Method for carrying out in-situ sterilization and treatment on microcarrier by using bioreactor
By using in-situ sterilization in a bioreactor and continuous perfusion replacement of PBS solution, the problems of poor sterilization effect and contamination risk of traditional microcarriers have been solved, achieving efficient and safe microcarrier treatment.
Patent Information
- Application Number
- CN202511761623.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-27
- Publication Date
- 2026-02-10
AI Technical Summary
Traditional microcarrier sterilization methods are ineffective because heat cannot penetrate directly, and subsequent processing is cumbersome and prone to microbial contamination.
In-situ sterilization is performed using a bioreactor, where steam directly enters the reactor and comes into contact with the microcarriers. A stirring system ensures uniform temperature, and a sedimentation device traps the microcarriers. Sterile culture medium is used to continuously replace the PBS solution.
It achieves thorough sterilization of microcarriers, avoids microbial contamination, simplifies the operation process, and improves sterilization efficiency and safety.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell culture, and specifically relates to a method for in-situ sterilization and treatment of microcarriers using a bioreactor. Background Technology
[0002] Microcarriers refer to tiny particulate carriers used for adherent cell culture, including but not limited to the Cytodex series of microcarriers. Their diameter is typically within a certain range (e.g., 60-250 μm), and the material can be natural polymers (e.g., dextran) or synthetic polymers (e.g., polystyrene). The surface of microcarriers possesses properties suitable for cell adhesion and growth, providing a large adhesion area for adherent cells, thereby enabling high-density cell culture. During cell culture, microcarriers can be suspended in the culture medium, and in conjunction with the stirring functions of a bioreactor, allow cells to grow uniformly in a suitable environment, further facilitating high-density cell culture.
[0003] Microcarriers, used as cell culture carriers, must be sterilized and processed before use. However, traditional microcarrier sterilization and processing methods often involve mixing the microcarriers with PBS solution in glass vials, then placing the vials in a sterilizer for heat penetration sterilization. Because this method doesn't allow direct contact between the steam and the microcarriers, heat cannot penetrate effectively, resulting in poor sterilization and wasted time and resources. Furthermore, post-sterilization liquid replacement methods often involve manually opening the sterilized vials, emptying the contents, and then refilling with culture medium. This process requires repeated operations to fully replace the PBS solution in the microcarriers, making it cumbersome, risky for aseptic techniques, and highly susceptible to microbial contamination.
[0004] Continuous perfusion bioreactor technology is an advanced cell culture method. This method ensures efficient cell production under optimal conditions by continuously adding fresh culture medium to the bioreactor while simultaneously removing culture medium containing metabolic waste. The core of this technology lies in the bioreactor equipped with a microcarrier retention and sedimentation device.
[0005] Therefore, it is of great significance to develop a method for in-situ sterilization and treatment of microcarriers to overcome the problems of poor sterilization effect and easy microbial contamination in subsequent processing of existing microcarrier sterilization methods. Summary of the Invention
[0006] To address the problems in existing technologies, this invention provides a method for in-situ sterilization and treatment of microcarriers using a bioreactor. This method involves adding the microcarriers into the reactor for in-situ sterilization. During this sterilization process, steam directly enters the reactor, ensuring full contact with the microcarriers without concern for heat penetration. Furthermore, throughout the sterilization process, the reactor's internal stirring system operates continuously, ensuring uniform sterilization temperature without dead zones. The internal temperature of the microcarriers can reach above 121°C, significantly improving the sterilization effect. After sterilization of the reactor and microcarriers, a certain volume of culture medium is continuously added to the reactor over a specific time period. Simultaneously, a sedimentation device removes the PBS solution from the reactor, ensuring effective retention and sedimentation of the microcarriers and preventing loss. Throughout the entire replacement process, all operations are performed under aseptic and closed conditions, effectively eliminating the risk of microbial contamination that may occur with traditional methods.
[0007] This invention provides a method for in-situ sterilization and treatment of microcarriers, including the steps of adding microcarriers, soaking and swelling, in-situ sterilization, and replacing the culture medium.
[0008] Specifically, the method uses a bioreactor as the carrier container.
[0009] Specifically, a bioreactor refers to a closed container used for biological reactions, providing suitable environmental conditions such as temperature, dissolved oxygen, pH value, and stirring speed to support cell or tissue growth and metabolism, enabling large-scale production of bioproducts. It typically includes components such as a reactor tank, control cabinet, stirring device, temperature control system, aeration system, and microcarrier sedimentation device.
[0010] Specifically, the microcarrier is a tiny particle carrier for cell culture with a diameter of 60-250 μm, such as the Cytodex series microcarriers. Preferably, the amount of the microcarrier added is 400-700 g, and more preferably, the amount added is 400-600 g.
[0011] Specifically, the soaking and swelling is performed by fully immersing the microcarrier in PBS solution for a period of not less than 30 minutes.
[0012] Specifically, the in-situ sterilization is carried out with pure steam at a temperature of 121°C or higher, and the sterilization time of the bioreactor and its microcarriers is not less than 30 minutes.
[0013] Specifically, the in-situ sterilization also includes sterilizing each branch of the reactor, with each branch sterilizing for no less than 15 minutes, preferably no less than 30 minutes.
[0014] Specifically, the culture medium replacement is performed using a continuous perfusion method and sterile cell culture medium.
[0015] Specifically, the volume of the sterile cell culture medium used is 80-150L. Preferably, the replacement rate is not less than three times the flow rate, the replacement time is not less than 24 hours, and the perfusion flow rate is not less than 1 liter / hour. Preferably, the perfusion flow rate is not less than 2 liters / hour.
[0016] Specifically, the bioreactor is equipped with a microcarrier retention and sedimentation device.
[0017] On the other hand, the present invention utilizes the method in adherent cell culture.
[0018] The present invention also provides a method for adhering cell culture, wherein, after employing the aforementioned method, cells (mammalian cells, such as Vero cells) are seeded into a bioreactor for cell culture.
[0019] Beneficial effects:
[0020] (1) The present invention first adds the microcarrier to the bioreactor, and then sterilizes the reactor and the microcarrier together in situ. This allows pure steam to directly enter the reactor and fully contact the microcarrier, so as to achieve the effect of fully sterilizing the microcarrier to the qualified level.
[0021] (2) By using filtered and sterilized sterile cell culture medium, the bioreactor can automatically replace the PBS solution under the protection of a sterile connector, thus avoiding the problem of microbial contamination caused by human operation. Attached Figure Description
[0022] Figure 1 This is a process flow diagram of using a bioreactor to sterilize and process microcarriers in situ.
[0023] Figure 2 This is the cell growth state after treatment using traditional methods.
[0024] Figure 3 This refers to the cell growth state after treatment using the method described in this invention. Detailed Implementation
[0025] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely illustrative and do not constitute any limitation on the scope of protection defined by the claims of the present invention.
[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0028] Example 1
[0029] This method utilizes a bioreactor for in-situ sterilization and treatment of microcarriers. Cytodex microcarriers were selected as the carriers for cell attachment and growth. Preparation included: a comprehensive inspection of all components of the bioreactor to ensure the stirring device, temperature control system, pH and dissolved oxygen electrodes, aeration system, microcarrier / cell retention device, and related interfaces were functioning correctly; and ensuring that the inlet / outlet solutions and cell inlet tubing were sterilized. The process flow is as follows: Figure 1 As shown.
[0030] A method for in-situ sterilization and treatment of cell microcarriers using a bioreactor includes the following steps:
[0031] (1) Weigh 400-500g of the microcarrier and add it to the bioreactor;
[0032] (2) Soak and swell the microcarriers thoroughly with PBS solution;
[0033] (3) Sterilize the bioreactor, the microcarrier inside the tank and each branch in situ. The reactor and the microcarrier inside the tank shall be sterilized with pure steam at 121°C for no less than 30 minutes, and each branch shall be sterilized for no less than 15 minutes. Then cool and let stand.
[0034] (4) Use about 100 liters of sterile cell culture medium and use continuous perfusion to replace the PBS solution in the reactor. The replacement rate should be no less than three times the flow rate and the replacement time should be no less than 24 hours. After the replacement is completed, prepare to inoculate cells.
[0035] Example 2: Comparison between the conventional method and the method of the present invention
[0036] The traditional method involves the following steps:
[0037] (1) Weigh about 500g of Cytodex microcarriers and add them to 5 glass bottles, each containing about 100g of microcarriers;
[0038] (2) Add about 3.5 liters of PBS solution to each glass bottle and soak for 30 minutes;
[0039] (3) Transfer the 5 glass bottles to a moist heat sterilizer and sterilize them for 2.5 hours;
[0040] (4) Place the sterilized glass bottle into the Class A laminar flow hood. After the microcarrier has settled sufficiently, pour the supernatant into the waste liquid container, trying to avoid losing the microcarrier. Then add 2 liters of cell culture medium, shake well and let stand for 30 minutes.
[0041] (5) Repeat (4) once. During the entire operation, handle the object gently and avoid excessive movements. Ensure a sterile environment.
[0042] (6) After all the treatments are completed, the microcarrier solution is stored at room temperature for 72 hours, then mixed with Vero cells and inoculated into a bioreactor for cell culture. After 5 days of culture, samples are taken to observe the cell status.
[0043] The processing procedure of the method described in this invention is as follows:
[0044] (1) Weigh approximately 500g of Cytodex microcarriers and add them to the bioreactor;
[0045] (2) Soak the microcarriers in approximately 20 liters of PBS solution for 30 minutes;
[0046] (3) Sterilize the bioreactor and the microcarrier inside the tank for 40 minutes, and each branch for 35 minutes. The pure steam sterilization temperature is 121℃. After sterilization, cool and let stand.
[0047] (4) After sterilization, the bioreactor discharges liquid through the sedimentation system to prevent the microcarriers from flowing out; the cell culture medium is perfused through the bioreactor inlet system to replace the PBS solution, the perfusion rate is about 2 liters / hour, and the perfusion time is about 24 hours.
[0048] (5) After the replacement is completed, the cells are stored at room temperature for 72 hours. Vero cells are then seeded into the bioreactor for cell culture. After 5 days of culture, samples are taken to observe the cell status.
[0049] Cell state after traditional treatment, such as Figure 2 As shown. The cell state after treatment by the method described in this invention is as follows. Figure 3As shown in the figure. A comparison reveals that the method of the present invention, while ensuring thorough sterilization of the microcarriers, does not affect cell growth status, and therefore does not affect product quality.
[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for in-situ sterilization and treatment of microcarriers, characterized in that, It includes steps such as adding microcarriers, soaking and swelling, in-situ sterilization, and replacing the culture medium; and uses a bioreactor as the carrier container.
2. The method according to claim 1, characterized in that, The microcarrier is a tiny particle carrier for cell culture with a diameter of 60-250 μm, such as the Cytodex series microcarriers. Preferably, the amount of the microcarrier added is 400-700 g, and more preferably, the amount added is 400-600 g.
3. The method according to claim 1, characterized in that, The soaking and swelling process involves thoroughly immersing the microcarrier in PBS solution for at least 30 minutes.
4. The method according to claim 1, characterized in that, The in-situ sterilization is carried out with pure steam at a temperature of 121°C or higher, and the sterilization time for the bioreactor and its microcarriers is no less than 30 minutes.
5. The method according to claim 4, characterized in that, The in-situ sterilization also includes sterilizing each branch of the reactor, with each branch sterilizing for no less than 15 minutes, preferably no less than 30 minutes.
6. The method according to claim 1, characterized in that, The culture medium replacement is performed using a continuous perfusion method, and the culture medium used is sterile cell culture medium.
7. The method according to claim 6, characterized in that, The volume of the sterile cell culture medium used is 70-150L, the replacement rate is not less than three times the flow rate, the replacement time is not less than 24 hours, the perfusion flow rate is not less than 1 liter / hour, preferably, the perfusion flow rate is not less than 2 liters / hour.
8. The method according to claim 1, characterized in that, The bioreactor is equipped with a microcarrier retention and sedimentation device.
9. The application of the method according to any one of claims 1-8 in adherent cell culture.
10. A method for adhering cell culture, characterized in that, After employing the method described in any one of claims 1 to 8, cells (mammalian cells, such as Vero cells) are seeded into a bioreactor for cell culture.