Cell in-vitro culture equipment, method and application thereof

Abstract

The invention discloses in-vitro cell culture equipment and method and application thereof, and the in-vitro cell culture equipment comprises a centrifugal culture device which is used for containing a cell sample and a culture substance to realize differentiation and amplification of cells and can realize radial layering of the cells and the culture substance through rotary centrifugal motion; the enriching and harvesting device is used for containing the cultured cells and culture substances and radially layering the cells and the culture substances through rotary centrifugal movement so as to enrich the cells after internal clear liquid is extracted; the liquid path control system is used for supplying a cell sample and a culture substance into the centrifugal culture device and realizing cell transfer and collection; the gas path control system is used for supplying mixed gas; the temperature control device comprises a heating unit and a refrigerating unit; and a monitoring feedback device. The in-vitro automatic and large-scale preparation of the cells is realized, the cost is lower, and the damage rate of the cells is lower by a rotary suspension culture mode.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to an in vitro cell culture device, method and application thereof. Background Art

[0002] Cell culture refers to the technology of culturing cells taken from the body in an in vitro environment to simulate the in vivo environment and make the cells survive and grow. From 1855 to the present, the study of cell culture has developed into a research hotspot in the biomedical field, and cell culture has also become a widely used technical means in biomedical research applications.

[0003] 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.

[0004] 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

[0005] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a cell in vitro culture device, method and application thereof, which have the advantages of realizing automated and large-scale preparation of cells in vitro.

[0006] 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 in vitro culture device, 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; A fluid control system, used for supplying cell samples and culture materials into the centrifugal culture device, transferring the cultured cells to the enrichment and harvesting device for enrichment, and finally supplying the cultured cells into a product bag for collection and storage; A gas circuit control system, used for mixing gas for cell culture to a predetermined concentration and then supplying the gas to the centrifugal culture device; The temperature control device comprises: a heating unit for heating the centrifugal culture device and the enrichment and harvesting device to a predetermined temperature range, and a refrigeration unit for performing low-temperature refrigeration treatment on cell samples, culture materials and product bags; and, A monitoring feedback device is used to monitor the pH value and DO value in the centrifugal culture device to provide feedback to the gas circuit control system to adjust the concentration of the supplied gas, and to perform stratification monitoring on the centrifugal culture device to provide feedback to the liquid circuit control system to discharge the clear liquid after stratification.

[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 liquid inlet component and a liquid outlet component 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] In some exemplary embodiments, the centrifugal culture chamber comprises: An upper bin body and a lower bin body sealed and matched with each other, wherein the storage space is formed between the upper bin body and the lower bin body; A rotating sealing body disposed on the upper storage body; A fixed joint seat rotatably mounted on the rotary seal body, wherein the fixed joint seat is provided with a gas-liquid channel and a liquid outlet channel, wherein the liquid outlet channel is connected to the liquid outlet assembly; and A blade shaft is rotatably assembled on the rotating seal body and connected to the fixed joint seat, the bottom of the blade shaft is connected to the lower warehouse body, and a through guide channel is provided on the blade shaft, the first end of the guide channel is connected to the gas-liquid channel, and the second end is connected to the liquid inlet component.

[0009] In some exemplary embodiments, the liquid inlet assembly includes: A liquid inlet ring seat, wherein the liquid inlet ring seat is provided with a rotation groove adapted to the bottom of the blade shaft, and the rotation groove is provided with a liquid through port connected to the guide channel; A guide plate connected to the side of the liquid inlet ring seat and extending in the radial direction of the liquid inlet ring seat; The lower bin body is provided with an installation groove adapted to the liquid inlet ring seat and the guide plate, and the installation groove is connected to the liquid through port, and a guide groove is formed between the liquid inlet ring seat and the guide plate, and a liquid inlet connected to the lower bin body is formed at the end of the guide groove.

[0010] In some exemplary embodiments, the liquid outlet assembly includes a liquid outlet guide plate fixed to the upper warehouse body, the liquid outlet guide plate extends from the rotating seal body to near the lower warehouse body, the rotating seal body is provided with a transition guide groove connected to the liquid outlet channel, the liquid outlet guide plate is provided with a liquid outlet guide groove connected to the transition guide groove, and the liquid outlet guide groove extends to one end near the lower warehouse body to form a liquid outlet.

[0011] To implement the above technical solution, when discharging liquid, the liquid flows from the liquid outlet into the liquid outlet guide groove, and under the drainage effect of the liquid outlet guide groove, enters the liquid outlet channel through the transfer guide groove to realize liquid discharge.

[0012] 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, the enrichment bin is connected to the centrifugal culture bin through the fluid circuit control system, and cells are harvested from the enrichment bin to a product bag through the fluid 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.

[0013] In some exemplary embodiments, the fluid circuit control system includes: Several reagent bags for storing cell samples and various culture materials; A peristaltic pump is connected to the reagent bag, the centrifugal culture chamber and the enrichment chamber 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, a sampling 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, each of the centrifugal culture chambers, the enrichment chamber and the collection unit; The peristaltic pump pumps the cell sample and culture material from the reagent bag into the centrifugal culture chamber, transfers the cultured cells to the enrichment chamber for enrichment, and finally supplies the cultured cells to the product bag for collection and preservation.

[0014] In some exemplary embodiments, the gas circuit control system includes: Multiple gas sources, used to provide carbon dioxide, oxygen / air, and nitrogen respectively; A mass flow controller connected to each of the gas sources, for measuring and controlling the gas mass flow; A static mixer connected to each of the mass flow controllers, used for mixing the gases supplied from each of the gas sources to form a mixed gas; A high efficiency filter connected to the static mixer; A gas concentration detection module connected to the high efficiency filter, used to detect the concentration of the mixed gas; A proportional control valve connected to the gas concentration detection module and arranged corresponding to each of the centrifugal culture bins is used to control the supply of mixed gas into each of the centrifugal culture bins.

[0015] In some exemplary embodiments, the centrifugal culture device is disposed in an openable / closable culture chamber, and the heating unit: A partition chamber is provided in the culture chamber, wherein the bottom of the partition chamber is provided with an air inlet and the top of the partition chamber is provided with an air outlet: A heating wire disposed in the partition compartment and located near the air outlet; and A cross-flow fan is disposed in the partition chamber, wherein the air inlet side of the cross-flow fan is close to the air inlet, and the air outlet side of the cross-flow fan is close to the heating wire; In addition, a temperature sensor is provided in the culture chamber for real-time detection and feedback of temperature information in the culture chamber.

[0016] In some exemplary embodiments, the refrigeration unit comprises: A refrigeration chamber, used for storing the reagent bags and the product bags; A semiconductor refrigerator fitted with the refrigeration bin, wherein the cold end of the semiconductor refrigerator fits with the refrigeration bin; A heat sink fitted with the hot end of the semiconductor refrigerator; and A cooling fan connected to the radiator is used to perform heat dissipation treatment on the radiator.

[0017] In some exemplary embodiments, the monitoring feedback device is disposed between the plurality of centrifugal culture chambers, and includes: Detection bracket; A rotating mounting seat rotatably mounted on the detection bracket; A detection sensor carried by the rotating mounting seat, used to correspond to the centrifugal culture chamber to detect pH value and DO value, and feed back the pH value and DO value to the gas circuit control system to adjust the concentration of the supplied gas; A detection camera carried on the rotating mounting seat is used to correspond to the centrifugal culture chamber to perform stratification monitoring and to feed back to the liquid circuit control system to discharge the inner clear liquid after stratification; and The rotation driving mechanism carried by the detection bracket is used to periodically drive the rotation mounting seat to rotate so that the detection sensor and the detection camera correspond to each of the centrifugal culture chambers in sequence.

[0018] According to a second aspect of an embodiment of the present disclosure, a method for culturing cells in vitro is provided. The method is implemented based on the in vitro cell culturing device according to the first aspect, and comprises: Supplying a cell sample, a culture substance and a mixed gas mixed to a predetermined concentration into a centrifugal culture device, and controlling the centrifugal culture device to be within a predetermined temperature range; 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 a rotating centrifugal motion to radially stratify the cells and the culture material to discharge the inner clear liquid for liquid replacement; After reaching a predetermined culture period, the cells are transferred to an enrichment and harvesting device, and culture material is injected into the enrichment and harvesting device to enrich the cells; The enriched cells are put into product bags for collection and preservation, and the product bags are refrigerated at low temperatures.

[0019] In some exemplary embodiments, when cell suspension culture is performed, the pH value and DO value in the centrifugal culture device are detected and fed back to the gas circuit control system to adjust the concentration of the supplied gas; When the rotating centrifugal movement is performed, the centrifugal culture device is stratified and the stratification is fed back to the liquid control system to discharge the inner clear liquid after the stratification is completed; When the cells are transferred to the enrichment harvesting device, they are also filtered and denucleated.

[0020] According to a third aspect of an embodiment of the present disclosure, there is provided a use of the in vitro cell culture device as described in the first aspect in in vitro culture of red blood cells or stem cells.

[0021] In summary, compared with the prior art, the present invention has the following beneficial effects: The embodiment of the present invention provides a cell in vitro culture device, method and application thereof. When performing cell culture, a cell sample and a culture material are supplied to a centrifugal culture device, and a mixed gas of a predetermined concentration is supplied to the centrifugal culture device through a gas path control system to meet the pH and oxygen required for cell growth. The centrifugal culture device is heated to a predetermined temperature range by a heating unit so that the cells are at an optimal culture temperature. During the culture process, the pH value and DO value of the centrifugal culture device are monitored in real time through a monitoring feedback device. The concentration of the supplied gas is adjusted by feeding back the pH value and DO value to the gas path control system so that the pH value and DO value are within an appropriate range to ensure that the culture process proceeds normally. At the same time, during the culture process, the centrifugal culture device can realize radial stratification of cells and culture materials through a rotating centrifugal motion, and the monitoring feedback device can be used to monitor the pH value and DO value of the centrifugal culture device in real time. The feed device monitors the cell stratification. After the stratification is completed, the liquid control system discharges the inner clear liquid, and then adds new culture medium to realize the liquid replacement operation. After the cells are cultured for a predetermined period, the liquid control system transfers the cells to 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 movement to radially stratify the cells and culture materials, and discharge the inner clear liquid through the liquid control system to achieve concentration and enrichment. 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. The cell samples and product bags can be refrigerated at low temperature through the refrigeration unit, so that the cells can be stored for a long time, and the overall cost of the device is lower. The rotational suspension culture method also makes the cell damage rate lower. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the structure of the in vitro cell culture device in an embodiment of the present invention.

[0023] Figure 2 This is a schematic diagram of the structure of the in vitro cell culture device without the culture chamber in an embodiment of the present invention.

[0024] Figure 3 It is a partial cross-sectional view of the in vitro cell culture device in an embodiment of the present invention.

[0025] Figure 4 Schematic diagram of the structure of the centrifugal culture device in an embodiment of the present invention.

[0026] Figure 5 It is a cross-sectional view of a centrifugal culture chamber in an embodiment of the present invention.

[0027] Figure 6 for Figure 5 Enlarged view of part A in the middle.

[0028] Figure 7 Schematic diagram of the structure of the upper storage body in an embodiment of the present invention.

[0029] Figure 8 This is a schematic structural diagram of the upper storage body from another perspective in an embodiment of the present invention.

[0030] Fig. 9 It is a schematic diagram of the half-section structure of the rotary seal body in an embodiment of the present invention.

[0031] Fig.10 It is a schematic diagram of the half-section structure of the fixed joint seat in the embodiment of the present invention.

[0032] Fig.11 Schematic diagram of the structure of the liquid outlet component in an embodiment of the present invention.

[0033] Fig.12 Schematic diagram of the structure of the lower bin body in an embodiment of the present invention.

[0034] Fig.13 Schematic diagram of the structure of the liquid inlet component in an embodiment of the present invention.

[0035] Fig.14 Schematic diagram of the control principle of the liquid circuit control system in an embodiment of the present invention.

[0036] Fig.15 Schematic diagram of the control principle of the gas path control system in the embodiment of the present invention.

[0037] Fig.16 Schematic diagram of the structure of a refrigeration unit in an embodiment of the present invention.

[0038] Fig.17 Schematic diagram of the structure of the monitoring feedback device in an embodiment of the present invention.

[0039] The numbers and letters in the figure represent the corresponding component names: 10. Centrifugal culture device; 11. Centrifugal support frame; 12. Centrifugal culture chamber; 121. Upper chamber body; 1211. Top plate; 1212. Upper connecting plate; 1213. Upper enclosure plate; 1214. Mounting port; 1215. Positioning flange; 1216. Liquid outlet groove; 1217. Guide hole; 122. Lower chamber body; 1221. Bottom plate; 1222. Lower connecting plate; 1223. Lower enclosure plate; 1224. Mounting groove; 1225. Guide groove; 1226. Liquid inlet; 123. Rotary sealing body; 1231. Transfer guide groove; 1232. Upper retaining ring; 1233. Positioning and mounting portion; 1234. First positioning hole ; 1235, first bearing surface; 1236, second bearing surface; 1237, flow channel step surface; 124, fixed joint seat; 1241, gas-liquid channel; 1242, liquid outlet channel; 1243, first step shaft; 1244, second step shaft; 1245, upper bearing; 1246, positioning shoulder; 1247, lower bearing; 1248, sealing ring; 125, blade shaft; 1251, guide channel; 1252, turbulent blade; 13, liquid inlet assembly; 131, liquid inlet ring seat; 132, rotating groove; 133, liquid port; 134, guide plate; 14, liquid outlet assembly; 141, liquid outlet guide plate; 142, outlet Liquid guide groove; 143, liquid outlet; 15, first drive motor; 20, enrichment harvesting device; 21, enrichment support frame; 22, enrichment bin; 23, denuclearization filter; 24, second drive motor; 30, liquid circuit control system; 31, reagent bag; 32, peristaltic pump; 33, pipeline system; 34, flow sensor; 35, collection unit; 351, waste liquid bag; 352, sampling bag; 353, product bag; 36, pinch valve; 37, reagent filter; 40, gas circuit control system; 41, gas source; 42, mass flow controller; 43, static mixer; 44, high efficiency filter; 45, gas concentration detection module; 451. Carbon dioxide sensor; 452. Oxygen sensor; 46. Proportional control valve; 51. Heating unit; 511. Partition chamber; 512. Air inlet; 513. Air outlet; 514. Heating wire; 515. Crossflow fan; 52. Refrigeration unit; 521. Refrigeration chamber; 522. Semiconductor refrigerator; 523. Radiator; 524. Cooling fan; 525. Wind shield; 60. Monitoring feedback device; 61. Detection bracket; 62. Rotating mounting seat; 63. Detection sensor; 64. Detection camera; 65. Rotating drive mechanism; 70. Rack; 71. Culture chamber; 711. Sealing box; 712. Outer cover. DETAILED DESCRIPTION

[0040] 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.

[0041] like Figures 1 to 17 As shown, a first aspect of an embodiment of the present invention provides a cell in vitro culture device, including: a centrifugal culture device 10, an enrichment and harvesting device 20, a liquid circuit control system 30, a gas circuit control system 40, a temperature control device, and a monitoring feedback device 60.

[0042] Specifically, the centrifugal culture device 10 is used to contain cell samples and culture materials to achieve cell differentiation and proliferation, and can achieve radial stratification of cells and culture materials through rotating centrifugal motion, such as Figure 4 As shown, the centrifugal culture device 10 specifically includes: a centrifugal support frame 11; a centrifugal culture chamber 12 rotatably assembled on the centrifugal support frame 11, a containing space is formed in the centrifugal culture chamber 12, and a liquid inlet component 13 and a liquid outlet component 14 are provided on the centrifugal culture chamber 12; and a first driving motor 15 carried by the centrifugal support frame 11 and used for driving the centrifugal culture chamber 12 to perform centrifugal rotational motion.

[0043] Among them, the cell in vitro culture equipment includes a frame 70, which serves as the installation base of each device. The centrifugal support frame 11 is fixed to the frame 70 for supporting and installing 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 rotation connection of the centrifugal culture chamber 12. The first drive motor 15 adopts a servo motor, so that the centrifugal culture chamber 12 can be driven to rotate intermittently, rotate forward and reverse, or rotate centrifugally at high speed; the storage space formed in the centrifugal culture chamber 12 can be used to store cells and culture materials, and the supply and discharge of cells and culture materials are realized through the liquid inlet component 13 and the liquid outlet component 14, and the centrifugal culture chamber 12 can be driven to rotate through the first drive motor 15 to realize the suspension culture and centrifugal stratification of cells.

[0044] like Figure 5As shown, the centrifugal culture chamber 12 includes: an upper chamber body 121 and a lower chamber body 122 that are sealed and matched with each other, and a storage space is formed between the upper chamber body 121 and the lower chamber body 122; a rotating sealing body 123 arranged on the upper chamber body 121; a fixed joint seat 124 rotatably assembled on the rotating sealing body 123, and a gas-liquid channel 1241 and a liquid outlet channel 1242 are provided on the fixed joint seat 124, and the liquid outlet channel 1242 is connected to the liquid outlet component 14; and a blade shaft 125 rotatably assembled on the rotating sealing body 123 and connected to the fixed joint seat 124, the bottom of the blade shaft 125 is connected to the lower chamber body 122, and a through guide channel 1251 is provided on the blade shaft 125, and the first end of the guide channel 1251 is connected to the gas-liquid channel 1241, and the second end is connected to the liquid inlet component 13.

[0045] Among them, Figures 6 to 10 As shown, the upper storage body 121 includes an integrally formed top plate 1211, an upper connecting plate 1212 and an upper enclosure plate 1213, the upper enclosure plate 1213 and the top plate 1211 are perpendicular to each other, the upper connecting plate 1212 is obliquely arranged between the top plate 1211 and the upper enclosure plate 1213 to form a bowl-shaped structure, the inclination angle of the upper connecting plate 1212 can be set to 40-50 degrees, preferably 45 degrees, a through installation opening 1214 is opened in the middle of the top plate 1211 for assembling the rotating seal body 123, the rotating seal body 123 and the upper storage body 121 are clamped and fixed by an upper retaining spring 1232, specifically: a positioning flange 1215 is arranged on the outer side of the top plate 1211, and the rotating seal body 123 is fixed to the upper storage body 121 by the upper retaining spring 1232. The upper end of the sealing body 123 is provided with a positioning and mounting portion 1233 that is compatible with the positioning flange 1215. When the rotating sealing body 123 is inserted into the mounting port 1214, the positioning and mounting portion 1233 is embedded in the positioning flange 1215, and an upper clamping groove is provided on the rotating sealing body 123. The upper retaining spring 1232 is embedded in the upper clamping groove and abuts against the inner wall of the top plate 1211, and corresponding first positioning holes 1234 are provided on the positioning and mounting portion 1233 and the top plate 1211. The installation position and direction of the rotating sealing body 123 are limited by the first positioning hole 1234; and an interlocking step is formed between the upper connecting plate 1212 and the upper enclosure plate 1213 for abutting and cooperating with the lower warehouse body 122.

[0046] like Figure 5 and Fig.12As shown, the lower warehouse body 122 includes an integrally formed bottom plate 1221, a lower connecting plate 1222 and a lower enclosure plate 1223, the lower enclosure plate 1223 and the bottom plate 1221 are perpendicular to each other, the lower connecting plate 1222 is obliquely arranged between the bottom plate 1221 and the lower enclosure plate 1223 to form a bowl-shaped structure, the inclination angle of the lower connecting plate 1222 can be set to 40-50°, for example, preferably to 45°, the outer diameter of the lower enclosure plate 1223 matches the inner diameter of the upper enclosure plate 1213, and the lower enclosure plate 1223 and the upper enclosure plate 1213 are interlocked, and the lower enclosure plate 1223 is in contact with the engaging step.

[0047] like Figure 6 , Fig. 9 and Fig.10 As shown, the fixed joint seat 124 and the rotating sealing body 123 are rotationally connected through a bearing, and the top of the gas-liquid channel 1241 is connected with an air circuit interface and a liquid circuit interface, the air circuit interface is used to connect with the air circuit control system 40, and the liquid circuit interface is used to connect with the liquid circuit control system 30. The air circuit interface and the liquid circuit interface are respectively used to introduce air flow and liquid flow and converge them to the gas-liquid channel 1241, and the top of the liquid outlet channel 1242 is connected with a liquid outlet interface.

[0048] A first step shaft 1243 and a second step shaft 1244 are formed on the fixed joint seat 124, the second step shaft 1244 extends downward and is connected to the blade shaft 125, a first bearing surface 1235 and a second bearing surface 1236 are formed in the rotary seal body 123, an upper bearing 1245 is provided between the first bearing surface 1235 and the first step shaft 1243, and a positioning shoulder 1246 is provided outside the fixed joint seat 124, the positioning shoulder 1246 is in conflict with the upper end of the upper bearing 1245 to limit the installation position of the fixed joint seat 124, the liquid outlet channel 1242 passes through the lower end surface of the first step shaft 1243, and a flow channel step surface 1237 is provided in the middle of the rotary seal body 123, the lower end surface of the first step shaft 1243 and the flow channel step surface 1237 form a transfer guide groove 1 connected to the liquid outlet channel 1242 231; the second bearing surface 1236 is located below the flow channel step surface 1237, and a lower bearing 1247 is provided between the second bearing surface 1236 and the second step shaft 1244, the gas-liquid channel 1241 passes through the lower end surface of the second step shaft 1244, and is connected to the blade shaft 125 through the second step shaft 1244 to achieve communication with the guide channel 1251; in some embodiments, a sealing ring 1248 is provided between the first step shaft 1243 and the rotating seal body 123, between the second step shaft 1244 and the rotating seal body 123, and between the second step shaft 1244 and the blade shaft 125 to improve the sealing performance, and a plurality of turbulent blades 1252 are also provided in the blade shaft 125 to form turbulence during the rotation of the centrifugal culture chamber 12, and the turbulent blades 1252 are preferably evenly arranged in four groups around the blade shaft 125.

[0049] The fixed joint seat 124 is used to connect the gas control system 40 and the liquid control system 30. The gas-liquid channel 1241 allows mixed gas, cell samples and culture materials to enter the centrifugal culture chamber 12. When supplied, they are transferred and drained through the guide channel 1251 on the blade shaft 125, and then introduced into the centrifugal culture chamber 12 by the liquid inlet component 13 to realize liquid inlet control, and the liquid outlet channel 1242 is connected to the liquid outlet component 14 for discharging the clear liquid. Since the fixed joint seat 124 is rotatably connected to the rotating sealing body 123, the connection with the gas control system 40 and the liquid control system 30 will not be affected when the centrifugal culture chamber 12 rotates, and the setting of the blade shaft 125 can provide turbulence during the rotation process of the centrifugal culture chamber 12 to promote the convective exchange of nutrients.

[0050] like Fig.12 and Fig.13 As shown, the liquid inlet assembly 13 includes: a liquid inlet ring seat 131, on which is provided a rotating groove 132 which is adapted to the bottom of the blade shaft 125, and in which is provided a liquid through port 133 which is connected to the guide channel 1251; a guide plate 134 which is connected to the side of the liquid inlet ring seat 131 and extends radially along the liquid inlet ring seat 131; and on the lower warehouse body 122, a mounting groove 1224 which is adapted to the liquid inlet ring seat 131 and the guide plate 134 is provided, and the mounting groove 1224 is connected to the liquid through port 133, and a guide groove 1225 is formed between the liquid inlet ring seat 131 and the guide plate 134, and a liquid inlet port 1226 which is connected to the lower warehouse body 122 is formed at the end of the guide groove 1225.

[0051] Specifically, the mounting groove 1224 includes: a central ring groove adapted to the liquid inlet ring seat 131, and a radial groove adapted to the guide plate 134. The liquid inlet component 13 is embedded in the mounting groove 1224 and fits with the mounting groove 1224 to achieve sealing, and can be sealed by ultrasonic welding or photosensitive adhesive bonding. The guide groove 1225 is arranged in the radial groove and extends from the center of the central ring groove to the edge of the bottom plate 1221, and the width of the guide groove 1225 is smaller than the width of the radial groove to limit the embedding depth of the guide plate 134. The guide groove 1225 extends out of the radial groove to form a liquid inlet 1226 at the end.

[0052] The rotating groove 132 cooperates with the bottom of the blade shaft 125 to achieve a stable connection with the blade shaft 125. The airflow and liquid flow are guided through the guide channel 1251 and enter the guide groove 1225 from the liquid port 133. Restricted by the liquid inlet ring seat 131 and the guide plate 134, the airflow and liquid flow are finally forced to enter the centrifugal culture chamber 12 from the liquid inlet 1226. At the same time, since the liquid inlet component 13 is arranged in the lower chamber body 122, the direction of the airflow and liquid flow is from bottom to top during the supply process, which reduces the damage to the cells caused by the airflow and liquid flow during the supply process, and further reduces the cell damage rate.

[0053] like Figures 7 to 11 As shown, the liquid outlet assembly 14 includes a liquid outlet guide plate 141 fixed to the upper warehouse body 121, the liquid outlet guide plate 141 extends from the rotating seal body 123 to near the lower warehouse body 122, the rotating seal body 123 is provided with a transfer guide groove 1231 connected to the liquid outlet channel 1242, the liquid outlet guide plate 141 is provided with a liquid outlet guide groove 142 connected to the transfer guide groove 1231, and the liquid outlet guide groove 142 extends to one end near the lower warehouse body 122 to form a liquid outlet 143, the liquid outlet 143 is rectangular, and the liquid outlet 143 is set to be extremely thin, for example, the width of the liquid outlet 143 is set to 0.5mm; it can be understood that a liquid outlet embedded groove 1216 adapted to the liquid outlet guide plate 141 is provided on the upper warehouse body 121, and the liquid outlet guide plate 141 is embedded in the liquid outlet embedded groove 1216 and passes through the ultra The sealing connection is performed by ultrasonic welding or photosensitive adhesive bonding, and a guide hole 1217 is also provided on the upper chamber body 121. The guide hole 1217 is correspondingly connected to the end of the liquid outlet channel 1242. One side of the transfer guide groove 1231 extends in the rotating seal body 123 to be correspondingly connected to the guide hole 1217. When discharging liquid, the liquid flows into the liquid outlet guide groove 142 from the liquid outlet port 143, and enters the liquid outlet channel 1242 through the transfer guide groove 1231 under the drainage effect of the liquid outlet guide groove 142 to achieve liquid discharge. After the centrifugal culture chamber 12 is centrifuged, the cells are centrifugally attached to the inner wall of the centrifugal culture chamber 12, and a very small concentration space is formed on the inner wall of the centrifugal culture chamber 12, which can meet the high-proportion concentration requirements, and then the inner clear liquid is extracted through the extremely thin liquid outlet port 143, thereby achieving a more accurate and thorough liquid replacement operation.

[0054] like Figure 2 and Fig.14 As shown, the enrichment and harvesting device 20 is used to contain the cultured cells and culture materials, and radially stratify the cells and culture materials through rotating centrifugal motion to enrich the cells after extracting the inner clear liquid. The enrichment and harvesting device 20 specifically 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, the enrichment bin 22 is connected to the centrifugal culture bin 12 through a liquid circuit control system 30, and the cells are harvested from the enrichment bin 22 to a 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 and used to drive the enrichment bin 22 to perform centrifugal rotational motion.

[0055] The enrichment support frame 21 is fixed to the frame 70 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 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. When the cells enter the enrichment bin 22, they are filtered and denuclearized by the denuclearization filter 23 to separate the nucleated red blood cells and the 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 material, and the inner layer of the clear liquid is pumped away to leave the cells in the enrichment bin 22, thereby achieving the concentration and enrichment of the cells.

[0056] like Fig.14 As shown, the liquid circuit control system 30 is used to supply cell samples and culture materials into the centrifugal culture device 10, transfer the cultured cells to the enrichment and harvesting device 20 for enrichment, and finally supply the cultured cells into the product bag 353 for collection and preservation. Specifically, the liquid circuit control system 30 includes: a plurality of reagent bags 31, which are used to store 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 a 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; The collecting unit 35 includes a waste liquid bag 351, a sampling bag 352 and a product bag 353; and a plurality of pinch valves 36 connected to the piping system 33 and correspondingly arranged with each reagent bag 31, each centrifugal culture chamber 12, the enrichment chamber 22 and the collecting unit 35; the peristaltic pump 32 pumps the cell sample and the culture material from the reagent bag 31 into the centrifugal culture chamber 12, 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.

[0057] 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 medium 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 sampling bag 352 is used for sampling quality control during the culture and enrichment process. It can be understood that the waste liquid bag 351 and the sampling bag 352 can also be connected to the outside of the centrifugal culture chamber 12, and the product bag 353 is used to collect the cultured cells.

[0058] 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. Each centrifugal culture chamber 12 and enrichment chamber 22 is provided with a pinch valve 36 corresponding to the gas-liquid channel 1241 and the liquid outlet channel 1242. 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, the multiple centrifugal culture chambers 12 or enrichment chambers 22 are connected in parallel to the pipeline system 33. In this embodiment, Four groups of centrifugal culture chambers 12 and one group of enrichment chambers 22; 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 in parallel to the pipeline system 33. 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 achieve sterile culture; in some embodiments, a reagent filter 37 can also be set in the liquid control system 30 to filter the reagent to improve the cleanliness of the reagent.

[0059] The pinch valve 36 controls the switching of each node in the piping 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 processes of cell culture, transfer, enrichment and collection are completed. The flow rate in the piping system 33 is monitored in real time by the flow sensor 34, and real-time feedback can be provided to regulate the speed of the peristaltic pump 32.

[0060] like Fig.15 As shown, the gas circuit control system 40 is used to mix the gas for cell culture to a predetermined concentration and then supply it into the centrifugal culture device 10, and specifically includes: a plurality of gas sources 41, which are used to respectively provide carbon dioxide, oxygen / air, and nitrogen; a mass flow controller 42 connected to each gas source 41, which is used to measure and control the gas mass flow; a static mixer 43 connected to each mass flow controller 42, which is used to mix the gas supplied by each gas source 41 to form a mixed gas; a high-efficiency filter 44 connected to the static mixer 43; a gas concentration detection module 45 connected to the high-efficiency filter 44, which is used to detect the concentration of the mixed gas; a proportional control valve 46 connected to the gas concentration detection module 45 and corresponding to each centrifugal culture bin 12, which is used to control the supply of the mixed gas into each centrifugal culture bin 12.

[0061] The gas source 41 may specifically be a high-pressure gas cylinder storing gas. In the present embodiment, three groups of high-pressure gas cylinders are provided, one group storing carbon dioxide, one group storing oxygen or air, and one group storing nitrogen, and each high-pressure gas cylinder is respectively connected to a pressure reducing valve for controlling the switch of the high-pressure gas cylinder. The mass flow controller 42 (Mass Flow Controller, MFC) can accurately measure and control the mass flow rate of the gas. Usually, the mass flow controller 42 is also connected to a one-way valve so that each type of gas can only flow in one direction. The static mixer 43 can evenly mix the various types of gases to form a mixed gas. For example, an SV-type static mixer 43 may be used, and a one-way valve may also be provided between the static mixer 43 and the high-efficiency filter 44 to control the one-way outflow of the mixed gas. The gas concentration detection module 45 includes a carbon dioxide sensor 451 and an oxygen sensor 452, which are respectively used to detect the carbon dioxide concentration and oxygen concentration in the mixed gas.

[0062] Carbon dioxide, oxygen / air and nitrogen are provided respectively by multiple gas sources 41, and the gas mass flow rate is measured in real time by the mass flow controller 42. The flow rate supplied by each gas source 41 is controlled according to the predetermined gas concentration. The supplied gas is mixed in the static mixer 43 to form a mixed gas. The mixed gas is passed into the centrifugal culture chamber 12 to adjust the pH and dissolved oxygen content to meet the growth requirements of the cells. The mixed gas is filtered by the high-efficiency filter 44 to ensure the cleanliness of the mixed gas, and the concentration of the mixed gas is detected by the gas concentration detection module 45. The detection value is further fed back to adjust the gas supply flow rate, so that the concentration of the mixed gas is more accurate, and finally the proportional control valve 46 controls the supply to each centrifugal culture chamber 12.

[0063] In some embodiments, the gas path control system 40 also includes an MCU control module, which is communicatively connected to the pressure reducing valve, the mass flow controller 42, the carbon dioxide sensor 451, and the oxygen sensor 452. For example, the MUC control module is communicatively connected to the mass flow controller 42 via an RS-232 interface, is communicatively connected to the pressure reducing valve via a GPIO general input and output port, and realizes data transmission with the carbon dioxide sensor 451 and the oxygen sensor 452 via a TTL method. The MUC control module is communicatively connected to the host computer via a CAN bus, the gas flow information is detected by the mass flow controller 42, and the gas concentration information is detected by the carbon dioxide sensor 451 and the oxygen sensor 452. The MCU control module calculates the intake volume of each mass flow controller 42 according to the gas concentration target value sent by the host computer, and corrects the intake volume in combination with the gas concentration information collected by the carbon dioxide sensor 451 and the oxygen sensor 452 to realize precise gas concentration control. When the gas concentration operation reaches a predetermined concentration threshold, the MCU control module can control the exhaust valve on the static mixer 43 to open and discharge the gas to the atmosphere for re-mixing.

[0064] like Figure 3 and Fig.16 As shown, the temperature control device includes: a heating unit 51 for heating the centrifugal culture device 10 and the enrichment and harvesting device 20 to a predetermined temperature range, and a refrigeration unit 52 for performing low-temperature refrigeration treatment on the cell sample, the culture material and the product bag 353.

[0065] Specifically, the centrifugal culture device 10 is arranged in an openable / closable culture chamber 71, and the culture chamber 71 includes: a sealing box 711 fixed to the frame 70, and an outer cover 712 hinged to the sealing box 711, the outer cover 712 can be set to be transparent, so as to observe the working state of the centrifugal culture device 10, the outer cover 712 is hingedly connected to the sealing box 711 through a hinge, and a rubber sealing belt is embedded on the top of the sealing box 711, and when the outer cover 712 is closed, it is tightly matched with the rubber sealing belt to open the culture chamber 71. A sealed space is formed inside to reduce heat loss. At the same time, in order to avoid heat loss in the culture chamber 71 as much as possible, the sealing box 711 and the outer cover 712 are made of materials with low thermal conductivity. For example, the material of the outer cover 712 is thermal insulation resin and the material of the sealing box 711 is engineering plastic. It can be understood that a wire hole should also be set on the side of the culture chamber 71 for the passage of pipelines and control lines. The enrichment and harvesting device 20 can also be set in a culture chamber 71 and equipped with a heating unit 51 for heating treatment.

[0066] The heating unit 51: a partition chamber 511 arranged in the culture chamber 71, the bottom of the partition chamber 511 is provided with an air inlet 512, and the top of the partition chamber 511 is provided with an air outlet 513; a heating wire 514 arranged in the partition chamber 511 and located near the air outlet 513; and a cross-flow fan 515 arranged in the partition chamber 511, the air inlet side of the cross-flow fan 515 is close to the air inlet 512, and the air outlet side is close to the heating wire 514; and a temperature sensor is provided in the culture chamber 71 for real-time detection and feedback of temperature information in the culture chamber 71.

[0067] Among them, the partition chamber 511 is fixed to one side of the culture chamber 71, the air inlet 512 and the air outlet 513 can be set to a rectangular long groove shape or a plurality of through-hole shapes, the heating wire 514 is used to generate heat, and the material of the heating wire 514 can be, for example, iron-chromium-aluminum alloy or nickel-chromium alloy, which has the advantages of high heat transfer efficiency and no harmful substances. One or more temperature sensors can be provided. For example, a temperature sensor can be provided near the air outlet 513 to detect the outlet air temperature. After maintaining the outlet air temperature within a predetermined temperature range for a certain period of time, the culture chamber 71 can reach a suitable temperature. The temperature sensor can specifically adopt an NTC thermistor temperature sensor.

[0068] The centrifugal culture chamber 12 is thermally insulated by the culture chamber 71. When heating, the heating wire 514 and the cross-flow fan 515 are started, and hot air is blown into the culture chamber 71 by the cross-flow fan 515. A circulating air convection environment is formed in the culture chamber 71, and the temperature in the culture chamber 71 is evenly heated, so that the centrifugal culture chamber 12 is in an environment within a predetermined temperature range. At the same time, the temperature information in the culture chamber 71 is detected in real time by the temperature sensor, so that the working power of the heating wire 514 can be adjusted in time.

[0069] The refrigeration unit 52 can be set in one or more groups as needed. For example, a refrigeration unit 52 specifically for storing reagent bags and a refrigeration unit 52 specifically for storing product bags can be set. The refrigeration unit 52 includes: a refrigeration bin 521 for storing reagent bags 31 and product bags 353; a semiconductor refrigerator 522 fitted with the refrigeration bin 521, and the cold end of the semiconductor refrigerator 522 fits with the refrigeration bin 521; a radiator 523 fitted with the hot end of the semiconductor refrigerator 522; and a cooling fan 524 connected to the radiator 523, for dissipating heat from the radiator 523.

[0070] The refrigeration bin 521 is provided with an openable bin cover, which is hingedly connected to the refrigeration bin 521 through a hinge, and a heat-insulating cotton layer can be provided in the refrigeration bin 521 to improve the heat-insulating performance of the refrigeration bin 521, so that the refrigeration bin 521 can maintain a low temperature for a long time. The semiconductor refrigerator 522 specifically adopts a semiconductor Peltier, and the semiconductor refrigerator 522 is fitted at the bottom of the refrigeration bin 521. The radiator 523 includes a substrate and a plurality of heat-dissipating fins evenly arranged on the substrate. The substrate and the semiconductor refrigerator 522 are The radiator 523 is fitted together, and a wind shield 525 is also provided at the bottom of the radiator 523. The wind shield 525 is connected and fixed to the radiator 523 via an L-shaped connecting piece arranged on the side of the radiator 523. The wind shield 525 is used to provide an installation basis for the cooling fan 524 and can form a wind gathering effect. The cooling fan 524 is fixed to the bottom of the wind shield 525. One or more cooling fans 524 can be provided, and ventilation holes are provided on the wind shield 525 corresponding to each cooling fan 524 for the wind of the cooling fan 524 to pass through.

[0071] When the semiconductor refrigerator 522 is started, its cold end cools the refrigeration bin 521, so that the refrigeration bin 521 is in a low-temperature environment, thereby realizing low-temperature refrigeration of the reagent bag 31 and the product bag 353, and its hot end is in contact with the radiator 523, and heat is conducted out through the radiator 523, and the radiator 523 is dissipated by the cooling fan 524, thereby realizing rapid cooling of the radiator 523; in some embodiments, one or more temperature sensors may also be provided in the refrigeration bin 521, and the temperature in the refrigeration bin 521 is detected by the temperature sensor, so as to control the semiconductor refrigerator 522 to adjust the working power in time and keep the refrigeration bin 521 in a suitable low-temperature environment.

[0072] In some embodiments, a preheating module is further provided on the rack 70, and the pipeline system 33 passes through the preheating module, which is used to preheat the cell sample and the culture reagent supplied to the centrifugal culture device 10. The preheating module may include, for example, a preheating box, and the preheating box is provided with a row of pipe holes for the pipeline system 33 to pass through, and a heating plate is provided in the preheating box, and the heating plate heats the preheating box to a predetermined temperature. When the liquid circuit control system 30 controls the cell sample and the culture reagent to be supplied to the centrifugal culture device 10, the preheating treatment can be performed, so that the cell sample and the culture reagent can quickly reach a suitable culture temperature after the centrifugal culture device 10. like Fig.17 As shown, the monitoring feedback device 60 is used to monitor the pH value and DO value in the centrifugal culture device 10 to feed back to the gas circuit control system 40 to adjust the concentration of the supplied gas, and to perform stratification monitoring on the centrifugal culture device 10 to feed back to the liquid circuit control system 30 to discharge the inner clear liquid after stratification. The monitoring feedback device 60 is arranged in the middle of a plurality of centrifugal culture chambers 12. This embodiment takes the arrangement of four groups of centrifugal culture chambers 12 as an example. The four centrifugal culture chambers 12 are arranged in a rectangular shape. The monitoring feedback device is arranged in the middle of the four centrifugal culture chambers 12.

[0073] Specifically, the monitoring feedback device 60 includes: a detection bracket 61; a rotating mounting seat 62 rotatably mounted on the detection bracket 61; a detection sensor 63 carried by the rotating mounting seat 62, which is used to correspond to the centrifugal culture chamber 12 to detect the pH value and DO value, and feed back the pH value and DO value to the gas circuit control system 40 to adjust the concentration of the supplied gas; a detection camera 64 carried by the rotating mounting seat 62, which is used to correspond to the centrifugal culture chamber 12 to perform stratification monitoring, and feed back to the liquid circuit control system 30 to discharge the inner clear liquid after stratification; and a rotating drive mechanism 65 carried by the detection bracket 61, which is used to periodically drive the rotating mounting seat 62 to rotate so that the detection sensor 63 and the detection camera 64 correspond to each centrifugal culture chamber 12 in turn.

[0074] The rotatable mounting seat 62 is rotatably connected to the detection bracket 61 through a rotating shaft. The detection sensor 63 adopts a non-contact pH and DO fiber optic probe, and a sensor patch is provided on the inner wall of the lower chamber 122. When the detection sensor 63 corresponds to the sensor patch, the pH value and dissolved oxygen level of the current culture medium in the centrifugal culture chamber 12 can be detected after data collection and processing. The collected pH value and DO value can be used as the basis for the gas path control system 40 to adjust the gas concentration. The detection camera 64 can, for example, adopt an industrial CCD camera, which collects image information in the centrifugal culture chamber 12 and compares it with a preset stratified image. When the comparison is consistent, it can be determined that the stratification is completed.

[0075] The rotation driving mechanism 65 can be, for example, a belt transmission mechanism, a sprocket transmission mechanism and a gear transmission mechanism, or a servo motor can be directly used to drive the rotation mounting seat 62 to rotate. In the present embodiment, the rotation driving mechanism 65 preferably adopts a belt transmission mechanism, which specifically includes: a rotating motor fixed to the detection bracket 61, a driving pulley fixed to the power output shaft of the rotating motor, a driven pulley fixed to the rotating shaft, and a belt sleeved on the driving pulley and the driven belt bracket to realize power transmission. The rotating motor adopts a servo motor, and the driving pulley is driven to rotate by the rotating motor, that is, the belt is driven by the belt. The belt transmission power drives the rotating mounting seat 62 to rotate; in some embodiments, a photoelectric detector is also provided on the detection bracket 61, and the photoelectric detector includes a light emitter and a light receiver. The light emitter and the light receiver are relatively arranged on a C-shaped bracket. The light emitter emits a detection light beam which is received by the light receiver, and a light shielding plate is fixed on the rotating shaft. The light shielding plate can intermittently block between the light emitter and the light receiver during the rotation of the rotating mounting seat 62. Each time the light shielding plate is blocked, a photoelectric detection signal can be generated, which means that the rotating mounting seat 62 rotates one circle, completing the monitoring feedback of the four centrifugal culture chambers 12.

[0076] The rotating mounting seat 62 is driven by the rotating driving mechanism 65, so that the detection sensor 63 and the detection camera 64 can correspond to each centrifugal culture chamber 12 in turn, thereby realizing the detection of each centrifugal culture chamber 12, and the pH value and DO value are monitored by the detection sensor 63, and the layered monitoring is performed by the detection camera 64, thereby realizing the regulation of the gas circuit control system 40 and the liquid circuit control system 30.

[0077] In some embodiments, a group of detection sensors 63 and detection cameras 64 may be respectively provided for each centrifugal culture chamber 12. In this case, there is no need to provide a rotating mounting seat 62 and a rotating drive mechanism 65. The detection sensor 63 and the detection camera 64 can be fixed to the detection bracket 61 at a predetermined angle and direction. Of course, a monitoring feedback device 60 may also be provided at the enrichment and harvesting device 20 for signal monitoring.

[0078] When cell culture is performed, cell samples and culture materials are supplied to the centrifugal culture device 10, and a mixed gas of a predetermined concentration is supplied to the centrifugal culture device 10 through the gas circuit control system 40 to meet the pH and oxygen required for cell growth. The centrifugal culture device 10 is heated to a predetermined temperature range by the heating unit 51 so that the cells are at an optimal culture temperature. During the culture process, the pH value and DO value of the centrifugal culture device 10 are monitored in real time through the monitoring feedback device 60. The concentration of the supplied gas is adjusted by feeding back the pH value and DO value to the gas circuit control system 40 so that the pH value and DO value are within an appropriate range to ensure that the culture process proceeds normally. At the same time, during the culture process, the centrifugal culture device 10 can achieve radial stratification of cells and culture materials through rotating centrifugal motion, and the cell stratification is monitored through the monitoring feedback device 60. After completion, the liquid control system 30 will discharge the inner clear liquid, and then add new culture medium to realize the liquid replacement operation; after the cells are cultured for a predetermined period, the liquid control system 30 will transfer the cells to 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 a rotational centrifugal motion to radially stratify the cells and the culture material, and discharge the inner clear liquid through the liquid control system 30 to achieve concentration and enrichment. After the enrichment is completed, the cells can be supplied to the product bag 353 for preservation through the liquid control system 30, thereby realizing the automated and large-scale preparation of cells in vitro, and the cell samples and the product bag 353 can be refrigerated at low temperature through the refrigeration unit 52, so that the cells can be stored for a long time, and the overall cost of the device is lower, and the rotating suspension culture method makes the cell damage rate lower.

[0079] A second aspect of an embodiment of the present invention provides a method for culturing cells in vitro. The method is implemented based on the in vitro cell culturing device as described in the first aspect, and includes: S100, supplying a cell sample, a culture material, and a mixed gas mixed to a predetermined concentration into the centrifugal culture device 10, and controlling the centrifugal culture device 10 to be within a predetermined temperature range.

[0080] Specifically, cell samples and culture materials are supplied into the centrifugal culture device 10 by the liquid control system 30, and mixed gas is supplied by the gas control system 40. The centrifugal culture device 10 is heated by the heating unit 51 so that the cells can be expanded at a suitable culture temperature.

[0081] 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 to discharge the inner clear liquid for liquid replacement. The mass transfer efficiency of the culture process can be improved by 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 control system 30 to achieve liquid replacement, so that the cells can be continuously cultured and expanded.

[0082] At the same time, when cell suspension culture is performed, the pH value and DO value in the centrifugal culture device 10 are detected and fed back to the gas circuit control system 40 to adjust the concentration of the supplied gas, thereby realizing feedback control of the gas circuit control system 40; and when rotating centrifugal motion is performed, the centrifugal culture device 10 is stratified and monitored, and feedback is given to the liquid circuit control system 30 to discharge the inner clear liquid after stratification and perform liquid replacement operation.

[0083] S300, after reaching the predetermined culture cycle, the cells are transferred to the enrichment and harvesting device 20 for cell enrichment. The cell transfer can also be performed through the liquid control system 30. When the cells are transferred to the enrichment and harvesting device 20, they are also filtered and denucleated through the denucleation filter 23 to achieve separation of nucleated red blood cells and mature red blood cells. During the enrichment process, the enrichment device can also perform centrifugal stratification and discharge the clear liquid to achieve concentrated enrichment.

[0084] S400 , the enriched cells are supplied to the product bag 353 for collection and preservation, and the product bag 353 is refrigerated at low temperature. The collection of cells can also be achieved by the liquid circuit control system 30 , and the low temperature refrigeration of the product bag 353 is achieved by the refrigeration unit 52 .

[0085] 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.

[0086] A third aspect of an embodiment of the present invention provides an application of the cell in vitro culture device as described in the first aspect in the in vitro culture of red blood cells or stem cells. For example, hematopoietic stem and progenitor cells are used as cell samples, and red blood cells are cultured in vitro using the cell in vitro culture device to achieve the integration of cell induction differentiation, amplification, denucleation, and harvesting processes.

[0087] 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 in vitro culture device, 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; A fluid control system, used for supplying cell samples and culture materials into the centrifugal culture device, transferring the cultured cells to the enrichment and harvesting device for enrichment, and finally supplying the cultured cells into a product bag for collection and storage; A gas circuit control system, used for mixing gas for cell culture to a predetermined concentration and then supplying the gas to the centrifugal culture device; The temperature control device comprises: a heating unit for heating the centrifugal culture device and the enrichment and harvesting device to a predetermined temperature range, and a refrigeration unit for performing low-temperature refrigeration treatment on cell samples, culture materials and product bags; and, A monitoring feedback device is used to monitor the pH value and DO value in the centrifugal culture device to provide feedback to the gas circuit control system to adjust the concentration of the supplied gas, and to perform stratification monitoring on the centrifugal culture device to provide feedback to the liquid circuit control system to discharge the clear liquid after stratification.

2. The in vitro cell culture device 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 liquid inlet component and a liquid outlet component 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 in vitro cell culture device according to claim 2, characterized in that: The centrifugal culture chamber comprises: An upper bin body and a lower bin body sealed and matched with each other, wherein the storage space is formed between the upper bin body and the lower bin body; A rotating sealing body disposed on the upper storage body; A fixed joint seat rotatably mounted on the rotary seal body, wherein the fixed joint seat is provided with a gas-liquid channel and a liquid outlet channel, wherein the liquid outlet channel is connected to the liquid outlet assembly; and A blade shaft is rotatably assembled on the rotating seal body and connected to the fixed joint seat, the bottom of the blade shaft is connected to the lower warehouse body, and a through guide channel is provided on the blade shaft, the first end of the guide channel is connected to the gas-liquid channel, and the second end is connected to the liquid inlet component.

4. The in vitro cell culture device according to claim 3, characterized in that: The liquid inlet assembly comprises: A liquid inlet ring seat, wherein the liquid inlet ring seat is provided with a rotation groove adapted to the bottom of the blade shaft, and the rotation groove is provided with a liquid through port connected to the guide channel; A guide plate connected to the side of the liquid inlet ring seat and extending in the radial direction of the liquid inlet ring seat; The lower bin body is provided with an installation groove adapted to the liquid inlet ring seat and the guide plate, and the installation groove is connected to the liquid through port, and a guide groove is formed between the liquid inlet ring seat and the guide plate, and a liquid inlet connected to the lower bin body is formed at the end of the guide groove.

5. The in vitro cell culture device according to claim 3 or 4, characterized in that: The liquid outlet assembly includes a liquid outlet guide plate fixed to the upper bin body, the liquid outlet guide plate extends from the rotating seal body to a position close to the lower bin body, the rotating seal body is provided with a transfer guide groove connected to the liquid outlet channel, the liquid outlet guide plate is provided with a liquid outlet guide groove connected to the transfer guide groove, and the liquid outlet guide groove extends to one end close to the lower bin body to form a liquid outlet.

6. The in vitro cell culture device according to claim 2, 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, the enrichment bin is connected to the centrifugal culture bin through the fluid circuit control system, and cells are harvested from the enrichment bin to a product bag through the fluid 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.

7. The in vitro cell culture device according to claim 6, characterized in that: The hydraulic control system comprises: Several reagent bags for storing cell samples and various culture materials; A peristaltic pump is connected to the reagent bag, the centrifugal culture chamber and the enrichment chamber 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, a sampling 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, each of the centrifugal culture chambers, the enrichment chamber and the collection unit; The peristaltic pump pumps the cell sample and culture material from the reagent bag into the centrifugal culture chamber, transfers the cultured cells to the enrichment chamber for enrichment, and finally supplies the cultured cells to the product bag for collection and preservation.

8. The in vitro cell culture device according to claim 2, characterized in that: The gas circuit control system comprises: Multiple gas sources, used to provide carbon dioxide, oxygen / air, and nitrogen respectively; A mass flow controller connected to each of the gas sources, for measuring and controlling the gas mass flow; A static mixer connected to each of the mass flow controllers, used for mixing the gases supplied from each of the gas sources to form a mixed gas; A high efficiency filter connected to the static mixer; A gas concentration detection module connected to the high efficiency filter, used to detect the concentration of the mixed gas; A proportional control valve connected to the gas concentration detection module and arranged corresponding to each of the centrifugal culture bins is used to control the supply of mixed gas into each of the centrifugal culture bins.

9. The in vitro cell culture device according to claim 2, characterized in that: The centrifugal culture device is arranged in a culture chamber which can be opened / closed, and the heating unit: A partition chamber is provided in the culture chamber, wherein the bottom of the partition chamber is provided with an air inlet and the top of the partition chamber is provided with an air outlet: A heating wire disposed in the partition compartment and located near the air outlet; and A cross-flow fan is disposed in the partition chamber, wherein the air inlet side of the cross-flow fan is close to the air inlet, and the air outlet side of the cross-flow fan is close to the heating wire; In addition, a temperature sensor is provided in the culture chamber for real-time detection and feedback of temperature information in the culture chamber.

10. The in vitro cell culture device according to claim 7, characterized in that: The refrigeration unit comprises: A refrigeration chamber, used for storing the reagent bags and the product bags; A semiconductor refrigerator fitted with the refrigeration bin, wherein the cold end of the semiconductor refrigerator fits with the refrigeration bin; A heat sink fitted with the hot end of the semiconductor refrigerator; and A cooling fan connected to the radiator is used to perform heat dissipation treatment on the radiator.

11. The in vitro cell culture device according to claim 2, characterized in that: The monitoring feedback device is arranged in the middle of the plurality of centrifugal culture chambers, and includes: Detection bracket; A rotating mounting seat rotatably mounted on the detection bracket; A detection sensor carried by the rotating mounting seat, used to correspond to the centrifugal culture chamber to detect pH value and DO value, and feed back the pH value and DO value to the gas circuit control system to adjust the concentration of the supplied gas; A detection camera carried on the rotating mounting seat is used to correspond to the centrifugal culture chamber to perform stratification monitoring and to feed back to the liquid circuit control system to discharge the inner clear liquid after stratification; and The rotation driving mechanism carried by the detection bracket is used to periodically drive the rotation mounting seat to rotate so that the detection sensor and the detection camera correspond to each of the centrifugal culture chambers in sequence.

12. A method for culturing cells in vitro, characterized in that: The method is implemented based on the in vitro cell culture device according to any one of claims 1 to 11, comprising: Supplying a cell sample, a culture substance and a mixed gas mixed to a predetermined concentration into a centrifugal culture device, and controlling the centrifugal culture device to be within a predetermined temperature range; 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 a rotating centrifugal motion to radially stratify the cells and the culture material to discharge the inner clear liquid for liquid replacement; After reaching a predetermined culture period, the cells are transferred to an enrichment and harvesting device for cell enrichment; The enriched cells are put into product bags for collection and preservation, and the product bags are refrigerated at low temperatures.

13. The in vitro cell culture method according to claim 12, characterized in that: When cell suspension culture is performed, the pH value and DO value in the centrifugal culture device are detected and fed back to the gas circuit control system to adjust the concentration of the supplied gas; When the rotating centrifugal movement is performed, the centrifugal culture device is stratified and the stratification is fed back to the liquid control system to discharge the inner clear liquid after the stratification is completed; When the cells are transferred to the enrichment harvesting device, they are also filtered and denucleated.

14. Use of the in vitro cell culture device according to any one of claims 1 to 11 in in vitro culture of red blood cells or stem cells.

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