Multi-site oxygen supply in-vitro brain slice incubation device

Abstract

The invention belongs to the related technical field of neuroscience experimental equipment and brain tissue slice incubation devices, and discloses a multi-site oxygen supply in-vitro brain slice incubation device.The in-vitro brain slice incubation device comprises an incubation container, a porous oxygen permeation membrane and a slice supporting piece, the incubation container is provided with a liquid bath, the liquid bath is provided with an oxygen inlet, and the porous oxygen permeation membrane is provided with an oxygen outlet; the porous oxygen permeation membrane is installed on the inner side wall of the liquid bath and corresponds to the oxygen inlet, the section supporting piece is installed in the liquid bath, located above the oxygen inlet and used for supporting a brain tissue section, and the porous oxygen permeation membrane surrounds the section supporting piece. According to the in-vitro brain slice incubation device, a large number of oxygen bubbles can be prevented from being detained below a brain tissue slice, vibration caused by oxygen bubble gathering in the incubation process of the brain tissue slice is reduced, oxygen can be distributed more evenly in incubation liquid, the brain tissue slice can be stably incubated, the keep-alive rate of the brain tissue slice is increased, and the incubation efficiency of the brain tissue slice is improved. The neuron activity rate is improved.

Description

Technical Field

[0001] The invention relates to the technical field related to neuroscience experimental equipment and brain tissue slice incubation devices, and in particular to an in vitro brain slice incubation device with multi-point oxygen supply. Background Art

[0002] In neuroscience research, patch clamp technology is an important research method. In order to obtain accurate experimental data, brain tissue slices need to be incubated so that the activity of their nerve cells reaches a certain standard. At present, the existing brain tissue slice incubation devices have developed to a certain extent in structure and function. For example, some incubation devices can basically meet the needs of providing incubation liquid and gas for brain tissue slices, and there are corresponding measures in temperature control to simulate the physiological environment in the body.

[0003] Existing rewarming incubation devices have many deficiencies in oxygen supply, mainly including: 1) the oxygen supply position is single, mostly at the bottom, which will cause brain tissue slices to roll in the incubation liquid and thus be damaged; 2) the oxygen supply area is small, and oxygen cannot be supplied quickly to allow the culture liquid to reach oxygen saturation. Summary of the invention

[0004] The object of the present invention is to provide an in vitro brain slice incubation device with multi-point oxygen supply, which can prevent a large number of oxygen bubbles from being retained under brain tissue slices, reduce the vibration caused by the aggregation of oxygen bubbles during the incubation of brain tissue slices, enable the brain tissue slices to be stably incubated, improve the survival rate of brain tissue slices, and make the distribution of oxygen in the incubation liquid more uniform, which is beneficial for the nerve cells on the surface of the brain tissue slices to better recover to a normal state and improve the activity rate of neurons.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] The present invention discloses an in vitro brain slice incubation device with multi-point oxygen supply, comprising: an incubation container, wherein the incubation container has a liquid bath, wherein the liquid bath is used to hold incubation liquid, and wherein the liquid bath has an oxygen inlet; a porous oxygen-permeable membrane, wherein the porous oxygen-permeable membrane is installed on the inner side wall of the liquid bath and is arranged corresponding to the oxygen inlet, wherein the porous oxygen-permeable membrane is used to guide bubbles entering the liquid bath from the oxygen inlet to rise along the inner side wall of the liquid bath; and a slice support, wherein the slice support is installed in the liquid bath and is located on a side of the porous oxygen-permeable membrane away from the inner side wall of the liquid bath, wherein the slice support is located above the oxygen inlet and is used to support brain tissue slices; wherein the porous oxygen-permeable membrane is arranged around the slice support.

[0007] In some embodiments, the in vitro brain slice incubation device with multi-site oxygen supply also includes a fixed base, which is installed on the bottom of the incubation container, and the fixed base is used to be installed in a water bath container.

[0008] In some embodiments, the in vitro brain slice incubation device with multi-site oxygen supply also includes an oxygen input tube, one end of which is connected to an external oxygen source, and the other end of which is connected to the oxygen inlet.

[0009] In some embodiments, the in vitro brain slice incubation device with multi-site oxygen supply also includes a fixed floating plate, and the fixed floating plate is provided with at least two fixed grooves, and the fixed grooves are used to accommodate the incubation container.

[0010] In some embodiments, the porous oxygen-permeable membrane has a pore size of 1.0 μm-2.8 μm.

[0011] In some embodiments, the bubbles passing through the porous oxygen-permeable membrane can generate small-size bubbles with an average particle size of less than 50 um.

[0012] In some embodiments, the incubation container is a cylindrical structure, and the porous oxygen-permeable membrane is a cylinder attached to the inner wall of the incubation container.

[0013] In some embodiments, the slice support is a porous mesh.

[0014] In some embodiments, the porous oxygen-permeable membrane is a porous ceramic membrane.

[0015] In some embodiments, along the height direction of the incubation container, the slice support is arranged corresponding to the middle of the porous oxygen-permeable membrane, and the distance between the top of the porous oxygen-permeable membrane and the top of the incubation container is smaller than the distance between the bottom of the porous oxygen-permeable membrane and the bottom of the incubation container.

[0016] The beneficial effects of the in vitro brain slice incubation device with multi-point oxygen supply of the present invention are as follows: by providing a porous oxygen-permeable membrane, a large number of oxygen bubbles can be prevented from being retained under the brain tissue slices, and the vibration caused by the aggregation of oxygen bubbles during the incubation of the brain tissue slices is reduced, so that the brain tissue slices can be stably incubated, and the survival rate of the brain tissue slices is improved. In addition, the porous oxygen-permeable membrane arranged around the slice support can provide dense and fine oxygen, so that the distribution of oxygen in the incubation liquid is more uniform, which is beneficial for the nerve cells on the surface of the brain tissue slices to better recover to a normal state, and the activity rate of neurons is improved, thereby laying a good foundation for subsequent neuron recording experiments.

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic structural diagram of an in vitro brain slice incubation device with multi-site oxygen supply according to an embodiment of the present invention;

[0019] Figure 2 It is a schematic diagram of the structure of the fixed floating plate and the incubation container in an embodiment of the present invention.

[0020] Reference numerals:

[0021] 100, incubation container; 110, liquid bath; 120, oxygen inlet; 200, porous oxygen permeable membrane; 300, slice support; 400, fixed base; 500, oxygen input pipe; 600, fixed floating plate; 610, fixed tank. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. It should also be noted that, for ease of description, only parts related to the present invention, rather than all structures, are shown in the accompanying drawings.

[0023] In the description of the present invention, unless otherwise clearly specified and limited, the terms "connected", "connected", and "fixed" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0024] In the description of this embodiment, the terms "upper", "lower", "left", "right", "front", "rear" and other directions or positional relationships are based on the directions or positional relationships shown in the drawings, and are only for the convenience of description and simplification of operation, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used to distinguish in the description and have no special meaning.

[0025] Reference below Figure 1-Figure 2 The specific structure of the in vitro brain slice incubation device with multi-site oxygen supply according to a specific embodiment of the present invention is described.

[0026] The present invention discloses an in vitro brain slice incubation device with multi-site oxygen supply, referring to Figure 1As shown, the in vitro brain slice incubation device with multi-site oxygen supply includes an incubation container 100, a porous oxygen-permeable membrane 200 and a slice support 300. The incubation container 100 has a liquid bath 110, the liquid bath 110 is used to hold incubation liquid, the liquid bath 110 has an oxygen inlet 120, the porous oxygen-permeable membrane 200 is installed on the inner wall of the liquid bath 110 and is arranged corresponding to the oxygen inlet 120, the porous oxygen-permeable membrane 200 is used to guide the bubbles entering the liquid bath 110 from the oxygen inlet 120 to rise along the inner wall of the liquid bath 110, the slice support 300 is installed in the liquid bath 110 and is located on the side of the porous oxygen-permeable membrane 200 away from the inner wall of the liquid bath 110, the slice support 300 is located above the oxygen inlet 120 for supporting brain tissue slices, and the porous oxygen-permeable membrane 200 is arranged around the slice support 300.

[0027] It is understandable that, in the actual working process, a suitable incubation container 100 is selected to install the porous oxygen permeable membrane 200 on the inner side of the incubation container 100, and then the output pipeline of the external oxygen source is sealed and connected to the oxygen inlet 120, and the oxygen flow rate is adjusted to a suitable value, and then the brain tissue slices are placed on the slice support 300, and an appropriate amount of artificial cerebrospinal fluid is poured as the incubation liquid, so that the brain tissue slices are completely immersed in the liquid, and the brain tissue slices are incubated. The incubation container 100 is used to hold the incubation liquid and provide a stable incubation environment for the brain tissue slices. Since the porous oxygen permeable membrane 200 has good oxygen permeability, the bubbles generated by the external oxygen source can rise along the inner wall of the liquid bath 110 (the principle is that the special pore structure of the porous oxygen permeable membrane 200 allows the bubbles to penetrate slowly and evenly, while preventing the bubbles from forming larger cavities inside the material, so as to achieve the refinement and uniform distribution of the bubbles). That is to say, by providing the porous oxygen-permeable membrane 200, a large number of oxygen bubbles can be prevented from being retained under the brain tissue slices, and the vibration caused by the aggregation of oxygen bubbles during the incubation of the brain tissue slices is reduced, so that the brain tissue slices can be stably incubated, and the survival rate of the brain tissue slices is improved. In addition, the porous oxygen-permeable membrane 200 arranged around the slice support 300 can provide dense and fine oxygen, so that the distribution of oxygen in the incubation liquid is more uniform, which is beneficial for the nerve cells on the surface of the brain tissue slices to better recover to a normal state and improve the neuronal activity rate, thereby laying a good foundation for subsequent neuronal recording experiments.

[0028] refer to Figure 1As shown, the in vitro brain slice incubation device with multi-site oxygen supply also includes a fixed base 400, which is installed at the bottom of the incubation container 100, and the fixed base 400 is used to be installed in a water bath container. It is understandable that the additional fixed base 400 can ensure that the incubation container 100 remains stable and does not tilt in the water bath container. By closely contacting the bottom of the water bath container, the fixed base 400 can resist the influence of liquid flow and external force on the liquid tank, thereby ensuring the stability of the incubation process. It should be noted that the material, shape, size and fixing method of the fixed base 400 to the water bath container can be selected according to actual needs, and the above parameters are not limited here. As long as the structure that can maintain the stability of the incubation container 100 in the water bath container can be used as the fixed base 400.

[0029] refer to Figure 1 As shown, the in vitro brain slice incubation device with multi-site oxygen supply also includes an oxygen input tube 500, one end of which is connected to an external oxygen source, and the other end is connected to the oxygen inlet 120. It can be understood that the oxygen input tube 500 connects the porous oxygen permeable membrane 200 and the external oxygen source, and can stably transport oxygen to the porous oxygen permeable membrane 200, ensuring that the porous oxygen permeable membrane 200 can continuously and stably provide dense and fine oxygen.

[0030] refer to Figure 2 As shown, the in vitro brain slice incubation device with multi-site oxygen supply also includes a fixed floating plate 600, and at least two fixed grooves 610 are provided on the fixed floating plate 600, and the fixed grooves 610 are used to accommodate the incubation container 100. Existing brain slice incubators usually have only one incubation groove, and it is impossible to conduct comparative observation tests on two or more experimental treatment conditions at the same time. In this embodiment, by setting a fixed floating plate 600, multiple incubation containers 100 can be used simultaneously through the fixed floating plate 600, so that multiple brain tissue slices can be rewarmed and restored to the in vivo state. The fixed floating plate 600 can be provided with multiple fixed grooves 610 as needed to place the incubation container 100, so that multiple incubation experiments under different conditions can be carried out simultaneously in the same device, thereby improving the experimental efficiency.

[0031] Optionally, the pore size of the porous oxygen permeable membrane 200 is 1.0 μm-2.8 μm. In the incubation tank in the prior art, due to the large pore size of the vent hole of the oxygen supply device, it is impossible to accurately control the bubble particle size, resulting in large bubbles in the incubation liquid, violent fluctuations or bubble bursting. In this embodiment, by controlling the pore size of the porous oxygen permeable membrane 200, the bubble particle size can be accurately controlled, which is conducive to the better recovery of the nerve cells on the surface of the brain tissue slice to a normal state and improves the activity rate of neurons.

[0032] Optionally, the bubbles passing through the porous oxygen permeable membrane 200 can generate small-sized bubbles with an average particle size of less than 50 um. Small-sized bubbles with an average particle size of less than 50 um are beneficial for the nerve cells on the surface of the brain tissue slice to recover to a normal state better, thereby improving the activity rate of neurons.

[0033] Optionally, the incubation container 100 is a cylindrical structure, and the porous oxygen-permeable membrane 200 is a cylinder attached to the inner wall of the incubation container 100. It is understandable that the cylindrical incubation container 100 is conducive to the uniform distribution and temperature control of the incubation liquid, thereby improving the success rate of incubation. Of course, in other embodiments of the present invention, the shape of the incubation container 100 can also be selected from other shapes according to actual needs.

[0034] Optionally, the slice support 300 is a porous mesh. It is understandable that the design of the porous mesh can allow the brain tissue slices to fully contact the incubation liquid and oxygen, while preventing the brain tissue slices from floating or accumulating in the liquid, ensuring that the brain tissue slices can be stably incubated.

[0035] Optionally, the porous oxygen-permeable membrane 200 is a porous ceramic membrane. Of course, in other embodiments of the present invention, the porous oxygen-permeable membrane 200 may also be made of other materials.

[0036] In some embodiments, along the height direction of the incubation container 100, the slice support 300 is arranged corresponding to the middle part of the porous oxygen permeable membrane 200, and the distance between the top of the porous oxygen permeable membrane 200 and the top of the incubation container 100 is smaller than the distance between the bottom of the porous oxygen permeable membrane 200 and the bottom of the incubation container 100. It can be understood that the slice support 300 is arranged corresponding to the middle part of the porous oxygen permeable membrane 200, so that the brain tissue slices can be fully contacted with the incubation liquid and oxygen, ensuring that the brain tissue slices can be stably incubated. It should be noted that the space between the bottom of the porous oxygen permeable membrane 200 and the bottom of the incubation container 100 is located in the area below the brain tissue slices. This area is small, which is conducive to the rapid rise of bubbles, avoiding the accumulation of bubbles in this area, and is conducive to improving the success rate of incubation.

[0037] The advantages of the in vitro brain slice incubation device with multi-site oxygen supply disclosed in the present invention are as follows:

[0038] First, the stability of brain tissue slices is improved: the bubbles are restricted on the inner wall of the incubation container 100 by a porous ceramic membrane, so that a large amount of oxygen bubbles are prevented from being retained under the brain tissue slices, and the vibration caused by the aggregation of oxygen bubbles during the incubation of the brain tissue slices is reduced, so that the brain tissue slices can be stably incubated, and the survival rate of the brain tissue slices is improved.

[0039] Second: Improve the oxygen supply effect: The porous ceramic membrane can provide dense and fine oxygen, making the distribution of oxygen in the incubation liquid more even, which is beneficial for the nerve cells on the surface of brain tissue slices to better recover to normal state and improve the activity rate of neurons, thus laying a good foundation for subsequent neuron recording experiments.

[0040] Third: improving experimental efficiency: the fixed floating plate 600 enables the simultaneous use of multiple incubation containers 100, and can simultaneously conduct comparative observation tests under multiple experimental treatment conditions, thereby reducing the number of experimental repetitions and saving experimental time and costs.

[0041] The following describes the process of performing single-condition incubation experiments and multi-condition incubation experiments using the in vitro brain slice incubation device with multi-site oxygen supply of the present invention.

[0042] Single condition incubation experiment:

[0043] Preparation stage: First, select a suitable cylindrical incubation container 100 (with an inner diameter of 42 mm, an outer diameter of 45 mm, and a height of 90 mm), and place it on a bottom fixed base 400 (a cylinder with a diameter of 45 mm and a height of 30 mm), and ensure that the incubation container 100 is stable in the water bath. Then, install the porous ceramic membrane on the inner side of the incubation container 100 (the height of the porous ceramic membrane is 50 mm, and the top spacing with the incubation container 100 is 30 mm, and the bottom spacing is 10 mm), and connect the external oxygen source through the oxygen input tube 500 (with a length of 20 mm and an inner diameter of 5 mm), and adjust the oxygen flow rate to a suitable value (for example, according to experience, it can be set to 5 ml-10 ml per minute). Next, place the brain tissue slices on the porous filter, pour in an appropriate amount of artificial cerebrospinal fluid as the incubation liquid, and immerse the brain tissue slices completely in the liquid.

[0044] Incubation stage: Set the temperature of the water bath to a temperature close to the physiological temperature of the body (for example, about 37°C) and start the incubation process. During the incubation process, observe the size and distribution of oxygen bubbles. Through the effect of the porous ceramic membrane, it can be seen that the bubbles rise evenly along the inner wall of the incubation container 100, and no large bubbles gather under the brain tissue slices. After incubation for 1 hour to 1.5 hours, observe the state of the brain tissue slices and use relevant instruments to detect the activity rate of nerve cells. If the activity rate meets the requirements (the number of neurons with complete morphology must be >50%), it means that the incubation is successful.

[0045] Multi-condition comparative incubation experiment:

[0046] Preparation stage: According to the number of experimental conditions to be compared, select four cylindrical incubation containers 100 and place them on different fixed grooves 610 (circular grooves with a diameter of 45 mm) of the fixed floating plate 600 (rectangular plate with a length of 160 mm and a width of 140 mm). According to the steps of the above-mentioned single-condition incubation experiment, each incubation container 100 is respectively installed with a porous ceramic membrane, connected to an oxygen source, placed with brain tissue slices, and poured with incubation liquid. Different incubation containers 100 can be set with different oxygen flow, temperature and other parameters to achieve comparison of different conditions.

[0047] Incubation stage: The incubation process of each incubation container 100 is started at the same time. During the incubation process, the size and distribution of oxygen bubbles and the state of brain tissue slices in each incubation container 100 are observed. After the incubation is completed, the activity rate of nerve cells in the brain tissue slices in each incubation container 100 is detected using relevant instruments, and the results under different conditions are compared and analyzed to obtain the influence of different conditions on the incubation effect of brain tissue slices.

[0048] The following points should be noted during the above experiment:

[0049] Installation of the porous ceramic membrane: Make sure that the porous ceramic membrane is installed tightly on the inner side of the incubation container 100 to avoid gaps, otherwise it will affect the oxygen permeation effect and the bubble restriction effect.

[0050] Adjustment of oxygen flow rate: The appropriateness of oxygen flow rate directly affects the incubation effect of brain tissue slices. If the oxygen flow rate is too large, it will cause too many bubbles and affect the stability of brain tissue slices; if the oxygen flow rate is too small, it may not be able to meet the oxygen demand of brain tissue slices. Therefore, before starting incubation, it is necessary to carefully adjust the oxygen flow rate based on experience and actual conditions.

[0051] Placement of brain tissue slices: Place brain tissue slices evenly on the porous mesh to avoid stacking or partial vacancies in the slices, so as to ensure that the slices can fully contact the incubation liquid and oxygen.

[0052] Prevent contamination: During the entire process, care should be taken to keep the equipment and experimental materials clean to avoid contamination of brain tissue slices and incubation liquids. For example, hands and experimental equipment should be disinfected before operation, and the incubation liquid used should be strictly filtered and sterilized.

[0053] Temperature control: The temperature of the water bath should be accurately controlled close to the physiological temperature of the body, and the deviation should not be too large, otherwise it will affect the activity recovery of brain tissue slices. At the same time, the temperature stability of the water bath should be checked regularly to ensure that the temperature remains constant during incubation.

[0054] In the description of this specification, the description with reference to the terms "some embodiments", "other embodiments", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least two embodiments or examples of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0055] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, various obvious changes, readjustments and substitutions can be made without departing from the protection scope of the present invention. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. An in vitro brain slice incubation device with multi-site oxygen supply, characterized in that: include: An incubation container (100), wherein the incubation container (100) has a liquid bath (110), wherein the liquid bath (110) is used to contain an incubation liquid, and wherein the liquid bath (110) has an oxygen inlet (120); a porous oxygen-permeable membrane (200), the porous oxygen-permeable membrane (200) being mounted on the inner side wall of the liquid bath (110) and arranged corresponding to the oxygen inlet (120), the porous oxygen-permeable membrane (200) being used to guide bubbles entering the liquid bath (110) from the oxygen inlet (120) to rise along the inner side wall of the liquid bath (110); a slice support (300), the slice support (300) being installed in the liquid bath (110) and being located on a side of the porous oxygen-permeable membrane (200) away from the inner wall of the liquid bath (110), the slice support (300) being located above the oxygen inlet (120) and used for supporting brain tissue slices; Wherein, the porous oxygen-permeable membrane (200) is arranged around the slice support (300).

2. The in vitro brain slice incubation device with multi-site oxygen supply according to claim 1, characterized in that: It also includes a fixed base (400), which is installed on the bottom of the incubation container (100), and the fixed base (400) is used to be installed in a water bath container.

3. The in vitro brain slice incubation device with multi-site oxygen supply according to claim 1, characterized in that: It also includes an oxygen input pipe (500), one end of which is connected to an external oxygen source, and the other end of which is connected to the oxygen inlet (120).

4. The in vitro brain slice incubation device with multi-site oxygen supply according to claim 1, characterized in that: It also includes a fixed floating plate (600), on which at least two fixed grooves (610) are provided, and the fixed grooves (610) are used to accommodate the incubation container (100).

5. The in vitro brain slice incubation device with multi-site oxygen supply according to any one of claims 1 to 4, characterized in that: The pore size of the porous oxygen-permeable membrane (200) is 1.0 μm-2.8 μm.

6. The in vitro brain slice incubation device with multi-site oxygen supply according to any one of claims 1 to 4, characterized in that: The bubbles passing through the porous oxygen-permeable membrane (200) can generate small-diameter bubbles with an average particle size of less than 50 um.

7. The in vitro brain slice incubation device with multi-site oxygen supply according to any one of claims 1 to 4, characterized in that: The incubation container (100) is a cylindrical structure, and the porous oxygen-permeable membrane (200) is a cylinder attached to the inner wall of the incubation container (100).

8. The in vitro brain slice incubation device with multi-site oxygen supply according to any one of claims 1 to 4, characterized in that: The slice support (300) is a porous mesh.

9. The in vitro brain slice incubation device with multi-site oxygen supply according to any one of claims 1 to 4, characterized in that: The porous oxygen-permeable membrane (200) is a porous ceramic membrane.

10. The in vitro brain slice incubation device with multi-site oxygen supply according to any one of claims 1 to 4, characterized in that: Along the height direction of the incubation container (100), the slice support (300) is arranged corresponding to the middle part of the porous oxygen-permeable membrane (200), and the distance between the top of the porous oxygen-permeable membrane (200) and the top of the incubation container (100) is smaller than the distance between the bottom of the porous oxygen-permeable membrane (200) and the bottom of the incubation container (100).

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