Apparatus and method for cell amplification and non-contact real-time measurement of cell amplification density

Abstract

Provided are an apparatus and method for cell amplification and non-contact real-time measurement of cell amplification density, used for measuring the density of a measured object in a cell culture bag. The apparatus comprises: a weighing sensor provided on a fixing plate and used for monitoring the weight in real time; a Y-axis swing mechanism provided on the weighing sensor and used for swinging in the Y-axis direction; an X-axis swing mechanism provided on the Y-axis swing mechanism and used for swinging in the X-axis direction; and a density sensor provided on the X-axis swing mechanism and used for measuring the density of the measured object in the cell culture bag. The apparatus and method can realize automated sterile culture of the cell culture bag without manual intervention in the whole process.

Description

A device and method for cell expansion and non-contact real-time measurement of cell expansion density

[0001] This application claims the benefit of priority to Chinese Patent Application Nos. 2023218446223, 2023108631538, 202310860536X, 202310864234X, 2023218491534, 2023218446257, and 2023218491708, filed on July 14, 2023, the disclosures of which are incorporated herein by reference as if fully written herein. Technical Field

[0002] The present invention relates to an automatic cell culture amplification device (usually called a swing table), and in particular to a device and method for online monitoring of cell amplification density and fluid replenishment according to amplification multiples. Background Art

[0003] GE has disclosed a cell amplifier that consists of a closed 2D swing table. The disadvantage is that it cannot be placed in a carbon dioxide incubator for use. It is necessary to separately transport carbon dioxide and dissolved oxygen into the interior and extract the exhaust gas, which is relatively complicated and prone to bacterial contamination. In addition, existing cell expansion and mixing devices all require a separate carbon dioxide pipeline to deeply contact the culture medium inside the cell culture bag for ventilation. The installation is inconvenient, the operation requirements are high, the use environment requirements are high, and the sterile culture of cells cannot be guaranteed. Some existing cell expansion devices or systems use mechanical stirring, which has a shear force on the cells, causing the cells to collide with the stirrer and break the cells. Some of them have a small swing mixing angle because the pipeline is deep into the cell culture bag, and cannot truly mix evenly, especially cannot fully mix with the culture medium. As mentioned above, they all rely on manual operation and have many disadvantages. The operating equipment for cell culture is not centralized and is not suitable for large-scale cell culture. Manual access to carbon dioxide gas intubation for ventilation is likely to contaminate the cells, resulting in the scrapping of the results of many days of culture.

[0004] There are two existing methods for measuring cell density: the first is to sample the cells by droplet, place them under a microscope for contact observation, take a photo, and then circle a certain area of ​​cells for a rough count; the second is to mix the cells, sample them, use a flow cytometer for contact measurement, and manually circle and count them, which is more accurate. Disadvantages: Both methods rely on manual operation and manual data upload, and cannot be automated. It is impossible to monitor cell growth data in real time, read data online, and obtain cell density in a timely manner. The operating equipment is complex and not suitable for large-scale cell culture. Manual counting is cumbersome and prone to cell contamination.

[0005] Moreover, the rehydration mode is single. Currently, there are manual rehydration, timed or quantitative rehydration, which cannot be truly automated.

[0006] Summary of the Invention

[0007] In view of the technical problems raised in the background art, the present invention provides a device for cell expansion and non-contact real-time measurement of cell expansion density, in order to at least partially solve at least one of the above technical problems.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] This invention relates to a device for cell expansion and non-contact real-time measurement of cell expansion density, which is used for cell expansion and detection of the density of the object being measured in a cell culture bag, including:

[0010] A weighing sensor is provided on the fixed plate and is used to monitor the weight in real time;

[0011] The Y-axis swing mechanism is provided on the weighing sensor and is used for swinging along the Y-axis direction;

[0012] An X-axis swing mechanism is provided on the Y-axis swing mechanism and is used for swinging along the X-axis direction;

[0013] The density sensor is arranged on the X-axis swing mechanism and is used to detect the density of the object being measured in the cell culture bag.

[0014] In a further preferred embodiment, the cell culture bag is provided with a cell density detection port, and the interior of the cell density detection port is communicated with the space in the cell culture bag.

[0015] In a further preferred embodiment, the density sensor includes an infrared light source, a beam splitter prism, a reference infrared receiver A, a detection infrared receiver B, and an optical signal processing circuit.

[0016] In a further preferred embodiment, the infrared light source emits 800-1100nm infrared parallel light, the dichroic prism is a 1:1 dichroic prism, and the infrared parallel light is divided into reference parallel light and detection parallel light after passing through the dichroic prism. The reference parallel light is detected by the reference infrared receiver A, and the detection parallel light is detected by the detection infrared receiver B after passing through the cell density detection port.

[0017] In a further preferred embodiment, the reference parallel light signal and the absorbed parallel light signal enter the optical signal processing circuit, the illumination values ​​of the two are compared and corrected, and the light absorbance value is calculated to characterize the density of the cells, thereby indirectly detecting the change in the density value of the object being measured.

[0018] In a further preferred embodiment, the Y-axis swing mechanism includes a Y-axis bracket, a first swing axis located on both sides of the Y-axis bracket, a connecting rod driving at least one side of the first swing axis to rotate, a first driving rod and a Y-axis motor, support frames are provided on both sides of the weighing sensor, and a first support shaft hole for supporting the first swing axis is provided on the support frame, the first swing axis is hinged to one end of the connecting rod, and the other end of the connecting rod is hinged to the first driving rod of the Y-axis motor.

[0019] In a further preferred embodiment, the X-axis swing mechanism includes an X-axis swing pallet, a second swing shaft located on one side or both sides of the X-axis swing pallet, and a second drive rod and an X-axis motor for driving the X-axis swing pallet to swing, wherein the second swing shaft is passed through a second support shaft hole on the Y-axis bracket that is perpendicular to the first swing shaft, and the second drive rod is hinged at the bottom of the X-axis swing pallet and deviates from the axis where the second swing shaft is located.

[0020] In a further preferred embodiment, the Y-axis bracket is in the form of a rectangular frame as a whole, and the X-axis swing support plate is arranged in the middle of the Y-axis bracket with gaps left around it.

[0021] In a further preferred embodiment, the Y-axis motor and the X-axis motor are electrically connected to an electric control board to control the rotation amplitude and frequency of the Y-axis motor and the X-axis motor.

[0022] In a further preferred embodiment, a control valve is provided on the X-axis support plate, and the control valve is used to control the on-off of the cell culture bag and the culture medium liquid pipeline. The control valve is a peristaltic pump or a pinch valve.

[0023] The present invention also protects a method for cell expansion and non-contact real-time measurement of cell expansion density, using the device of any of the above embodiments, comprising the following steps:

[0024] S01: Place the entire device in a CO2 incubator, fix the cell culture bag to the X-axis swing support, fix the tubing of the cell culture bag to the control valve, and fix the cell density detection port of the cell culture bag to the density sensor;

[0025] S02: Start the machine and power on the electric control board. Return the Y-axis motor and the X-axis motor to zero position. Adjust the X-axis swing plate and the Y-axis bracket to a completely horizontal position. The weighing sensor reads the weight. The machine is ready.

[0026] S03: Inject the coated cells into the cell culture bag and culture them horizontally for a certain period of time;

[0027] S04: Open the control valve to inject the culture medium, and according to the swing amplitude and frequency of the Y-axis motor and / or the X-axis motor set by the user, the Y-axis motor and / or the X-axis motor can be turned on simultaneously or individually to gently swing and mix the cells and the culture medium, and mix the carbon dioxide that permeates the breathable cell culture bag into the cell culture bag.

[0028] S05: Turn on the density sensor, record the cell density data in real time and upload it to the electronic control board. After the cells grow for a certain number of days, the cells will expand in large quantities.

[0029] S06: After the culture is completed, the data of the weighing sensor is read to obtain the accurate cell weight.

[0030] In a further preferred embodiment, in step S05, the control valve is opened again to inject the same or different culture medium according to the user's needs or program settings, and the culture medium and cells are continued to be mixed according to the new density setting or the predetermined swing amplitude and frequency, so that the cells can fully mix the culture medium and carbon dioxide and absorb nutrients to grow and expand.

[0031] The present invention overcomes the shortcomings of the existing technology and provides an automated cell production device that fully mixes cells by selecting the appropriate rocking angle, amplitude, and frequency level according to cell type. This device can achieve automated sterile culture in cell culture bags without human intervention. The cell expansion system integrates a density sensor that can monitor cell expansion multiples in real time and provides real-time fluid replenishment based on cell expansion multiples, truly enabling fluid replenishment (such as culture medium) based on cell expansion multiples or cell status, and offers multiple configurable fluid replenishment modes. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a schematic structural diagram of a cell expansion device according to an embodiment of the present invention;

[0033] FIG2 is a schematic structural diagram of the other side of a cell expansion device according to an embodiment of the present invention;

[0034] FIG3 is a schematic structural diagram of a density sensor and a cell culture bag according to an embodiment of the present invention;

[0035] FIG4 is a schematic diagram of the detection principle of a density sensor according to an embodiment of the present invention;

[0036] [Corrected 24.07.2024 in accordance with Rule 91] FIG5 is a statistical graph of cell growth data using a cell expansion device according to an embodiment of the present invention; [0036.1] [Corrected 24.07.2024 according to Rule 91] Figure 6 is a flow chart of the cell expansion culture and detection steps according to an embodiment of the present invention.

[0037] In the figure: 1-fixed plate, 2-Y-axis bracket, 21-first swing axis, 22-connecting rod, 23-second support shaft hole, 3-density sensor, 4-cell culture bag, 41-cell density detection port; 5-X-axis swing support plate, 51-second swing axis, 6-peristaltic pump / pinch valve, 7-Y-axis motor, 71-first drive rod, 8-electronic control board, 9-weighing sensor, 10-X-axis motor, 101-second drive rod, 12-support frame, 121-first support shaft hole, 122-U-shaped horizontal support plate. DETAILED DESCRIPTION

[0038] The features and exemplary embodiments of various aspects of the present invention will be described in detail below. In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only configured to explain the present invention and are not configured to limit the present invention. For those skilled in the art, the present invention can be implemented without the need for some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present invention by illustrating examples of the present invention.

[0039] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, the elements defined by the phrase "comprising..." do not exclude the presence of other identical elements in the process, method, article, or device comprising the elements.

[0040] Referring to Figures 1 and 2 , a cell expansion device according to the present invention includes a fixed plate 1, a load cell 9, a Y-axis bracket 2, a Y-axis motor 7, an X-axis swing plate 5, an X-axis motor 10, a cell culture bag 4, a density sensor 3, and an electronic control board 8. The entire cell expansion device can be placed in a CO2 incubator.

[0041] The load cell 9 is mounted on a fixed plate 1. Support frames 12 are provided on both sides of the load cell 9. A first support shaft hole 121 is provided near the top of the support frame 12. The Y-axis bracket 2 is generally a rectangular frame. A first swing shaft 21 is provided in the middle of the outer sides of the two long sides of the Y-axis bracket 2. The first swing shaft 21 passes through the first support shaft hole 121. The first swing shaft 21 and the first support shaft hole 121 cooperate to allow the Y-axis bracket 2 to rotate / swing relative to the support frame 12. Furthermore, one side of the first swing shaft 21 is hingedly connected to one end of a connecting rod 22. The other end of the connecting rod 22 is hingedly connected to a first drive rod 71 of the Y-axis motor 7. The Y-axis motor 7 is electrically connected to an electronic control board 8. The Y-axis motor 7 drives the first drive rod 71 to extend and retract, and drives the connecting rod 22 to swing. The swing of the connecting rod 22 is converted into rotation of the first swing shaft 21, thereby achieving left and right swing of the Y-axis bracket 2. The rotation amplitude and frequency of the Y-axis motor 7, and thus the swing amplitude and frequency of the Y-axis bracket 2, can be controlled by the control board 8.

[0042] The X-axis swing support plate 5 is plate-shaped, substantially similar in shape to the rectangular frame of the Y-axis bracket 2, but slightly smaller in overall size than the Y-axis bracket 2. The X-axis swing support plate 5 is disposed in the middle of the rectangular frame of the Y-axis bracket 2, and a gap is left on all sides with the Y-axis bracket 2 to ensure that the X-axis swing support plate 5 can swing within the Y-axis bracket. A second swing axis 51 is disposed in the middle of the short sides of the X-axis swing support plate 5, and second support axis holes 23 are disposed at positions corresponding to the short sides of the Y-axis bracket 2. The second swing axis 51 on each side of the X-axis swing support plate 5 respectively penetrates into the second support axis holes 23 in the middle of the short sides of the Y-axis bracket 2. By virtue of the cooperation between the second swing axis 51 and the second support axis hole 23, the X-axis swing support plate 5 also rotates / sways relative to the Y-axis bracket 2. Preferably, the second swing axis 51 and the second support axis hole 23 are disposed only on one side. Further preferably, a U-shaped transverse support plate 122 is provided extending downwardly on both sides of the Y-axis bracket 2 (i.e., the position of the first swing axis 21), and an X-axis motor 10 is provided on the transverse plate of the U-shaped transverse support plate 122 at a position close to the support frame 12 on one side, that is, the X-axis motor 10 is a certain distance away from the center of the transverse support plate 122. The output end of the X-axis motor 10 is connected to the second drive rod 101, and the other end of the second drive rod 101 is hinged to the X-axis swing support plate 5, and the hinge position deviates from the axis of the second swing axis 51 on both sides by a certain distance. In this way, the second drive rod 101 is pushed to move telescopically under the drive of the X-axis motor 10, and the X-axis swing support plate 5 can swing back and forth around the second swing axis 51. Preferably, the X-axis motor 10 is electrically connected to the electric control board 8, and the rotation amplitude and frequency of the X-axis motor 10 can be controlled by the control board 8, thereby controlling the swing amplitude and frequency of the X-axis swing support plate 5.

[0043] The cell expansion device of the present invention can bring the following beneficial technical effects:

[0044] 1. The coated cells need to be cultured horizontally for multiple days; for the subsequent large-scale amplified cells, a variety of mixing modes and angles are provided to achieve differentiated cell production; a vortex swing mixing mode without shear force, a wave swing mixing mode to achieve cell clustering without aggregation, and a small amplitude mixing mode can be provided; the cells can be completely fixed on the swinging support table; other sensors can be integrated to monitor the cell culture process.

[0045] 2. The entire setup is the key part to achieve fully automated cell production, with the highest degree of automation and integration.

[0046] 3. Provides a variety of key technologies required for cell coating and culture processes, such as vibration detachment of primary adherent or semi-suspended cells, and gravity-driven tilted flow of primary cells; multiple individually preset modes such as horizontal mode, vortex non-shear mode, and wave-based agglomeration mode for swing mixing during culture; real-time monitoring of the total volume of cell culture bags, collection of cells upon completion of culture, providing weight data, and weighing and measurement after culture completion.

[0047] 4. Throughout the entire culture process, a highly customizable swing mixing mode and multiple adjustable parameters facilitate the development of culture plans for different cell types. This allows for multiple summaries of culture experience, significantly improving the efficiency of different cell cultures. The control system provides real-time control of the cell culture process, allows for remote control, and can be expanded to include cameras for real-time monitoring of cell morphology.

[0048] The following are data on cell mixing and growth using various rocking modes.

[0049] Furthermore, referring to Figure 3 , the cell culture bag 4 is provided with a cell density detection port 41, which is welded and integrated into the edge of the cell culture bag 4. The interior of the cell density detection port 41 communicates with the space within the cell culture bag 4. When the swing table swings, the liquid in the cell culture bag 4 can flow into the cell density detection port 41. The density sensor 3 is disposed on the X-axis swing support 5. When the cell culture bag 4 is secured to the X-axis swing support 5, the cell density detection port 41 can be inserted into the density sensor 3. Preferably, the density sensor 3 is disposed at a corner of the X-axis swing support 5.

[0050] Furthermore, a control valve 6 is provided on the X-axis swing support plate 5 . Preferably, the control valve 6 is a peristaltic pump or a pinch valve, and the pipeline of the cell culture bag 4 is fixed on the control valve 6 .

[0051] The optical principle, structure and circuit principle of the density sensor 3 are shown in FIG4 .

[0052] 1. Using 800-1100nm infrared light source, providing a stable and reliable parallel light source. The light in this band has a very high absorption rate to the object being measured, which is convenient for detecting the absorption rate of the object being measured to infrared light.

[0053] 2. The parallel light of the infrared light source passes through a 1:1 beam splitter prism, which splits the light source into two, providing them as a reference light source and a detection light source respectively, for use in detection and light source intensity correction.

[0054] 3. Reference infrared receiver A monitors the stability of the light source in real time, providing a correction basis for subsequent calculations of changes in incident light intensity.

[0055] 4. The cell density detection port 41 is welded and integrated into the edge of the cell culture bag 4. The interior of the cell density detection port 41 is connected to the space inside the cell culture bag 4. When the swing table swings, the liquid in the cell culture bag 4 can flow into the cell density detection port 41. Preferably, the detection area of ​​the cell density detection port 41 is highly transparent and can transmit light.

[0056] 5. After passing through the 1:1 spectroscope, the detection parallel light passes through the cell density detection port 41, and after being absorbed by the object to be measured, enters the detection infrared receiver B.

[0057] 6. The reference parallel light signal received by the reference infrared receiver A and the absorbed parallel light signal received by the detection infrared receiver B enter, and the illumination values ​​obtained by the reference infrared receiver A and the detection infrared receiver B are compared and corrected, and the incident light intensity and the received light intensity at this moment are calculated, and then the absorbance value is calculated to represent the density of the cells, thereby indirectly detecting the density value change of the object being measured.

[0058] The density sensor of the present invention can bring the following beneficial technical effects:

[0059] 1) The entire sensor is completely enclosed and has no contact with the outside world, avoiding the risk of light and circuit being affected by the outside world, and can be used repeatedly.

[0060] 2) The cell density detection port is completely independently integrated into the cell culture bag of the cell culture system. It has been sterilized at high temperature and has no risk of contamination. It is disposable.

[0061] 3) The entire cell culture process data is recorded in real time, which makes it easy to read the data, obtain curve changes, and obtain the amplitude and growth of cell proliferation, so that the operator can understand the situation in real time and make targeted operations, such as adding nutrient solution or terminating culture.

[0062] 4) The cell growth density can be intuitively fed back, which is convenient for summarizing the growth of different cells during the culture process, classifying different cells and formulating different culture plans, greatly improving cell culture efficiency.

[0063] Figure 5 shows the data of different cells grown for 14 days. It can be seen from the figure that the linearity is very high.

[0064] The following describes the cell expansion culture and detection method in detail in conjunction with the cell expansion device and density sensor of the present invention. The specific steps include:

[0065] S01: Place the entire cell expansion device together with the fixed plate 1 into a carbon dioxide incubator, fix the cell culture bag 4 to the X-axis swing support 5, fix the pipeline of the cell culture bag 4 to the peristaltic pump / pinch valve 6, and fix the cell density detection port 41 of the cell culture bag 4 to the density sensor 3.

[0066] S02: When the machine is turned on, the electric control board 8 is powered on, the Y-axis motor 7 and the X-axis motor 10 return to zero position, that is, the X-axis swing support plate 5 and the Y-axis bracket 2 return to a completely horizontal position, the weighing sensor 9 reads the weight, and the whole machine is ready.

[0067] S03: When culturing cells, the coated cells are injected into the cell culture bag 4 and cultured horizontally for a certain period of time.

[0068] S04: After horizontal incubation, according to the needs of the user, the peristaltic pump / pinch valve 6 is turned on to inject the culture medium, and according to the swing amplitude and angle set by the user, the Y-axis motor 7 and / or the X-axis motor 10 can be turned on simultaneously or individually to swing the X-axis swing support plate 5 in one or two directions, thereby achieving gentle swing mixing of the cells and the culture medium, and mixing the carbon dioxide that permeates the breathable cell culture bag 4 into the bag.

[0069] S05: Turn on the density sensor 3, record the cell density data in real time and upload it to the electronic control board 8. After the cells grow for a certain number of days, the cells will proliferate in large quantities. Turn on the peristaltic pump / pinch valve 6 again to inject the same or different culture medium according to the user's needs / program settings, and continue to mix the culture medium and cells according to the new density setting / predetermined swing amplitude and angle (or maintain the previous swing amplitude and angle), frequency, so that the cells can fully mix the culture medium and carbon dioxide, absorb nutrients, grow and proliferate.

[0070] S06: After the culture is completed, the data of the weighing sensor 9 is read to obtain the accurate cell weight.

[0071] Furthermore, as shown in FIG6 , taking coated NK cells as an example, a specific embodiment of cell expansion culture is performed from day 5 to day 11 using the device and method of the present invention.

[0072] Obviously, those skilled in the art may make various changes and modifications to the embodiments of the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A device for cell expansion and non-contact real-time measurement of cell expansion density, the device is used for cell expansion and detection of the density of the object to be measured in a cell culture bag, characterized in that: include: A weighing sensor is arranged on the fixed plate and is used to monitor the weight in real time; A Y-axis swing mechanism is provided on the weighing sensor and is used for swinging along the Y-axis direction; An X-axis swing mechanism is arranged on the Y-axis swing mechanism and is used for swinging along the X-axis direction; The density sensor is arranged on the X-axis swing mechanism and is used to detect the density of the object to be measured in the cell culture bag.

2. The device according to claim 1, characterized in that The cell culture bag is provided with a cell density detection port, and the interior of the cell density detection port is communicated with the space in the cell culture bag.

3. The device according to claim 2, characterized in that The density sensor comprises an infrared light source, a beam splitter, a reference infrared receiver A, a detection infrared receiver B and an optical signal processing circuit.

4. The device according to claim 3, characterized in that The infrared light source emits 800-1100nm infrared parallel light, the beam splitter prism is a 1:1 beam splitter prism, and the infrared parallel light is divided into reference parallel light and detection parallel light after passing through the beam splitter prism. The reference parallel light is detected by the reference infrared receiver A, and the detection parallel light is detected by the detection infrared receiver B after passing through the cell density detection port.

5. The device according to claim 4, characterized in that The reference parallel light signal and the absorbed parallel light signal enter the optical signal processing circuit, and the illumination values ​​of the two are compared and corrected to calculate the light absorbance value to characterize the density of the cell, thereby indirectly detecting the density value change of the object being measured.

6. The device according to claim 1, characterized in that The Y-axis swing mechanism includes a Y-axis bracket, a first swing shaft located on both sides of the Y-axis bracket, a connecting rod driving at least one side of the first swing shaft to rotate, a first driving rod and a Y-axis motor. Support frames are arranged on both sides of the weighing sensor, and a first supporting shaft hole for supporting the first swing shaft is arranged on the support frame. The first swing shaft is hinged to one end of the connecting rod, and the other end of the connecting rod is hinged to the first driving rod of the Y-axis motor.

7. The device according to claim 6, characterized in that The X-axis swing mechanism includes an X-axis swing pallet, a second swing shaft located on one side or both sides of the X-axis swing pallet, and a second driving rod and an X-axis motor for driving the X-axis swing pallet to swing, wherein the second swing shaft is penetrated into a second supporting shaft hole on the Y-axis bracket that is perpendicular to the first swing shaft, and the second driving rod is hinged at the bottom of the X-axis swing pallet and deviates from the axis where the second swing shaft is located.

8. The device according to claim 7, characterized in that The Y-axis bracket is in the form of a rectangular frame as a whole, and the X-axis swing support plate is arranged in the middle of the Y-axis bracket with gaps left around it.

9. The device according to claim 7 or 8, characterized in that The Y-axis motor and the X-axis motor are electrically connected to an electric control board to control the rotation amplitude and frequency of the Y-axis motor and the X-axis motor.

10. The device according to claim 7, characterized in that The X-axis support plate is provided with a control valve, which is used to control the on-off of the cell culture bag and the culture medium liquid pipeline. The control valve is a peristaltic pump or a pinch valve.

11. A method for cell expansion and non-contact real-time measurement of cell expansion density, using the device according to any one of claims 1 to 10, comprising the following steps: S01: Place the entire device in a carbon dioxide incubator, fix the cell culture bag to the X-axis swing support, fix the pipe of the cell culture bag to the control valve, and fix the cell density detection port of the cell culture bag to the density sensor; S02: Start the machine and power on the electric control board. The Y-axis motor and the X-axis motor return to zero position. The X-axis swing support and the Y-axis bracket are adjusted to a completely horizontal position. The weighing sensor reads the weight. The machine is ready. S03: injecting the coated cells into the cell culture bag and completing horizontal static culture for a certain period of time; S04: Open the control valve to inject the culture medium, and according to the swing amplitude and frequency of the Y-axis motor and / or the X-axis motor set by the user, the Y-axis motor and / or the X-axis motor can be turned on simultaneously or individually, so as to gently swing and mix the cells and the culture medium, and mix the carbon dioxide that permeates the breathable cell culture bag into the cell culture bag. S05: Turn on the density sensor, record the cell density data in real time and upload it to the electronic control board. After the cells grow for a certain number of days, the cells will proliferate in large quantities. S06: After the culture is completed, the data of the weighing sensor is read to obtain the accurate cell weight.

12. The method according to claim 11, characterized in that In step S05, the control valve is opened again to inject the same or different culture medium according to the needs of the user or the program settings, and the culture medium and cells are continued to be mixed according to the new density setting or the predetermined swing amplitude and frequency, so that the cells can fully mix the culture medium and carbon dioxide and absorb nutrients to grow and expand.

Images