, the DF-1 cells are passaged to a quantity of 5 × 10⁶. 6 -1×10 7 indivual.

[0035] More specifically, the initial suspension acclimatization is performed using a shake-flask culture method. The initial suspension acclimatization is carried out in shake flasks.

[0036] More specifically, the large-scale suspension acclimatization adopts a 3D culture method. The large-scale suspension acclimatization is carried out in a bioreactor, preferably a quadruple parallel stirred bioreactor.

[0037] Further specifying, the reaction conditions for the large-scale suspension acclimatization include: (1) The temperature is 37~41℃.

[0038] (2) The pH value is 7.2~7.4. This range is close to the optimal physiological environment of DF-1, which can reduce the effects of lactic acid and ammonia toxicity on cells, maintain good proliferative activity and support stable suspension growth.

[0039] (3) Dissolved oxygen value is 60%~80%. Since DF-1 has a high oxygen requirement, 60%–80% DO can provide sufficient oxygen supply, while avoiding oxidative stress caused by high oxygen, which is beneficial to maintaining cell proliferation rate and metabolic stability.

[0040] (4) The culture volume shall not be less than 100 mL. This invention adopts a small-volume culture system. The small volume makes it easier to form a uniform flow field and a stable mass transfer environment, which can reduce the risk of agglomeration, hypoxia and sedimentation in the early stage of suspension, and improve the controllability and success rate of the acclimatization process.

[0041] More specifically, the rotation speed ramping method includes: increasing the speed from an initial 50-60 rpm to 75-85 rpm at an increase of 8-12 rpm / day over 2-3 days; and increasing the speed from 16-24 rpm / day to 95-105 rpm over 2-3 days. This invention, by gradually increasing the stirring speed, enables the naturally adherent and shear-resistant DF-1 cells to gradually adapt to the suspension environment, reducing cell death and aggregation caused by instantaneous high shear, thereby forming a stable and homogeneous suspension.

[0042] Further specified, the composition of the complete culture medium includes: Pluronic F-68 at a final concentration of 0.05% to 0.2%, Tween 20 at a final concentration of 0.01% to 0.05%, ITS at an addition amount of 0.5× to 5×, β-mercaptoethanol (β-ME) at a final concentration of 50 to 500 μM, and L-ascorbic acid-2-phosphate at a final concentration of 50 to 500 μM.

[0043] More specifically, in the low-serum suspension acclimatization culture method for DF-1 cells, after resuspension in the complete culture medium and after the proliferation of DF-1 cells has stabilized, the serum content in the complete culture medium is 2-3%.

[0044] More specifically, in the low serum suspension acclimation culture method for DF-1 cells, the basal culture medium is DMEM / F-12 medium.

[0045] The present invention also provides the application of the above-mentioned low serum suspension acclimatization culture method of DF-1 cells in cell engineering, vaccine preparation, and cultured meat.

[0046] To further illustrate the present invention, the following describes a method for low-serum suspension culture of DF-1 cells and its application, in conjunction with embodiments. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. They are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims of the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0047] Example 1: The low-serum suspension culture method for DF-1 cells provided in this embodiment includes the following steps: S1: Remove the cell cryopreservation tubes from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the mouth of the cryopreservation tubes with alcohol. S2: Transfer the cell suspension from the cryopreservation tube to a 15mL centrifuge tube and centrifuge at 1000rpm for 3min; S3: After centrifugation, remove the supernatant and resuspend the cells in fresh basal culture medium; S4: Passage DF-1 cells to a density of 5 × 10⁶ cells / year. 6 -1×10 7 One sample was inoculated into a shake flask and culture medium was added for initial suspension acclimatization. S5: After one week of initial suspension acclimatization, cells were transferred to a bioreactor for large-scale suspension acclimatization culture, initially using 10% FBS basal medium. Culture conditions included: temperature controlled at 37℃; pH controlled at 7.2; dissolved oxygen controlled at 60%. Culture was conducted using a rotation speed escalation method: increasing the speed from an initial 50-60 rpm to 75-85 rpm at a rate of 8-12 rpm / day for 2-3 days; then increasing the speed to 95-105 rpm at a rate of 16-24 rpm / day for 2-3 days.

[0048] S6: After 5 days of large-scale suspension acclimatization, DF-1 cells were passaged again for the first time and resuspended in complete medium containing 5% FBS. The cells were cultured at 100 rpm until cell proliferation stabilized, at which point the serum level was reduced to 2%. The complete medium consisted of: 0.1% Pluronic F-68, 0.03% Tween 20, 3× ITS, 250 μM β-mercaptoethanol (β-ME), and 250 μM L-ascorbic acid-2-phosphate.

[0049] Example 2: The low-serum suspension culture method for DF-1 cells provided in this embodiment includes the following steps: S1: Remove the cell cryopreservation tubes from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the mouth of the cryopreservation tubes with alcohol. S2: Transfer the cell suspension from the cryopreservation tube to a 15mL centrifuge tube and centrifuge at 1000rpm for 3min; S3: After centrifugation, remove the supernatant and resuspend the cells in fresh basal culture medium; S4: Passage DF-1 cells to a density of 5 × 10⁶ cells / year. 6 -1×107 One sample was inoculated into a shake flask and culture medium was added for initial suspension acclimatization. S5: After one week of initial suspension acclimatization, cells were transferred to a bioreactor for large-scale suspension acclimatization culture, initially using 10% FBS basal medium. Culture conditions included: temperature controlled at 39℃; pH controlled at 7.3; dissolved oxygen controlled at 72%. Culture was conducted using a rotation speed escalation method: increasing the speed from an initial 50-60 rpm to 75-85 rpm at a rate of 8-12 rpm / day for 2-3 days; then increasing the speed to 95-105 rpm at a rate of 16-24 rpm / day for 2-3 days.

[0050] S6: After 6 days of large-scale suspension acclimatization, DF-1 cells were passaged again for the first time and resuspended in complete medium containing 5% FBS. The cells were cultured at 100 rpm until cell proliferation stabilized, at which point the serum level was reduced to 3%. The complete medium consisted of: 0.1% Pluronic F-68, 0.03% Tween 20, 3× ITS, 250 μM β-mercaptoethanol (β-ME), and 250 μM L-ascorbic acid-2-phosphate.

[0051] Example 3: The low-serum suspension culture method for DF-1 cells provided in this embodiment includes the following steps: S1: Remove the cell cryopreservation tubes from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the mouth of the cryopreservation tubes with alcohol. S2: Transfer the cell suspension from the cryopreservation tube to a 15mL centrifuge tube and centrifuge at 1000rpm for 3min; S3: After centrifugation, remove the supernatant and resuspend the cells in fresh basal culture medium; S4: Passage DF-1 cells to a density of 5 × 10⁶ cells / year. 6 -1×10 7 One sample was inoculated into a shake flask and culture medium was added for initial suspension acclimatization. S5: After one week of initial suspension acclimatization, cells were transferred to a bioreactor for large-scale suspension acclimatization culture, initially using 10% FBS basal medium. Culture conditions included: temperature controlled at 41℃; pH controlled at 7.4; dissolved oxygen controlled at 80%. Culture was conducted using a progressive rotation speed approach: increasing the speed from an initial 50-60 rpm to 75-85 rpm at a rate of 8-12 rpm / day for 2-3 days; then increasing the speed to 95-105 rpm at a rate of 16-24 rpm / day for 2-3 days.

[0052] S6: After 6 days of large-scale suspension acclimatization, DF-1 cells were passaged again for the first time and resuspended in complete medium containing 5% FBS. The cells were cultured at 100 rpm until cell proliferation stabilized, at which point the serum level was reduced to 2%. The complete medium consisted of: 0.1% Pluronic F-68, 0.03% Tween 20, 3× ITS, 250 μM β-mercaptoethanol (β-ME), and 250 μM L-ascorbic acid-2-phosphate.

[0053] Example 1: The low-serum suspension culture method for DF-1 cells provided in this example includes the following steps: First, remove the cell cryovials from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the cryovial opening with alcohol. Transfer the DF-1 cell suspension from the cryovials to a 15mL centrifuge tube and centrifuge at 1000rpm for 3 minutes. Aspirate the supernatant and resuspend the cells in fresh culture medium. Seed the thawed DF-1 cells into culture dishes for expansion. Once the cells reach a suitable density, digest and count the cells.

[0054] Next, 5×10 6 Cells were seeded into a parallel bioreactor and cultured with a stirring speed of 60 rpm, initially using 10% FBS medium. The stirring speed was increased by 10 rpm every 24 hours. After 24 hours, the stirring speed was increased to 70 rpm; after 48 hours, it was increased to 80 rpm; after 72 hours, it was increased to 90 rpm; and after 96 hours, it was increased to the target stirring speed of 100 rpm.

[0055] After 120 h of culture, the cells were passaged for the first time and resuspended in a complete medium containing 5% FBS and all additives (Pluronic F-68, Tween 20, ITS, β-mercaptoethanol, L-ascorbic acid-2-phosphate). The cells were cultured at 100 rpm for 96 h, after which the serum concentration was reduced to 2%. The complete medium consisted of: 0.1% Pluronic F-68, 0.03% Tween 20, 3× ITS, 250 μM β-mercaptoethanol (β-ME), and 250 μM L-ascorbic acid-2-phosphate.

[0056] Example 2: The low-serum suspension culture method for DF-1 cells provided in this example includes the following steps: First, remove the cell cryovials from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the cryovial opening with alcohol. Transfer the DF-1 cell suspension from the cryovials to a 15mL centrifuge tube and centrifuge at 1000rpm for 3 minutes. Aspirate the supernatant and resuspend the cells in fresh culture medium. Seed the thawed DF-1 cells into culture dishes for expansion. Once the cells reach a suitable density, digest and count the cells.

[0057] Next, 5×10 6 One DF-1 cell was seeded into a shake flask, and culture medium was added for initial suspension acclimatization. After one week of initial acclimatization, the cells were transferred to a bioreactor and cultured with stirring at 100 rpm, initially using 10% FBS culture medium.

[0058] After 120 h of culture, the cells were passaged for the first time and resuspended in a complete medium containing 5% FBS and all additives (Pluronic F-68, Tween 20, ITS, β-mercaptoethanol, L-ascorbic acid-2-phosphate). The cells were cultured at 100 rpm for 96 h, after which the serum concentration was reduced to 2%. The complete medium consisted of: 0.1% Pluronic F-68, 0.03% Tween 20, 3× ITS, 250 μM β-mercaptoethanol (β-ME), and 250 μM L-ascorbic acid-2-phosphate.

[0059] Example 3: The low-serum suspension culture method for DF-1 cells provided in this example includes the following steps: First, remove the cell cryovials from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the cryovial opening with alcohol. Transfer the DF-1 cell suspension from the cryovials to a 15mL centrifuge tube and centrifuge at 1000rpm for 3 minutes. Aspirate the supernatant and resuspend the cells in fresh culture medium. Seed the thawed DF-1 cells into culture dishes for expansion. Once the cells reach a suitable density, digest and count the cells.

[0060] Next, 5×10 6 Cells were seeded into shake flasks and cultured in medium for initial suspension acclimatization. After one week of initial acclimatization, the cells were transferred to a bioreactor and cultured at 60 rpm with 10% FBS medium initially. The stirring speed was increased by 10 rpm every 24 hours. After 24 hours, the stirring speed was increased to 70 rpm; after 48 hours, it was increased to 80 rpm; after 72 hours, it was increased to 90 rpm; and after 96 hours, it was increased to the target stirring speed of 100 rpm.

[0061] After 120 hours of culture, the cells were passaged for the first time and resuspended in a complete medium containing 2% FBS and all additives (Pluronic F-68, Tween 20, ITS, β-mercaptoethanol, and L-ascorbic acid-2-phosphate). The medium was cultured at 100 rpm. The complete medium consisted of: 0.1% Pluronic F-68, 0.03% Tween 20, 3× ITS, 250 μM β-mercaptoethanol (β-ME), and 250 μM L-ascorbic acid-2-phosphate.

[0062] Example 4: The low-serum suspension culture method for DF-1 cells provided in this example includes the following steps: First, remove the cell cryovials from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the cryovial opening with alcohol. Transfer the DF-1 cell suspension from the cryovials to a 15mL centrifuge tube and centrifuge at 1000rpm for 3 minutes. Aspirate the supernatant and resuspend the cells in fresh culture medium. Seed the thawed DF-1 cells into culture dishes for expansion. Once the cells reach a suitable density, digest and count the cells.

[0063] Next, 5×10 6 Cells were seeded into shake flasks and cultured in medium for initial suspension acclimatization. After one week of initial acclimatization, the cells were transferred to a bioreactor and cultured at 60 rpm with 10% FBS medium initially. The stirring speed was increased by 10 rpm every 24 hours. After 24 hours, the stirring speed was increased to 70 rpm; after 48 hours, it was increased to 80 rpm; after 72 hours, it was increased to 90 rpm; and after 96 hours, it was increased to the target stirring speed of 100 rpm.

[0064] After 120 hours of culture, the cells were passaged for the first time, resuspended in medium containing 2% FBS, and cultured at 100 rpm.

[0065] Example 5: The low-serum suspension culture method for DF-1 cells provided in this example includes the following steps: First, remove the cell cryovials from liquid nitrogen and thaw them in a 37°C water bath. After thawing, wipe the cryovial opening with alcohol. Transfer the DF-1 cell suspension from the cryovials to a 15mL centrifuge tube and centrifuge at 1000rpm for 3 minutes. Aspirate the supernatant and resuspend the cells in fresh culture medium. Seed the thawed DF-1 cells into culture dishes for expansion. Once the cells reach a suitable density, digest and count the cells.

[0066] Next, 5×10 6Cells were seeded into shake flasks and cultured in medium for initial suspension acclimatization. After one week of initial acclimatization, the cells were transferred to a bioreactor and cultured at 60 rpm with 10% FBS medium initially. The stirring speed was increased by 10 rpm every 24 hours. After 24 hours, the stirring speed was increased to 70 rpm; after 48 hours, it was increased to 80 rpm; after 72 hours, it was increased to 90 rpm; and after 96 hours, it was increased to the target stirring speed of 100 rpm.

[0067] After 120 h of culture, the cells were passaged for the first time and resuspended in a complete medium containing 5% FBS and all additives (Pluronic F-68, Tween 20, ITS, β-mercaptoethanol, L-ascorbic acid-2-phosphate). The cells were cultured at 100 rpm for 96 h, after which the serum concentration was reduced to 2%. The complete medium consisted of: 0.1% Pluronic F-68, 0.03% Tween 20, 3× ITS, 250 μM β-mercaptoethanol (β-ME), and 250 μM L-ascorbic acid-2-phosphate.

[0068] Test 1: The proliferation of DF-1 cells in Examples 1, 2, and 3 was measured, and the results are shown in the figure. Figure 3 As shown.

[0069] Test 2: The viability of DF-1 cells was measured after culture in Examples 1, 2, and 3. The results are shown in the table below. Figure 4 As shown.

[0070] Test 3: After the DF-1 cells in Examples 1, 2, and 3 were cultured, they were observed under a microscope. The results are shown in the figure below. Figure 5 As shown.

[0071] Example 1 involves directly seeding DF-1 cells into a bioreactor for suspension acclimatization; Example 2 involves first performing suspension acclimatization in shake flasks for one week, and then transferring the cells to the bioreactor for culture, but without setting a speed ramping process; Example 5 also involves first completing suspension acclimatization in shake flasks for one week, but a speed ramping strategy is introduced after transferring the cells to the bioreactor.

[0072] Figure 3-5 The results showed that, compared with the method of direct acclimatization in the reactor, the transition through shake flasks can significantly improve the adaptability of cells to the suspension environment, while the speed ramping in the reactor stage further promotes cell proliferation and survival. The combination of the two can achieve higher cell growth efficiency, survival rate and dispersibility.

[0073] Test 4: The proliferation of DF-1 cells in Examples 3, 4, and 5 was measured, and the results are shown in the figure. Figure 6 As shown.

[0074] Test 5: The viability of DF-1 cells was measured after culture in Examples 3, 4, and 5. The results are shown in the table below. Figure 7 As shown.

[0075] Test 6: After the DF-1 cells in Examples 3, 4, and 5 were cultured, they were observed under a microscope. The results are shown in the figure below. Figure 8 As shown.

[0076] Example 3 employed a strategy of first acclimating DF-1 cells to suspension in shake flasks for one week, and then transferring the cells to a bioreactor for culture, but without implementing a gradient serum reduction. Example 4 also involved acclimation to suspension in shake flasks followed by transfer to a bioreactor for culture, and although a gradient serum reduction was performed, no additives were added during the serum reduction process. In contrast, Example 5, after completing the shake flask acclimation, not only employed a gradient serum reduction strategy in the bioreactor, but also added the necessary additives during the serum reduction process.

[0077] Figure 6-8 The results showed that the combined use of gradient-degraded serum and complete culture medium containing key additives could significantly improve the adaptability and dispersibility of cells in a low-serum environment, and jointly promote efficient cell proliferation and high survival rate.

[0078] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A low serum suspension domestication culture method of DF-1 cells, characterized in that, The application relates to a low-serum suspension domestication culture method of DF-1 cells. After the frozen DF-1 cells are thawed, the suspension is centrifuged, the supernatant is sucked off, and the DF-1 cells are resuspended by adding a basic culture medium; After the DF-1 cells are subcultured, the basic culture medium is added for preliminary suspension domestication; After the preliminary suspension domestication for one week, the DF-1 cells are resuspended by adding a basic culture medium containing 10% FBS and adopting a rotation speed climbing mode for large-scale suspension domestication; After the large-scale suspension domestication for 5-6 days, the DF-1 cells are subcultured for the first time, resuspended by a complete culture medium containing 5% FBS, and collected.

2. The low serum suspension acclimation culture method of DF-1 cells according to claim 1, characterized in that, The DF-1 cells are subcultured to a number of 5 x 10 6 -1 x 10 7 cells.

3. The low serum suspension acclimation culture method of DF-1 cells according to claim 1, characterized in that, The preliminary suspension domestication adopts a shake flask culture mode.

4. The low serum suspension acclimation culture method of DF-1 cells according to claim 1, characterized in that, The large-scale suspension domestication adopts a 3D culture mode.

5. The low serum suspension acclimation culture method of DF-1 cells according to claim 1, characterized in that, The reaction conditions of the large-scale suspension domestication include: (1) the temperature is 37-41 DEG C; (2) the pH value is 7.2-7.4; (3) the dissolved oxygen value is 60%-80%; (4) the culture volume is not less than 100 mL. 6.The low serum suspension acclimation culture method of DF-1 cells according to claim 1, wherein, The rotation speed climbing mode includes: The initial rotation speed is 50-60 rpm, and the rotation speed is increased to 75-85 rpm at an increase rate of 8-12 rpm / d for 2-3 days; The rotation speed is increased to 95-105 rpm at an increase rate of 16-24 rpm / d for 2-3 days.

7. The method for low-serum suspension acclimatization culture of DF-1 cells according to claim 1, characterized in that, The composition of the complete culture medium includes Pluronic F-68 with a concentration of 0.05%-0.2%, Tween20 with a concentration of 0.01%-0.05%, ITS with an added amount of 0.5x-5x, beta-mercaptoethanol with a concentration of 50-500 muM, and L-ascorbic acid-2-phosphate with a concentration of 50-500 muM. 8.The low serum suspension acclimation culture method of DF-1 cells according to claim 1, wherein, After the resuspension in the complete culture medium is completed, the DF-1 cells are stably proliferated, and the serum content in the complete culture medium is 2-3%. 9.The low serum suspension acclimation culture method of DF-1 cells according to claim 1, wherein, The basic culture medium is a DMEM / F-12 culture medium.

10. The application of the low-serum suspension domestication culture method of the DF-1 cells according to any one of claims 1-9 in cell engineering, vaccine preparation and cell culture meat.

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