CN117187060A - High-flux 3D microsphere synthesis, manufacturing and distribution system and method - Google Patents

Abstract

The application discloses a system and a method for synthesizing, manufacturing and distributing high-flux 3D microspheres, comprising the following steps: the pressure output module is used for providing a power source; the microfluidic shearing module is used for mixing cells and biological glue into cell suspension by using a power source and shearing and forming to form 3D microspheres; the visual identification and quality inspection module is used for recording and marking the cell micelle number of each 3D microsphere by utilizing an image identification technology, and judging the qualification state of each 3D microsphere according to the cell micelle number; the three-axis printing distribution module is used for carrying out printing distribution on each 3D microsphere according to the qualified state of the microsphere; and the automatic liquid adding module is used for adding culture solution to the printed 3D microspheres. The application realizes the construction of in vitro microenvironment; meanwhile, the pressure output module, the microfluidic shearing module, the visual identification and quality inspection module, the triaxial printing distribution module and the automatic liquid adding module are combined, so that the automation of in-vitro model construction is further realized.

Description

High-flux 3D microsphere synthesis, manufacturing and distribution system and method

Technical Field

The application relates to the technical field of biological manufacturing, in particular to a system and a method for synthesizing, manufacturing and distributing high-flux 3D microspheres.

Background

At present, the traditional in-vitro biological 3D model manufacturing and culturing technology mainly relies on manual work, and hydrogel suspension mixed with cells is planted in a culturing pore plate in a manual dispensing mode. However, the traditional in-vitro 3D biological model manufacturing method has high labor cost and large influence of human factors, and the product cannot realize standardized manufacturing. Meanwhile, the size of the manual manufacturing scale is limited to about 2.5 mu l, and the manufacturing of smaller scale cannot be achieved. Taking clinical drug screening as an example, in order to effectively distinguish the sensitivity of tumor cells to different drugs, it is often necessary to set concentration gradients and multiple wells for a single drug tested, thus requiring a large number of drug detection units. The enormous number of requirements presents a greater challenge to manual dispensing manufacturing.

The international in-vitro biological 3D model culture technology is rapid in development, the research range based on the 3D biological model is larger and larger, and the method has wide application in the aspects of mechanism research, disease modeling and drug screening, and mass, integrated and standardized model manufacturing becomes a bottleneck which needs to be broken through in the industry.

The automatic liquid-transferring workstation can effectively realize automatic liquid distribution. However, many extracellular matrices, such as: matrigel, collagen and the like are very sensitive to temperature, and a huge refrigeration system is needed to be carried in order to construct a low-temperature operation environment, so that the matrigel, collagen and the like cannot be effectively popularized in small and medium-sized laboratories. In addition, an automatic pipetting workstation usually has a dead volume of more than tens of microliters, and a pipetting gun head cannot sample trace, so that preparation and distribution cannot be carried out on trace samples such as puncture, biopsy and the like, and the application range of the pipetting workstation is greatly limited.

The preparation of 3D microspheres based on the microfluidic technology becomes an important means for constructing an in-vitro biological 3D model, and the prepared 3D microspheres have the characteristics of small volume and large specific surface area, can effectively promote sample growth, and shortens culture time. However, the significant heterogeneity of primary tumor samples, how to maintain effective inter-well consistency, presents a significant challenge to the 3D microsphere structure of microfluidic preparation. The presently disclosed research results are mainly achieved by increasing the cell density or increasing the number of microspheres in the well. The former sacrifices good light transmittance of the 3D microspheres under the condition of improving the density, and sample analysis based on images cannot be realized; the latter overcomes the difference between holes by increasing the number of microspheres in the holes, but in the actual operation process, the uniformity of the number between holes with high precision cannot be realized, thereby limiting the application of the method in accurate printing of samples and accurate medicine screening.

Disclosure of Invention

The application aims to provide a high-flux 3D microsphere synthesis, manufacturing and distribution system and method, which realize in-vitro microenvironment construction of A-B type, wrapped type and A-B wrapped type solid microspheres. Meanwhile, the micro-manufacturing of the 3D microspheres can be effectively realized, the difference between holes is overcome, and the automation and standardization of in-vitro model construction are further realized.

The technical scheme adopted by the application is as follows:

a high-throughput 3D microsphere synthesis, manufacturing, and dispensing system comprising:

the pressure output module is used for providing a power source;

the microfluidic shearing module is used for mixing cells and biological glue into cell suspension by using a power source and shearing and forming to form 3D microspheres; the microfluidic shearing module comprises a refrigerating unit, wherein a carrier unit and a sample adding chip unit are arranged in the refrigerating unit, the sample adding chip unit is arranged above the carrier unit, and the carrier unit and the sample adding chip unit are connected with the pressure output module through pipelines;

the visual identification and quality inspection module is used for recording and marking the cell micelle number of each 3D microsphere by utilizing an image identification technology, and judging the qualification state of each 3D microsphere according to the cell micelle number;

the three-axis printing distribution module is used for carrying out printing distribution on each 3D microsphere according to the qualified state of the microsphere;

and the automatic liquid adding module is used for adding culture solution to the printed 3D microspheres.

Preferably, the pressure output module comprises a pressure pump and a pressure valve, wherein the output end of the pressure pump is connected with the microfluidic shearing module through a pipeline and provides a power source for the microfluidic shearing module, and the pressure valve is arranged on the pressure pump and is used for adjusting the size of the power source.

Preferably, the carrier unit specifically includes:

the carrier is provided with a placing groove and a chip groove;

the oil storage device comprises an oil storage pipe and an oil phase connecting cover, wherein the top end of the oil storage pipe is in threaded connection with the oil phase connecting cover, and the oil storage pipe is in fit connection with the placing groove; the oil phase connecting cover is also provided with an air pipe connecting hole and an oil phase output hole, the air pipe connecting hole is connected with the pressure output module through a pipeline, and the oil phase output hole is connected with the sample adding chip unit through a pipeline;

the chip fixing device comprises a chip fixer, wherein the chip fixer is in fit connection with the chip groove, and the chip fixer is used for fixing the sample adding chip unit in the chip groove through a fixing piece.

Preferably, the sample adding chip unit specifically includes:

the micro-channel shearing layer is provided with an oil phase channel and a micro-channel; the oil phase flow channel and the micro flow channel are combined to form a combined flow channel, and the other end of the combined flow channel extends out of the micro flow channel shear layer;

the chip packaging layer is provided with a sample adding conical tube, the sample adding conical tube and the chip packaging layer are integrally formed, and the chip packaging layer is also provided with an oil phase input port; the chip packaging layer is stacked above the micro-channel shear layer, and the oil phase input port is communicated with the oil phase channel; the top of the sample adding conical tube is connected with a connector, the connector is externally connected with the pressure output module through a pipeline, and the bottom of the sample adding conical tube is communicated with the micro-channel.

Preferably, the visual identification and quality inspection module specifically includes:

the high-speed camera is used for capturing and photographing the 3D microspheres and transmitting photographing information to the image analysis system;

and the image analysis system is used for recording and acquiring the cell micelle number of each 3D microsphere in the photographing information by utilizing an image recognition technology, and judging the qualification state of each 3D microsphere according to the cell micelle number.

Preferably, the triaxial print distribution module specifically includes:

a three-axis printing platform;

the printing spray head is arranged at the bottom end of the triaxial printing platform, is connected with the microfluidic shearing module through a pipeline and is used for printing the 3D microspheres manufactured by the microfluidic shearing module on the pore plate;

the liquid adding spray head is arranged at the bottom end of the triaxial printing platform and is close to the printing spray head, and an inlet of the liquid adding spray head is connected with the automatic liquid adding module through a pipeline and is used for adding liquid;

the hue photosensitive camera set up in triaxial print platform, hue photosensitive camera's camera lens aim at print the liquid outlet of shower nozzle for hue discernment.

Preferably, the automatic liquid adding module specifically includes:

a plurality of liquid storage pipes for loading different liquids;

the vibration carrier is used for carrying and vibrating the liquid storage tube so as to uniformly mix the liquid in the liquid storage tube;

the liquid pumps are used for connecting the liquid storage pipes and carrying out liquid pumping;

and the fluid switching valve is used for being connected with the liquid pump so as to switch the liquid to realize intelligent liquid separation.

The application also provides a method for synthesizing, manufacturing and distributing the high-flux 3D microsphere, which at least comprises the following steps:

step S1: the pressure output module is respectively connected with the oil storage pipe and the sample loading conical pipe through pipelines, and the refrigerating unit is started to control the temperature within a preset temperature range;

step S2: transferring the water phase mixed with the cells into a sample adding conical tube, and regulating the pressure output values of the water phase and the oil phase by using a pressure output module;

step S3: providing a power source through the pressure output module, pushing the oil phase and the water phase into the sample adding chip unit to form 3D microspheres, and enabling the 3D microspheres to enter the triaxial printing distribution module through a pipeline;

step S4: the 3D microspheres pass through a light source in the visual identification and quality inspection module, are captured and photographed by a high-speed camera, and transmit photographing information to an image analysis system for identification and analysis, and the qualification state of each 3D microsphere is judged according to preset conditions;

step S5: the hue photosensitive cameras in the triaxial printing distribution module identify sample hues of liquid outlet of the printing spray head, and intelligent distribution printing is carried out according to a judgment result of whether the sample hues are in a preset hue interval or not;

step S6: different liquids are loaded in different liquid storage tubes in the automatic liquid adding module, the liquid storage tubes are installed in the vibration carrier to vibrate, each liquid storage tube is connected with a corresponding liquid pumping pump, the other end of each liquid pumping pump is connected with a fluid switching valve, and liquid in the liquid storage tubes is added to the pore plate through the liquid adding spray head by adjusting the fluid switching valve.

As a preferred alternative to this,

in the step S2, the pressure output value of the water phase is 10-80mbar, and the pressure output value of the oil phase is 10-300mbar;

in the step S6, the time interval between the dispensing and printing of the 3D microspheres and the liquid adding of the pore plate is 5-15S.

Preferably, the step S4 specifically includes the following substeps:

step S41: presetting hue parameters of the 3D microspheres, and capturing and photographing the 3D microspheres by a high-speed camera when the 3D microspheres pass through a light source in a visual identification and quality inspection module;

step S42: transmitting the photographing information to an image analysis system, obtaining the cell micelle number of the 3D microspheres, and marking;

step S43: obtaining the size and the number of the cell clusters according to the number of the cell clusters of the 3D microspheres;

step S44: and dividing the 3D microspheres into qualified and unqualified according to whether the size and the number of the cell clusters meet preset conditions.

The beneficial effects of the application are as follows:

1. the application combines the micro-fluidic biological manufacturing technology, can be suitable for manufacturing and printing 5 mu l and above samples, and the minimum volume of a single 3D microsphere can reach 0.02 mu l, so that the application can be compatible with micro-scale sample modeling, and effectively solves the in-vitro modeling problem of puncture/biopsy samples.

2. The application combines with visual recognition technology, can highly adapt to sample printing, and improves flexibility of organoid printing.

3. According to the application, based on image analysis and evaluation of single printed microspheres, quality inspection is carried out on the prepared 3D microspheres through images, and an allocation mode is optimized according to requirements, so that the difference between holes can be reduced, initial culture data in single sample holes can be effectively quantized, original reference data can be conveniently provided for subsequent image analysis and organoid growth, and the analysis scale is accurate from single holes to single organoids.

4. The microfluidic shearing module designed by the application can synthesize a plurality of microsphere models containing different matrixes and different cell types based on a microfluidic technology, and is widely applicable to the construction of complex in-vitro models.

5. The automatic liquid adding module designed by the application is compatible with the addition of various liquids such as cell suspension, tissue suspension cytokines, medicines and the like, and is beneficial to constructing an in-vitro co-culture model or an in-vitro microenvironment in a high-throughput manner; prevent the organoid from losing water, effectively ensure the activity of a single sample in the long-term and high-flux printing process.

Drawings

FIG. 1 is a schematic diagram of a high throughput 3D microsphere synthesis, manufacturing and dispensing system according to the present application;

FIG. 2 is a schematic view of a carrier unit according to the present application;

FIG. 3 is a schematic diagram of a sample application chip unit according to the present application;

FIG. 4 is a schematic illustration of the manufacture of A-B microspheres of the present application;

FIG. 5 is a diagram of an example of an A-B microsphere according to the present application;

FIG. 6 is a schematic illustration of the manufacture of coated microspheres according to the present application;

FIG. 7 is an exemplary view of a coated microsphere of the present application;

FIG. 8 is a schematic illustration of the manufacture of A-B coated microspheres of the present application;

FIG. 9 is an overhead view of a print zone of the present application;

FIG. 10 is a diagram illustrating analysis of a visual recognition and quality control module according to the present application;

FIG. 11 is a sample identification chart of a hue photosensitive camera of the present application;

FIG. 12 is a schematic view of an automatic liquid feeding module according to the present application;

FIG. 13 is a diagram of an example liquid-based culture microenvironment in accordance with the present application;

FIG. 14 is a flow chart of a method of synthesizing, manufacturing and dispensing high throughput 3D microspheres according to the present application;

FIG. 15 is a flow chart of the visual recognition and quality control module according to the present application;

FIG. 16 is a graph comparing the morphology of droplet organoids according to the application with that of hand organoids.

Description of the reference numerals

1-refrigerating unit, 2-carrier unit, 21-carrier, 211-standing groove, 212-chip groove, 22-oil storage device, 221-oil storage pipe, 222-oil phase connecting cover, 2221-air pipe connecting hole, 2222-oil phase output hole, 23-chip fixing device, 231-chip fixer, 232-fixing piece, 3-sample adding chip unit, 31-micro-channel shearing layer, 311-oil phase flow channel, 312-micro-channel, 313-converging flow channel, 32-chip packaging layer, 321-sample adding conical pipe, 322-oil phase input port, 323-connector, 4-high speed camera, 5-image analysis system, 6-triaxial print platform, 7-print nozzle, 8-liquid adding nozzle, 9-hue photosensitive camera, 10-liquid storage pipe, 20-vibration carrier, 30-liquid pump and 40-fluid switching valve.

Detailed Description

The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the application, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Referring to fig. 1, a high-throughput 3D microsphere synthesis, manufacturing, and dispensing system comprising:

the pressure output module is used for providing a power source;

the pressure output module comprises a pressure pump and a pressure valve, wherein the output end of the pressure pump is connected with the microfluidic shearing module through a pipeline and provides a power source for the microfluidic shearing module, and the pressure valve is arranged on the pressure pump and is used for adjusting the size of the power source.

The microfluidic shearing module is used for mixing cells and biological glue into cell suspension by using a power source and shearing and forming to form 3D microspheres; the microfluidic shearing module comprises a refrigerating unit 1, a carrier unit 2 and a sample adding chip unit 3 are arranged in the refrigerating unit 1, the sample adding chip unit 3 is arranged above the carrier unit 2, and the carrier unit 2 and the sample adding chip unit 3 are connected with the pressure output module through pipelines;

the carrier unit 2 is placed in a low temperature environment to prevent the cell gel, which is the aqueous phase mixed with cells, from solidifying within the sample loading cone 321, preferably at a temperature ranging from 2 to 10 c, preferably 5 c.

Referring to fig. 2, the carrier unit 2 specifically includes:

a carrier 21, wherein a placement groove 211 and a chip groove 212 are arranged on the carrier 21;

the oil storage device 22 comprises an oil storage pipe 221 and an oil phase connection cover 222, wherein the top end of the oil storage pipe 221 is in threaded connection with the oil phase connection cover 222, and the oil storage pipe 221 is in fit connection with the placing groove 211; the oil phase connection cover 222 is further provided with an air pipe connection hole 2221 and an oil phase output hole 2222, the air pipe connection hole 2221 is connected with the pressure output module through a pipeline, and the oil phase output hole 2222 is connected with the sample adding chip unit through a pipeline;

the chip fixing device 23 comprises a chip fixer 231, the chip fixer 231 is in fit connection with the chip slot 212, and the chip fixer 231 fixes the sample adding chip unit 3 in the chip slot 212 through a fixing piece 232.

Referring to fig. 3, the sample loading chip unit 3 specifically includes:

a micro flow channel shear layer 31 on which an oil phase flow channel 311 and a micro flow channel 312 are provided; the oil phase flow channel 311 and the micro flow channel 312 are combined to form a combined flow channel 313, and the other end of the combined flow channel 313 extends out of the micro flow channel shear layer 31;

the chip packaging layer 32 is provided with a sample adding conical tube 321, the sample adding conical tube 321 and the chip packaging layer 32 are integrally formed, and the chip packaging layer 32 is also provided with an oil phase input port 322; the chip packaging layer 32 is stacked above the micro-channel shear layer 31, and the oil phase input port 322 is communicated with the oil phase channel 311; the top of the sample adding conical tube 321 is connected with a connector 323, the connector 323 is externally connected with the pressure output module through a pipeline, and the bottom of the sample adding conical tube 321 is communicated with the micro-channel 312.

The number of the sample addition conical tubes 321 can be multiple, and different substrate suspensions can be loaded, and the sample addition conical tubes 321 can be placed and sheared in different manners to obtain the sample addition conical tubes comprising: a-B type microsphere, a coated microsphere, a-B coated microsphere;

referring to FIG. 4, a schematic diagram of manufacturing an A-B microsphere is shown, wherein the sample loading conical tubes 321 are symmetrically distributed by a converging flow channel 313; as shown in FIG. 5, which is a diagram of an example of A-B type microspheres, the upper right corner cells are immune T cells, the lower left corner cells are tumor cells, and an in vitro tumor-immune model is established based on the A-B type microspheres. .

Referring to fig. 6, for manufacturing a schematic diagram of a coated microsphere, a sample-adding conical tube 321 is first provided, then two sample-adding conical tubes 321 are symmetrically provided with the sample-adding conical tube 321 as a center, and at this time, matrix suspension in the three sample-adding conical tubes 321 is converged and enters the converging flow channel 313 together with oil phase; as shown in fig. 7, an example of a coated microsphere is shown, wherein the central deep black tissue is primary gastric carcinoma organoids, the surrounding spindle cells are fibroblasts, and the model is intended to construct an in vitro tumor-stromal cell model.

Referring to fig. 8, a-B encapsulated microspheres are provided that are configured in accordance with the schematic illustration of the encapsulated microsphere manufacture, and the matrix suspensions in the three loading cone 321 are different, collectively converging and co-entering the converging channel 313 with the oil phase.

The fluorine oil is arranged in the oil storage pipe 221, the water phase mixed with cells, namely cell gel, is arranged in the sample adding conical pipe 321, and the power sources of the oil storage pipe 221 and the sample adding conical pipe 321 come from compressed air, and the sizes of the power sources are controlled through a pressure valve.

The visual identification and quality inspection module is used for recording and marking the cell micelle number of each 3D microsphere by utilizing an image identification technology, and judging the qualification state of each 3D microsphere according to the cell micelle number;

the visual identification and quality inspection module specifically comprises:

the high-speed camera 4 is used for capturing and photographing the 3D microspheres and transmitting photographing information to the image analysis system 5;

and the image analysis system 5 is used for recording and acquiring the cell micelle number of each 3D microsphere in the photographing information by utilizing an image recognition technology, and judging the qualification state of each 3D microsphere according to the cell micelle number.

When the 3D microsphere passes through the pipeline, the 3D microsphere is captured and photographed by the high-speed camera 4, and the picture information is transmitted to the image analysis system 5 for recognition and analysis. Each 3D microsphere passing through the high-speed camera 4 is marked, after passing through the image analysis system 5, the qualified 3D microspheres are printed into the micro-porous plate, and the unqualified 3D microspheres are printed into the unqualified sample collection holes, as shown in FIG. 9; the photographs taken by the high-speed camera 4 are analyzed by the image analysis system 5, and the number of cell clusters is marked, as shown in fig. 10.

The image analysis system 5 marks the 3D microspheres in sequence and classifies them into two categories, pass and fail. Wherein, in the qualified 3D microsphere, the cell micelle number is recorded. Before the initial sample is printed, a user can customize the number range of cell micro-clusters in a single culture micropore, then the image analysis system 5 effectively arranges and combines the marked samples, and transmits a printing instruction to the three-axis printing platform 6 and the hue photosensitive camera 9, the hue photosensitive camera 9 recognizes the printed samples, and the three-axis printing platform 6 is combined to realize intelligent distribution of the samples.

The three-axis printing distribution module is used for carrying out printing distribution on each 3D microsphere according to the qualified state of the microsphere;

the triaxial print distribution module specifically comprises:

a triaxial print platform 6; the printing can be made based on a preset printing program including, but not limited to: single-point, straight, multi-segment, circular arc, rectangular, etc. printing travel paths. Meanwhile, the number of the 3D microspheres printed at a time can be controlled by a program.

The printing spray head 7 is arranged at the bottom end of the triaxial printing platform 6, and the printing spray head 7 is connected with the microfluidic shearing module through a pipeline and is used for printing the 3D microspheres manufactured by the microfluidic shearing module on a pore plate;

the liquid adding spray head 8 is arranged at the bottom end of the triaxial printing platform 6 and is close to the printing spray head 7, and an inlet of the liquid adding spray head 8 is connected with the automatic liquid adding module through a pipeline and is used for realizing liquid adding;

the hue photosensitive camera 9 is arranged on the triaxial printing platform 6, and a lens of the hue photosensitive camera 9 is aligned with a liquid outlet of the printing nozzle 7 and used for hue recognition, as shown in fig. 11, a sample recognition diagram of the hue photosensitive camera 9.

And the automatic liquid adding module is used for adding culture solution to the printed 3D microspheres.

Referring to fig. 12, the automatic liquid adding module specifically includes:

a plurality of liquid storage tubes 10 for loading different liquids; such as culture medium, drug, cytokine, cell suspension, and micro-tissue suspension, etc., for constructing a liquid-based culture microenvironment, as shown in fig. 13, which is an example graph of a liquid-based culture microenvironment in which large cell clusters are tumor cells and dispersed small cells are immune cells, the model is intended to construct a tumor immune microenvironment, and observe the tumor infiltration process of immune cells in vitro.

The vibration carrier 20 is used for carrying and vibrating the liquid storage tube 10 so as to uniformly mix the liquid in the liquid storage tube 10;

a plurality of liquid pumps 30 for connecting the liquid storage tubes 10 and performing liquid pumping;

and the fluid switching valve 40 is used for being connected with the liquid pump 30 to switch the liquid to realize intelligent liquid separation.

i. The automatic liquid adding module is used for constructing an in-vitro microenvironment. When used to allocate as: when the liquid storage tube 10 is used for preparing medicines, cytokines, cell suspensions, micro-tissue suspensions and other suspensions, the vibration carrier 20 connected with the liquid storage tube 10 can vibrate according to a set frequency, the set frequency is recommended to be 50-500rpm, preferably 200rpm, and the vibration is performed to prevent the cells or the micro-tissues in the liquid storage tube 10 from sinking, so that the distribution consistency of samples among holes is ensured. In operation, the liquid pump 30 pumps out the liquid in the liquid storage tube 10, and sends the liquid to the liquid adding spray head 8 through the fluid switching valve 40 to finish liquid separation.

And ii, the automatic liquid adding module is used for automatically separating liquid. After the 3D microspheres are printed on the micro-pore plate, the oil phase can volatilize rapidly, after the oil phase volatilizes, the liquid adding spray head 8 moves to the position where the 3D microspheres are printed, a culture medium is added, and the interval between the 3D microsphere printing and the liquid adding of the culture medium is recommended to be 5s-15s, preferably 10s. The method can effectively prevent the influence of the residual fluorine oil on imaging and the influence on the firmness of the 3D microsphere attaching bottom, and simultaneously prevent the 3D microsphere from losing water, thereby effectively protecting the activity of a sample and the integrity of a culture medium.

Referring to fig. 14, a method for synthesizing, manufacturing and dispensing high-throughput 3D microspheres comprises at least the steps of:

step S1: the pressure output module is respectively connected with an oil storage pipe 221 and a sample adding conical pipe 321 through pipelines, and the refrigerating unit 1 is started to control the temperature within a preset temperature range;

step S2: transferring the water phase mixed with the cells into a sample adding conical tube 321, and regulating the pressure output values of the water phase and the oil phase by using a pressure output module; the pressure output value of the water phase is 10-80mbar, and the pressure output value of the oil phase is 10-300mbar;

step S3: the pressure output module is used for providing a power source, the oil phase and the water phase are pushed into the sample adding chip unit 3 to form 3D microspheres, and the 3D microspheres enter the triaxial printing distribution module through a pipeline;

referring to fig. 15, step S4: the 3D microspheres pass through a light source in the visual identification and quality inspection module, are captured and photographed by the high-speed camera 4, and transmit photographing information to the image analysis system 5 for identification and analysis, and the qualification state of each 3D microsphere is judged according to preset conditions;

step S41: presetting hue parameters of the 3D microspheres, and capturing and photographing the 3D microspheres by the high-speed camera 4 when the 3D microspheres pass through a light source in the visual identification and quality inspection module;

step S42: transmitting the photographing information to an image analysis system 5, obtaining the cell micelle number of the 3D microspheres, and marking;

step S43: obtaining the size and the number of the cell clusters according to the number of the cell clusters of the 3D microspheres; step S44: and dividing the 3D microspheres into qualified and unqualified according to whether the size and the number of the cell clusters meet preset conditions.

The preset conditions are as follows: the cell mass size is more than 20 mu m, the number is more than 3, and the cell mass is adjusted according to actual requirements.

Step S5: the hue photosensitive cameras 9 in the triaxial printing distribution module identify sample hues of liquid outlet of the printing spray head 7, and intelligent distribution printing is carried out according to a judgment result of whether the sample hues are in a preset hue interval;

step S6: different liquids are loaded in different liquid storage tubes 10 in the automatic liquid adding module, the liquid storage tubes 10 are installed in the vibration carrier 20 to vibrate, each liquid storage tube 10 is connected with a corresponding liquid drawing pump 30, the other end of each liquid drawing pump 30 is connected with a fluid switching valve 40, and liquid in the liquid storage tubes 10 is added through the liquid adding spray head 8 by adjusting the fluid switching valve 40. The time interval between the dispensing and printing of the 3D microspheres and the liquid adding of the pore plate is 5-15s.

Referring to fig. 16, a comparison of the growth morphology of the microdroplet organoids and the hand-site organoids prepared by the 3D microsphere technique of the present application shows that the growth of the microdroplet organoids is significantly superior to that of the conventional hand-site organoids with the same cell number. This is mainly because the 3D microsphere technology produces smaller droplets of organoids with a larger specific surface area, which is more conducive to cell growth. Meanwhile, the cell density in the micro-droplet organoids is larger, and paracrine action in the cell growth process further stimulates the organoids to grow.

The working process of the application is described in detail below: the pressure output module is used for introducing compressed gas into the sample loading conical pipe 321 and the oil storage pipe 221 to provide liquid driving force. The added cell and biological glue mixed suspension is sheared, manufactured and molded by a sample adding chip unit 3, and the prepared 3D microsphere is conveyed to a printing spray head 7 through a microtube. And the visual identification and quality inspection module shoots and marks each prepared 3D microsphere, and marks the microspheres as qualified and unqualified. The marked 3D microspheres are output to a triaxial printing platform 6 through microtubes, the 3D microspheres marked as qualified are distributed to the microplates, and the unqualified 3D microspheres are printed into unqualified sample collection holes. After the 3D microspheres are printed on the micro-pore plate, the fluorine oil can drop into the pore plate along with the micro-droplets and volatilize rapidly, and at the moment, the 3D microspheres are exposed to the air and rapidly lose water. In order to prevent the 3D microspheres from losing water, an automatic liquid adding module is arranged in the triaxial printing platform 6, and each printed micropore is automatically added with liquid (including but not limited to a culture medium, a cell suspension, a tissue suspension and the like) by setting a time interval between printing and liquid adding.

For example: the user defines the acceptable product as: the number of cell micelles satisfying a diameter of 20 μm or more in a single microsphere is 3 or more, and the microsphere is regarded as being acceptable.

The three-axis printing platform 6 mainly comprises an aseptic laminar flow hood, an ultraviolet lamp, a three-axis moving platform, a printing nozzle 7 and a hue photosensitive camera 9. The sterile laminar flow hood and the ultraviolet lamp can provide sterile conditions for the working environment to avoid sample pollution. The hue photosensitive camera 9 can be set according to the sample by color, usually phenol red. The hue photosensitive camera 9 recognizes the 3D microsphere signals at the liquid adding spray head 8 and transmits the signals to the triaxial printing platform 6, so that sample printing is completed according to the printing mode planned by the image analysis system 5.

In summary, the application utilizes microfluidic technology to realize micro-scale and high-flux biological manufacture. The integrated combination of the sample-adding conical tube 321 and the chip packaging layer 32 effectively solves the technical difficulty of modeling a small sample (5 mu l or more) organoid, and the high-speed camera 4 and the image analysis system 5 can be used for shooting, recording and marking the cell micelle number of each 3D microsphere, and performing preliminary quality inspection and intelligent accurate printing on the prepared 3D microsphere, so that the obtained data can be used as an important reference for subsequent image analysis. Secondly, the problem of water loss of the 3D microsphere with large specific surface area and the problem of co-culture outside the high-flux 3D microsphere in the high-flux and long-time printing process are all the difficulties to be solved in an in-vitro medicine sieve model. The method aims at the pain point, an automatic liquid adding module is arranged, a timely automatic dynamic liquid adding mode is realized, the vibration carrier 20 can continuously vibrate to maintain cells or micro-tissues in a homogeneous suspension state all the time, and consistency among holes in the liquid adding process is ensured. As a composite organoid manufacturing and printing system, the application breaks the technical barrier of micro sample manufacturing in traditional biological manufacturing, and provides a finer and better solution for high-throughput and intelligent micro-scale organoid manufacturing and imaging analysis.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (10)

1. A system for high-throughput 3D microsphere synthesis, fabrication, and distribution comprising:

the pressure output module is used for providing a power source;

the microfluidic shearing module is used for mixing cells and biological glue into cell suspension by using a power source and shearing and forming to form 3D microspheres; the microfluidic shearing module comprises a refrigerating unit (1), wherein a carrier unit (2) and a sample adding chip unit (3) are arranged in the refrigerating unit (1), the sample adding chip unit (3) is arranged above the carrier unit (2), and the carrier unit (2) and the sample adding chip unit (3) are connected with the pressure output module through pipelines;

the visual identification and quality inspection module is used for recording and marking the cell micelle number of each 3D microsphere by utilizing an image identification technology, and judging the qualification state of each 3D microsphere according to the cell micelle number;

the three-axis printing distribution module is used for carrying out printing distribution on each 3D microsphere according to the qualified state of the microsphere;

and the automatic liquid adding module is used for adding culture solution to the printed 3D microspheres.

2. The high-throughput 3D microsphere synthesis, manufacturing and distribution system according to claim 1, wherein the pressure output module comprises a pressure pump and a pressure valve, the output end of the pressure pump is connected with the microfluidic shearing module through a pipeline and provides a power source for the microfluidic shearing module, and the pressure valve is arranged on the pressure pump and is used for adjusting the size of the power source.

3. A system for high-throughput 3D microsphere synthesis, manufacturing and distribution according to claim 1, characterized in that said carrier unit (2) comprises in particular:

a carrier (21), wherein a placement groove (211) and a chip groove (212) are arranged on the carrier (21);

the oil storage device (22) comprises an oil storage pipe (221) and an oil phase connecting cover (222), wherein the top end of the oil storage pipe (221) is in threaded connection with the oil phase connecting cover (222), and the oil storage pipe (221) is in fit connection with the placing groove (211); the oil phase connecting cover (222) is also provided with an air pipe connecting hole (2221) and an oil phase output hole (2222), the air pipe connecting hole (2221) is connected with the pressure output module through a pipeline, and the oil phase output hole (2222) is connected with the sample adding chip unit through a pipeline;

chip fixing device (23), including chip fixer (231), chip fixer (231) with chip groove (212) is fit and is connected, just chip fixer (231) will add appearance chip unit (3) are fixed in through mounting (232) in chip groove (212).

4. The high throughput 3D microsphere synthesis, manufacturing and distribution system of claim 1, wherein said sample application chip unit (3) comprises:

a micro flow channel shear layer (31) on which an oil phase flow channel (311) and a micro flow channel (312) are arranged; the oil phase flow channel (311) and the micro flow channel (312) are combined to form a combined flow channel (313), and the other end of the combined flow channel (313) extends out of the micro flow channel shear layer (31);

the chip packaging layer (32) is provided with a sample adding conical tube (321), the sample adding conical tube (321) and the chip packaging layer (32) are integrally formed, and the chip packaging layer (32) is also provided with an oil phase input port (322); the chip packaging layer (32) is stacked above the micro-channel shearing layer (31), and the oil phase input port (322) is communicated with the oil phase channel (311); the top of the sample adding conical tube (321) is connected with a connector (323), the connector (323) is externally connected with the pressure output module through a pipeline, and the bottom of the sample adding conical tube (321) is communicated with the micro-channel (312).

5. The system for high-throughput 3D microsphere synthesis, manufacturing and distribution according to claim 1, wherein said visual identification and quality control module comprises:

the high-speed camera (4) is used for capturing and photographing the 3D microspheres and transmitting photographing information to the image analysis system (5);

and the image analysis system (5) is used for recording and acquiring the cell micelle number of each 3D microsphere in the photographing information by utilizing an image recognition technology, and judging the qualification state of each 3D microsphere according to the cell micelle number.

6. The high throughput 3D microsphere synthesis, manufacturing and distribution system of claim 1, wherein said tri-axial print distribution module comprises:

a triaxial printing platform (6);

the printing spray head (7) is arranged at the bottom end of the triaxial printing platform (6), and the printing spray head (7) is connected with the microfluidic shearing module through a pipeline and is used for printing the 3D microspheres manufactured by the microfluidic shearing module on a pore plate;

the liquid adding spray head (8) is arranged at the bottom end of the triaxial printing platform (6) and is close to the printing spray head (7), and an inlet of the liquid adding spray head (8) is connected with the automatic liquid adding module through a pipeline and is used for adding liquid;

the hue photosensitive camera (9) is arranged on the triaxial printing platform (6), and a lens of the hue photosensitive camera (9) is aligned with a liquid outlet of the printing nozzle (7) and used for hue identification.

7. The high throughput 3D microsphere synthesis, manufacturing and distribution system of claim 1, wherein said automated liquid feeding module comprises:

a plurality of liquid storage pipes (10) for loading different liquids;

the vibration carrier (20) is used for carrying and vibrating the liquid storage tube (10) so as to uniformly mix the liquid in the liquid storage tube (10);

the liquid pumps (30) are used for connecting the liquid storage pipes (10) and pumping liquid;

and the fluid switching valve (40) is used for being connected with the liquid pump (30) so as to switch the liquid to realize intelligent liquid separation.

8. A method for synthesizing, manufacturing and distributing high-throughput 3D microspheres, comprising at least the steps of:

step S1: the pressure output module is respectively connected with an oil storage pipe (221) and a sample adding conical pipe (321) through pipelines, and a refrigerating unit (1) is started to control the temperature within a preset temperature range;

step S2: transferring the water phase mixed with the cells into a sample adding conical tube (321), and regulating the pressure output values of the water phase and the oil phase by using a pressure output module;

step S3: the pressure output module is used for providing a power source, the oil phase and the water phase are pushed into the sample adding chip unit (3) to form 3D microspheres, and the 3D microspheres enter the triaxial printing distribution module through a pipeline;

step S4: the 3D microspheres pass through a light source in the visual identification and quality inspection module, are captured and photographed by a high-speed camera (4), and transmit photographing information to an image analysis system (5) for identification and analysis, and the qualification state of each 3D microsphere is judged according to preset conditions;

step S5: a hue photosensitive camera (9) in the triaxial printing distribution module identifies the sample hue of the liquid outlet of the printing spray head (7), and intelligent distribution printing is performed according to the judgment result of whether the sample hue is in a preset hue interval;

step S6: different liquids are loaded in different liquid storage pipes (10) in an automatic liquid adding module, the liquid storage pipes (10) are installed in a vibration carrier (20) to vibrate, each liquid storage pipe (10) is connected with a corresponding liquid drawing pump (30), the other end of each liquid drawing pump (30) is connected with a fluid switching valve (40), and liquid in the liquid storage pipe (10) is added through a liquid adding spray nozzle (8) through adjustment of the fluid switching valve (40).

9. The method of claim 8, wherein the high throughput 3D microsphere is synthesized, fabricated and dispensed,

in the step S2, the pressure output value of the water phase is 10-80mbar, and the pressure output value of the oil phase is 10-300mbar;

in the step S6, the time interval between the dispensing and printing of the 3D microspheres and the liquid adding of the pore plate is 5-15S.

10. The method of high throughput 3D microsphere synthesis, fabrication and distribution according to claim 8, wherein step S4 comprises the following steps:

step S41: presetting hue parameters of the 3D microspheres, and capturing and photographing the 3D microspheres by a high-speed camera (4) when the 3D microspheres pass through a light source in a visual identification and quality inspection module;

step S42: transmitting the photographing information to an image analysis system (5), obtaining the cell micelle number of the 3D microspheres, and marking;

step S43: obtaining the size and the number of the cell clusters according to the number of the cell clusters of the 3D microspheres;

step S44: and dividing the 3D microspheres into qualified and unqualified according to whether the size and the number of the cell clusters meet preset conditions.