System and method for producing a target substance

Abstract

The present application relates to a system and method for producing a target substance. The system includes a culture device, a first culture solution supply device, a second culture solution supply device, and a culture supply unit. The culture device is partitioned into a growth region and a culture solution circulation region via a filtration membrane, the first culture solution supply device is configured to be in fluid communication with the growth region via a first inlet and a first outlet, the second culture solution supply device is configured to be in fluid communication with the culture solution circulation region via a second inlet and a second outlet, and the culture supply unit is configured to be in fluid communication with the growth region via the first inlet. The system can efficiently produce a target substance with a high yield using a simple process.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a Chinese patent application filed on November 16, 2022, bearing application number 2022114373492 and entitled "System and method for producing target substance," the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] This application relates to the field of biotechnology, and more particularly to systems and methods for producing a substance of interest. [Background technology]

[0003] The production of target substances is usually a process of rapidly growing a target substance using artificially created culture conditions (such as a suitable medium and culture temperature). Taking cell culture as an example, it is a process in which cells are incubated and cultured under specific in vitro conditions that mimic the physiological environment in a living body, allowing the cells to survive and proliferate. To ensure smooth cell growth, the cell growth environment in the cell culture process must be periodically monitored. In conventional cell culture, cells are generally inoculated into a reactor, and then culture medium is supplied to the cells or tissues through a transport pipe to maintain their growth.

[0004] Currently, flasks are often used as a culture device to produce cells or their target metabolites (e.g., extracellular vesicles (EVs)). However, the drawbacks are that the culture device takes up a large amount of space, the concentration of cells or their target metabolites in the produced culture solution is too low, and complex downstream processing is required to concentrate the produced cells or their target metabolites. At the same time, this culture method requires complex manual operations, which can contaminate the cell culture solution and cannot guarantee batch-to-batch consistency of the product. Additionally, current production processes have drawbacks, such as a small surface-to-volume ratio, material transport that relies solely on diffusion, unfavorable local conditions, and a tendency for waste to accumulate.

[0005] Therefore, there remains a need for a system and method that efficiently obtains high content, low contamination of target materials using a simple process. Summary of the Invention [Problem to be solved by the invention]

[0006] SUMMARY OF THE INVENTION An object of the present embodiment is to provide a system and method for producing a target substance. [Means for solving the problem]

[0007] In one aspect, the present application provides a system for producing a target substance, the system comprising: a culture device a, which is partitioned into a growth region and a culture solution circulation region via a filtration membrane and includes a first inlet, a first outlet, and a second inlet, a second outlet; a first culture solution supply device b configured to be in fluid communication with the growth region via the first inlet and the first outlet; a second culture solution supply device c configured to be in fluid communication with the culture solution circulation region via the second inlet and the second outlet; a culture supply unit d configured to be in fluid communication with the growth region via the first inlet.

[0008] In an embodiment of the present application, the system further includes a plurality of perfusion pumps e configured to be positioned between the culture device and the first culture solution supply device and / or between the culture device and the second culture solution supply device.

[0009] In an embodiment of the present application, a first valve is provided between the first culture solution supply device and the first inlet, and a second valve is provided between the first culture solution supply device and the first outlet.

[0010] In an embodiment of the present application, a third valve is provided between the second culture solution supply device and the second inlet, and a fourth valve is provided between the second culture solution supply device and the second outlet.

[0011] In an embodiment of the present application, the culture supply unit and the first culture solution supply device are in fluid communication with each other via a fifth valve; The first culture solution supply device and the second culture solution supply device are in fluid communication via a sixth valve.

[0012] In some embodiments, the system further includes a plurality of sensors configured to sense, for example, a fluid temperature, concentration, flow rate, etc., between the culture device and the first culture solution supply device, for example, disposed in a first flow path between the culture device and the first culture solution supply device; and / or The sensor is configured to sense, for example, the temperature, concentration, flow rate, etc. of the fluid between the culture device and the second culture solution supply device, and is, for example, positioned in a second flow path between the culture device and the second culture solution supply device.

[0013] In an embodiment of the present application, the sensor may be selected from one or more of a pressure sensor, a temperature sensor, a dissolved oxygen sensor, a pH acidity / alkalinity sensor, and an air bubble detector.

[0014] In an embodiment of the present application, the system of the present application further includes an oxygenator configured to supply oxygen to the flow path between the second culture solution supply device and the culture device, for example, disposed in the second flow path between the second culture solution supply device and the culture device.

[0015] In an embodiment of the present application, the system of the present application further includes a plurality of sampling units, the plurality of sampling units being in fluid communication with one or more of the first inlet, the first outlet, the second inlet, or the second outlet.

[0016] In embodiments of the present application, the system further includes a waste collection unit configured to be in fluid communication with the incubation device via the first outlet or the second outlet.

[0017] In an embodiment of the present application, the first culture solution supply device includes a first culture medium supply unit and a first pretreatment solution supply unit, and / or The second culture solution supplying device includes a second culture medium supplying unit and a second pretreatment solution supplying unit.

[0018] In an embodiment of the present application, the first culture medium supply unit stores a first culture medium, and the first pretreatment liquid supply unit stores a first pretreatment liquid. The second culture medium supply unit stores a second culture medium, and the second pretreatment liquid supply unit stores a second pretreatment liquid. The first culture medium and the second culture medium may be the same or different, and the first pretreatment liquid and the second pretreatment liquid may be the same or different.

[0019] In embodiments of the present application, the filtration membrane may be a microfiltration membrane, an ultrafiltration membrane, or a nanofiltration membrane. In a specific embodiment, the filtration membrane is a hollow fiber tube.

[0020] In a specific embodiment, the growth zone is the exterior space of the hollow fiber tube and the medium circulation zone is the interior space of the hollow fiber tube.

[0021] In a specific embodiment, the growth zone is the interior space of the hollow fiber tube and the nutrient medium circulation zone is the exterior space of the hollow fiber tube.

[0022] In an embodiment of the present application, the surface of the filtration membrane corresponding to the growth region is treated with a coating solution.

[0023] In an embodiment of the present application, the coating solution is an aqueous solution or salt buffer containing one or more of fibronectin, vitronectin, osteopontin, collagen, gelatin, laminin, or polylysine.

[0024] In another aspect, the present application provides a method for producing a substance of interest, the method comprising: (a) providing a culture to a growth region of a culture device; (b) providing a first culture solution to the growth region; and (c) supplying the second culture solution to a culture solution circulation region of the culture device, the culture solution circulation region and the growth region being separated by a filtration membrane.

[0025] In an embodiment of the present application, the method includes, prior to step (a), rinsing the culture device with a pretreatment solution.

[0026] In a specific embodiment, the pretreatment solution may be an aqueous solution or a salt buffer containing one or more of fibronectin, vitronectin, osteopontin, collagen, gelatin, laminin, or polylysine.

[0027] In an embodiment of the present application, the method further includes a step of inverting the culture device after step (a) and before step (b), thereby achieving sufficient adhesion of the culture, e.g., cells. In a specific embodiment, the inversion includes inverting the culture device 6 to 10 times at a frequency of 20 to 40 minutes per inversion.

[0028] In an embodiment of the present application, the first culture solution is supplied to the growth region at a flow rate of 0.05 to 0.30 mL / min.

[0029] In an embodiment of the present application, the second culture solution is supplied to the culture solution circulation region at a flow rate of 50 to 500 mL / min.

[0030] In an embodiment of the present application, during the cell culture period, the concentration of the target substance in the growth region is detected at a frequency of 1 to 4 days, and the contents of lactic acid and glucose in the growth region and the culture medium circulation region are detected, and / or the acidity / alkalinity, dissolved oxygen rate, and chamber pressure in the growth region and the culture medium circulation region are monitored online in real time.

[0031] In embodiments of the present application, the culture includes, but is not limited to, bacteria, adherent cells (mesenchymal stem cells, fibroblasts, endothelial cells, etc.), or suspension cells (immune cells, hematopoietic stem cells, neural stem cells, etc.). The culture may be a microcarrier that supports or encases the cells.

[0032] In an embodiment of the present application, the target substance may be the culture itself or a metabolic product of the culture, including, but not limited to, cells, extracellular vesicles, viruses, vaccines, antibodies, proteins, and other metabolic products. In a specific embodiment of the present application, the target substance is an exosome.

[0033] In an embodiment of the present application, the culture period may vary depending on the target substance being cultured, and may be, for example, from about several minutes to several days, or even several weeks. For example, it may be about 30 minutes, about 1 hour, about 5 hours, about 10 hours, about 15 hours, about 24 hours, or about 2 to 13 days, for example, about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days. In a specific embodiment, the culture period is 7 days. In a specific embodiment, the culture period is 9 days. In a specific embodiment, the culture period is 11 days.

[0034] In a specific embodiment of the present application, the density is about 1×10 6 cells / ml ~ 1 x 10 8 cells / ml, e.g., about 1-5 x 10 6 cells / ml, e.g., about 1-5 x 10 7 cells / ml, e.g., about 1 x 10 6 cells / ml, e.g., about 5 x 10 6 cells / ml, e.g., about 1 x 10 7 cells / ml, e.g., about 5 x 10 7 Inoculate the growth area with cells at 0.5x cells / ml.

[0035] The systems and methods of the present application are applicable to the cultivation of any material suitable for cultivation within the system, and are not limited to specific mesenchymal stem cells, including human mesenchymal stem cells, animal mesenchymal stem cells, and the like.

[0036] Additional aspects and advantages of the present application will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the present application. [Brief explanation of the drawings]

[0037] Other aspects and advantages of the present application will become more apparent from the following description, which is given by way of illustration only and not by way of limitation, with reference to the exemplary drawings in which:

[0038] [Figure 1] 1 is a diagram showing a system for producing a target substance according to one embodiment of the present application. [Figure 2] 1 is a schematic diagram showing a system for producing a target substance according to a specific embodiment of the present application. [Figure 3] 1 is a schematic diagram showing a system for producing a target substance according to an embodiment of the present application. [Figure 4] 1 is a flowchart illustrating a method for producing a target substance according to one embodiment of the present application. [Figure 5] 1 is a flow chart showing a method for producing a target substance according to another embodiment of the present application. [Figure 6] 1 is a graph showing the total number of viable cells harvested using flasks and the system of the present application. [Figure 7] 1 is a graph showing fold cell expansion relative to the initial cell number of viable cells harvested using flasks and the system of the present application. [Figure 8] 1 is a graph showing the viability of viable cells harvested using flasks and the system of the present application relative to the total cell number. [Figure 9] FIG. 1 shows the exosome yield expressed as protein per million cells harvested using flasks and the system of the present application. [Figure 10]FIG. 1 shows the exosome yield per 48 hours, expressed as protein, harvested using flasks and the system of the present application. [Figure 11] FIG. 1 shows the exosome yield expressed as particle counts per million cells harvested using flasks and the system of the present application. [Figure 12] FIG. 1 shows the exosome yield per 48 hours, expressed as particle number, harvested using flasks and the system of the present application. [Figure 13] FIG. 1 shows the chemotactic effect of exosomes harvested using a flask and the system of the present application on fibroblasts. [Figure 14] FIG. 1 shows that exosomes harvested using flasks and the system of the present application promote fibroblast proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0039] Hereinafter, the embodiments of the present application will be described in detail. Examples of the embodiments are illustrated in the drawings, and the same or similar reference numerals throughout the drawings indicate the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are merely illustrative and are intended to explain the present application, and should not be understood as limiting the present application.

[0040] In conventional technology, flasks are commonly used as cell reactors to produce target substances such as extracellular vesicles (EVs). Flask-based production methods have several characteristics: a small surface area-to-volume ratio, large incubator space, diffusion-only material transport, unfavorable local conditions, and a tendency for waste to accumulate. The harvested cell solution typically contains a large amount of medium, and the concentration of cells or their target metabolites is very low, often requiring complex purification processes to concentrate the product, resulting in yield loss.

[0041] To solve the above technical problems, embodiments of the present application provide an improved system for producing a target substance. Embodiments of the present application also provide a method for producing a target substance using the system of the present application. The system and method of the present application can efficiently produce a higher yield of a target substance, such as a target cell or a target metabolite thereof, using a simple process.

[0042] Specifically, the system of the present application is a membrane-based bioreactor. The membrane may be a microfiltration membrane, an ultrafiltration membrane, or a nanofiltration membrane. The membrane is specifically a hollow fiber tube. The hollow fiber tube allows the culture to attach and grow on the membrane, thereby maximizing the space utilization efficiency of the bioreactor and enabling efficient production of target substances, such as target cells and their target metabolites.

[0043] In the system of the present application, the culture solution in which the culture is cultivated flows cyclically, and as the culture solution flows, waste products generated by cell metabolism can be removed in a timely and sufficient manner, thereby maintaining high metabolic activity of the culture throughout the cultivation period and ensuring efficient production of target metabolites. More specifically, by using the system, a first medium is supplied to the growth region at a low flow rate and a second medium is supplied to the culture solution circulation region at a high flow rate, and the flow rate difference allows the nutrients and oxygen required for the culture to be permeated and supplied to the growth region, removing metabolic waste products and ensuring stable growth of the culture.

[0044] Furthermore, the system is sealed during production and the production process is automated, significantly reducing contamination from the external environment or human intervention and improving batch-to-batch consistency of the product.

[0045] Furthermore, the filtration membrane structure of the system concentrates the culture and the target metabolites produced by it in the growth area, thereby achieving a concentration effect, effectively reducing downstream purification treatment and simplifying the production process.

[0046] Furthermore, the system provides two independent sets of culture solution supply devices for the region inside the filtration membrane, specifically the inner region of the hollow fiber tube, and the region outside the filtration membrane, specifically the outer region of the hollow fiber tube, thereby enabling flexible and real-time adjustment of the culture solution based on the state of the culture cultivated in the growth region, ensuring optimal culture conditions, promoting the growth of the culture, and achieving a higher growth rate and higher yield of target substances, such as target cells or their target metabolic products.

[0047] Furthermore, compared with a system using only one culture solution supply device, the system using two culture solution supply devices in the present application can adjust the culture conditions in the growth area and the culture solution circulation area more sensitively and independently, ensuring better culture conditions and resulting in a higher growth rate and higher yield.

[0048] Hereinafter, a system and a method for producing a target substance according to an embodiment of the present application will be described in detail with reference to the drawings.

[0049] FIG. 1 is a diagram showing a system for producing a target substance according to one embodiment of the present application.

[0050] 1, the system 1 includes a culture device 100, a first culture solution supply device 200, a second culture solution supply device 300, and a culture supply unit 400. The culture device 100 includes a growth region 101 and a culture solution circulation region 102. The culture device 100 is partitioned into the growth region 101 and the culture solution circulation region 102 by a filtration membrane 103.

[0051] The first culture solution supply device 200 is in fluid communication with the growth region 101 via a first inlet 1011 and a first outlet 1012 and is configured to supply a first culture solution to the growth region 101 .

[0052] The second culture solution supply device 300 is in fluid communication with the culture solution circulation region 102 via a second inlet 1021 and a second outlet 1022 and is configured to supply a second culture solution to the culture solution circulation region 102.

[0053] The culture supply unit 400 is in fluid communication with said growth region 101 via a first inlet 1011 and is configured to supply a culture to the growth region 101 .

[0054] In an embodiment of the present application, the system 1 may further include a plurality of perfusion pumps. The plurality of perfusion pumps are located between the culture device 100 and the first culture solution supply device 200, and between the culture device 100 and the second culture solution supply device 300, and are configured to supply the culture, the first culture solution, and the second culture solution to the culture device 100, respectively. The system enables circulatory flow of the cell culture solution by the perfusion pumps.

[0055] In a specific embodiment, the filter membrane is a hollow fiber tube. Hereinafter, a culture device made of a hollow fiber tube is referred to as a hollow fiber tube reactor. By using the hollow fiber tube, the culture can adhere to and grow on the hollow fiber membrane, thereby improving the space utilization efficiency of the hollow fiber tube reactor and enabling efficient production of the target substance.

[0056] According to an embodiment of the present application, the hollow fiber tube has a pore size of about 500 nm or less. According to an embodiment of the present application, the hollow fiber tube has a pore size of about 20 to 200 nm, for example, 20 to 180 nm, for example, 20 to 170 nm, for example, 20 to 160 nm, for example, 20 to 150 nm, for example, 20 to 140 nm, for example, 20 to 130 nm, for example, 20 to 120 nm, for example, 20 to 110 nm, for example, 20 to 100 nm, for example, 20 to 90 nm, for example, 20 to 80 nm, for example, 20 to 70 nm, for example, 20 to 60 nm, for example, 20 to 50 nm, for example, 20 to 40 nm. In a specific embodiment, the hollow fiber tube has a pore size of 40 nm or less. In a specific embodiment, the hollow fiber tube has a pore size of about 80 nm or less. In a specific embodiment, the hollow fiber tube has a pore size of about 100 nm or less. In a specific embodiment, the hollow fiber tube has a pore size of about 120 nm or less. In a specific embodiment, the hollow fiber tube has a pore size of about 150 nm or less. In another embodiment, the pore size of the hollow fiber tube is adjustable depending on the size of the desired target substance.

[0057] The growth zone may be located in the exterior space of the hollow fiber tube, and the nutrient circulation zone may be located in the interior space of the hollow fiber tube.

[0058] In an alternative embodiment, the growth zone may be located in the interior space of the hollow fiber tube, while the nutrient circulation zone may be located in the exterior space of the hollow fiber tube.

[0059] If necessary, multiple hollow fiber tubes can be arranged in the hollow fiber tube reactor to ensure sufficient growth space, thereby allowing for the production of large amounts of amplified target substances, such as target cells or their target metabolites, in a limited environmental space.

[0060] According to an embodiment of the present application, a perfusion pump pumps the culture or a first culture solution into the growth region, and a perfusion pump pumps the second culture solution into the culture solution circulation region. In a specific embodiment, the perfusion pump is a peristaltic pump that generates a pulsed flow of the culture solution in the culture solution circulation channel, thereby achieving high mass exchange efficiency between the culture solution in the culture solution circulation region and the culture in the growth region.

[0061] According to an embodiment of the present application, a first valve is provided between a first culture solution supply device and a first inlet of a corresponding growth zone. A second valve is provided between the first culture solution supply device and a first outlet of a corresponding growth zone. A third valve is provided between a second culture solution supply device and a second inlet of a corresponding culture solution circulation zone. A fourth valve is provided between the second culture solution supply device and a second outlet of a corresponding culture solution circulation zone.

[0062] In the present embodiment, specific valve configurations allow for independent control of the culture solution supplied to the growth zone and the culture solution supplied to the culture solution circulation zone. This valve configuration also allows for adjustment of the circulating flow of the culture solution. Controlling the opening and closing of the valves also allows for adjustment of the pressure difference between the growth zone and the culture solution circulation zone, allowing for more flexible control of material exchange between the growth zone and the culture solution circulation zone.

[0063] This valve arrangement allows the culture, such as cells, to adhere more uniformly to the hollow fiber membrane. Specifically, after inoculating the prepared culture into the growth zone, closing the valves corresponding to both ends of the growth zone before starting the automatic culture mode and inverting the hollow fiber tube reactor at a set time interval can ensure more uniform adhesion of the culture.

[0064] Specifically, the inversion treatment involves inverting the culture device 6 to 10 times at a frequency of about 20 to 40 minutes per inversion, for example, 30 minutes per inversion, which significantly reduces aggregation and death of the cultured material, such as cells, and advantageously promotes high growth rate of the cultured material and efficient production of target substances, such as target cells or their target metabolites.

[0065] In the present embodiment, the culture supply unit and the first culture solution supply device are in fluid communication through a fifth valve, which allows the culture suspension and culture solution to be independently supplied to the growth zone of the hollow fiber tube reactor as needed.

[0066] In this embodiment, the first culture solution supplying device that supplies the culture solution to the growth zone and the second culture solution supplying device that supplies the culture solution to the culture solution circulation zone are fluidly connected via a sixth valve, which allows for more flexible adjustment of the culture solution supplying method to the hollow fiber tubes.

[0067] According to the above embodiment of the present application, the culture solution in the system for culturing a culture flows cyclically, supplying a first culture solution to the growth zone at a low flow rate of approximately 0.05 to 0.30 mL / min and a second culture solution to the culture solution circulation zone at a high flow rate of approximately 50 to 500 mL / min. The flow of the culture solution allows metabolic waste generated by the metabolism of the culture in the growth zone to be removed in a timely and sufficient manner, maintaining high metabolic activity of the culture throughout the culture period and ensuring efficient production. Furthermore, the system is sealed during production and the production process is automated, significantly reducing contamination from the external environment or human intervention and improving batch-to-batch consistency of the product.

[0068] In an embodiment of the present application, the system further includes a plurality of sensors. The sensors are configured to sense between the culture device and the first culture solution supply device, for example, disposed in a first flow path between the culture device and the first culture solution supply device. In another embodiment, the sensors are configured to sense between the culture device and the second culture solution supply device, for example, disposed in a second flow path between the culture device and the second culture solution supply device. The sensors may be selected from one or more of a pressure sensor, a temperature sensor, a dissolved oxygen sensor, a pH / acidity / alkalinity sensor, and an air bubble detector.

[0069] According to an embodiment of the present application, the pressure difference between the culture medium circulation zone and the growth zone can be adjusted to match the specific pore size of the hollow fiber tube, so that the cultured medium and the target metabolites produced by the culture medium can be concentrated in the growth zone, thereby achieving a concentration effect, which can effectively reduce downstream purification processes and simplify the production process.

[0070] In an embodiment of the present application, the system further includes an oxygenator. The oxygenator adds oxygen to the culture solution to provide sufficient oxygen to the culture in the growth zone. In a specific embodiment, the oxygenator is configured to add oxygen to the second flow path between the second culture solution supply device and the culture device to replenish oxygen as needed. Oxygen is transferred from the culture solution circulation zone to the growth zone via a filtration membrane. By adding gas to the system, the oxygenator can also adjust the pressure in the growth zone, adjusting the pressure difference between the culture solution and the growth zone within an appropriate range and further controlling the mass exchange between the growth zone and the culture solution circulation zone.

[0071] The "first flow path" and "second flow path" in this application are components that fluidly connect each device or unit, such as transport piping, and their structures and functions are well known in the art and will not be described in detail here.

[0072] In an embodiment of the present application, the system further includes a sampling unit. The system includes a plurality of sampling units. The plurality of sampling units are in fluid communication with one or more of the first inlet, the first outlet, the second inlet, or the second outlet. The system may include two to eight sampling units. For example, the system may include four sampling units. Specifically, a sampling unit is disposed at the first inlet 1011 adjacent to the corresponding growth region 101. Another sampling unit is disposed at the first outlet 1012 adjacent to the corresponding growth region 101. Yet another sampling unit is disposed at the second inlet 1021 adjacent to the corresponding nutrient solution circulation region 102. Yet another sampling unit is disposed at the second outlet 1022 adjacent to the corresponding nutrient solution circulation region 102.

[0073] The sampling unit can sample the culture solution in the culture solution circulation area and detect the lactate and glucose contents, thereby monitoring the quality of the culture solution in the system. The sampling unit can sample the culture solution in the growth area and measure the lactate and glucose contents, the number of cultures cultured, or the protein and particle counts of their target metabolites, thereby accurately monitoring the culture process in real time. Furthermore, after the culture is completed, the sampling unit collects the target substances accumulated in the growth area.

[0074] In an embodiment of the present application, the system further includes a waste collection unit, which may be in fluid communication with the incubation device via the first outlet or the second outlet and configured to collect waste liquid discharged from the incubation device.

[0075] In a specific embodiment, a sampling unit is disposed between the culture device and the waste collection unit to determine whether nutrients in the flowing culture solution are fully utilized. In a specific embodiment, one or more valves are disposed between the culture device and the waste collection unit to control whether the culture solution in the growth zone or culture solution circulation zone is recirculated or discharged as waste.

[0076] In an embodiment of the present application, the first culture solution supplying device includes a first culture medium supplying unit containing a first culture medium and a first pretreatment solution supplying unit containing a first pretreatment solution. This allows nutrients necessary for the culture to be easily supplied to the growth area via piping. At the same time, a pretreatment solution such as a buffer solution may be supplied to the growth area as needed to clean the growth area. In a specific embodiment, multiple first culture medium supplying units are provided. The first culture medium may include a medium for culturing the culture, such as a cell culture medium. Therefore, the first culture solution supplying device may include a supplying unit containing a pretreatment solution and a supplying unit containing a cell culture medium.

[0077] In a specific embodiment, the pretreatment solution comprises a culture solution, such as sterile water, saline, or a buffer solution such as phosphate buffer (PBS), that rinses the growth area.

[0078] The pretreatment liquid may be a coating solution. Treating the surface of the filtration membrane corresponding to the growth region with the coating solution enhances the adhesion ability of cultured cells, etc. In a specific embodiment, the coating solution is an aqueous solution or a salt buffer containing one or more of fibronectin, vitronectin, osteopontin, collagen, gelatin, laminin, or polylysine.

[0079] In an embodiment of the present application, the cell culture medium is a medium suitable for culturing cells. In a specific embodiment, the culture includes, but is not limited to, mesenchymal stem cells (MSCs). Thus, the cell culture medium includes, but is not limited to, MSC medium.

[0080] In this application, the system is applicable to the cultivation of any substance suitable for cultivation in the system.

[0081] In embodiments of the present application, the culture includes, but is not limited to, bacteria, adherent cells (mesenchymal stem cells, fibroblasts, endothelial cells, etc.), or suspension cells (immune cells, hematopoietic stem cells, neural stem cells, etc.). The culture may be a microcarrier that supports or encases the cells.

[0082] In an embodiment of the present application, the second culture solution supplying device includes a second culture medium supplying unit and a second pretreatment solution supplying unit. This allows the culture solution to be easily supplied to the culture solution circulation region through piping. The first culture medium and the second culture medium may be the same or different. The first pretreatment solution and the second pretreatment solution may be the same or different.

[0083] In a specific embodiment, multiple second culture medium supply units are provided. For example, the second culture medium may include, but is not limited to, Dulbecco's Minimum Essential Medium (DMEM), MSC medium, cell growth promoting factors, fetal bovine serum, sugars, amino acids, vitamins, inorganic ions, trace elements, etc. The second treatment solution may include, but is not limited to, sterile water, physiological saline, phosphate (PBS) buffer, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, trishydroxymethylaminomethane (Tris) buffer, etc. Therefore, the second culture solution supply device may include multiple second culture medium supply units and multiple second treatment solution supply units to independently supply nutrients to the culture solution circulation region. In this embodiment, each culture solution supply unit may have an independent valve at its outlet to independently supply the necessary nutrients to the culture device.

[0084] In an embodiment of the present application, the first culture solution supplying device may include, in addition to the first culture medium supplying unit and the first treatment solution supplying unit, a plurality of units that are advantageous for circulating and flowing the first culture solution in the growth region, such as a liquid storage unit, a gas exchange unit, etc. Specifically, the liquid storage unit is configured to store the solution flowing out from the first culture medium supplying unit or the first treatment solution supplying unit, or the culture solution flowing out from the growth region.

[0085] In an embodiment of the present application, the second culture solution supply device may include, in addition to the second culture medium supply unit and the second pretreatment solution supply unit, multiple units that are advantageous for the second culture solution to circulate and flow in the culture solution circulation area, such as a liquid storage unit, a gas exchange unit, etc.

[0086] In embodiments of the present application, the culture includes, but is not limited to, animal cells. In specific embodiments, the culture includes, but is not limited to, bacteria, adherent cells (mesenchymal stem cells, fibroblasts, endothelial cells, etc.), or suspension cells (immune cells, hematopoietic stem cells, neural stem cells, etc.). The culture may be a microcarrier that supports or encases cells.

[0087] In embodiments of the present application, the target substance may include the culture itself or metabolic products of the culture, including, but not limited to, cells, extracellular vesicles, viruses, vaccines, antibodies, proteins, and other metabolic products. In a specific embodiment, the target substance is an exosome.

[0088] In an embodiment of the present application, extracellular vesicles are secreted from cells, and a hollow fiber tube reactor provides a substrate for cell growth. The porous hollow fiber membrane allows cell culture materials and water to pass through to the growth region while preventing large cells and extracellular vesicles from exiting the growth region, thereby concentrating the extracellular vesicles. When the target metabolite is an exosome, the pore size of the hollow fiber membrane is less than 40 nm.

[0089] FIG. 2 is a schematic diagram showing a system for producing a target substance according to a specific embodiment. As shown in FIG. 2, in the system of the present application, the culture device is a hollow fiber tube reactor (HFBB). The first culture solution supplying device includes a culture medium supplying unit (i.e., EC culture medium), a pretreatment solution supplying unit (i.e., PBS supplying unit), and a storage unit. The second culture solution supplying device includes a culture medium supplying unit (i.e., IC culture medium) and a pretreatment solution supplying unit (reagent supplying unit). The PBS supplying unit and the EC culture medium supplying unit are connected to the hollow fiber tube reactor via one of the pipings. The IC culture medium supplying unit and the reagent supplying unit are connected to the hollow fiber tube reactor via the other piping. The culture supplying unit supplies the culture to the growth region of the hollow fiber tube reactor. Between the first nutrient solution supply device and the hollow fiber tube reactor are multiple valves (V1, V2, V5, V15, V16), a bubble detector, multiple peristaltic pumps, temperature sensors, pressure sensors, pH acidity / alkalinity sensors, dissolved oxygen sensors, and multiple sampling units. Between the second nutrient solution supply device and the hollow fiber tube reactor are multiple valves (V3, V4, V5, V12, V13, V17), multiple bubble detectors, multiple peristaltic pumps, pressure sensors, and multiple sampling units. The system further includes a waste liquid collection unit. Between the waste liquid collection unit and the hollow fiber tube reactor are multiple valves (V9) and a sampling unit.

[0090] In another aspect, embodiments of the present application provide a method for producing a target substance, the method being capable of being carried out using a system described in any of the embodiments of the above aspects.

[0091] 4 is a flowchart showing a method for producing a target substance according to an embodiment of the present application. As shown in FIG. 4, the method includes steps S101 to S103.

[0092] In step S101, a culture is supplied to a growth region of a culture device.

[0093] In step S102, a first culture solution is supplied to the growth region.

[0094] In step S103, the second culture solution is supplied to the culture solution circulation region of the culture device, and the culture solution circulation region and the growth region are separated by a filtration membrane.

[0095] In an embodiment of the present application, the method further comprises, before step (a), step S104 of rinsing the culture device with a pretreatment solution.

[0096] In a specific embodiment, the growth area and the nutrient circulation area are rinsed multiple times with a pretreatment solution to thoroughly remove impurities, which may be a buffer solution such as sterile water, saline, phosphate buffer (PBS), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, or Trishydroxymethylaminomethane (Tris) buffer.

[0097] In a specific embodiment, the pretreatment solution may be a coating solution. In a specific embodiment, the coating solution is an aqueous solution or a salt buffer solution containing one or more of fibronectin, vitronectin, osteopontin, collagen, gelatin, laminin, and polylysine. Specifically, the coating solution is a mixture of 5 mL of deionized water containing 1 mg to 10 mg of fibronectin and 45 mL of phosphate buffer. For example, the coating solution is a mixture of 5 mL of deionized water containing 5 mg of fibronectin and 45 mL of phosphate buffer.

[0098] In a specific embodiment, a pre-coating treatment is performed on the growth area before inoculation with a culture such as cells. More specifically, a coating solution is used to pre-coat the membrane surface corresponding to the growth area of ​​the filtration membrane, thereby improving the cell adhesion ability.

[0099] In an embodiment of the present application, the pre-coating process comprises: flushing the growth area and the medium circulation area with a buffer solution; removing the buffer in the growth zone and any medium circulation zone; and injecting the prepared coating solution into the growth area and holding for 6 to 24 hours.

[0100] In a specific embodiment, the prepared coating solution is injected into the growth area and maintained for 6 to 24 hours, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, to ensure sufficient fibronectin attachment to the hollow fiber membrane.

[0101] By using the coating solution, cultures such as cells can adhere more uniformly to the membrane surface of the growth area, thereby significantly reducing cell aggregation and allowing cells to grow efficiently.

[0102] In an embodiment of the present application, after step (a) and before step (b), the method further includes step S105 of performing an inversion process on the culture device, thereby achieving sufficient adhesion of the culture, e.g., cells.

[0103] The inversion treatment involves inverting the culture device 6 to 10 times at a frequency of 20 to 40 minutes per inversion. For example, the culture device is rotated once every 30 minutes, and this is repeated 8 times to allow the attachment of cultured matter such as cells for 4.5 hours.

[0104] By using a specific inversion treatment, it is possible to facilitate the attachment of cultures such as cells, which significantly reduces cell aggregation and apoptosis, advantageously promoting cell growth at a high growth rate and efficient production of cells and target metabolites.

[0105] In an embodiment of the present application, the first culture solution is supplied to the growth region at a flow rate of about 0.05 to 0.30 mL / min, e.g., 0.05 to 0.25 mL / min, e.g., 0.05 to 0.20 mL / min, e.g., 0.05 to 0.15 mL / min, e.g., 0.05 to 0.10 mL / min. In one embodiment, the first culture solution is supplied to the growth region at a flow rate of about 0.08 to 0.12 mL / min. In another embodiment, the first culture solution is supplied to the growth region at a flow rate of 0.05 mL / min, 0.06 mL / min, 0.07 mL / min, 0.08 mL / min, 0.09 mL / min, 0.10 mL / min, 0.11 mL / min, or 0.12 mL / min.

[0106] In an embodiment of the present application, the second culture solution is supplied to the culture solution circulation region at a flow rate of about 50 to 500 mL / min, for example, 50 to 150 mL / min, for example, 50 to 120 mL / min, for example, 50 to 100 mL / min, for example, 150 to 500 mL / min, for example, 150 to 450 mL / min, for example, 150 to 400 mL / min, for example, 150 to 350 mL / min, for example, 150 to 300 mL / min, for example, 150 to 250 mL / min, for example, 150 to 200 mL / min. In one embodiment, the second culture solution is supplied to the culture solution circulation region at a flow rate of about 100 mL / min to about 300 mL / min. In another embodiment, the second culture solution is supplied to the culture solution circulation region at a flow rate of 100 mL / min, 110 mL / min, 120 mL / min, 150 mL / min, 180 mL / min, 200 mL / min, 250 mL / min, or 300 mL / min.

[0107] The first culture solution is supplied to the growth region at a low flow rate, and the second culture solution is supplied to the culture medium circulation region at a high flow rate. This allows the nutrients and oxygen required for the culture to be passed through to the growth region based on the flow rate difference, and the culture medium that flows quickly through the culture medium circulation region removes metabolic waste and ensures that the culture obtains sufficient nutrients for stable growth.

[0108] In this embodiment, during the culture period, the concentration of the target substance in the growth zone is detected approximately once every 1 to 4 days, for example, once every 1 to 3 days, for example, once every 1 to 2 days, for example, once a day. The lactic acid and glucose contents in the growth zone and the nutrient solution circulation zone are also detected. During the culture period, the acidity / alkalinity, dissolved oxygen percentage, and chamber pressure in the growth zone and the nutrient solution circulation zone are monitored online in real time. When the lactic acid level reaches 4 mM, more frequent detection is performed, and the nutrient solution supply protocol is adjusted in real time according to the detection results. This ensures that the culture is growing under optimal culture conditions.

[0109] In the embodiment of the present application, target substances such as target cells and / or target metabolites thereof are collected via the sampling unit at the first inlet and / or the first outlet.

[0110] In an embodiment, the target substance may be the culture itself or a metabolic product of the culture. In a specific embodiment, the culture includes, but is not limited to, mesenchymal stem cells. The target substance may be an extracellular vesicle. In a specific embodiment, the extracellular vesicle is an exosome.

[0111] In a specific embodiment, the first culture medium is a medium suitable for culturing the culture. In a specific embodiment, the culture is a mesenchymal stem cell. Thus, the first culture medium is an MSC medium.

[0112] In embodiments of the present application, the culture includes, but is not limited to, bacteria, adherent cells (mesenchymal stem cells, fibroblasts, endothelial cells, etc.), or suspension cells (immune cells, hematopoietic stem cells, neural stem cells, etc.). The culture may be a microcarrier that supports or encases the cells.

[0113] In embodiments of the present application, the second medium provides the cultures in the growth area with the nutrients and oxygen necessary for growth. The second medium may be the same as or different from the first medium. The composition of the second medium may be adjusted as needed.

[0114] In a specific embodiment, the second culture medium may contain, but is not limited to, DMEM medium, MSC medium, cell growth promoting factors, fetal bovine serum, sugars, amino acids, vitamins, inorganic ions, trace elements, etc., which can provide sufficient nutrients to the cultures in the growth region.

[0115] In embodiments of the present application, the filtration membrane may be a microfiltration membrane, an ultrafiltration membrane, or a nanofiltration membrane. In a specific embodiment, the filtration membrane is a hollow fiber tube.

[0116] In a specific embodiment, the growth zone is the exterior space of the hollow fiber tube and the medium circulation zone is the interior space of the hollow fiber tube.

[0117] In a specific embodiment, the growth zone is the interior space of the hollow fiber tube and the nutrient medium circulation zone is the exterior space of the hollow fiber tube.

[0118] According to embodiments of the present application, the hollow fiber tube has a pore size of about 500 nm or less. In specific embodiments, the hollow fiber tube has a pore size of about 40 nm or less. In specific embodiments, the hollow fiber tube has a pore size of about 80 nm or less. In specific embodiments, the hollow fiber tube has a pore size of about 100 nm or less. In specific embodiments, the hollow fiber tube has a pore size of about 120 nm or less. In other embodiments, the pore size of the hollow fiber tube can be adjusted depending on the size of the desired target substance.

[0119] In a specific embodiment, when the target substance is exosomes, the pore size of the filter membrane of the culture device is set to less than about 40 nm, and about 1 × 10 6 cells / ml ~ approx. 1 x 10 8Mesenchymal stem cells are inoculated at a cell density of cells / ml, the first medium is supplied to the growth area at a flow rate of approximately 0.01 mL / min, and the second medium is supplied to the culture medium circulation area at a flow rate of approximately 100 mL / min to approximately 300 mL / min. The culture period can be several minutes, several hours, several days, or even several weeks, for example, 30 minutes, 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 48 ​​hours, 3 to 13 days, or even longer. The first and second media may be MSC media. This allows for high-quality exosomes to be obtained in high yields.

[0120] The system and method of the present application can be applied to the cultivation of any substance suitable for cultivation within the system, including but not limited to mesenchymal stem cells exemplified below, as well as human mesenchymal stem cells or animal mesenchymal stem cells. Example

[0121] Example 1 - Measurement of mesenchymal stem cells In this example, mesenchymal stem cells were cultured, and the culture method was as follows.

[0122] 1.1 Preparation of cell culture medium and cell culture consumables Preconditioning medium: PBS (1% penicillin-streptomycin double antibiotics), DMEM medium (1% penicillin-streptomycin double antibiotics), and 4 L of MSC medium (MSC medium supplemented with 10% EV-depleted MSC medium, 1% penicillin-streptomycin double antibiotics).

[0123] Cell culture medium: MSC cell culture medium was thawed in a refrigerator at 4°C, centrifuged in an ultracentrifuge, and the supernatant of the MSC cell culture medium was collected. The supernatant was filtered through a 0.22 μm filter and placed in a refrigerator at 4°C for use.

[0124] Sterilize cell culture consumables and tubing in an autoclave, then place the sterilized cell culture consumables in an oven to dry out the moisture inside. At the same time, sterilize several 2 mL ultracentrifuge tubes in an autoclave for 15 minutes, then place them in an oven to dry and prepare for use.

[0125] 1.2. Pre-coating treatment of hollow fiber tube reactor Phosphate buffer solution (PBS) was supplied to the intramembrane region (i.e., the culture medium circulation region) of the hollow fiber tube reactor using a buffer supply unit. Next, the phosphate buffer solution was supplied to the extramembrane region (i.e., the growth region) of the hollow fiber tube reactor. The liquid in the growth region was then drained as much as possible. A coating solution (5 mg of fibronectin in 5 mL of deionized water and 45 mL of PBS) was prepared using a 60 mL Luer-lock syringe and injected into the corresponding inlet of the growth region. The coating protocol was carried out to coat the membrane of the growth region with fibronectin overnight, after which the pre-coated hollow fiber tube reactor was stored in a refrigerator at 4°C.

[0126] 1.3. System assembly Sterilized consumables, a coated hollow fiber reactor, a medium supply device, a peristaltic pump, an FX10 coating box, a media bag, sensors, and piping were assembled. PBS buffer, DMEM medium, and finally MSC medium were injected into the medium circulation and growth zones of the hollow fiber reactor, allowing the reactor and piping to be completely flushed with medium. The medium in the liquid bottle was then discarded and replaced with fresh MSC medium.

[0127] Step 1.4: Inoculation of mesenchymal stem cells to be cultured Mesenchymal stem cells (approximately 1 × 10) pre-expanded in flasks 6 cells ~ approx. 1 x 10 8 The 1000 cells were digested and resuspended in approximately 40–50 mL of MSC medium (EV-removed), then transferred into a 60 mL Luer-lock syringe.

[0128] The prepared mesenchymal stem cells to be cultured were inoculated into the growth zone of the hollow fiber bioreactor. Specifically, the hollow fiber reactor was placed horizontally. The inlet and outlet piping of the growth zone were clamped with hemostats, and simultaneously valves V17 and V13 at both ends of the medium circulation zone were closed. The inlet or outlet piping of the medium circulation zone was clamped to stop the circulation of medium between the growth zone and the medium circulation zone. The prepared cell suspension was injected into the growth zone through the inlet of the growth zone using a syringe. Simultaneously, the medium replaced with the cell suspension was aspirated from the outlet of the growth zone using another syringe. The medium aspirated from the outlet was then injected back into the inlet, and the replaced cell mixture was aspirated from the outlet using a syringe. This procedure was repeated 5–6 times using syringes at the inlet and outlet until the turbidity of the cell mixture in the two syringes became approximately the same. The hemostats at the inlet or outlet of the medium circulation zone were opened. The cell mixture in the syringe was injected into the growth zone.

[0129] 1.5. To ensure better and more uniform cell adhesion, the hollow fiber reactor was periodically inverted. Specifically, with the inlet and outlet pipes of the growth zone closed, the reactor was rotated once every 30 minutes at an ambient temperature of 37°C, and this was repeated eight times to allow cells to adhere to the hollow fiber membrane for 4.5 hours. During this time, the culture medium was circulated in the culture medium circulation zone at a flow rate of 100 mL / min.

[0130] 1.6 Cells were cultured in automatic mode, with the culture medium and oxygen circulating through the piping to the cells in the growth area, while waste materials were removed during the circulation. The entire system automatically replenished the culture medium. Specifically, the hemostats at the inlet and outlet of the growth area were opened, and the culture medium was supplied to the growth area at a flow rate of 0.1 mL / min, achieving automatic circulation mode.

[0131] 1.7. Cell metabolic indicators were monitored periodically. Specifically, glucose, lactate, and cell levels were measured periodically in the circulating culture medium. At the beginning of the culture, measurements were taken once every two days. When the lactate level reached 4 mM, measurements were taken more frequently, and the culture medium supply protocol was adjusted in real time based on the results. For sampling and measurement, the hollow fiber reactor was set up, the sampling ports corresponding to the inlet of the growth zone were positioned upward, and 2 mL of crude culture medium was aspirated with a syringe from each of the sampling ports in the growth zone and the culture medium circulation zone. 0.5 mL of crude culture medium was used to measure lactate and glucose, and 1.5 mL of crude culture medium was used to measure cell particle count and protein concentration.

[0132] After sampling of the culture medium circulation area was completed, the IC peristaltic pump corresponding to the culture medium circulation area was stopped, valve V15 was closed, the tubing at the outlet of the culture medium circulation area was clamped with a hemostat, and the EC peristaltic pump was stopped.

[0133] 1.8. The generated mesenchymal stem cells (MSCs) were collected, and the cells and culture medium were sampled for detection. Specifically, a 0.22 μm filter was connected to the sampling port at the inlet of the growth zone (the side where the culture medium was supplied), followed by a 60 mL syringe. Filtered air was injected into the growth zone using the syringe, while approximately 40 mL of the MSC-containing culture medium was aspirated from the sampling port at the outlet of the growth zone (the side farthest from the culture medium supply port). The resulting MSC-containing culture medium was collected in a storage bottle and mixed. 2 mL of the mixture was left for measuring lactate, glucose, cell particle count, and protein concentration.

[0134] 1.9. The flow rate of the peristaltic pump for supplying fluid to the growth zone was reset, and fresh medium was injected into the inlet of the growth zone while simultaneously aspirating air through the sampling port at the outlet of the growth zone with a syringe. The flow rate of the peristaltic pump for supplying fluid to the growth zone was reset to 0.2 mL / min until the growth zone was filled with medium again.

[0135] After collecting the cultured mesenchymal stem cells (MSCs) in step 1.10, the system was returned to cell culture mode. Specifically, the hemostat at the outlet of the culture medium circulation area was loosened, valve V15 was opened, and the IC culture medium circulation was resumed.

[0136] At the same time, as a comparative example, cells were cultured in a T175 flask as a cell reactor using the same cell inoculation density and cell growth medium. Specific experimental results are shown below.

[0137] 1.11. Measurement of total viable cells Throughout the culture period, from the start of culture (day 0) to day 8, a certain amount of cell culture was taken out every day from the growth area of ​​the culture device of the present invention and from the T175 flask, and the total number of viable cells was measured under a microscope.

[0138] The cell count results are shown in Figure 6. As can be seen from the results, cells in the growth zone of the present system increased exponentially, and the cell number could reach more than 400 million cells. Compared to using a T175 flask as a cell reactor, the number of cells produced by the present system was more than 10 times the number of cells produced by the T175 flask, indicating that the present system has significantly higher cell production efficiency.

[0139] 1.12. Measurement of live cell amplification fold From day 0 to day 8, the total number of viable cells obtained each day was divided by the initial cell number to obtain the cell amplification fold.

[0140] The results of the cell expansion fold are shown in Figure 7. As can be seen from the results, compared to when a T175 flask was used as a cell reactor, the cell expansion fold obtained with the system of the present invention was 10 to 30 times higher than the cell expansion fold obtained with a T175 flask, indicating that the system of the present invention has significantly higher cell production efficiency.

[0141] 1.13. Measurement of cell viability From day 0 to day 8, cell viability was calculated by dividing the total number of live cells obtained each day by the total cells obtained (including live and dead cells).

[0142] The results of cell viability are shown in Figure 8. As can be seen from the results, the cell viability obtained with the present system is comparable to that obtained using a T175 flask as a cell reactor. It can be seen that using the present system as a cell reactor does not affect the vitality of the cells.

[0143] Example 2 - Mesenchymal stem cells (MSCs) and extracellular vesicles (EVs) production In this example, human mesenchymal stem cells and extracellular vesicles, which are the target metabolites produced by the stem cells, were designed and produced using the method of Example 1, and steps 1.8 to 1.10 of Example 1 were replaced with the following steps.

[0144] In 1.8-1, the produced mesenchymal stem cells (MSCs) and extracellular vesicles (EVs) were collected and measured by sampling the cells and culture medium. Specifically, a 0.22 μm filter was connected to the sampling port at the inlet of the growth zone (the side where the culture medium is supplied), followed by a 60 mL syringe. Filtered air was injected into the growth zone using the syringe, while approximately 40 mL of the MSC-containing culture medium was aspirated from the sampling port at the outlet of the growth zone (the side farthest from the culture medium supply port). The resulting MSC-containing culture medium was collected in a storage bottle and mixed. 2 mL of the mixture was left for measuring lactate, glucose, cell particle count, and protein concentration.

[0145] 1.9-1. Reset the flow rate of the peristaltic pump to supply the growth zone and inject fresh medium into the inlet of the growth zone. At the same time, use a syringe to aspirate air from the sampling port at the outlet of the growth zone. Reset the flow rate of the peristaltic pump to supply the growth zone to 0.2 mL / min until the growth zone is filled with medium again.

[0146] After collecting the cultured mesenchymal stem cells (MSCs) and extracellular vesicles (EVs) in step 1.10-1, the system was returned to cell culture mode. Specifically, the hemostat at the outlet of the culture medium circulation area was loosened, valve V15 was opened, and the circulation of the IC culture medium was resumed.

[0147] The experimental test process for the design verification of this embodiment was carried out by the Oxford University Laboratory. The measurement results of this embodiment are as follows:

[0148] 2.1. Measurement of exosome yield produced per million cells In this example, the content of exosomes produced from mesenchymal stem cells cultured in the present system and in a T175 flask was measured according to the method described in Example 2.

[0149] The exosome yield per million cells is expressed as protein content. The results are shown in Figure 9. As can be seen from the results, on day 3, the exosome yield per million mesenchymal stem cells harvested using the system of the present invention was slightly lower than the yield of exosomes produced in T175 flasks. From day 5 onwards, the exosome yield per million mesenchymal stem cells harvested using the system of the present invention was not significantly different from the yield of exosomes produced in T175 flasks. As can be seen from the results, the system of the present invention can be used to produce exosomes.

[0150] The exosome yield per million cells is expressed as particle count. The results are shown in Figure 11. As can be seen from the results, the exosome yield per million mesenchymal stem cells harvested using the present system is even higher than the exosome yield produced in T175 flasks. As can be seen from the results, the present system can be used to produce exosomes.

[0151] 2.2 Measurement of the total amount of exosomes produced per 48 hours In this example, the total amount of exosomes produced per 48 hours was measured. The total amount of exosomes produced per 48 hours is expressed as protein. The results are shown in Figure 10. As can be seen, over the entire 13-day growth period, the total amount of exosomes harvested per 48 hours using the present system was significantly higher than the total amount of exosomes obtained per 48 hours using a single T175 flask with the same cell culture volume. The present system provides a larger growth area for cells, allowing for the cultivation of larger numbers of cells. Over the entire 13-day growth period, the yield of exosomes harvested using the present system was more than 18-fold higher than the yield of exosomes harvested using a T175 flask.

[0152] The total amount of exosomes produced per 48 hours is expressed as particle counts. The results are shown in Figure 12. As can be seen, over the entire 13-day growth period, the total amount of exosomes harvested per 48 hours using our system is significantly higher than the total amount of exosomes obtained per 48 hours using a single T175 flask with the same cell culture volume. Over the entire 13-day growth period, the exosome yield harvested with our system is more than 250-fold higher than the exosome yield harvested in a T175 flask.

[0153] Example 3 Measurement of the biological activity of the produced cell exosomes We have requested Oxford University to produce human mesenchymal stem cell exosomes according to the method of Example 2 and measure their biological activity.

[0154] 3.1. Measurement of cell migration Exosomes produced using the present system and exosomes produced in a T175 flask were co-cultured with normal human fibroblasts.

[0155] The results of the fibroblast migration experiment are shown in Figure 13. As can be seen from the experimental results, both exosomes produced in the present system and exosomes produced in T175 flasks promoted fibroblast migration, and the migration-promoting effects of both were equivalent, indicating that exosomes produced in the present system and exosomes produced in T175 flasks have comparable biological activities.

[0156] 3.2. Measurement of cell proliferation In this example, exosomes produced using the present system and exosomes produced using a T175 flask were co-cultured with normal human fibroblasts. While T175 flasks can only produce exosomes at a maximum concentration of 30 μg / mL, the present system can produce exosomes at a high concentration of 70 μg / mL. The proliferative effect of exosomes on fibroblasts was measured using a CCK-8 cell activity assay.

[0157] The results of the cell proliferation experiment are shown in Figure 14. As can be seen from the experiment results, both the exosomes produced in the present system and the exosomes produced in a T175 flask can effectively promote the proliferation of fibroblasts.

[0158] In the description of this application, orientations or positional relationships indicated by terms such as "center," "longitudinal direction," "lateral direction," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial direction," "radial direction," "circumferential direction," etc. are orientations or positional relationships shown in the drawings, and are used only to facilitate or simplify the description of this application, and it should be understood that these do not represent or imply that the devices or parts shown necessarily have a specific orientation or a specific oriented structure and operation, and therefore should not be construed as limiting this application.

[0159] The terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply a relative importance or to implicitly indicate the number of technical features indicated. Thus, a feature qualified as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of this application, unless explicitly and specifically limited, "plurality" means at least two, e.g., two, three, etc.

[0160] In this application, unless otherwise clearly specified or limited, the terms "attached," "coupled," "connected," "fixed," etc. should be understood in a broad sense, and may refer to, for example, a fixed connection, a detachable connection, an integral connection, a mechanical connection, an electrical connection, communication with each other, a direct connection, an indirect connection via an intermediate substrate, internal communication between two elements, or an interactive relationship between two elements. Those skilled in the art can understand the specific meanings of the above terms in this application according to specific circumstances.

[0161] In this application, unless otherwise clearly specified and limited, when a first feature is "above" or "below" a second feature, it means that the first and second features may be in direct contact with each other, or that the first and second features may be in indirect contact with each other via an intermediate substrate. Furthermore, when a first feature is "above," "above," or "on the upper surface" of a second feature, it simply means that the first feature may be directly above or diagonally above the second feature, or that the horizontal height of the first feature is higher than that of the second feature. When a first feature is "below," "below," or "on the lower surface" of a second feature, it simply means that the first feature may be directly below or diagonally below the second feature, or that the horizontal height of the first feature is lower than that of the second feature.

[0162] In the description herein, references to terms such as "one embodiment," "some embodiments," "example," "embodiment," or "some examples" mean that the specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. General references to such terms in the description herein do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0163] The technical features of the above-described embodiments can be combined in any manner, and for the sake of brevity, not all possible combinations of the technical features in the above-described embodiments are described, but as long as there is no contradiction in the combination of these technical features, they should be considered within the scope described in this specification.

[0164] The above examples only describe some embodiments of the present application, and although the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent of the present invention. It should be noted that those skilled in the art may make various modifications and improvements to the present application without departing from the spirit of the present application, and both of these modifications and improvements fall within the scope of protection of the present application. Therefore, the scope of protection of the patent of the present application should be determined based on the scope of the attached claims.

Claims

1. A system for producing a target substance, comprising: a culture device a configured to be partitioned into a growth region and a culture solution circulation region via a filtration membrane, the culture device a including a first inlet, a first outlet, and a second inlet, a second outlet; a first culture solution supply device b configured to be in fluid communication with the growth region via the first inlet and the first outlet; a second culture solution supply device c configured to be in fluid communication with the culture solution circulation region via the second inlet and the second outlet; a culture supply unit d configured to be in fluid communication with the growth region via the first inlet.

2. The system comprises: The system of claim 1, further comprising a plurality of perfusion pumps e configured to be positioned between the culture device and the first culture solution supply device and / or between the culture device and the second culture solution supply device.

3. 3. The system according to claim 1, wherein a first valve is provided between the first culture solution supply device and the first inlet, and a second valve is provided between the first culture solution supply device and the first outlet.

4. 3. The system according to claim 1, wherein a third valve is provided between the second culture solution supply device and the second inlet, and a fourth valve is provided between the second culture solution supply device and the second outlet.

5. the culture supply unit and the first culture solution supply device are in fluid communication with each other via a fifth valve; 3. The system according to claim 1, wherein the first culture solution supply device and the second culture solution supply device are in fluid communication with each other via a sixth valve.

6. The system further includes a plurality of sensors configured to sense between the incubation device and the first incubation solution supply device; and / or The system according to claim 1 or 2, wherein the sensor is configured to perform sensing between the culture device and the second culture solution supply device.

7. 3. The system of claim 1, further comprising an oxygenator configured to supply oxygen to a flow path between the second culture solution supply device and the culture device.

8. 3. The system of claim 1, further comprising a plurality of sampling units, the plurality of sampling units being in fluid communication with one or more of the first inlet, the first outlet, the second inlet, or the second outlet.

9. 3. The system of claim 1 or 2, further comprising a waste collection unit configured to be in fluid communication with the incubation device via the first outlet or the second outlet.

10. the first culture solution supply device includes a first culture medium supply unit and a first pretreatment solution supply unit; and / or 3. The system according to claim 1, wherein the second culture solution supply device includes a second culture medium supply unit and a second pretreatment solution supply unit.

11. the filtration membrane is a hollow fiber tube; 3. The system of claim 1, wherein the growth area is the external space of the hollow fiber tube and the nutrient solution circulation area is the internal space of the hollow fiber tube; or, alternatively, the growth area is the internal space of the hollow fiber tube and the nutrient solution circulation area is the external space of the hollow fiber tube.

12. 3. The system of claim 1, wherein the surface of the filtration membrane corresponding to the growth area is treated with a coating solution.

13. 13. The system of claim 12, wherein the coating solution is an aqueous solution or salt buffer containing one or more of fibronectin, vitronectin, osteopontin, collagen, gelatin, laminin, or polylysine.

14. A method for producing a target substance, comprising: (a) providing a culture to a growth region of a culture device; (b) providing a first culture solution to the growth region; and (c) supplying a second culture solution to a culture medium circulation region of the culture device, the culture medium circulation region and the growth region being separated by a filtration membrane.

15. supplying the first culture solution to the growth region at a flow rate of 0.05 to 0.30 mL / min; and / or The method according to claim 14, wherein the second culture solution is supplied to the culture solution circulation region at a flow rate of 50 to 500 mL / min.

16. 16. The method of claim 14 or 15, wherein prior to step (a), the culture device is rinsed with a pretreatment solution.

17. The method further includes a step of performing an inversion process on the culture device after step (a) and before step (b), The method according to claim 14 or 15, wherein the inversion treatment comprises inverting the culture device 6 to 10 times at a frequency of 20 to 40 minutes per inversion.

18. During the culture period, the concentration of the target substance in the growth area is detected at a frequency of 1 to 4 days, and the contents of lactic acid and glucose in the growth area and the culture medium circulation area are detected, and / or 16. The method according to claim 14 or 15, wherein the acidity / alkalinity, the dissolved oxygen percentage, and the chamber pressure in the growth zone and the culture medium circulation zone are monitored online in real time.

19. The culture is a mesenchymal stem cell, and The method according to claim 14 or 15, wherein the target substance is an extracellular vesicle.

20. The system / method according to any one of claims 1 to 19, wherein the culture and the target substance comprise human cells, animal cells, human extracellular vesicles, and / or animal extracellular vesicles.

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