Rotary cell culture system and liquid path control method

Abstract

The invention discloses a cell rotary culture system and a liquid path control method, and the cell rotary culture system comprises a centrifugal culture device; an enrichment harvesting device; the liquid path control system is used for controlling the flowing of the cell sample and the culture substance; wherein the liquid path control system comprises a plurality of reagent bags arranged in parallel; a peristaltic pump; a flow sensor; the collecting unit comprises a waste liquid bag and a product bag; and a plurality of pinch valves; the peristaltic pump is used for pumping a cell sample and a culture substance into the centrifugal culture device from the reagent bag, or extracting inner clear liquid after centrifugal layering into the waste liquid bag, transferring the cultured cells into the enrichment harvesting device for enrichment, and finally supplying the cultured cells into the product bag for collection and storage. The in-vitro automatic and large-scale preparation of cells can be realized, the overall cost of the system is lower, and the damage rate of the cells is lower due to the rotary suspension culture mode.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a cell rotation culture system and a liquid circuit control method. Background Art

[0002] With the aging of the population and the continuous updating of medical technology, the blood supply is becoming increasingly tight, and the demand for red blood cells is increasing. The blood used in clinical practice mainly comes from citizens' free blood donations, but the demand for clinical blood is huge, and blood donations alone cannot meet the demand for blood. In addition, traditional blood transfusions may also have risks such as disease transmission and allogeneic immunity. The production of safe red blood cells in vitro through cell culture technology is an effective way to solve this problem.

[0003] At present, laboratory or small-scale cell culture equipment mainly includes culture plates, rotating bottles, etc., while large-scale cell production is mainly achieved through bioreactors, mainly including perfusion bioreactors and stirred suspension bioreactors. However, culture based on perfusion bioreactors has problems such as large volume, easy clogging or rupture, and high cost, while culture based on stirred bioreactors has problems such as high cell damage rate and inability to achieve automatic fluid replacement and harvesting. Therefore, existing cell culture equipment is difficult to meet the current needs of large-scale cell culture. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a cell rotation culture system and a liquid circuit control method, which have the advantages of being able to realize automated and large-scale preparation of cells in vitro.

[0005] The purpose of the present invention is achieved by the following technical solutions: According to a first aspect of an embodiment of the present disclosure, there is provided a cell rotation culture system, comprising: A centrifugal culture device, used to contain cell samples and culture materials to achieve cell differentiation and expansion, and capable of achieving radial stratification of cells and culture materials through rotating centrifugal motion; An enrichment and harvesting device is used to contain the cultured cells and culture materials, and to radially separate the cells and culture materials through a rotating centrifugal motion so as to enrich the cells after extracting the inner supernatant; and Liquid control system, used to control the flow of cell samples and culture materials; Wherein, the fluid circuit control system comprises: A number of reagent bags arranged in parallel, used to store cell samples and various culture materials respectively; A peristaltic pump is connected to the reagent bag, the centrifugal culture device and the enrichment and harvesting device through a pipeline system to provide liquid flow driving power; Flow sensor, used to monitor the flow in the pipeline system in real time; a collection unit, the collection unit comprising a waste liquid bag and a product bag; and, A plurality of pinch valves connected to the pipeline system and arranged corresponding to each of the reagent bags, the centrifugal culture device, the enrichment and harvesting device, and the collection unit; The peristaltic pump pumps the cell sample and culture material from the reagent bag into the centrifugal culture device, or extracts the inner clear liquid after centrifugal stratification into the waste liquid bag, and transfers the cultured cells to the enrichment and harvesting device for enrichment, and finally supplies the cultured cells to the product bag for collection and storage.

[0006] To implement the above technical solution, when cell culture is carried out, the liquid circuit control system supplies the cell sample and the culture material into the centrifugal culture device, so that the cells are cultured and expanded in the centrifugal culture device. During the culture process, the centrifugal culture device can realize radial stratification of cells and culture materials by rotating centrifugal motion. After the stratification is completed, the liquid circuit control system discharges the inner clear liquid, thereby realizing the liquid replacement operation; after the cells are cultured for a predetermined period, the liquid circuit control system transfers the cells to the enrichment and harvesting device, and injects the culture material into the enrichment and harvesting device, so that the cells are enriched in the enrichment and harvesting device. During the enrichment process, the enrichment and harvesting device can also perform rotating centrifugal motion to radially stratify the cells and culture materials. The liquid replacement operation is performed through the liquid control system. After the enrichment is completed, the cells can be supplied to the product bag for preservation through the liquid control system, thereby realizing the automated and large-scale preparation of cells in vitro, and the overall cost of the system is lower. The rotating suspension culture method also makes the cell damage rate lower. When the liquid control system is working, the clamp valve controls the switch of each node in the pipeline system, and the peristaltic pump is used as a power source to provide liquid flow driving power. By controlling the opening and closing of the clamp valve, the rotation direction and speed of the peristaltic pump, the processes of cell culture, transfer, enrichment and collection are completed. The flow sensor monitors the flow in the pipeline system in real time, and real-time feedback can be provided to regulate the speed of the peristaltic pump.

[0007] In some exemplary embodiments, the centrifugal culture device comprises: Centrifugal support frame; a centrifugal culture chamber rotatably mounted on the centrifugal support frame, wherein a containing space is formed in the centrifugal culture chamber, and a first liquid inlet and a first liquid outlet connected to the liquid circuit control system are provided on the centrifugal culture chamber; and A first driving motor is carried by the centrifugal support frame and is used to drive the centrifugal culture chamber to perform centrifugal rotational motion.

[0008] To implement the above technical solution, the containing space formed in the centrifugal culture chamber can be used to contain cells and culture materials. The cells and culture materials can be supplied and discharged through the first liquid inlet and the first liquid outlet, and the centrifugal culture chamber can be driven to rotate by the first driving motor to realize suspension culture and centrifugal stratification of cells.

[0009] In some exemplary embodiments, the centrifugal culture devices are provided in multiple groups, and the multiple groups of centrifugal culture devices are connected in parallel to the liquid circuit control system.

[0010] By implementing the above technical solution, synchronous culture and amplification of multiple groups of centrifugal culture devices can be achieved, thereby improving the amplification efficiency.

[0011] In some exemplary embodiments, the enrichment and harvesting device comprises: Enrichment support frame; An enrichment bin rotatably mounted on the enrichment support frame, wherein an enrichment space is formed in the enrichment bin, and a second liquid inlet and a second liquid outlet connected to the liquid circuit control system are provided on the enrichment bin, the enrichment bin is connected to the centrifugal culture bin through the liquid circuit control system, and cells are harvested from the enrichment bin to a product bag through the liquid circuit control system; A denucleation filter connected to the fluid control system, used for filtering and denucleating cells; and A second driving motor is carried by the enrichment support frame and is used to drive the enrichment bin to perform centrifugal rotational motion.

[0012] To implement the above technical solution, the enrichment space formed in the enrichment bin is used for cell enrichment, and the transfer of cells and the supply and discharge of culture materials are realized through the second liquid inlet and the second liquid outlet. When the cells enter the enrichment bin, they are filtered and denucleated through the denucleation filter to separate the nucleated red blood cells and the mature red blood cells. The second drive motor can drive the enrichment bin to perform centrifugal rotation to radially stratify the cells and the culture material, and the inner layer of the clear liquid is pumped away to leave the cells in the enrichment bin, thereby realizing cell enrichment.

[0013] In some exemplary embodiments, the first liquid inlet and the first liquid outlet on the centrifugal culture chamber, and the second liquid inlet and the second liquid outlet on the enrichment chamber are respectively provided with the pinch valve.

[0014] The above technical solution is implemented to achieve separate control of liquid inlet and liquid outlet.

[0015] In some exemplary embodiments, the collection unit further comprises a sampling bag, which is connected to the centrifugal culture chamber and the enrichment chamber and is used for sampling quality control during the culture process.

[0016] In some exemplary embodiments, the liquid control system is further provided with a reagent filter for filtering the reagent.

[0017] The above technical solution is implemented to improve the cleanliness of the reagents.

[0018] In some exemplary embodiments, the flow sensor is an ultrasonic non-contact flow sensor.

[0019] In some exemplary embodiments, both the first driving motor and the second driving motor are servo motors.

[0020] According to a second aspect of an embodiment of the present disclosure, a method for controlling a fluid path of a cell rotation culture system is provided. The method is based on the cell rotation culture system according to the first aspect, and comprises: The liquid control system supplies cell samples and culture materials into the centrifugal culture device so that the cells are cultured and expanded in the centrifugal culture device; The centrifugal culture device operates in any of the working modes of low-speed unidirectional rotation, intermittent rotation and forward and reverse rotation to carry out cell suspension culture, and periodically performs rotary centrifugal motion to radially stratify cells and culture materials, so as to discharge the inner clear liquid through the liquid control system for liquid replacement; After reaching the predetermined culture cycle, the liquid control system transfers the cells to the enrichment and harvesting device for cell enrichment, and during the enrichment process, a rotating centrifugal motion is performed to radially separate the cells and the culture material, and the inner clear liquid is discharged through the liquid control system for concentration and enrichment; After enrichment is completed, the liquid control system supplies the cells into the product bag for collection and storage.

[0021] The implementation of the above technical solution realizes the automated and large-scale preparation of cells in vitro with lower culture costs, and the cell damage rate is also reduced through the rotating suspension culture method.

[0022] In summary, compared with the prior art, the present invention has the following beneficial effects: The embodiment of the present invention provides a cell rotation culture system and a fluid circuit control method. When cell culture is performed, the fluid circuit control system supplies cell samples and culture materials into a centrifugal culture device, so that the cells are cultured and expanded in the centrifugal culture device. During the culture process, the centrifugal culture device can achieve radial stratification of cells and culture materials through rotational centrifugal motion. After the stratification is completed, the fluid circuit control system discharges the inner clear liquid, thereby realizing the liquid replacement operation. After the cells are cultured for a predetermined period, the fluid circuit control system transfers the cells to an enrichment and harvesting device, and injects culture materials into the enrichment and harvesting device, so that the cells are enriched in the enrichment and harvesting device. During the enrichment process, the enrichment and harvesting device can also perform rotational centrifugal motion to make the cells The culture material is radially layered, and the liquid is replaced through the liquid control system. After the enrichment is completed, the cells can be supplied to the product bag for preservation through the liquid control system, thereby realizing the automated and large-scale preparation of cells in vitro, and the overall cost of the system is lower. The rotary suspension culture method also makes the cell damage rate lower. When the liquid control system is working, the clamp valve controls the switch of each node in the pipeline system, and the peristaltic pump is used as the power source to provide liquid flow driving power. By controlling the opening and closing of the clamp valve, the rotation direction and speed of the peristaltic pump, the processes of cell culture, transfer, enrichment and collection are completed. The flow sensor monitors the flow in the pipeline system in real time, and real-time feedback can be performed to regulate the speed of the peristaltic pump. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the structure of the cell rotation culture system in an embodiment of the present invention.

[0024] Figure 2 Schematic diagram of the electrical control principle of the liquid circuit control system in an embodiment of the present invention.

[0025] Figure 3 This is a control principle diagram of a liquid circuit control method when a group of centrifugal culture devices are provided in an embodiment of the present invention.

[0026] Figure 4 This is a control principle diagram of a liquid circuit control method when multiple groups of centrifugal culture devices are provided in an embodiment of the present invention.

[0027] The numbers and letters in the figure represent the corresponding component names: 10. Centrifugal culture device; 11. Centrifugal support frame; 12. Centrifugal culture chamber; 13. First liquid inlet; 14. First liquid outlet; 15. First drive motor; 20. Enrichment harvesting device; 21. Enrichment support frame; 22. Enrichment chamber; 23. Denuclearization filter; 24. Second drive motor; 25. Second liquid inlet; 26. Second liquid outlet; 30. Liquid control system; 31. Reagent bag; 32. Peristaltic pump; 33. Pipe system; 34. Flow sensor; 35. Collection unit; 351. Waste liquid bag; 352. Sampling bag; 353. Product bag; 36. Pinch valve; 37. Reagent filter. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] like Figure 1 As shown, a first aspect of an embodiment of the present invention provides a cell rotation culture system, comprising: a centrifugal culture device 10, used to contain cell samples and culture materials to achieve cell differentiation and expansion, and capable of radially stratifying cells and culture materials through rotational centrifugal motion; an enrichment and harvesting device 20, used to contain cultured cells and culture materials, and radially stratify cells and culture materials through rotational centrifugal motion to enrich cells after extracting the inner clear liquid; and a liquid control system 30, used to control the flow of cell samples and culture materials.

[0030] Specifically, the centrifugal culture device 10 includes: a centrifugal support frame 11; a centrifugal culture chamber 12 rotatably mounted on the centrifugal support frame 11, a containing space is formed in the centrifugal culture chamber 12, and a first liquid inlet portion 13 and a first liquid outlet portion 14 connected to the liquid circuit control system 30 are provided on the centrifugal culture chamber 12; and a first driving motor 15 carried by the centrifugal support frame 11 and used to drive the centrifugal culture chamber 12 to perform centrifugal rotational motion.

[0031] The centrifugal support frame 11 is used to support and install the centrifugal culture chamber 12. A bearing can be arranged between the bottom of the centrifugal culture chamber 12 and the centrifugal support frame 11 to realize the rotational connection of the centrifugal culture chamber 12. The first drive motor 15 adopts a servo motor, so that it can drive the centrifugal culture chamber 12 to perform intermittent rotation, forward and reverse rotation or high-speed centrifugal rotation. The first liquid inlet portion 13 and the first liquid outlet portion 14 are respectively connected to the centrifugal culture chamber 12; the storage space formed in the centrifugal culture chamber 12 can be used to store cells and culture materials, and the cells and culture materials are supplied and discharged through the first liquid inlet portion 13 and the first liquid outlet portion 14, and the centrifugal culture chamber 12 can be driven to rotate through the first drive motor 15 to realize cell suspension culture and centrifugal stratification.

[0032] In some embodiments, the centrifugal culture device 10 can be provided with multiple groups, and the multiple groups of centrifugal culture devices 10 are connected in parallel to the liquid circuit control system 30, so that the synchronous culture and amplification of the multiple groups of centrifugal culture devices 10 can be achieved, and the amplification efficiency can be improved. The system schematic diagram of a single group of centrifugal culture devices 10 is as follows: Figure 3 As shown, the system schematic diagram of setting multiple groups of centrifugal culture devices 10 is as follows Figure 4 shown.

[0033] The enrichment and harvesting device 20 includes: an enrichment support frame 21; an enrichment bin 22 rotatably mounted on the enrichment support frame 21, an enrichment space is formed in the enrichment bin 22, and a second liquid inlet 25 and a second liquid outlet 26 connected to the liquid circuit control system 30 are provided on the enrichment bin 22, the enrichment bin 22 is connected to the centrifugal culture bin 12 through the liquid circuit control system 30, and the cells are harvested from the enrichment bin 22 to the product bag 353 through the liquid circuit control system 30; a denuclearization filter 23 connected to the liquid circuit control system 30, for filtering and denuclearizing the cells; and a second drive motor 24 carried on the enrichment support frame 21, for driving the enrichment bin 22 to perform centrifugal rotational motion.

[0034] The enrichment support frame 21 is used to support and install the enrichment bin 22. The structure of the enrichment bin 22 is the same as that of the centrifugal culture bin 12, which will not be elaborated here. The second drive motor 24 also adopts a servo motor. The denuclearization filter 23 is connected in series in the pipeline system 33 between the centrifugal culture bin 12 and the enrichment bin 22. The enrichment space formed in the enrichment bin 22 is used for cell enrichment. The second liquid inlet 25 and the second liquid outlet 26 are used to realize cell transfer and the supply and discharge of culture materials. When the cells enter the enrichment bin 22, they are filtered and denuclearized by the denuclearization filter 23 to separate nucleated red blood cells and mature red blood cells. The second drive motor 24 can drive the enrichment bin 22 to perform centrifugal rotation to radially stratify the cells and the culture materials, and the inner layer of the clear liquid is pumped away to leave the cells in the enrichment bin 22, thereby realizing cell enrichment.

[0035] The liquid circuit control system 30 includes: a plurality of reagent bags 31 arranged in parallel, for storing cell samples and various culture materials respectively; a peristaltic pump 32, which is connected to the reagent bag 31, the centrifugal culture chamber 12 and the enrichment chamber 22 through the pipeline system 33, and is used to provide liquid flow driving power; a flow sensor 34, which is used to monitor the flow in the pipeline system 33 in real time; a collection unit 35, the collection unit 35 includes a waste liquid bag 351 and a product bag 353; and a plurality of pinch valves 36 connected to the pipeline system 33 and correspondingly arranged with each reagent bag 31, the centrifugal culture device 10, the enrichment harvesting device 20 and the collection unit 35; the peristaltic pump 32 pumps the cell sample and culture material from the reagent bag 31 into the centrifugal culture chamber 12, or extracts the inner clear liquid after centrifugal stratification to the waste liquid bag 351, and transfers the cultured cells to the enrichment chamber 22 for enrichment, and finally supplies the cultured cells to the product bag 353 for collection and preservation.

[0036] Among them, several reagent bags 31 can be specifically used to store buffer solutions, cell samples, culture media, culture reagents and expansion reagents, etc. respectively. The buffer solution is used for cleaning the pipeline. When preparing red blood cells, the cell sample can be, for example, a pre-treated hematopoietic stem cell sample. The culture reagents and expansion reagents can include, for example, induction differentiation reagents, cytokines, microcarriers, etc. The culture media is used to provide nutrients for cell growth and expansion; the waste liquid bag 351 is used to collect waste liquid generated during the liquid exchange process, and the product bag 353 is used to collect cultured cells. In some embodiments, the collection unit 35 also includes a sampling bag 352, which is connected to the centrifugal culture chamber 12 and the enrichment chamber 22 for sampling quality control during the culture process.

[0037] The pinch valve 36 is a normally closed valve. It can be understood that each reagent bag 31 is provided with a pinch valve 36 to control the opening and closing of different reagent bags 31 as needed. The first liquid inlet 13 and the first liquid outlet 14 on each centrifugal culture chamber 12, the second liquid inlet 25 and the second liquid outlet 26 on the enrichment chamber 22 are respectively provided with a pinch valve 36. The centrifugal culture chamber 12 and the enrichment chamber 22 can be provided with one or more. When multiple centrifugal culture chambers 12 or enrichment chambers 22 are provided, multiple centrifugal culture chambers 12 or enrichment chambers 22 are provided with a pinch valve 36. In parallel, the pipeline system 33 is connected. In this embodiment, four groups of centrifugal culture chambers 12 and one group of enrichment chambers 22 are arranged. The waste liquid bag 351, the sampling bag 352 and the product bag 353 are respectively connected to a clamping valve 36, and are also connected to the pipeline system 33 in parallel. The pipeline system 33 is used to realize the transportation of liquid flow. The pipeline system 33 is closed and sterile. Usually, the whole system is sterilized after the system is assembled to realize sterile culture. The flow sensor 34 is an ultrasonic non-contact flow sensor 34.

[0038] When the liquid circuit control system 30 is working, the pinch valve 36 controls the switch of each node in the pipeline system 33, and the peristaltic pump 32 is used as a power source to provide liquid flow driving power. By controlling the opening and closing of the pinch valve 36 and the rotation direction and speed of the peristaltic pump 32, the cell culture, transfer, enrichment and collection processes are completed. The flow sensor 34 monitors the flow in the pipeline system 33 in real time, and real-time feedback can be performed to adjust the speed of the peristaltic pump 32. At the same time, a single peristaltic pump 32 is used to realize a complex liquid circuit control process through the combined action of the pump and valve, simplifying the control process, facilitating the realization of automatic control, and reducing costs.

[0039] Furthermore, a reagent filter 37 is provided in the liquid circuit control system 30 for filtering the reagent to improve the cleanliness of the reagent. The reagent filter 37 is arranged between the reagent bag 31 and the centrifugal culture chamber 12. At the same time, a clamping valve 36 can be connected at both ends of the filter to control whether the liquid circuit flows through the reagent filter 37.

[0040] like Figure 2 As shown, Figure 2 The electronic control schematic diagram of the liquid circuit control system 30 is shown. The liquid circuit control system 30 also includes an MCU control module, which is connected to the host computer through a CAN bus to receive control signals from the host computer to implement liquid circuit control and feedback the operating status of the liquid circuit control system 30. The flow sensor 34 is communicated with the MCU control module through an RS485 interface, each clamping valve 36 is communicated with the MCU control module through an IO interface, and the peristaltic pump 32 is communicated with the MCU control module through an RS485 interface.

[0041] When cell culture is performed, the liquid circuit control system 30 supplies cell samples and culture materials into the centrifugal culture device 10, so that the cells are cultured and expanded in the centrifugal culture device 10. During the culture process, the centrifugal culture device 10 can realize radial stratification of cells and culture materials through rotational centrifugal motion. After the stratification is completed, the liquid circuit control system 30 discharges the inner clear liquid, thereby realizing the liquid replacement operation; after the cells are cultured for a predetermined period, the liquid circuit control system 30 transfers the cells to the enrichment and harvesting device 20, and injects the culture material into the enrichment and harvesting device 20, so that the cells are enriched in the enrichment and harvesting device 20. During the enrichment process, the enrichment and harvesting device 20 can also perform rotational centrifugal motion to radially stratify the cells and culture materials, and perform the liquid replacement operation through the liquid circuit control system 30. After the enrichment is completed, the cells can be supplied to the product bag 353 for storage through the liquid circuit control system 30, thereby realizing the in vitro automated and large-scale preparation of cells, and the overall cost of the system is lower. During the culture process, the rotation culture method is adopted, and the microcarrier is used to suspend the cells in the liquid to realize three-dimensional culture, increase the contact area between the cells and the culture medium, and improve the mass transfer efficiency, thereby realizing high-density culture.

[0042] A second aspect of an embodiment of the present invention provides a liquid circuit control method of a cell rotation culture system. The method is based on the cell rotation culture system as described in the first aspect, and includes: S100 , a cell sample and a culture material are supplied to the centrifugal culture device 10 by the liquid path control system 30 so that the cells are cultured and expanded in the centrifugal culture device 10 .

[0043] S200, the centrifugal culture device 10 operates in any working mode of low-speed unidirectional rotation, intermittent rotation and forward and reverse rotation to perform cell suspension culture, and periodically performs rotational centrifugal motion to radially stratify the cells and the culture material, so as to discharge the inner clear liquid through the liquid circuit control system 30 for liquid replacement. The mass transfer efficiency of the culture process can be improved by low-speed unidirectional rotation, intermittent rotation or forward and reverse rotation. Since the nutrients in the culture liquid will be consumed after a period of cultivation, the cells and the inner clear liquid are separated by centrifugal stratification at this time, and the inner clear liquid is discharged while the cells remain in the centrifugal culture device 10. New culture material is re-injected through the liquid circuit control system 30 to achieve liquid replacement, so that the cells can be continuously cultured and expanded.

[0044] After reaching the predetermined culture cycle, the liquid control system transfers the cells to the enrichment and harvesting device for cell enrichment, and during the enrichment process, a rotating centrifugal motion is performed to radially separate the cells and the culture material, and the inner clear liquid is discharged through the liquid control system for concentration and enrichment; S300, after reaching the predetermined culture cycle, the liquid control system 30 transfers the cells to the enrichment and harvesting device 20 for cell enrichment, and during the enrichment process, a rotational centrifugal motion is performed to radially stratify the cells and the culture material, so that the inner clear liquid is discharged through the liquid control system 30 for concentration and enrichment; the transfer of cells can also be carried out by the liquid control system 30. When the cells are transferred to the enrichment and harvesting device 20, they are also filtered and denuclearized through the denuclearization filter 23 to achieve separation of nucleated red blood cells and mature red blood cells. During the enrichment process, the enrichment device performs centrifugal stratification and then discharges the inner clear liquid to achieve concentration and enrichment.

[0045] S400, after the enrichment is completed, the liquid control system 30 supplies the cells into the product bag 353 for collection and storage.

[0046] The above process realizes the automated and large-scale preparation of cells in vitro, with lower culture costs, and the cell damage rate is also reduced through the rotating suspension culture method.

[0047] In view of setting up a single centrifugal culture device 10, the liquid circuit control process of the present application is described in detail below with a specific example, and its specific principle diagram is as follows: Figure 3 As shown in the figure, the numbers ~ Both represent pinch valves. The liquid inlet into the centrifugal chamber and the enrichment chamber is controlled by the pinch valves with corresponding numbers.

[0048] 1) Add sample: open pinch valves 2, 6, 11, and 10, and rotate the peristaltic pump forward to allow the sample to enter the centrifugal culture chamber 1, then close the peristaltic pump and pinch valve; 2) Cleaning: Open pinch valves 1, 6, 12, and 15, rotate the peristaltic pump forward, allow the buffer to clean the pipeline, and then close the peristaltic pump and pinch valves; 3) Add culture medium: open pinch valves 3, 6, 11, and 10, rotate the peristaltic pump forward to allow the culture medium to enter the centrifugal culture chamber, and then close the peristaltic pump and pinch valves; 4) Add reagents: open pinch valves 4, 6, 11, and 10, rotate the peristaltic pump forward to allow the reagents to enter the centrifugal culture chamber, and then close the peristaltic pump and pinch valves; 5) Culture expansion: During the culture process, the centrifugal culture chamber rotates unidirectionally at a low speed, rotates forward and reverse, or rotates intermittently to promote cell expansion; 6) Centrifugal separation: The centrifugal culture chamber rotates at high speed to separate the cell suspension into layers, then the pinch valves 9, 11, 8, and 15 are opened, the peristaltic pump is reversed, and the clear liquid after centrifugation is discharged into the waste liquid bag, and then the peristaltic pump and the pinch valve are closed; 7) Washing: Open the pinch valves 5, 11, 8, and 15, reverse the peristaltic pump, flush the pipeline, and then close the peristaltic pump and pinch valve; 8) Denucleation: Open the pinch valves 9, 11, 7, and 14, reverse the peristaltic pump, and the cells enter the enrichment chamber after being denucleated by the denucleation filter, and then close the peristaltic pump and the pinch valve; 9) Enrichment: The enrichment chamber rotates at high speed to separate the cell suspension into layers, then the pinch valves 13, 8, and 15 are opened, the peristaltic pump is reversed, the clear liquid is discharged into the waste liquid bag, and then the peristaltic pump and the pinch valve are closed; 10) Harvesting: Open the pinch valves 13, 8, and 17, reverse the peristaltic pump, and store the cell products in the product bag, then close the peristaltic pump and pinch valves; 11) Sampling: Open pinch valves 9, 11, 8, 16 (or open pinch valves 13, 8, 16), reverse the peristaltic pump, collect the sample into the sampling bag, and then close the peristaltic pump and pinch valves.

[0049] The automatic liquid replacement operation process is: a) Centrifugal separation: The centrifugal culture chamber rotates at high speed to separate the cell suspension into layers, then the pinch valves 9, 11, 8, and 15 are opened, the peristaltic pump is reversed to separate the clear liquid, then it stops, and the peristaltic pump and the pinch valve are closed; b) Washing: Open the pinch valves 5, 11, 8, and 15, reverse the peristaltic pump, flush the pipeline, and then close the peristaltic pump and pinch valves; c) Add culture medium: open pinch valves 3, 6, 11, and 10, rotate the peristaltic pump forward, and let the cell matrix enter the centrifugal culture chamber. Then close the peristaltic pump and pinch valves; d) Open pinch valves 1, 6, 12, and 15, rotate the peristaltic pump forward, flush the pipeline, and then close the peristaltic pump and pinch valves.

[0050] In view of setting up multiple groups of centrifugal culture devices 10, the liquid circuit control process of the present application is described in detail below with a specific example, and its specific principle diagram is as follows: Figure 4 As shown in the figure, the numbers ~ All represent pinch valves. The centrifugal chamber and the enrichment chamber are both controlled by the pinch valves with corresponding numbers. The following description takes the liquid supply and liquid discharge to one of the centrifugal culture chambers as an example. The liquid circuit control process of other centrifugal culture chambers is the same.

[0051] 1) Add sample: open pinch valves 2, 6, 11, and 13, and rotate the peristaltic pump forward to allow the sample to enter the centrifugal culture chamber 1, then close the peristaltic pump and pinch valve; 2) Cleaning: Open the pinch valves 1, 6, 10, and 22, and rotate the peristaltic pump forward to allow the buffer solution to clean the pipeline, and then close the peristaltic pump and pinch valve; 3) Add culture medium: open pinch valves 3, 6, 11, and 13, rotate the peristaltic pump forward, allow the culture medium to enter the centrifugal culture chamber 1, and then close the peristaltic pump and pinch valve; 4) Add reagent: open pinch valves 4 / 5, 6, 11, and 13, and rotate the peristaltic pump forward to allow the reagent to enter the centrifugal culture chamber 1, then close the peristaltic pump and pinch valve; 5) Culture expansion: During the culture process, the centrifugal culture chamber rotates unidirectionally at a low speed, rotates forward and reversely, or rotates intermittently to promote cell expansion; 6) Centrifugal separation: The centrifugal culture chamber rotates at high speed to separate the cell suspension into layers, then the pinch valves 12, 11, 9, and 22 are opened, the peristaltic pump is reversed, and the clear liquid after centrifugation is discharged into the waste liquid bag, and then the peristaltic pump and the pinch valve are closed; 7) Washing: Open the pinch valves 7, 11, 9, and 22, reverse the peristaltic pump, flush the pipeline, and then close the peristaltic pump and pinch valve; 8) Denucleation: Open the pinch valves 13, 11, 8, and 21, reverse the peristaltic pump, and the cells enter the enrichment chamber after being denucleated by the denucleation filter, and then close the peristaltic pump and the pinch valve; 9) Enrichment: The enrichment chamber rotates at high speed to separate the cell suspension into layers, then the pinch valves 21, 10, 9, and 22 are opened, the peristaltic pump is reversed, the clear liquid is discharged into the waste liquid bag, and then the peristaltic pump and the pinch valve are closed; 10) Harvesting: Open the pinch valves 20, 9, and 24, reverse the peristaltic pump, and store the cell products in the product bag. Then close the peristaltic pump and pinch valves. 11) Sampling: Open pinch valves 12, 11, 9, 23 (or open pinch valves 20, 9, 23), reverse the peristaltic pump, collect the sample into the sampling bag, and then close the peristaltic pump and pinch valves.

[0052] The automatic liquid replacement operation process is: a) Centrifugal separation: The centrifugal culture chamber rotates at high speed to separate the cell suspension into layers, then the pinch valves 12, 11, 9, and 22 are opened, the peristaltic pump is reversed to separate the clear liquid, then it stops, and the peristaltic pump and pinch valve are closed; b) Washing: Open the pinch valves 7, 11, 9, and 22, reverse the peristaltic pump, flush the pipeline, and then close the peristaltic pump and pinch valves; c) Add culture medium: open pinch valves 3, 6, 11, and 13, the peristaltic pump rotates forward, the cell matrix enters the centrifugal culture chamber, and then close the peristaltic pump and pinch valves; d) Open pinch valves 1, 6, 10, and 22, rotate the peristaltic pump forward, flush the pipeline, and then close the peristaltic pump and pinch valves.

[0053] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which are equivalent modifications and improvements made to the above embodiments based on the essential technology of the present invention, and all of them belong to the protection scope of the present invention.

Claims

1. A cell rotation culture system, characterized in that: include: A centrifugal culture device, used to contain cell samples and culture materials to achieve cell differentiation and expansion, and capable of achieving radial stratification of cells and culture materials through rotating centrifugal motion; An enrichment and harvesting device is used to contain the cultured cells and culture materials, and to radially separate the cells and culture materials through a rotating centrifugal motion so as to enrich the cells after extracting the inner supernatant; as well as, Liquid control system, used to control the flow of cell samples and culture materials; Wherein, the fluid circuit control system comprises: A number of reagent bags arranged in parallel, used to store cell samples and various culture materials respectively; A peristaltic pump is connected to the reagent bag, the centrifugal culture device and the enrichment and harvesting device through a pipeline system to provide liquid flow driving power; Flow sensor, used to monitor the flow in the pipeline system in real time; a collection unit, the collection unit comprising a waste liquid bag and a product bag; and, A plurality of pinch valves connected to the pipeline system and arranged corresponding to each of the reagent bags, the centrifugal culture device, the enrichment and harvesting device, and the collection unit; The peristaltic pump pumps the cell sample and culture material from the reagent bag into the centrifugal culture device, or extracts the inner clear liquid after centrifugal stratification into the waste liquid bag, and transfers the cultured cells to the enrichment and harvesting device for enrichment, and finally supplies the cultured cells to the product bag for collection and storage.

2. The cell rotation culture system according to claim 1, characterized in that: The centrifugal culture device comprises: Centrifugal support frame; a centrifugal culture chamber rotatably mounted on the centrifugal support frame, wherein a containing space is formed in the centrifugal culture chamber, and a first liquid inlet and a first liquid outlet connected to the liquid circuit control system are provided on the centrifugal culture chamber; and A first driving motor is carried by the centrifugal support frame and is used to drive the centrifugal culture chamber to perform centrifugal rotational motion.

3. The cell rotation culture system according to claim 2, characterized in that: The centrifugal culture devices are provided with a plurality of groups, and the plurality of groups of centrifugal culture devices are connected in parallel to the liquid circuit control system.

4. The cell rotation culture system according to claim 2 or 3, characterized in that: The enrichment and harvesting device comprises: Enrichment support frame; An enrichment bin rotatably mounted on the enrichment support frame, wherein an enrichment space is formed in the enrichment bin, and a second liquid inlet and a second liquid outlet connected to the liquid circuit control system are provided on the enrichment bin, the enrichment bin is connected to the centrifugal culture bin through the liquid circuit control system, and cells are harvested from the enrichment bin to a product bag through the liquid circuit control system; A denucleation filter connected to the fluid control system, used for filtering and denucleating cells; and A second driving motor is carried by the enrichment support frame and is used to drive the enrichment bin to perform centrifugal rotational motion.

5. The cell rotation culture system according to claim 4, characterized in that: The first liquid inlet and the first liquid outlet on the centrifugal culture chamber, and the second liquid inlet and the second liquid outlet on the enrichment chamber are respectively and correspondingly provided with the pinch valve.

6. The cell rotation culture system according to claim 5, characterized in that: The collection unit also includes a sampling bag, which is connected to the centrifugal culture chamber and the enrichment chamber and is used for sampling quality control during the culture process.

7. The cell rotation culture system according to claim 1, characterized in that: The liquid circuit control system is also provided with a reagent filter for filtering the reagent.

8. The cell rotation culture system according to claim 1, characterized in that: The flow sensor is an ultrasonic non-contact flow sensor.

9. The cell rotation culture system according to claim 4, characterized in that: The first drive motor and the second drive motor are both servo motors.

10. A method for controlling a liquid path of a cell rotation culture system, the method being based on the cell rotation culture system according to any one of claims 1 to 9, characterized in that: include: The liquid control system supplies cell samples and culture materials into the centrifugal culture device so that the cells are cultured and expanded in the centrifugal culture device; The centrifugal culture device operates in any of the working modes of low-speed unidirectional rotation, intermittent rotation and forward and reverse rotation to carry out cell suspension culture, and periodically performs rotary centrifugal motion to radially stratify cells and culture materials, so as to discharge the inner clear liquid through the liquid control system for liquid replacement; After reaching the predetermined culture cycle, the liquid control system transfers the cells to the enrichment and harvesting device for cell enrichment, and during the enrichment process, a rotating centrifugal motion is performed to radially separate the cells and the culture material, and the inner clear liquid is discharged through the liquid control system for concentration and enrichment; After enrichment is completed, the liquid control system supplies the cells into the product bag for collection and storage.

Images