Vertical shaft stirring bioreactor for preparing mesenchymal stem cell spheres

Abstract

The utility model discloses a vertical shaft stirring bioreactor for preparing mesenchymal stem cell spheres, which comprises a base, a motor arranged on the base, a magnetic coupling device connected with the motor, a container bottle arranged at the upper part of the base and a stirring device arranged in the container bottle, a magnetic coupling device is arranged in the container bottle, a magnetic transmission part matched with the magnetic coupling device is arranged in the container bottle, the magnetic transmission part is connected with a stirring device, the stirring device comprises a vertically arranged stirring shaft and multiple layers of stirring blades fixed on the stirring shaft, a liquid inlet / outlet is formed in the container bottle, a diaphragm valve is arranged on the liquid inlet / outlet, and a control circuit is arranged in the base. The diameter uniformity of mesenchymal stem cell spheres cultured by using the device disclosed by the utility model is greatly improved, the cell activity and the culture efficiency can be effectively improved, and a powerful tool is provided for the application of mesenchymal stem cells in the fields of tissue engineering and regenerative medicine.

Description

Technical Field

[0001] This utility model relates to the field of biomedical equipment technology, specifically to a vertical axis stirred bioreactor for preparing mesenchymal stem cell spheres. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with multi-lineage differentiation potential and self-renewal capacity, showing broad application prospects in tissue engineering, cell therapy, and regenerative medicine. Traditional MSC culture methods primarily involve two-dimensional planar culture, but this method differs significantly from the three-dimensional microenvironment in vivo and cannot fully simulate the physiological state of cells within the body. To better simulate the in vivo microenvironment, three-dimensional cell culture technology has become a research hotspot, with MSC spheroid culture being an important three-dimensional culture method.

[0003] Currently, the main methods for preparing mesenchymal stem cell spheres include the hanging drop method, the low-adsorption plate method, and the stirred suspension culture method. Among these methods, the stirred suspension culture method has attracted widespread attention due to its advantages such as strong scalability and simple operation. However, existing stirred bioreactors have the following shortcomings: first, most adopt a horizontal stirring shaft structure, resulting in uneven stirring and inconsistent cell sphere size; second, poor sealing, making them prone to contamination; third, difficulty in controlling the stirring intensity, causing cell damage; and fourth, difficulty in achieving large-scale culture, resulting in limited yield.

[0004] Therefore, there is an urgent need to develop a novel bioreactor capable of efficiently and stably preparing mesenchymal stem cell spheres. Utility Model Content

[0005] The purpose of this utility model is to overcome the shortcomings of the prior art and provide a vertical axis stirred bioreactor for the preparation of mesenchymal stem cell spheres. The reactor adopts a vertical axis stirring structure and magnetic coupling transmission method, which has good sealing performance, uniform stirring effect, adjustable stirring speed and convenient operation process, and can efficiently and stably prepare mesenchymal stem cell spheres with uniform quality and high biological activity.

[0006] To achieve the above objectives, this utility model provides the following technical solution:

[0007] A vertical-axis stirred bioreactor for preparing mesenchymal stem cell spheres is characterized by comprising: a base, a motor mounted on the base, a magnetic coupling device connected to the motor, a container bottle disposed on the upper part of the base, and a stirring device disposed inside the container bottle; the container bottle is made of transparent material and has a magnetic transmission component matching the magnetic coupling device inside, the magnetic transmission component being connected to the stirring device; the stirring device includes a vertically arranged stirring shaft and multiple layers of stirring blades fixed on the stirring shaft; the container bottle has inlet and outlet ports, each equipped with a diaphragm valve; the base contains a control circuit for controlling the motor speed and running time.

[0008] Preferably, the stirring shaft is a vertical shaft, and its length accounts for 50%-80% of the height of the container, ensuring that the liquid inside the container can be stirred in all directions.

[0009] Preferably, the stirring blades are multi-layered, with each layer including 2-4 blades. The blades of adjacent layers are staggered, and the blades are arc-shaped or spiral-shaped, which is beneficial for generating vertical convection and uniform stirring effect.

[0010] Preferably, the magnetic coupling device includes a driving magnet and a driven magnet. The driving magnet is connected to a motor, and the driven magnet is connected to a stirring shaft. The two achieve non-contact magnetic transmission through an isolation layer at the bottom of the container, ensuring the sealing and sterile environment inside the container.

[0011] Preferably, the container is made of a biocompatible material, specifically glass or medical-grade polycarbonate, and the inner wall of the container is specially treated to prevent cells from adhering to the wall surface.

[0012] Preferably, the control circuit includes a speed regulator and a timer, which can adjust the stirring speed and mode according to the cell type and culture stage, with a speed range of 10-200 rpm and an intermittent stirring mode.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention provides a vertical-axis stirred bioreactor for preparing mesenchymal stem cell spheres. Through its vertical-axis stirring structure design combined with magnetic coupling transmission technology, it achieves a high degree of sealing and stability of the culture environment. The multi-layered, staggered stirring blades generate a uniform liquid flow field, avoiding dead zones and localized high-shear-force areas, promoting cell sphere formation and ensuring their size uniformity. Adjustable stirring speeds and modes meet the needs of different culture stages, improving the efficiency and quality of cell sphere formation. The special design of the container facilitates observation, sampling, and harvesting of cell spheres, simplifying the operation process and reducing the risk of contamination. Experimental verification shows that the mesenchymal stem cell spheres cultured using this device exhibit significantly improved diameter uniformity, and both cell viability and culture efficiency are effectively enhanced, providing a powerful tool for the application of mesenchymal stem cells in tissue engineering and regenerative medicine. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of this utility model.

[0016] Figure 2 This is a schematic diagram of the internal structure of the container bottle.

[0017] In the diagram: 1-base, 2-motor, 3-container, 4-magnetic coupling, 5-inlet / outlet, 6-stirring shaft, 7-stirring blade, 8-diaphragm valve, 9-control panel. Detailed Implementation

[0018] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0019] A vertical axis stirred bioreactor for preparing mesenchymal stem cell spheres includes a base 1, a motor 2, a container 3, a magnetic coupling 4, an inlet / outlet 5, a stirring shaft 6, stirring blades 7, and a diaphragm valve 8.

[0020] The base 1 serves as the supporting structure for the entire device, made of stainless steel or engineering plastic, providing sufficient stability. The motor 2 and control circuitry are installed inside the base 1, and a support structure for placing the container bottle 3 is located on the upper part of the base 1. The control panel 9 is located on the front of the base 1 and includes a power switch, a speed adjustment knob, a timer setting button, and a digital display screen.

[0021] Motor 2 is a precision stepper motor with a speed range of 10-200 rpm, featuring high-precision control and stable operation. The shaft of motor 2 is connected to the drive section of magnetic coupling 4.

[0022] The magnetic coupling 4 consists of two parts: a driving magnet and a driven magnet. The driving magnet is fixed on the output shaft of the motor 2, and the driven magnet is located inside the bottom of the container 3 and connected to the lower end of the stirring shaft 6. The two achieve non-contact magnetic transmission through the isolation layer (2-5mm thick) at the bottom of the container 3, eliminating the leakage and contamination risks that may be caused by traditional shaft seal structures.

[0023] Container 3 is made of a transparent, biocompatible material, specifically borosilicate glass or medical-grade polycarbonate, and its capacity is available in 100ml, 250ml, 500ml, or 1000ml sizes to suit different culture needs. The inner wall of container 3 is specially treated with a hydrophilic coating to prevent cell adhesion. Container 3 has a sealing cap at the top, with inlet / outlet ports 5 and a gas exchange port, all equipped with diaphragm valves 8 for sealing and aseptic operation.

[0024] The stirring shaft 6 is a vertically positioned cylindrical structure made of medical-grade stainless steel or high-strength engineering plastic. Its length accounts for approximately 60% of the height of the container bottle 3, and its diameter is 3-8 mm. Three to four layers of stirring blades 7 are evenly distributed on the stirring shaft 6.

[0025] The stirring blades 7 feature an arc or spiral design, with 2-4 blades per layer, and adjacent layers staggered at 45° or 90°. The blade material is the same as the stirring shaft 6, ensuring good biocompatibility. The size and tilt angle of the blades are optimized to generate a fluid shear force field suitable for cell spheroid formation, preventing cell sedimentation without causing excessive mechanical damage to the cells.

[0026] The operating steps for culturing mesenchymal stem cell spheres using the device of this invention are as follows:

[0027] Step S1: Under aseptic conditions, inject the mixture containing cell suspension and culture medium into container bottle 3 through inlet / outlet 5;

[0028] Step S2: Tighten the sealing cap of container bottle 3, ensuring all valves are closed;

[0029] Step S3: Place container bottle 3 on base 1, aligning the driven magnet at the bottom of container bottle 3 with the driving magnet on base 1;

[0030] Step S4: Set the appropriate stirring speed (generally 20-60 rpm in the initial stage) and operating mode (continuous or intermittent operation can be selected) through the control panel 9;

[0031] Step S5: Start the device and place the entire system in a 37°C, 5% CO2 incubator for incubation;

[0032] Step S6: During the culture process, the formation of cell spheres can be observed through the transparent wall of container 3. If necessary, samples can be taken for analysis through inlet / outlet 5.

[0033] Step S7: After the culture is completed, harvest the cell spheres through the inlet / outlet 5, or directly remove the container bottle 3 for further processing.

[0034] Compared to traditional horizontal axis structures, the vertical axis stirring structure of this invention generates more uniform vertical convection, preventing cell accumulation at the bottom of the container. The multi-layered, staggered arrangement of the stirring blades further optimizes the hydrodynamic characteristics, creating a microenvironment suitable for cell ball formation. The magnetic coupling drive method solves the leakage and contamination problems that may exist at the shaft seal of traditional stirrers, greatly improving the system's sealing performance and ease of aseptic operation.

[0035] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A vertical shaft stirring bioreactor for mesenchymal stem cell sphere preparation, characterized by, include: The base (1), the motor (2) mounted on the base (1), the magnetic coupling (4) connected to the motor (2), the container bottle (3) set on the upper part of the base (1), and the stirring device set in the container bottle (3); the container bottle (3) is made of transparent material and has a magnetic transmission component that matches the magnetic coupling (4) inside, and the magnetic transmission component is connected to the stirring device; the stirring device includes a vertically set stirring shaft (6) and a multi-layer stirring blade (7) fixed on the stirring shaft (6); the container bottle (3) is provided with an inlet and outlet (5), and a diaphragm valve (8) is provided on the inlet and outlet (5); the base (1) is provided with a control circuit for controlling the speed and running time of the motor (2).

2. The vertical axis stirred bioreactor for preparing mesenchymal stem cell spheroids according to claim 1, wherein, The stirring shaft (6) is a vertical shaft, and its length accounts for 50%-80% of the height of the container bottle (3).

3. The vertical-axis stirred bioreactor for preparing mesenchymal stem cell spheres according to claim 1, characterized in that, The stirring blades (7) are multi-layered, with each layer including 2-4 blades. The blades of adjacent layers are arranged in an alternating pattern, and the blades are arc-shaped or spiral-shaped.

4. The vertical axis stirred bioreactor for preparing mesenchymal stem cell spheroids according to claim 1, wherein, The magnetic coupling (4) includes a driving magnet and a driven magnet. The driving magnet is connected to the motor (2), and the driven magnet is connected to the stirring shaft (6). The two achieve non-contact magnetic transmission through the isolation layer at the bottom of the container bottle (3).

5. The vertical axis stirred bioreactor for preparing mesenchymal stem cell spheroids according to claim 1, wherein, The container bottle (3) is made of a biocompatible material, specifically glass or medical-grade polycarbonate.

6. The vertical axis stirred bioreactor for preparing mesenchymal stem cell spheroids according to claim 1, wherein, The control circuit includes a speed regulator and a timer, which can adjust the stirring speed and mode according to the cell type and culture stage. The speed range is 10-200 rpm, and an intermittent stirring mode can be set.

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