3D suspension cell culture device and application thereof

Abstract

The invention relates to a 3D suspension cell culture device and application thereof.The 3D suspension cell culture device comprises a base (1) and a reactor (2) installed on the base, and the reactor is fixed to the base through a U-shaped groove (005) protruding out of the middle position of a base shell (001); a driving device is arranged in the U-shaped groove and is used for driving a mixer 2 (026) in the reactor to rotate in a non-contact manner. The invention also relates to application of the 3D suspension cell culture device in culture of embryonic stem cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to a 3D suspension cell culture device and its application. Background Technology

[0002] Over the past 30 years, stem cells have shown remarkable promise in regenerative medicine, drug screening, and in vitro toxicology, bringing tremendous technological breakthroughs and hope to the scientific and medical communities. However, reproducibility and robustness of production remain significant bottlenecks for regulatory approval. The therapeutic application of stem cells requires the generation of large quantities of cells under highly robust, well-defined, and economically feasible conditions. Generally, the number of stem cells obtainable from donors is very limited. A pressing issue for advancing stem cells towards clinical and industrial applications is how to achieve large-scale in vitro expansion of stem cells to meet the production requirements for clinical treatment.

[0003] Stem cell culture in the presence of serum, feeder layers, or matrix gel is a traditional 2D culture method. The need for feeder layers poses a risk of pathogen contamination, is difficult to manage, and results in phenotypic or genotypic variations in the cultured stem cells, limiting cell yield. Even with matrix gel instead of feeder cells, 2D culture introduces excessive human intervention, making it difficult to scale up pharmaceutical production from laboratory settings. Furthermore, the culture method differs significantly from the natural expansion and development pathways of 3D cell clusters.

[0004] Compared to 2D culture, bioreactor-based processes reduce labor and operating costs, improve process monitoring and control, create a well-mixed environment, and promote increased cell density per unit volume. Currently, bioreactors provide a highly efficient method for stem cell culture, enabling large-scale expansion and effectively maintaining stem cell stemness and pluripotency, thus meeting the requirements of clinical cell therapy. In addition to scalability, bioreactors also allow for process monitoring, control, and reproducibility, which helps improve the quality, safety, and efficacy of clinical stem cell applications.

[0005] The most commonly used stem cell culture bioreactors include sway bioreactors, rotary bioreactors, and stirred bioreactors. The characteristics of each type of stem cell culture bioreactor are briefly described below: (1) Swing bioreactor: The design and operating costs of the reactor are high, the biological system is easily contaminated, the reactor scale is limited, it lacks scalability, the production capacity is low, and the reaction process cannot be monitored in real time. (2) Rotary bioreactor: The operation is complex and poses certain technical challenges to users. It is not suitable for continuous media replacement and real-time monitoring. (3) Stirred bioreactor: Its performance is affected by the collision between cells and impellers and the initiation of turbulence, which causes non-physiological mechanical and hydrodynamic shear stress on the cells and leads to cell damage. These adverse conditions affect cell growth rate and metabolism, interfere with stem cell pluripotency, and limit the efficiency and reproducibility of the culture process.

[0006] Swing and rotary bioreactors suffer from high manufacturing costs and operational complexity, hindering their widespread applicability in stem cell culture. Commercially available stirred bioreactors offer better applicability and adaptability, suitable for small, medium, and large-scale stem cell culture processes, and are easily scaled up. Their simplicity lowers the barrier to entry, attracting researchers from various fields. However, the mechanical shear forces within the reactor can easily damage cells, leading to reduced cell culture yields. Furthermore, daily medium changes and replenishment require repeated pipetting, increasing the risk of contamination and operational complexity. Additionally, each reactor base can only control one reactor, increasing time and space costs.

[0007] Currently, bioreactors used for expanding stem cells on the market include those using horizontal impellers, paddlewheels, or rocking wave mixing methods. Impeller-type bioreactors are a common method in traditional biological treatments, but the high shear stress transmitted by their stirring mechanism is unsuitable for culturing shear-sensitive cells such as stem cells. Most other bioreactors avoid this problem by not using impellers, but this can impair mass transfer and potentially affect the homogeneity of the culture.

[0008] Based on this, the present invention is proposed. Summary of the Invention

[0009] The present invention relates to a 3D suspended cell culture device, which includes a base (1) and a reactor (2) mounted on the base. The reactor is fixed on the base by a U-shaped groove (005) protruding from the middle of the base shell (001). A driving device is provided inside the U-shaped groove to drive the mixer 2 (026) inside the reactor to rotate in a non-contact manner.

[0010] Furthermore, the base also includes: a power connector (007) located on the rear side of the base housing, a speed display (002) and a speed knob (003) located on the front side of the base housing, and a power switch (004) located on the side of the base housing.

[0011] Furthermore, the base also includes reactor base supports (008) located on both sides of the U-shaped groove for supporting the reactor; and shock-absorbing pads (006) disposed on the lower part of the base shell.

[0012] Furthermore, the driving device includes: A fixing plate (013) is fixed on the base. A motor (015) is installed on one side of the fixing plate. On the other side of the fixing plate, there is a rotating shaft 1 (014) connected to the motor, a rotating shaft 2 (011) with a bearing (010) installed, and a transmission belt (012) connecting the rotating shaft 1 and the rotating shaft 2. The transmission belt is on the same plane as the rotating shaft 1 and the rotating shaft 2. Multiple magnets (009) are installed in the rotating shaft 2. The driving device drives the rotating shaft 1 through the motor and drives the rotating shaft 2 to rotate through the transmission belt, and drives the mixer 2 (026) inside the reactor to rotate in a non-contact manner by magnetic attraction conduction.

[0013] Furthermore, the mixer 2 (026) includes an impeller (021) and a set of magnets (009) disposed on the impeller. The magnets in the mixer 2 are matched with the magnets in the mixer 1 to transmit the rotation of the mixer 1 to the mixer 2 in a non-contact manner to rotate the impeller.

[0014] Furthermore, the impeller includes blades that extend radially inward from the blades and are adapted to each other. The impeller is also provided with inclined inner blades. These blades rotate around a horizontal axis in a vertical plane, so that the impeller generates both axial and radial flow. The impeller is provided with a plurality of magnets corresponding to the magnets on the rotating shaft 2. Preferably, the blades that make up the impeller are hollow.

[0015] Furthermore, the reactor includes an integral culture bottle body (020) and a sealing cap (019) fixed to the upper part of the culture bottle body. The sealing cap is provided with a feed inlet (018) integrally formed with the sealing cap. The impeller (021) is disposed inside the reactor and is fixed with a bearing plug (022). The lower arc edge of the impeller matches the arc-shaped lower structure of the culture bottle body. Preferably, the capacity of the culture bottle body is 500ml.

[0016] Furthermore, the reactor also includes a cap (017) for sealing the feed inlet. Preferably, the cap is provided with an integrally formed exhaust filter (025), which is used to ensure gas exchange while preventing contaminants from entering. Furthermore, the reactor also includes an integrally formed outlet port (024) located on the upper part of the culture bottle body. Preferably, a set of bottle caps containing an integrally formed exhaust filter is also provided to seal the outlet port, ensuring gas exchange while preventing contaminants from entering.

[0017] The present invention also relates to a method for culturing embryonic stem cells using the aforementioned 3D suspension cell culture device, the method comprising the following steps: (1) Resuspend the stem cells at a density of 1-5*10^5 / mL in mTeSR1+Y27632 medium, and then inoculate them into the 500ml volume 3D suspension culture device. Set the speed of mixer 1 to 30-50 rpm for 3D suspension culture. The culture parameters are 37℃ and 5% CO2. (2) Culture continuously for 5-7 days. The day of inoculation is D0. Do not change the medium on D1. Change the medium halfway from D2 to DN. Change the medium every 24±2 hours.

[0018] Preferably, the stem cells are pluripotent stem cells or induced pluripotent stem cells.

[0019] The beneficial effects of this invention are as follows: The vertical wheel culture apparatus of this invention overcomes the shortcomings of existing stem cell culture apparatuses by using a gentler and more efficient stirring mechanism to provide a uniform hydrodynamic environment. The invention focuses on optimizing the inoculation, stirring, oxygen, and nutrient availability for culturing stem cells into aggregates in a disposable, low-shear, vertical wheel stem cell culture apparatus.

[0020] Using the stem cell culture apparatus base and 500ml disposable container of this invention, 3D cell culture and suspension can be rapidly scaled up. The vertical impeller of this invention provides gentle yet efficient mixing, eliminating the need for antifoaming agents or shear protectants to expand shear-sensitive stem cells. The use of a side-mounted vent cap port allows for quick and convenient medium and material replenishment. Stem cells are cultured within a compact base and sealed reaction vessel, and the stem cell culture system can be easily controlled by adjusting the rotation speed button on the culture apparatus base and the digital display on the speedometer.

[0021] The disposable stem cell culture device (500ml) of this invention features a homogeneous environment, simple monitoring, direct control of key culture parameters, and, most importantly, scalability. The stem cell culture device consists of a cylindrical culture container equipped with an impeller or stirrer, which can be directly or indirectly driven by a motor. Hydrodynamics are generated by two types of impellers: axial and radial flow impellers. The blades of the axial flow impeller are angled to guide the airflow to the bottom of the container, while the blades of the radial flow impeller are perpendicular to the impeller axis, creating a flow pattern directed towards the container wall. The U-shaped bottom of the container and the relatively large impeller diameter create a large sweep volume, avoiding a "dead zone" below the impeller, limiting the sedimentation of stem cell aggregates, and providing a more efficient mixing effect.

[0022] The geometry of the 3D suspension culture device (500ml) of the present invention consists of a large vertical wheel with inclined inner blades that rotate about a horizontal axis along a vertical plane. This system combines radial and axial flow, providing agitation and particle suspension while achieving a more uniform hydrodynamic distribution.

[0023] Compared to traditional horizontal blade stirred suspension bioreactors, the 3D suspension culture device of this invention offers more efficient mixing while exhibiting lower shear rates and power input. The impeller and U-shaped container of this invention work together to promote strong, sweeping liquid flow at the bottom of the 3D suspension culture device; axial blades in opposite directions generate cutting and folding fluid flow to enhance mixing; and the relatively large impeller area with a sweeping volume collectively produces a relatively uniform hydrodynamic distribution, making it an ideal tool for stem cell culture and bioprocess design.

[0024] The disposable stem cell culture device of the present invention has excellent mixing performance, gentle and intact particle suspension, and minimal shear force in a homogeneous mixing environment; similar hydrodynamic conditions can be achieved at all volumes, from benchtop to clinical and commercial scale; the development and optimization of the process in small containers can predict cell culture performance at larger scales and enable the transition to process scale-up. Attached Figure Description

[0025] Figure 1 A schematic diagram of the reactor after it is assembled with the base, showing both the front and back sides.

[0026] Figure 2 Front and back views of the reactor base.

[0027] Figure 3 Schematic diagram of the internal structure of the base.

[0028] Figure 4 Schematic diagram of reactor consumables.

[0029] Figure 5 Schematic diagram of reactor consumable impeller assembly.

[0030] Figure 6 Morphological diagram of hPSCs cultured in a plane.

[0031] Figure 7 Flow cytometry results for detecting stemness in planar cultured cells.

[0032] Figure 8 Cell morphology diagram of hPSCs 3D culture.

[0033] Figure 9 Flow cytometry results of 3D culture stemness detection of hPSCs cells.

[0034] Figure 10 Cell morphology diagram of hPSCs during 3D domestication.

[0035] Figure 11 A statistical chart of the amplification folds across five generations during the domestication process.

[0036] Figure 12 Flow cytometry results of each generation of dryness detection during the domestication process.

[0037] Figure 13 Cell morphology diagram of hPSCs after cryopreservation and 3D culture.

[0038] Figure 14 Flow cytometry results of 3D culture of hPSCs after cryopreservation and resuscitation to detect dryness.

[0039] Appendix: Explanation of Icon Numbers Detailed Implementation

[0040] The present application will be further described in detail below with reference to the accompanying drawings.

[0041] Example 1: Structure and assembly of a 3D stem cell suspension culture device Taking a 500ml volume one (base) control two (reactor) stem cell culture device as an example, Reference Figure 1 The specific structure of the stem cell 3D suspension culture device includes a base (1) and a reactor (2) mounted on the base (1). The base includes a speed display and a speed knob on its front side, a switch on the right side of the bottom of the base, a power connector on the rear of the bottom of the base, and the reactor is fixed to the base by a U-shaped groove (005) protruding from the middle of the base shell (001).

[0042] Reference Figure 2 and Figure 3 Describe the structure of the base of the 3D suspension culture device.

[0043] like Figure 2 The diagram shows the main structure of the base. The power connector (007) is sealed and moisture-proof, fixed to the rear side of the base housing (001). The speed indicator (002) is sealed and moisture-proof, fixed to the front side of the base housing. The speed knob (003) is also sealed and moisture-proof, fixed to the front side of the base housing. The power switch (004) is sealed and moisture-proof, located on the side of the base housing. The U-shaped groove (005) protrudes from the middle of the base housing. The reactor base support (008) is located on both sides of the U-shaped groove, used to support the reactor. The shock-absorbing pad (006) is located at the bottom of the base housing.

[0044] like Figure 3 The structure shown is that of the motor and the rotating shaft, which is set inside the base and the U-shaped groove. The specific structure includes: a fixing plate (013) that can be fixed to the base by screws or riveting; a motor (015) is set on one side of the fixing plate; on the other side of the fixing plate are: a rotating shaft 1 (014) connected to the motor (015), and a rotating shaft 2 (011) with a bearing (010) installed; a transmission belt (012) connecting the rotating shaft 1 and the rotating shaft 2, and the transmission belt is on the same plane as the rotating shaft 1 and the rotating shaft 2. Magnets (009) are evenly arranged in the rotating shaft 2. The overall transmission structure composed of the motor, the rotating shaft 1, the rotating shaft 2 containing magnets, and the transmission belt is a mixer 1 (016).

[0045] Reference Figure 4 and Figure 5 Describe the structure of the reactor.

[0046] like Figure 4 The reactor and its internal impeller are shown. The culture flask body (020) and its sealing cap (019) are made of medical-grade PC material. The culture flask body can be fused together using laser welding to ensure no gaps, dead corners, or contaminants. The complete impeller (021) is located in the culture flask body and is fixed with a bearing plug (022). The lower arc edge of the impeller matches the arc-shaped lower structure of the culture flask body. The sealing cap (019) is fixed to the upper part of the culture flask body and can also be connected using laser welding to prevent the sealing plug from loosening and liquid from seeping into the body. The sealing cap is provided with an inlet (018) integrally formed with the sealing cap. The bottle cap (017) is used to seal the inlet. The bottle cap (017) is provided with an integrally formed exhaust filter (025). The bottle cap and exhaust filter can be formed using a hot pressing process. The exhaust filter is used to diffuse gas into the container to provide the necessary process gas for the cells, while preventing pathogenic microorganisms from entering the container and contaminating the cells. The outlet port (024) is integrally formed and located on the upper part of the culture bottle body. A bottle cap containing an integrally formed exhaust filter is also provided to seal the outlet port, ensuring gas exchange while preventing contaminants from entering.

[0047] like Figure 5The mixer 2 (026) shown has an impeller (21) comprising blades that extend radially inward from the blades and are adapted to each other. Magnets (009) are also mounted on the impeller. The two radially opposing blades are configured to generate biaxial fluid flow when the impeller rotates. The paddle is hollow. An impeller containing blades and a set of magnets constitutes a mixer 2 (026). The magnets in mixer 2 are matched with the magnets in mixer 1, transmitting the rotation of mixer 1 to mixer 2 non-contactly to rotate the impeller.

[0048] Example 2: 3D suspension culture of stem cells using a 3D stem cell culture device 1. hPSCs cell planar culture Preparation method of required reagents: mTeSR1 medium (mTeSRTM1 complete medium, STEMCELL, 85850). Accutase (AccutaseTM, STEMCELL, 07920); Vitronectin (VTN-N, Gibco, A14700); Y-27632 dihydrochloride, Rhokinase Inhibitor (Y27632, abcam, ab120129): Prepare a 10 mM stock solution with DMSO. The final concentration for use is 10 μM, i.e., it is used as 1000X. mTeSR1+Y27632 medium: Add 50 μL of 10 mM Y27632 to 50 mL of mTeSR1, mix well, store at 4℃ when not in use, and use within two weeks after preparation.

[0049] ① Remove DMEM / F12 and rewarm. Prepare mTeSR1 complete culture medium (containing 10 μM Y27632) and rewarm. Turn on the water bath and set it to 37℃ for later use.

[0050] ② Dilute VTN-N to prepare a working solution and add it to a six-well plate at a rate of 0.5-1.5 mL / well. Incubate at room temperature / 37°C for at least 1 hour.

[0051] ③ Quickly immerse the cryovials taken from the liquid nitrogen into a 37°C water bath and shake them rapidly to thaw them within 1-2 minutes.

[0052] ④ Transfer the cell suspension into a 50mL centrifuge tube. Using a Pasteur pipette, add DMEM / F12 dropwise to the 50mL centrifuge tube containing the cell suspension at a ratio of 1:9 (cell suspension:DMEM / F12 medium).

[0053] ⑤ Centrifuge the centrifuge tubes at 300-800g in a centrifuge at room temperature for 3-5 minutes. After centrifugation, discard the supernatant.

[0054] ⑥ Add 0.5-2 mL of mTeSR1+Y27632 complete culture medium and gently mix.

[0055] ⑦ Take the cell suspension into a 1.5 mL EP tube, add an equal volume of AO / PI staining solution to the EP tube, mix well, and then count the cells using a fluorescence cell analyzer.

[0056] ⑧ Remove the coated culture plate from the biosafety cabinet, discard the matrix gel in the six-well plate, and add mTeSR1 (containing 10 μM Y27632) medium to each well at a rate of 2-4 mL / well. Based on the cell count results, culture the cells at a rate of 1.0-3.0 x 10⁻⁶ cells / well. 4 / cm 2 The inoculum was seeded into six-well plates at a density of [amount missing], shaken well, and then placed in a 37°C, 5% CO2 incubator for static incubation.

[0057] ⑨ Change the fluid: Change the fluid every 24-48 hours starting from the time of resuscitation.

[0058] ⑩ During the planar culture process, the cell morphology was normal, as shown in the microscope photograph. Figure 6 The indicators and results of stem cell pluripotency detection during the planar culture process are shown in [reference needed]. Figure 7 The results showed that hPSCs cultured in planar environment using the above method could maintain the clonal morphology of stem cells well, and the expression of stemness-related genes was very good. Flow cytometry results showed that the proportion of Oct3 / 4 and SSEA4 double-positive cells was over 95%, indicating that stemness was well maintained during the culture process; no cell differentiation was observed.

[0059] 2. hPSCs were passaged into a 3D suspension culture device for cultivation. ① Washing: Remove the original culture medium, slowly add 0.5-2 mL of DMEM / F12 and gently shake, then discard the waste liquid.

[0060] ② Digestion: Add 0.5-2 mL / well Accutase to a 6-well plate to cover the bottom of the plate, and incubate at 37℃ for 2-5 min; ③ Neutralization: Gently tap the bottom of the dish to detach the cells, add 2-4 mL of DMEM / F12 medium to neutralize, gently pipette 3-5 times, and transfer to a 15 mL centrifuge tube; ④ Counting: Centrifuge at 300-800 g for 3-5 min; discard the supernatant, pipette the cells 5-10 times with stem cell culture medium, and take a portion of the cell suspension for counting; ⑤ Inoculation: Based on the counting results, resuspend 1-5*10^5 / mL in mTeSR1+Y27632 medium, and then inoculate it into the 3D suspension culture device described in this invention, using a culture system of 300-500 mL. Secure the reactor consumables in the slot in the center of the base, turn on the switch, and set the rotation speed to 30-50 rpm using the speed control button to perform 3D suspension culture. Set the incubator parameters to a constant temperature of 37℃, 5% CO2, and 100% humidity.

[0061] ⑥ Culture continuously for 5-7 days (subculture is recommended when the cell cluster size is approximately 250-400 μm). The day of inoculation is D0, do not change the medium on D1, and change the medium halfway from D2 to DN. Change the medium every 24±2 hours.

[0062] ⑦ Digestion: Collect all cell clusters in the reactor using a reversible filter. Invert the reversible filter into a 50mL centrifuge tube, add 15-40mL of warmed Accutase, and flush the cell clusters from the reversible filter into the centrifuge tube. Gently mix the cell suspension, divide it into two equal 50mL centrifuge tubes, tighten the caps, and incubate at 37°C for 10-30 minutes, until the cell clusters are no longer visible to the naked eye. Add twice the volume of DMEM / F12 medium to each centrifuge tube to terminate the digestion.

[0063] ⑧ After centrifugation, count the cells. Based on the counting results, inoculate the single cells back into the reactor and continue culturing for 5-7 days.

[0064] ⑨ Cryopreservation: Centrifuge the remaining cells again, discard the supernatant, and add cryopreservation solution according to the counting results. The cryopreservation density is 5*10^5-1*10^7 / mL. Place the cryopreservation tubes in a programmed cooling box and then place them in a -80°C freezer overnight. The next day, transfer them to a liquid nitrogen tank.

[0065] ⑩ See the microscope image of 3D culture of hPSCs. Figure 8 .

[0066] ⑪ Flow cytometry results of stemness-related genes in 3D culture of hPSCs cells are shown in [the table below]. Figure 9 .

[0067] 3. Analysis of the results of 3D domestication of hPSC ① After acclimatizing for 3-5 generations in the 3D suspension culture device, and through continuous subculturing and amplification, it was determined that hPSC can maintain its dryness and be effectively amplified in the 3D system.

[0068] ②The cell culture process during the domestication process is shown in the following figure. Figure 10 It is evident that pluripotent stem cells can proliferate well during the domestication process. The cell count was calculated at harvest, and the fold increase per generation was recorded. The results are shown in [Table / Reference]. Figure 11During the domestication process, hPSCs were also able to express stemness-related genes well, and flow cytometry results showed that the proportion of Oct3 / 4 and SSEA4 double-positive cells was over 95%. Figure 12 This also indicates that the stemness of 3D pluripotent stem cells was well maintained during the domestication process.

[0069] ③ After cryopreservation and thawing, the acclimatized cells were directly inoculated into the reactor for culture, during which the cell morphology remained well maintained. Figure 13 At harvest, cell quantity was calculated, with an expansion fold of 3-5. Flow cytometry results showed that the proportion of Oct3 / 4 and SSEA4 double-positive cells was above 95%. Figure 14 This indicates that the pluripotent stem cells cultured in this system are in a stable state and can maintain their stemness and proliferate normally after cryopreservation and thawing.

[0070] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention. The descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention.

Claims

1. A 3D suspension cell culture device, comprising: The base (1) and the reactor (2) mounted on the base are fixed to the base by a U-shaped groove (005) protruding from the middle of the base shell (001); a driving device is provided inside the U-shaped groove to drive the mixer 2 (026) inside the reactor to rotate in a non-contact manner.

2. The 3D suspension cell culture device according to claim 1, characterized in that, The base also includes: a power connector (007) located on the rear side of the base housing, a speed display (002) and a speed knob (003) located on the front side of the base housing, and a power switch button (004) located on the side of the base housing.

3. The 3D suspension cell culture device according to claim 2, characterized in that, The base also includes reactor base supports (008) located on both sides of the U-shaped groove for supporting the reactor; and shock-absorbing pads (006) disposed on the lower part of the base shell.

4. The 3D suspension cell culture device according to any one of claims 1-3, characterized in that, The driving device includes: A fixing plate (013) is fixed on the base. A motor (015) is installed on one side of the fixing plate. On the other side of the fixing plate, there is a rotating shaft 1 (014) connected to the motor, a rotating shaft 2 (011) with a bearing (010) installed, and a transmission belt (012) connecting the rotating shaft 1 and the rotating shaft 2. The transmission belt is on the same plane as the rotating shaft 1 and the rotating shaft 2. Multiple magnets (009) are installed in the rotating shaft 2. The driving device drives the rotating shaft 1 through the motor and drives the rotating shaft 2 to rotate through the transmission belt, and drives the mixer 2 (026) inside the reactor to rotate non-contactly by magnetic attraction conduction.

5. The 3D suspension cell culture device according to any one of claims 1-4, characterized in that, The mixer 2 (026) includes an impeller (021) and a set of magnets (009) disposed on the impeller. The magnets in the mixer 2 are matched with the magnets in the mixer 1 to transmit the rotation of the mixer 1 to the mixer 2 in a non-contact manner to rotate the impeller.

6. The 3D suspension cell culture device according to claim 5, characterized in that, The impeller includes blades that extend radially inward from the blades and are adapted to each other. The impeller is also provided with inclined inner blades. These blades rotate about a horizontal axis along a vertical plane, so that the impeller generates both axial and radial flow. The impeller is provided with a plurality of magnets corresponding to the magnets on the rotating shaft 2. Preferably, the blades that make up the impeller are hollow.

7. The 3D suspension cell culture device according to any one of claims 1-6, characterized in that, The reactor includes an integral culture bottle body (020) and a sealing cap (019) fixed to the upper part of the culture bottle body. The sealing cap is provided with a feed inlet (018) integrally formed with the sealing cap. The impeller (021) is disposed inside the reactor and is fixed with a bearing plug (022). The lower arc edge of the impeller matches the arc-shaped lower structure of the culture bottle body. Preferably, the capacity of the culture bottle body is 500ml.

8. The 3D suspension cell culture device according to any one of claims 1-7, characterized in that, The reactor also includes a cap (017) for sealing the feed inlet; preferably, the cap is provided with an integrally formed exhaust filter (025).

9. The 3D suspension cell culture device according to any one of claims 1-8, characterized in that, The reactor also includes an integrally formed outlet port (024) located on the upper part of the culture bottle body. Preferably, a set of bottle caps containing an integrally formed exhaust filter is also provided to seal the outlet port.

10. A method for culturing embryonic stem cells using the 3D suspension cell culture apparatus according to any one of claims 1-9, the method comprising the following steps, (1) Resuspend the stem cells at a density of 1-5*10^5 / mL in mTeSR1+Y27632 medium, and then inoculate them into the 500ml volume 3D suspension culture device. Set the speed of mixer 1 to 30-50rpm for 3D suspension culture. The culture parameters are 37℃ and 5% CO2. (2) Culture continuously for 5-7 days. The day of inoculation is D0. Do not change the medium on D1. Change the medium halfway from D2 to DN. Change the medium every 24±2 hours. Preferably, the stem cells are pluripotent stem cells or induced pluripotent stem cells.

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