Method beneficial to culture of hollow fiber bioreactor

Abstract

The invention discloses a hollow fiber bioreactor culture method, which is characterized in that one or more groups of hollow fiber bioreactors are arranged in a parallel array, and the number of one group of hollow fiber bioreactors meets the requirement that an in-vitro liver support system can be simultaneously used at a time; in the initial planting stage after the seed cells are injected, all the hollow fiber bioreactors are kept in a parallel array state with the ground for culture; and at a growth culture stage after the seed cells are uniformly attached to the fiber side walls of the hollow fiber bioreactors, all the hollow fiber bioreactors are obliquely arranged at an included angle of 30-60 degrees with the ground. Wherein the seed cells are C3A cells. The time of the initial planting stage is 12-24 hours, the bioreactor is arranged on a culture rack with an adjustable and locked angle, and the culture rack is adjusted to maintain a horizontal or inclined state so as to meet the requirement of controlling the initial culture stage and the growth culture stage of all the bioreactors. Through the arrangement, the homogeneity of the same batch of hollow fiber bioreactors is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cell culture equipment, and in particular relates to a culture device for a hollow fiber bioreactor, in particular to a culture rack which is conducive to the culture of the hollow fiber bioreactor. Background Art

[0002] CN112755288B is an extracorporeal liver support system, which is currently used temporarily by patients with liver failure or waiting for liver source to replace the liver. The system uses C3A hollow fiber bioreactor to play the role of liver replacement. In order to provide sufficient and qualified hollow fiber bioreactors for extracorporeal liver support, it is necessary to carry out scientific and effective cultivation and treatment of the hollow fiber bioreactor.

[0003] Among them, how to reasonably and effectively set up the hollow fiber bioreactor to ensure that the hollow fiber bioreactors produced in a uniform batch have good homogeneity, and then ensure the stable therapeutic effect of the hollow fiber bioreactor during subsequent treatment, is a real technical problem that needs to be solved.

[0004] In view of the above technical problems, the present invention provides a culture rack which is beneficial to the culture of hollow fiber bioreactor. Summary of the invention

[0005] The hollow fiber bioreactor placement rack in the prior art is an inclined placement plate, on which multiple hollow fiber bioreactors are placed to ensure that the hollow fiber bioreactors used for a single in vitro liver support system come from hollow fiber bioreactors cultured at the same time, to ensure that the support efficiency provided by the in vitro liver support system used for treatment is the same, and to avoid problems caused by differences.

[0006] However, it was found during the specific culture process that if a fixed tilt angle is maintained for the entire process of hollow fiber bioreactor culture, it is easy for the cells planted between the hollow fibers of the hollow fiber bioreactor to move rapidly to the bottom of the hollow fiber bioreactor under the action of gravity and the flow of culture fluid, resulting in uneven distribution of planted cells, which in turn leads to different states of uneven cell distribution in different hollow fiber bioreactors, resulting in insufficient growth uniformity of cell reactors of the same batch, which may affect the treatment effect during subsequent use. However, once the initial cell planting is completed, the tilt setting forms better liquid pressure due to the action of gravity, so that cells far away from the liquid input end can also get sufficient nutrient supply.

[0007] Therefore, for the initial cell planting stage of the hollow fiber bioreactor, it is best to place the hollow fiber bioreactor horizontally to ensure that the pumped liquid quickly drives the seeded cells to quickly diffuse throughout the entire hollow fiber bioreactor and adhere to the outer wall of the hollow fiber. Once evenly distributed, the hollow fiber bioreactor needs to be cultured in an inclined state to ensure that effective liquid pressure can be formed to promote cell reproduction. Therefore, a culture method and culture rack for a hollow fiber bioreactor with adjustable angles are required, so that it can meet the requirements of being in a horizontal state in the initial cell planting stage and maintaining the required inclined state in the growth culture stage. The above settings ensure that the cell reactor maintains different inclined states in different culture stages, so as to further optimize the homogeneity of each hollow fiber bioreactor after culture.

[0008] The present invention provides a hollow fiber bioreactor culture method.

[0009] One or more groups of hollow fiber bioreactors are arranged in parallel array, and the number of hollow fiber bioreactors in one group is the number that can be used simultaneously by the extracorporeal liver support system at one time;

[0010] The stage after the seed cells are injected is the initial planting stage, and all hollow fiber bioreactors are maintained parallel to the ground for cultivation;

[0011] The stage after the seed cells are uniformly attached to the fiber sidewalls of the hollow fiber bioreactor is the growth culture stage, and all hollow fiber bioreactors are maintained at an angle of 30-60 degrees to the ground for culture.

[0012] Furthermore, the seed cells are C3A cells used in an in vitro liver support system.

[0013] Furthermore, the initial planting phase lasts for 12-24 hours.

[0014] It should be noted that the seed cells are implanted between the hollow fibers, and the nutrient liquid flows through the cavity in the hollow fibers to provide nutrients to the cells disposed between the hollow fibers, so that the implanted seed cells adhere to the side walls of the hollow fibers, completing the initial implantation.

[0015] The growth culture stage is when the seed cells are attached to the side walls between the hollow fibers and need to continue to multiply in order to grow to a sufficient cell volume to meet the cell volume required by the in vitro liver support system. This stage is the growth culture stage, during which the seed cells are stabilized on the side walls between the hollow fibers.

[0016] Furthermore, the hollow fiber bioreactor is arranged on a culture rack with an adjustable and lockable angle. The culture rack is adjusted and locked to maintain a horizontal state so that the hollow fiber bioreactor completes the culture in the initial planting stage; the culture rack is adjusted and locked to maintain an inclined state so that the hollow fiber bioreactor completes the culture in the initial growth culture stage; by adjusting and locking the culture rack to maintain a horizontal or inclined state, all hollow fiber bioreactors are controlled to meet the needs of the initial culture stage and the growth culture stage.

[0017] Furthermore, the hollow fiber bioreactor is placed on the culture rack and fixed in a closed culture box. By placing it in the closed culture box, it can be ensured that the culture process is not affected by the external environment.

[0018] Furthermore, a liquid tank for supplying nutrients to the hollow fiber bioreactor is also provided in the incubator. The liquid tank is provided with a liquid inlet and a liquid outlet connected to the outside. The liquid inlet completes the supply of fresh liquid, and the liquid outlet completes the output of the liquid in the liquid tank. The liquid inlet and the liquid outlet maintain a balance. The liquid tank is also provided with an output pipe for supplying nutrients to the hollow fiber bioreactor and an input pipe for returning nutrients to the liquid tank through the hollow fiber bioreactor. The supply of nutrients is achieved through the rapid liquid circulation of the liquid tank and the hollow fiber reactor inside the incubator. Through the input of fresh liquid outside the liquid tank and the output of liquid in the liquid tank, the liquid in the liquid tank is satisfied to be a kind of liquid that can meet the needs of culturing nutrients in the hollow fiber bioreactor.

[0019] Furthermore, a culture rack with adjustable and lockable angles is arranged in the incubator, which includes a support frame fixed in the incubator and a culture plate with a hollow fiber bioreactor arranged thereon, the culture plate is arranged on the support frame, and the angles of the culture plate and the support frame can be adjusted and locked, so that the culture plate can reach a parallel state and an inclined state.

[0020] Technical Effects

[0021] By maintaining the horizontal state of the hollow fiber bioreactor during the initial planting stage, the uniform planting of seed cells can be completed more quickly and evenly. By maintaining the inclined state of the bioreactor during the growth and culture stage, it can be ensured that the liquid can effectively enter the end of the hollow fiber bioreactor, making the growth rate of the entire hollow fiber bioreactor more uniform, and ultimately achieving good homogeneity in the hollow fiber bioreactors cultured in the same group, avoiding risks during the treatment process.

[0022] By placing the hollow fiber bioreactor on a culture rack with adjustable and lockable angles, the hollow fiber bioreactor can be quickly and effectively adjusted between a horizontal state and an inclined state to meet culture needs.

[0023] The provision of the liquid tank can meet the supply of nutrients during the cultivation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic structural diagram of an implementation method of the culture rack of the present invention being arranged in an incubator;

[0025] Figure 2 It is a longitudinal cross-sectional structural schematic diagram of an embodiment of the culture rack of the present invention being arranged in a culture box;

[0026] Figure 3 It is a partial structural schematic diagram of the culture rack of the present invention being fixed to the culture box;

[0027] Figure 4 It is a schematic diagram of the structure of the culture rack of the present invention from the front side;

[0028] Figure 5 It is a schematic diagram of the structure of the rear side view of the culture rack of the present invention;

[0029] Figure 6 It is a partial enlarged structural schematic diagram of the support structure with a locking rod of the present invention;

[0030] Figure 7 This is a schematic diagram of the support structure of the present invention;

[0031] Figure 8 It is a schematic diagram of the support frame structure with horizontal blocking protrusions and inclined blocking protrusions of the present invention;

[0032] Fig. 9 It is a schematic diagram of the partial structure of the culture plate of the present invention;

[0033] Fig.10 This is a schematic diagram of the locking rod structure of the present invention;

[0034] Fig.11 This is a schematic diagram of the structure of a culture plate with a convenient adjustment structure but without an elastic clip according to the present invention;

[0035] Fig.12 It is a schematic diagram of the flow structure of the liquid circulation system 1 and the liquid circulation system 2 of the present invention;

[0036] Fig.13 It is a structural schematic diagram of the liquid tank of the present invention being arranged on the weighing structure;

[0037] Fig.14 It is a schematic diagram of the weighing structure of the present invention;

[0038] Fig.15 It is a schematic diagram of the longitudinal cross-sectional structure of the weighing structure of the present invention;

[0039] Description of main reference numerals

[0040] 1. Culture plate; 11. Top plate; 121. First plate; 122. Second plate; 13. Rotating column; 21. Adapter groove; 22. Guide groove; 31. First support structure; 32. Second support structure; 311. Vertical section; 312. Inclined section; 313. Setting part; 33. Transverse support structure; 41. Inclined blocking protrusion; 42. Horizontal blocking protrusion; 43. Extension plate; 44. Locking hole 1; 45. Locking hole 2; 46. Locking rod; 461. Locking section 1; 462. Locking section 2; 463. Protective ring; 464. Holding operation section; 51. Guide section; 52. Arc section; 53. Vertical section; 54. Horizontal plate; 61, longitudinal rod; 62, transverse rod; 63, short vertical rod; 7, incubator; 8, liquid tank; 81, liquid inlet; 82, liquid outlet; 83, output pipe; 84, input pipe; 9, hollow fiber bioreactor; 101, tank body; 102, tank cover; 1031, support rod; 1032, inclined support foot; 1033, fixing ring; 1034, connecting ring; 1035, abutment screw; 1041, center area; 1042, recessed ring; 1043, blocking ring; 1044, circular hole; 1045, containing structure; 1046, track one; 1047, track two; 1048, weighing module. DETAILED DESCRIPTION

[0041] Example 1

[0042] refer to Figure 1-12 The present invention provides a hollow fiber bioreactor 9 culture method,

[0043] One or more groups of hollow fiber bioreactors 9 are arranged in parallel array, and the number of hollow fiber bioreactors 9 in one group is the number that can be used simultaneously by the extracorporeal liver support system at one time;

[0044] In the initial planting stage after the seed cells are injected, all hollow fiber bioreactors 9 are maintained in an array state parallel to the ground for cultivation;

[0045] In the growth and cultivation stage after the seed cells are uniformly attached to the fiber sidewalls of the hollow fiber bioreactors 9, all hollow fiber bioreactors 9 are tilted at an angle of 30-60° to the ground. The seed cells are C3A cells.

[0046] The initial planting phase lasts for 12-24 hours. The hollow fiber bioreactor 9 is placed on a culture rack with an adjustable and lockable angle. By adjusting the culture rack to maintain a horizontal or tilted state, all hollow fiber bioreactors 9 are controlled to meet the needs of the initial culture phase and the growth culture phase.

[0047] The hollow fiber bioreactor 9 is placed on the culture rack and fixed in the closed culture box 7. By placing it in the closed culture box 7, it can be ensured that the culture process is not affected by the external environment.

[0048] refer to Figure 1 and Fig.12 A liquid tank for supplying nutrients to the hollow fiber bioreactor 9 is also provided in the incubator 7. The liquid tank is provided with a liquid inlet 81 and a liquid outlet 82 connected to the outside. The liquid inlet 81 completes the supply of fresh liquid, and the liquid outlet 82 completes the output of the liquid in the liquid tank 8. The liquid inlet amount of the liquid inlet 81 and the liquid outlet amount of the liquid outlet 82 maintain a balance. The liquid tank is also provided with an output pipe 83 for supplying nutrients to the hollow fiber bioreactor 9 and an input pipe 84 for returning to the liquid tank through the hollow fiber bioreactor 9. The supply of nutrients is achieved through the rapid liquid circulation of the liquid tank and the hollow fiber reactor inside the incubator 7. Through the input of fresh liquid outside the liquid tank and the output of liquid in the liquid tank 8, the liquid in the liquid tank 8 is satisfied to be a liquid that can meet the needs of culturing the nutrients in the hollow fiber bioreactor 9.

[0049] The specific culture process is as follows: first, one or more groups, 2-3 groups of hollow fiber bioreactors 9 are set on the culture rack, and the pipelines are connected in parallel. A liquid inlet and a liquid outlet are set on each hollow fiber bioreactor 9. The liquid inlet and the liquid outlet are distributed and connected with parallel pipelines. The parallel pipelines gather a main branch of the liquid inlet and a main branch of the liquid outlet. The main branch of the liquid inlet is connected to the output tank of the liquid tank. The main branch of the liquid outlet is connected to the input pipe 84 of the liquid tank, and the liquid inlet 81 on the liquid tank is connected to the external nutrient solution input system. The liquid outlet 82 on the liquid tank is connected to the waste liquid collection system. In this way, two liquid circulation systems are constructed, one is a liquid circulation system 1 for providing nutrition to the hollow fiber bioreactor 9 and the other is a liquid circulation system 2 for completing the liquid renewal in the liquid tank 8. Both the liquid circulation system 1 and the liquid circulation system 2 are provided with power pumps for providing power. The power pump of the liquid circulation system 1 can be a gear pump, and the power pump of the liquid circulation system 2 can be a peristaltic pump. The power pump is set to different speeds in different cultivation stages to meet the demand for nutrients in each stage.

[0050] In a more specific embodiment, a culture rack with adjustable and lockable angle is arranged in the incubator 7, which includes a support frame fixed in the incubator 7 and a culture plate 1 with a hollow fiber bioreactor 9, the culture plate 1 is arranged on the support frame, and the angle between the culture plate 1 and the support frame can be adjusted and locked.

[0051] A more specific implementation method is to refer to Figure 3-5; An elastic clamp for elastically clamping the hollow fiber bioreactor 9 is arranged on the culture rack, and each hollow fiber bioreactor 9 is provided with two elastic clamps, and the stability of the clamping can be ensured by the arrangement of the two elastic clamps. The elastic clamp includes two arc segments 52 passing through the center of the circle, and the first end of each arc segment 52 is connected to a guide segment 51 inclined toward the outside, and the second end of each arc segment 52 is connected to a vertical segment 53, and the two vertical segments 53 are connected by a cross plate 54, and the cross plate 54 is provided with a fixing hole for fixing the elastic clamp to the culture plate 1. Through the above arrangement, the entire hollow fiber bioreactor 9 can be clamped and fixed at the position of the culture plate 1, and the hollow fiber biological reaction will not be damaged due to over-tight clamping. In addition, because the arrangement of the inclined segment 312 can ensure that the hollow fiber bioreactor 9 is quickly introduced into the corresponding area of ​​the arc segment 52 to complete the fixation, and the arrangement of the vertical segment 53 can also reserve a certain buffer space below to avoid the risk of accidental contact of the hollow fiber bioreactor 9 directly contacting the culture plate 1.

[0052] refer to Figure 4 , Figure 5 and Fig. 9 In a more specific embodiment, the culture plate 1 includes a top plate 11 and surrounding plates, and the surrounding plates are arranged around the top plate 11. This arrangement can ensure that the culture plate 1 remains in a relatively light state, maintains the stability of the entire culture plate 1, and will not deform, thereby ensuring the stability of the positional relationship of the hollow fiber bioreactor 9 above it.

[0053] In a more specific embodiment, the four-sided plate includes a first plate 121 and a second plate 122, each of which has a rotating column 13 extending therefrom, and a rotating groove for accommodating the rotating column 13 is correspondingly provided on the support frame; further, the rotating groove includes a guide groove 22 with a larger opening at the top and an adapting groove 21 at the bottom adapted to the rotating column 13, so that the culture plate 1 can be conveniently placed on the support frame. Finally, a combination mode is formed in which the rotating column 13 can be adjusted after being placed in the rotating groove.

[0054] A more preferred embodiment is to refer to Figure 6-8 The support frame includes a first support structure 31, a second support structure 32, and a transverse support structure 33 connecting the first support structure 31 and the second support structure 32, and the support frame is fixed to the incubator 7 through the transverse support structure 33. A rotation groove is respectively provided on the first support structure 31 and the second support structure 32.

[0055] A more preferred embodiment is that the angle adjustment and locking structure is arranged on the first support structure 31 of the support frame and the corresponding first plate 121 or on the second support structure 32 or the second plate 122. The angle adjustment and locking structure can be a threaded rotating column 13 and a locking nut sleeved on the rotating column 13. The locking nut is rotated along the rotating column 13 so that the first support structure 31 and the corresponding first plate 121 or the second support structure 32 and the corresponding second plate 122 are in close contact to achieve locking. The reverse rotation releases the close contact locking relationship to adjust the angle. (Not shown in the figure) Further, refer to Figure 8 ; A horizontal maintenance blocking protrusion and a tilt maintenance blocking protrusion are respectively provided on both sides of the tilting section 312. When the culture plate 1 is adjusted to touch the horizontal maintenance blocking protrusion, the culture plate 1 reaches a horizontal state and is locked in the horizontal state by tightening the locking nut; when the culture plate 1 is adjusted to touch the tilt maintenance blocking protrusion, the culture plate 1 reaches a tilted state and is locked in the tilted state by tightening the locking nut. In this way, accurate and effective horizontal and tilt maintenance states can be quickly given.

[0056] Alternatively, refer to Figure 4-6 and Fig. 9 The angle adjustment and locking structure includes: an extension plate 43 is arranged on the first plate 121 and / or the second plate 122, two locking holes 44 for maintaining a horizontal state and an inclined state are arranged on the extension plate 43, a locking hole 45 matching with the locking hole 44 is arranged on one of the first supporting structure 31 and the second supporting structure 32, and a locking rod 46 is also arranged. When a locking hole 44 is aligned with the locking hole 45, the locking rod 46 is inserted into the hole to achieve locking of the angle of the culture plate 1.

[0057] A more preferred embodiment is as follows: the distances between the two locking holes 44 and the axis of the rotating column 13 are equal, and the locking hole 45 is arranged on the locking plate outside the rotating groove.

[0058] A more preferred embodiment is that the rotating column 13 is set in the middle position of the culture plate 1, the entire culture plate 1 is a plane-symmetrical figure, and the central axis of the rotating column 13 is on the longitudinal symmetry plane; this arrangement can ensure the arbitrariness of the setting of the culture plate 1 without specially distinguishing the up and down directions.

[0059] A more preferred embodiment is to set two sets of locking holes 44 on the first plate 121 and the second plate 122 with the longitudinal symmetry plane as the mirror plane. This arrangement can ensure that any placement method can find an effective horizontal maintenance state and an inclined maintenance state.

[0060] A more preferred embodiment is to refer to Figure 6; The first supporting structure 31 and the second supporting structure 32 both include a vertical section 311 and an inclined section 312, and the inclined section 312 is inclined upward; the vertical section 311 is used to connect the transverse supporting structure 33, and a rotation groove is arranged on the inclined section 312. By setting the inclined section 312, an effective rotation space can be provided for the rotation of the culture plate 1. In addition, the inclined section 312 can introduce the entire culture plate 1 into the space above, ensuring that it can be effectively fixed on the side wall and introduced into the space above, which can also increase the ease of operation.

[0061] A more preferred embodiment is to refer to Figure 6 ; The setting portion 313 of the locking hole 2 45 is tilted downward on the inclined section 312 of one of the first supporting structure 31 and the second supporting structure 32, and the downward tilt angle is the same as the angle at which the culture plate 1 maintains the inclined state. This setting can ensure that when the culture plate 1 and the setting portion 313 of the locking hole 2 45 are parallel as a whole, in order to maintain the locking hole 1 44 and the locking hole 2 45 in the inclined state in the aligned state, the locking rod 46 is inserted in this way. The aligned state can be found very conveniently through the above setting.

[0062] A more preferred embodiment is to refer to Fig.10 The diameter of the locking hole 44 is smaller than that of the locking hole 45. A locking section 461 adapted to the locking hole 44 and a locking section 462 adapted to the locking hole 45 are provided on the locking rod 46. The stepped setting can ensure that the position relationship after locking is more stable.

[0063] In a more preferred embodiment, the first end of the locking segment 462 is connected to the locking segment 1 461 , and the second end of the locking segment 462 is provided with a gripping operation segment 464 , so that the convenience of operation is maintained by gripping the operation segment 464 .

[0064] A more preferred implementation is to provide a protective ring 463 with a diameter larger than that of the gripping operation segment 464 at both ends of the gripping operation segment 464. By providing the protective ring 463, it can be ensured that during operation, the hand is confined between the protective rings 463, avoiding the hand from directly touching the inclined segment 312 during operation, thereby ensuring the safety of the operation.

[0065] A more preferred embodiment is to set a convenient adjustment structure on the culture plate 1, and the convenient adjustment structure includes a rod-shaped structure symmetrically set on the culture plate 1. By setting the rod-shaped structure, one person can control the hole action and one person can complete the jack locking action.

[0066] A more preferred embodiment is that the convenient adjustment structure includes two parallel longitudinal rods 61 and a transverse rod 62 connecting the two longitudinal rods 61. The transverse rod 62 is arranged in the peripheral space of the culture plate 1. The arrangement of the transverse rod 62 can make it easier to implement the hole-matching action.

[0067] A more preferred embodiment is that two short vertical rods 63 are set on both side edges of the longitudinal rod 61, and the two short vertical rods 63 form a group. A group of short vertical rods 63 constructs a hanging space. By setting up the hanging space, the culture plate 1 can be hung and fixed to a specific position before and after use, saving space.

[0068] When finally used, the rotating column 13 of the culture plate 1 is set in the rotating groove, and the angle adjustment and locking structure is used to complete the locking of maintaining the horizontal state and the inclined state. The two angle adjustment and locking structures can achieve state maintenance and conversion, ensuring effective cultivation of the hollow fiber bioreactor 9.

[0069] Example 2

[0070] A hollow fiber bioreactor 9 culture system, comprising a sealed box, an angle-adjustable and lockable culture rack in the box, and a liquid tank system, the liquid tank system comprising

[0071] The liquid tank includes a tank body 101 and a tank cover 102. The tank cover 102 is provided with various openings for arranging various connectors. The volume of the tank body 101 is sufficient to meet the volume of the nutrient solution required for culturing at least one group of hollow fiber bioreactors 9. The connector includes a liquid inlet connector 1 for

[0072] The weighing structure is used to weigh the weight of the liquid in the liquid tank and the tank body 101 in real time to obtain the actual weight value;

[0073] The control structure receives the actual weight value of the weighing structure in real time and compares it with the theoretical weight value. The theoretical weight value is the sum of the weight of the tank body 101 and the weight of the liquid required for culturing the required number of hollow fiber bioreactors 9, and there is a fluctuation value of the theoretical weight. When the actual weight value is the difference between the theoretical weight value and the fluctuation value, the control structure controls the input of nutrient solution into the tank body 101. When the actual weight value is the sum of the theoretical weight value and the fluctuation value, the control structure controls the liquid in the tank body 101 to flow out, and the process repeats continuously.

[0074] In a more preferred embodiment, the fluctuation value ranges from 50 to 100 g, which can meet the needs of culture medium fluctuations without causing any impact.

[0075] In a more preferred embodiment, the time required to complete a round of liquid replacement is adjusted by adjusting the speed of inputting and outputting the nutrient solution into and out of the tank body 101 according to the nutritional components in the tank body 101. In this way, the rate of completing a round of liquid replacement is adjusted.

[0076] A more preferred implementation manner is to carry out real-time weighing throughout the entire process, and in order to ensure the stability of the overall structure during weighing, a support frame is provided outside the liquid tank, and inclined support feet 1032 are provided at the bottom of the support frame. By providing the inclined support feet 1032, the stability of the liquid tank when placed can be increased by increasing the supporting area, and the weight of the support frame is also within the weighing range during weighing, as part of the initial weight of the liquid tank.

[0077] In a more preferred embodiment, the support frame includes a fixing ring 1033 arranged on the top, the inner diameter of the fixing ring 1033 is smaller than the outer diameter of the edge of the tank body 101, and the diameter of the fixing ring 1033 is equal to the outer diameter of the tank cover 102, and a sealing ring is also arranged on the tank body 101; the fixing ring 1033 and the tank cover 102 are fixed together, and the sealing ring realizes the sealing of the tank cover 102 and the tank body 101; in this way, the support frame and the tank body 101 are stably combined together to form a whole that can be weighed together. In this way, the stability of the liquid tank when placed, as well as the effectiveness and operability of the weighing can be maximized.

[0078] In a more preferred embodiment, corresponding fixing holes are set on the fixing ring 1033 and the tank cover 102, and locking screws and nuts are set. The locking screws are passed through the fixing holes, and the positions of the fixing ring 1033 and the tank cover 102 are locked by the screws and nuts.

[0079] In a more preferred embodiment, the support frame includes multiple support rods 1031, an inclined rod-shaped support foot is arranged under each support rod 1031, a fixing ring 1033 is arranged above the support rod 1031, and a connecting ring 1034 fixed to the inner side of the multiple support rods 1031 is arranged under the support rod 1031. The upper fixing ring 1033 and the lower connecting ring 1034 are combined with the support rod 1031 to construct a stable support frame.

[0080] In a more preferred embodiment, abutment screws 1035 are further provided at the bottom of each support rod 1031 for abutting the tank body 101 at the bottom, so that the support frame and the tank body 101 form a more stable structure.

[0081] In a more preferred embodiment, the weighing structure includes a weighing module 1048, on which a receiving plate is arranged for receiving the liquid tank. The receiving plate is arranged as a circular plate. A central area 1041 and a recessed ring 1042 which is lower than the central area 1041 and is arranged on the circular plate for receiving the supporting feet of the supporting frame. A blocking ring 1043 which is arranged perpendicular to the recessed ring 1042 is also arranged on the outer side of the recessed ring 1042; the supporting feet are arranged in the recessed ring 1042, and the bottom of the tank body 101 is arranged in the central area 1041. In this way, the liquid tank can be well fixed on the receiving plate.

[0082] In a more preferred embodiment, in order to prevent the receiving plate from shaking during use, a circular hole 1044 is provided with the same outer diameter as the blocking ring 1043, and a receiving plate is provided inside the circular hole 1044, and the side wall of the circular hole 1044 limits the shaking of the receiving plate; through this arrangement, the stability of the entire liquid tank during use is further maintained by maintaining the stability of the receiving plate.

[0083] In a more preferred embodiment, a containing structure 1045 for accommodating a weighing structure is provided, a cavity is provided in the containing structure 1045, a bearing structure is provided in the cavity, and a circular hole 1044 which is consistent with the outer diameter of the blocking ring 1043 is provided on the containing structure 1045. Through this arrangement, the weighing structure can be uniformly fixed or movably set to the bottom of the incubator 7.

[0084] In a more preferred embodiment, the containing structure 1045 is configured as a structure capable of translation; a track groove equal to the width of the containing structure 1045 is provided at the bottom of the incubator 7, the track groove is the groove body in the middle of the track structure, and a track 1046 is provided on both side walls of the track structure, and a track 2 1047 adapted to the track 1 1046 is correspondingly provided on the containing structure 1045, and the purpose of moving the containing structure 1045 with the liquid tank out of the incubator 7 is achieved by the relative movement of the track 1046 with respect to the track 2 1047.

[0085] The technical solutions in the embodiments of the present invention are clearly and completely described above through specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation methods. In the absence of conflict, the above embodiments and features in the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A hollow fiber bioreactor culture method, characterized in that: One or more groups of hollow fiber bioreactors are arranged in parallel array, and the number of hollow fiber bioreactors in one group is the number that can be used simultaneously by the extracorporeal liver support system at one time; The stage after the seed cells are injected is the initial planting stage, and all hollow fiber bioreactors are maintained in a parallel state with the ground for cultivation; The stage after the seed cells are uniformly attached to the fiber sidewalls of the hollow fiber bioreactor is the growth culture stage, and all hollow fiber bioreactors are maintained at an angle of 30-60 degrees to the ground for culture.

2. The hollow fiber bioreactor culture method according to claim 1, characterized in that: The initial planting phase lasts 12-24 hours.

3. The hollow fiber bioreactor culture method according to claim 1, characterized in that: The hollow fiber bioreactor is set on a culture rack with an adjustable and lockable angle. The culture rack is adjusted and locked to maintain a horizontal state, so that the hollow fiber bioreactor completes the culture in the initial planting stage; the culture rack is adjusted and locked to maintain an inclined state, so that the hollow fiber bioreactor completes the culture in the initial growth culture stage. 4 . The hollow fiber bioreactor culture method according to claim 3 , wherein the hollow fiber bioreactor is placed on a culture rack and then fixed in a closed culture box for culture.

5. The hollow fiber bioreactor culture method according to claim 4, characterized in that: A liquid tank for supplying nutrients to the hollow fiber bioreactor is also provided in the incubator. A liquid inlet and a liquid outlet connected to the outside are provided on the liquid tank. The liquid inlet completes the supply of fresh liquid, and the liquid outlet completes the output of the liquid in the liquid tank. The liquid inlet amount and the liquid outlet amount of the liquid inlet are balanced. The liquid tank is also provided with an output pipe for supplying nutrients to the hollow fiber bioreactor and an input pipe for returning nutrients to the liquid tank through the hollow fiber bioreactor. Two liquid circulation systems are constructed, one is a liquid circulation system 1 for providing nutrition to the hollow fiber bioreactor and the other is a liquid circulation system 2 for completing the renewal of the liquid in the liquid tank. Both the liquid circulation system 1 and the liquid circulation system 2 are provided with a power pump for providing power. Preferably, an elastic clamp for elastically clamping the hollow fiber bioreactor is provided on the culture rack, and each hollow fiber bioreactor is provided with two elastic clamps, and the stability of clamping can be ensured by the provision of the two elastic clamps; Preferably, the elastic clip includes two arc segments passing through the center of the circle, the first end of each arc segment is connected to a guide segment inclined toward the outside, the second end of each arc segment is connected to a vertical segment, the two vertical segments are connected by a cross plate, and the cross plate is provided with a fixing hole for fixing the elastic clip to the culture plate.

6. The hollow fiber bioreactor culture method according to claim 4, characterized in that: The angle-adjustable and lockable culture rack is arranged in the incubator, which includes a support frame fixed in the incubator and a culture plate with a hollow fiber bioreactor. The culture plate is arranged on the support frame, and an angle adjustment and locking structure is also arranged for adjusting and locking the angle between the culture plate and the support frame.

7. The hollow fiber bioreactor culture method according to claim 6, characterized in that: The culture plate includes a top plate and surrounding plates, and the surrounding plates are arranged around the top plate; the surrounding plates include a first plate and a second plate, and a rotating column extends from each of the first plate and the second plate, and a rotating groove for accommodating the rotating column is correspondingly arranged on the support frame; Preferably, the rotating groove includes a guide groove with a larger opening at the top and an adapting groove at the bottom adapted to the rotating column, so that the culture plate can be placed on the support frame very conveniently; Preferably, the support frame includes a first support structure, a second support structure, and a transverse support structure connecting the first support structure and the second support structure. The support frame is fixed to the incubator through the transverse support structure, and a rotating groove is respectively provided on the first support structure and the second support structure.

8. The hollow fiber bioreactor culture method according to claim 6, characterized in that: The angle adjustment and locking structure is arranged on the first support structure of the support frame and the corresponding first plate or on the second support structure or the second plate. The angle adjustment and locking structure can be a threaded rotating column and a locking nut sleeved on the rotating column. The locking nut is rotated along the rotating column so that the first support structure and the corresponding first plate or the second support structure and the corresponding second plate are in close contact to achieve locking. The close contact locking relationship is released by reverse rotation to adjust the angle. Alternatively, an extension plate is provided on the first plate and / or the second plate, two locking holes 1 for maintaining a horizontal state and a tilted state are provided on the extension plate, a locking hole 2 cooperating with the locking hole 1 is provided on one of the first supporting structure and the second supporting structure, and a locking rod is provided, and when one locking hole 1 is aligned with the locking hole 2, the locking rod is inserted into the hole to achieve locking of the angle of the culture plate; the distances between the two locking holes 1 and the axis of the rotating column are equal, and the locking hole 2 is provided on the locking plate outside the rotating groove, the rotating column is provided in the middle position of the culture plate, the entire culture plate is a plane-symmetrical figure, and the central axis of the rotating column is on the longitudinal symmetry plane; two groups of locking holes 1 are provided on the first plate and the second plate with the longitudinal symmetry plane as a mirror plane; Preferably, the first supporting structure and the second supporting structure both include a vertical section and an inclined section, the inclined section is inclined upward; the vertical section is used to connect the transverse supporting structure, and a rotation groove is provided on the inclined section; the setting part of the locking hole 2 which is inclined downward on the inclined section of one of the first supporting structure and the second supporting structure has the same downward inclined angle as the angle at which the culture plate maintains its inclined state, Preferably, the diameter of the locking hole 1 is smaller than that of the locking hole 2, and a locking section 1 adapted to the locking hole 1 and a locking section 2 adapted to the locking hole 2 are provided on the locking rod, and the stepped arrangement can ensure that the position relationship after locking is more stable; Preferably, the first end of the locking segment 2 is connected to the locking segment 1, and the second end of the locking segment 2 is provided with a gripping operation segment.

9. A culture rack with adjustable and lockable angle for hollow fiber bioreactor culture, characterized in that: It is arranged in an incubator, and includes a support frame fixed in the incubator and a culture plate on which a hollow fiber bioreactor is arranged, the culture plate is arranged on the support frame, and an angle adjustment and locking structure is also arranged, which is used to adjust and lock the angle between the culture plate and the support frame; the culture plate includes a top plate and surrounding plates, and the surrounding plates are arranged around the top plate; the surrounding plates include a first plate and a second plate, and a rotating column extends from each of the first plate and the second plate; the support frame includes a first supporting structure, a second supporting structure, and also includes a transverse supporting structure connecting the first supporting structure and the second supporting structure, and the support frame is fixed to the incubator through the transverse supporting structure, and a rotating groove is respectively arranged on the first supporting structure and the second supporting structure; The angle adjustment and locking structure is arranged on the first supporting structure of the supporting frame and the corresponding first plate or on the second supporting structure or the second plate. The angle adjustment and locking structure may be a threaded rotating column and a locking nut sleeved on the rotating column, and the locking nut is rotated along the rotating column so that the first support structure and the corresponding first plate or the second support structure and the corresponding second plate are in close contact to achieve locking, and the angle adjustment is achieved by reverse rotation to release the close contact locking relationship; Alternatively, an extension plate is arranged on the first plate and / or the second plate, two locking holes 1 for maintaining a horizontal state and a tilted state are arranged on the extension plate, a locking hole 2 cooperating with the locking hole 1 is arranged on one of the first supporting structure and the second supporting structure, and a locking rod is also arranged. When the locking hole 1 is aligned with the locking hole 2, the locking rod is inserted into the hole to lock the angle of the culture plate.

10. A hollow fiber bioreactor culture system, comprising a sealed box, a culture rack with adjustable and lockable angles for implementing the culture method according to any one of claims 1 to 8, and a liquid tank system, the liquid tank system comprising The liquid tank comprises a tank body and a tank cover. Various openings are arranged on the tank cover for arranging various connectors. The volume of the tank body is sufficient to meet the volume of the nutrient solution required for culturing at least one group of hollow fiber bioreactors. The connectors include a liquid inlet connector 1 for A weighing structure is used to weigh the liquid tank and the liquid in the tank in real time to obtain the actual weight value; The control structure receives the actual weight value of the weighing structure in real time and compares it with the theoretical weight value. The theoretical weight value is the sum of the tank weight and the liquid weight required for the hollow fiber bioreactor to culture the required number of groups. There is a fluctuation value of the theoretical weight. When the actual weight value is the difference between the theoretical weight value and the fluctuation value, the control structure The control structure controls the input of nutrient solution into the tank. When the actual weight value is the sum of the theoretical weight value and the fluctuation value, The control structure controls the liquid in the tank to flow out, repeatedly.

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