Cell activation reactor and cell activation method

Abstract

The present invention discloses a cell activation reactor and a cell activation method. The cell activation reactor comprises a body, a rotating member, an upper cover, a microporous film, and a plurality of baffles. The body has a storage space suitable for accommodating a plurality of cells and a plurality of magnetic beads. The rotating member is disposed in the storage space and includes a plurality of impellers. The microporous film is disposed in the storage space and covers a plurality of transparent holes in the storage space. The plurality of baffles are disposed on the body. When the rotating member is driven to rotate, the interaction between the plurality of baffles and the plurality of impellers separates the cells from the magnetic beads.

Description

Technical Field

[0001] The present invention relates to a cell activation reactor and a cell activation method, and in particular to a cell activation reactor and a cell activation method for promoting cell proliferation. Background Art

[0002] Cell culture is a key technology in the biomedical sciences field. Immunotherapy, a field that has been actively researched and optimized in recent years, involves isolating T cells from a patient's biological specimen, culturing and amplifying them to a sufficient concentration, and then implementing immunotherapy methods to specifically attack specific cancer cells with minimal side effects.

[0003] During the cell culture process (especially T cells), in addition to general routine conditions (such as placing in a stable cell culture incubator and adding nutrient solution), multi-point magnetic beads can also be used to increase the surface area in contact with the cells to stimulate cell activation, thereby achieving better quality and quantity expansion effects. In this technical process, the cells that are closely attached to the magnetic beads need to be separated from each other, and after separation, the cells can be purified for the next stage of the expansion culture cycle. In existing technologies, cell separation is generally performed manually, and operators need to use micropipettes and plastic containers to transfer these liquids between various machines. However, manual operation is likely to lead to problems such as cell cross-contamination, concerns about infection of biological waste, longer operation time, and higher labor costs, thereby limiting the research site of cell culture to laboratory mode. When applied to immunotherapy, it is not popular in current cancer treatment methods due to its high price.

[0004] Based on the above, it is becoming increasingly important to improve experimental techniques for artificially isolating cells and optimize cell culture production processes to shorten cell culture time, improve cell culture quality, and thus reduce costs. Summary of the Invention

[0005] The present invention provides a cell activation reactor and a cell activation method, which are suitable for improving the experimental technique of artificially separating cells, can optimize the cell culture production process, reduce manual operation errors, and improve experimental reproducibility.

[0006] The cell activation reactor of the present invention includes a body, a rotating member, an upper cover, a microporous film, and a plurality of baffles. The body has a storage space formed by a closed end, a side, and an open end, suitable for accommodating a cell culture medium containing a plurality of cells and a plurality of magnetic beads. The rotating member is arranged in the storage space, includes an axis and a plurality of impellers, and is suitable for connecting to a driver and being driven to rotate. The upper cover is detachably arranged above the body to cover the open end of the storage space. The upper cover has an opening, which is arranged corresponding to the position of the rotating member so that the axis of the rotating member is passed through the opening and the end of the axis of the rotating member is exposed. The driver is connected to the rotating member through the end. The microporous film is arranged at the closed end of the storage space, and the closed end has a plurality of transparent holes extending therethrough. The microporous film covers the plurality of transparent holes. The plurality of baffles are arranged on the sides of the storage space. When the rotating member is driven to rotate, the interaction between the plurality of baffles and the plurality of impellers of the rotating member separates the cells from the magnetic beads.

[0007] The cell activation method of the present invention uses the above-mentioned cell activation reactor to perform cell activation, and its steps include: starting a programmable driver to intermittently drive the rotating member to rotate to disperse multiple cells and multiple magnetic beads; and removing and collecting the multiple cells after static activation.

[0008] Based on the above, the cell activation reactor of the present invention includes a main body, a rotating part, an upper cover, a microporous film and a plurality of baffles. Through the interaction of the plurality of baffles and the rotating part, the cells and the magnetic beads can be separated automatically. In addition, the microporous film located at the closed end of the main body accommodating space has a plurality of transparent holes, and the gas is autonomously exchanged inside and outside the accommodating space through the transparent holes, so that the main body accommodating space can be placed in an oxygen-rich environment. In this way, static cell culture can be carried out, and the experimental method of artificially separating cells can be improved. At the same time, the cell culture production process can be optimized, manual operation errors can be reduced, and experimental reproducibility can be improved. In addition, the possibility of automation can be improved, and the quantitative production process can accelerate the development of the cell production system, thereby shortening the cell culture time and improving the quality of cell culture, and reducing the high labor costs in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 FIG1 is a schematic diagram of a partial structure of a cell activation reactor according to an embodiment of the present invention;

[0010] Figure 2 Schematic diagram of the structure of a cell activation reactor according to one embodiment of the present invention;

[0011] Figure 3 is a schematic cross-sectional view of the interface between the body and the upper cover of a cell activation reactor according to one embodiment of the present invention;

[0012] Figure 4 For the Figure 3Schematic diagram of the cross section along the midline L-L';

[0013] Figure 5 is a schematic cross-sectional view of the interface between the body and the lower cover of a cell activation reactor according to one embodiment of the present invention;

[0014] Figures 6A to 6C is a schematic cross-sectional view of the operation of a cell activation reactor according to one embodiment of the present invention;

[0015] Figure 7 This is a microscopic image of a magnetic bead cell dispersion test using a cell activation reactor according to one embodiment of the present invention;

[0016] Figure 8 1 is a graph showing the cell activity and number measurements of a magnetic bead cell dispersion test using a cell activation reactor according to an embodiment of the present invention. DETAILED DESCRIPTION

[0017] The following examples are listed and described in detail with reference to the accompanying drawings, but the examples provided are not intended to limit the scope of the present invention. In addition, the drawings are for illustrative purposes only and are not drawn to their original size. For ease of understanding, the same components will be described with the same symbols in the following description. In addition, the terms "include", "including", "have", etc. used in the text are all open terms, which means "including but not limited to". Furthermore, the directional terms mentioned in the text, such as "upper", "lower", etc., are only used to refer to the directions of the drawings and are not used to limit the present invention. In addition, the quantities and shapes mentioned in the specification are only used to specifically illustrate the present invention in order to facilitate understanding of its contents, and are not used to limit the present invention.

[0018] In this document, the term "range from one value to another value" is used as a summary to avoid listing all values within the range. Therefore, a description of a specific numerical range encompasses any value within that range and any smaller numerical range defined by that value, just as if the value and smaller numerical range were listed in the specification.

[0019] Figure 1 FIG. 1 is a schematic diagram of a partial structure of a cell activation reactor according to an embodiment of the present invention. Figure 2 FIG. 1 is a schematic structural diagram of a cell activation reactor according to an embodiment of the present invention. Figure 3 FIG1 is a schematic cross-sectional view of the interface between the body and the upper cover of a cell activation reactor according to one embodiment of the present invention. Figure 4 For the Figure 3 Schematic cross-section of the midline L-L'. Figure 5 FIG1 is a schematic cross-sectional view of the junction between the body and the lower cover of a cell activation reactor according to one embodiment of the present invention.

[0020] Please refer to Figure 1 The cell activation reactor mainly comprises a body 10, an upper cover 20 and a lower cover 30. The upper cover 20 is detachably mounted on the upper portion of the body 10 and the lower cover 30 is mounted below the body 10. The material of the body 10, the upper cover 20 and the lower cover 30 may comprise polycarbonate (PC). Figure 1 and Figure 2 ,Apart from Figure 1 In addition to the main components shown in FIG, the cell activation reactor may further include a driver 40, a bracket 42, and a base 50. The base 50 is disposed below the body 10, and the lower cover 30 can be inserted into a groove in the base 50. The driver 40 is disposed above the upper cover 20. The bracket 42 connects the driver 40 and the base 50 to support the driver 40 and maintain its position above the upper cover 20. The driver 40 may include a motor, but the present invention is not limited to this.

[0021] Please also refer to Figure 1 、 Figure 2 and Figure 3 The main body 10 has a accommodating space 12. The accommodating space 12 of the main body 10 is formed by a closed end 12A, a side 12B and an open end 12C. The accommodating space 12 is suitable for accommodating a cell culture fluid containing a plurality of cells and a plurality of magnetic beads. In one embodiment, the cavity volume of the accommodating space 12 is, for example, 20 mL. In other embodiments, the cavity volume of the accommodating space 12 may be 10-1000 mL. A rotating member 70 is disposed in the accommodating space 12, which includes an axis A and a plurality of impellers 72. The rotating member 70 is suitable for being connected to the driver 40 and driven to rotate. In this embodiment, the upper cover 20 covers the open end 12C of the accommodating space 12. The upper cover 20 can limit the position of the rotating member 70 through the opening 22, and can prevent the cell culture fluid from leaking when the rotating member 70 is driven to rotate. Specifically, the upper cover 20 has an opening 22 positioned corresponding to the position of the rotating member 70. This allows the axis A of the rotating member 70 to protrude from the exterior of the body 10 and pass through the opening 22, exposing an end M of the axis A of the rotating member 70. Thus, the driver 40 is connected to the rotating member 70 via the end M, allowing the rotating member 70 to be driven by the driver 40 for rotation. The rotation speed of the rotating member 70 can be, for example, 0 to 100 rpm, and can be programmably rotated. A plurality of baffles 60 are disposed on the side 12B of the accommodating space 12. The number of impellers 72 can correspond to the number of baffles 60, but is not limited thereto. When the rotating member 70 is driven to rotate, the narrow gaps between the baffles 60 and the impellers 72 of the rotating member 70 allow the baffles 60 to interact with the impellers 72 of the rotating member 70, thereby separating cells from magnetic beads. Furthermore, the baffles 60 and impellers 72 can increase fluid shear force. The detailed operation process of cell and magnetic bead separation will be described in detail below.

[0022] Please also refer to Figure 1 、 Figure 2 and Figure 3 , the minimum distance d between the impeller 72 and the baffle 60 is, for example, 1 mm. In other embodiments, the minimum distance d between the impeller 72 and the baffle 60 may be 1-8 mm. In this embodiment, the main body 10 is, for example, cylindrical, and the axis of the main body 10 may coincide with the axis A of the rotating member 70. Each impeller 72 extends from the axis A of the rotating member 70 toward the side 12B of the accommodating space 12 and its thickness gradually decreases. Each baffle 60 extends from the side 12B of the accommodating space 12 toward the axis A of the rotating member 70 and its thickness gradually decreases to reduce cell rupture. The number of the plurality of baffles 60 is, for example, more than 4, and the number of the plurality of impellers 72 is, for example, more than 4. Although Figure 3 The number of the baffles 60 and the number of the impellers 72 shown in FIG. 7 are six respectively, but the present disclosure is not limited thereto, and the number of the baffles 60 and the impellers 72 can be an odd number or an even number.

[0023] Please also refer to Figure 1 、 Figure 2 、 Figure 3 and Figure 4 The distance h between the rotating member 70 and the closed end 12A of the accommodating space 12 is, for example, 0.05 mm to 1 mm, which can enhance laminar flow and evenly distribute force on the fluid (ie, cell culture fluid). In this embodiment, the cross-sectional shape of the impeller 72 is, for example, a rectangle.

[0024] Please also refer to Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 and Figure 5 . It should be noted that in Figure 4In response to the need for explanation, only some components are illustrated, and each component is only for illustration, mainly showing the configuration relationship between the components, but not representing the proportional relationship of the actual component size. In this embodiment, the microporous film 80 is arranged at the closed end 12A of the accommodating space 12, which is made of a breathable and water-tight film material, such as a material including polytetrafluoroethylene (PTFE). In detail, the closed end 12A of the accommodating space 12 has a plurality of through holes 14 that pass through from top to bottom, and the microporous film 80 is arranged at the closed end 12A of the accommodating space 12 and covers these through holes 14. Since the thickness of the microporous film 80 is very thin, the closed end 12A of the accommodating space 12 can be made of a supportive material, such as PC or nylon, to prevent the microporous film 80 from collapsing due to the cell culture medium contained in the accommodating space 12. By configuring the microporous film 80 and the through holes 14, gases can be autonomously exchanged inside and outside the accommodating space 12, so that the cell culture medium can be maintained in an oxygen-rich environment. In one embodiment, the diameter of the through holes 14 is, for example, 1.2 mm, and the area of the microporous film 80 is, for example, 490.87 mm. 2 In other embodiments, the diameter of the through hole 14 may be 0.6-1.5 mm, and the area of the microporous film 80 may be 324-14400 mm. 2 .

[0025] In one embodiment, the lower cover 30 has a plurality of legs 32 that elevate the body 10 to expose the through holes 14. This allows sufficient space for gas to flow into and out of the accommodating space 12 through the through holes 14 when the cell activation reactor is stationary, thereby providing the accommodating space 12 with an autonomous oxygen-rich environment for cell activation. In another embodiment, the cell activation reactor may further include a base 50 disposed beneath the body 10. The lower cover 30 may be recessed into a groove in the base 50, making the cell activation reactor more stable when stationary while providing sufficient space for gas flow.

[0026] Figures 6A to 6C FIG. 1 is a cross-sectional view of the operation of a cell activation reactor according to one embodiment of the present invention.

[0027] The present invention also provides a cell activation method, using the above cell activation reactor to activate cells, the detailed operation process is as follows Figures 6A to 6C Please refer to Figure 6A The cell culture medium 100 contains a plurality of cells 102 and a plurality of magnetic beads 104. At this time, the cells 102 and the magnetic beads 104 are tightly combined. The initial amount of cells 102 in the cell culture medium 100 is, for example, 5×10 5 to 10×10 5 Please refer to Figure 6B When the rotating member 70 is connected to the driver 40 and driven to rotate, the multiple impellers 72 of the rotating member 70 interact to separate the cells 102 from the magnetic beads 104. The rotating member 70 is driven to rotate by the driver 40, and the baffle 60 and impellers 72 provide shear force to separate the cells 102 and the magnetic beads 104, replacing the conventional process of manually operating a pipette to perform cell separation. In one embodiment, the driver 40 can be programmably activated, such as intermittently driving the rotating member 70 to rotate, thereby dispersing the magnetic beads and cells.

[0028] In this case, please refer to Figure 6C , the cells 102 can be separated from the magnetic beads 104. Figures 6A to 6C The cross-sectional shape of each impeller 72 and each baffle 60 is, for example, a rectangle, and the height of each impeller 72 and each baffle 60 is the same. The liquid level of the cell culture medium 100 is, for example, higher than the height of each impeller 72 .

[0029] The cell activation reactor and the cell activation method proposed in the present invention are described in detail below through experimental examples. However, the following experimental examples are not intended to limit the present invention.

[0030] Experimental example

[0031] In order to demonstrate that the cell activation reactor and the cell activation method of the present invention can effectively separate cells from magnetic beads, the following experimental example is specifically described.

[0032] Experimental Example 1: Magnetic Bead Cell Dispersion Test with Different Numbers of Baffles and Impellers

[0033] Figure 7 The following is a microscope image of a magnetic bead cell dispersion test using a cell activation reactor according to an embodiment of the present invention. Magnetic bead cell dispersion tests were performed using cell activation reactors with different numbers of baffles and impellers. 5 In the case of the rotating part, the rotation speed is 100 rpm, and the rotation is performed 5 times under the conditions of 30 seconds of rotation and 30 seconds of rest. After that, a part of the field of view is taken for photography using a microscope. Figure 7 It can be seen that when the number of baffles is 4 or more and the number of impellers is 4 or more, a good magnetic bead cell dispersion effect is achieved, and the magnetic beads and cells can be fully dispersed. In this embodiment, when 6 baffles and 6 impellers are used, the effect is good.

[0034] Experimental Example 2: Comparison of magnetic bead cell dispersion test

[0035] Figure 8 This is a graph showing the cell activity and number measurements of a magnetic bead cell dispersion test using a cell activation reactor according to an embodiment of the present invention. In this experimental example, the number of cells was 5×105 T cells and iKNOBEAD magnetic beads were separated and dispersed using a manual pipette and the cell activation reactor of the present invention when the T cells and magnetic beads were tightly bound. After three days of static activation, the magnetic beads were removed and the T cells were collected. Afterwards, the T cells collected in the experimental group were removed from the cell activation reactor and statically cultured in a cell culture device with the T cells in the control group for four days, and the cell activity and number were measured. Please refer to Figure 8 , it can be seen that compared with the manual operation of the pipette (corresponding to Figure 8 The cell activation reactor of the present invention can make T cells and magnetic beads have good dispersion, can reduce manual errors, and has high reproducibility.

[0036] In summary, the cell activation reactor of the present invention includes a main body, a rotating part, an upper cover, a microporous film and a plurality of baffles. Through the interaction of the plurality of baffles and the rotating part, the cells and the magnetic beads can be separated automatically, and the fluid shear force can be enhanced and the viscosity can be cut off. The baffles can also accelerate liquid convection. In addition, the microporous film located at the closed end of the main body accommodating space has a plurality of transparent holes, and the gas is autonomously exchanged inside and outside the accommodating space through the transparent holes. Therefore, the main body accommodating space can be enriched with oxygen to activate the cells. In this way, static cell culture can be carried out, and the experimental method of artificially separating cells can be improved. At the same time, the cell culture production process can be optimized, the manual operation error can be reduced, and the experimental reproducibility can be improved. In addition, the possibility of automation can be improved, and the quantitative production process can accelerate the development of the cell production system, thereby shortening the cell culture time and improving the cell culture quality, and reducing the high labor cost in the existing technology.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell activation reactor, characterized in that include: The body has a receiving space formed by a closed end, a side portion, and an open end, suitable for receiving a cell culture solution containing a plurality of cells and a plurality of magnetic beads; A rotating member is disposed in the accommodating space, the rotating member includes a shaft and a plurality of impellers, and the rotating member is suitable for being connected to a driver to be driven to rotate; an upper cover detachably disposed above the body to cover the open end of the accommodating space, the upper cover having an opening, the opening being arranged corresponding to the position of the rotating member so that the axis of the rotating member passes through the opening and an end of the axis of the rotating member is exposed, and the driver is connected to the rotating member through the end; A microporous film is disposed at the closed end of the accommodating space, the closed end having a plurality of through holes therethrough, and the microporous film covers the plurality of through holes; a plurality of baffles disposed on the side of the accommodating space, wherein when the rotating member is driven to rotate, the interaction between the plurality of baffles and the plurality of impellers separates the cells from the magnetic beads; as well as The lower cover is arranged below the main body to elevate the main body, and the main body is connected to the lower cover through the microporous film. The main body is cylindrical, the axis of the main body coincides with the axis of the rotating member, and each impeller extends from the axis of the rotating member toward the side of the accommodating space and gradually decreases in thickness. Each of the baffles extends from the side of the accommodating space toward the axis of the rotating member and has a gradually decreasing thickness. Wherein, the heights of the impellers and the baffles are the same.

2. The cell activation reactor according to claim 1, characterized in that The minimum distance between the impeller and the baffle is 1-8 mm.

3. The cell activation reactor according to claim 1, characterized in that The number of the plurality of baffles is 4 or more, and the number of the plurality of impellers is 4 or more.

4. The cell activation reactor according to claim 1, characterized in that The cross-sectional shape of each impeller and each baffle is a rectangle.

5. The cell activation reactor according to claim 1, characterized in that The distance between the rotating member and the closed end of the accommodating space is 0.05 mm to 1 mm.

6. The cell activation reactor according to claim 1, characterized in that The diameter of the plurality of through holes is 0.6 to 1.5 mm.

7. The cell activation reactor according to claim 1, characterized in that The area of the microporous film is 324 to 14400 mm 2 .

8. The cell activation reactor according to claim 1, characterized in that It also includes a base body, which is arranged below the main body, and the lower cover is embedded in the groove of the base body.

9. The cell activation reactor according to claim 1, characterized in that The liquid level of the cell culture fluid is higher than the height of each impeller.

10. A cell activation method, comprising: performing cell activation using the cell activation reactor according to any one of claims 1 to 9; and comprising: A programmable start drive intermittently drives the rotating member to rotate to disperse the plurality of cells and the plurality of magnetic beads; and After static activation, a plurality of the cells are removed and collected.

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