An in vitro life-sustaining perfusion culture system and control method thereof

Abstract

The invention relates to the technical field of in vitro life culture, and discloses an in vitro life-sustaining perfusion culture system, comprising: a premixing unit, which comprises a premixing chamber, into which nutrient solution and gas are added, and mixed in the premixing chamber to form culture solution; a culture unit, which comprises a culture chamber, and the culture chamber is connected with the premixing chamber; an exchange unit, which comprises an exchange chamber and a membrane component, and the exchange chamber comprises a first exchange chamber and a second exchange chamber, a membrane component is arranged between the first exchange chamber and the second exchange chamber, and the first exchange chamber and the premixing chamber are connected through the membrane component, and the culture chamber and the premixing chamber are both connected with the first exchange chamber, and the first exchange chamber can not only receive the culture solution flowing out of the culture chamber, but also transport the culture solution to the premixing chamber and the culture chamber; a fluid replenishing unit, which is connected with the second exchange chamber, and the fluid replenishing unit can directionally transport one or more components required for the culture into the second exchange chamber, and the components in the nutrient solution can penetrate into the first exchange chamber through the membrane component.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro life culture, and in particular to an in vitro life-sustaining perfusion culture system and a control method thereof. Background Art

[0002] As a 3D cell tissue, organoids have a very high accuracy rate in the evaluation of the efficacy of tumor drugs. At present, the culture method of organoids is mainly static culture in the well plate. The operator cuts the primary sample into pieces, then coats it in matrix gel at low temperature, and then adds culture medium to the incubator for culture. The medium is changed three times a week and the culture is performed every 7-10 days. There are many problems with this culture mode: first, the growth rate of organoids is slow and the culture cycle is long. During the operation, the culture chamber needs to be opened regularly for manual liquid change, which has a high risk of contamination. When the number of culture samples increases, the cost of manpower, equipment, space, etc. will be greatly increased. Second, static culture in the culture plate will lead to insufficient exchange between organoids and culture medium, especially the internal nutrients of 3D cell tissues cannot be absorbed, which will eventually limit the growth of organoids, limit the growth degree of organoids and the similarity of simulating human organs.

[0003] In order to solve the above technical problems, the prior art adopts perfusion culture, in which the culture fluid in the culture plate flows out and is discharged at any time. However, this perfusion culture method has a low utilization rate of the culture fluid, resulting in a high culture cost.

[0004] Therefore, there is an urgent need for an in vitro life-sustaining perfusion culture system to solve the above technical problems. Summary of the invention

[0005] The object of the present invention is to provide an in vitro life-sustaining perfusion culture system and a control method thereof, which realizes the perfusion culture of the culture while realizing the recycling of the culture fluid, thereby improving the utilization rate of the culture fluid and reducing the culture cost.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, an in vitro life-sustaining perfusion culture system is provided, comprising:

[0008] A premixing unit, comprising a premixing chamber, into which a nutrient solution and a gas are added and mixed to form a culture solution;

[0009] A culture unit, comprising a culture chamber, the culture chamber being in communication with the premix chamber, the culture solution in the premix chamber being transportable to the culture chamber, the culture chamber being used to hold a culture and cultivate the culture;

[0010] An exchange unit comprises an exchange chamber and a membrane assembly, wherein the exchange chamber comprises a first exchange chamber and a second exchange chamber, the membrane assembly is arranged between the first exchange chamber and the second exchange chamber, and the first exchange chamber is connected through the membrane assembly, the first exchange chamber is connected with the culture chamber, the first exchange chamber can both receive the culture fluid flowing out of the culture chamber and reflux the culture fluid to the culture chamber or the premixing chamber, and the membrane assembly is used to intercept and / or pass some components in the culture fluid.

[0011] As an optimal technical solution for an in vitro life-sustaining perfusion culture system, the premixing unit also includes a fluid replenishment chamber, which is connected to the premixing chamber and is used to transport nutrient solution into the premixing chamber. The fluid replenishment chamber can directionally transport one or more components required for the culture into the premixing chamber.

[0012] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, the first exchange chamber is provided with a first interface and a second interface, the first interface is communicated with the culture chamber, and the second interface is communicated with the premixing chamber and / or the culture chamber;

[0013] The first exchange chamber can receive the culture fluid flowing out of the culture chamber through the first interface, and the first exchange chamber can flow back the culture fluid to the culture chamber and / or the premixing chamber through the second interface.

[0014] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, it further includes a power unit, and the power unit is arranged between the premixing chamber and the culture chamber; and / or,

[0015] The power unit is arranged between the culture chamber and the first interface; and / or,

[0016] The power unit is arranged between the culture chamber or the premixing chamber and the second interface.

[0017] In a second aspect, an in vitro life-sustaining perfusion culture system is provided, comprising:

[0018] A premixing unit, comprising a premixing chamber, into which a nutrient solution and a gas are added and mixed to form a culture solution;

[0019] A culture unit, comprising a culture chamber, the culture chamber being in communication with the premix chamber, the culture solution in the premix chamber being transportable to the culture chamber, the culture chamber being used to hold a culture and cultivate the culture;

[0020] An exchange unit, comprising an exchange chamber and a membrane assembly, wherein the exchange chamber comprises a first exchange chamber and a second exchange chamber, the membrane assembly is disposed between the first exchange chamber and the second exchange chamber, and the first exchange chamber and the second exchange chamber are connected through the membrane assembly, the culture chamber and the premix chamber are both connected to the first exchange chamber, the first exchange chamber can both receive the culture solution flowing out of the culture chamber and transport the culture solution to the premix chamber and / or the culture chamber, and the membrane assembly is used to intercept and / or pass through some components in the culture solution;

[0021] A rehydration unit is connected to the second exchange chamber and is used to transport nutrient solution to the second exchange chamber. The rehydration unit can directionally transport one or more components required for the culture into the second exchange chamber, and at least some components in the nutrient solution can penetrate the membrane assembly into the first exchange chamber.

[0022] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, the first exchange chamber is provided with a first interface and a second interface, the first interface is communicated with the culture chamber, and the second interface is communicated with the premixing chamber and / or the culture chamber;

[0023] The culture solution in the culture chamber can flow to the first exchange chamber through the first interface, and the culture solution in the first exchange chamber can flow to the premixing chamber and / or the culture chamber through the second interface.

[0024] As a preferred technical solution for an in vitro life-sustaining perfusion culture system, it also includes a power unit, which is arranged between the premixing chamber and the culture chamber, between the culture chamber and the first interface, and between the premixing chamber and the second interface and / or between the culture chamber and the second interface.

[0025] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, it also includes a collecting unit, which is connected to the second exchange chamber and is used to collect the liquid in the second exchange chamber.

[0026] As a preferred technical solution for an in vitro life-sustaining perfusion culture system, at least one of the premixing unit, the culture chamber, the exchange unit and the collection unit is a disposable consumable.

[0027] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, there are multiple culture chambers, and the multiple culture chambers are all connected to the premixing chamber;

[0028] Each of the culture chambers is connected to one of the exchange units, or all of the culture chambers are connected to one of the exchange units.

[0029] In a third aspect, an in vitro life-sustaining perfusion culture system is provided, comprising:

[0030] A premixing unit, comprising a premixing chamber, into which a nutrient solution and a gas are added and mixed to form a culture solution;

[0031] A culture unit, comprising a culture chamber, the culture chamber being in communication with the premix chamber, the culture solution in the premix chamber being transportable to the culture chamber, the culture chamber being used to hold a culture and cultivate the culture;

[0032] a temperature control unit, comprising a refrigeration assembly and a temperature control module, wherein the refrigeration assembly is electrically connected to the temperature control module, and the temperature control module is used to control the refrigeration assembly to cool the culture chamber to a first preset temperature, wherein the first preset temperature is a liquefaction temperature of the culture support structure;

[0033] The culture chamber is provided with a sampling port, and the sampling port is used to take out the culture after the culture support structure is liquefied.

[0034] As an optimal technical solution for an in vitro life-sustaining perfusion culture system, the temperature control unit also includes a heating component, which is electrically connected to the temperature control module, and the temperature control module is used to control the heating component to heat the culture chamber and / or the premixing chamber.

[0035] As an optimal technical solution for an in vitro life-sustaining perfusion culture system, the heating component includes a plurality of heating plates, and the cooling component includes a plurality of cooling plates. The plurality of heating plates are arranged at intervals, and the plurality of cooling plates are arranged at intervals. One cooling plate is arranged between two adjacent heating plates, and one heating plate is arranged between two adjacent cooling plates. The energy of the heating plates can be transferred to the cooling plates.

[0036] As a preferred technical solution for an in vitro life-sustaining perfusion culture system, the temperature control unit further comprises a temperature detection element electrically connected to the temperature control module, and the temperature detection element is used to detect the temperature of the culture solution in the culture chamber.

[0037] In a fourth aspect, an in vitro life-sustaining perfusion culture system is provided, comprising:

[0038] A premixing unit, comprising a premixing chamber, into which a nutrient solution and a gas are added and mixed to form a culture solution;

[0039] A culture unit, comprising a culture chamber, the culture chamber being in communication with the premix chamber, the culture solution in the premix chamber being transportable to the culture chamber, the culture chamber being used to hold a culture and cultivate the culture;

[0040] A microscopic observation module, comprising a stage and an observation component, the culture chamber is placed on the stage, the stage can hold at least one culture chamber, and the observation component is used to observe the culture in the culture chamber;

[0041] The microscopic observation module also includes a frame, the object platform is swingably arranged on the frame, and the object platform can drive the culture chamber to swing synchronously.

[0042] As an optimal technical solution for an in vitro life-sustaining perfusion culture system, the microscopic observation module also includes an automatic sample injector and a sample injection track. The automatic sample injection track is arranged on the frame, and the automatic sample injector is slidably arranged on the sample injection track. The automatic sample injector is used to add samples to the culture chamber.

[0043] In a fifth aspect, an in vitro life-sustaining perfusion culture system is provided, comprising:

[0044] A premixing unit, comprising a premixing chamber, into which a nutrient solution and a gas are added and mixed to form a culture solution;

[0045] A culture unit, comprising a culture chamber, the culture chamber being in communication with the premix chamber, the culture solution in the premix chamber being transportable to the culture chamber, the culture chamber being used to hold a culture and cultivate the culture;

[0046] The sterile control module comprises a sterile workroom, a filtering component and a sterilizing component, wherein at least the culture chamber is arranged in the sterile workroom, the filtering component is used for filtering the gas entering the sterile workroom, and the sterilizing component is used for sterilizing the sterile workroom.

[0047] As a preferred technical solution for an in vitro life-sustaining perfusion culture system, the premixing unit also includes a pH detector, a dissolved oxygen detector and a signal detector. The pH detector and the dissolved oxygen detector are both arranged in the premixing chamber. The signal detector can sense the signals fed back by the pH detector and the dissolved oxygen detector to obtain the pH value and dissolved oxygen content of the culture solution in the premixing chamber.

[0048] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, the pH detection element is a pH electrode sheet, and the dissolved oxygen detection element is a dissolved oxygen electrode sheet;

[0049] The pH electrode sheet is arranged on the inner wall of the premixing chamber or in the premixing chamber, and the dissolved oxygen detector is arranged on the inner wall of the premixing chamber or immersed in the liquid in the premixing chamber.

[0050] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, the premixing unit further includes a gas premixing control unit, which is connected to the premixing chamber and is used to transport gas into the premixing chamber.

[0051] As an optimal technical solution for an in vitro life-sustaining perfusion culture system, there are multiple premixing chambers and one gas premixing control unit. The gas premixing control unit is simultaneously connected to the multiple premixing chambers and can control the gas concentrations in the multiple premixing chambers.

[0052] As a preferred technical solution of an in vitro life-sustaining perfusion culture system, the culture unit further includes a mixing module for shaking the culture solution in the culture chamber.

[0053] In a sixth aspect, a control method for an in vitro life-sustaining perfusion culture system is provided, which is directed to the in vitro life-sustaining perfusion culture system as described above, comprising the following steps:

[0054] The concentration of the components required for the culture in the culture chamber is detected and / or the growth of the culture is determined, and the speed at which the premixing chamber delivers the culture solution to the culture chamber is controlled according to the detected concentration and / or the determined growth of the culture.

[0055] In a seventh aspect, a control method for an in vitro life-sustaining perfusion culture system is provided, which is directed to the in vitro life-sustaining perfusion culture system as described above, comprising the following steps:

[0056] Detect the concentration of the components required for the culture in the culture fluid in the culture chamber and / or determine the growth of the culture, and control the speed at which the premixing chamber transports the culture fluid to the culture chamber and the speed at which the fluid replenishing unit transports the nutrient solution to the second exchange chamber based on the detected concentration and / or the determined growth of the culture.

[0057] Beneficial effects of the present invention:

[0058] The culture solution in the premix chamber can be transported to the culture chamber, the culture solution in the culture chamber can flow out to the first exchange chamber, and the culture solution in the first exchange chamber can flow back to the premix chamber, so as to realize the circulation of the culture solution, and then realize the perfusion culture of the culture. The membrane assembly can retain some components in the culture solution required for the growth of the culture in the first exchange chamber, and the waste generated by the metabolism of the culture in the culture solution can penetrate into the second exchange chamber through the membrane assembly. The culture solution in the first exchange chamber can flow back to the premix chamber and then be transported to the culture chamber for continued use by the culture, that is, the recycling of the culture solution is realized, the utilization rate of the culture solution is improved, and the culture cost is reduced. Moreover, the nutrient solution provided by the liquid replenishment unit to the second exchange chamber, at least some of the components in the nutrient solution can penetrate into the first exchange chamber through the membrane assembly, and the components can be replenished in a directional manner according to the consumption of the components in the culture solution, thereby reducing the amount of nutrient solution used and reducing the culture cost.

[0059] Moreover, the gas premixing control unit can introduce gas into the premixing chamber to adjust the dissolved oxygen concentration of the culture medium; the temperature control unit can control the temperature of the premixing chamber and the culture chamber, and adjust the pH value of the culture medium by adding the amount of nutrient solution and alkaline solution. The temperature, pH value and dissolved oxygen concentration of the culture medium can be controlled in the premixing chamber, so that the system is freed from the constraints of the carbon dioxide incubator and the scale of culture can be expanded arbitrarily. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is a schematic structural diagram of an in vitro life-sustaining perfusion culture system provided in Example 1 of the present invention;

[0061] Figure 2 This is a schematic diagram of the structure of the temperature control unit provided in the first embodiment of the present invention. Figure 1 ;

[0062] Figure 3 This is a schematic diagram of the structure of the temperature control unit provided in the first embodiment of the present invention. Figure 2 ;

[0063] Figure 4 is a schematic diagram of the structure of a heating component and a cooling component provided in Embodiment 1 of the present invention;

[0064] Figure 5 The structure of the microscopic observation module provided in the first embodiment of the present invention is shown in FIG. Figure 1 ;

[0065] Figure 6 The structure of the microscopic observation module provided in the first embodiment of the present invention is shown in FIG. Figure 2 ;

[0066] Figure 7 The structure of the microscopic observation module provided in the first embodiment of the present invention is shown in FIG. Figure 3 ;

[0067] Figure 8 is a schematic structural diagram of a sterile control module provided in Embodiment 1 of the present invention;

[0068] Fig. 9 The structure of the microscopic observation module provided in the third embodiment of the present invention is shown in FIG. Figure 1 ;

[0069] Fig.10 The structure of the microscopic observation module provided in the third embodiment of the present invention is shown in FIG. Figure 2 ;

[0070] Fig.11 is a schematic structural diagram of an in vitro life-sustaining perfusion culture system provided by a fourth embodiment of the present invention;

[0071] Fig.12 is a schematic structural diagram of an in vitro life-sustaining perfusion culture system provided in Embodiment 5 of the present invention;

[0072] Fig.13 It is a schematic diagram of the structure of the in vitro life-sustaining perfusion culture system provided in Example 6 of the present invention.

[0073] In the figure: 1, premixing unit; 101, premixing chamber; 102, pH detection element; 103, dissolved oxygen detection element; 104, liquid replenishment chamber; 105, gas premixing control unit;

[0074] 2. Cultivation chamber; 201. Sample addition port; 202. Sampling port; 3. Exchange unit; 301. Membrane assembly; 302. First exchange chamber; 303. Second exchange chamber;

[0075] 4. Collection unit; 5. Power unit; 6. Temperature control unit; 61. Heating plate; 62. Refrigeration plate; 63. Temperature detection element; 7. Mixing module;

[0076] 8. Microscopic observation module; 801. Stage; 802. Rack; 803. Objective lens; 804. Eyepiece; 805. First mechanical track; 806. Second mechanical track; 807. Camera; 808. Connecting part; 809. Automatic sampler; 810. Sample injection track;

[0077] 9. Sterile control module; 901. Sterile workroom; 902. Air intake fan; 903. Filter component; 904. Sterilization component. DETAILED DESCRIPTION

[0078] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0079] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0081] In the description of the present invention, it should be noted that the terms "upper", "lower", "left", "right", "vertical", "horizontal", "inside", "outside", etc. indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, or are the positions or positional relationships in which the inventive product is usually placed when in use. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0082] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "disposed" and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0083] In the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes that the first feature is directly below and obliquely below the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0084] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0085] Embodiment 1

[0086] like Figure 1 As shown, this embodiment discloses an in vitro life-sustaining perfusion culture system, which includes a premixing unit 1 , a culture unit, an exchange unit 3 , a collection unit 4 and a power unit 5 .

[0087] Among them, at least one of the premixing unit 1, the culture chamber 2 of the culture unit, the exchange unit 3 and the collection unit 4 is a disposable consumable, which is treated as waste after one use, thereby ensuring the sanitary conditions during the perfusion culture process and avoiding the contamination of the premixing unit 1, the culture chamber 2, the exchange unit 3 and the collection unit 4, which may affect the normal perfusion culture.

[0088] The premixing unit 1 includes a premixing chamber 101, a liquid replenishing chamber 104 and a gas premixing control unit 105. The nutrient solution contains nutrients required for the growth of the culture, such as culture medium, factors, drugs and enzymes. The liquid replenishing chamber 104 is connected to the premixing chamber 101. The nutrient solution is stored in the liquid replenishing chamber 104, and the liquid replenishing chamber 104 is used to transport the nutrient solution to the premixing chamber 101. A power unit 5 can be provided between the liquid replenishing chamber 104 and the premixing chamber 101 as needed, and the power unit 5 is used to transport the nutrient solution in the liquid replenishing chamber 104 to the premixing chamber 101 according to the use requirements.

[0089] The gas premixing control unit 105 is connected to the premixing chamber 101 and is used to transport gas (specifically including oxygen, carbon dioxide and nitrogen, etc.) into the premixing chamber 101. Nutrient solution and gas are added to the premixing chamber 101 through the liquid replenishing chamber 104 and the gas premixing control unit 105, and the introduced nutrient solution and gas are mixed in the premixing chamber 101 to form a culture solution. The introduced oxygen can increase the dissolved oxygen content of the culture solution to meet the growth requirements of the culture; the introduced carbon dioxide reduces and adjusts the pH value of the culture solution to meet the growth requirements of the culture. Preferably, in this embodiment, the premixing chamber 101 and the gas premixing control unit 105 are both one, and in other embodiments, the premixing chamber 101 is multiple, the gas premixing control unit 105 is one, and the gas premixing control unit 105 is simultaneously connected to multiple premixing chambers 101, and can control the gas concentration in multiple premixing chambers 101, specifically controlling the dissolved oxygen concentration of the culture solution in the premixing chamber 101.

[0090] Preferably, the premixing unit 1 further comprises a pH detector 102, a dissolved oxygen detector 103 and a signal detector, wherein the pH detector 102 and the dissolved oxygen detector 103 are both arranged in the premixing chamber 101.

[0091] The signal detector can sense the signals of the pH detection element 102 and the dissolved oxygen detection element 103, and calculate and analyze the sensed signals to obtain the pH value and dissolved oxygen content of the culture solution in the premix chamber 101. As the pH value and dissolved oxygen content in the premix chamber 101 change, the signals detected by the pH detection element 102 and the dissolved oxygen detection element 103 will change. Specifically, the optical signals of the pH detection element 102 and the dissolved oxygen detection element 103 will change. The signal detector will periodically receive the optical signals on the surfaces of the pH detection element 102 and the dissolved oxygen detection element 103 to obtain data feedback, and calculate the pH value and dissolved oxygen content based on the feedback data. The signal detector can feed back the detected information to the rehydration chamber 104 and the gas premixing control unit 105. If the dissolved oxygen content is too high, the rehydration chamber 104 is controlled to deliver more nutrient solution to the premixing chamber 101 for dilution to reduce the dissolved oxygen content of the culture solution in the premixing chamber 101; if the dissolved oxygen content is too low, the gas premixing control unit 105 is controlled to pass oxygen into the premixing chamber 101 to increase the dissolved oxygen content of the culture solution in the premixing chamber 101. If the pH value is too high, the gas premixing control unit 105 is controlled to pass carbon dioxide to reduce the pH value of the culture solution in the premixing chamber 101; if the pH value is too low, the rehydration chamber 104 is controlled to deliver more nutrient solution to the premixing chamber 101 for dilution or pass alkali solution into the premixing chamber 101 to increase the pH value of the culture solution in the premixing chamber 101.

[0092] Specifically, the pH detector 102 is a pH electrode sheet, and the dissolved oxygen detector 103 is a dissolved oxygen electrode sheet, both of which are patch materials; the pH electrode sheet is arranged on the inner wall of the premixing chamber 101 or in the premixing chamber 101, and the dissolved oxygen detector 103 is arranged on the inner wall of the premixing chamber 101 or in the premixing chamber 101.

[0093] The premix chamber 101 is also provided with an exhaust port, which can exhaust the gas in the premix chamber 101 to ensure the stability of the air pressure in the premix chamber 101. The premix chamber 101 is provided with a stirring component, which can stir the liquid in the premix chamber 101 to mix it evenly and allow the gas to fully immerse into the liquid. The premix chamber 101 is also provided with an alkali addition port, through which alkali solution can be added to the premix chamber 101 to adjust the pH value of the culture solution in the premix chamber 101.

[0094] In the premixing chamber 101, the nutrient solution and oxygen are premixed to form a culture solution, and by controlling the corresponding variables (such as pH value and dissolved oxygen content), the culture solution formed by the mixture of the nutrient solution and oxygen is more suitable for the growth of the culture, and can fully provide the culture with the nutrients required for growth. Compared with the prior art of directly introducing the nutrient solution and oxygen into the culture chamber, the design of complex pipelines, valves and detectors is saved, and the system cost is reduced.

[0095] The culture unit includes a culture chamber 2, which is connected to the premix chamber 101, and the culture solution in the premix chamber 101 can be transported to the culture chamber 2. According to the relative setting position of the premix chamber 101 and the culture chamber 2 and the requirements of the transportation speed and transportation amount of the culture solution, it can be determined whether to set a power unit 5 between the premix chamber 101 and the culture chamber 2, and use the power unit 5 to transport the culture solution in the premix chamber 101 to the culture chamber 2. Preferably, in this embodiment, there is one culture chamber 2, that is, one premix chamber 101 corresponds to one culture chamber. In other embodiments, there are multiple culture chambers 2, and multiple culture chambers 2 are all connected to the premix chamber 101, that is, multiple culture chambers 2 are connected in parallel, and each culture chamber 2 can be connected to an exchange unit 3, and all culture chambers 2 can be connected to an exchange unit, or several culture chambers 2 supply one exchange unit 3. It can culture multiple cultures at the same time, which is convenient for ensuring the consistency of the cultures in the subsequent test process and can ensure the accuracy of the comparison of test results.

[0096] The culture chamber 2 is used to hold the culture and cultivate the culture. Specifically, the culture chamber 2 is provided with a sample addition port 201 and a sampling port 202. The culture can be added to the culture chamber 2 through the sample addition port 201. In addition, according to the experimental requirements, growth factors, drugs, etc. can be added to the culture chamber 2 through the sample addition port 201 during the culture process to promote or inhibit the growth of the culture. Enzymes, matrix gel treatment reagents, staining reagents, etc. can also be added to the system after the culture is completed to collect, process or characterize the culture. The sampling port 202 can be used to take samples of the culture or culture fluid for analysis during the culture process.

[0097] The exchange unit 3 includes an exchange chamber and a membrane assembly 301. The exchange chamber includes a first exchange chamber 302 and a second exchange chamber 303. The membrane assembly 301 is arranged between the first exchange chamber 302 and the second exchange chamber 303, and the first exchange chamber 302 and the second exchange chamber 303 are connected through the membrane assembly 301. The first exchange chamber 302 can not only receive the culture solution flowing out of the culture chamber 2, but also can flow the culture solution back to the culture chamber 2. The membrane assembly 301 is used to intercept and / or pass through some components in the culture solution. Specifically, the membrane assembly 301 can intercept the nutrients required by the culture in the culture solution in the first exchange chamber 302, such as serum, growth factors and enzymes, etc. The metabolites generated by the growth of the culture in the first exchange chamber 302, such as urea, carbon dioxide, etc., can penetrate into the second exchange chamber 303 through the membrane assembly 301, and then be discharged from the second exchange chamber 303.

[0098] The exchange unit 3 and the membrane assembly 301 can be arranged to intercept useful components in the culture solution while realizing perfusion culture, and flow back to the culture chamber 2 for reabsorption and utilization, thereby improving the utilization rate of the culture solution and reducing the culture cost. The exchange unit 3 is arranged outside the culture chamber 2, so that the effective area of ​​the membrane assembly 301 can be arranged larger and the structure is more flexible, thereby improving the exchange efficiency.

[0099] The collecting unit 4 is in communication with the second exchange chamber 303 and is used to collect the culture solution in the second exchange chamber 303. Whether to set a power unit 5 between the second exchange chamber 303 and the collecting unit 4 can be determined according to the relative setting positions of the second exchange chamber 303 and the collecting unit 4 and the requirements of the conveying speed and conveying amount of the culture solution. Preferably, in this embodiment, a power unit 5 is set between the collecting unit 4 and the second exchange chamber 303, and the power unit 5 is used to convey the culture solution in the second exchange chamber 303 to the collecting unit 4.

[0100] Specifically, the first exchange chamber 302 is provided with a first interface and a second interface, and both the first interface and the second interface are connected to the culture chamber 2. The first exchange chamber 302 can receive the culture solution flowing out of the culture chamber 2 through the first interface, and the first exchange chamber 302 can flow the culture solution back to the culture chamber 2 through the second interface. Preferably, in this embodiment, a power unit 5 is provided between the culture chamber 2 and the first interface and between the culture chamber 2 and the second interface. The power unit 5 between the culture chamber 2 and the first interface can transport the culture solution in the culture chamber 2 to the first exchange chamber 302 through the first interface; the power unit 5 between the culture chamber 2 and the second interface can transport the culture solution in the first exchange chamber 302 to the culture chamber 2 through the second interface.

[0101] Since the consumption rates of various components in the culture medium are different during the culture process, the solution of this embodiment can increase one or more components in a directional manner through the rehydration chamber 104, ensuring the balance of various components while reducing the overall liquid replacement steps, reducing costs, and reducing the probability of culture contamination. The rehydration chamber 104 replenishes the premix chamber 101, and then the premix chamber 101 transports the nutrient solution to the culture chamber 2; therefore, the concentration and components of the nutrient solution in the rehydration chamber 104 do not need to be formulated to be the same as the concentration and components of the culture medium required by the culture, and the volume of the rehydration chamber 104 can be made smaller. At the same time, the perfusion speed of the premix chamber 101 can be higher without worrying about the excessive consumption of the culture medium due to the increase in the perfusion speed, thereby improving the culture efficiency and increasing the types of cultures that can be cultured.

[0102] Specifically, the concentration of the culture solution components flowing out of the culture chamber 2 can be detected, and the components can be replenished through the rehydration chamber 104 in a targeted manner. The concentration detection can be performed by sampling detection, or by in-situ detection (such as infrared spectroscopy technology, fluorescence detection technology). Preferably, the present embodiment adopts fluorescence detection technology. The system can also be provided with an automatic sample addition module to automatically add nutrient components to the rehydration chamber 104 based on the above-mentioned concentration detection results.

[0103] Preferably, the culture unit further comprises a temperature control unit 6, which is used to control the temperature of the premixing chamber 101 and the culture chamber 2. Specifically, the premixing chamber 101 and the culture chamber 2 can be heated to raise the temperature of the culture solution in the premixing chamber 101 and the culture chamber 2 to a physiological temperature suitable for the growth of the culture, so as to culture the culture; when the temperature is too high, the premixing chamber 101 and the culture chamber 2 can be cooled to lower the temperature, so as to lower the temperature of the culture solution in the premixing chamber 101 and the culture chamber 2 to a physiological temperature suitable for the growth of the culture, so as to culture the culture. After the culture is completed, when the matrix gel needs to be removed and the culture is collected, the temperature control unit 6 can control the temperature of the culture solution in the culture chamber 2 to a first preset temperature, wherein the first preset temperature is the liquefaction temperature of the culture support structure, and the culture can be taken out through the sampling port 202 after the culture support structure is liquefied. The culture support structure forms a network support for the culture, which is conducive to the 3D growth of the culture. Culture support structures include but are not limited to matrix gel (liquefies at 2-8 degrees) and artificial synthetic gel (liquefies at room temperature, 25 degrees).

[0104] Specifically, the temperature control unit 6 includes a heating component, a cooling component and a temperature control module. The heating component and the cooling component are both electrically connected to the temperature control module. The temperature control module is used to control the heating component to heat the culture chamber 2 and to control the cooling component to cool the culture chamber 2.

[0105] The outer walls of the premixing chamber 101 and the culture chamber 2 are both provided with a heating component and a cooling component. The heating component includes a plurality of heating sheets 61, which are provided on the outer wall of the culture chamber 2. The heating sheet 61 can be a transparent heating plate, a metal heating plate, a semiconductor heating plate, a heating blanket or a heating sheet, etc. The cooling component includes a plurality of cooling sheets 62, which are provided on the outer wall of the culture chamber 2. The cooling sheets 62 can be semiconductor cooling sheets 62 or other cooling materials.

[0106] The heating plate 61 and the cooling plate 62 can be located at different positions of the culture chamber 2, for example: Figure 2 As shown, the heating plate 61 is located at the bottom of the culture chamber 2, and the cooling plate 62 is located at the side of the culture chamber 2; or, as shown in Figure 3 As shown, the heating sheet 61 is located at the side of the culture chamber 2, and the cooling sheet 62 is located at the bottom of the culture chamber 2. Of course, the heating sheet 61 and the cooling sheet 62 can also be located at the same position of the culture chamber 2, for example, both are located at the same side or bottom of the culture chamber 2.

[0107] As a preferred embodiment, in this embodiment, Figure 4As shown, the heating sheet 61 and the cooling sheet 62 are located on the same side or bottom wall of the culture chamber 2. Specifically, a plurality of heating sheets 61 are arranged at intervals, a plurality of cooling sheets 62 are arranged at intervals, a cooling sheet 62 is arranged between two adjacent heating sheets 61, and a heating sheet 61 is arranged between two adjacent cooling sheets 62. Moreover, the heating sheet 61 is in close contact with the cooling sheet 62 adjacent thereto, and the cooling sheet 62 is in close contact with the heating sheet 61 adjacent thereto, so that the energy of the heating sheet 61 can be transferred to the cooling sheet 62. The heating sheet 61 and the cooling sheet 62 are both made of materials with good thermal conductivity, and are structurally processed into an embeddable structure, so that the heating sheet 61 can be embedded between the cooling sheets 62, and the cooling sheet 62 can be embedded between the heating sheets 61.

[0108] When the culture chamber 2 needs to be heated, the heating sheet 61 is heated to a specified temperature under the control of the temperature control module. Since the cooling sheet 62 is a heat-conducting material and the heat of the heating sheet 61 can be transferred to the cooling sheet 62, the cooling sheet 62 is heated while the culture chamber 2 is heated, and the temperature of the cooling sheet 62 is quickly raised to the specified temperature, and then the heating sheet 61 and the cooling sheet 62 jointly heat the culture chamber 2. When the culture chamber 2 needs to be cooled, the cooling sheet 62 is cooled to a specified temperature under the control of the temperature control module. Since the heating sheet 61 is a heat-conducting material and the heat of the heating sheet 61 can be transferred to the cooling sheet 62, the cooling sheet 62 cools the culture chamber 2 while cooling the heating sheet 61, so that the temperature is quickly reduced to the specified temperature, and then the cooling sheet 62 and the heating sheet 61 jointly cool the culture chamber 2.

[0109] The heating fins 61 and the cooling fins 62 are arranged alternately and can transfer heat, so that the two can heat and cool the culture chamber 2 together, thereby increasing the heat exchange area and improving the heat exchange efficiency.

[0110] The temperature control unit 6 also includes a temperature detection member 63, which can be a temperature sensor. The temperature detection member 63 is electrically connected to the temperature control module, and can be arranged on the inner wall of the culture chamber 2 or on the outer wall of the culture chamber 2, for detecting the temperature of the culture liquid in the culture chamber, and feeding back the detection value to the temperature control module, and the temperature control module compares the temperature value detected by the temperature detection member 63 with the actual required set value, so as to control the heating component to heat the temperature culture chamber 2 or control the refrigeration component to cool the culture chamber 2.

[0111] The structure and arrangement of the heating component, the cooling component and the temperature detection element 63 in the premixing chamber 101 are the same as those in the culture chamber 2 and will not be described in detail here.

[0112] Optionally, continue to refer to Figure 1The culture unit also includes a mixing module 7, which is specifically a mechanical shaking mechanism, which is used to shake the culture solution in the culture chamber 2, specifically by shaking the culture chamber 2 to shake the culture solution in the culture chamber 2, so as to provide more sufficient exchange opportunities for the culture solution and the culture. The shaking amplitude and speed of the mixing module 7 can be matched with the speed of conveying the culture solution into the culture chamber 2. For example, a higher culture solution conveying speed can promote the exchange opportunities between the culture solution and the culture, but will cause excessive consumption of the culture solution. A lower culture solution conveying speed combined with large-scale mechanical shaking can simultaneously achieve the purpose of renewing the culture solution and increasing the exchange opportunities between the nutrient solution and the culture, thereby promoting the exchange of nutrients.

[0113] Alternatively, if Figure 5 and Figure 6 As shown, the culture unit further includes a microscopic observation module 8, which includes a stage 801 and an observation assembly. The culture chamber 2 is placed on the stage 801, specifically, the culture chamber 2 is placed on the mixing module 7, and the mixing module 7 is placed on the stage 801. The stage 801 can place at least one culture chamber 2. In this embodiment, one placement station is provided on the stage 801, and the mixing module 7 is placed on the placement station; in other embodiments, a plurality of placement stations are provided on the stage 801 (see Figure 7 ), a mixing module 7 can be placed on each placement station.

[0114] The observation assembly is used to observe the culture in the culture chamber 2. Specifically, the observation assembly includes an objective lens 803, an eyepiece 804, and a camera 807 (specifically a CCD or CMOS camera). The microscopic observation module 8 also includes a frame 802, the objective lens 803 is arranged on the frame 802, and it can move in the vertical direction. The eyepiece 804 is also arranged on the frame 802, and the culture in the culture chamber 2 can be observed through the eyepiece 804.

[0115] The rack 802 is provided with a first mechanical track 805, and a second mechanical track 806 is slidably provided on the first mechanical track 805, wherein the first mechanical track 805 and the second mechanical track 806 are arranged perpendicular to each other and are both located in a horizontal plane. The stage 801 is slidably provided on the second mechanical track 806, and the stage 801 can be adjusted to any position in the horizontal plane by sliding the stage 801 relative to the second mechanical track 806 and the second mechanical track 806 relative to the first mechanical track 805, so that the objective lens 803 faces the observation chamber, and the eyepiece 804 is used to focus on the culture in the culture chamber 2 to observe the culture. If image data needs to be recorded during the observation process, the camera 807 can be used to take pictures and record the image data.

[0116] Preferably, if Figure 7As shown, the microscopic observation module 8 also includes an automatic sample injector 809 and a sample injection track 810, wherein the automatic sample injection track 810 is arranged on the frame 802, and the automatic sample injector 809 is slidably arranged on the sample injection track 810, and the automatic sample injector 809 is driven by the sample injection driving member to slide on the sample injection track 810 to perform the sample injection work. The automatic sample injector 809 is mainly used for adding samples when the culture, reagents or drugs need to be added to the culture chamber 2, avoiding manual sample addition, ensuring the sanitary conditions of the culture chamber 2, and making the culture not easily contaminated.

[0117] Alternatively, if Figure 8 As shown, the in vitro life-sustaining perfusion culture system also includes a sterile control module 9, which includes a sterile workroom 901, a filtration component 903 and a sterilization component 904. At least the culture chamber 2 is arranged in the sterile workroom 901. In this embodiment, the premixing unit 1, the culture unit, the exchange unit 3, the collection unit 4 and the power unit 5 are all arranged in the sterile workroom 901, that is, the culture system of the culture except the sterile control module 9 is all located in the sterile workroom 901.

[0118] The sterile studio 901 is provided with a vent, a filter assembly 903 is provided at the vent, and an air intake fan 902 is provided on the vent. Gas is introduced into the sterile studio 901 through the air intake fan 902. The filter assembly 903 is used to filter the gas introduced into the sterile studio 901, so that dust and impurities cannot enter the sterile studio 901. The sterilization assembly 904 is used to sterilize and disinfect the sterile studio 901. Specifically, the sterilization assembly 904 is an ultraviolet lamp, which is provided at the air intake. The sterilization assembly 904 can sterilize the sterile studio 901 and the gas introduced into the sterile studio 901, which can prevent the sterile gas from entering the sterile studio 901.

[0119] This embodiment also discloses a control method for an in vitro life-sustaining perfusion culture system, which is directed to the above-mentioned in vitro life-sustaining perfusion culture system and includes the following steps:

[0120] The concentration of each component required for the culture in the culture solution in the culture chamber 2 is detected and / or the growth condition of the culture is determined. According to the detected concentration and / or the determined growth condition of the culture, the speed at which the premixing chamber 101 delivers the culture solution to the culture chamber 2 is controlled. At the same time, the speed at which the replenishing chamber 104 delivers the nutrient solution to the premixing chamber 1 is controlled, and the speed at which the gas premixing control unit 105 delivers the gas to the premixing chamber 101 is controlled.

[0121] Embodiment 2

[0122] This embodiment discloses an in vitro life-sustaining perfusion culture system, which includes a premixing unit 1 , a culture unit, an exchange unit 3 , a collection unit 4 and a power unit 5 .

[0123] The in vitro life-sustaining perfusion culture system in this embodiment is basically the same in structure as the in vitro life-sustaining perfusion culture system in Example 1, except that the culture unit does not include the temperature control unit 6 and the mixing unit, and the culture unit in this embodiment also includes a mixing temperature control module, which is used to control the temperature of the culture chamber 2 and shake the culture fluid in the culture chamber 2.

[0124] The mixing and temperature control module includes a mechanical shaking mechanism and a temperature control component, and the mechanical shaking mechanism is provided with a mounting surface. The temperature control component is used to control the temperature of the culture chamber 2, and can specifically heat the premixing chamber 101 and the culture chamber 2, so that the temperature of the culture solution in the premixing chamber 101 and the culture solution in the culture chamber 2 rises to a physiological temperature suitable for the growth of the culture, so as to cultivate the culture; after the culture is completed, when the matrix gel needs to be removed and the culture is collected, the temperature control component can make the culture solution in the culture chamber 2 drop to the liquefaction temperature of the matrix gel in the culture solution, so as to collect the culture. Specifically, the temperature control component includes a heating component, a cooling component and a temperature control module, and the heating component and the cooling component are both electrically connected to the temperature control module, and the temperature control module is used to control the heating component to heat the culture chamber 2, and is also used to control the cooling component to cool the culture chamber 2.

[0125] The heating component includes a plurality of heating sheets 61, and the heating sheets 61 are arranged on the outer wall of the culture chamber 2. The heating sheet 61 can be a transparent heating plate, a metal heating plate, a semiconductor heating plate, a heating blanket or a heating sheet, etc. The cooling component includes a plurality of cooling sheets 62, and the cooling sheets 62 are arranged on the outer wall of the culture chamber 2. The cooling sheets 62 can be semiconductor cooling sheets 62, or other cooling materials. Specifically, a plurality of heating sheets 61 are arranged at intervals, and a plurality of cooling sheets 62 are arranged at intervals. A cooling sheet 62 is arranged between two adjacent heating sheets 61, and a heating sheet 61 is arranged between two adjacent cooling sheets 62. Moreover, the heating sheet 61 is in close contact with the cooling sheet 62 adjacent thereto, and the cooling sheet 62 is in close contact with the heating sheet 61 adjacent thereto, so that the energy of the heating sheet 61 can be transferred to the cooling sheet 62. The heating sheet 61 and the cooling sheet 62 are both made of materials with good thermal conductivity, and are structured to be embeddable, so that the heating sheet 61 can be embedded between the cooling sheets 62, and the cooling sheets 62 can be embedded between the heating sheets 61. The heating sheet 61 and the cooling sheet 62 are arranged on the mounting surface in sequence, and are mainly used to heat or cool the bottom surface of the culture chamber 2, and then control the temperature of the culture solution in the culture chamber 2.

[0126] When the culture chamber 2 needs to be heated, the heating sheet 61 is heated to a specified temperature under the control of the temperature control module. Since the cooling sheet 62 is a heat-conducting material and the heat of the heating sheet 61 can be transferred to the cooling sheet 62, the cooling sheet 62 is heated while the culture chamber 2 is heated, and the temperature of the cooling sheet 62 is quickly raised to the specified temperature, and then the heating sheet 61 and the cooling sheet 62 jointly heat the culture chamber 2. When the culture chamber 2 needs to be cooled, the cooling sheet 62 is cooled to a specified temperature under the control of the temperature control module. Since the heating sheet 61 is a heat-conducting material and the heat of the heating sheet 61 can be transferred to the cooling sheet 62, the cooling sheet 62 cools the culture chamber 2 while cooling the heating sheet 61, so that the temperature is quickly reduced to the specified temperature, and then the cooling sheet 62 and the heating sheet 61 jointly cool the culture chamber 2.

[0127] The heating fins 61 and the cooling fins 62 are arranged alternately and can transfer heat, so that the two can heat and cool the culture chamber 2 together, thereby increasing the heat exchange area and improving the heat exchange efficiency.

[0128] The temperature control component also includes a temperature detection component 63, which can be a temperature sensor. The temperature detection component 63 is electrically connected to the temperature control module. The temperature detection component 63 can be set on the inner wall of the culture chamber 2 or on the outer wall of the culture chamber 2 to detect the temperature of the culture liquid in the culture chamber and feed the detection value back to the temperature control module. The temperature control module compares the temperature value detected by the temperature detection component 63 with the actual required set value to control the heating component to heat the temperature culture chamber 2 or control the refrigeration component to cool the culture chamber 2.

[0129] Embodiment 3

[0130] This embodiment discloses an in vitro life-sustaining perfusion culture system, which includes a premixing unit 1 , a culture unit, an exchange unit 3 , a collection unit 4 and a power unit 5 .

[0131] The in vitro life-sustaining perfusion culture system in this embodiment is substantially the same in structure as the in vitro life-sustaining perfusion culture system in Example 1, except that the culture unit in this embodiment does not include the mixing module 7, and the structure of the microscopic observation unit is also different. Fig. 9 As shown, the microscopic observation module 8 in this embodiment includes a stage 801, an observation component and a frame 802. The culture chamber 2 is placed on the stage 801, and the stage 801 is swingably set on the frame 802. The swinging stage 801 can shake the culture liquid in the culture chamber 2.

[0132] The culture chamber 2 is placed on the stage 801. The stage 801 can hold at least one culture chamber 2. In this embodiment, one placement station is provided on the stage 801, and the culture chamber 2 is placed on the placement station. In other embodiments, multiple placement stations are provided on the stage 801, and one culture chamber 2 can be placed on each placement station.

[0133] The observation assembly is used to observe the culture in the culture chamber 2. Specifically, the observation assembly includes an objective lens 803, an eyepiece 804 and a camera 807 (specifically a CCD camera). The microscopic observation module 8 also includes a frame 802, on which the objective lens 803 is arranged and can move in a vertical direction, and an eyepiece 804 is also arranged on the frame 802, through which the culture in the culture chamber 2 can be observed.

[0134] A first mechanical track 805 is provided on the frame 802, and a second mechanical track 806 is slidably provided on the first mechanical track 805, wherein the first mechanical track 805 and the second mechanical track 806 are arranged perpendicular to each other and are both located in a horizontal plane. A connecting portion 808 is installed at the bottom of the stage 801, and a rotating shaft is provided on the connecting portion 808. The stage 801 is rotatably connected to the rotating shaft, and the stage 801 can swing relative to the connecting portion 808. The connecting portion 808 is slidably provided on the second mechanical track 806 and can slide along the length direction of the second mechanical track 806. The microscopic observation module 8 also includes a driving member, whose output end is connected to the stage 801, for driving the stage 801 to shake, so as to shake the culture solution in the culture chamber 2. Provide more sufficient exchange opportunities for the culture solution and the culture. The shaking amplitude and speed of the stage 801 can be coordinated with the speed of delivering the culture solution to the culture chamber 2. For example, a higher culture solution delivery speed can promote the exchange opportunities between the culture solution and the culture, but it will cause excessive consumption of the culture solution. A lower culture solution delivery speed combined with large-scale mechanical shaking can simultaneously achieve the purpose of refreshing the culture solution and increasing the exchange opportunities between the nutrient solution and the culture, thereby promoting the exchange of nutrients.

[0135] The stage 801 can be adjusted to any position in the horizontal plane by sliding the stage 801 relative to the second mechanical track 806 and the second mechanical track 806 relative to the first mechanical track 805, so that the objective lens 803 faces the observation chamber, and the eyepiece 804 is used to focus on the culture in the culture chamber 2 to observe the culture. If image data needs to be recorded during the observation process, the camera 807 can be used to take pictures and record the image data. During the observation process, the stage 801 can be shaken to put the culture in a better observation state for easy observation.

[0136] like Fig.10As shown, preferably, the microscopic observation module 8 further includes an automatic sample injector 809 and a sample injection track 810, wherein the automatic sample injection track 810 is arranged on the frame 802, and the automatic sample injector 809 is slidably arranged on the sample injection track 810, and the automatic sample injector 809 is driven by the sample injection driving member to slide on the sample injection track 810 to perform the sample injection work. The automatic sample injector 809 is mainly used for adding samples when the culture, reagents or drugs need to be added to the culture chamber 2, avoiding manual sample addition, ensuring the sanitary conditions of the culture chamber 2, and making the culture not easily contaminated.

[0137] Embodiment 4

[0138] like Fig.11 As shown, this embodiment discloses an in vitro life-sustaining perfusion culture system, including a premixing unit 1 , a culture unit, an exchange unit 3 , a collection unit 4 and a power unit 5 .

[0139] The in vitro life-sustaining perfusion culture system in this embodiment is basically the same in structure as the in vitro life-sustaining perfusion culture system in Example 1, except that the first exchange chamber 302 does not directly reflux the culture fluid to the culture chamber 2, but directly refluxes the culture fluid to the premixing chamber 101, and a power unit 5 is arranged between the first exchange chamber 302 and the premixing chamber 101, and the power unit 5 is used to transport the culture fluid in the second exchange chamber 302 to the premixing chamber 101.

[0140] Embodiment 5

[0141] like Fig.12 As shown, this embodiment discloses an in vitro life-sustaining perfusion culture system, which includes a premixing unit 1, a culture unit, an exchange unit 3, a fluid infusion unit 11, a collection unit 4 and a power unit 5.

[0142] Among them, at least one of the premixing unit 1, the culture chamber 2 of the culture unit, the exchange unit 3 and the collection unit 4 is a disposable consumable, which is treated as waste after one use, thereby ensuring the sanitary conditions during the perfusion culture process and avoiding the contamination of the premixing unit 1, the culture chamber 2, the exchange unit 3 and the collection unit 4, which may affect the normal perfusion culture.

[0143] The premixing unit 1 includes a premixing chamber 101 and a gas premixing control unit 105. The culture solution contains nutrients required for the growth of the culture, such as culture medium, factors, drugs and enzymes. The gas premixing control unit 105 is connected to the premixing chamber 101 and is used to transport gas (specifically including oxygen, carbon dioxide and nitrogen, etc.) into the premixing chamber 101. The premixing chamber 101 can receive a nutrient solution. The specific method of receiving the nutrient solution will be introduced later and will not be described in detail here. Gas is added to the premixing chamber 101 through the gas premixing control unit 105, and the nutrient solution and gas introduced are mixed in the premixing chamber 101 to form a culture solution. The oxygen introduced can increase the dissolved oxygen content of the culture solution to meet the growth requirements of the culture; the carbon dioxide introduced reduces and adjusts the pH value of the culture solution to meet the growth requirements of the culture. Preferably, in the present embodiment, there is one premixing chamber 101 and one gas premixing control unit 105. In other embodiments, there are multiple premixing chambers 101 and one gas premixing control unit 105. The gas premixing control unit 105 is connected to multiple premixing chambers 101 at the same time, and can control the gas concentration in multiple premixing chambers 101, specifically control the dissolved oxygen concentration of the culture solution in the premixing chamber 101.

[0144] Preferably, the signal detector can sense the signals of the pH detector 102 and the dissolved oxygen detector 103, and calculate and analyze the sensed signals to obtain the pH value and dissolved oxygen content of the culture solution in the premix chamber 101. As the pH value and dissolved oxygen content in the premix chamber 101 change, the signals detected by the pH detector 102 and the dissolved oxygen detector 103 will change. Specifically, the optical signals of the pH detector 102 and the dissolved oxygen detector 103 will change. The signal detector will regularly receive the optical signals on the surface of the pH detector 102 and the dissolved oxygen detector 103 to obtain data feedback, and calculate the pH value and dissolved oxygen content based on the feedback data. The signal detector can feed back the detected information to the gas premix control unit 105. If the dissolved oxygen content is too low, the gas premix control unit 105 will introduce oxygen into the premix chamber 101 to increase the dissolved oxygen content of the culture solution in the premix chamber 101. If the pH value is too high, the gas premix control unit 105 will be controlled to introduce carbon dioxide to reduce the pH value of the culture solution in the premix chamber 101. If the pH value is too low, an alkaline solution is introduced into the premixing chamber 101 to increase the pH value of the culture solution in the premixing chamber 101 .

[0145] Specifically, the pH detector 102 is a pH electrode sheet, and the dissolved oxygen detector 103 is a dissolved oxygen electrode sheet, both of which are patch materials; the pH electrode sheet is arranged on the inner wall of the premixing chamber 101 or in the premixing chamber 101, and the dissolved oxygen detector 103 is arranged on the inner wall of the premixing chamber 101 or in the premixing chamber 101.

[0146] The premix chamber 101 is also provided with an exhaust port, which can exhaust the gas in the premix chamber 101 to ensure the stability of the air pressure in the premix chamber 101. The premix chamber 101 is provided with a stirring component, which can stir the liquid in the premix chamber 101 to mix it evenly and allow the gas to fully immerse into the liquid. The premix chamber 101 is also provided with an alkali addition port, through which alkali solution can be added to the premix chamber 101 to adjust the pH value of the culture solution in the premix chamber 101.

[0147] In the premixing chamber 101, the nutrient solution and oxygen are premixed to form a culture solution, and by controlling the corresponding variables (such as pH value and dissolved oxygen content), the culture solution formed by the mixture of the nutrient solution and oxygen is more suitable for the growth of the culture, and can fully provide the culture with the nutrients required for growth. Compared with the prior art of directly introducing the nutrient solution and oxygen into the culture chamber, the design of complex pipelines, valves and detectors is saved, and the system cost is reduced.

[0148] The culture unit includes a culture chamber 2, which is connected to the premix chamber 101, and the culture solution in the premix chamber 101 can be transported to the culture chamber 2. According to the relative setting position of the premix chamber 101 and the culture chamber 2 and the requirements of the transportation speed and transportation amount of the culture solution, it can be determined whether a power unit 5 is provided between the premix chamber 101 and the culture chamber 2, and the power unit 5 is used to transport the culture solution in the premix chamber 101 to the culture chamber 2. Preferably, in this embodiment, there is one culture chamber 2, that is, one premix chamber 101 corresponds to one culture chamber. In other embodiments, there are multiple culture chambers 2, and multiple culture chambers 2 are all connected to the premix chamber 101, that is, multiple culture chambers 2 are connected in parallel, and each culture chamber 2 can be connected to an exchange unit 3, and all culture chambers 2 can be connected to one exchange unit, or several culture chambers 2 supply one exchange unit 3. It can culture multiple cultures at the same time, which is convenient for ensuring the consistency of the cultures in the subsequent test process and can ensure the accuracy of the comparison of test results.

[0149] The culture chamber 2 is used to hold the culture and cultivate the culture. Specifically, the culture chamber 2 is provided with a sample addition port 201 and a sampling port 202. The culture can be added to the culture chamber 2 through the sample addition port 201. In addition, according to the experimental requirements, growth factors, drugs, etc. can be added to the culture chamber 2 through the sample addition port 201 during the culture process to promote or inhibit the growth of the culture. Enzymes, matrix gel treatment reagents, staining reagents, etc. can also be added to the system after the culture is completed to collect, process or characterize the culture. The sampling port 202 can be used to take samples of the culture or culture fluid for analysis during the culture process.

[0150] The exchange unit 3 includes an exchange chamber and a membrane assembly 301. The exchange chamber includes a first exchange chamber 302 and a second exchange chamber 303. The membrane assembly 301 is disposed between the first exchange chamber 302 and the second exchange chamber 303, and the first exchange chamber 302 and the second exchange chamber 303 are connected through the membrane assembly 301. The membrane assembly 301 is used to intercept and / or permeate components inside the culture solution. Specifically, the membrane assembly 301 can intercept useful components in the culture solution in the first exchange chamber 302 in the first exchange chamber 302, such as serum, growth factors, and enzymes, etc. Metabolites generated by the growth of the culture in the first exchange chamber 302, such as urea, carbon dioxide, etc., can penetrate through the membrane assembly 301 into the second exchange chamber 303, and then be discharged from the second exchange chamber 303.

[0151] The culture chamber 2 and the premix chamber 101 are both connected to the first exchange chamber 302. The first exchange chamber 302 can not only receive the culture solution flowing out of the culture chamber 2, but also flow the culture solution back to the premix chamber 101. Specifically, the first exchange chamber 302 is provided with a first interface and a second interface. The first interface is connected to the culture chamber 2, and the second interface is connected to the premix chamber 101. The first exchange chamber 302 can receive the culture solution flowing out of the culture chamber 2 through the first interface, and the first exchange chamber 302 can flow the culture solution back to the premix chamber 101 through the second interface. Preferably, in this embodiment, a power unit 5 is provided between the culture chamber 2 and the first interface and between the premix chamber 101 and the second interface. The power unit 5 between the culture chamber 2 and the first interface can transport the culture solution in the culture chamber 2 to the first exchange chamber 302 through the first interface; the power unit 5 between the premix chamber 101 and the second interface can transport the culture solution in the first exchange chamber 302 to the premix chamber 101 through the second interface.

[0152] The exchange unit 3 and the membrane assembly 301 can be arranged to intercept useful components in the culture solution while realizing perfusion culture, and flow back to the premixing chamber 101, and then be transported to the culture chamber 2 for reabsorption and utilization, thereby improving the utilization rate of the culture solution and reducing the culture cost. The exchange unit 3 is arranged outside the culture chamber 2, so that the effective area of ​​the membrane assembly 301 can be arranged larger and the structure is more flexible, thereby improving the exchange efficiency.

[0153] The collecting unit 4 is in communication with the second exchange chamber 303 and is used to collect the culture solution in the second exchange chamber 303. Whether a power unit 5 is provided between the second exchange chamber 303 and the collecting unit 4 can be determined according to the relative arrangement positions of the second exchange chamber 303 and the collecting unit 4 and the requirements for the delivery speed and delivery amount of the culture solution. Preferably, in this embodiment, a power unit 5 is provided between the collecting unit 4 and the second exchange chamber 303, and the power unit 5 is used to deliver the culture solution in the second exchange chamber 303 to the collecting unit 4.

[0154] The rehydration unit 11 is connected to the second exchange chamber 303 and is used to transport nutrient solution to the second exchange chamber 303. At least some components in the nutrient solution in the second exchange chamber 303 can penetrate the membrane assembly 301 into the first exchange chamber 302. According to the relative setting position of the second exchange chamber 303 and the rehydration unit 11 and the requirements of the transport speed and transport amount of the nutrient solution, it can be determined whether to set a power unit 5 between the rehydration unit 11 and the second exchange chamber 303. As a preference, in this embodiment, a power unit 5 is set between the rehydration unit 11 and the second exchange chamber 303, and the power unit 5 is used to transport nutrient solution into the second exchange chamber 303. The concentration of the nutrient solution is greater than the concentration of the culture solution in the first exchange chamber 302. The concentration difference between the two can drive the components in the nutrient solution to move through the membrane assembly 301 to the first exchange chamber 302, and then transported to the nutrient solution in the premix chamber 101, where the nutrient solution and gas are mixed to form the culture solution.

[0155] Since the consumption rates of various components in the culture medium are different during the culture process, the solution of this embodiment can increase one or more components in a directional manner through the rehydration unit 11, ensure the balance of various components, reduce the overall liquid replacement steps, reduce costs, and reduce the probability of culture contamination. The rehydration unit 11 replenishes the second exchange chamber 303, and then the first exchange chamber 302 transports the nutrient solution to the culture chamber 2; therefore, the concentration and components of the nutrient solution in the rehydration unit 11 do not need to be formulated to be the same as the concentration and components of the culture medium required by the culture, and the volume of the rehydration unit 11 can be made smaller. At the same time, the perfusion speed of the premix chamber 101 can be higher without worrying about the excessive consumption of the culture medium due to the increase in the perfusion speed, thereby improving the culture efficiency and increasing the types of cultures that can be cultured.

[0156] Specifically, the concentration of the culture solution components flowing out of the culture chamber 2 can be detected and replenished in a targeted manner through the rehydration unit 11. The concentration detection can be performed by sampling detection, or by in-situ detection (such as infrared spectroscopy technology, fluorescence detection technology). Preferably, the present embodiment adopts fluorescence detection technology. The system can also be provided with an automatic sample addition module to automatically add nutrient components to the rehydration unit 11 based on the above-mentioned concentration detection results.

[0157] Preferably, the culture unit further comprises a temperature control unit 6 and a microscopic observation module 8. The temperature control unit 6 and the microscopic observation module 8 have the same structure as the temperature control unit 6 and the microscopic observation module 8 in the first embodiment, and will not be described in detail here.

[0158] Optionally, the in vitro life-sustaining perfusion culture system further includes a sterility control module 9 . The structure of the sterility control module 9 is the same as that of the sterility control module 9 in the first embodiment, and will not be described in detail here.

[0159] This embodiment also discloses a control method for an in vitro life-sustaining perfusion culture system, which is directed to the above-mentioned in vitro life-sustaining perfusion culture system and includes the following steps:

[0160] Detect the concentration of the components required for the culture in the culture solution in the culture chamber 2 and / or determine the growth of the culture, and control the speed at which the premixing chamber 101 delivers the culture solution to the culture chamber 2 and the speed at which the liquid replenishing unit 11 delivers the nutrient solution to the second exchange chamber 303 according to the detected concentration and / or the determined growth of the culture.

[0161] Embodiment 6

[0162] like Fig.13 As shown, this embodiment discloses an in vitro life-sustaining perfusion culture system, which includes a premixing unit 1, a culture unit, an exchange unit 3, a fluid infusion unit 11, a collection unit 4 and a power unit 5.

[0163] The in vitro life-sustaining perfusion culture system in this embodiment is basically the same in structure as the in vitro life-sustaining perfusion culture system in Example 1, except that the first exchange chamber 302 does not directly flow the culture fluid back to the premixing chamber 101, but directly flows the culture fluid back to the culture chamber 2, and a power unit 5 is arranged between the first exchange chamber 302 and the culture chamber 2, and the power unit 5 is used to transport the culture fluid in the second exchange chamber 302 to the culture chamber 2. At this time, the premixing chamber 101 also keeps transporting the culture fluid to the culture chamber 2, and continuously transports the gas required for the growth of the culture into the culture chamber 2.

[0164] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An in vitro life-sustaining perfusion culture system, characterized in that: include: A premixing unit (1), comprising a premixing chamber (101), into which a nutrient solution and a gas are added, and mixed in the premixing chamber (101) to form a culture solution; A culture unit, comprising a culture chamber (2), the culture chamber (2) being in communication with the premixing chamber (101), the culture liquid in the premixing chamber (101) being capable of being transported into the culture chamber (2), the culture chamber (2) being used to contain a culture and cultivate the culture; An exchange unit (3), comprising an exchange chamber and a membrane assembly (301), wherein the exchange chamber comprises a first exchange chamber (302) and a second exchange chamber (303), wherein the membrane assembly (301) is disposed between the first exchange chamber (302) and the second exchange chamber (303), and the first exchange chamber (302) and the second exchange chamber (303) are connected via the membrane assembly (301), wherein the culture chamber (2) and the premix chamber (101) are both connected to the first exchange chamber (302), wherein the first exchange chamber (302) can both receive the culture fluid flowing out of the culture chamber (2) and transport the culture fluid to the premix chamber (101) and / or the culture chamber (2), and wherein the membrane assembly (301) is used for intercepting and / or transmitting some components in the culture fluid; a liquid replenishing unit (11), which is in communication with the second exchange chamber (303) and is used to transport nutrient solution to the second exchange chamber (303); the liquid replenishing unit (11) is capable of directionally transporting one or more components required by the culture into the second exchange chamber (303); at least part of the components in the nutrient solution is capable of permeating through the membrane assembly (301) into the first exchange chamber (302); A microscopic observation module (8), comprising a stage (801) and an observation component, wherein the culture chamber (2) is placed on the stage (801), the stage (801) is capable of placing at least one culture chamber (2), and the observation component is used to observe the culture in the culture chamber (2); The microscopic observation module (8) further comprises a frame (802), the object stage (801) being swingably arranged on the frame (802), and the object stage (801) being able to drive the culture chamber (2) to swing synchronously; The shaking amplitude and speed of the loading platform (801) are coordinated with the speed of conveying the culture medium into the culture chamber (2). A lower culture medium conveying speed combined with large-scale mechanical shaking can simultaneously renew the culture medium and increase the exchange opportunities between the nutrient solution and the culture medium, thereby promoting the exchange of nutrients.

2. The in vitro life-sustaining perfusion culture system according to claim 1, characterized in that: The first exchange chamber (302) is provided with a first interface and a second interface, the first interface is in communication with the culture chamber (2), and the second interface is in communication with the premixing chamber (101) and / or the culture chamber (2); The culture solution in the culture chamber (2) can flow to the first exchange chamber (302) through the first interface, and the culture solution in the first exchange chamber (302) can flow to the premixing chamber (101) and / or the culture chamber (2) through the second interface.

3. The in vitro life-sustaining perfusion culture system according to claim 2, characterized in that: The invention also comprises a power unit (5), wherein the power unit (5) is arranged between the premixing chamber (101) and the culture chamber (2), the power unit (5) is arranged between the culture chamber (2) and the first interface, and the power unit (5) is arranged between the premixing chamber (101) and the second interface and / or between the culture chamber (2) and the second interface.

4. The in vitro life-sustaining perfusion culture system according to claim 1, characterized in that: It also comprises a collecting unit (4), which is in communication with the second exchange chamber (303) and is used to collect liquid in the second exchange chamber (303).

5. The in vitro life-sustaining perfusion culture system according to claim 4, characterized in that: At least one of the premixing unit (1), the culture chamber (2), the exchange unit (3) and the collection unit (4) is a disposable consumable.

6. The in vitro life-sustaining perfusion culture system according to claim 4, characterized in that: There are a plurality of culture chambers (2), and the plurality of culture chambers (2) are all connected to the premixing chamber (101); Each of the culture chambers (2) is connected to one of the exchange units (3), or all of the culture chambers (2) are connected to one of the exchange units.

7. The in vitro life-sustaining perfusion culture system according to claim 1, characterized in that: Also includes: a temperature control unit (6), comprising a refrigeration component and a temperature control module, wherein the refrigeration component is electrically connected to the temperature control module, and the temperature control module is used to control the refrigeration component to cool the culture chamber (2) to a first preset temperature, wherein the first preset temperature is a liquefaction temperature of a culture support structure; The culture chamber (2) is provided with a sampling port (202), and the sampling port (202) is used to take out the culture after the culture support structure is liquefied.

8. The in vitro life-sustaining perfusion culture system according to claim 1, characterized in that: The premixing unit (1) further comprises a pH detector (102), a dissolved oxygen detector (103) and a signal detector. The pH detector (102) and the dissolved oxygen detector (103) are both arranged in the premixing chamber (101). The signal detector can sense the signals fed back by the pH detector (102) and the dissolved oxygen detector (103) to obtain the pH value and dissolved oxygen content of the culture solution in the premixing chamber (101).

9. The in vitro life-sustaining perfusion culture system according to claim 8, characterized in that: The pH detection element (102) is a pH electrode sheet, and the dissolved oxygen detection element (103) is a dissolved oxygen electrode sheet; The pH electrode sheet is arranged on the inner wall of the premixing chamber (101) or in the premixing chamber (101), and the dissolved oxygen detector (103) is arranged on the inner wall of the premixing chamber (101) or immersed in the liquid in the premixing chamber (101).

10. The in vitro life-sustaining perfusion culture system according to claim 1, characterized in that: The premixing unit (1) further comprises a gas premixing control unit (105), which is in communication with the premixing chamber (101) and is used for conveying gas into the premixing chamber (101).

11. The in vitro life-sustaining perfusion culture system according to claim 10, characterized in that: There are a plurality of premixing chambers (101), and there is one gas premixing control unit (105). The gas premixing control unit is simultaneously connected to the plurality of premixing chambers (101) and is capable of controlling the gas concentrations in the plurality of premixing chambers (101).

12. The in vitro life-sustaining perfusion culture system according to claim 1, characterized in that: Also includes: A sterile control module (9), comprising a sterile workroom (901), a filtering component (903) and a sterilizing component (904), wherein at least the culture chamber (2) is arranged in the sterile workroom (901), the filtering component (903) is used to filter the gas entering the sterile workroom (901), and the sterilizing component (904) is used to sterilize the sterile workroom (901).

13. A control method for an in vitro life-sustaining perfusion culture system, characterized in that: The in vitro life-sustaining perfusion culture system according to any one of claims 1 to 12 comprises the following steps: The concentration of the components required for the culture in the culture solution of the culture chamber (2) is detected and / or the growth condition of the culture is determined, and the speed at which the premixing chamber (101) delivers the culture solution to the culture chamber and the speed at which the liquid replenishing unit (11) delivers the nutrient solution to the second exchange chamber (303) are controlled according to the detected concentration and / or the determined growth condition of the culture.

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