An in situ variable volume culture chamber, three-dimensional culture apparatus and culture method

Abstract

This invention discloses an in-situ variable-volume culture chamber, a three-dimensional culture device, and a culture method. The device includes a variable-volume culture chamber, which is hollow inside, with an opening at one end and a cell inlet / outlet at the other. A culture chamber tension rod is positioned within the variable-volume culture chamber, with its end edge slidably connected to the inner wall of the chamber. A fixing component is also mounted on the variable-volume culture chamber. This device uses the fixing component to fix the position of the tension rod after movement, thereby changing the volume of the culture medium and adjusting the number of cells or organoids. This device can meet the three-dimensional growth conditions of cells and can also regulate the stress level of the culture medium according to the needs of cell growth, aggregation, and functional differentiation during the culture process, thereby inducing cell function or directed differentiation, and growing into cells or organoids with specific structures and functions.

Description

Technical Field

[0001] This invention relates to the field of bioculture technology, specifically to an in-situ variable volume culture chamber, a three-dimensional culture device, and a culture method. Background Technology

[0002] Three-dimensional (3D) cell or organoid culture is a cell culture method that simulates the three-dimensional growth environment in vivo. By allowing cells to aggregate into 3D spheres or by allowing cells to adhere, extend, and grow on a three-dimensional structural carrier with a composition and structure similar to solid tissues, cell proliferation and differentiation are jointly regulated in time and space, so that tissue structure and function can be preserved to a greater extent.

[0003] Currently, most three-dimensional cell culture in vitro uses cylindrical culture vessels. The problem is that the volume of most three-dimensional cell culture vessels is fixed, meaning that the internal volume of the culture vessel cannot be changed. Therefore, in practical use, when conducting cell or organoid culture experiments of different volumes, different volume culture vessels must be used. This cannot meet the requirement of being applicable to different cell numbers and corresponding changes in culture medium volume, causing various inconveniences in application. Summary of the Invention

[0004] The purpose of this invention is to provide an in-situ variable volume culture chamber, a three-dimensional culturer, and a culture method to solve the technical problem of inconvenience caused by the fixed volume of the culturer in the prior art.

[0005] To solve the above-mentioned technical problems, the present invention specifically provides the following technical solution:

[0006] An in-situ variable volume culture chamber, characterized in that it comprises,

[0007] A variable volume culture chamber is hollow inside, with an opening at one end and a cell inlet / outlet at the other end. A culture chamber extension rod is installed inside the variable volume culture chamber, and the culture chamber extension rod extends outward through the opening.

[0008] The end edge of the culture chamber tension rod is slidably connected to the inner wall of the variable volume culture chamber, and a culture chamber is formed between the end face of the culture chamber tension rod and the inner end face of the variable volume culture chamber.

[0009] The variable volume culture chamber is also equipped with a fixing component for fixing the position of the culture chamber tension rod.

[0010] In a preferred embodiment of the present invention, the variable volume culture chamber is used for culturing cells or organoids. The culture chamber stretching rod includes a stretching rod body and a stretching rod top cover. The stretching rod top cover is disposed in the variable volume culture chamber and is movably connected to the stretching rod body. A sliding sealing ring is provided on the side edge of the stretching rod top cover, and the sliding sealing ring is slidably and sealingly connected to the inner wall of the variable volume culture chamber.

[0011] As a preferred embodiment of the present invention, the top cover of the stretching rod is provided with a vent hole, and a vent membrane is provided on the vent hole, and the vent membrane is disposed in the culture chamber;

[0012] The tension rod body is hollow inside, and a hole is opened at the end of the tension rod body away from the top cover of the tension rod, which is connected to the external space.

[0013] As a preferred embodiment of the present invention, a sealing element is provided at the cell inlet and outlet.

[0014] In a preferred embodiment of the present invention, the fixing component includes a fixing part and a fixing bolt. The fixing part is integrally fixedly installed on the outer wall of the variable volume culture chamber. A first compression rubber ring is provided inside the fixing part. The first compression rubber ring contacts the outer surface of the tension rod body. The fixing bolt is connected to the fixing part by thread engagement, and the first compression rubber ring is disposed between the fixing bolt and the fixing part. The first compression rubber ring fixes the tension rod body by the compression of the fixing bolt.

[0015] In a preferred embodiment of the present invention, the fixing part includes a fixing nut, a T-shaped sleeve, and a second compression rubber ring. The fixing nut is fitted onto the outer wall of the variable volume culture chamber near the opening. There is a gap between the inner wall of the fixing nut with internal threads and the outer wall of the variable volume culture chamber. The second compression rubber ring is disposed in the gap. One end of the T-shaped sleeve is connected to the internal thread of the fixing nut in the gap by threaded engagement. The T-shaped sleeve presses the second compression rubber ring by screwing in to fix the fixing nut onto the outer wall of the variable volume culture chamber.

[0016] The inner wall of the T-shaped sleeve is provided with an annular external protrusion, which is fitted to the outer wall of the tension rod body. A first compression rubber ring is provided on one side of the annular external protrusion, and a fixing bolt is provided on one side of the first compression rubber ring. The fixing bolt is spirally connected to the inner wall of the T-shaped sleeve by thread engagement. The first compression rubber ring is fixed to the tension rod body by the screwing in of the fixing bolt.

[0017] A three-dimensional culture apparatus having the aforementioned in-situ variable volume culture chamber includes,

[0018] A culture medium stand, the culture medium stand including a base, a support provided on the base, and a rotating mechanism provided on one side of the support;

[0019] An in-situ variable volume culture chamber is provided on the other side of the support, and the tension rod of the in-situ variable volume culture chamber is mainly connected to the rotating mechanism.

[0020] The rotating mechanism drives the in-situ variable volume culture chamber to rotate for culturing cells and organoids.

[0021] As a preferred embodiment of the present invention, the culture medium seat is further provided with a liquid supply-drainage system for changing the internal pressure distribution of the in-situ variable volume culture chamber, and an air supply-exhaust system for adjusting the air supply and exhaust rates inside the in-situ variable volume culture chamber. The air supply-exhaust system supplies air to the air chamber at a flow rate of 0.4-10 ml / min, and the liquid supply-drainage system supplies liquid at a flow rate of 0.1-10 ml / min.

[0022] A method for culturing cells or organoids based on the aforementioned three-dimensional culture dish, wherein the culture and fresh culture medium are loaded into the culture chamber of the present invention through the cell inlet and outlet according to conventional culture processes, and the culture conditions are the same as those of conventional processes. The method is characterized in that the culture chamber is rotatable, and the rotation speed of the culture chamber changes with the formation of cell aggregates during the cell culture process. The rotation speed of the culture chamber is such that the shear stress in the culture chamber gradually changes from approaching zero to the physiological stress of the cultured cells / tissues.

[0023] As a preferred embodiment of the present invention, the rotational speed of the culture chamber is determined by the size of different cultures suspended in the culture medium, and is greater than or equal to the minimum angular velocity of the culture suspension.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] This invention comprises a variable-volume culture chamber, a tension rod body, and a fixing assembly. The culture apparatus, composed of these components, is mounted on a rotating device. The volume of the culture chamber is expanded or reduced by sliding the tension rod body within the variable-volume culture chamber, and the fixing assembly secures the position of the tension rod body after movement. This alters the volume of the culture medium, thereby increasing or decreasing the number of cells or organoids. This device can meet the three-dimensional growth conditions of cells and can regulate the stress level of the culture medium according to the needs of cell growth, aggregation, and functional differentiation during culture, thus inducing directed differentiation of cells and their growth into cells with specific structures and functions. Attached Figure Description

[0026] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0028] Figure 2 This is a cross-sectional view of the variable volume culture chamber in this invention;

[0029] Figure 3 This is an exploded view of the components of the variable volume culture chamber in this invention.

[0030] The labels in the diagram represent the following:

[0031] 1. Variable volume culture chamber; 2. Fixing nut; 3. Second clamping rubber ring; 4. T-sleeve; 5. Tensioner rod body; 6. Fixing bolt; 7. Tensioner rod top cover; 8. Cell inlet / outlet; 9. Sealing element; 10. Sliding sealing ring; 11. First clamping rubber ring; 12. Base; 13. Support; 14. Rotating mechanism; 15. Culture chamber tensioner rod; 16. Culture chamber; 17. Vent hole; 18. Fixing part; 19. Annular external protrusion; 20. Culture medium seat. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] like Figures 1 to 2 As shown, the present invention provides an in-situ variable volume culture chamber, a three-dimensional culture device, and a culture method. In existing three-dimensional cell culture, the culture volume of the culture device is fixed, which not only limits the number of cultures in the culture device, but also limits the growth of the cultures already in the culture device. Therefore, the present invention proposes a three-dimensional culture device that can change the volume.

[0034] A variable volume culture chamber 1 is hollow inside, with one end set as an opening and the other end set as a cell inlet / outlet 8. A culture chamber tension rod 15 is set inside the variable volume culture chamber 1, and the culture chamber tension rod 15 extends outward through the opening.

[0035] The end edge of the culture chamber tension rod 15 is slidably connected to the inner wall of the variable volume culture chamber 1, and a culture chamber 16 is formed between the end face of the culture chamber tension rod 15 and the inner end face of the variable volume culture chamber 1.

[0036] The variable volume culture chamber 1 is also provided with a fixing component for fixing the position of the culture chamber tension rod 15;

[0037] A culture medium stand 20 is provided with a rotating mechanism 14. A variable volume culture chamber 1 is installed on the culture medium stand 20 and is connected to the rotating mechanism 14. The rotating mechanism 14 drives the variable volume culture chamber 1 to rotate in order to culture cells and organoids.

[0038] This device primarily addresses the issue of fixed volume in existing three-dimensional culturers by sliding the culture chamber tension rod 15 within the variable-volume culture chamber 1. Within the variable-volume culture chamber 1, the end of the culture chamber tension rod 15 and the inner end face of the chamber form a culture chamber 16 for culturing cells. As the culture chamber tension rod 15 moves within the chamber, the distance between its end and the inner end face changes, thereby altering the volume of the culture chamber 16. This allows for increasing the amount of cultured material before cultivation begins and for changing the volume during cultivation.

[0039] In-situ variable volume culture chambers can achieve the expansion / growth of cells or organoids during the culture process. By changing the volume during the culture process, the culture chamber volume can be expanded to grow more cells or organoids.

[0040] Furthermore, the culture chamber stretching rod 15 includes a stretching rod body 5 and a stretching rod top cover 7. The stretching rod top cover 7 is disposed in the variable volume culture chamber 1. The stretching rod top cover 7 is movably connected to the stretching rod body 5. A sliding sealing ring 10 is provided on the side edge of the stretching rod top cover 7. The sliding sealing ring 10 is slidably sealed to the inner wall of the variable volume culture chamber 1.

[0041] The top cover 7 of the stretching rod has a vent hole 17, and a vent membrane is provided on the vent hole 17. The vent membrane is placed inside the culture chamber 16.

[0042] The tension rod body 5 is hollow inside, and a hole is opened at the end of the tension rod body 5 away from the tension rod top cover 7 and communicates with the external space.

[0043] By utilizing the sliding sealing ring 10 on the side of the top cover 7 of the tension rod to slide and connect with the inner wall of the variable volume culture chamber 1, the culture chamber 16 can become a sealed space when the culture chamber tension rod 15 slides in the variable volume culture chamber 1, preventing the outflow of culture medium.

[0044] Furthermore, a vent hole 17 is provided on the top cover 7 of the stretching rod, and the interior of the stretching rod body 5 is made hollow, so that the culture chamber 16 forms an air circuit with the outside through the vent hole 17 and the hollow stretching rod body 5, allowing external gas to enter the culture chamber 16 for use by the culture, and also allowing the waste gas generated by the culture in the culture chamber 16 to flow out from the air circuit, realizing online ventilation.

[0045] Furthermore, a sealing element 9 is provided on the cell inlet / outlet 8. In this device, the variable volume culture chamber 1 has a cell inlet / outlet 8 at the end away from the opening for injecting cells to be cultured, and the cell inlet / outlet 8 can be configured as follows: Figure 1 The diagram shows a protruding nozzle, on which a sealing element 9 can be installed. Specifically, the sealing element 9 can be as follows: Figure 1 The sealing cap shown is used to seal the culture chamber 16.

[0046] like Figure 1 As shown, the fixing assembly includes a fixing part 18 and a fixing bolt 6. The fixing part 18 is fixedly installed on the outer wall of the variable volume culture chamber 1. A first compression rubber ring 11 is provided inside the fixing part 18. The first compression rubber ring 11 contacts the outer surface of the tension rod body 5. The fixing bolt 6 is connected to the fixing part 18 by thread engagement. The first compression rubber ring 11 is located between the fixing bolt 6 and the fixing part 18. The first compression rubber ring 11 fixes the tension rod body 5 by the compression of the fixing bolt 6.

[0047] The fixing part 18 includes a fixing nut 2, a T-shaped sleeve 4, and a second compression rubber ring 3. The fixing nut 2 is fitted onto the outer wall of the variable volume culture chamber 1 near the opening. There is a gap between the inner wall of the fixing nut 2 with internal threads and the outer wall of the variable volume culture chamber 1. The second compression rubber ring 3 is provided in the gap. One end of the T-shaped sleeve 4 is connected to the inner thread of the fixing nut 2 in the gap by thread engagement. The T-shaped sleeve 4 squeezes the second compression rubber ring 3 by screwing in to fix the fixing nut 2 on the outer wall of the variable volume culture chamber 1.

[0048] An annular protrusion 19 is provided on the inner wall of the T-shaped sleeve 4. The annular protrusion 19 is fitted to the outer wall of the tension rod body 5. A first compression rubber ring 11 is provided on one side of the annular protrusion 19, and a fixing bolt 6 is provided on one side of the first compression rubber ring 11. The fixing bolt 6 is spirally connected to the inner wall of the T-shaped sleeve 4 by thread engagement. The first compression rubber ring 11 is fixed to the tension rod body 5 by the screwing in of the fixing bolt 6.

[0049] In this device, the tension rod body 5 is fixed by a first clamping rubber ring 11 and a second clamping rubber ring 3. For example... Figure 1 As shown, when the fixing bolt 6 rotates and moves within the T-sleeve 4, it compresses the first clamping rubber ring 11 when it reaches the bottom. The first clamping sealing ring, which has a circular cross-section, is then flattened, forming an elliptical cross-section. Furthermore, the first clamping rubber ring 11 presses against the outer wall of the tension rod body 5, thereby increasing the friction with the tension rod body 5 and fixing the culture chamber tension rod 15 in place. The fixing nut 2, which is fixed to the variable volume culture chamber 1, utilizes the same principle. Moreover, only the fixing bolt 6 needs to be rotated; the rotation of the fixing bolt 6 drives the T-sleeve 4 to move, thereby compressing the second clamping rubber ring 3, achieving the purpose of securely connecting the outer wall of the variable volume culture chamber 1.

[0050] In addition, a method for three-dimensional culture of cells or organoids using a three-dimensional culture vessel with a variable volume culture chamber and a culture method is also provided. The culture and fresh culture medium are loaded into the culture chamber 16 of the present invention through the cell inlet and outlet 8 according to the conventional culture process. The culture conditions are the same as those of the conventional process. The culture chamber 16 is rotatable. The rotation speed of the culture chamber 16 changes with the formation of cell aggregates during the cell culture process. The rotation speed of the culture chamber 16 is required to make the shear stress in the culture chamber 16 gradually change from approaching zero to the physiological stress of the cultured cells / tissues.

[0051] The rotational speed of the culture chamber 16 is determined by the size of different cultures suspended in the culture medium, and is greater than or equal to the minimum angular velocity of the culture suspension.

[0052] In addition, during the cultivation process, the gas supply flow rate of the gas supply-exhaust system to the gas chamber can be set to 0.4-10 ml / min, and the liquid supply flow rate of the liquid supply-exhaust system can be set to 0.1-10 ml / min.

[0053] like Figure 2 As shown, the rotation of this device can be performed by mounting a rotating device. Specifically, the rotating device includes a base 12, a support 13 is provided on the base 12, a rotating mechanism 14 is provided on one side of the support 13, and a culture vessel is installed on the other side. The tension rod body 5 of the culture vessel is connected to the rotating mechanism 14. The support 13 is also provided with a liquid supply-drainage system (not shown in the figure) for changing the pressure distribution inside the culture vessel, and a gas supply-exhaust system (not shown in the figure) for adjusting the gas supply and exhaust rates inside the culture vessel.

[0054] The above embodiments are merely exemplary embodiments of this application and are not intended to limit this application. The scope of protection of this application is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to this application within its substance and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of this application.

Claims

1. An in-situ variable volume culture chamber, characterized in that, include, A variable volume culture chamber (1) is hollow inside, with one end set as an opening and the other end set as a cell inlet / outlet (8). A culture chamber stretching rod (15) is set inside the variable volume culture chamber (1), and the culture chamber stretching rod (15) extends outward through the opening. The end edge of the culture chamber tension rod (15) is slidably connected to the inner wall of the variable volume culture chamber (1), and a culture chamber (16) is formed between the end face of the culture chamber tension rod (15) and the inner end face of the variable volume culture chamber (1). The variable volume culture chamber (1) is also provided with a fixing component for fixing the position of the culture chamber tension rod (15); The variable volume culture chamber (1) is used to culture cells or organoids. The culture chamber stretching rod (15) includes a stretching rod body (5) and a stretching rod top cover (7). The stretching rod top cover (7) is disposed in the variable volume culture chamber (1). The stretching rod top cover (7) is movably connected to the stretching rod body (5). A sliding sealing ring (10) is provided on the side edge of the stretching rod top cover (7). The sliding sealing ring (10) is slidably and sealingly connected to the inner wall of the variable volume culture chamber (1). The top cover (7) of the stretching rod is provided with a vent hole (17), and a vent membrane is provided on the vent hole (17). The vent membrane is placed inside the culture chamber (16). The tension rod body (5) is hollow inside, and a hole is opened at one end of the tension rod body (5) away from the tension rod top cover (7) and communicates with the external space.

2. The in-situ variable volume culture chamber according to claim 1, characterized in that, A sealing element (9) is provided on the cell inlet / outlet (8).

3. The in-situ variable volume culture chamber according to claim 1, characterized in that, The fixing component includes a fixing part (18) and a fixing bolt (6). The fixing part (18) is fixedly installed on the outer wall of the variable volume culture chamber (1). A first compression rubber ring (11) is provided inside the fixing part (18). The first compression rubber ring (11) contacts the outer surface of the tension rod body (5). The fixing bolt (6) is connected to the fixing part (18) by thread engagement. The first compression rubber ring (11) is located between the fixing bolt (6) and the fixing part (18). The first compression rubber ring (11) fixes the tension rod body (5) by the compression of the fixing bolt (6).

4. The in-situ variable volume culture chamber according to claim 3, characterized in that, The fixing part (18) includes a fixing nut (2), a T-shaped sleeve (4), and a second compression rubber ring (3). The fixing nut (2) is fitted on the outer wall of the variable volume culture chamber (1) near the opening. There is a gap between the inner wall of the fixing nut (2) with internal threads and the outer wall of the variable volume culture chamber (1). The second compression rubber ring (3) is provided in the gap. One end of the T-shaped sleeve (4) is connected to the internal thread of the fixing nut (2) in the gap by thread engagement. The T-shaped sleeve (4) squeezes the second compression rubber ring (3) by screwing in to fix the fixing nut (2) on the outer wall of the variable volume culture chamber (1). The inner wall of the T-shaped sleeve (4) is provided with an annular external protrusion (19), which is fitted to the outer wall of the tension rod body (5). A first compression rubber ring (11) is provided on one side of the annular external protrusion (19), and a fixing bolt (6) is provided on one side of the first compression rubber ring (11). The fixing bolt (6) is spirally connected to the inner wall of the T-shaped sleeve (4) by thread engagement. The first compression rubber ring (11) is fixed to the tension rod body (5) by the screwing and pressing of the fixing bolt (6).

5. A three-dimensional culture apparatus having an in-situ variable volume culture chamber as described in any one of claims 1-4, comprising, Culture medium stand (20), the culture medium stand (20) includes a base (12), a support (13) is provided on the base (12), and a rotating mechanism is provided on one side of the support (13); An in-situ variable volume culture chamber is provided on the other side of the support, and the tension rod body (5) of the in-situ variable volume culture chamber is connected to the rotating mechanism (14). The rotating mechanism (14) drives the in-situ variable volume culture chamber to rotate for culturing cells and organoids.

6. A three-dimensional culture device according to claim 5, characterized in that, The culture medium stand (20) is also provided with a liquid supply-drainage system for changing the internal pressure distribution of the in-situ variable volume culture chamber, and an air supply-exhaust system for adjusting the air supply and exhaust rates inside the in-situ variable volume culture chamber. The air supply-exhaust system supplies air to the air chamber at a flow rate of 0.4-10 ml / min, and the liquid supply-drainage system supplies liquid at a flow rate of 0.1-10 ml / min.

7. A method for culturing cells or organoids using the three-dimensional culture device described in claim 6, wherein the culture and fresh culture medium are loaded into the culture chamber (16) through the cell inlet / outlet (8) according to conventional culture processes, and the culture conditions are the same as those of conventional processes, characterized in that, The culture chamber (16) is rotatable, and the rotation speed of the culture chamber (16) changes with the formation of cell aggregates during the cell culture process. The rotation speed of the culture chamber (16) is such that the shear stress in the culture chamber (16) gradually changes from approaching zero to the physiological stress of the cultured cells / tissues.

8. The method for culturing cells or organoids according to claim 7, characterized in that, The rotational speed of the culture chamber (16) is determined by the size of different cultures suspended in the culture medium and is greater than or equal to the minimum angular velocity of the culture suspension.

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