Radiofrequency therapy system for organoids

Abstract

The present application provides a radiofrequency physiotherapy system for organoids, which includes a porous cell culture sub-device, a microelectrode array sub-device, an analysis device and a radiofrequency control device. After placing the organoid in the truncated cone through-hole in the porous cell culture sub-device, the analysis device obtains the electrical impedance value through the resistance measurement point in the conductive layer in the microelectrode array sub-device, thereby determining the size of the organoid placed in the truncated cone through-hole according to the electrical impedance value, and then determining the radiofrequency control information according to the size of the organoid. After that, the radiofrequency control device electrically stimulates the organoid according to the radiofrequency control information through the input end and the electrophysiological stimulation site connected to the input end, and records the electrophysiological activity of the organoid through an oscilloscope. It makes it possible to develop the complete structure of the vaginal wall in vitro, thereby promoting related research work such as artificial vaginal repair.

Description

Technical Field

[0001] The present application relates to the field of biomedical engineering, and in particular to a radiofrequency therapy system for organoids. Background Art

[0002] Organoids are three-dimensional (3D) cultured cells with stem potential. The tissue analogs formed have a certain spatial structure and can well reflect the characteristics of the source tissue in terms of morphology, structure and epigenetics. However, compared with human organs, their geometry and volume are very different. This means that if the RF device used for the human body is directly applied to the organoid model, the expected energy transfer and distribution may not be achieved, resulting in unsatisfactory treatment effects or damage to the model. Secondly, the biophysical properties of the organoid model, such as conductivity, dielectric constant and thermal conductivity, may be different from those of human tissue. These differences affect the propagation and absorption of RF energy in the model, and thus affect the treatment effect. Therefore, the direct use of human RF devices may not achieve the best treatment effect. In addition, the culture environment and conditions of the organoid model are also different from those inside the human body. For example, the organoid model may require a specific culture medium and gas environment, and these conditions may affect the transfer and distribution of RF energy. Summary of the invention

[0003] In view of this, the present application provides a radiofrequency therapy system for organoids, which makes it possible to develop the complete structure of the vaginal wall in vitro, thereby promoting related research work such as artificial vaginal repair.

[0004] The present application provides a radiofrequency therapy system for organoids, comprising:

[0005] A porous cell culture sub-device, a microelectrode array sub-device, an analysis device and a radio frequency control device; wherein the porous cell culture sub-device includes N truncated cone-shaped through holes; the microelectrode array sub-device includes a conductive layer, an insulating layer and a base layer; the conductive layer includes N core sites and (2M+2)×N pad sites; each of the core sites includes M electrophysiological detection sites, M electrophysiological stimulation sites, a resistance measurement site and a reference electrode site; the radio frequency control device includes M input terminals and M output terminals;

[0006] For each truncated cone through hole, the truncated cone through hole is connected to one of the core sites by bonding; each of the core sites is respectively connected to a corresponding pad site by a wire; wherein the truncated cone through hole is used to place the organoid; the reference electrode point is used for zero calibration; the pad site corresponding to the resistance measurement point is connected to the analysis device; the pad site corresponding to each electrophysiological stimulation site is connected to the corresponding input end; the pad site corresponding to each electrophysiological detection site is connected to the corresponding output end; the output end is connected to an oscilloscope;

[0007] The analysis device is used to obtain an electrical impedance value through a resistance measurement point; determine the size of the organoid placed in the truncated cone-shaped through hole according to the electrical impedance value; determine radio frequency control information according to the size of the organoid; wherein the radio frequency control information includes radio frequency frequency, radio frequency power, temperature and cycle;

[0008] The radio frequency control device electrically stimulates the organoid according to the radio frequency control information through the input end, and the oscilloscope is used to record the electrophysiological activities of the organoid.

[0009] Optionally, the insulating layer covers the conductive layer; the core site and the pad site are exposed outside the insulating layer.

[0010] Optionally, the base layer is used as a support for microelectrodes.

[0011] Optionally, the dimensions of the truncated cone-shaped through hole are as follows: the upper hole diameter is 7 mm, the lower hole diameter is 5 mm, and the hole depth is 10 mm; the lateral distance between every two through holes is 10 mm, and the longitudinal distance is 10 mm.

[0012] Optionally, the bottom surface of the truncated cone-shaped through hole is made of U-shaped low-adsorption material.

[0013] Optionally, the base layer material is quartz glass.

[0014] Optionally, the base layer is 120 mm long, 80 mm wide and 0.55 mm thick.

[0015] Optionally, the insulating layer material is SU-8.

[0016] Optionally, the insulating layer has a thickness of 2 μm.

[0017] Optionally, the diameter of the electrophysiological site in the conductive layer is 100 μm, and the diameter of the reference electrode point is 300 μm; the size of the pad site is 2.2 mm×2.2 mm; the lateral distance between each two electrophysiological sites is 600 μm, and the longitudinal distance is 600 μm; the electrophysiological sites include electrophysiological detection sites and electrophysiological stimulation sites.

[0018] It can be seen from the above scheme that the present application provides a radiofrequency physiotherapy system for organoids, which includes a porous cell culture sub-device, a microelectrode array sub-device, an analysis device and a radiofrequency control device. After the organoid is placed in the truncated cone through-hole in the porous cell culture sub-device, the analysis device obtains the electrical impedance value through the resistance measurement point in the conductive layer in the microelectrode array sub-device, thereby determining the size of the organoid placed in the truncated cone through-hole according to the electrical impedance value, and then determining the radiofrequency control information according to the size of the organoid. After that, the radiofrequency control device electrically stimulates the organoid through the input end and the electrophysiological stimulation site connected to the input end according to the radiofrequency control information, and records the electrophysiological activity of the organoid through an oscilloscope. It makes it possible to develop the complete structure of the vaginal wall in vitro, thereby promoting related research work such as artificial vaginal repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 A schematic diagram of a radiofrequency therapy system for organoids provided in an embodiment of the present application;

[0021] Figure 2 A schematic diagram of a multi-well cell culture sub-device provided in another embodiment of the present application;

[0022] Figure 3 A schematic diagram of a truncated cone-shaped through hole provided in another embodiment of the present application;

[0023] Figure 4 A schematic diagram of a truncated cone-shaped through hole provided in another embodiment of the present application;

[0024] Figure 5 A schematic diagram of a bonded multi-well cell culture sub-device and a microelectrode array sub-device provided in another embodiment of the present application;

[0025] Figure 6 A schematic diagram of a bonded multi-well cell culture sub-device and a microelectrode array sub-device provided in another embodiment of the present application;

[0026] Figure 7 A schematic diagram of a microelectrode array sub-device provided in another embodiment of the present application;

[0027] Figure 8 A bright field microscope image of a cultured organoid provided in another embodiment of the present application;

[0028] Fig. 9 A diameter growth histogram of a cultured organoid provided in another embodiment of the present application;

[0029] Fig.10 An area growth histogram of a cultured organoid provided in another embodiment of the present application;

[0030] Fig.11 A bright field image of an organoid drug provided in another embodiment of the present application;

[0031] Fig.12 A vaginal tissue and epithelial organoids derived therefrom, H&E staining and immunofluorescence electron microscopy scanning images provided in another embodiment of the present application;

[0032] Fig.13 A schematic diagram of a microelectrode array sub-device manufacturing process provided in another embodiment of the present application. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0034] The term "including" and its variations used herein are open inclusions, i.e., "including but not limited to". The term "based on" means "based at least in part on". The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments". The relevant definitions of other terms will be given in the following description.

[0035] It should be noted that the concepts such as "first" and "second" mentioned in this application are only used to distinguish different devices, modules or units, and are not used to limit the order or interdependence of the functions performed by these devices, modules or units.

[0036] It should be noted that the modifications of "one" and "plurality" mentioned in the present application are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0037] The present application embodiment provides a radiofrequency therapy system for organoids, such as Figure 1 As shown, specifically including:

[0038] The multi-well cell culture sub-device 1000 , the microelectrode array sub-device 2000 , the analysis device 3000 and the radio frequency control device 4000 .

[0039] In the specific implementation of the present application, the multi-porous cell culture sub-device 1000 includes N truncated cone-shaped through holes 1100; taking N as 6 as an example, Figure 2 , which is a schematic diagram of a multi-well cell culture sub-device 1000 provided in the present application.

[0040] In the actual application process of the present application, for a single truncated cone through hole 1100, such as Figure 3 and Figure 4 As shown, its dimensions can be but are not limited to: the upper hole diameter is 7mm, the lower hole diameter is 5mm, the hole depth is 10mm, the lateral distance between every two through holes is 10mm, and the longitudinal distance is 10mm, which is not limited here.

[0041] In the actual application process of the present application, the porous cell culture sub-device 1000 can be manufactured by PDMS casting using, but not limited to, a PMMA mold.

[0042] Optionally, in another embodiment of the present application, the bottom surface of the truncated cone through hole 1100 is made of a U-shaped low-adsorption material, thereby reducing mechanical damage to the organoids when collecting them.

[0043] In the specific implementation of the present application, the microelectrode array sub-device 2000 includes a conductive layer 2100, an insulating layer 2200 and a base layer 2300; the conductive layer 2100 includes N core sites 2110 and (2M+2)×N pad sites 2120; each core site 2110 includes M electrophysiological detection sites 2111, M electrophysiological stimulation sites 2112, a resistance measurement point 2113 and a reference electrode point 2114; the radio frequency control device 4000 includes M input terminals 4100 and M output terminals 4200.

[0044] Among them, the truncated cone-shaped through hole 1100 is used for placing organoids; the reference electrode point 2114 is used for zero calibration; the pad site 2120 corresponding to the resistance measurement point 2113 is connected to the analysis device 3000; the pad site 2120 corresponding to each electrophysiological stimulation site 2112 is connected to the corresponding input terminal 4100; the pad site 2120 corresponding to each electrophysiological detection site 2111 is connected to the corresponding output terminal 4200; and the output terminal 4200 is connected to an oscilloscope.

[0045] For each truncated cone through hole 1100, the truncated cone through hole 1100 is connected to a core site 2110 by bonding. For example, N is 6 and M is 4. Figure 5 and Figure 6As shown, it is a schematic diagram of the bonded multi-well cell culture sub-device 1000 and the microelectrode array sub-device 2000; each point in the core site 2110 is connected to a corresponding pad site 2120 through a wire. Figure 7 , which is a schematic diagram of a microelectrode array sub-device 2000 provided by the present application. The core site 2110 includes a reference electrode site 2114, a resistance measurement site 2113, four electrophysiological detection sites 2111 and four electrophysiological stimulation sites 2112. It should be noted that the arrangement of the resistance measurement site 2113, the electrophysiological detection site 2111 and the electrophysiological stimulation site 2112 can be designed in advance by technicians, experts, etc. Figure 7 Only a schematic diagram is given in the figure, and no limitation is made here.

[0046] In the specific implementation process of the present application, the analysis device 3000 and the radio frequency control device 4000 can be coupled to the microelectrode array sub-device 2000 through, but not limited to, a coaxial cable, which is not limited here.

[0047] The analysis device 3000 is used to obtain an electrical impedance value through the resistance measurement point 2113; determine the size of the organoid placed in the truncated cone-shaped through hole 1100 according to the electrical impedance value; and determine the radio frequency control information according to the size of the organoid.

[0048] The RF control information includes but is not limited to RF frequency, RF power, temperature, cycle, etc., which are not limited here.

[0049] It is understandable that organoids of different sizes cause different changes in electrical impedance between microelectrodes. Therefore, the size of the organoid can be determined according to the electrical impedance value through, but not limited to, a pre-set calibration curve of the organoid size and the electrical impedance value, which is not limited here.

[0050] It should be noted that the size of the organoid will change with the passage of culture time. Therefore, the present application determines the size of the organoid in real time through the change of the electrical impedance value, and determines the current optimal RF control information in real time to achieve the best effect.

[0051] In the specific implementation process of the present application, the method for obtaining the calibration curve of organoid size and electrical impedance value can be, but is not limited to, obtained through multiple experimental measurements, training through a neural network model, and predictive analysis, which is not limited here.

[0052] In the specific implementation process of the present application, the optimal radio frequency control information for the size of the organoid can be determined by observing the changes in the electrical signals of the organoid after being stimulated by radio frequency electricity.

[0053] It is understandable that if the RF frequency, power, temperature and periodicity are appropriate, the electrical activity of the organoid should show a certain regularity and stability, and adaptive changes related to the stimulation may occur; if the RF power is too high or too low, the electrical signal may become disordered, the amplitude may abnormally decrease or increase, and normal action potentials may not be generated, reflecting the adaptability of the organoid to the current RF power.

[0054] The radio frequency control device 4000 electrically stimulates the organoid according to the radio frequency control information through the input terminal 4100, and the oscilloscope is used to record the electrophysiological activities of the organoid.

[0055] It can be understood that in the actual application process of this application, not only organoids but also human organs can be targeted, but the corresponding radio frequency control information needs to be adjusted to adapt to human organs, and instruments integrated with microelectronic array sub-devices can be used to treat human organs, which is not limited here.

[0056] In the specific implementation process of the present application, a culture medium for culturing human vaginal organoids is also provided, the culture medium includes a basal culture medium and added components, the added components include FGF (fibroblast growth factor), EGF (epidermal growth factor), B27 (serum substitute), N2 (serum substitute), Wnt-3a (Wnt / β-catenin signaling pathway stimulator), Noggin (bone morphogenetic protein 4 antagonist), A83-01 (transforming growth factor β kinase type 1 inhibitor), Y-27632 (ROCK inhibitor). These nutritional factors can promote cell proliferation, maintain cell stemness and cell apoptosis, and effectively maintain the proliferation activity and long-term viability of human vaginal organoids.

[0057] The vaginal organoid model construction method includes:

[0058] (1) Tissue enzymatic hydrolysis: The human vaginal wall surgical tissue was cleaned and digested with dispase at 4°C overnight. The next day, it was chopped into a paste and digested with collagenase IV for about 40 minutes.

[0059] (2) Filter and collect cells: Filter the cells after enzymatic hydrolysis, wash the cells with PBS (phosphate buffered saline), centrifuge, and discard the supernatant. Then, lyse the red blood cells with red blood cell lysis buffer, centrifuge, and discard the supernatant to obtain the vaginal wall tissue cell pellet.

[0060] (3) Primary culture of organoids: The cell pellet was resuspended in vaginal wall tissue organoid culture medium and mixed with Matrigel, then added to a 24-well cell culture plate and incubated in a 37°C, 5% CO2 incubator to form a gel. When the organoids grew to a diameter of 100 um, they were subcultured. After 4 days of growth, organoids of uniform diameter were selected through a filter and placed in a multi-well cell culture device.

[0061] like Figure 8 Shown is a bright field microscope image of vaginal organoids cultured for one week using the above protocol, scale bar: 50um.

[0062] like Fig. 9 Shown is a histogram of diameter growth of vaginal organoids cultured using the above protocol.

[0063] like Fig.10 Shown is a histogram of area growth of vaginal organoids cultured using the above protocol.

[0064] like Fig.11 As shown, it is a bright field image of organoid drugs. It can be seen that the vaginal organoids cultured using the above scheme can respond normally to estrogen and progesterone.

[0065] like Fig.12 As shown, it is the H&E staining and immunofluorescence electron microscopy scanning of vaginal tissue and epithelial organoids derived from it, among which P63 is a basal cell marker and CK13 is a suprabasal cell marker. It can be seen that the vaginal organoids cultured by the above scheme are very close to human organs.

[0066] Optionally, in another embodiment of the present application, the diameter of the electrophysiological site in the conductive layer 2100 is 100 μm, and the diameter of the reference electrode point is 300 μm; the size of the pad site 2120 is 2.2 mm×2.2 mm; the lateral distance between each two electrophysiological sites is 600 μm, and the longitudinal distance is 600 μm; the electrophysiological sites include electrophysiological detection sites 2111 and electrophysiological stimulation sites 2112.

[0067] Optionally, in another embodiment of the present application, the insulating layer 2200 covers the conductive layer 2100 ; the core site 2110 and the pad site 2120 are exposed outside the insulating layer 2200 .

[0068] In the specific implementation process of the present application, the material of the insulating layer 2200 may be but is not limited to SU-8, which is not limited here.

[0069] In the specific implementation process of the present application, the thickness of the insulating layer 2200 can be but is not limited to 2 μm, which is not limited here.

[0070] In the specific implementation process of the present application, the base layer 2300 is used as a support for the microelectrode, and its material may be but is not limited to quartz glass, which is not limited here.

[0071] Optionally, in another embodiment of the present application, the size of the base layer 2300 may be, but is not limited to, 120 mm in length, 80 mm in width, and 0.55 mm in thickness.

[0072] In the specific implementation process of the present application, the microelectrode array sub-device 2000 can be manufactured by, but not limited to, micro-nano processing technology, such as Fig.13 As shown, the specific steps include:

[0073] (1) ITO is deposited on the surface of clean quartz glass to form a metal layer with a thickness of 185 nm. The metal layer is laser etched to form electrophysiological detection sites, electrophysiological stimulation sites, reference electrodes, pad sites and wire structures.

[0074] (2) Spin-coat positive photoresist SU-8 on the surface of the metal layer, and expose the electrophysiological detection site, electrophysiological stimulation site, reference electrode and pad site after photolithography and development to form an insulating layer with a thickness of 1 μm.

[0075] (3) Electroplating IrOx on the ITO electrode to reduce resistance and enhance conductivity. The electroplating is done using a three-electrode system.

[0076] It can be seen from the above scheme that the present application provides a radiofrequency physiotherapy system for organoids, which includes a porous cell culture sub-device, a microelectrode array sub-device, an analysis device and a radiofrequency control device. After the organoid is placed in the truncated cone through-hole in the porous cell culture sub-device, the analysis device obtains the electrical impedance value through the resistance measurement point in the conductive layer in the microelectrode array sub-device, thereby determining the size of the organoid placed in the truncated cone through-hole according to the electrical impedance value, and then determining the radiofrequency control information according to the size of the organoid. After that, the radiofrequency control device electrically stimulates the organoid through the input end and the electrophysiological stimulation site connected to the input end according to the radiofrequency control information, and records the electrophysiological activity of the organoid through an oscilloscope. It makes it possible to develop the complete structure of the vaginal wall in vitro, thereby promoting related research work such as artificial vaginal repair.

[0077] The functions described above herein may be performed at least in part by one or more hardware logic components. For example, without limitation, exemplary types of hardware logic components that may be used include: field programmable gate arrays (FPGAs), application specific integrated circuits (ASICs), application specific standard products (ASSPs), systems on chips (SOCs), complex programmable logic devices (CPLDs), and the like.

[0078] Although the subject matter has been described in language specific to structural features and / or method logic actions, it should be understood that the subject matter defined in this application is not necessarily limited to the specific features or actions described above. On the contrary, the specific features and actions described above are merely example forms of implementing this application.

[0079] Although several specific implementation details are included in the above discussion, these should not be interpreted as limiting the scope of the present application. Certain features described in the context of a separate embodiment can also be implemented in a single embodiment in combination. On the contrary, the various features described in the context of a single embodiment can also be implemented in multiple embodiments individually or in any suitable sub-combination.

[0080] The above description is only a preferred embodiment of the present application and an explanation of the technical principles used. Those skilled in the art should understand that the scope of application involved in the present application is not limited to the technical solution formed by a specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above application concept. For example, the above features are replaced with (but not limited to) technical features with similar functions applied in the present application.

Claims

1. A radiofrequency therapy system for organoids, characterized in that: include: A porous cell culture sub-device, a microelectrode array sub-device, an analysis device and a radio frequency control device; wherein the porous cell culture sub-device includes N truncated cone-shaped through holes; the microelectrode array sub-device includes a conductive layer, an insulating layer and a base layer; the conductive layer includes N core sites and (2M+2)×N pad sites; each of the core sites includes M electrophysiological detection sites, M electrophysiological stimulation sites, a resistance measurement site and a reference electrode site; the radio frequency control device includes M input terminals and M output terminals; For each truncated cone through hole, the truncated cone through hole is connected to one of the core sites by bonding; each of the core sites is respectively connected to a corresponding pad site by a wire; wherein the truncated cone through hole is used to place the organoid; the reference electrode point is used for zero calibration; the pad site corresponding to the resistance measurement point is connected to the analysis device; the pad site corresponding to each electrophysiological stimulation site is connected to the corresponding input end; the pad site corresponding to each electrophysiological detection site is connected to the corresponding output end; the output end is connected to an oscilloscope; The analysis device is used to obtain an electrical impedance value through a resistance measurement point; determine the size of the organoid placed in the truncated cone-shaped through hole according to the electrical impedance value; determine radio frequency control information according to the size of the organoid; wherein the radio frequency control information includes radio frequency frequency, radio frequency power, temperature and cycle; The radio frequency control device electrically stimulates the organoid according to the radio frequency control information through the input end, and the oscilloscope is used to record the electrophysiological activities of the organoid.

2. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The insulating layer covers the conductive layer; the core site and the pad site are exposed outside the insulating layer.

3. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The base layer is used as a support for the microelectrodes.

4. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The dimensions of the truncated cone-shaped through hole are as follows: the upper hole diameter is 7 mm, the lower hole diameter is 5 mm, and the hole depth is 10 mm; the lateral distance between every two through holes is 10 mm, and the longitudinal distance is 10 mm.

5. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The bottom surface of the truncated cone through hole is made of U-shaped low-adsorption material.

6. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The base layer material is quartz glass.

7. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The base layer is 120 mm long, 80 mm wide and 0.55 mm thick.

8. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The insulating layer material is SU-8.

9. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The thickness of the insulating layer is 2 μm.

10. The radiofrequency therapy system for organoids according to claim 1, characterized in that: The diameter of the electrophysiological sites in the conductive layer is 100 μm, and the diameter of the reference electrode point is 300 μm; the size of the pad site is 2.2 mm×2.2 mm; the lateral distance between every two electrophysiological sites is 600 μm, and the longitudinal distance is 600 μm; the electrophysiological sites include electrophysiological detection sites and electrophysiological stimulation sites.

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