CN109593703B - Cell culture method and system - Google Patents
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Abstract
The invention discloses a cell culture method and a cell culture system, wherein the method comprises the following steps: s10, a liquid interface expanding step, namely expanding one liquid interface of the cell factory into a plurality of liquid expanding interfaces; s20, an inoculation step, namely injecting a first culture solution containing inoculated cells into a cell factory through a first liquid expansion interface; s30, a first liquid expansion interface is closed, and after the first culture solution is injected, the first liquid expansion interface is closed and is not opened any more; s40, a culture step; s50, a digestion step, namely identifying an unused liquid expansion interface, and finishing liquid exchange of the cell factory through the unused liquid expansion interface. The system comprises a cell factory having at least one fluid interface; a liquid interface expanding device; the liquid changing device can be in butt joint or separation with the liquid expanding interface to complete liquid exchange of the cell factory. The invention ensures that the whole liquid changing process of cell culture is not polluted by the outside and ensures aseptic operation.
Description
Technical Field
The invention is used in the field of cell culture, and particularly relates to a cell culture method and a cell culture system.
Background
At present, knowledge automation driven by data based on big data is the most advanced development direction in the automation field, which is considered as a revolution in the third automation field after mechanical automation and electrical and electronic automation, is a necessary requirement for people to be embedded in automation, and is also a basis for turning from automation control in the physical world to intelligent management in the human society. In the industrial automation industry, knowledge automation can obtain knowledge from big data generated in the production process, the intelligence of traditional equipment is improved, and the problems that the traditional industrial equipment depends on people, the labor cost is high, and the inheritance of knowledge and experience is blocked are solved. Under the environment of rapid development of big data technology, a big data environment for high-speed acquisition, reliable transmission, storage, analysis and decision of intelligently manufactured data is constructed, and the knowledge and experience of people are fused and extracted to form the knowledge for guiding production, namely the intelligent manufacturing knowledge is automated, so that a new opportunity is brought to the intellectualization of production equipment.
The intellectualization is the core of the fourth industrial revolution and is also the key for accelerating the transformation and upgrading of the traditional industry. The current cell culture industry mainly uses manpower culture, and the difference between product batches is big, mainly embodies:
1) experience dependence on humans is large. The cell culture process involves a number of discrete tasks, each of which sets process parameters (time, broth, angle of shaking, etc.) of the response. The whole process requires manual adjustment of various processes, so that the quality of the final product is greatly dependent on operators, and the stability of the product is affected.
2) The pollution risk coefficient is high. In the cell culture process, the influence of human participation on the operation environment is large, and the pollution probability of manual operation is 3% -5% according to experience, so that the production process is interrupted.
3) Experience is poorly transmissible. The understanding of cells and the operation habit between operators are not necessary, and training can only solve the problem of operation from no understanding, but not from culturing ordinary operators to culturing excellent operators.
4) The production rhythm is not uniform. The different working efficiency of each operator leads to the fact that the operation time can not be unified, the rhythm is fast or slow, and the quality control can not be kept at the same level; because the cell factory device is very heavy, and the manual operation degree of difficulty is very big, can only operate a cell factory at one time everyone in the operation scene, need to be equipped with a plurality of operating personnel in order to realize the simultaneous operation of a plurality of cell factories, consequently the cell quality is uneven.
5) The labor cost is high. The current level of human cost for a typical operator is 12 ten thousand per year, with experienced and highly qualified operators costing well above this. The production benefit is directly proportional to the professional culture of operators, and high production benefit requires high investment of labor cost.
6) Cell passage is a manual operation process, and data management cannot be achieved.
7) The cell culture production efficiency is low.
8) The cell factory has a fixed structure, and is easily polluted by the outside in the liquid changing process of the cell factory.
Disclosure of Invention
The invention aims to provide a cell culture method and a cell culture system, which adopt a brand new design concept aiming at a cell factory with a given structure, so that the whole cell culture liquid changing process is not polluted by the outside, and the aseptic operation is ensured.
The technical scheme adopted by the invention for solving the technical problems is as follows: a cell culture method comprising the steps of:
s10, a liquid interface expanding step, namely expanding one liquid interface of the cell factory into a plurality of liquid expanding interfaces;
s20, an inoculation step, namely injecting a first culture solution containing inoculated cells into a cell factory through a first liquid expansion interface;
s30, a first liquid expansion interface is closed, and after the first culture solution is injected, the first liquid expansion interface is closed and is not opened any more;
s40, a culture step;
s50, a digestion step, namely identifying an unused liquid expansion interface, and finishing liquid exchange of the cell factory through the unused liquid expansion interface.
Further as an improvement of the technical scheme of the invention, the method also comprises an S05. assembling step, wherein a plurality of cell factories are placed on the carrier in the same posture to form a cell factory group.
As a further improvement of the technical solution of the present invention, in step S10, a port cover assembled on the carrier is used to expand one liquid interface of the cell factory into a plurality of liquid expansion interfaces, the port cover is provided with an inner interface in butt joint with the liquid interface of the corresponding cell factory and an outer interface separated from the inner interface to form the plurality of liquid expansion interfaces, and each outer interface is provided with an automatic closing device.
Further as the improvement of the technical scheme of the invention, the method also comprises S25. the step of inoculation shaking.
Further as an improvement of the technical scheme of the invention, S40, a digestion step is carried out to identify an unused second liquid expansion interface, and S40, the digestion step comprises S41, a liquid evacuation step is carried out, and liquid in the cell factory is evacuated through the second liquid expansion interface.
Further as an improvement of the technical scheme of the invention, S40, the digestion step comprises S42, a washing liquid injection step, S43, a washing liquid shaking step and S44, a secondary liquid emptying step,
s42, a washing liquid injection step, namely injecting washing liquid through the second liquid expansion interface or an unused third liquid expansion interface;
and S44, secondary liquid emptying, namely emptying liquid in the cell factory through a liquid expansion interface for injecting washing liquid.
Further as an improvement of the technical scheme of the invention, S40, the digestion step comprises S45, a digestion liquid injection step, S46, a digestion liquid shaking step and S47, a third liquid emptying step,
s45, a digestive juice injecting step, namely injecting digestive juice through the second liquid expanding interface or an unused fourth liquid expanding interface;
and S47, a third liquid emptying step, wherein liquid in the cell factory is emptied through a liquid expansion interface for injecting digestive juice.
Further as an improvement of the technical scheme of the invention, S40, the digestion step comprises S48, a second culture solution injection step, S49, a second culture solution shaking step and S410, four liquid emptying steps,
s45, injecting a second culture solution, namely injecting the second culture solution through a second liquid expansion interface or an unused fifth liquid expansion interface;
and S410, four liquid emptying steps, namely emptying the liquid in the cell factory through a liquid expansion interface for injecting the second culture solution.
As a further improvement of the technical scheme of the invention, the cell factory group is in a straight parallelepiped shape, four corners at the top are ABDC, four corners at the bottom are EFHG, four corners at the front side are BDHF, four corners at the rear side are ACGE, four corners at the left side are ABFE, four corners at the right side are CDHG, and liquid interfaces of a plurality of cell factories are upward and placed on the carrier in the same posture.
Further as an improvement of the technical scheme of the invention, S25, an inoculation shaking step, S43, a washing liquid shaking step, S46, a digestion liquid shaking step and S49, a second culture liquid shaking step all adopt the following operations: the cell factory group takes a DH edge as an axis, the BDHF surface is turned to be in a vertical state, and the CDHG surface is turned to be in a bottom horizontal state; the cell factory group takes the HG side as an axis, the CDHG surface is turned to be in a vertical state, and the EFHG surface is turned to be in a bottom horizontal state; shaking the cell factory group while maintaining the EFHG surface horizontal state; the cell factory group was turned to the vertical state with the FH side as the axis, and the vertical state with the EFHG side and the horizontal state with the BDHF side.
Further as an improvement of the technical scheme of the invention, S20, an inoculation step, wherein the liquid changing device is matched with the cell factory set, and a liquid outlet of the liquid changing device is butted with the first liquid expanding interface; the cell factory group takes the FH edge as an axis, the EFHG surface is turned to a vertical state, the BDHF surface is turned to a bottom horizontal state, and the liquid changing device injects a first culture solution containing inoculated cells into the cell factory through the first liquid expanding interface; s30, a first liquid expansion interface closing step, wherein the liquid changing device is separated from the cell factory group, and the first liquid expansion interface is closed and is not opened any more; s40, a digestion step, namely matching the liquid changing device with the cell factory set, identifying an unused second liquid expansion interface, and butting a liquid outlet of the liquid changing device with the second liquid expansion interface; s41, a primary liquid evacuation step, wherein the cell factory group takes the BD edge as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device evacuates liquid in the cell factory through a second liquid expanding interface; s42, a washing liquid injection step, namely injecting the washing liquid into the liquid changing device through a second liquid expansion interface; s44, secondary liquid emptying, namely, the cell factory group takes the BD side as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device empties liquid in the cell factory through a liquid expanding interface for injecting washing liquid; s45, injecting digestive juice, namely injecting the digestive juice into the liquid changing device through a second liquid expanding interface; s47, a third liquid emptying step, wherein the cell factory group takes the BD side as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device empties liquid in the cell factory through a liquid expanding interface for injecting digestive juice; s45, injecting a second culture solution, namely injecting the second culture solution into the solution changing device through a second liquid expanding interface; s410, a liquid emptying step is carried out for four times, and liquid in the cell factory is emptied by the liquid changing device through a liquid expanding interface for injecting second culture solution; the liquid changing device is separated from the cell factory group, and the corresponding liquid expanding interface is closed and is not opened any more.
As a further improvement of the technical scheme of the invention, the liquid changing device comprises a liquid supply pipeline, a liquid collecting system pipeline and a confluence main pipe, wherein the liquid supply pipeline and the liquid collecting system pipeline are both connected with the confluence main pipe, pipeline control valves are respectively arranged on the liquid supply pipeline and the liquid collecting system pipeline, the confluence main pipe is connected with a liquid outlet of the liquid changing device, a liquid outlet of the liquid changing device is provided with a liquid outlet control valve, when a first culture solution, a washing solution, a digestive juice or a second culture solution is injected, the liquid outlet control valve and the pipeline control valve corresponding to the liquid supply pipeline are opened, and the liquid is injected into the cell factory through the liquid changing device; when the liquid in the cell factory is emptied, the liquid outlet control valve and the pipeline control valve of the liquid collecting system pipeline are opened, and the liquid is discharged out of the cell factory through the liquid changing device.
The liquid changing device comprises a cleaning liquid system pipeline and a waste liquid treatment system pipeline which are both connected with the confluence main pipe, wherein the cleaning liquid system pipeline and the waste liquid treatment system pipeline are both provided with pipeline control valves, and the cleaning step of the liquid changing device comprises the steps of closing the liquid outlet control valve and other pipeline control valves, opening the pipeline control valve of the cleaning liquid system pipeline, introducing cleaning liquid to clean the liquid changing device, closing the pipeline control valve of the cleaning liquid system pipeline, opening the pipeline control valve of the waste liquid treatment system pipeline and closing the pipeline control valve of the waste liquid treatment system pipeline after the cleaning is finished.
A cell culture system comprising
A cell factory having at least one fluid interface;
the liquid interface expanding device can expand one liquid interface of the cell factory into a plurality of liquid expanding interfaces;
the liquid changing device can be in butt joint or separation with the liquid expanding interface to complete liquid exchange of the cell factory.
Further as an improvement of the technical scheme of the invention, the cell factory set further comprises a carrier, and a plurality of cell factories can be placed on the carrier in the same posture to form a cell factory set.
As a further improvement of the technical scheme of the invention, the liquid interface expanding device comprises an opening cover, an inner interface which is in butt joint with the liquid interface of the corresponding cell factory and an outer interface which is separated from the inner interface to form a plurality of liquid expanding interfaces are arranged on the opening cover, and each outer interface is provided with an automatic closing device.
As a further improvement of the technical scheme of the present invention, the liquid changing device includes a liquid supply pipeline, a liquid collecting system pipeline and a confluence main pipe, the liquid supply pipeline and the liquid collecting system pipeline are both connected to the confluence main pipe, the liquid supply pipeline and the liquid collecting system pipeline are both provided with pipeline control valves, the confluence main pipe is connected to a liquid outlet of the liquid changing device, and the liquid outlet of the liquid changing device is provided with a liquid outlet control valve.
As a further improvement of the technical scheme of the invention, the liquid changing device comprises a cleaning liquid system pipeline and a waste liquid treatment system pipeline which are both connected with the confluence main pipe, and the cleaning liquid system pipeline and the waste liquid treatment system pipeline are both provided with pipeline control valves.
As further improvement of the technical scheme of the invention, the liquid changing device is provided with an identification device.
Further as an improvement of the technical scheme of the invention, the liquid changing device further comprises a driving device which can drive the liquid changing device to move so as to enable a liquid outlet of the liquid changing device and a corresponding liquid expansion interface.
The invention has the beneficial effects that: when a cell factory is adopted to culture cells, the cell factory has a plurality of liquid expansion interfaces which can be selected in the liquid injection process, and the liquid expansion interfaces can not be used after being used, so that the unused new liquid expansion interfaces are ensured to be used every time, the whole liquid exchange process is free from external pollution, and the aseptic operation is ensured.
Drawings
The invention will be further described with reference to the accompanying drawings in which:
FIG. 1 is a schematic diagram of a cell factory according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a cell factory set according to an embodiment of the present invention;
FIG. 3 is an expanded view of a fluid interface of a cell factory set according to an embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a liquid changing device according to an embodiment of the present invention;
FIG. 5 is a flow chart of cell culture according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to the present preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout.
In the present invention, if directions (up, down, left, right, front, and rear) are described, reference is made to the structure shown in fig. 1 and 2, but it is only for convenience of describing the technical solution of the present invention, and it is not intended or implied that the technical features referred to must have a specific orientation, be constructed in a specific orientation, and operate, and thus it is not to be construed as limiting the present invention.
In the present invention, "a plurality" means one or more, "a plurality" means two or more, "more than", "less than", "more than", and the like are understood as not including the number; the terms "above", "below", "within" and the like are to be understood as including the number. In the description of the present invention, if there is description of "first" and "second" for the purpose of distinguishing technical features only, it is not to be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features or implicitly indicating the precedence of the indicated technical features.
In the present invention, unless explicitly defined otherwise, the terms "disposed," "mounted," "connected," and the like are to be understood in a broad sense, and for example, may be directly connected or indirectly connected through an intermediate; can be fixedly connected, can also be detachably connected and can also be integrally formed; may be mechanically coupled, may be electrically coupled or may be capable of communicating with each other; either as communication within the two elements or as an interactive relationship of the two elements. The specific meaning of the above-mentioned words in the present invention can be reasonably determined by those skilled in the art in combination with the detailed contents of the technical solutions.
The structure of the conventional cell factory is shown in fig. 1, the cell factory 1 is used as a main structure of cell culture, a liquid interface 11 and a gas interface 12 are arranged on the cell factory 1, the liquid interface 11 is used for liquid exchange in the cell culture process of the cell factory 1, and the gas interface 12 is used for gas exchange in the cell culture process of the cell factory 1.
The invention provides a cell culture method, which comprises the following steps:
s10, a liquid interface expanding step, namely expanding one liquid interface of the cell factory 1 into a plurality of liquid expanding interfaces;
s20, an inoculation step, namely injecting a first culture solution containing inoculated cells into the cell factory 1 through a first liquid expansion interface;
s30, a first liquid expansion interface is closed, and after the first culture solution is injected, the first liquid expansion interface is closed and is not opened any more;
s40, a culture step;
s50, a digestion step, namely identifying an unused liquid expansion interface and finishing liquid exchange of the cell factory 1 through the unused liquid expansion interface.
When the cell factory 1 is adopted to culture cells, the cell factory 1 has a plurality of liquid expansion interfaces to be selected in the liquid injection process, and the liquid expansion interfaces cannot be reused after being used, so that the unused new liquid expansion interfaces are ensured to be used every time, the whole liquid exchange process is free from external pollution, and aseptic operation is ensured.
In some embodiments, the cell culture may be performed using one cell factory 1 or a plurality of cell factories 1, and when the cell culture is performed using a plurality of cell factories 1, the cell culture may further include an S05. assembling step of placing a plurality of cell factories 1 on the carrier 2 in the same posture to form a cell factory group. The combination of a plurality of cell factories into a factory group is equivalent to the operation of a plurality of cell factories 1 at a time, and the culture efficiency and the working efficiency are improved.
In some embodiments, in step S10, a liquid interface of the cell factory 1 is expanded into a plurality of liquid expansion interfaces by using a port cover 3 assembled on the carrier 2, an inner interface butted with the liquid interface of the corresponding cell factory 1 and an outer interface separated by the inner interface to form the plurality of liquid expansion interfaces are disposed on the port cover 3, the port cover 3 is equivalent to a medium of a liquid exchange system and a cell factory group, the port cover 3 is matched with the carrier 2 while realizing the liquid interface expansion function, so as to limit the cell factory 1 in the carrier 2, and the cell factory group can be matched with the liquid changing device 4. In addition, each external interface is provided with an automatic closing device, the automatic closing devices can adopt switches, valves, blocking pieces and the like, when the liquid changing operation is completed, the automatic closing devices are triggered in the separation process of the liquid changing device 4 and the cell factory group, and the corresponding liquid expansion interfaces are automatically closed. In the operation process of liquid changing each time, the system automatically identifies a new liquid expansion interface for connection.
Whether to shake the cell factory set is selected as required, and in some embodiments, an S25. inoculation shaking step is further included. The main purpose of the shaking operation is to accelerate the diffusion of the first culture medium in the cell factory group and to make it uniformly distributed.
In some embodiments, s40, a digestion step, which identifies an unused second liquid expansion interface, that is, identifies a liquid expansion interface, determines whether the liquid expansion interface is a new interface, if the interface is a new interface, performs accurate positioning and docking, and if the new interface is not to be found, s40, the digestion step includes s41, a liquid evacuation step, in which liquid inside the cell factory 1 is evacuated through the second liquid expansion interface.
In some embodiments, s40. the digestion step comprises s42. the wash solution injection step, s43. the wash solution shaking step, and s44. the secondary liquid evacuation step,
s42, a washing liquid injection step, namely injecting washing liquid through the second liquid expansion interface or an unused third liquid expansion interface;
and S44, a secondary liquid emptying step, wherein liquid in the cell factory 1 is emptied through a liquid expansion interface for injecting washing liquid.
In some embodiments, s40. the digestion step comprises s45. the digestion liquid injection step, s46. the digestion liquid shaking step, and s47. the three liquid evacuation steps,
s45, a digestive juice injecting step, namely injecting digestive juice through the second liquid expanding interface or an unused fourth liquid expanding interface;
and S47, a liquid evacuation step for three times, wherein liquid in the cell factory 1 is evacuated through a liquid expansion interface for injecting digestive juice.
In some embodiments, S40. the digestion step comprises S48. the second broth injection step, S49. the second broth shaking step, and S410. the four liquid evacuation steps,
s45, injecting a second culture solution, namely injecting the second culture solution through a second liquid expansion interface or an unused fifth liquid expansion interface;
and S410, four liquid evacuation steps, wherein liquid in the cell factory 1 is evacuated through a liquid expansion interface for injecting a second culture solution.
The cell factory group is in a square shape, a special shape and the like, referring to fig. 1 and fig. 2, in some embodiments, the cell factory group is in a straight parallelepiped shape, four corners at the top are ABDC, four corners at the bottom are EFHG, four corners at the front side are BDHF, four corners at the rear side are ACGE, four corners at the left side are ABFE, four corners at the right side are CDHG, eight corners of the cell factory 1 are abcdefgh respectively, liquid interfaces of a plurality of cell factories 1 are placed on the carrier 2 upwards in the same posture, for example, but not limited to, points a-H of each cell factory 1 are placed corresponding to points a-H of the cell factory group.
The shaking step can adopt forward and backward and left and right translation shaking or overturning movement shaking, and in some embodiments, the following operations are adopted in the S25. inoculation shaking step, the S43. washing liquid shaking step, the S46. digestive juice shaking step and the S49. second culture solution shaking step: the cell factory group takes a DH edge as an axis, the BDHF surface is turned to be in a vertical state, and the CDHG surface is turned to be in a bottom horizontal state; the cell factory group takes the HG side as an axis, the CDHG surface is turned to be in a vertical state, and the EFHG surface is turned to be in a bottom horizontal state; shaking the cell factory group while maintaining the EFHG surface horizontal state; the cell factory group was turned to the vertical state with the FH side as the axis, and the vertical state with the EFHG side and the horizontal state with the BDHF side.
In some embodiments, s20. an inoculation step, the liquid changing device 4 is matched with the cell factory set, and a liquid outlet of the liquid changing device 4 is in butt joint with the first liquid expansion interface; the cell factory group takes the FH edge as an axis, the EFHG surface is turned to a vertical state, the BDHF surface is turned to a bottom horizontal state, and the liquid changing device 4 injects a first culture solution containing inoculated cells into the cell factory 1 through the first liquid expanding interface; s30, a first liquid expansion interface closing step, wherein the liquid changing device 4 is separated from the cell factory group, and the first liquid expansion interface is closed and is not opened any more; s40, a digestion step, namely matching the liquid changing device 4 with a cell factory group, identifying an unused second liquid expansion interface, and butting a liquid outlet of the liquid changing device 4 with the second liquid expansion interface; s41, a primary liquid evacuation step, wherein the cell factory group takes the BD edge as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device 4 evacuates liquid in the cell factory 1 through a second liquid expanding interface; s42, a washing liquid injection step, in which the liquid changing device 4 injects washing liquid through a second liquid expansion interface; s44, secondary liquid emptying, namely, the cell factory group takes the BD side as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device 4 empties liquid in the cell factory 1 through a liquid expanding interface for injecting washing liquid; s45, injecting digestive juice, namely injecting the digestive juice into the liquid changing device 4 through a second liquid expanding interface; s47, a third liquid emptying step, wherein the cell factory group takes the BD side as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device 4 empties liquid in the cell factory 1 through a liquid expanding interface for injecting digestive juice; s45, injecting a second culture solution, namely injecting the second culture solution into the solution changing device 4 through a second liquid expanding interface; s410, a liquid emptying step is carried out for four times, and the liquid changing device 4 empties liquid in the cell factory 1 through a liquid expanding interface for injecting second culture liquid; the liquid changing device 4 is separated from the cell factory group and the corresponding liquid expansion interface is closed and not opened any more.
Referring to fig. 4, in some embodiments, the liquid changing apparatus 4 includes a liquid supply source pipeline, a liquid collecting system pipeline, and a confluence main pipe, the liquid supply source pipeline and the liquid collecting system pipeline are both connected to the confluence main pipe, the liquid supply source pipeline and the liquid collecting system pipeline are both provided with pipeline control valves 41, the confluence main pipe is connected to a liquid outlet of the liquid changing apparatus 4, the liquid outlet of the liquid changing apparatus 4 is provided with a liquid outlet control valve 42, when the first culture solution, the washing solution, the digestion solution, or the second culture solution is injected, the liquid outlet control valve 42 and the pipeline control valve 41 corresponding to the liquid supply source pipeline are opened, and the liquid is injected into the cell factory 1 through the liquid changing apparatus 4; when the liquid inside the cell factory 1 is to be drained, the liquid outlet control valve 42 and the pipe control valve 41 of the pipe of the liquid collecting system are opened, and the liquid is discharged from the cell factory 1 through the liquid changing device 4.
In some embodiments, the liquid changing device 4 includes a cleaning liquid system pipeline and a waste liquid treatment system pipeline both connected to the confluence main, the cleaning liquid system pipeline and the waste liquid treatment system pipeline are provided with pipeline control valves 41, the cleaning step of the liquid changing device 4 is further included, the liquid outlet control valve 42 and the rest of pipeline control valves 41 are closed, the pipeline control valve 41 of the cleaning liquid system pipeline is opened, the cleaning liquid is introduced to clean the liquid changing device 4, after the cleaning is completed, the pipeline control valve 41 of the cleaning liquid system pipeline is closed, the pipeline control valve 41 of the waste liquid treatment system pipeline is opened, and the pipeline control valve 41 of the waste liquid treatment system pipeline is closed after the emptying.
The invention also provides a cell culture system comprising
A cell factory 1, the cell factory 1 having at least one fluid interface;
the liquid interface expanding device can expand one liquid interface of the cell factory 1 into a plurality of liquid expanding interfaces;
the liquid changing device 4 can be in butt joint with or separated from the liquid expanding interface to complete liquid exchange of the cell factory 1.
When the cell factory 1 is adopted to culture cells, the cell factory 1 has a plurality of liquid expansion interfaces to be selected in the liquid injection process, and the liquid expansion interfaces cannot be reused after being used, so that the unused new liquid expansion interfaces are ensured to be used every time, the whole liquid exchange process is free from external pollution, and aseptic operation is ensured.
In some embodiments, the cell factory further comprises a carrier 2, the carrier 2 may be a box structure, a tray structure, a frame structure, etc., and a plurality of cell factories 1 can be placed on the carrier 2 in the same posture to form a cell factory group. The combination of a plurality of cell factories into a factory group is equivalent to the operation of a plurality of cell factories 1 at a time, and the culture efficiency and the working efficiency are improved.
In some embodiments, the liquid interface expanding device comprises a distributing cover 3, an inner interface which is connected with the liquid interface of the corresponding cell factory 1 in a butt joint mode and an outer interface which is separated from the inner interface to form a plurality of liquid expanding interfaces are arranged on the distributing cover 3, and each outer interface is provided with an automatic closing device.
Trade liquid device 4 and include that supply liquid source pipeline, liquid collecting system pipeline and the house steward that converges, supply liquid source pipeline, liquid collecting system pipeline all with converge the house steward and be connected, supply liquid source pipeline, all be equipped with pipeline control valve 41 on the liquid collecting system pipeline, converge the house steward and be connected with the liquid outlet of trading liquid device 4, the liquid outlet of trading liquid device 4 is equipped with liquid outlet control valve 42. The liquid changing device 4 comprises a cleaning liquid system pipeline and a waste liquid treatment system pipeline which are both connected with the confluence main pipe, and the cleaning liquid system pipeline and the waste liquid treatment system pipeline are both provided with pipeline control valves 41. The liquid changing device 4 forms an integrated structure, all liquid sources are connected with the operation unit, the number and the disorder degree of front-section pipelines are reduced, and hose access is changed into hard pipe access, so that automatic operation is facilitated; the liquid changing device 4 can be selectively provided with the functions of on-line washing and on-line disinfection. The liquid changing cover can be repeatedly used by adopting a mode of separating the liquid changing device 4 from the cell factory group, so that resources are saved.
In some embodiments, the liquid changing device 4 is provided with an identification device. The identification device can adopt a visual device, a sensor and the like, can identify the liquid expansion interface, judge whether the liquid expansion interface is a new interface or not, and if the liquid expansion interface is accurately positioned and butted, if the liquid expansion interface is not the new interface, the liquid expansion interface is searched. The liquid exchange device further comprises a driving device which can drive the liquid exchange device 4 to move so as to enable the liquid outlet of the liquid exchange device 4 to be connected with the corresponding liquid expansion interface. The spatial orientation of the liquid changer 4 is controlled by a multi-degree-of-freedom driving device which enables the liquid changer 4 to rotate, move and position at various angles and controls the engagement and disengagement of the liquid changer 4 and the cell factory group. The liquid changing device 4 and the cell factory set realize positioning butt joint through a high-precision positioning device, and the high-precision positioning device comprises but is not limited to a bolt. The driving device finishes primary positioning, error correction is realized through the bolt device, and accurate positioning is realized, so that the cell factory set and the interface on the liquid changing device 4 cannot touch other parts in the connection process. After the connection is completed, the cell factory set is embedded with the liquid changing device 4, so that the phenomenon of liquid leakage can not occur in the following operation process, and the cooperative motion of the liquid changing device 4 and the cell factory set is realized.
Referring to fig. 5, the present invention is further illustrated by an adherent cell culture process:
1. sterilizing the working environment and the working equipment (before and after each use);
2. the six cell factories 1 are sequentially placed by mechanical means at corresponding positions of the carriers 2, wherein the cell factories 1 are oriented in the same direction, and the cell factory groups are covered with the split-lid lids 3. The cell factory set after assembly is shown in FIG. 2, where Ii1(i＝1，2，……6)、Ii2(i ═ 1, 2, … … 6) are all liquid expansion interfaces;
3. conveying the cell factory group to a liquid changing station through a mechanical device;
4. the mechanical device of the liquid changing station moves the cell factory group to the vicinity of the liquid changing device 4;
5. the station mechanical device and the driving device are controlled, the liquid changing device 4 is connected and embedded with the cell factory set through the positioning device, and a liquid outlet of the liquid changing device 4 is in butt joint with the first liquid expanding interface;
6. the cell factory set turns over the EFHG surface to a vertical state and the BDHF surface to a bottom horizontal state with the FH side as an axis, opens the pipe control valve 41 and the liquid outlet control valve 42 of the liquid supply pipe,the liquid changing device 4 expands the interface I through the first liquidi1(i-1, 2, … … 6) feeding the first culture solution containing the seeded cells to the cell factory 1, and closing the line control valve 41 and the port control valve 42 of the liquid supply line after the feeding is completed;
keeping the BDFH surface horizontally standing for 5 seconds until the liquid is uniformly distributed;
8. the cell factory group takes a DH edge as an axis, the BDHF surface is turned to be in a vertical state, and the CDHG surface is turned to be in a bottom horizontal state; the cell factory group takes the HG side as an axis, the CDHG surface is turned to be in a vertical state, and the EFHG surface is turned to be in a bottom horizontal state; shaking the cell factory group while maintaining the EFHG surface horizontal state; the cell factory group takes the FH side as an axis, the EFHG surface is turned to be in a vertical state, and the BDHF surface is turned to be in a bottom horizontal state;
9. the liquid changing device 4 is separated from the cell factory group, and the first liquid expands the interface Ii1(i ═ 1, 2, … … 6) closed and no longer open;
10. transporting the cell factory group to a conveying device through a mechanical device arm, and conveying the cell factory group to a specified place for standing culture;
11. after the cell culture is finished, the cell factory group is conveyed to a liquid changing station again through a conveying device, a station mechanical device and a driving device are controlled, and the liquid changing device 4 and the cell factory group are connected and embedded through a positioning device;
12. the cell factory set takes BD edge as an axis, the BDHF surface is turned over to an inclined state, for example, the BDHF surface is inclined at 5 degrees to the horizontal plane, a pipeline control valve 41 and a liquid outlet control valve 42 of a waste liquid treatment system pipeline are opened, and the liquid changing device 4 passes through a second liquid expansion interface Ii2(i ═ 1, 2, … … 6) evacuating the liquid inside the cell factory 1, and closing the line control valve 41 and the port control valve 42 of the waste liquid disposal system line;
13. opening a pipeline control valve 41 of a cleaning liquid system pipeline, introducing cleaning liquid to clean the liquid changing device 4, closing the pipeline control valve 41 of the cleaning liquid system pipeline after cleaning is finished, opening a pipeline control valve 41 of a waste liquid treatment system pipeline, and closing the pipeline control valve 41 of the waste liquid treatment system pipeline after emptying;
14. opening the line control valve 41 and outlet of the supply lineA liquid port control valve 42, a liquid changing device 4 which expands a port I through a second liquidi2(i-1, 2, … … 6) supplying a washing solution to the cell factory 1, and closing the line control valve 41 and the outlet control valve 42 of the liquid supply line after the supply of the washing solution is completed;
keeping the BDFH surface horizontally standing for 5 seconds until the liquid is uniformly distributed;
16. the cell factory group takes a DH edge as an axis, the BDHF surface is turned to be in a vertical state, and the CDHG surface is turned to be in a bottom horizontal state; the cell factory group takes the HG side as an axis, the CDHG surface is turned to be in a vertical state, and the EFHG surface is turned to be in a bottom horizontal state; shaking the cell factory group while maintaining the EFHG surface horizontal state; the cell factory group takes the FH side as an axis, the EFHG surface is turned to be in a vertical state, and the BDHF surface is turned to be in a bottom horizontal state;
17. the cell factory set takes BD edge as an axis, the BDHF surface is turned over to an inclined state, for example, the BDHF surface is inclined at 5 degrees to the horizontal plane, a pipeline control valve 41 and a liquid outlet control valve 42 of a waste liquid treatment system pipeline are opened, and the liquid changing device 4 passes through a second liquid expansion interface Ii2(i ═ 1, 2, … … 6) evacuating the liquid inside the cell factory 1, and closing the line control valve 41 and the port control valve 42 of the waste liquid disposal system line;
18. opening a pipeline control valve 41 of a cleaning liquid system pipeline, introducing cleaning liquid to clean the liquid changing device 4, closing the pipeline control valve 41 of the cleaning liquid system pipeline after cleaning is finished, opening a pipeline control valve 41 of a waste liquid treatment system pipeline, and closing the pipeline control valve 41 of the waste liquid treatment system pipeline after emptying;
19. opening the pipe control valve 41 and the liquid outlet control valve 42 of the liquid supply pipe, the liquid changing device 4 expands the interface I through the second liquidi2(i ═ 1, 2, … … 6) the cell factory 1 was filled with the digestion solution, and after completion of the filling, the line control valve 41 and the liquid outlet control valve 42 of the liquid supply line were closed;
keeping the BDFH surface horizontally standing for 5 seconds until the liquid is uniformly distributed;
21. the cell factory group takes a DH edge as an axis, the BDHF surface is turned to be in a vertical state, and the CDHG surface is turned to be in a bottom horizontal state; the cell factory group takes the HG side as an axis, the CDHG surface is turned to be in a vertical state, and the EFHG surface is turned to be in a bottom horizontal state; shaking the cell factory group while maintaining the EFHG surface horizontal state; the cell factory group takes the FH side as an axis, the EFHG surface is turned to be in a vertical state, and the BDHF surface is turned to be in a bottom horizontal state;
22. the cell factory set takes BD edge as an axis, the BDHF surface is turned over to an inclined state, for example, the BDHF surface is inclined at 5 degrees to the horizontal plane, a pipeline control valve 41 and a liquid outlet control valve 42 of a waste liquid treatment system pipeline are opened, and the liquid changing device 4 passes through a second liquid expansion interface Ii2(i ═ 1, 2, … … 6) evacuating the liquid inside the cell factory 1, and closing the line control valve 41 and the port control valve 42 of the waste liquid disposal system line;
23. opening a pipeline control valve 41 of a cleaning liquid system pipeline, introducing cleaning liquid to clean the liquid changing device 4, closing the pipeline control valve 41 of the cleaning liquid system pipeline after cleaning is finished, opening a pipeline control valve 41 of a waste liquid treatment system pipeline, and closing the pipeline control valve 41 of the waste liquid treatment system pipeline after emptying;
24. opening the pipe control valve 41 and the liquid outlet control valve 42 of the liquid supply pipe, the liquid changing device 4 expands the interface I through the second liquidi2(i-1, 2, … … 6) feeding the second culture solution to the cell factory 1, and closing the line control valve 41 and the port control valve 42 of the liquid supply line after the feeding;
25, keeping the BDFH surface horizontally standing for 5 seconds until the liquid is uniformly distributed;
26. the cell factory group takes a DH edge as an axis, the BDHF surface is turned to be in a vertical state, and the CDHG surface is turned to be in a bottom horizontal state; the cell factory group takes the HG side as an axis, the CDHG surface is turned to be in a vertical state, and the EFHG surface is turned to be in a bottom horizontal state; shaking the cell factory group while maintaining the EFHG surface horizontal state; the cell factory group takes the FH side as an axis, the EFHG surface is turned to be in a vertical state, and the BDHF surface is turned to be in a bottom horizontal state;
27. the cell factory set takes BD edge as an axis, the BDHF surface is turned over to an inclined state, for example, the BDHF surface is inclined at 5 degrees to the horizontal plane, a pipeline control valve 41 and a liquid outlet control valve 42 of a liquid collecting system pipeline are opened, and the liquid changing device 4 passes through a second liquid expansion interface Ii2(i ═ 1, 2, … … 6) collect liquid, closing conduit control valve 41 and outlet control valve 42 of the liquid collection system conduits;
28. the liquid changing device 4 is separated from the cell factory group, and the second liquid expands the interface Ii1(i ═ 1, 2, … … 6) is closed and no longer open.
In the invention, the whole cell culture process can realize industrial automation, the production rhythm can be controlled, the productivity evaluation is more accurate, and the industrial planning and expansion are facilitated; the whole process does not need manual participation, realizes the industrial automation of the cell culture production line, thus not only keeping the whole operation process in a sterile environment to avoid pollution, but also improving the working efficiency and liberating the labor force.
The invention is not limited to the above embodiments, and those skilled in the art can make equivalent modifications or substitutions without departing from the spirit of the invention, and such equivalent modifications or substitutions are included in the scope defined by the claims of the present application.
Claims (14)
1. A method of cell culture comprising the steps of:
s05, an assembling step, namely placing a plurality of cell factories on a carrier in the same posture, and sequentially placing the cell factories at corresponding positions of the carrier through a mechanical device to form a cell factory group;
s10, a liquid interface expanding step, namely expanding one liquid interface of a cell factory into a plurality of liquid expanding interfaces, expanding one liquid interface of the cell factory into a plurality of liquid expanding interfaces by adopting a branch port cover assembled on a carrier, wherein the branch port cover is provided with an inner interface butted with the liquid interface of the corresponding cell factory and an outer interface separated from the inner interface to form the plurality of liquid expanding interfaces, and each outer interface is provided with an automatic closing device;
s20, an inoculation step, namely conveying the cell factory group to a liquid changing station through a mechanical device, moving the cell factory group to the position near a liquid changing device through the mechanical device of the liquid changing station, controlling the station mechanical device and a driving device, connecting and embedding the liquid changing device and the cell factory group through a positioning device, butting a liquid outlet of the liquid changing device with a first liquid expansion interface, and injecting a first culture solution containing inoculated cells into the cell factory through the first liquid expansion interface;
s30, a first liquid expansion interface is closed, and after the first culture solution is injected, the first liquid expansion interface is closed and is not opened any more;
s40, a culture step, namely transporting the cell factory group to a conveying device through a mechanical device arm, and conveying the cell factory group to a specified place for standing culture;
s50, a digestion step, namely conveying the cell factory group to a liquid changing station again through a conveying device, controlling a station mechanical device and a driving device, connecting and embedding the liquid changing device and the cell factory group through a positioning device, wherein the liquid changing device is provided with an identification device and can identify a liquid expansion interface, judging whether the liquid expansion interface is a new interface or not, if the liquid expansion interface is accurately positioned and butted, if the liquid expansion interface is not a new interface, identifying an unused liquid expansion interface, and finishing liquid exchange of the cell factory through the unused liquid expansion interface.
2. The cell culture method according to claim 1, characterized in that: s25, inoculating and shaking steps are further included.
3. The cell culture method according to claim 2, characterized in that: s40, a digestion step, namely identifying an unused second liquid expansion interface, wherein the S40 digestion step comprises S41 a primary liquid emptying step, and liquid in the cell factory is emptied through the second liquid expansion interface.
4. The cell culture method according to claim 3, characterized in that: s40, the digestion step comprises S42, a washing liquid injection step, S43, a washing liquid shaking step and S44, a secondary liquid emptying step,
s42, a washing liquid injection step, namely injecting washing liquid through the second liquid expansion interface or an unused third liquid expansion interface;
and S44, secondary liquid emptying, namely emptying liquid in the cell factory through a liquid expansion interface for injecting washing liquid.
5. The cell culture method according to claim 4, characterized in that: s40, the digestion step comprises S45, a digestion liquid injection step, S46, a digestion liquid shaking step and S47, a third liquid emptying step,
s45, a digestive juice injecting step, namely injecting digestive juice through the second liquid expanding interface or an unused fourth liquid expanding interface;
and S47, a third liquid emptying step, wherein liquid in the cell factory is emptied through a liquid expansion interface for injecting digestive juice.
6. The cell culture method according to claim 5, characterized in that: s40, the digestion step comprises S48, a second culture solution injection step, S49, a second culture solution shaking step and S410, a fourth liquid emptying step,
s45, injecting a second culture solution, namely injecting the second culture solution through a second liquid expansion interface or an unused fifth liquid expansion interface;
and S410, four liquid emptying steps, namely emptying the liquid in the cell factory through a liquid expansion interface for injecting the second culture solution.
7. The cell culture method according to claim 6, characterized in that: the cell factory group is in a straight parallel hexahedron shape, four corners at the top are ABDC, four corners at the bottom are EFHG, four corners at the front side are BDHF, four corners at the rear side are ACGE, four corners at the left side are ABFE, four corners at the right side are CDHG, and liquid interfaces of a plurality of cell factories are placed on the carrier in the same posture upwards.
8. The cell culture method according to claim 7, characterized in that: s25, an inoculation shaking step, S43, a washing liquid shaking step, S46, a digestion liquid shaking step and S49, a second culture liquid shaking step all adopt the following operations: the cell factory group takes a DH edge as an axis, the BDHF surface is turned to be in a vertical state, and the CDHG surface is turned to be in a bottom horizontal state; the cell factory group takes the HG side as an axis, the CDHG surface is turned to be in a vertical state, and the EFHG surface is turned to be in a bottom horizontal state; shaking the cell factory group while maintaining the EFHG surface horizontal state; the cell factory group was turned to the vertical state with the FH side as the axis, and the vertical state with the EFHG side and the horizontal state with the BDHF side.
9. The cell culture method according to claim 8, characterized in that: s20, inoculating, namely matching the liquid changing device with the cell factory set, and butting a liquid outlet of the liquid changing device with the first liquid expansion interface; the cell factory group takes the FH edge as an axis, the EFHG surface is turned to a vertical state, the BDHF surface is turned to a bottom horizontal state, and the liquid changing device injects a first culture solution containing inoculated cells into the cell factory through the first liquid expanding interface; s30, a first liquid expansion interface closing step, wherein the liquid changing device is separated from the cell factory group, and the first liquid expansion interface is closed and is not opened any more; s40, a digestion step, namely matching the liquid changing device with the cell factory set, identifying an unused second liquid expansion interface, and butting a liquid outlet of the liquid changing device with the second liquid expansion interface; s41, a primary liquid evacuation step, wherein the cell factory group takes the BD edge as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device evacuates liquid in the cell factory through a second liquid expanding interface; s42, a washing liquid injection step, namely injecting the washing liquid into the liquid changing device through a second liquid expansion interface; s44, secondary liquid emptying, namely, the cell factory group takes the BD side as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device empties liquid in the cell factory through a liquid expanding interface for injecting washing liquid; s45, injecting digestive juice, namely injecting the digestive juice into the liquid changing device through a second liquid expanding interface; s47, a third liquid emptying step, wherein the cell factory group takes the BD side as an axis, the BDHF surface is turned over to be in an inclined state, and the liquid changing device empties liquid in the cell factory through a liquid expanding interface for injecting digestive juice; s45, injecting a second culture solution, namely injecting the second culture solution into the solution changing device through a second liquid expanding interface; s410, a liquid emptying step is carried out for four times, and liquid in the cell factory is emptied by the liquid changing device through a liquid expanding interface for injecting second culture solution; the liquid changing device is separated from the cell factory group, and the corresponding liquid expanding interface is closed and is not opened any more.
10. The cell culture method according to claim 9, characterized in that: the liquid changing device comprises a liquid supply pipeline, a liquid collecting system pipeline and a confluence main pipe, wherein the liquid supply pipeline and the liquid collecting system pipeline are both connected with the confluence main pipe, pipeline control valves are respectively arranged on the liquid supply pipeline and the liquid collecting system pipeline, the confluence main pipe is connected with a liquid outlet of the liquid changing device, the liquid outlet of the liquid changing device is provided with a liquid outlet control valve, when a first culture solution, a washing solution, a digestive juice or a second culture solution is injected, the liquid outlet control valve and the pipeline control valve corresponding to the liquid supply pipeline are opened, and the liquid is injected into a cell factory through the liquid changing device; when the liquid in the cell factory is emptied, the liquid outlet control valve and the pipeline control valve of the liquid collecting system pipeline are opened, and the liquid is discharged out of the cell factory through the liquid changing device.
11. The cell culture method according to claim 10, characterized in that: trade the liquid device including all with the clean liquid system pipeline and the waste liquid treatment system pipeline that the header pipe is connected, clean liquid system pipeline and waste liquid treatment system pipeline all are equipped with the pipeline control valve, still including trading clean step of liquid device, close liquid outlet control valve and all the other pipeline control valves, open the pipeline control valve of clean liquid system pipeline, let in the washing liquid and wash trading the liquid device, the back that finishes washs, close the pipeline control valve of clean liquid system pipeline, open the pipeline control valve of waste liquid treatment system pipeline, close the pipeline control valve of waste liquid treatment system pipeline after the evacuation.
12. A cell culture system, comprising: comprises that
A cell factory having at least one fluid interface;
a carrier on which a plurality of cell factories can be placed in the same posture to form a cell factory group;
the liquid interface expanding device can expand one liquid interface of the cell factory into a plurality of liquid expanding interfaces, the liquid interface expanding device comprises a port cover, an inner interface which is in butt joint with the liquid interface of the corresponding cell factory and an outer interface which is separated from the inner interface to form the plurality of liquid expanding interfaces are arranged on the port cover, and each outer interface is provided with an automatic closing device;
the liquid changing device can be in butt joint with or separated from the liquid expanding interface to complete liquid exchange of the cell factory, and an identification device is arranged on the liquid changing device;
the driving device drives the liquid changing device to move so that a liquid outlet of the liquid changing device is in butt joint with the corresponding liquid expansion interface;
the mechanical device is used for sequentially placing a plurality of cell factories at corresponding positions of the carrier, conveying the cell factory groups to the liquid changing station and moving the cell factory groups to the vicinity of the liquid changing device;
a mechanical device arm for transporting the cell factory group to the conveying device to a designated place for static culture;
and the conveying device is used for conveying the cell factory group to the liquid changing station again.
13. The cell culture system of claim 12, wherein: the liquid changing device comprises a liquid supply source pipeline, a liquid collecting system pipeline and a confluence main pipe, wherein the liquid supply source pipeline and the liquid collecting system pipeline are all connected with the confluence main pipe, pipeline control valves are arranged on the liquid supply source pipeline and the liquid collecting system pipeline, the confluence main pipe is connected with a liquid outlet of the liquid changing device, and the liquid outlet of the liquid changing device is provided with a liquid outlet control valve.
14. The cell culture system of claim 13, wherein: the liquid changing device comprises a cleaning liquid system pipeline and a waste liquid treatment system pipeline which are both connected with the confluence main pipe, and the cleaning liquid system pipeline and the waste liquid treatment system pipeline are both provided with pipeline control valves.
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| US8895291B2 (en) * | 2010-10-08 | 2014-11-25 | Terumo Bct, Inc. | Methods and systems of growing and harvesting cells in a hollow fiber bioreactor system with control conditions |
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| DE102014214076A1 (en) * | 2014-07-18 | 2016-01-21 | Hamilton Bonaduz Ag | A fluid conduit assembly comprising a plurality of fluid conduit elements, valve conduit assembly, comprising a fluid conduit assembly and a switching assembly, and a handle assembly having a valve conduit assembly |
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| US4172013A (en) * | 1976-05-28 | 1979-10-23 | Dr. Rentschler Arzneimittel Gmbh & Co. | Process for the mass growth of cells and system of chambers for the carrying out thereof |
| CN102203240A (en) * | 2008-10-31 | 2011-09-28 | 株式会社淋巴细胞技术 | Kit for preparation of antigen-specific cytotoxic t lymphocyte |
| CN104937093B (en) * | 2013-01-23 | 2018-04-24 | 哈美顿博纳图斯股份公司 | Interface is supplied for cultivating the cell cultivation equipment of intrinsic cell and the fluid with cell culture container |
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