WO2024027717A1

WO2024027717A1 - Total system and method for full automatic preparation, sorting, and distribution and culturing of in-vitro organ microsphere

Info

Publication number: WO2024027717A1
Application number: PCT/CN2023/110588
Authority: WO
Inventors: 田甜; 富国祥
Original assignee: 丹望医疗科技(上海)有限公司
Priority date: 2022-08-02
Filing date: 2023-08-01
Publication date: 2024-02-08
Also published as: CN115369033A

Abstract

The present disclosure relates to the technical field of in-vitro organs, and in particular to a total system and a method for full automatic preparation, sorting, and distribution and culturing of in-vitro organ microspheres.

Description

Total system and method for fully automated in vitro organ microsphere preparation, sorting, and distribution culture

priority information

This disclosure claims the priority and rights of patent application 202210919531.5 filed with the State Intellectual Property Office of China on August 2, 2022, and the full text of which is incorporated herein by reference.

Technical field

The present disclosure relates to the field of in vitro organ technology. Specifically, the present disclosure relates to a total system and method for fully automated in vitro organ microsphere preparation, sorting, and distribution culture.

Background technique

According to statistics, the average research and development of a new drug costs more than 1 billion US dollars and takes more than 10 years. However, the actual investment of money and time is much higher than this figure. Currently, the most commonly used methods for drug screening are mainly pharmacodynamic models based on animal and cell experiments. However, due to the long cycle of animal experiments, high labor intensity, high operational technical requirements and high costs, and due to the racial differences between animals and humans, there is still a long distance between the screened drugs and clinical applications, so development is slow and has not yet been completed. It can carry out large-scale screening, but it has also been controversial because of issues such as animal rights protection. In traditional cell experiments, most of the cells are two-dimensional adherent culture and single cell culture, which is quite different from the growth microenvironment of the cells in the body, and the cells in this state cannot show the complete functions of the cells in the body, so As a result, the response of the in vitro cell model to the drug cannot reflect the true response of the drug in the body.

The emergence of organoids in recent years provides a good in vitro model for drug screening. Organoids are multicellular structures with a three-dimensional structure that are functionally close to organs, formed from embryonic stem cells, pluripotent stem cells or adult somatic cells under a certain culture environment and the support of extracellular matrix. Compared with cell line culture, organoids have a three-dimensional microstructure that is closer to the state of in vivo tissues. They can retain tumor heterogeneity in tumor models and have wide application in many fields such as drug screening, pathological research, and precision medicine. potential. At present, the traditional organoid culture method mainly uses Matrigel as the supporting matrix. The organoid seeds are mixed with Matrigel and then inoculated into the culture dish through a pipette. The Matrigel is solidified at 37°C to achieve the effect of three-dimensional culture. . However, the size of the organoids cultured by this method and the number of organoid microspheres formed in the culture dish are uncontrollable, and the entire process is manual operation, which has low efficiency. The culture state of the organoids is greatly affected by the differences between operators and cannot be controlled. It is conducive to the standardization and throughput culture of organoids, but it also cannot meet the needs of drug screening. Ultra-low adhesion U-shaped well plates are also used for organoid culture. This method modifies the surface material of the well plate so that cells will not grow adherently on its surface, and then uses the bottom of the prototype well to allow cells to grow due to gravity. They gather at the bottom of the well plate and grow into balls to achieve the purpose of three-dimensional culture. However, this method is inconvenient to use Matrigel and therefore cannot provide the nutrients needed for organoid growth. In addition, there is also the use of 3D printing technology to construct three-dimensional cells and then simulate the three-dimensional structure of organs in the body. However, this method does not have enough understanding and knowledge of the structure of organs and cell types in the body, so it cannot be completely simulated in vitro, and the reconstructed model is also The function of organs in the body cannot be reproduced.

Patent CN110004111B discloses a method for preparing organoid spheroids. This method uses a syringe pump and a three-way device to prepare organoid droplet microspheres. The oil phase is fluorine oil, and the water phase is a mixture of matrigel and cells. Inject the water phase and oil phase into a syringe respectively. The syringe is connected to the tee device and a syringe pump is used to drive the syringe. The syringe pump is placed in the refrigerator (4°C) to maintain the matrigel. Coagulation occurs and organoid microspheres are prepared in a three-way device via a syringe driven by a syringe pump. Patent CN110042077B discloses a high-throughput culture method for organoid spheroids. This method is a continuation of the previous patent. The above-mentioned patent is used to obtain organoid microspheres, then heated, and linked with the 3D printing platform to convert the organoid spheroids. Make allocations.

The disadvantages of the above two patented technologies are: 1. The entire system is simply built with multiple instruments (such as refrigerators), which is large in size, troublesome to operate, and is not conducive to promotion; 2. The system cannot be placed in a biological safety cabinet for operation. A sterile environment cannot be guaranteed, and the prepared organoids may have the risk of contamination; 3. The system lacks a real-time observation module and cannot realize observation and quality monitoring functions; 4. The system lacks an automatic liquid addition function and it is difficult to achieve high-pass performance in the true sense. The survival rate of organoids is not guaranteed due to high-volume culture; 5. Since syringe pumps and syringes are involved, the replacement and addition of oil and water phase samples are cumbersome and there are interfering factors such as bubbles; 6. The organoids are distributed using a 3D printing platform. During the movement, it is easy to cause disturbance of the organoids in the tubing, thereby affecting the accuracy of distribution.

Contents of the invention

The present disclosure aims to solve one of the technical problems in the related art, at least to a certain extent. To this end, one object of the present disclosure is to provide a fully automated overall system and method for in vitro organ microsphere preparation, sorting, and distribution culture. The modular system constructed by this disclosure can realize Nowadays, the automated preparation, detection, sorting, and distribution of hair fluid for culture of VOS are simple to operate and pollution-free. It provides a reproducible in vitro biological model for drug screening and efficacy evaluation, and has good application prospects and promotion value.

To this end, on the one hand, the present disclosure provides a fully automated total system for in vitro organ microsphere preparation, sorting, and distribution culture. According to an embodiment of the present disclosure, the total system for fully automated in vitro organ microsphere preparation, sorting and distribution culture includes:

Preparation system, the preparation system is used to prepare in vitro organ microspheres, the preparation system includes a microsphere preparation flow channel provided on a microfluidic chip, the microsphere preparation flow channel includes a microsphere fluid flow channel;

Detection and sorting system, the detection and sorting system includes a defective microsphere fluid channel and a qualified microsphere fluid channel formed by the bifurcation of the microsphere fluid channel, the defective microsphere fluid channel and the qualified microsphere fluid channel The microsphere fluid channels are all arranged on the microfluidic chip. The microsphere fluid channels are provided with asymmetric electrodes on the corresponding microfluidic chip before bifurcation, and one side of the defective microsphere fluid channel The electric field generated by the corresponding electrode is greater than the electric field generated by the corresponding electrode on one side of the qualified microsphere fluid channel. The detection and sorting system also includes a signal generator, a detection and sorting control unit and an optical detection unit. The optical detection unit The unit is arranged above the microfluidic chip corresponding to the microsphere fluid channel before bifurcation, and the signal generator is connected to the asymmetric electrode and the detection and sorting control unit respectively, and the detection and sorting control unit The control unit is connected to the optical detection unit and the signal generator respectively;

Dispensing hair liquid system, the hair liquid distributing system is used for distributing the qualified microsphere fluid sorted by the detection and sorting system into the orifice plate and adding the qualified microsphere fluid packed in the orifice plate. Culture fluid.

According to the overall system of fully automated in vitro organ microsphere preparation, sorting, and distribution culture according to the above embodiments of the present disclosure, the modular system constructed by the present disclosure can realize the automated preparation, detection, sorting, and distribution of hair fluid culture of VOS, and is simple to operate and It is pollution-free, provides a reproducible in vitro biological model for drug screening and efficacy evaluation, and has good application prospects and promotion value. At the same time, the detection and sorting function is integrated on the chip to screen VOS with more dead cells or no cells to ensure that all VOS obtained are highly active and achieve effective sorting of target VOS.

The total system for fully automated in vitro organ microsphere preparation, sorting, and distribution culture according to embodiments of the present disclosure may also have the following additional technical features:

According to an embodiment of the present disclosure, the microsphere preparation flow channel includes: a cell fluid flow channel of a shell structure, a cell fluid flow channel of a core structure, and an oil phase flow channel. The cell fluid flow channel of the shell structure and the core structure are The cell fluid flow channels of the structure merge to form a merged flow channel, and the merged flow channel merges with the oil phase flow channel to form a microsphere fluid flow channel. Between the cell fluid flow channel of the shell structure and the core structure, At the confluence of the cell fluid flow channels, the cell fluid flow channels of the shell structure are arranged on both sides of the cell fluid flow channels of the core structure. At the confluence of the merge flow channel and the oil phase flow channel, the The oil phase flow channel is arranged on both sides of the converging flow channel.

According to an embodiment of the present disclosure, the preparation system further includes: a first liquid storage device, the liquid outlet of the first liquid storage device is connected to the inlet of the cell fluid flow channel of the shell structure, the first liquid storage device The fluid device is used to store cellular fluids in the shell structure;

a second liquid storage device, the liquid outlet of the second liquid storage device is connected to the inlet of the cell fluid flow channel of the nuclear structure, and the second liquid storage device is used to store the cell fluid of the nuclear structure;

A third liquid storage device, the liquid outlet of the third liquid storage device is connected to the inlet of the oil phase flow channel, and the third liquid storage device is used to store the oil phase;

A pressure controller and a diaphragm pump. The pressure controller is connected to the first liquid storage device, the second liquid storage device and the third liquid storage device respectively. The diaphragm pump is connected to the pressure controller. .

According to an embodiment of the present disclosure, the preparation system further includes: a flow rate control unit connected to the pressure controller.

According to an embodiment of the present disclosure, the preparation system further includes: a refrigeration module, the refrigeration module is used to combine the microfluidic chip, the first liquid storage device, the second liquid storage device and the third liquid storage device. The temperature of the three liquid storage devices is maintained at a certain set temperature.

According to an embodiment of the present disclosure, the preparation system further includes: a heat preservation unit, the microfluidic chip, the first liquid storage device, the second liquid storage device and the third liquid storage device are all arranged in inside the insulation unit.

According to an embodiment of the present disclosure, the width of the cell fluid flow channel of the shell structure is 100-150 μm, and the flow rate of the cell fluid of the shell structure in the cell fluid flow channel of the shell structure is 0.5-5 μL/min.

According to an embodiment of the present disclosure, the width of the cellular fluid channel of the nuclear structure is 100-150 μm, and the flow rate of the cellular fluid of the nuclear structure in the cellular fluid channel of the nuclear structure is 0.5-5 μL/min.

According to an embodiment of the present disclosure, the width of the oil phase flow channel is 75-300 μm, and the flow rate of the oil phase in the oil phase flow channel is 1-20 μL/min.

According to an embodiment of the present disclosure, the cellular fluid of the shell structure within the cellular fluid channel of the shell structure includes cells of the shell structure and the first hydrogel, and the cellular fluid of the core structure within the cellular fluid channel of the core structure includes The cells of the core structure and the second hydrogel, the cells of the shell structure are different from the cells of the core structure, the first hydrogel and the second hydrogel are different, and the cells of the shell structure are selected From at least one of umbilical cord vein endothelial cells and immune cells, the cells of the nuclear structure are selected from at least one of tumor cells and stem cells, and the first hydrogel is selected from the group consisting of fibrinogen, collagen and alginate At least one of the second hydrogel is selected from at least one of Matrigel, fibrinogen and collagen.

According to an embodiment of the present disclosure, the microsphere preparation flow channel includes: a cell fluid flow channel and an oil phase flow channel, and the cell fluid flow channel and the oil phase flow channel merge to form a microsphere fluid flow channel.

According to an embodiment of the present disclosure, the total system further includes a heating unit for forming the qualified microspheres into a solid state, and one end of the heating unit is connected to the outlet of the qualified microsphere fluid flow channel;

The hair liquid dispensing system includes:

A support plate with a slideway provided on the support plate;

Mobile unit, the mobile unit includes a mobile platform, an X-direction slide rail and a Y-direction slide rail. The Y-direction slide rail is vertically arranged on the slide rail. The Y-direction slide rail can move along the Y-direction on the slide rail. move, the X-direction slide rail is vertically arranged on the Y-direction slide rail, the X-direction slide rail can move along the X-direction on the Y-direction slide rail, and the mobile platform is fixed on the X-direction slide rail. above the rail;

A well plate, the well plate is arranged on the mobile platform, and the well plate is provided with a plurality of culture wells;

Microsphere fluid distribution unit, the microsphere fluid distribution unit includes a microsphere fluid distribution tube, a first connecting rod and a first vertical rod, the first vertical rod is arranged on the support plate, the microsphere fluid distribution unit The pipe is supported above the culture hole through a first connecting rod. The microsphere fluid distribution tube includes a distribution head. The distribution head is provided at the lower end of the microsphere fluid distribution tube. The microsphere fluid distribution tube has The upper end is connected to the other end of the heating unit;

Culture liquid pipetting unit, the culture liquid pipetting unit includes the culture liquid pipette, a second connecting rod and a second vertical rod, the second vertical rod is arranged on the support plate, the culture liquid pipetting unit The pipette is supported above the culture hole through a second connecting rod, and a plurality of pipette tips are provided at the bottom of the culture liquid pipette.

According to an embodiment of the present disclosure, the hair liquid dispensing system further includes: a hair liquid distributing control unit, which is respectively connected to the moving unit, the microsphere fluid dispensing unit and the culture liquid pipetting unit. units are connected.

According to an embodiment of the present disclosure, the first connecting rod is configured to rotate circumferentially about the axis of the first vertical rod, the first vertical rod is configured to be telescopic up and down, and the second vertical rod is configured to be able to move up and down. Telescopic.

Another aspect of the present disclosure provides a method for in vitro organ microsphere preparation, sorting, and distribution culture using the overall system described in the above embodiments. According to an embodiment of the present disclosure, the method includes:

(1) In vitro organ microspheres are prepared in the microsphere preparation flow channel, and the in vitro organ microspheres are wrapped in an oil phase to form a pre-sorting microsphere fluid;

(2) Before the pre-sorting microsphere fluid bifurcates, an optical detection unit is used to observe the pre-sorting microspheres, and the optical detection unit sends the observation results to the detection and sorting control unit. If the sorting If there is a defect in the front microsphere, the detection and sorting control unit sends an instruction to the signal generator, and the signal generator turns on the asymmetric electrode to generate an uneven electric field. The uneven electric field causes the defective microsphere to deviate. Enter the defective microsphere fluid flow channel;

If there are no defects in the microspheres before sorting, the asymmetric electrode is not connected so that qualified microspheres enter the qualified microsphere fluid flow channel;

(3) Use a liquid dispensing system to distribute the qualified microsphere fluid in the well plate, and add culture fluid to the qualified microspheres distributed in the well plate.

The fully automated in vitro organ microsphere preparation, sorting and distribution culture method according to the embodiments of the present disclosure has all the advantages of the fully automated in vitro organ microsphere preparation, sorting and distribution culture system described in the above embodiments. This will not be described again.

Additional aspects and advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure.

Description of the drawings

The above and/or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which middle:

Figure 1 shows a schematic diagram of the overall system for fully automated in vitro organ microsphere preparation, sorting and distribution culture according to an embodiment of the present disclosure;

Figure 2 shows a schematic structural diagram of a microfluidic chip according to an embodiment of the present disclosure;

Figure 3 shows a schematic structural diagram of a preparation system according to an embodiment of the present disclosure;

Figure 4 shows a schematic structural diagram of a detection and sorting system according to an embodiment of the present disclosure;

Figure 5 shows a schematic structural diagram of a hair liquid dispensing system according to an embodiment of the present disclosure;

Figure 6 shows a schematic diagram of the principle of preparing double emulsion core-shell structure microspheres according to an embodiment of the present disclosure;

Figure 7 shows a schematic diagram of the principle of preparing single emulsion core-shell structure microspheres according to an embodiment of the present disclosure;

Figure 8 shows a schematic diagram of preparing double emulsion core-shell structure microspheres according to an embodiment of the present disclosure;

Figure 9 shows a schematic diagram of in vitro organ microspheres of different sizes according to embodiments of the present disclosure;

Figure 10 shows a schematic diagram of the movement path of the mobile unit according to the embodiment of the present disclosure;

Figure 11 shows the fluorescence image of VOS dead and alive cells stained after sorting in Example 1 of the present disclosure;

Figure 12 shows a schematic diagram of matrigel microspheres distributed into well plates according to Example 1 of the present disclosure;

Figure 13 shows the VOS state diagram cultivated in Example 1 of the present disclosure;

Figure 14 shows a comparison of Example 1 of the present disclosure and the traditional manual method of preparing cultured VOS;

Figure 15 shows the growth curve of VOS prepared and cultured in Example 1 of the present disclosure and traditional manual methods;

Figure 16 shows the fluorescence image of the core-shell structure VOS cultured in Example 1 of the present disclosure.

Reference signs:
1000-preparation system, 100-microfluidic chip, 1100-microsphere preparation flow channel, 1110-oil phase flow channel, 1120-shell structure cell fluid flow channel, 1130-core structure cell fluid flow channel, 1140-sink Combined flow channel, 1150-microsphere fluid flow channel, 1160-cell fluid flow channel, 1200-flow rate control unit, 1300-pressure controller, 1400-diaphragm pump, 1500-first liquid storage device, 1600-second liquid storage device , 1700-third liquid storage device;
2000-detection and sorting system, 2100-defective microsphere fluid flow channel, 2200-qualified microsphere fluid flow channel, 2300-asymmetric electrode, 2400-
Optical detection unit, 2500-signal generator, 2600-detection and sorting control unit;
3000-hair liquid distribution system, 3100-support plate, 3200-slide, 3300-Y direction slide rail, 3400-X direction slide rail, 3500-mobile platform, 3600-first vertical pole, 3700-first connecting rod, 3800-microsphere fluid distribution tube, 3810-distribution head, 3900-second vertical rod, 3910-second connecting rod, 3920-culture liquid pipette, 3921-pipettor head.

Detailed description of the invention

Embodiments of the present disclosure are described in detail below. The embodiments described below are illustrative and are only used to explain the present disclosure and are not to be construed as limitations of the present disclosure.

In addition, the terms “first” and “second” are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.

The terms "center", "lengthwise", "crosswise", "length", "width", "thickness", "top", "bottom", "front", "back", "left", "right", " The orientation or positional relationships indicated by "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc. are based on those shown in the accompanying drawings. The orientations or positional relationships shown are only to facilitate the description of the present disclosure and simplify the description, but do not indicate or imply that the indicated elements must have specific orientations, be constructed and operated in specific orientations, and therefore cannot be understood as limiting the disclosure.

In this disclosure, unless otherwise clearly stated and limited, terms such as "installation", "connection", "connection", and "fixing" should be understood in a broad sense. For example, it can be a fixed connection or a detachable connection. or integrated into one; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be an internal connection between two elements or an interactive relationship between two elements, unless otherwise specified limited. For those of ordinary skill in the art, the specific meanings of the above terms in this disclosure can be understood according to specific circumstances.

In this disclosure, unless otherwise expressly stated and limited, a first feature being "on" or "below" a second feature may mean that the first and second features are in direct contact, or the first and second features may be in indirect contact through an intermediary. touch. Furthermore, the terms "above", "above" and "above" the first feature is above the second feature may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is higher in level than the second feature. The first characteristic is “Below”, “below” and “below” the second feature may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature has a smaller horizontal height than the second feature.

In the embodiments of the present disclosure, in order to solve the problem of uneven and uncontrollable sizes of in vitro organ microspheres prepared by existing preparation methods, the present disclosure uses microfluidic microdroplet generation and gel solidification technology to achieve uniform and controllable sizes. Preparation of in vitro organoid microspheres. In view of the problem that the prepared VOS cannot be detected and sorted by existing methods, the present disclosure combines an optical detection unit and dielectrophoretic sorting technology on a microfluidic chip to achieve an effective sorting function of VOS and realize automated detection and sorting of VOS. selection, providing an effective quality control method for the preparation of VOS. In view of the problems of low efficiency and poor accuracy caused by manual distribution in the existing distribution method, the present disclosure utilizes a mobile unit whose movement path is controllable to realize automatic distribution of organ microspheres outside the body. In view of the fact that existing culture methods cannot meet the needs of automated culture, the present disclosure implements automatic liquid addition based on a culture liquid pipetting unit. In view of the problem that the existing preparation system is bulky and cannot be operated in a biological safety cabinet, the present disclosure integrates a diaphragm pump and pressure controller, a refrigeration module, a heating unit, a mobile unit, a microsphere fluid distribution unit, a culture fluid pipetting unit and a detection analyzer. The selection system has the advantages of small size, convenient operation, full automation, and easy promotion. In view of the cumbersome sample addition caused by the use of syringe pumps and syringes in the existing preparation methods and the presence of interference factors such as bubbles, the present disclosure is based on a diaphragm pump driving method, which enables one-button operation after one sample addition.

In view of this, in one aspect of the present disclosure, the present disclosure proposes a fully automated overall system for the preparation, sorting and distribution of organ microspheres in vitro. With reference to Figure 1, the fully automated in vitro organ microsphere preparation, sorting and The total system for distributing culture includes: preparation system 1000, detection and sorting system 2000, and distribution system 3000.

According to an embodiment of the present disclosure, the preparation system 1000 is used to prepare organ microspheres in vitro. The preparation system 1000 includes a microsphere preparation flow channel 1100 disposed on a microfluidic chip 100. The microsphere preparation flow channel 1100 Microsphere fluid flow channels 1150 are included. To prepare microspheres with different structures, the corresponding microsphere preparation flow channels 1100 are different, specifically:

When preparing microspheres with a double emulsion core-shell structure, refer to Figures 2 and 6. The microsphere preparation flow channel 1100 includes: a cell fluid flow channel 1120 with a shell structure, a cell fluid flow channel 1130 with a core structure, and an oil phase flow. Channel 1110, the cell fluid channel 1120 of the shell structure contains the cell fluid of the shell structure, the cell fluid channel 1130 of the core structure contains the cell fluid of the core structure, and the oil phase channel 1110 contains the oil phase. The cell fluid flow channel 1120 of the shell structure and the cell fluid flow channel 1130 of the core structure merge to form a merged flow channel 1140, and between the cell fluid flow channel 1120 of the shell structure and the cell fluid flow channel 1130 of the core structure, At the confluence of the channels 1130, the cell fluid flow channel 1120 of the shell structure is disposed on both sides of the cell fluid flow channel 1130 of the core structure. In the converging flow channel 1140, the cellular fluid of the shell structure and the cellular fluid of the core structure do not dissolve, but form a three-layer laminar flow including the cellular fluid of the shell structure, the cellular fluid of the core structure, and the cellular fluid of the shell structure in sequence. , the cell fluid of the shell structure is distributed on both sides of the cell fluid of the core structure, so that the cell fluid of the shell structure is wrapped on both sides of the cell fluid of the core structure, so that microspheres of the core-shell structure can be formed later. The converging flow channel 1140 and the oil phase flow channel 1110 merge to form a microsphere fluid flow channel 1150. At the confluence of the converging flow channel 1140 and the oil phase flow channel 1110, the oil phase flow channel 1110 are provided on both sides of the converging flow channel 1140. At the confluence, the above-mentioned three-layer laminar flow including the cellular fluid of the shell structure, the cellular fluid of the core structure and the cellular fluid of the shell structure are sheared by the oil phase flowing in from both sides, thereby forming microspheres with a core-shell structure, and The core-shell structure in vitro organ microspheres are wrapped in the oil phase. The structure of the core-shell structure in vitro organ microsphere is shown in Figure 8, wherein Figure 8a is a schematic diagram of the core structure, Figure 8b is a schematic diagram of the shell structure, and Figure 8c is a schematic diagram of the core-shell structure. This core-shell structure can realize vascularized co-culture of VOS, further improve the performance of VOS to simulate the organs in the body, lay a foundation for the application of VOS as an in vitro model in the fields of clinical medicine, precision medicine and drug screening, and solve the current problems. The VOS prepared by this method has a simple structure and cannot form vascular structures or co-culture with immune cells.

According to embodiments of the present disclosure, the width of the cell fluid flow channel 1120 of the shell structure and the flow rate of the cell fluid of the shell structure within the cell fluid flow channel 1120 of the shell structure are not particularly limited, and those in the art can proceed according to actual needs. Design, as a preferred solution, the width of the shell-structured cell fluid flow channel 1120 is 100-150 μm, and the flow rate of the shell-structured cell fluid in the shell-structured cell fluid flow channel 1120 is 0.5-5 μL/min. .

According to embodiments of the present disclosure, the width of the cellular fluid flow channel 1130 of the nuclear structure and the flow rate of the cellular fluid of the nuclear structure within the cellular fluid channel 1130 of the nuclear structure are not particularly limited, and those in the art can proceed according to actual needs. Design, as a preferred solution, the width of the nuclear structure cell fluid channel 1130 is 100-150 μm, and the flow rate of the nuclear structure cell fluid in the nuclear structure cell fluid channel 1130 is 0.5-5 μL/min. .

According to the embodiment of the present disclosure, the width of the oil phase flow channel 1110 and the flow rate of the oil phase in the oil phase are not particularly limited. Persons in the art can design according to actual needs. As a preferred solution, the oil phase The width of the flow channel 1110 is 75-300 μm, and the flow rate of the oil phase in the oil phase flow channel 1110 is 1-20 μL/min.

According to an embodiment of the present disclosure, the cell fluid of the shell structure within the cell fluid channel 1120 of the shell structure includes cells of the shell structure and The first hydrogel serves as a dispersion medium for the cells of the shell structure. The cell fluid of the nuclear structure in the cell fluid channel 1130 of the nuclear structure includes the cells of the nuclear structure and a second hydrogel, and the second hydrogel serves as a dispersion medium for the cells of the nuclear structure. The cells of the shell structure are different from the cells of the core structure, and the first hydrogel and the second hydrogel are different, so that an incompatible multi-layer laminar flow can be formed. It should be noted that the specific types of cells with the shell structure, the specific types of cells with the core structure, the specific types of the first hydrogel, and the specific types of the second hydrogel are not particularly limited. As a specific example, The cells of the shell structure are selected from at least one of umbilical cord vein endothelial cells and immune cells, the cells of the nuclear structure are selected from at least one of tumor cells and stem cells, and the first hydrogel is selected from fibrin. The second hydrogel is at least one selected from the group consisting of Matrigel, fibrinogen and collagen.

According to a specific embodiment of the present disclosure, with reference to Figure 3, the preparation system 1000 further includes: a first liquid storage device 1500, the liquid outlet of the first liquid storage device 1500 is connected with the cell fluid flow of the shell structure. The inlet of the channel 1120 is connected, and the first liquid storage device 1500 is used to store the cell fluid of the shell structure; the second liquid storage device 1600, the liquid outlet of the second liquid storage device 1600 is connected with the cell fluid of the core structure. The inlet of the flow channel 1130 is connected, the second liquid storage device 1600 is used to store the cellular fluid of the nuclear structure; the third liquid storage device 1700, the liquid outlet of the third liquid storage device 1700 is connected with the oil phase flow channel 1110 is connected to the inlet, and the third liquid storage device 1700 is used to store the oil phase.

Further, referring to Figure 3, the preparation system 1000 also includes: a pressure controller 1300 and a diaphragm pump 1400. The pressure controller 1300 is connected to the first liquid storage device 1500 and the second liquid storage device 1600 respectively. It is connected to the third liquid storage device 1700, and the diaphragm pump 1400 is connected to the pressure controller 1300. Specifically, the diaphragm pump 1400 is connected to the pressure controller 1300, and the pressure controller 1300 is connected to the liquid storage device. The liquid storage device is connected to the pressure controller 1300 through a tube and connected to the microfluidic chip 100 through a capillary tube extending under the liquid surface. The function of the diaphragm pump 1400 is to generate pressure, and the function of the pressure controller 1300 is to adjust the pressure generated by the diaphragm pump 1400. The fluid in the liquid storage device is pressed out by the air pressure adjusted by the pressure controller 1300 and then enters the microfluidic chip 100 for VOS. preparation. Thus, by adjusting the air pressure of the pressure controller 1300, the flow rate of the fluid in each channel is adjusted, thereby adjusting the size and spacing distance of VOS and other parameters; at the same time, it also solves the problem of flux in the current preparation process of organ microspheres in vitro The problem is that it is low, the size is uneven and uncontrollable, and it is difficult to achieve standardized preparation by relying on manual operations. In addition, when the concentration of cells or tissue micro-blocks is constant, the number of cells or tissue micro-blocks can be further determined by adjusting the volume of the microspheres, as shown in Figure 9.

Further, referring to Figure 3, the preparation system 1000 also includes: a flow rate control unit 1200, which is connected to the pressure controller 1300. The flow rate control unit 1200 controls the storage by controlling the pressure controller 1300. The air pressure in the liquid device is used to control the flow rate of the fluid in the flow channel. The flow rate control unit 1200 may be an electronic device, such as a mobile phone or a computer.

Further, the preparation system 1000 also includes: a refrigeration module (not shown in the figure), the refrigeration module is used to combine the microfluidic chip 100, the first liquid storage device 1500, the second liquid storage device 1500 and the second liquid storage device 1500. The temperatures of the liquid storage device 1600 and the third liquid storage device 1700 are maintained at a certain set temperature (for example, 4°C). Specifically, the microfluidic chip 100 can be disposed on a refrigeration module (such as a refrigeration plate), in the first liquid storage device 1500 , the second liquid storage device 1600 and the third liquid storage device 1700 The refrigeration module is wrapped around the outside.

Further, the preparation system 1000 also includes: a heat preservation unit (not shown in the figure), the microfluidic chip 100, the first liquid storage device 1500, the second liquid storage device 1600 and the The third liquid storage device 1700 is disposed in the heat preservation unit, so that the microfluidic chip 100 , the first liquid storage device 1500 , the second liquid storage device 1600 and the third liquid storage device 1700 All are maintained at a low temperature (for example, maintained at 4°C) to avoid Matrigel solidification.

When preparing single emulsion microspheres, refer to Figure 7. The microsphere preparation flow channel includes: cell fluid flow channel 1160 and oil phase flow channel 1110. The cell fluid flow channel 1160 and the oil phase flow channel 1110 are in phase. They merge to form a microsphere fluid flow channel. As a continuous phase, the oil phase uses shear force to cut off the cell fluid, forming water-in-oil VOS. Specifically, the oil phase can cut off the cell fluid from one side, as shown in Figure 7a; the oil phase can also be distributed on both sides of the cell fluid, so that the cell fluid is sheared by the oil phase flowing in from both sides, such as As shown in Figure 7b, water-in-oil VOS is formed.

It should be noted that the above-mentioned preparation system 1000 for preparing single emulsion microspheres also includes a device for storing cell fluid and a device for storing oil phase, a pressure controller 1300, a diaphragm pump 1400, a refrigeration module, a heat preservation unit, etc., and their connection methods are as follows: The function is the same as the structure of the preparation system 1000 for preparing double emulsified microspheres, and will not be described again here.

According to an embodiment of the present disclosure, with reference to Figures 2 and 4, a detection and sorting system is provided. The detection and sorting system includes a defective microsphere fluid channel 2100 formed by the bifurcation of the microsphere fluid channel 1150 and qualified microspheres. fluid channel 2200, the defective microsphere fluid channel 2100 and The qualified microsphere fluid flow channels 2200 are all arranged on the microfluidic chip 100, that is, the preparation flow channels and detection and sorting flow channels of microspheres are both arranged on the microfluidic chip 100, integrating preparation and detection. In one. The microsphere fluid channel is provided with an asymmetric electrode 2300 on the corresponding microfluidic chip 100 before bifurcation, and the electric field generated by the corresponding electrode on one side of the defective microsphere fluid channel 2100 is greater than that of the qualified microsphere fluid. The electric field generated by the corresponding electrode on one side of the flow channel 2200. The detection and sorting system also includes a signal generator 2500, a detection and sorting control unit 2600 and an optical detection unit 2400. The optical detection unit 2400 is arranged on the corresponding microfluidic chip before the microsphere fluid channel bifurcates. Above 100, the signal generator 2500 is respectively connected to the asymmetric electrode 2300 and the detection and sorting control unit 2600. The detection and sorting control unit 2600 is respectively connected to the optical detection unit 2400 and the signal generator. 2500 is connected.

The process of detecting and sorting using the above detection and sorting system is as follows: before the pre-sorting microsphere fluid is bifurcated, an optical detection unit 2400 (such as a microscope lens) is used to observe the pre-sorting microspheres, such as simultaneously observing fluorescence. and side light scattering imaging for observation, and the optical detection unit 2400 sends the observation results to the detection and sorting control unit 2600. If the microspheres are defective before sorting, for example, when a fluorescence signal is captured and/or a void is detected, When soaking, the detection and sorting control unit 2600 issues an instruction to the signal generator 2500, and the signal generator 2500 turns on the asymmetric electrode 2300 to generate an uneven electric field. The uneven electric field causes the generation of defective microspheres. deviation into the defective microsphere fluid channel 2100; if there are no defects in the microspheres before sorting, the asymmetric electrode 2300 is not connected so that qualified microspheres enter the qualified microsphere fluid channel 2200. Therefore, the detection and sorting system is an automated operation and uses a combination of fluorescence and bright field detection methods. It can not only screen out dead cells or tissue blocks, but also screen out vacuoles, which improves detection efficiency and Accuracy.

Therefore, by combining the above preparation system 1000 with the detection and sorting system, the automated and standardized preparation of highly active VOS can be achieved.

It should be noted that the above-mentioned detection and sorting control unit 2600 is an electronic device, such as a mobile phone or a computer. Preferably, the above-mentioned detection and sorting control unit 2600 and the above-mentioned flow rate control unit may be on the same electronic device.

The total system also includes a heating unit (not shown in the figure) for forming qualified microspheres into a solid state (the oil phase of the dispersion medium is still liquid at this time). One end of the heating unit is connected to the qualified microspheres. The outlets of the fluid flow channels 2200 are connected. The qualified VOS fluid flows out from the outlet of the qualified microsphere fluid channel 2200 and enters the heating unit. Under the heating effect of the heating unit (for example, heated to 25°C), the qualified microspheres form a solid state, and then flow into the distribution distribution together with the oil phase. liquid system.

According to an embodiment of the present disclosure, the hair liquid dispensing system is used to distribute the qualified microsphere fluid sorted by the detection and sorting system into the orifice plate and to distribute the qualified microsphere fluid packed in the orifice plate. Add culture medium to the ball. Referring to Figure 5, the hair liquid dispensing system 3000 includes: a support plate 3100, a slide 3200 is provided on the support plate 3100; a mobile unit, the mobile unit includes a mobile platform 3500, an X-direction slide rail 3400 and a Y-direction slide rail 3400. Slide rail 3300. The Y-direction slide rail 3300 is vertically disposed on the slideway 3200. The Y-direction slide rail 3300 can move along the Y-direction on the slideway 3200. The X-direction slide rail 3400 is vertically disposed. On the Y-direction slide rail 3300, the X-direction slide rail 3400 can move in the X-direction on the Y-direction slide rail 3300, and the mobile platform 3500 is fixed above the X-direction slide rail 3400; holes plate (not shown in Figure 5), the well plate is arranged on the mobile platform 3500, and a plurality of culture wells are provided on the well plate; a microsphere fluid distribution unit, the microsphere fluid distribution unit includes Microsphere fluid distribution tube 3800, first connecting rod 3700 and first vertical rod 3600. The first vertical rod 3600 is provided on the support plate 3100. The microsphere fluid distribution tube 3800 is supported by the first connecting rod 3700. Above the culture well, the microsphere fluid distribution tube 3800 includes a distribution head 3810. The distribution head 3810 is disposed at the lower end of the microsphere fluid distribution tube 3800. The upper end of the microsphere fluid distribution tube 3800 is connected to the upper end of the microsphere fluid distribution tube 3800. The other end of the heating unit is connected to a culture liquid pipetting unit, which includes the culture liquid pipette 3920, a second connecting rod 3910, and a second vertical rod 3900. The second vertical rod 3900 is arranged on the support plate 3100. The culture fluid pipette 3920 is supported above the culture hole through the second connecting rod 3910. The culture fluid pipette 3920 is provided with multiple pipetting tips at the bottom. 3921. Further, the hair liquid distribution system further includes: a hair liquid distribution control unit, which is respectively connected to the moving unit, the microsphere fluid distribution unit and the culture liquid pipetting unit, so The distribution liquid control unit respectively controls the moving path of the mobile unit, the number of VOS distributed in each culture well, and the amount of culture liquid added in each culture well.

Specifically, the heated and solidified VOS fluid is connected to the microsphere fluid distribution tube 3800 through a PTFE tube, and the heated and solidified VOS fluid is distributed through the distribution head to each culture well of the well plate according to actual needs. This distribution method can avoid Due to the vibration caused by the movement of the distribution head, the VOS in the microsphere fluid distribution tube 3800 may be bonded in multiple ways, ensuring the stability of VOS distribution. The microsphere fluid distribution tube 3800 is vertically downward and has a fixed position, thereby reducing the unstable influence of VOS distribution due to movement. During the distribution process, the orifice plate is placed above the mobile platform 3500, and by moving the mobile platform 3500 according to a preset route, the cured VOS flows out into each hole of the orifice plate through the distribution head. The number of VOS in each hole can be adjusted according to actual needs.

Specifically, the mobile unit can move according to a certain path according to actual needs, wherein the Y-direction slide rail 3300 can move along the Y-direction on the slide, thereby controlling the movement of the mobile platform 3500 in the Y-direction, and the X-direction slide rail 3300 can move along the Y-direction. The rail 3400 can move along the X direction on the Y-direction slide rail 3300, thereby controlling the movement of the mobile platform 3500 in the X direction. This ensures that VOS is allocated to each required hole, and there is no need to change the plate midway. operate. The moving path of the well plate can be set by the distribution liquid control unit, and can be personalized according to the well plate and test requirements to ensure that the distributed VOS can meet the needs of later experiments. Figure 10 is a schematic diagram of one of the paths.

After the well plate completes the distribution of VOS, the culture liquid pipetting unit can be controlled to move to the top of the well plate to add culture liquid. The bottom of the culture medium pipette 3920 can be connected with pipette tips of different specifications and quantities. Move the culture medium pipette 3920 to the top of the liquid storage tank. The pipette head will draw a certain amount of liquid from the culture liquid storage tank through negative pressure. After adding culture medium, move to the top of the well plate and release the negative pressure to allow the culture medium to flow out into the well plate. The amount of liquid added and the number and specifications of the pipette tips connected to the bottom of the culture medium pipette 3920 can be matched according to the specifications of the well plate used. Automatic addition of liquid is performed immediately after VOS distribution to avoid damage to cell activity caused by Matrigel dehydration. Thus, the hair liquid dispensing system operates automatically.

Further, the first connecting rod 3700 is configured to rotate circumferentially with the first vertical rod 3600 as an axis. After the microsphere fluid distribution tube 3800 completes distributing the heated and formed microsphere fluid, the first vertical rod can be rotated. 3600 is a shaft that rotates the first connecting rod 3700 circumferentially to move the microsphere fluid distribution tube 3800 away from the top of the orifice plate to avoid affecting the subsequent operation of the culture fluid pipetting unit. Furthermore, the first vertical rod 3600 is configured to be telescopic up and down, thereby enabling the movement of the first vertical rod 3600 on the Z-axis, thereby realizing the movement of the microsphere fluid distribution tube 3800 on the Z-axis.

Further, the second vertical rod 3900 is configured to be telescopic up and down, which can realize the movement of the second vertical rod 3900 on the Z-axis, thereby realizing the movement of the culture medium pipette 3920 on the Z-axis; in addition, when the microsphere When the fluid distribution unit distributes the heated and formed microsphere fluid, the second vertical rod 3900 is raised, thereby driving the culture solution pipette 3920 and the second connecting rod to rise to avoid affecting the distribution operation of the microsphere fluid distribution unit; when After the distribution of the microsphere fluid distribution unit is completed, the second vertical rod 3900 is lowered, thereby driving the culture solution pipette 3920 and the second connecting rod to lower, so that the culture solution pipette 3920 is just above the culture well, and the dispensing Add culture fluid to the qualified microspheres in the well plate.

According to the fully automated in vitro organ microsphere preparation, sorting and distribution culture system described in the embodiments of the present disclosure, the overall system solves the problems of low throughput, non-uniform and uncontrollable size, poor reproducibility and relatively large size in traditional preparation methods. It is difficult to achieve automated preparation and distribution, which in turn limits its application in basic research and clinical diagnosis. This disclosure uses a microfluidic droplet generation and gel solidification system to prepare VOS with uniform and controllable sizes, and realizes its automated preparation; at the same time, a detection and sorting function is integrated on the chip to detect VOS with a lot of dead cells or no cells. Screening is ensured to ensure that all the VOS obtained are highly active, achieving effective sorting of the target VOS; further utilizing the microsphere fluid distribution unit and the controllable motion platform to achieve automatic distribution of VOS, solving the low efficiency and low efficiency in manual spotting. Problems such as poor accuracy and great influence by the operator; finally, the culture medium pipetting unit is used to realize automated medium or drug injection to reduce the risk of contamination during the operation. The modular system constructed in this disclosure can realize the automated preparation and culture of VOS, is simple to operate and pollution-free, provides a reproducible in vitro biological model for drug screening and efficacy evaluation, and has good application prospects and promotion value. At the same time, the above-mentioned system can greatly save raw materials and dosage of drugs, greatly reduce costs and labor, and improve the efficiency of drug screening and new drug research and development, which has great economic benefits.

S100: Prepare in vitro organ microspheres in the microsphere preparation flow channel. The in vitro organ microspheres are wrapped in the oil phase to form pre-sorting microsphere fluid, which specifically includes the following steps:

S110: Pass the cell fluid of the shell structure into the cell fluid channel 1120 of the shell structure, pass the cell fluid of the core structure into the cell fluid channel 1130 of the core structure, and pass the oil phase into the oil phase channel 1110;

S120: Combine the cellular fluid of the shell structure and the cellular fluid of the core structure to form a merged fluid, and distribute the cellular fluid of the shell structure outside the cellular fluid of the core structure;

S130: Combine the combined fluid and the oil phase to form core-shell structure extracorporeal organ microspheres, and wrap the core-shell structure extracorporeal organ microspheres in the oil phase.

S200: Before the pre-sorting microsphere fluid is bifurcated, an optical detection unit is used to observe the pre-sorting microspheres. The optical detection unit sends the observation results to the detection and sorting control unit. If the pre-sorting microsphere fluid is bifurcated, If there are defects in the microspheres, the detection and sorting control unit will issue instructions to the signal generator, and the signal generator will turn on the asymmetric electrode to generate an uneven electric field, and the uneven electric field will cause the defects to The microspheres deviate and enter the defective microsphere fluid channel 2100; if there are no defects in the microspheres before sorting, the asymmetric electrode is not connected so that qualified microspheres enter the qualified microsphere fluid channel 2200.

Specifically, after VOS flows to the sorting area, a microscope lens can be used to observe fluorescence and side light scattering imaging at the same time, and the computer control system analyzes the observation results. When the fluorescence signal is captured, an instruction is issued to the signal generator. The signal generator connects the electrodes integrated on the chip to generate a non-uniform electric field, causing the VOS that generates the signal to deviate into the defective microsphere fluid channel 2100 for separation. At the same time, side scatter detection is performed to detect the particle size of VOS. When cavitation is detected, the signal generator is triggered to separate the VOS. The principle is the same as above. Dual-channel detection can simultaneously separate low-activity VOS and vacuoles, ensuring that all VOS obtained are highly active. In addition, the detection method provided by the disclosed method is to detect non-target VOS, that is, to detect low activity and vacuoles. In view of the fact that the target VOS accounts for a large proportion of the generated VOS, and the fluorescent dye can easily affect the cell activity. Therefore, this method chooses to stain non-target VOS, which avoids the damage caused by labeling to the target, has a protective effect on high-activity VOS, and has a higher separation efficiency. The optical detection unit can simultaneously detect fluorescence and side scattering of VOS, and apply an asymmetric electric field through electrodes integrated on the chip to shift the VOS to achieve separation.

The VOS that has passed the test will continue to flow out from the outlet through the flow channel in the chip, and then pass through the 25°C heating plate for solidification treatment before entering the liquid distribution system.

S300: Use a liquid dispensing system to distribute the qualified microsphere fluid in the well plate, and add culture fluid to the qualified microspheres distributed in the well plate.

Specifically, the heated and solidified VOS fluid is connected to the microsphere fluid distribution tube through a PTFE tube, and the heated and solidified VOS fluid is distributed through the distribution head to each culture well of the well plate according to actual needs. During the distribution process, the orifice plate is placed above the moving platform, and the solidified VOS flows out through the distribution head into each hole of the orifice plate by making the moving platform move along a preset route. After the well plate completes the distribution of VOS, the culture liquid pipetting unit can be controlled to move to the top of the well plate to add culture liquid. The bottom of the culture medium pipette can be connected to pipette tips of different specifications and quantities. Move the culture medium pipette to the top of the liquid storage tank. The pipette head will draw a certain amount of culture medium from the culture liquid storage tank through negative pressure. Then, move to the top of the well plate and release the negative pressure to allow the culture medium to flow out into the well plate. The amount of liquid added and the number and specifications of the pipette tips connected to the bottom of the culture medium pipette can be matched according to the specifications of the well plate used. Automatic addition of liquid is performed immediately after VOS distribution to avoid damage to cell activity caused by Matrigel dehydration.

If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

Example 1

The preparation system shown in Figure 3 and Figure 7 is used to prepare microspheres with a single emulsion structure. The method is as follows:

Add the dead cell fluorescent dye to the intestinal tumor cells from the patient, mix them evenly and wait for the staining reaction. After the reaction, mix the stained cells with matrigel evenly and add it to the storage device. Add the oil phase fluorine oil HFE7000 to the oil phase. In the storage device, connect the chip. After the sample addition is completed, pressurize the liquid storage tank and inject the sample, turn on the diaphragm pump, and adjust the flow controller so that the pressure of the cell liquid is 15 mbar and the pressure of the oil phase is 25 mbar, thereby preparing microspheres with a single emulsion structure.

The detection and sorting system described in Figures 2 and 4 is used to screen the preparation emulsified microspheres prepared above, and the microspheres with red fluorescence or vacuoles are screened out and flow into the defective microsphere fluid channel, and the active The higher microspheres flow into the qualified microsphere fluid channel, and the sorted VOS is stained for dead and alive cells again. The fluorescence display is shown in Figure 11. The fluorescence picture is all green (that is, the white points or gray points in the picture are green Fluorescence), there is no red color, indicating that the activity of VOS after sorting is higher.

The qualified VOS fluid flows out from the outlet of the qualified microsphere fluid channel and enters the heating unit. Under the heating effect of the heating unit (25°C), the qualified microspheres form a solid state, and then flow into the hair liquid distribution system together with the oil phase. The detection and sorting system described in Figure 5 is used to automatically distribute the qualified microsphere fluid to each culture well in the 384-well plate, as shown in Figure 12. Then use a culture medium pipetting unit to add culture medium to each culture well, and finally put it into a cell culture incubator for culture. VOS was photographed and observed every day, as shown in Figure 13. It can be seen from Figure 13 that the size of each microsphere is uniform. At the same time, it was compared with the VOS prepared and cultured by traditional manual methods. The results are shown in Figure 14. Draw a growth curve, as shown in Figure 15. It can be seen from Figures 14 and 15 that after 5 days of culture, the volume of organ microspheres can be observed to be significantly larger in the VOS prepared and cultured in this embodiment, which is comparable to the VOS prepared and cultured by traditional manual methods. faster than the growth rate.

Example 2

Double emulsion core-shell structure microspheres are prepared using the preparation system shown in Figure 2 and Figure 3. The method is as follows:

The intestinal tumor cells in the nuclear layer from the patient were transfected with a virus carrying the GFP reporter gene, and then mixed with matrigel evenly and then added to the nuclear layer cell fluid storage device. The human umbilical vein endothelial cells in the shell layer were transfected with a virus carrying the RFP reporter gene. Then mix it evenly with collagen and add it to the shell cell liquid storage device. Add the oil phase fluorine oil HFE7000 to the oil phase storage device and connect the chip. After the sample addition is completed, pressurize the liquid storage tank and inject the sample, turn on the diaphragm pump, and adjust the flow controller so that the pressure of the nuclear layer cell fluid is 15 mbar, the pressure of the shell layer cell fluid is 15 mbar, and the oil phase pressure is 25 mbar, thus preparing Microspheres with double emulsified core-shell structure were obtained.

The above-mentioned double emulsion core-shell structure microspheres were distributed into each culture well in a 384-well plate. Then use a culture medium pipetting unit to add culture medium to each culture well, and finally put it into a cell culture incubator for culture.

Then observe through a fluorescence microscope and collect images. The results are shown in Figure 16. It can be seen from Figure 16 that the cells in the middle nuclear layer are intestinal tumor cells from the patient. The fluorescent staining is green, and the outer shell is human umbilical vein. The vascular structure formed by endothelial cells is fluorescently stained red, with red distributed in the outer layer (as shown in B) and green in the middle layer (as shown in A). Therefore, it can be proved that the VOS prepared by this method has a core-shell structure. At the same time, it can also be seen from Figure 16 that the VOS has a vascular structure (as shown in C).

In the description of this specification, reference to the terms "one embodiment," "some embodiments," "an example," "specific examples," or "some examples" or the like means that specific features are described in connection with the embodiment or example. , structures, materials, or features are included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

Although the embodiments of the present disclosure have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present disclosure. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present disclosure. The embodiments are subject to changes, modifications, substitutions and variations.

Claims

A fully automated total system for in vitro organ microsphere preparation, sorting and distribution culture, including:

Preparation system, the preparation system is used to prepare in vitro organ microspheres, the preparation system includes a microsphere preparation flow channel provided on a microfluidic chip, the microsphere preparation flow channel includes a microsphere fluid flow channel;

Detection and sorting system, the detection and sorting system includes a defective microsphere fluid channel and a qualified microsphere fluid channel formed by the bifurcation of the microsphere fluid channel, the defective microsphere fluid channel and the qualified microsphere fluid channel The microsphere fluid channels are all arranged on the microfluidic chip. The microsphere fluid channels are provided with asymmetric electrodes on the corresponding microfluidic chip before bifurcation, and one side of the defective microsphere fluid channel The electric field generated by the corresponding electrode is greater than the electric field generated by the corresponding electrode on one side of the qualified microsphere fluid channel. The detection and sorting system also includes a signal generator, a detection and sorting control unit and an optical detection unit. The optical detection unit The unit is arranged above the microfluidic chip corresponding to the microsphere fluid channel before bifurcation, and the signal generator is connected to the asymmetric electrode and the detection and sorting control unit respectively, and the detection and sorting control unit The control unit is connected to the optical detection unit and the signal generator respectively;

Dispensing hair liquid system, the hair liquid distributing system is used for distributing the qualified microsphere fluid sorted by the detection and sorting system into the orifice plate and adding the qualified microsphere fluid packed in the orifice plate. Culture fluid.
The total system according to claim 1, wherein the microsphere preparation flow channel includes: a cell fluid flow channel with a shell structure, a cell fluid flow channel with a core structure, and an oil phase flow channel, and the cell fluid flow channel with the shell structure The channel and the cell fluid flow channel of the core structure merge to form a merged flow channel, and the merged flow channel merges with the oil phase flow channel to form the microsphere fluid flow channel. In the cell fluid flow channel of the shell structure, The confluence of the channel and the cell fluid flow channel of the core structure. The cell fluid flow channel of the shell structure is arranged on both sides of the cell fluid flow channel of the core structure. Between the confluence channel and the oil phase At the confluence of flow channels, the oil phase flow channels are arranged on both sides of the merged flow channels.
The total system of claim 2, wherein the preparation system further includes:

A first liquid storage device, the liquid outlet of the first liquid storage device is connected to the inlet of the cell fluid flow channel of the shell structure, and the first liquid storage device is used to store the cell fluid of the shell structure;

a second liquid storage device, the liquid outlet of the second liquid storage device is connected to the inlet of the cell fluid flow channel of the nuclear structure, and the second liquid storage device is used to store the cell fluid of the nuclear structure;

A third liquid storage device, the liquid outlet of the third liquid storage device is connected to the inlet of the oil phase flow channel, and the third liquid storage device is used to store the oil phase;

A pressure controller and a diaphragm pump. The pressure controller is connected to the first liquid storage device, the second liquid storage device and the third liquid storage device respectively. The diaphragm pump is connected to the pressure controller. .
The overall system according to claim 3, wherein the preparation system further includes: a flow rate control unit, the flow rate control unit is connected to the pressure controller.
The total system according to claim 3 or 4, wherein the preparation system further includes:

A refrigeration module, the refrigeration module is used to maintain the temperatures of the microfluidic chip, the first liquid storage device, the second liquid storage device and the third liquid storage device at a certain set temperature.
The total system according to claim 3 or 4, wherein the preparation system further includes:

The heat preservation unit, the microfluidic chip, the first liquid storage device, the second liquid storage device and the third liquid storage device are all arranged in the heat preservation unit.
The total system according to claim 2, wherein the width of the cell fluid channel of the shell structure is 100-150 μm, and the flow rate of the cell fluid of the shell structure in the cell fluid channel of the shell structure is 0.5-5 μL/ min.
The total system according to claim 2, wherein the width of the cellular fluid channel of the nuclear structure is 100-150 μm, and the flow rate of the cellular fluid of the nuclear structure in the cellular fluid channel of the nuclear structure is 0.5-5 μL/ min.
The total system according to claim 2, wherein the width of the oil phase flow channel is 75-300 μm, and the flow rate of the oil phase in the oil phase flow channel is 1-20 μL/min.
The total system of claim 2, wherein the cell fluid of the shell structure within the cell fluid flow channel of the shell structure includes cells of the shell structure and a first hydrogel, and the core within the cell fluid flow channel of the core structure The cellular fluid of the structure includes cells of a core structure and a second hydrogel, the cells of the shell structure are different from the cells of the core structure, the first hydrogel and the second hydrogel are different, the The cells of the shell structure are selected from at least one of umbilical cord vein endothelial cells and immune cells, the cells of the nuclear structure are selected from at least one of tumor cells and stem cells, and the first hydrogel is selected from fibrinogen, At least one of collagen and alginate, the second hydrogel is selected from At least one of Matrigel, fibrinogen and collagen.
The total system according to claim 1, wherein the microsphere preparation flow channel includes: a cell fluid flow channel and an oil phase flow channel, and the cell fluid flow channel and the oil phase flow channel merge to form a microsphere fluid flow channel.
The total system according to claim 1, wherein the total system further includes a heating unit for forming qualified microspheres into a solid state, and one end of the heating unit is connected to the outlet of the qualified microsphere fluid flow channel;

The hair liquid dispensing system includes:

A support plate, the support plate is provided with a slideway;

Mobile unit, the mobile unit includes a mobile platform, an X-direction slide rail and a Y-direction slide rail. The Y-direction slide rail is vertically arranged on the slide rail. The Y-direction slide rail can move along the Y-direction on the slide rail. move, the X-direction slide rail is vertically arranged on the Y-direction slide rail, the X-direction slide rail can move along the X-direction on the Y-direction slide rail, and the mobile platform is fixed on the X-direction slide rail. above the rail;

A well plate, the well plate is arranged on the mobile platform, and the well plate is provided with a plurality of culture wells;

Microsphere fluid distribution unit, the microsphere fluid distribution unit includes a microsphere fluid distribution tube, a first connecting rod and a first vertical rod, the first vertical rod is arranged on the support plate, the microsphere fluid distribution unit The pipe is supported above the culture hole through a first connecting rod. The microsphere fluid distribution tube includes a distribution head. The distribution head is provided at the lower end of the microsphere fluid distribution tube. The microsphere fluid distribution tube has The upper end is connected to the other end of the heating unit;

Culture liquid pipetting unit, the culture liquid pipetting unit includes the culture liquid pipette, a second connecting rod and a second vertical rod, the second vertical rod is arranged on the support plate, the culture liquid pipetting unit The pipette is supported above the culture hole through a second connecting rod, and a plurality of pipette tips are provided at the bottom of the culture liquid pipette.
The total system according to claim 12, wherein the hair liquid dispensing system further includes: a hair liquid distributing control unit, the hair liquid distributing control unit is connected to the moving unit, the microsphere fluid dispensing unit and the hair liquid distributing unit respectively. The culture medium pipetting unit is connected.
The total system according to claim 12, wherein the first connecting rod is configured to rotate circumferentially about the axis of the first vertical rod, the first vertical rod is configured to be telescopic up and down, and the second vertical rod is configured to be telescopic up and down. The pole is configured to telescope up and down.
A method for preparing, sorting, and distributing culture of organ microspheres in vitro using the total system according to any one of claims 1 to 14, which includes:

(1) In vitro organ microspheres are prepared in the microsphere preparation flow channel, and the in vitro organ microspheres are wrapped in an oil phase to form a pre-sorting microsphere fluid;

(2) Before the pre-sorting microsphere fluid bifurcates, an optical detection unit is used to observe the pre-sorting microspheres, and the optical detection unit sends the observation results to the detection and sorting control unit. If the sorting If there is a defect in the front microsphere, the detection and sorting control unit sends an instruction to the signal generator, and the signal generator turns on the asymmetric electrode to generate an uneven electric field. The uneven electric field causes the defective microsphere to deviate. Enter the defective microsphere fluid flow channel;

If there are no defects in the microspheres before sorting, the asymmetric electrode is not connected so that qualified microspheres enter the qualified microsphere fluid flow channel;

(3) Use a liquid dispensing system to distribute the qualified microsphere fluid in the well plate, and add culture fluid to the qualified microspheres distributed in the well plate.