3D printing biotissue perfusion culture and light sheet microscope imaging integrated device

Abstract

The application relates to a 3D printing biological tissue perfusion culture and light sheet microscope imaging integrated device, wherein a perfusion culture unit is arranged in an imaging cavity, and the upper end of the perfusion culture unit is connected with a displacement platform connection unit through a hollow connecting shaft; the perfusion culture unit comprises a visible module and a temperature control module, wherein a temperature control flow channel is arranged in the temperature control module, a sample cavity for accommodating 3D printing biological tissues is formed between the temperature control module and the visible module, and a laser and an imaging objective are aligned with the visible module; the upper end of the perfusion culture unit is provided with a liquid inlet pipe and a liquid outlet pipe, the liquid inlet pipe is communicated with the upper end of the temperature control flow channel, and the liquid outlet pipe is communicated with the upper end of the sample cavity; a connecting pipe is arranged in the lower end of the perfusion culture unit, and the lower end of the temperature control flow channel and the lower end of the sample cavity are communicated through the connecting pipe. The perfusion culture unit is integrated in the imaging cavity of the light sheet microscope, long-term survival of biological tissues is ensured, and real-time observation of the biological tissues is realized.

Description

Technical Field

[0001] This invention relates to the field of 3D printed biological tissue perfusion culture technology, specifically an integrated device for 3D printed biological tissue perfusion culture and light slide microscopy imaging. Background Technology

[0002] With the rapid development of tissue engineering and 3D bioprinting technologies, constructing in vitro biomimetic tissue models with physiological functions has become an important means of studying cell behavior, tissue development, and disease mechanisms. However, achieving long-term stable culture of 3D-printed biological tissues and conducting non-invasive dynamic monitoring of their structure and function is one of the key challenges currently facing research in this process.

[0003] Light-sheet fluorescence microscopy (LSFM) has been widely used in the field of three-dimensional imaging of live samples due to its advantages such as low phototoxicity, fast imaging speed, and strong optical slicing capabilities. However, most sample culture systems currently used for LSFM are unable to meet the physiological needs of 3D-printed biological tissues during long-term imaging, such as continuous nutrient perfusion, gas exchange, and removal of metabolic waste, which can lead to a decrease in cell viability in the core tissue region. Therefore, there is a need to develop an integrated device that is compatible with light-sheet microscopy and has dynamic perfusion culture capabilities, so as to achieve long-term, stable, high-resolution real-time observation of cell activity, structural development, and functional dynamics of 3D-printed biological tissues under the premise of simulating the in vivo physiological environment. Summary of the Invention

[0004] The purpose of this invention is to provide an integrated device for 3D printing biological tissue perfusion culture and light-sheet microscopy imaging. It integrates the perfusion culture unit into the imaging cavity of the light-sheet microscope, which not only ensures the long-term survival of biological tissues, but also realizes real-time observation of biological tissues. This provides reliable technical support for the study of cell proliferation, differentiation and tissue dynamic evolution.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] An integrated device for 3D-printed biological tissue perfusion culture and light-sheet microscopy imaging includes a displacement platform connecting unit, a hollow connecting shaft, an imaging cavity, and a perfusion culture unit. The perfusion culture unit is located within the imaging cavity, and its upper end is connected to the displacement platform connecting unit via the hollow connecting shaft. The perfusion culture unit includes a viewing module and a temperature control module. The temperature control module has a temperature control channel inside, and a sample cavity for accommodating the 3D-printed biological tissue is formed between the temperature control module and the viewing module. Lasers are located on both sides of the imaging cavity in the X direction, and an imaging objective is located on one side in the Y direction. Both the lasers and the imaging objective are aligned with the viewing module. The upper end of the perfusion culture unit has an inlet pipe and an outlet pipe. The inlet pipe is connected to the upper end of the temperature control channel, and the outlet pipe is connected to the upper end of the sample cavity. Both the inlet pipe and the outlet pipe are located within the hollow connecting shaft. The lower end of the perfusion culture unit has a connecting pipe inside, and the lower end of the temperature control channel and the lower end of the sample cavity are connected via the connecting pipe.

[0007] The perfusion culture unit includes a unit cover and a unit base. The visual module and the temperature control module are embedded and fixed by the unit cover and the unit base. The unit cover is provided with an inlet pipe and an outlet pipe, and the unit base is provided with a connecting pipe inside.

[0008] The visual module has an upper support plate at the top and a lower support plate at the bottom. The visual module has module side plates on both sides, and the upper support plate, lower support plate and module side plates on both sides enclose the sample cavity. The temperature control module has a side protrusion on one side, and the side protrusion is embedded in the opening of the sample cavity.

[0009] The temperature control module has an upper protrusion at the top and a lower protrusion at the bottom. The upper support plate of the visual module has an upper stop on the outside and the lower support plate has a lower stop on the outside. The lower side of the unit cover has a first mounting groove, and one side of the first mounting groove cooperates with the outer side of the upper protrusion and the other side cooperates with the upper stop. The upper side of the unit base has a second mounting groove, and one side of the second mounting groove cooperates with the outer side of the lower protrusion and the other side cooperates with the lower stop.

[0010] The upper side of the unit cover is provided with an insertion groove that is fixedly connected to the hollow connecting shaft. The bottom of the insertion groove is provided with an upper cover liquid inlet connecting pipe and an upper cover liquid outlet connecting pipe. The upper end of the upper cover liquid inlet connecting pipe is connected to the liquid inlet pipe and the lower end is connected to the temperature control channel. The upper end of the upper cover liquid outlet connecting pipe is connected to the liquid outlet pipe and the lower end is connected to the sample chamber.

[0011] When the 3D printed biological tissue is placed into the sample chamber, the liquid outlet tube is connected to the biological tissue liquid outlet of the 3D printed biological tissue, and the connecting tube is connected to the biological tissue liquid inlet of the 3D printed biological tissue. The 3D printed biological tissue has multiple biological tissue channels inside between the biological tissue liquid outlet and the biological tissue liquid inlet.

[0012] The temperature control module includes a heating chamber and a detection chamber, which are located on opposite sides of the temperature control channel. The heating chamber contains a heating element, and the detection chamber contains a temperature sensor.

[0013] The displacement platform connection unit includes a positioning knob and a fine-tuning device. The lower end of the positioning knob is provided with a flange that is connected to the fine-tuning device. The upper end of the hollow connecting shaft passes through the fine-tuning device and is inserted into the positioning knob for fixation. The lower side of the fine-tuning device is provided with a connecting plate, and the connecting plate is connected to the triaxial displacement platform of the light sheet microscope.

[0014] The positioning knob is equipped with fastening screws that fix the hollow connecting shaft evenly on its outer side, and the positioning knob has a knob through hole in the middle.

[0015] The flange is fixedly connected to the fine-tuning device by fixing bolts, and the flange is provided with a locking groove for the fixing bolts to pass through; the connecting plate is provided with a positioning groove and a positioning hole that cooperate with the three-axis displacement platform of the light sheet microscope for positioning and connection, and the connecting plate is provided with a fine-tuning controller for controlling the fine-tuning device.

[0016] The beneficial effects of this invention are as follows:

[0017] 1. This invention integrates a biological tissue perfusion culture unit into the imaging cavity of a light-sheet microscope. By continuously inputting culture medium, it can continuously perfuse nutrient solution and remove metabolic waste from 3D-printed biological tissues under simulated physiological conditions, ensuring the long-term survival of biological tissues. At the same time, the real-time observation of biological tissues is realized through the cooperation of relevant components of the light-sheet microscope, which provides reliable technical support for the study of cell proliferation, differentiation and tissue dynamic evolution.

[0018] 2. The perfusion culture unit of the present invention includes a visual module and a temperature control module, and a sample cavity for accommodating biological tissue is formed between the visual module and the temperature control module. The laser and imaging objective lens outside the imaging cavity are aligned with the visual module to realize the real-time observation function of biological tissue. In addition, the upper end of the perfusion culture unit is connected to the displacement platform connection unit through a hollow connecting shaft. The displacement platform connection unit is fixedly connected to the three-axis displacement platform of the light sheet microscope. In this way, the present invention can achieve the initial position adjustment of the perfusion culture unit through the three-axis displacement platform of the light sheet microscope, and can also achieve precise fine adjustment of the position of the perfusion culture unit through the displacement platform connection unit to ensure that the laser and imaging objective lens are aligned with the visual module, thereby ensuring the final imaging effect.

[0019] 3. The present invention utilizes a visual module and a temperature control module to form a sample cavity for containing biological tissue, and uses a unit cover and a unit base to fix the visual module and the temperature control module. In this way, the imaging effect of components such as laser and imaging objective lens is not affected, while the sealing performance of the sample cavity is guaranteed. At the same time, it is also convenient to disassemble and assemble to put in or take out biological tissue.

[0020] 4. The visual module and temperature control module of the present invention are embedded in each other. At the same time, the upper end of the visual module and the temperature control module after being fastened together are also embedded in the unit cover and the lower end is embedded in the unit base. This ensures the fixed sealing of the entire perfusion culture unit, facilitates the alignment of related through holes and pipelines, improves assembly efficiency, and ensures the continuous and smooth flow of culture medium inside the perfusion culture unit.

[0021] 5. The temperature control module of the present invention is equipped with a heating element and a temperature sensor for real-time temperature control. The heat generated by the heating element can be transferred not only to the temperature control channel of the temperature control module, but also to the biological tissue in the sample chamber, thereby avoiding local overheating or overcooling inside the perfusion culture unit and maintaining the temperature stability inside the perfusion culture unit. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the present invention.

[0023] Figure 2 for Figure 1 Overall sectional view of the present invention.

[0024] Figure 3 for Figure 1 Schematic diagram of the structure of the intermediate displacement platform connection unit.

[0025] Figure 4 for Figure 2 Schematic diagram of the structure of the medium-perfusion culture unit.

[0026] Figure 5 for Figure 4 Top view of the medium-perfusion culture unit Figure 1 ,

[0027] Figure 6 for Figure 5 AA view in

[0028] Figure 7 for Figure 4 Top view of the medium-perfusion culture unit Figure 2 ,

[0029] Figure 8 for Figure 7 BB view in

[0030] Figure 9 for Figure 4 Top view of the medium-perfusion culture unit Figure 3 ,

[0031] Figure 10 for Figure 9 The CC view in the middle.

[0032] Among them, 1 is a positioning knob, 101 is a fastening screw, 102 is a knob through hole, 2 is a flange, 201 is a locking groove, 3 is a fine-tuning device, 301 is a connecting plate, 302 is a fine-tuning controller, 303 is a positioning groove, 304 is a positioning hole, 4 is a hollow connecting shaft, 5 is an imaging cavity, 6 is a laser, 7 is an imaging objective lens, 8 is a 3D printed biological tissue, 801 is a biological tissue outlet, 802 is a biological tissue channel, 803 is a biological tissue inlet, 9 is a perfusion culture unit, 901 is an inlet pipe, 902 is a unit cover, 902... 1 is the liquid inlet connecting pipe of the upper cover, 9022 is the liquid outlet connecting pipe of the upper cover, 9023 is the insertion slot, 9024 is the fixing hole, 903 is the temperature control module, 9031 is the temperature control flow channel, 9032 is the upper protrusion, 9033 is the lower protrusion, 9034 is the side protrusion, 904 is the unit base, 9041 is the connecting pipe, 905 is the visual module, 9051 is the upper support plate, 9052 is the lower support plate, 9053 is the upper stop, 9054 is the lower stop, 906 is the liquid outlet pipe, 907 is the sample chamber, 10 is the heating element, and 11 is the temperature sensor. Detailed Implementation

[0033] The invention will now be described in further detail with reference to the accompanying drawings.

[0034] like Figures 1-10 As shown, the present invention includes a displacement platform connecting unit, a hollow connecting shaft 4, an imaging cavity 5, and a perfusion culture unit 9, wherein... Figure 2As shown, the perfusion culture unit 9 is disposed in the imaging cavity 5, and the upper end of the perfusion culture unit 9 is connected to the displacement platform connecting unit via a hollow connecting shaft 4. The displacement platform connecting unit is used to fine-tune the position of the perfusion culture unit 9 within the imaging cavity 5; as shown Figure 6 As shown, the perfusion culture unit 9 includes a visualization module 905 and a temperature control module 903, wherein the temperature control module 903 has a temperature control flow channel 9031 inside, and a sample cavity 907 for accommodating the 3D printed biological tissue 8 is formed between the temperature control module 903 and the visualization module 905; Figure 1 As shown, lasers 6 are provided on both sides of the imaging cavity 5 in the X direction, and an imaging objective lens 7 is provided on one side in the Y direction. Both the lasers 6 and the imaging objective lens 7 are aligned with the visual module 905. Figures 4 to 10 As shown, the perfusion culture unit 9 is provided with an inlet pipe 901 and an outlet pipe 906 at its upper end. The inlet pipe 901 is connected to the upper end of the temperature control channel 9031, and the outlet pipe 906 is connected to the upper end of the sample chamber 907. The inlet pipe 901 and the outlet pipe 906 are both located in the hollow connecting shaft 4 and are led out to the outside of the device and connected to the corresponding equipment via the hollow connecting shaft 4. The perfusion culture unit 9 is provided with a connecting pipe 9041 at its lower end, and the lower end of the temperature control channel 9031 and the lower end of the sample chamber 907 are connected through the connecting pipe 9041.

[0035] The laser 6 and imaging objective 7 are well-known technologies in the art and are standard components of light-sheet microscopes. Unlike conventional microscopes, light-sheet microscopes have incident light on both sides and an imaging objective 7 on only one side. Their advantages are: 1. They illuminate only the current imaging focal plane of the sample, resulting in extremely low background noise; 2. They reduce phototoxicity in other areas, allowing for long-term culture and imaging; 3. They are suitable for imaging thick samples, ranging from millimeters to centimeters. This invention is specifically designed to address these characteristics of light-sheet microscopes.

[0036] like Figure 6As shown, in this embodiment, the visual module 905 has an upper support plate 9051 at its upper end and a lower support plate 9052 at its lower end. Both sides of the visual module 905 have module side plates, and the upper support plate 9051, lower support plate 9052, and the module side plates on both sides enclose the sample cavity 907. The temperature control module 903 has a side protrusion 9034 on one side, and the side protrusion 9034 is embedded in the opening of the sample cavity 907, thereby forming a sealed cavity. In this embodiment, the visual module 905 is made of high-transmittance glass with a refractive index close to that of biological tissue to ensure uninterrupted penetration of the laser and imaging optical path. During operation, the light emitted by the lasers 6 on both sides of the imaging cavity 5 in the X direction enters through the corresponding module side plates of the visual module 905, then enters the 3D-printed biological tissue 8 within the sample cavity 907, and finally exits from the Y direction side of the visual module 905 into the imaging objective lens 7.

[0037] like Figures 4-6 As shown, in this embodiment, the perfusion culture unit 9 includes a unit cover 902 and a unit base 904. The visual module 905 and the temperature control module 903 are fitted together and then clamped and fixed by the unit cover 902 and the unit base 904. The temperature control module 903 has an upper protrusion 9032 at its upper end and a lower protrusion 9033 at its lower end. The visual module 905 has an upper stop 9053 on the outer side of its upper support plate 9051 and a lower stop 9054 on the outer side of its lower support plate 9052. The unit cover 902 has a lower... The unit base 904 has a first mounting groove, one side of which mates with the upper protrusion 9032 and the other side with the upper stop 9053. A second mounting groove is provided on the upper side of the unit base 904, one side of which mates with the lower protrusion 9033 and the other side with the lower stop 9054. The unit cover 902 is fixed to the upper end of the visual module 905 and the upper end of the temperature control module 903 by a first bolt. The unit base 904 is fixed to the lower end of the visual module 905 and the lower end of the temperature control module 903 by a second bolt. This invention, through the above structural design, ensures imaging in conjunction with the laser 6 and the imaging objective lens 7 while also fully guaranteeing the sealing performance of the sample chamber 907, thereby ensuring the culture of the 3D printed biological tissue 8. Furthermore, the above structure facilitates installation and disassembly, making it convenient to insert or remove the 3D printed biological tissue 8.

[0038] like Figure 6 As shown, in this embodiment, the upper side of the unit cover 902 is provided with an insertion groove 9023, and the groove wall of the insertion groove 9023 is provided with a fixing hole 9024. The lower end of the hollow connecting shaft 4 is inserted into the insertion groove 9023 and fixed to the unit cover 902 by bolts. The bolts pass through the corresponding fixing holes 9024 to achieve the connection.

[0039] like Figure 6 As shown, in this embodiment, the bottom of the insertion slot 9023 of the unit cover 902 is provided with a liquid inlet pipe 9021 and a liquid outlet pipe 9022. The upper end of the liquid inlet pipe 9021 is connected to the liquid inlet pipe 901 and the lower end is connected to the temperature control channel 9031. The upper end of the liquid outlet pipe 9022 is connected to the liquid outlet pipe 906 and the lower end is connected to the sample chamber 907. The connecting pipe 9041 is provided inside the unit base 904.

[0040] Other examples Figures 7-8 As shown, when the 3D printed biological tissue 8 is placed into the sample chamber 907, the liquid outlet pipe 906 is connected to the biological tissue outlet 801 of the 3D printed biological tissue 8 through the liquid outlet connecting pipe 9022 on the upper cover, and the connecting pipe 9041 is connected to the biological tissue inlet 803 of the 3D printed biological tissue 8. Multiple biological tissue channels 802 are provided between the biological tissue outlet 801 and the biological tissue inlet 803 to realize the flow of culture medium in the tissue.

[0041] and Figures 9-10 As shown, in this embodiment, the temperature control module 903 includes a heating chamber and a detection chamber, which are located on opposite sides of the temperature control channel 9031. The heating chamber contains a heating element 10, which can be a heating rod or similar component. The detection chamber contains a temperature sensor 11 for real-time temperature detection. Both the heating rod and the temperature sensor 11 are commercially available products. Furthermore, the relevant wiring for the heating element 10 and the temperature sensor 11 is housed within the hollow connecting shaft 4.

[0042] like Figures 1-3 As shown, in this embodiment, the displacement platform connection unit includes a positioning knob 1 and a fine-tuning device 3. The lower end of the positioning knob 1 is provided with a flange 2 connected to the fine-tuning device 3. The upper end of the hollow connecting shaft 4 passes through the fine-tuning device 3 and is inserted into the positioning knob 1 for fixation. The fine-tuning device 3 drives the positioning knob 1 and the hollow connecting shaft 4 to rotate together through the flange 2, thereby fine-tuning the position and angle of the perfusion culture unit 9 to ensure that the incident light emitted by the laser 6 can accurately enter the visual module 905 of the perfusion culture unit 9 and accurately exit from the imaging objective lens 7. At the same time, during the culture process, the operator can also fine-tune the position of the perfusion culture unit 9 as needed and observe the 3D printed biological tissue 8.

[0043] In this embodiment, the fine-tuning device 3 employs a piezoelectric rotary actuator, a technology known in the art, as can be found in patent CN110445415B, or other suitable commercially available products can be used. In this embodiment, the piezoelectric rotary actuator, through piezoelectric drive, can achieve high-precision XYZ three-axis movement and 360° horizontal rotation, with a movement accuracy of 100nm, which can precisely control the movement of the perfusion culture unit 9 during imaging.

[0044] Other examples Figure 1 and Figure 3 As shown, in this embodiment, the lower side of the fine-tuning device 3 is provided with a connecting plate 301 for connecting with the triaxial displacement platform of the light slide microscope to achieve preliminary adjustment of the position of the perfusion culture unit 9. The connecting plate 301 is provided with a positioning groove 303 and a positioning hole 304 to cooperate with the triaxial displacement platform of the light slide microscope for positioning connection. At the same time, the fine-tuning controller 302 (piezoelectric rotation controller) of the fine-tuning device 3 (piezoelectric rotation actuator) can also be installed on the connecting plate 301.

[0045] like Figure 3 As shown, in this embodiment, fastening screws 101 are evenly distributed on the outer circumference of the positioning knob 1 to lock the upper end of the hollow connecting shaft 4. At the same time, the positioning knob 1 has a knob through hole 102 in the middle for the liquid inlet pipe 901, liquid outlet pipe 906, and related lines of the heating element 10 and temperature sensor 11 to pass through and connect to related external equipment.

[0046] like Figure 3 As shown, in this embodiment, the flange 2 is fixedly connected to the fine-tuning device 3 by fixing bolts, and the flange 2 is provided with a locking groove 201 for the fixing bolts to pass through.

[0047] The working principle of this invention is as follows:

[0048] In this embodiment, the 3D-printed biological tissue 8 is constructed using dual-material 3D printing technology. A cross-linkable hydrogel is mixed with cells to create a bio-ink, which is then extruded and printed layer by layer. Ultraviolet light irradiation is used to achieve rapid cross-linking and curing of the material, resulting in a 3D-printed biological tissue 8 with a complex three-dimensional structure. Furthermore, during the printing process, a removable gelatin hydrogel material is used as a support structure to construct the internal channel system. After printing, the entire structure is heated to dissolve and remove the gelatin hydrogel, ultimately forming biological tissue channels 802 within the biological tissue. These channels not only mimic the vascular network function of natural tissues but also enhance the survival rate and functionality of the biological tissue during culture, thus exhibiting excellent physiological activity and structural biomimicry.

[0049] The installation process of this invention is as follows:

[0050] Step 1: First, place the 3D printed biological tissue 8 in the visual module 905, and then place the temperature control module 903 on the opening side of the sample chamber 907. The side protrusion 9034 on one side of the temperature control module 903 can cooperate to position the 3D printed biological tissue 8.

[0051] Other examples Figure 8 As shown, when the 3D printed biological tissue 8 is placed, it is necessary to ensure that the biological tissue inlet 803 of the 3D printed biological tissue 8 is aligned with the through hole on the lower support plate 9052 through which the connecting pipe 9041 passes, and the biological tissue outlet 801 is aligned with the through hole on the upper support plate 9051 through which the upper cover outlet connecting pipe 9022 passes.

[0052] Step 2: Install the unit base 904 onto the lower end of the visual module 905 and the lower end of the temperature control module 903. During installation... Figure 6 As shown, it is necessary to ensure that one end of the connecting pipe 9041 is inserted into the temperature control channel 9031 of the temperature control module 903, and the other end is inserted into the lower support plate 9052 of the visual module 905 and aligned and connected with the biological tissue inlet 803 of the 3D printed biological tissue 8. Then, the unit base 904 is fixed to the visual module 905 and the temperature control module 903 by bolts.

[0053] Step 3: As Figure 10 As shown, a heating element 10 and a temperature sensor 11 are placed inside the temperature control module 903.

[0054] Step 4: Install the unit cover 902 on the upper end of the visual module 905 and the upper end of the temperature control module 903. During installation, ensure that the liquid inlet connecting pipe 9021 of the cover is inserted into the temperature control flow channel 9031, and that the liquid outlet connecting pipe 9022 of the cover is aligned and connected with the liquid outlet 803 of the 3D printed biological tissue 8. At the same time, the relevant lines of the heating element 10 and the temperature sensor 11 should be led out from the corresponding through holes on the unit cover 902. Then, fix the unit cover 902 to the visual module 905 and the temperature control module 903 with bolts.

[0055] Step 5: Connect the inlet pipe 901 to the inlet connecting pipe 9021 of the upper cover, and connect the outlet pipe 906 to the outlet connecting pipe 9022 of the upper cover. Then, pass the relevant lines of the inlet pipe 901, the outlet pipe 906, the heating element 10, and the temperature sensor 11 through the hollow connecting shaft 4. After completing the wiring, insert the lower end of the hollow connecting shaft 4 into the insertion slot 9023 of the unit upper cover 902 and fix it.

[0056] Step 6: First, pass the relevant lines of the inlet pipe 901, outlet pipe 906, heating element 10 and temperature sensor 11 led out from the hollow connecting shaft 4 through the fine-tuning device 3 and positioning knob 1 of the displacement platform connecting unit. After completing the threading, pass the upper end of the hollow connecting shaft 4 through the fine-tuning device 3 and then fix it by the positioning knob 1.

[0057] Step 7: Place the perfusion culture unit 9 into the imaging cavity 5, and then connect the connecting plate 301 of the displacement platform connecting unit to the triaxial displacement platform of the light sheet microscope.

[0058] Among them, such as Figure 6 As shown, in steps two and four above, since the unit base 904 and unit top cover 902 are fitted into the visual module 905 and temperature control module 903 after being fastened together, this can assist in positioning and aligning the various through holes and related pipelines, improving assembly efficiency. Furthermore, this invention vertically positions the 3D-printed biological tissue 8 in the sample chamber 907 of the perfusion culture unit 9, allowing the culture medium to evenly penetrate into the tissue under gravity, improving nutrient delivery efficiency and promoting the excretion of metabolic waste.

[0059] And such Figure 1 As shown, after installation, the present invention first uses the triaxial displacement platform of the light-sheet microscope to initially adjust the position of the perfusion culture unit 9 within the imaging cavity 5. Then, the fine-tuning device 3 controls the fine-tuning of the position of the perfusion culture unit 9 within the imaging cavity 5 to ensure that the laser 6 and the imaging objective 7 are aligned with the perfusion culture unit 9. After the position of the perfusion culture unit 9 is adjusted, the inlet pipe 901 starts to input the culture medium, and the culture medium is sequentially input into the 3D printed biological tissue 8 through the temperature-controlled flow channel 9031 and the connecting pipe 9041, and then flows out through the outlet pipe 906. During the above process, the lasers 6 on both sides of the imaging cavity 5 can be activated to cooperate in imaging. In this way, continuous three-dimensional in vivo imaging can be performed through the imaging objective 7 of the light-sheet microscope, thereby realizing real-time observation of the proliferation, differentiation, and dynamic evolution of biological tissue cells.

[0060] In addition, during the perfusion process, the 3D printed biological tissue 8 in the temperature control channel 9031 and sample chamber 907 can be heated by the heating element 10, and the temperature can be controlled in real time by the temperature sensor 11 to avoid local overheating or overcooling inside the perfusion culture unit 9 and maintain the temperature stability inside the perfusion culture unit 9.

[0061] This invention enables continuous perfusion of nutrient solution and removal of metabolic waste from 3D-printed biological tissues under simulated physiological conditions by continuously introducing culture medium, effectively maintaining tissue activity. In addition, this invention not only ensures the long-term survival of biological tissues, but also enables real-time observation of biological tissues through the use of light-sheet microscope components, which provides reliable technical support for the study of cell proliferation, differentiation and dynamic evolution of tissues.

Claims

1. A device integrating 3D-printed biological tissue perfusion culture and light-section microscopy imaging, characterized in that: The system includes a displacement platform connecting unit, a hollow connecting shaft (4), an imaging cavity (5), and a perfusion culture unit (9). The perfusion culture unit (9) is located in the imaging cavity (5), and the upper end of the perfusion culture unit (9) is connected to the displacement platform connecting unit through the hollow connecting shaft (4). The perfusion culture unit (9) includes a visual module (905) and a temperature control module (903). The temperature control module (903) has a temperature control channel (9031) inside. A sample cavity (907) for accommodating 3D printed biological tissue (8) is formed between the temperature control module (903) and the visual module (905). Lasers (6) are provided on both sides of the imaging cavity (5) in the X direction and on one side in the Y direction. An imaging objective (7) is provided, and both the laser (6) and the imaging objective (7) are aligned with the visual module (905); the perfusion culture unit (9) is provided with an inlet pipe (901) and an outlet pipe (906) at its upper end, and the inlet pipe (901) is connected to the upper end of the temperature control channel (9031), and the outlet pipe (906) is connected to the upper end of the sample chamber (907). The inlet pipe (901) and the outlet pipe (906) are both located in the hollow connecting shaft (4); the perfusion culture unit (9) is provided with a connecting pipe (9041) inside its lower end, and the lower end of the temperature control channel (9031) and the lower end of the sample chamber (907) are connected through the connecting pipe (9041); The perfusion culture unit (9) includes a unit cover (902) and a unit base (904). The visual module (905) and the temperature control module (903) are embedded and fixed by the unit cover (902) and the unit base (904). The unit cover (902) is provided with an inlet pipe (901) and an outlet pipe (906). The unit base (904) is provided with a connecting pipe (9041). The visual module (905) has an upper support plate (9051) at its upper end and a lower support plate (9052) at its lower end. Both sides of the visual module (905) have module side plates. The upper support plate (9051), the lower support plate (9052), and the module side plates on both sides enclose the sample cavity (907). The temperature control module (903) has a side protrusion (9034) on one side, and the side protrusion (9034) is embedded in the opening of the sample cavity (907).

2. The integrated device for 3D printed biological tissue perfusion culture and light-section microscopy imaging according to claim 1, characterized in that: The temperature control module (903) has an upper protrusion (9032) at its upper end and a lower protrusion (9033) at its lower end. The upper support plate (9051) of the visual module (905) has an upper stop (9053) on its outer side and the lower support plate (9052) has a lower stop (9054) on its outer side. The unit cover (902) has a first mounting groove on its lower side, and one side of the first mounting groove cooperates with the outer side of the upper protrusion (9032) and the other side cooperates with the upper stop (9053). The unit base (904) has a second mounting groove on its upper side, and one side of the second mounting groove cooperates with the outer side of the lower protrusion (9033) and the other side cooperates with the lower stop (9054).

3. The integrated device for 3D printed biological tissue perfusion culture and light-section microscopy imaging according to claim 1, characterized in that: The upper side of the unit cover (902) is provided with an insertion groove (9023) that is fixedly connected to the hollow connecting shaft (4). The bottom of the insertion groove (9023) is provided with an upper cover liquid inlet connecting pipe (9021) and an upper cover liquid outlet connecting pipe (9022). The upper end of the upper cover liquid inlet connecting pipe (9021) is connected to the liquid inlet pipe (901) and the lower end is connected to the temperature control channel (9031). The upper end of the upper cover liquid outlet connecting pipe (9022) is connected to the liquid outlet pipe (906) and the lower end is connected to the sample chamber (907).

4. The integrated device for 3D printed biological tissue perfusion culture and light-section microscopy imaging according to claim 1, characterized in that: When the 3D printed biological tissue (8) is placed into the sample chamber (907), the liquid outlet tube (906) is connected to the biological tissue liquid outlet (801) of the 3D printed biological tissue (8), and the connecting tube (9041) is connected to the biological tissue liquid inlet (803) of the 3D printed biological tissue (8). The 3D printed biological tissue (8) has multiple biological tissue channels (802) between the biological tissue liquid outlet (801) and the biological tissue liquid inlet (803).

5. The integrated device for 3D printed biological tissue perfusion culture and light-section microscopy imaging according to claim 1, characterized in that: The temperature control module (903) is provided with a heating chamber and a detection chamber, and the heating chamber and the detection chamber are respectively located on both sides of the temperature control channel (9031). The heating chamber is provided with a heating element (10), and the detection chamber is provided with a temperature sensor (11).

6. The integrated device for 3D printed biological tissue perfusion culture and light-section microscopy imaging according to claim 1, characterized in that: The displacement platform connection unit includes a positioning knob (1) and a fine-tuning device (3). The lower end of the positioning knob (1) is provided with a flange (2) which is connected to the fine-tuning device (3). The upper end of the hollow connecting shaft (4) passes through the fine-tuning device (3) and is inserted into the positioning knob (1) for fixation. The lower side of the fine-tuning device (3) is provided with a connecting plate (301), and the connecting plate (301) is connected to the triaxial displacement platform of the light sheet microscope.

7. The integrated device for 3D printed biological tissue perfusion culture and light-section microscopy imaging according to claim 6, characterized in that: The positioning knob (1) is evenly distributed with fastening screws (101) for fixing the hollow connecting shaft (4) on the outside, and the positioning knob (1) is provided with a knob through hole (102) in the middle.

8. The integrated device for 3D printed biological tissue perfusion culture and light-section microscopy imaging according to claim 6, characterized in that: The flange (2) is fixed to the fine-tuning device (3) by fixing bolts, and the flange (2) is provided with a locking groove (201) for the fixing bolts to pass through; the connecting plate (301) is provided with a positioning groove (303) and a positioning hole (304) that are matched and connected with the three-axis displacement platform of the light sheet microscope; in addition, the connecting plate (301) is provided with a fine-tuning controller (302) for controlling the fine-tuning device (3).

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