An in vitro perfusion device for isolated human liver tissue

Abstract

This invention discloses an in vitro perfusion device for isolated human liver tissue, belonging to the field of medical experimental equipment technology. The key technical features are: a receiving box, a puncture needle, and a peristaltic pump. The receiving box has expansion zones on its four walls, with fixed points symmetrically arranged on both sides of the expansion zones. An angle adjuster is connected to the center of each fixed point, and a needle hole is located at the center of the angle adjuster for the puncture needle to pass through. A connector is connected to the tail of the puncture needle. The peristaltic pump has an outlet on one side and an inlet on the top. A container holding perfusion fluid and collagenase is connected to the inlet. The outlet is connected to a manifold connector, which is connected to a connecting pipe. The other end of the connecting pipe is connected to the connector. A control keyboard is provided on the peristaltic pump. A fixed platform is located at the bottom center of the receiving box, and a fixing structure is provided on the fixed platform. This invention solves the problems of long perfusion time, high operational difficulty, and high safety risks existing in the prior art.

Description

Technical Field

[0001] This invention relates to the field of medical experimental equipment technology, and more specifically, to an in vitro perfusion device for isolated human liver tissue. Background Technology

[0002] The liver, as the largest solid organ and gland in the human body, is often referred to as the "human chemical factory," playing a central role in the body's physiological functions. Hepatocytes, as the main functional units of the liver, are the basic carriers for performing various complex physiological functions. They synthesize clotting factors and serum albumin, participate in endocrine regulation, efficiently metabolize nutrients, store glucose, and remove toxins. Therefore, the isolation and culture of hepatocytes has extremely high value in basic life science research and clinical medical applications, helping to reveal the molecular mechanisms of life processes such as cell differentiation and metabolic regulation, and advancing research on the diagnosis, treatment, and prevention of liver diseases such as hepatitis, cirrhosis, and liver cancer.

[0003] The development of hepatocyte separation technology is a crucial driving force behind the continuous advancement of hepatocyte research. Among existing technologies, perfusion is the mainstream technique for hepatocyte separation. Perfusion involves injecting fluid into the liver's blood vessels, loosening the connections between hepatocytes and thus achieving more efficient and less invasive hepatocyte separation. Among these techniques, multi-point puncture collagenase perfusion is an extension of traditional perfusion. It involves perfusing collagenase into the liver tissue through multiple punctures to increase the contact area between the collagenase and the liver tissue, thereby improving the efficiency of hepatocyte separation.

[0004] Although multi-point puncture collagenase perfusion technology has been applied to some extent in hepatocyte isolation research, it still has the following problems:

[0005] 1. Irrigation efficiency is low and costs are high.

[0006] In the processing of ex vivo liver tissue, the procedure cannot be completed quickly via intravenous perfusion as it can with in vivo liver tissue, making multi-point puncture collagenase perfusion a very time-consuming process. Furthermore, the prolonged perfusion procedure requires large amounts of collagenase and perfusion fluid, which are expensive reagents, further increasing the overall cost of the experiment.

[0007] 2. The operation process is complex and the technical requirements are stringent.

[0008] The multi-point puncture collagenase perfusion technique involves numerous steps and demands a high level of professional skill from the operators. From the collection and fixation of excised liver tissue, to the selection of puncture sites and control of puncture depth, and then to the adjustment of collagenase perfusion rate and dosage, each step requires operators to have extensive experience and precise operational abilities.

[0009] 3. High operational risk

[0010] Multiple puncture procedures require operators to perform multiple punctures on the excised liver tissue, which can easily result in hand injuries and further complications such as infection. Summary of the Invention

[0011] The purpose of this invention is to provide an in vitro perfusion device for ex vivo human liver tissue to solve the problems of long perfusion time, high operation difficulty, and high safety risks in the existing technology.

[0012] The above-mentioned technical objective of the present invention is achieved through the following technical solution: an in vitro perfusion device for ex vivo human liver tissue, comprising a receiving box, a puncture needle and a peristaltic pump, wherein the four walls of the receiving box are provided with an expansion area, and each expansion area is symmetrically provided with a fixing point on both sides, an angle adjuster is connected to the center of the fixing point, a needle hole is provided at the center of the angle adjuster, the needle hole is for the puncture needle to pass through, and a connector is connected to the tail of the puncture needle;

[0013] The peristaltic pump has an outlet on one side and an inlet on the top. The inlet is connected to a container holding the infusion fluid and collagenase. The outlet is connected to a manifold connector, which is connected to a connecting pipe. The other end of the connecting pipe is connected to a connector. The peristaltic pump is equipped with a control keyboard.

[0014] A fixed platform is provided at the bottom center of the container, and a fixing structure is provided on the fixed platform.

[0015] The present invention is further configured such that the extended region is made of rubber or silicone material with elastic deformation capability.

[0016] The present invention is further configured such that the pipe connector is a 1-to-4 connector, a 1-to-5 connector, or a 1-to-6 connector.

[0017] The present invention is further configured such that: a control valve is provided at the connection between the branch pipe joint and the connecting pipe, and the control keyboard is used to regulate the opening and closing of the control valve and the switching on and off of the peristaltic pump, thereby regulating the flow rate and flow rate of the perfusion fluid and collagenase flowing out of the outlet.

[0018] The invention is further configured such that: the fixing structure is disposed at the four corners of the fixing platform; the fixing structure includes a fixing base, a rotating disk, and a triangular piece connected to the fixing platform; a column is connected to the top of the fixing base; a height control thread is provided on the top area of ​​the column; a threaded area adapted to the height control thread is provided on the inner side of the rotating disk; a connecting slot is provided on the rotating disk; the triangular piece is rotatably connected to the rotating disk through the connecting slot; two fixing rods are fixedly connected to the triangular piece; a fixing disk is provided at the bottom of the fixing rods.

[0019] The present invention is further configured such that: the triangular piece includes a triangular region and a circular region, the triangular piece is connected to the connecting bayonet through the circular region, and the fixing rod is connected through the triangular region.

[0020] The present invention is further configured such that the included angle between the two fixed rods is 30°-60°.

[0021] The present invention is further configured such that anti-slip particles are provided on the bottom of the fixed plate.

[0022] In summary, the present invention has the following beneficial effects:

[0023] 1. Significantly improves irrigation efficiency and reduces experimental costs.

[0024] The improved ex vivo liver tissue perfusion technique, compared to the traditional multi-point puncture collagenase perfusion method, achieves simultaneous perfusion at multiple sites by optimizing the perfusion path and reagent delivery mechanism, effectively shortening the perfusion time and improving perfusion efficiency.

[0025] Regarding reagent consumption, the control valve can be adjusted at any time via the control keyboard during the perfusion process, thereby precisely controlling the delivery dosage of collagenase and perfusion fluid, avoiding reagent waste caused by prolonged perfusion and inaccurate operation in traditional methods.

[0026] 2. Simplify operation procedures and lower technical barriers.

[0027] In the stage of ex vivo liver tissue sampling and fixation, a specialized fixation structure was developed. This eliminates the need for operators to repeatedly adjust based on experience; standardized procedures allow for rapid tissue fixation, effectively improving fixation accuracy and stability. For the puncture procedure, eight fixation points are set. Operators only need to select 4-6 of these points to perform the puncture, eliminating the need for extensive trial and error and enabling precise puncture with simple operation.

[0028] 3. Comprehensively reduce operational risks

[0029] In this invention, during the puncture process, the operator manipulates the puncture needle outside the receiving box, while the needle's support is located inside the box, effectively avoiding the risk of hand injury from the puncture needle in traditional procedures. Simultaneously, the direct contact between the operator and the tissue and reagents during the procedure is significantly reduced, lowering the probability of complications such as infection. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the device in an embodiment of the present invention;

[0031] Figure 2 This is a top view of the housing box in an embodiment of the present invention;

[0032] Figure 3 This is a schematic diagram showing the connection between the fixed structure and the fixed platform in an embodiment of the present invention;

[0033] Figure 4 This is a schematic diagram of the fixed structure in an embodiment of the present invention;

[0034] Figure 5 This is a schematic diagram of the triangular piece in an embodiment of the present invention.

[0035] In the diagram: 1. Receptacle box; 2. Expansion area; 3. Fixing point; 4. Angle adjuster; 5. Pinhole; 6. Connector; 7. Puncture needle; 8. Connecting pipe; 9. Pipe connector; 10. Outlet; 11. Peristaltic pump; 12. Inlet; 13. Control keyboard; 14. Fixing platform; 15. Fixing structure; 1501. Fixing base; 1502. Column; 1503. Height control thread; 1504. Rotating disk; 1505. Connecting bayonet; 1506. Triangular piece; 15061. Triangular area; 15062. Circular area; 1507. Fixing rod; 1508. Fixing disc; 16. Control valve. Detailed Implementation

[0036] The following is in conjunction with the appendix Figure 1-5 The present invention will be described in further detail below.

[0037] Example 1: An in vitro perfusion device for isolated human liver tissue includes a container 1, a puncture needle 7, and a peristaltic pump 11. The four walls of the container 1 are provided with expansion zones 2. The expansion zones 2 are made of rubber or silicone with elastic deformation capability. Waste liquid flowing out during perfusion is stored in the container 1 and will not flow out and contaminate the experimental table. When the amount of waste liquid is large due to special circumstances, the expansion zones 2 will deform, which increases the volume that the container 1 can hold, so that it can meet the experimental requirements.

[0038] Each extended area 2 has symmetrical fixed points 3 on both sides, so there will be a total of 8 fixed points 3. The experimental parameters of in vitro perfusion of isolated human liver tissue in the prior art are retrieved, and the corresponding analysis model is constructed. The perfusion positions in the four directions of up, down, left and right after the isolated human liver tissue is placed are analyzed. Two points with the best perfusion effect are selected in each direction. These 8 points are the positions of fixed points 3.

[0039] An angle adjuster 4 is connected to the center of the fixing point 3. A needle hole 5 is located at the center of the angle adjuster 4, through which the puncture needle 7 passes. The angle adjuster 4 is constructed with a rigid center and an outer ring that can elastically deform under pressure. By bending the angle adjuster 4, it compresses against the inner wall of the fixing point 3, thus adjusting the orientation of the needle hole 5 and consequently adjusting the angle of the puncture needle 7 passing through it.

[0040] The peristaltic pump 11 has an outlet 10 on one side and an inlet 12 on the top. The inlet 12 is connected to a container holding the perfusion fluid and collagenase. The outlet 10 is connected to a manifold connector 9. Although there are a total of 8 fixed points (3 positions), multiple experiments have shown that only 4-6 points are usually needed to complete the perfusion of isolated human liver tissue in actual operation. Therefore, a 1-to-4, 1-to-5, or 1-to-6 connector can be used for the manifold connector 9. For experienced operators, 4 points are usually sufficient for adequate perfusion of isolated human liver tissue, and even beginners can generally achieve adequate perfusion with 5 or 6 points.

[0041] The peristaltic pump 11 is equipped with a control keypad 13, and a control valve 16 is installed at the connection between the branch connector 9 and the connecting pipe 8. The control keypad 13 is used to regulate the opening and closing of the control valve 16 and the on / off state of the peristaltic pump 11, thereby regulating the flow rate and volume of the perfusion fluid and collagenase flowing out of the outlet 10. The branch connector 9 is connected to the connecting pipe 8, and the other end of the connecting pipe 8 is connected to the connector 6 at the tail of the puncture needle 7. The perfusion fluid and collagenase flow through the connecting pipe 8 and then through the puncture needle 7 to achieve perfusion of isolated human liver tissue.

[0042] A fixed platform 14 is located at the bottom center of the housing 1, and a fixing structure 15 is provided on the fixed platform 14. The fixing structure 15 is located at the four corners of the fixed platform 14 and includes a fixed base 1501, a rotating disk 1504, and a triangular piece 1506 connected to the fixed platform 14. A column 1502 is connected to the top of the fixed base 1501. The top area of ​​the column 1502 is provided with a height control thread 1503. The inner side of the rotating disk 1504 is provided with a threaded area that matches the height control thread 1503. The rotating disk 1504 can adjust its height by moving on the height control thread 1503. A connecting slot 1505 is provided on the rotating disk 1504, and the triangular piece 1506 is rotatably connected to the rotating disk 1504 through the connecting slot 1505. The triangular piece 1506 is fixedly connected to two fixing rods 1507. A fixing plate 1508 is located at the bottom of each fixing rod 1507, and the bottom of the fixing plate 1508 is provided with anti-slip particles. The triangular piece 1506 includes a triangular region 15061 and an annular region 15062. The triangular piece 1506 is connected to the connecting bayonet 1505 through the annular region 15062 and to the fixing rods 1507 through the triangular region 15061. When using this device, the fixing plate 1508 is used to press out the isolated human liver tissue. The anti-slip particles at its bottom effectively prevent it from detaching from the fixing plate 1508. Then, rotating the rotating disk 1504 causes it to move downwards, and the fixing plate 1508, guided by the rotating disk, compresses the isolated human liver tissue, making it more securely fixed. The triangular piece 1506 can rotate relative to the rotating disk, so that when the rotating disk rotates with the height control thread 1503, the triangular piece 1506 always maintains its orientation. The included angle between the two fixing rods 1507 is 30°-60°. The bottom of the two fixing rods 1507 and the extension of the bottom fulcrum of the column 1502 form a triangle. The structure of the triangle is relatively stable, which can effectively improve the stability of fixing the isolated human liver tissue.

[0043] Working Principle: When using this device, the sampled human liver tissue is placed on the fixed platform 14, and the isolated human liver tissue is fixed by the fixing structures 15 at the four corners of the fixed platform 14. Then, based on the shape, size, and other characteristics of the sampled human liver tissue, the puncture site and the number of puncture needles 7 are determined. The corresponding fixing point 3 is then selected, and the puncture needle 7, connected to the connecting pipe 8, is inserted into the needle hole 5 at the center of the corresponding fixing point 3. The angle of the puncture needle 7 is adjusted using the angle adjuster 4 to puncture into the isolated human liver tissue. The peristaltic pump 11 is turned on by the control keyboard 13, and the perfusion fluid and collagenase enter the sampled human liver tissue. Based on the changes in the isolated human liver tissue during perfusion, the control valve 16 is adjusted to regulate the flow rate and velocity of the perfusion fluid and collagenase, thereby improving perfusion efficiency and saving the amount of perfusion fluid and collagenase used. When the amount of waste fluid generated during perfusion is large, the expansion area 2 can be expanded to increase the capacity of the container 1, thereby accommodating a larger amount of waste fluid.

[0044] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An in vitro perfusion device for isolated human liver tissue, characterized in that: The device includes a receiving box (1), a puncture needle (7), and a peristaltic pump (11). The receiving box (1) has four expansion areas (2) on its four walls. Each expansion area (2) has a fixed point (3) symmetrically arranged on both sides. An angle adjuster (4) is connected to the center of the fixed point (3). A needle hole (5) is provided at the center of the angle adjuster (4). The needle hole (5) is for the puncture needle (7) to pass through. A connector (6) is connected to the tail of the puncture needle (7). The peristaltic pump (11) has an outlet (10) on one side and an inlet (12) on the top. The inlet (12) is connected to a container holding the perfusion fluid and collagenase. The outlet (10) is connected to a manifold connector (9). The manifold connector (9) is connected to a connecting pipe (8). The other end of the connecting pipe (8) is connected to a connector (6). The peristaltic pump (11) is equipped with a control keyboard (13). A fixed platform (14) is provided at the bottom center of the container (1), and a fixing structure (15) is provided on the fixed platform (14).

2. The ex vivo human liver tissue in vitro perfusion device according to claim 1, characterized in that: The extended area (2) is made of rubber or silicone material with elastic deformation capability.

3. The ex vivo human liver tissue in vitro perfusion device according to claim 1, characterized in that: The pipe connector (9) is a 1-to-4 connector, a 1-to-5 connector, or a 1-to-6 connector.

4. The ex vivo human liver tissue in vitro perfusion device according to claim 3, characterized in that: A control valve (16) is provided at the connection between the branch pipe joint (9) and the connecting pipe (8). The control keyboard (13) is used to regulate the opening and closing of the control valve (16) and the switching on and off of the peristaltic pump (11), thereby regulating the flow rate and flow of the perfusion fluid and collagenase flowing out of the outlet (10).

5. The ex vivo human liver tissue in vitro perfusion device according to claim 1, characterized in that: The fixing structure (15) is located at the four corners of the fixing platform (14). The fixing structure (15) includes a fixing base (1501), a rotating disk (1504), and a triangular piece (1506) connected to the fixing platform (14). The top of the fixing base (1501) is connected to a column (1502). The area near the top of the column (1502) is provided with a height control thread (1503). The inner side of the rotating disk (1504) is provided with a threaded area that matches the height control thread (1503). The rotating disk (1504) is provided with a connecting slot (1505). The triangular piece (1506) is rotatably connected to the rotating disk (1504) through the connecting slot (1505). The triangular piece (1506) is fixedly connected to two fixing rods (1507). The bottom of the fixing rods (1507) is provided with a fixing disk (1508).

6. The ex vivo human liver tissue in vitro perfusion device according to claim 5, characterized in that: The triangular piece (1506) includes a triangular region (15061) and an annular region (15062). The triangular piece (1506) is connected to the connecting slot (1505) through the annular region (15062) and to the fixing rod (1507) through the triangular region (15061).

7. The ex vivo human liver tissue in vitro perfusion device according to claim 5, characterized in that: The included angle between the two fixing rods (1507) is 30°-60°.

8. The ex vivo human liver tissue in vitro perfusion device according to claim 5, characterized in that: The bottom of the fixed plate (1508) is provided with anti-slip particles.

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