In-vitro oxygen-glucose deprivation simulation reoxygenation device

Abstract

The utility model relates to the field of in-vitro simulation oxygen-glucose deprivation re-reoxygenation experiments, and discloses an in-vitro simulation oxygen-glucose deprivation re-reoxygenation device which comprises a sealing box, a plurality of sets of pore plate placing frames are arranged on the inner wall of the sealing box, and an air inlet pipe and an air outlet pipe are arranged on the two opposite side faces of the sealing box respectively. The end part, far away from the sealing box, of the air outlet pipe is connected with an oxygen detector; the end part, far away from the sealing box, of the gas inlet pipe is connected with a mixed gas supply source; a needle valve is arranged on the air outlet pipe, and an adjusting valve is arranged on the air inlet pipe. According to the in-vitro oxygen-glucose deprivation simulation and reoxygenation device provided by the utility model, the sealed box, the gas inlet pipe, the gas outlet pipe, the oxygen detector and the mixed gas supply source are simple models which are built by simple instruments commonly existing in the existing experiment; the device can be widely applied to simulation of the hypoxia state of intravascular cell culture in experiments, and the experiment cost of in-vitro simulation of oxygen-glucose deprivation and reoxygenation is greatly reduced.

Description

Technical Field

[0001] This utility model relates to the field of oxygen-glucose deprivation and reoxygenation experimental technology, specifically to an in vitro simulated oxygen-glucose deprivation and reoxygenation device. Background Technology

[0002] A thorough understanding and study of the mechanisms of intravascular resorption (IRI) is of significant clinical value for the rehabilitation of patients with cardiovascular and cerebrovascular diseases. In vitro induced cell hematopoiesis (H / R) can serve as a model for exploring IRI in cardiovascular and cerebrovascular diseases. Currently, H / R models both domestically and internationally mainly employ physical and chemical methods. The most common physical model is the three-gas chamber, but this instrument is expensive and difficult to widely implement. Even in laboratories that possess this instrument, continuous gas refilling is required during use, which is inconvenient for laboratory applications. Utility Model Content

[0003] The purpose of this invention is to provide an in vitro simulated oxygen-glucose deprivation and reoxygenation device to solve the technical problems of existing experimental simulation equipment being difficult to popularize and having high operating costs.

[0004] To solve the above-mentioned technical problems, this utility model specifically provides the following technical solution:

[0005] An in vitro simulated oxygen-glucose deprivation and reoxygenation device includes:

[0006] A sealed box, wherein multiple sets of perforated plate placement racks are provided on the inner wall of the sealed box, and an air inlet pipe and an air outlet pipe are respectively provided on two opposite sides of the sealed box;

[0007] An oxygen detector is connected to the end of the outlet pipe away from the sealed box; a mixed gas supply source is connected to the end of the inlet pipe away from the sealed box.

[0008] A needle valve is installed on the air outlet pipe, and a regulating valve is installed on the air inlet pipe.

[0009] As a preferred embodiment of this utility model, the sealing box includes a box-shaped body and a cover, with one side surface of the box-shaped body open to form an opening;

[0010] A membrane is provided on the opening, the membrane is used to cover the opening, and the edge of the membrane extends to the outer wall of the box-shaped body;

[0011] The cover seals the box-shaped body by engaging with the opening that covers the membrane.

[0012] As a preferred embodiment of this utility model, a transparent window panel is provided on the surface of the cover, and the cover is provided with a mounting groove for installing the transparent window panel.

[0013] As a preferred embodiment of the present invention, the perforated plate placement rack includes a horizontal bottom plate protruding from the inner wall surface of the box-shaped main body, an inclined baffle plate that is inclined relative to the surface of the box-shaped main body is connected to one side of the horizontal bottom plate, a horizontal top plate is connected to the end of the inclined baffle plate away from the horizontal bottom plate, and the horizontal bottom plate is engaged with the bottom corner of the perforated plate.

[0014] The distance between the horizontal upper plate and the cover is greater than half the length of the perforated plate.

[0015] As a preferred embodiment of this utility model, a handle is provided on the outer wall of the box-shaped body, and magnets are provided at both ends of the handle. Iron pieces that cooperate with the two magnets are provided on both sides of the perforated plate.

[0016] In a preferred embodiment of this utility model, a syringe body is connected to the side wall of the box-shaped main body. One end of the syringe body is closed, and the other end is connected to the interior of the box-shaped main body. A piston body is provided inside the syringe body, and an operating rod is connected to the piston body. One end of the operating rod passes through the closed end of the syringe body. A connecting terminal is provided on the syringe body near the closed end of the syringe body. A detection port is provided on the syringe body near the box-shaped main body. The air outlet is connected to the connecting terminal.

[0017] Compared with the prior art, this utility model has the following advantages:

[0018] The in vitro simulated oxygen-glucose deprivation and reoxygenation device provided by this utility model is a simple model constructed using relatively sealed boxes, air inlet pipes, air outlet pipes, oxygen detectors, and mixed gas supply sources, all of which are simple instruments commonly found in existing experiments. It can be widely used in experiments to simulate the hypoxic state of intravascular cell culture, greatly reducing the experimental cost of in vitro simulated oxygen-glucose deprivation and reoxygenation. Attached Figure Description

[0019] To more clearly illustrate the embodiments of this utility model 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 in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the overall structure of an embodiment of the present utility model;

[0021] Figure 2 This is a schematic diagram of the membrane covering opening portion of an embodiment of the present invention;

[0022] Figure 3 This is a schematic cross-sectional view of the box-shaped main body without a membrane in an embodiment of the present invention.

[0023] The labels in the diagram represent the following:

[0024] 1-Sealed box; 2-Orifice plate holder; 3-Inlet pipe; 4-Outlet pipe; 5-Regulating valve; 6-Oxygen detector; 7-Mixed gas supply source; 8-Needle valve;

[0025] 11-Box-shaped main body; 12-Lid; 13-Opening; 14-Membrane; 15-Transparent window plate; 17-Syringe body; 18-Piston body; 19-Operating lever; 20-Connecting terminal; 21-Horizontal base plate; 22-Inclined baffle; 23-Horizontal upper plate; 24-Detection port. Detailed Implementation

[0026] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments. Based on the embodiments of the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of the present utility model.

[0027] like Figures 1 to 3 As shown, this utility model provides an in vitro simulated oxygen-glucose deprivation and reoxygenation device. Its purpose is to construct an oxygen-deficient environment using a self-made simple sealed box, establish a H / R damage model of HUVECs, and explore the feasibility and repeatability of this modeling method.

[0028] A sealed box 1, with multiple sets of perforated plate placement racks 2 provided on the inner wall of the sealed box 1, and an air inlet pipe 3 and an air outlet pipe 4 respectively provided on two opposite sides of the sealed box 1;

[0029] An oxygen detector 6 is connected to the end of the air outlet pipe 4 away from the sealing box 1; a mixed gas supply source 7 is connected to the end of the air inlet pipe 3 away from the sealing box 1.

[0030] A needle valve 8 is provided on the air outlet pipe 4, and a regulating valve 5 is provided on the air inlet pipe 3.

[0031] This embodiment provides an example of forming an experimental apparatus for a sealed box 1. The hypoxic environment in this experiment is constructed using a milk powder box to form the sealed box and an oxygen detector. The milk powder box itself has good sealing properties. Holes are drilled on the opposite side surface of the milk powder box (sealed box). The infusion tubing, i.e., the air inlet tube 3, is tightly connected to the holes using silicone. The 6-well plate of the experimental group is placed inside the milk powder box. The plate placement rack 2 is covered with a layer of plastic wrap, and the lid is closed. Beeswax is applied to the lid and the sides of the box body for sealing.

[0032] The inlet pipe 3 is filled with a mixture of 94% nitrogen, 5% carbon dioxide, and 1% oxygen. The outlet pipe 4 is connected to an oxygen detector 6. When the oxygen detector indicates <1%, the infusion pipeline regulator (the regulating valve 5 on the inlet pipe 3) is closed. Then, the infusion pipeline is folded and clamped with tweezers (the needle valve 8 on the outlet pipe 4 closes the outlet pipe). The self-made sealed box is then in a state of no gas convection, i.e., a state of oxygen deficiency.

[0033] The in vitro simulated oxygen-glucose deprivation and reoxygenation device provided by this utility model adopts a simple model constructed from relatively sealed box 1, air inlet pipe 3, air outlet pipe 4, oxygen detector 8, and mixed gas supply source 7, all of which are simple instruments commonly found in existing experiments. It can be widely used in experiments to simulate the hypoxic state of intravascular cell culture, greatly reducing the experimental cost of in vitro simulated oxygen-glucose deprivation and reoxygenation.

[0034] The sealed box 1 includes a box-shaped body 11 and a cover 12, with one side surface of the box-shaped body 11 open to form an opening 13;

[0035] A membrane 14 is provided on the opening 13, the membrane 14 is used to cover the opening 13, and the edge of the membrane 14 extends to the outer wall of the box-shaped body 11.

[0036] The cover 12 seals the box-shaped body 11 by engaging with the opening 13 that covers the membrane 14.

[0037] In this embodiment, HUVECs were digested in the cultured cells on the well plates. When 75% of the cells became rounded, complete culture medium was added, and the mixture was thoroughly mixed by pipetting. The cells were then seeded into the well plates. When the cells reached approximately 80% confluence, the experimental group was replaced with low-glucose serum-free culture medium and placed in a self-made sealed container. The sealed container was then placed in a CO2 incubator for hypoxia treatment for 3, 6, 9, and 12 hours, respectively, according to experimental requirements. After the hypoxia treatment, the cells were washed, and complete culture medium was added. The cells were then incubated for 2 hours. The control group (Control group) cells were cultured normally with medium changes and no other treatments were performed.

[0038] Existing models lack a unified standard, with the main metric being the induction of stable cell damage, such as cell structure and apoptosis rate. The duration of hypoxia varies; in this study, we found that after 12 hours of hypoxia, many cells became rounded and floated, potentially leading to significant errors. Therefore, in this implementation, the hypoxia time for all groups was set within 12 hours during the experiment.

[0039] Furthermore, this embodiment aims to enable process observation under hypoxic conditions within the sealed box 1, and to observe changes in cells in the culture dish on the inner perforated plate of the sealed box 1. To this end, a transparent window plate 15 is provided on the surface of the cover 12, and a mounting groove for mounting the transparent window plate 15 is provided on the cover 12.

[0040] To avoid the potential impact of airflow entering the box-shaped body 11 on the culture medium, such as airflow directly flowing into the culture medium and causing additional influence on the cells in the culture medium, the well plate holder 2 is tilted so that multiple well plates are arranged at equal intervals inside the box-shaped body 11, and the airflow entering the box-shaped body 11 blows towards the back side of the well plate. Of course, this tilt angle is mainly to avoid affecting the morphology of the cells in the culture dish.

[0041] Therefore, in this embodiment, the perforated plate placement rack 2 includes a horizontal bottom plate 21 protruding from the inner wall surface of the box-shaped body 11, an inclined baffle 22 that is inclined relative to the surface of the box-shaped body 11 is connected to one side of the horizontal bottom plate 21, a horizontal top plate 23 is connected to the end of the inclined baffle 22 away from the horizontal bottom plate 21, and the horizontal bottom plate 21 is engaged with the bottom corner of the perforated plate.

[0042] The distance between the horizontal upper plate 23 and the cover 12 is greater than half the length of the perforated plate.

[0043] That is, when the perforated plate is placed into the box-shaped body 11, the bottom edges of both sides of the perforated plate contact the inclined baffle 22 and then rest against the inclined baffle 22.

[0044] When it is necessary to observe the cell hypoxia of a well plate through the transparent window plate 15, a handle can be provided on the outer wall of the box-shaped body 11, and magnets are provided at both ends of the handle. Iron pieces that cooperate with the two magnets are provided on both sides of the well plate. The person holds the two handles and moves the well plate from the tilted state to the horizontal upper plate 23 to observe the cells in the culture dish.

[0045] Of course, if the petri dishes are placed horizontally, then a large-volume box-shaped body 11 is required for multiple experimental well plates, which is inconvenient for obtaining materials in the laboratory.

[0046] In this embodiment, it is necessary to consider the influence of the gas in the internal environment of the sealed box 1 after the experimental material is sealed. Therefore, it is necessary to purge the sealed box 1. The purging process requires the removal of residual air in the box body 11 as soon as possible. This means that the purged gas will have additional influence on the cells in the culture dish in the well plate as described above.

[0047] Meanwhile, when it is necessary to transport the gas inside the box-shaped body 11 to the detection end through pipes (outlet pipe 4, inlet pipe 3), such as a handheld oxygen meter, the detection of the gas present in the pipes and the gas after sealing cannot accurately characterize the oxygen content inside the box-shaped body 11. At the same time, during the hypoxia maintenance stage, the oxygen consumption of the cells cannot be accurately reflected, and it is necessary to provide the sealed detection of the box-shaped body 11.

[0048] Therefore, in this embodiment, a syringe body 17 is connected to the side wall of the box-shaped main body 11. Specifically, a syringe barrel can be used as an existing material. One end of the syringe body 17 is closed (the end opposite to the injection end), and the other end (i.e., the injection end of the syringe) is connected to the interior of the box-shaped main body 11. A piston body 18 is provided inside the syringe body 17, and an operating rod 19 is connected to the piston body 18. One end of the operating rod 19 passes through the closed end of the syringe body 17. A connecting terminal 20 is provided on the syringe body 17 near the closed end, and a detection port 24 is provided on the part of the syringe body 17 near the box-shaped main body 11. The air outlet 4 is connected to the connecting terminal 20.

[0049] When purging the air inside the box-shaped body 11, the piston body 18 is pulled to the closed end near the syringe body 17 by the operating lever 19, so that the connecting terminal 20, which is a quick-release interface, is connected to the inside of the box-shaped body 11. At this time, the detection port 24 is sealed and closed. The detection port 24 is similar to the valve structure of a car tire. The gas in the sealed box 1 enters the air outlet pipe 4 or is discharged through the air outlet pipe 4.

[0050] Before closing the infusion pipeline regulator (the regulating valve 5 installed on the air inlet pipe 3) when the oxygen meter reading is <1%, push the operating lever 19 to push the piston body 18 to a position close to the detection port 24 to close the connection between the box-shaped body 11 and the connecting terminal 20. At this time, the oxygen content in the gas closest to the box-shaped body 11 can be obtained by inserting the probe of the handheld oxygen meter into the detection port 24.

[0051] By freeing the operating lever 19, after the closed box-shaped body 11 and the connecting terminal 20 are connected, the displacement change of the piston body 18 due to the air pressure inside the sealed box 1 can be used to consider the respiration of cells under hypoxia.

[0052] Colleagues can also judge the sealing performance of the sealing box 1 by whether the piston body 18 moves.

[0053] 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 vitro simulated oxygen-glucose deprivation and reoxygenation device, characterized in that, include: A sealed box (1) is provided with multiple sets of perforated plate placement racks (2) on the inner wall of the sealed box (1), and an air inlet pipe (3) and an air outlet pipe (4) are respectively provided on two opposite sides of the sealed box (1). An aerobic detector (6) is connected to the end of the outlet pipe (4) away from the sealing box (1); a mixed gas supply source (7) is connected to the end of the inlet pipe (3) away from the sealing box (1). A needle valve (8) is provided on the air outlet pipe (4), and a regulating valve (5) is provided on the air inlet pipe (3).

2. The in vitro simulated oxygen-glucose deprivation and reoxygenation device according to claim 1, characterized in that, The sealed box (1) includes a box-shaped body (11) and a cover (12), with one side surface of the box-shaped body (11) open to form an opening (13). A membrane (14) is provided on the opening (13), the membrane (14) is used to cover the opening (13), and the edge of the membrane (14) extends to the outer wall of the box-shaped body (11); The cover (12) seals the box-shaped body (11) by engaging with the opening (13) covering the membrane (14).

3. The in vitro simulated oxygen-glucose deprivation and reoxygenation device according to claim 2, characterized in that, A transparent window panel (15) is provided on the surface of the cover (12), and a mounting groove for installing the transparent window panel (15) is provided on the cover (12).

4. The in vitro simulated oxygen-glucose deprivation and reoxygenation device according to claim 2, characterized in that, The perforated plate holder (2) includes a horizontal bottom plate (21) protruding from the inner wall surface of the box-shaped body (11), an inclined baffle (22) is connected to one side of the horizontal bottom plate (21) and is inclined relative to the surface of the box-shaped body (11), and a horizontal top plate (23) is connected to the end of the inclined baffle (22) away from the horizontal bottom plate (21), and the horizontal bottom plate (21) is engaged with the bottom corner of the perforated plate; The distance between the horizontal upper plate (23) and the cover (12) is greater than half the length of the perforated plate.

5. The in vitro simulated oxygen-glucose deprivation and reoxygenation device according to claim 4, characterized in that, A handle is provided on the outer side wall of the box-shaped body (11), and magnets are provided at both ends of the handle. Iron pieces that cooperate with the two magnets are provided on both sides of the perforated plate.

6. The in vitro simulated oxygen-glucose deprivation and reoxygenation device according to claim 2, characterized in that, A syringe body (17) is connected to the side wall of the box-shaped body (11). One end of the syringe body (17) is closed, and the other end is connected to the interior of the box-shaped body (11). A piston body (18) is provided inside the syringe body (17). An operating rod (19) is connected to the piston body (18). One end of the operating rod (19) passes through the closed end of the syringe body (17). A connecting terminal (20) is provided on the syringe body (17) near the closed end. A detection port (24) is provided on the syringe body (17) near the box-shaped body (11). The air outlet (4) is connected to the connecting terminal (20).

Images