Microcarrier adherent cell pancreatin digestion device for bioreactor
By replacing the original trypsin digester with 5L and 20L reaction flasks, and combining a multi-channel tubing design with cell observation tubes, the problems of high cost and cell damage in existing technologies have been solved, achieving more efficient cell digestion and a more stable production process.
Patent Information
- Application Number
- CN202520332489.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2026-02-06
- Estimated Expiration
- 2035-02-28
AI Technical Summary
In the existing technology, the bioreactor pancreatic enzyme digestion process in the production of Sabin strain inactivated polio vaccine is costly and complex to operate. Furthermore, the original pancreatic enzyme digester damages cells and affects cell growth in the next stage.
A 5L reaction flask replaces the cell digestion container of a 7L bioreactor, and a 20L reaction flask replaces the cell digestion container of a 75L bioreactor. Combined with a multi-channel pipeline design and cell observation tubes, the operation is simplified and damage to cells is reduced.
It reduced costs, simplified operation, improved the performance and stability of digestion equipment, increased the uniformity of cell suspension, improved cell recovery rate and cell adhesion rate, and enhanced work efficiency and product yield.
Smart Images
Figure CN223879763U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model belongs to cell trypsin digestion technical field, concretely relates to a kind of biological reactor microcarrier adherent cell trypsin digestion device. BACKGROUND
[0002] In prior art, trypsin digestion of biological reactor in Sabin strain poliomyelitis inactivated vaccine production is a key step.Vero cell is separated from microcarrier by trypsin digestion of 7L biological reactor and 75L biological reactor.
[0003] In prior art, trypsin digestion of biological reactor of Sabin strain poliomyelitis inactivated vaccine uses 1 dish-shaped trypsin digester as cell digestion container of 7L biological reactor to 75L biological reactor, and uses 3 butterfly-shaped trypsin digesters as cell digestion container of 75L biological reactor to 550L biological reactor, which is high in cost, and the original trypsin digester pipeline is more, and the digestion operation is more complex, the bottom filter screen of trypsin digester and the oscillator used when dispersing cell can cause certain damage to cell, increase the damage of external force to cell, and be not conducive to the growth of cell in next level. UTILITY MODEL CONTENT
[0004] In order to realize the solution to the above problems, the utility model takes the following technical solutions:
[0005] A kind of biological reactor microcarrier adherent cell trypsin digestion device, comprising:
[0006] Biological reactor;
[0007] Trypsin digestion unit connected with the biological reactor, the trypsin digestion unit is connected with the bottom valve of the biological reactor by second connecting pipe to carry out biological reactor trypsin digestion microcarrier adherent cell.
[0008] Further, the trypsin digestion unit includes trypsin digestion reaction bottle, washing liquid container, sampling container, transfer container, trypsin container, waste liquid collection container and termination liquid container;The first connecting pipe is connected on the trypsin digestion reaction bottle;The bottom valve in the bottom of the biological reactor is connected with the second connecting pipe;Five-way pipe is arranged between the first connecting pipe and the second connecting pipe;The first interface of the first connecting pipe is connected with five-way pipe;The second interface of the second connecting pipe is connected with the five-way pipe;The third interface of the five-way pipe is connected with the washing liquid container by the third connecting pipe;The fourth interface of the five-way pipe is connected with the trypsin container by the fourth connecting pipe;The fifth interface of the five-way pipe is connected with the transfer container by the fifth connecting pipe;
[0009] The sixth connecting pipe is connected to the trypsin digestion reaction bottle; the seventh connecting pipe is connected to the waste liquid collecting container; a four-way pipe is arranged between the seventh connecting pipe and the sixth connecting pipe; one end of the seventh connecting pipe away from the waste liquid collecting container is connected to a first interface of the four-way pipe; and one end of the sixth connecting pipe away from the trypsin digestion reaction bottle is connected to a second interface of the four-way pipe.
[0010] An eighth connecting pipe is arranged between a third interface of the four-way pipe and the transfer container; and a cell observation pipe is connected to the eighth connecting pipe.
[0011] A ninth connecting pipe is arranged between a fourth interface of the four-way pipe and the termination liquid containing container.
[0012] The second connecting pipe, the third connecting pipe, the fourth connecting pipe, the fifth connecting pipe, the sixth connecting pipe, the seventh connecting pipe, the eighth connecting pipe and the ninth connecting pipe are all provided with a pipe clamping device.
[0013] Further, a first end of the first connecting pipe is connected to a first interface of the five-way pipe, and a second end of the first connecting pipe is connected to the trypsin digestion reaction bottle and extends into the trypsin digestion reaction bottle by at least two-thirds.
[0014] Further, a first end of the sixth connecting pipe is connected to a second interface of the four-way pipe, and a second end of the sixth connecting pipe is connected to the trypsin digestion reaction bottle; the second end of the sixth connecting pipe extends into the trypsin digestion reaction bottle by at least two-thirds, and the second end of the sixth connecting pipe is bent and in contact with a side wall of the trypsin digestion reaction bottle.
[0015] Further, a bottom pipe is arranged in the trypsin digestion reaction bottle; a first end of the bottom pipe is connected to a first interface of a three-way pipe, and a second end of the bottom pipe is connected to the trypsin digestion reaction bottle and extends into the trypsin digestion reaction bottle to contact a bottom of the trypsin digestion reaction bottle; a sampling container is arranged on the three-way pipe; a tenth connecting pipe is arranged between the sampling container and a second interface of the three-way pipe to make the trypsin digestion reaction bottle communicate with the sampling container; and the tenth connecting pipe is provided with the pipe clamping device.
[0016] Further, an air pipe is arranged on the trypsin digestion reaction bottle; a first end of the air pipe is connected to an air filter, and a second end of the air pipe is connected to the trypsin digestion reaction bottle and extends into the trypsin digestion reaction bottle by at least one-tenth.
[0017] Further, an inner diameter of the cell observation pipe is 80 mm, and a wall thickness of the cell observation pipe is 2 mm.
[0018] Beneficial effects:
[0019] The utility model provides a kind of bioreactor microcarrier adherent cell trypsin digestion device, with 5L reaction bottle replaces previous dish-shaped trypsin digester as 7L bioreactor is transferred 75L bioreactor cell digestion container, with 20L reaction bottle replaces 3 dish-shaped trypsin digester as 75L bioreactor is transferred 550L bioreactor cell digestion container, the device has following advantages:
[0020] (1) from 1-3 several hundred thousand original dish-shaped trypsin digester is improved to several ten thousand 5L bottle and 20L bottle saving money, reduce cost;Original trypsin digester pipeline is more, and digestion operation is more complex, and the installation of 5L bottle and 20L bottle is simpler than original trypsin digester, and operation is more convenient, reduces preparation link, and reduces pollution risk energy saving and consumption reduction, effectively improves digestion equipment performance and stability;
[0021] (2) cell suspension after digestion is directly contained by 5L or 20L bottle, also increase the uniformity of cell digestion suspension;In addition, the bottom filter screen of original trypsin digester and the oscillator used when dispersing cell have certain harm to cell, and using 5L bottle and 20L bottle digestion reduces the harm of external force to cell, and it is more beneficial to the growth of cell in next level;
[0022] (3) after using 5L bottle and 20L bottle as cell digestion container for Sabin strain poliomyelitis inactivated vaccine cell digestion, the cell digestion state is observed in real time through cell observation tube, and digestion cell is terminated in time, and cell counting data statistics and analysis are carried out, the recovery rate of digestion cell is calculated, cell adhesion rate in next level bioreactor and cell growth condition are observed, and the culture parameter data of production batch using improved dish-shaped trypsin digester for cell digestion are compared, recovery rate and adhesion rate are stably increased, and work efficiency and product yield are preferably improved. BRIEF DESCRIPTION OF DRAWINGS
[0023] Fig. 1 It is partial structure schematic drawing of the utility model bioreactor microcarrier adherent cell trypsin digestion device;
[0024] Fig. 2 It is whole structure schematic drawing of the utility model bioreactor microcarrier adherent cell trypsin digestion device and bioreactor connection;
[0025] Wherein, 1, bioreactor; 2, bottom valve; 3, trypsin digestion reaction bottle; 4, waste liquid collection container; 5, termination liquid container; 6, washing liquid container; 7, sampling container; 8, transfer container; 9, trypsin container; 10, cell observation tube; 11, first connecting pipe; 12, second connecting pipe; 13, third connecting pipe; 14, fourth connecting pipe; 15, fifth connecting pipe; 16, sixth connecting pipe; 17, seventh connecting pipe; 18, eighth connecting pipe; 19, ninth connecting pipe; 20, five-way pipe; 21, four-way pipe; 22, three-way pipe; 23, bottom pipe; 24, vent pipe; 25, tenth connecting pipe. DETAILED DESCRIPTION
[0026] To make the objects, technical solutions, and advantages of the embodiments of the present application clearer, the following will be a clear and complete description of the technical solutions in the embodiments of the present application in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.
[0027] In the description of the present application, it should be noted that the orientations or positional relationships indicated by the terms "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the orientations or positional relationships shown in the drawings, or the orientations or positional relationships in which the product of the present application is usually placed, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third", etc. are only used for differentiation in description and cannot be understood as indicating or implying relative importance. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.
[0028] In the description of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "provided" and "connected" should be understood broadly, for example, can be fixedly connected, can be detachably connected, or integrally connected; can be mechanically connected, or electrically connected. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present application according to the specific circumstances.
[0029] The embodiments of this utility model are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this utility model, and should not be construed as limiting this utility model.
[0030] Example 1
[0031] like Figs. 1-2 As shown, a bioreactor microcarrier adherent cell trypsin digestion device includes:
[0032] The pancreatic enzyme digestion unit is connected to the bioreactor. The pancreatic enzyme digestion unit is connected to the bottom valve 2 of the bioreactor through a thermoplastic tube to perform pancreatic enzyme digestion of microcarrier adherent cells in the bioreactor.
[0033] In this embodiment, the pancreatic enzyme digestion unit includes a pancreatic enzyme digestion reaction bottle 3, a washing solution container 6, a sampling container 7, a transfer container 8, a pancreatic enzyme container 9, a waste liquid collection container 4, and a termination solution container 5. A first connecting pipe 11 is connected to the pancreatic enzyme digestion reaction bottle 3. The bottom valve 2 at the bottom of the bioreactor 1 is connected to a second connecting pipe 12. A five-way connector 20 is provided between the first connecting pipe 11 and the second connecting pipe 12. The first connecting pipe 11 is connected to the first interface of the five-way connector 20. The second connecting pipe 12 is connected to the second interface of the five-way connector 20. The third interface of the five-way connector 20 is connected to the washing solution container 6 through a third connecting pipe 13. The fourth interface of the five-way connector 20 is connected to the pancreatic enzyme container 9 through a fourth connecting pipe 14. The fifth interface of the five-way connector 20 is connected to the transfer container 8 through a fifth connecting pipe 15.
[0034] A sixth connecting tube 16 is connected to the pancreatic enzyme digestion reaction flask 3; a seventh connecting tube 17 is connected to the waste liquid collection container 4; a four-way tube 21 is provided between the seventh connecting tube 17 and the sixth connecting tube 16; the end of the seventh connecting tube 17 away from the waste liquid collection container 4 is connected to the first interface of the four-way tube 21; the end of the sixth connecting tube 16 away from the pancreatic enzyme digestion reaction flask 3 is connected to the second interface of the four-way tube 21.
[0035] The third port of the four-way tube 21 and the transfer container 8 are connected by the eighth connecting tube 18; the cell observation tube 10 is connected to the eighth connecting tube 18.
[0036] The fourth port of the four-way pipe 21 and the stop liquid container 5 are connected by the ninth connecting pipe 19.
[0037] Pipe clamps are installed on the second connecting pipe 12, the third connecting pipe 13, the fourth connecting pipe 14, the fifth connecting pipe 15, the sixth connecting pipe 16, the seventh connecting pipe 17, the eighth connecting pipe 18, and the ninth connecting pipe 19.
[0038] Through the technical scheme, the trypsin digestion reaction bottle 3 selects a 20L reaction bottle as a trypsin digestion reaction container, replaces three butterfly-shaped trypsin digesters as cell digestion containers of a 75L bioreactor to a 550L bioreactor, saves funds and reduces costs from tens of thousands of original butterfly-shaped trypsin digesters to tens of thousands of 20L bottles, the installation of the 20L bottle is simpler than that of the original trypsin digester, the operation is more convenient, the preparation link is reduced, the pollution risk is reduced, energy is saved, consumption is reduced, and the performance and stability of the digestion equipment are effectively improved.
[0039] In the embodiment, the first end of the first connecting pipe 11 is connected with the first interface of the five-way pipe 20, the second end is connected with the trypsin digestion reaction bottle 3, and at least two-thirds of the second end extends into the trypsin digestion reaction bottle 3.
[0040] In the embodiment, the first end of the sixth connecting pipe 16 is connected with the second interface of the four-way pipe 21, and the second end is connected with the trypsin digestion reaction bottle 3; the end of the second end of the sixth connecting pipe 16 extends into the trypsin digestion reaction bottle 3 by at least two-thirds, and the end of the second end is bent and in contact with the side wall (not the bottom) of the trypsin digestion reaction bottle 3.
[0041] In the embodiment, the trypsin digestion reaction bottle 3 is provided with a bottom pipe 23; the first end of the bottom pipe 23 is connected with the first interface of the three-way pipe 22, the second end is connected with the trypsin digestion reaction bottle 3, and extends into the trypsin digestion reaction bottle 3 and is in contact with the bottom of the trypsin digestion reaction bottle 3; the three-way pipe 22 is provided with a sampling container 7; the sampling container 7 and the second interface of the three-way pipe 22 are connected through a tenth connecting pipe 25, so that the trypsin digestion reaction bottle 3 is in communication with the sampling container 7; the tenth connecting pipe 25 is provided with an eighth valve.
[0042] In the embodiment, the trypsin digestion reaction bottle 3 is provided with a vent pipe 24; the first end of the vent pipe 24 is connected with an air filter, the second end is connected with the trypsin digestion reaction bottle 3, and at least one-tenth of the second end extends into the trypsin digestion reaction bottle 3.
[0043] In the embodiment, the cell observation pipe 10 is a hollow glass pipe, and preferably, the inner diameter is 80mm and the wall thickness is 2mm.
[0044] The utility model provides a kind of biological reactor microcarrier adherent cell trypsin digestion device, and the biological reactor microcarrier adherent cell trypsin digestion method of the device is as follows:
[0045] S1, the trypsin digestion reaction bottle 3 as the main body, in the sterile environment of C+A trypsin digestion reaction bottle 3, the termination liquid container 5, waste liquid container, trypsin container 9, washing liquid container 6, sampling container 7 and other containers connected into a closed system, the above separate containers are through the off-line moist heat sterilization method to achieve sterile state;
[0046] S2, with sterile pipe connecting machine trypsin digestion reaction bottle 3 and the bottom valve 2 of the bioreactor 1 through the heat plastic tube connection;
[0047] S3, the bioreactor cell microcarrier suspension is discharged through the filter tube to the volume that the trypsin digestion reaction bottle 3 can accommodate, and then discharged to the trypsin digestion reaction bottle 3 through the pipeline connected with the bottom valve 2 of the bioreactor 1, and the microcarrier is completely settled;
[0048] S4, the supernatant is discharged through the sixth connecting pipe 16 to the waste liquid container, the first connecting pipe 11 is installed in the trypsin digestion reaction bottle 3 above the position of the microcarrier settlement after collection 1-2cm position, and the loss caused by the floating of the microcarrier is avoided as far as possible when discharging;
[0049] S5, after the supernatant is discharged, the washing liquid in the washing liquid container 6 is injected into the trypsin digestion reaction bottle 3 through positive pressure, and then mixed and settled;
[0050] S6, the operation steps of S3 to S5 are repeated 2-3 times, and the serum in the cell microcarrier suspension is washed as far as possible, and finally the waste liquid is discharged;
[0051] S7, the trypsin digestion solution is added through the trypsin container 9, and then mixed, and a part of the cell microcarrier suspension is discharged through the observation tube to the transfer container 8 every N minutes (in this embodiment, preferably 3 minutes) through positive pressure, the observation tube is placed under the microscope, and the whole process of cell digestion from the microcarrier can be clearly observed, until the cell is completely digested from the microcarrier, the termination liquid is added into the trypsin digestion reaction bottle 3 through the ninth connecting pipe 19, the four-way pipe 21 and the sixth connecting pipe 16, and the digestion is terminated.
[0052] Among them, the three-way pipe 22, the four-way pipe 21 and the five-way pipe 20 are preferably made of stainless steel.
[0053] Through the technical scheme, the well-digested cell suspension is directly contained in the 20L reaction bottle, and the uniformity of the cell digestion suspension is also increased; in addition, the bottom filter screen of the original trypsin digester and the oscillator used when dispersing the cells have certain damage to the cells, and the use of the 20L bottle for digestion reduces the damage of external force to the cells, and is more beneficial to the growth of the cells in the next stage; after the 20L bottle is used as the cell digestion container for the cell digestion of the Sabin strain poliomyelitis inactivated vaccine, the cell digestion state is observed in real time through the cell observation tube, the cell digestion is terminated in time, the cell counting data are statistically analyzed, the recovery rate of the digested cells is calculated, the cell adhesion rate and the cell growth in the next stage of the bioreactor are observed, and the culture parameter data of the production batch using the improved butterfly-shaped trypsin digester for cell digestion are compared; the recovery rate and the adhesion rate are stably increased, and the work efficiency and the product yield are preferably improved.
[0054] The device for trypsin digestion of microcarrier adherent cells in a bioreactor is used for the trypsin digestion of microcarrier adherent cells in a bioreactor, the process is simple, the digestion process can be observed in real time, cell damage and loss caused by long trypsin action are avoided, the screen is not jammed when the cell surface is cleaned, and the recovery rate of the cell digestion is improved, thereby creating good conditions for the cells to enter the next stage of culture.
[0055] The above is only a preferred embodiment of the present application, and does not limit the technical scope of the present application, so any slight modification, equivalent change and modification made according to the technical essence of the present application are still within the scope of the technical scheme of the present application.
Claims
1. A bioreactor microcarrier adherent cell trypsinization apparatus, characterized by, include: Bioreactor; A pancreatic enzyme digestion unit is connected to the bioreactor, and the pancreatic enzyme digestion unit is connected to the bottom valve of the bioreactor via a second connecting pipe to perform pancreatic enzyme digestion of microcarrier adherent cells in the bioreactor.
2. The bioreactor microcarrier adherent cell trypsinization device of claim 1, wherein, The pancreatic enzyme digestion unit includes a pancreatic enzyme digestion reaction flask, a washing solution container, a sampling container, a transfer container, a pancreatic enzyme container, a waste liquid collection container, and a termination solution container. A first connecting pipe is connected to the pancreatic enzyme digestion reaction flask. A bottom valve at the bottom of the bioreactor is connected to a second connecting pipe. A five-way connector is provided between the first and second connecting pipes. The first connecting pipe is connected to the first port of the five-way connector. The second connecting pipe is connected to the second port of the five-way connector. The third port of the five-way connector is connected to the washing solution container via a third connecting pipe. The fourth port of the five-way connector is connected to the pancreatic enzyme container via a fourth connecting pipe. The fifth port of the five-way connector is connected to the transfer container via a fifth connecting pipe. A sixth connecting tube is connected to the pancreatic enzyme digestion reaction flask; a seventh connecting tube is connected to the waste liquid collection container; a four-way pipe is provided between the seventh connecting tube and the sixth connecting tube; the end of the seventh connecting tube away from the waste liquid collection container is connected to the first interface of the four-way pipe; the end of the sixth connecting tube away from the pancreatic enzyme digestion reaction flask is connected to the second interface of the four-way pipe. The third port of the four-way tube and the transfer container are connected by an eighth connecting tube; a cell observation tube is connected to the eighth connecting tube. The fourth port of the four-way pipe and the stop liquid container are connected by a ninth connecting pipe. Pipe clamps are provided on the second connecting pipe, the third connecting pipe, the fourth connecting pipe, the fifth connecting pipe, the sixth connecting pipe, the seventh connecting pipe, the eighth connecting pipe, and the ninth connecting pipe.
3. The bioreactor microcarrier adherent cell trypsinization device of claim 2, wherein, The first end of the first connecting tube is connected to the first interface of the five-way tube, and the second end is connected to the pancreatic enzyme digestion reaction flask, extending at least two-thirds of the way into the pancreatic enzyme digestion reaction flask.
4. The bioreactor microcarrier adherent cell trypsinization device of claim 2, wherein, The first end of the sixth connecting tube is connected to the second interface of the four-way tube, and the second end is connected to the pancreatic enzyme digestion reaction bottle; the end of the second end of the sixth connecting tube extends into the pancreatic enzyme digestion reaction bottle by at least two-thirds, and the end of the second end is bent and contacts the side wall of the pancreatic enzyme digestion reaction bottle.
5. The bioreactor microcarrier adherent cell trypsinization device of claim 2, wherein, The pancreatic enzyme digestion reaction flask is equipped with a bottom tube; the first end of the bottom tube is connected to the first interface of the three-way tube, and the second end is connected to the pancreatic enzyme digestion reaction flask and extends into the pancreatic enzyme digestion reaction flask to contact the bottom of the pancreatic enzyme digestion reaction flask; a sampling container is provided on the three-way tube; the sampling container and the second interface of the three-way tube are connected by a tenth connecting tube to allow the pancreatic enzyme digestion reaction flask to communicate with the sampling container; the pipeline clamp is provided on the tenth connecting tube.
6. The bioreactor microcarrier adherent cell trypsinization device of claim 2, wherein, The trypsin digestion reaction bottle is provided with an air pipe; a first end of the air pipe is connected with an air filter, and a second end of the air pipe is connected with the trypsin digestion reaction bottle and extends into the trypsin digestion reaction bottle by at least one tenth.
7. The bioreactor microcarrier adherent cell trypsinization device of claim 2, wherein, The inner diameter of the cell observation tube is 80 mm, and the wall thickness is 2 mm.