WO2024016404A1 - 一种类器官高通量培养方法 - Google Patents

一种类器官高通量培养方法 Download PDF

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WO2024016404A1
WO2024016404A1 PCT/CN2022/111624 CN2022111624W WO2024016404A1 WO 2024016404 A1 WO2024016404 A1 WO 2024016404A1 CN 2022111624 W CN2022111624 W CN 2022111624W WO 2024016404 A1 WO2024016404 A1 WO 2024016404A1
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organoid
syringe
dispersed phase
inlet pipe
pipe
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English (en)
French (fr)
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马少华
汤继元
曹远雄
杨浩威
赵浩然
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清华大学深圳国际研究生院
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
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    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
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    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
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    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
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    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0691—Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention relates to the field of medical devices, and specifically relates to a high-throughput culture method for organoids.
  • the culture of primary organoids is helpful for the development of anti-cancer drugs and the screening of precise drugs for cancer patients.
  • the culture method of primary organoids is mainly divided into two steps: first, the preparation of tumor organoids.
  • the preparation of tumor organoids usually relies on the skills of the laboratory technician; second, the laboratory technician manually converts the prepared Tumor organoids are transferred to the holes of a well plate for culture. This process is time-consuming and labor-intensive, and the operation process is uncontrollable.
  • the size of the prepared organoids is uneven, and the consistency of the organoids cannot be guaranteed. There are large differences between individual organoids, and Culturing in the holes of a well plate cannot simulate the microenvironment in the human body, and the growth and development of organoids takes a long time.
  • the present invention provides a high-throughput culture method for organoids, which has good controllability, saves time and effort, ensures the consistency of organoids, reduces differences between individual organoids, and can simulate the human body
  • the microenvironment shortens the growth and development time of organoids.
  • An organoid high-throughput culture method including the following steps: S2, wrapping cells in dispersed phase 1; S4, injecting dispersed phase 1 and dispersed phase 2 wrapped with cells into the organoid precursor of the microfluidic system respectively
  • dispersed phase 1 is sheared by dispersed phase 2 into organoid precursor droplets of uniform size.
  • the sheared organoid precursor droplets can sequentially enter the organoid droplet maturation area of the microfluidic system and can Mature into organoid spheroids in the organoid droplet maturation area; S6.
  • the control unit Control the action of the driving unit of the 3D printing system through the control unit, and drive the printing nozzle downward through the driving unit to the vascular bed culture of the first vascular bed microfluidic chip.
  • the first organoid spheroids will be adsorbed to the bottom of the vascular bed culture chamber of the first vascular bed microfluidic chip, so that the first organoid spheroids are printed to the first In the vascular bed culture chamber of a vascular bed microfluidic chip, after completion, the printing nozzle is driven by the drive unit to return to the initial position; S12, follow the method from step S6 to step S10 to sequentially print the remaining parts in the organoid droplet maturation area.
  • the organoid spheroids are printed into the vascular bed culture chambers of the remaining vascular bed microfluidic chips; S14. Culture the organoid spheroids in the vascular bed culture chambers of all vascular bed microfluidic chips.
  • the organoid precursor preparation area is a Y-shaped structure.
  • the Y-shaped structure includes a first inlet pipe, a second inlet pipe and a connecting pipe.
  • the first end of the first inlet pipe and The first end of the second inlet pipe is connected to a first syringe and a second syringe respectively.
  • the first syringe is connected to a first syringe pump.
  • the second syringe is connected to a second syringe pump.
  • the second end of the first inlet pipe is connected to a first syringe and a second syringe.
  • the end and the second end of the second inlet pipe are both connected to the first end of the connecting pipe, the organoid droplet maturation zone is an outlet pipe, and the second end of the connecting pipe is connected to the outlet pipe;
  • the step S4 includes the following steps; S42, place the dispersed phase 1 and the dispersed phase 2 in the first syringe and the second syringe respectively; S44, inject the dispersed phase 1 in the first syringe into the first syringe through the first syringe pump. In an inlet pipe, the dispersed phase 2 in the second syringe is injected into the second inlet pipe through the second syringe pump.
  • the dispersed phase 1 is sheared by the dispersed phase 2 into organoid precursor droplets of uniform size.
  • the sheared organoid precursor droplets can sequentially enter the outlet pipe and mature into organoid spheres in the outlet pipe.
  • the organoid precursor preparation area is a T-shaped structure.
  • the T-shaped structure includes a first inlet pipe, a second inlet pipe and a connecting pipe.
  • the first end of the first inlet pipe and The first end of the second inlet pipe is connected to the first syringe and the second syringe respectively.
  • the first syringe is connected to the first syringe pump.
  • the second syringe is connected to the second syringe pump.
  • the third end of the first inlet pipe is connected to the first syringe and the second syringe respectively.
  • the two ends and the second end of the second inlet pipe are respectively connected to the first end of the connecting pipe, the organoid droplet maturation zone is an outlet pipe, and the second end of the connecting pipe is connected to the outlet pipe.
  • the step S4 includes the following steps: S42, place the dispersed phase 1 and the dispersed phase 2 in the first syringe and the second syringe respectively; S44, inject the dispersed phase 1 in the first syringe into In the first inlet pipe, the dispersed phase 2 in the second syringe is injected into the second inlet pipe through the second syringe pump.
  • the dispersed phase 1 is sheared by the dispersed phase 2 into organoid precursor liquid of uniform size. Drops, the sheared organoid precursor droplets can sequentially enter the outlet pipe and mature into organoid spheres in the outlet pipe.
  • the organoid precursor preparation area is a cross-shaped structure.
  • the cross-shaped structure includes a first inlet pipe, two oppositely arranged second inlet pipes and a connecting pipe.
  • the first inlet The pipe and the connecting pipe are arranged oppositely, the first end of the first inlet pipe is connected to the first syringe, the second end of the first inlet pipe is connected to the first end of the connecting pipe, and the first syringe is connected to In the first syringe pump, the first ends of the two second inlet pipes are respectively connected to the two second syringes, the second ends of the two second inlet pipes are respectively connected to the first ends of the connecting pipes, and the two The second syringes are respectively connected to two second syringe pumps, the organoid droplet maturation area is an outlet pipe, and the second end of the connecting pipe is connected to the outlet pipe;
  • the step S4 includes the following steps: S42 , place the dispersed phase 1 in the first syring
  • the dispersed phase 1 is injected into the dispersed phase in the two second inlet pipes. 2. Cut into organoid precursor droplets of uniform size. The sheared organoid precursor droplets can sequentially enter the outlet pipeline and mature into organoid spheres in the outlet pipeline.
  • the cells are primary tumor tissue cells, stem cells or cell lines.
  • the material of the dispersed phase 1 includes Matrigel, collagen or gelatin.
  • the material of the dispersed phase 2 includes fluorine oil or vegetable oil.
  • the method for maturing organoid precursor droplets into organoid spheroids includes temperature cross-linking, enzymatic cross-linking, ion cross-linking or photo-cross-linking.
  • the organoid high-throughput culture method provided by the present invention completes the preparation of organoids through a microfluidic system, and combines the microfluidic system with a 3D printing system to easily, quickly and accurately
  • the organoids are printed and transferred to the bottom of the vascular bed culture chamber of the vascular bed microfluidic chip for culture. This process is highly controllable, saves time and effort, has good stability, and the organoids are uniform in size, ensuring the consistency of the organoids. sex, reducing the differences between individual organoids.
  • printing organoids into the vascular bed culture chamber of the vascular bed microfluidic chip for culture can simulate the microenvironment in the human body, and within a short period of time (such as 3-5 days)
  • a complete vascular network can be built inside the organoid, shortening the growth and development time of the organoid, and improving the efficiency and accuracy of drug screening.
  • Figure 1 is a flow chart of a high-throughput culture method for organoids provided by an embodiment of the present invention
  • Figure 2 is a schematic flow chart of a high-throughput culture method for organoids shown in Figure 1;
  • Figure 3 is a schematic diagram of the extraction of primary tumor tissue cells from primary tumor tissue
  • Figure 4 is a schematic structural diagram of the first syringe and the second syringe
  • Figure 5 is a schematic plan view of the organoid precursor preparation area and organoid droplet maturation area of the first solution of the microfluidic system;
  • Figure 6 is a schematic plan view of the organoid precursor preparation area and organoid droplet maturation area of the second solution of the microfluidic system;
  • Figure 7 is a schematic plan view of the organoid precursor preparation area and organoid droplet maturation area of the third solution of the microfluidic system;
  • Figure 8 is a schematic plan view of the organoid precursor preparation area and organoid droplet maturation area of the fourth solution of the microfluidic system;
  • Figure 9 is a schematic diagram of the effect of culturing organoid spheroids in the vascular bed culture chamber of the vascular bed microfluidic chip.
  • an organoid high-throughput culture method provided by one embodiment of the present invention includes the following steps:
  • the cells are primary tumor tissue cells extracted from primary tumor tissue, as shown in Figure 3. It is understood that the cells can also be, for example, stem cells, cell lines, etc., and the type and number of cells can be set according to the actual situation.
  • the density of cells wrapped in dispersed phase 1 is 1 ⁇ 10 7 /ml.
  • the cell densities compatible with the present invention include but are not limited to 1 ⁇ 10 5 /ml to 1 ⁇ 10 9 /ml. It can be flexibly adjusted according to culture needs and cell activity.
  • the material of dispersed phase 1 includes Matrigel. It can be understood that the material of the dispersed phase 1 can also include natural materials such as collagen and gelatin, and the material of the dispersed phase 1 can also include synthetic materials, which can be set according to actual conditions.
  • the material of the dispersed phase 2 includes fluorine oil.
  • Fluorine oil can better produce droplet phenomena and has better shear force, so that the dispersed phase 1 wrapped with cells forms organoid precursor droplets 100 of uniform size.
  • the dispersed phase 2 may also include materials that are well compatible with cells and mutually immiscible with water, such as vegetable oil, and may be set according to actual conditions.
  • Methods for maturing organoid precursor droplets 100 into organoid spheroids 100 include temperature cross-linking, enzymatic cross-linking, ion cross-linking, photo-cross-linking, and other methods.
  • the organoid precursor preparation area is a T-shaped structure, as shown in FIG. 5 .
  • the T-shaped structure includes a first inlet pipe 42 , a second inlet pipe 43 and a connecting pipe 44 .
  • the first end of the first inlet pipe 42 and the first end of the second inlet pipe 43 are respectively connected with the first syringe 32 (see Figure 4) and the second syringe 34 (see Figure 4).
  • the first syringe 32 is connected with the third A syringe pump
  • the second syringe 34 is connected to the second syringe pump
  • the second end of the first inlet pipe 42 and the second end of the second inlet pipe 43 are respectively connected to the first end of the connecting pipe 44.
  • the axis of the first inlet pipe 42 and the axis of the connecting pipe 44 are located on the same horizontal line, and the second inlet pipe 43 is arranged vertically with the first inlet pipe 42 and the connecting pipe 44 .
  • the second inlet pipe 43 is located above the first inlet pipe 42 and the connecting pipe 44 .
  • the organoid droplet maturation area is an outlet pipe 50, and the second end of the connecting pipe 44 is connected to the outlet pipe 50.
  • the connecting pipe 44 and the outlet pipe 50 are connected as one body.
  • Step S4 specifically includes the following steps:
  • the flow rate of dispersed phase 1 is usually set to 20 ⁇ l/min, and the flow rate of dispersed phase 2 is usually set to 120 ⁇ l/min.
  • the present invention can adjust the flow rate and flow rate ratio of the two dispersed phases according to the needs, thereby adjusting the shape of the generated organoid precursor droplets (for example, circular or elliptical with unequal lengths), the distance between the organoid precursor droplets 100 and the organoid precursor droplets. Overall speed of preparation of 100 body fluid droplets.
  • the organoid precursor preparation area is also a T-shaped structure. What is different from the organoid precursor preparation area in Figure 5 is that the axis of the first inlet pipe 42 and the first The axes of the two inlet pipes 43 are located on the same horizontal line.
  • the connecting pipe 44 is arranged vertically with the first inlet pipe 42 and the second inlet pipe 43 .
  • the connecting pipe 44 is located below the first inlet pipe 42 and the second inlet pipe 43 .
  • the organoid precursor preparation area is a Y-shaped structure.
  • the Y-shaped structure includes a first inlet pipe 42 , a second inlet pipe 43 and a connecting pipe 44 .
  • the first end of the first inlet pipe 42 and the first end of the second inlet pipe 43 are connected to the first syringe 32 and the second syringe 34 respectively.
  • the second end of the first inlet pipe 42 and the second end of the second inlet pipe 43 Both ends are connected to the first end of the connecting pipe 44.
  • the first inlet pipe 42 and the second inlet pipe 43 form a V-shaped structure, and the connecting pipe 44 is located below the first inlet pipe 42 and the second inlet pipe 43 .
  • the organoid droplet maturation area is an outlet pipe 50, and the second end of the connecting pipe 44 is connected to the outlet pipe 50.
  • the connecting pipe 44 and the outlet pipe 50 are connected as one body.
  • Step S4 includes the following steps:
  • the organoid precursor preparation area is a cross-shaped structure.
  • the cross-shaped structure includes a first inlet pipe 42, two opposite second inlet pipes 43 and a connecting pipe 44.
  • the first inlet pipe 42 and the connecting pipe 44 are arranged oppositely.
  • the first end of the first inlet pipe 42 is connected to the first syringe 32
  • the second end of the first inlet pipe 42 is connected to the first end of the connecting pipe 44 .
  • the first syringe 32 is connected to a first syringe pump.
  • the first ends of the two second inlet pipes 43 are connected to the two second syringes 34 respectively, and the second ends of the two second inlet pipes 43 are connected to the first ends of the connecting pipes 44 respectively.
  • the two second syringes 34 are respectively connected to two second syringe pumps.
  • the organoid droplet maturation area is an outlet pipe 50, and the second end of the connecting pipe 44 is connected to the outlet pipe 50.
  • the connecting pipe 44 and the outlet pipe 50 are connected integrally.
  • Step S4 specifically includes the following steps:
  • step S6 the 3D printing system is a 3D bioprinter.
  • organoid recognition and detection unit 64 of the 3D printing system uses the organoid recognition and detection unit 64 of the 3D printing system to detect whether there are organoid spheroids 102 at the printing nozzle 62.
  • the organoid recognition and detection unit 64 detects that there are organoid spheroids 102 at the printing nozzle 62, the organoid recognition and detection unit 64 outputs the organoid signal to the control unit of the 3D printing system.
  • the organoid recognition and detection unit 64 is opposite to the printing nozzle 62. As shown in FIG. 2, the organoid recognition and detection unit 64 detects, for example, the color, geometric outline, fluorescence signal, etc. of the organoid sphere 102, thereby detecting There are organoid spheres 102 at the printing nozzle 62 .
  • the organoid identification and detection unit 64 is, for example, a CCD camera.
  • the moving speed range of the printing nozzle 62 is generally 0-100 mm/s (millimeters per second) on the XY axis, and the Z-axis speed is 0-80 mm/s.
  • the moving speed and acceleration of the printing nozzle 62 can be flexibly adjusted in real time according to the printing speed and accuracy requirements.
  • step S10 the vascular bed culture chamber 202 has an opening, and the printing nozzle 62 moves to the bottom of the vascular bed culture chamber 202 through the opening.
  • step S12 follow the method from step S6 to step S10 to sequentially print the remaining organoid spheroids 102 in the organoid droplet maturation area into the vascular bed culture chamber 202 of the remaining vascular bed microfluidic chip 200. This is completed. High-throughput printing of organoids.
  • step S14 when the organoid spheroids 102 are cultured, since the material of the dispersed phase 2 includes fluorine oil, the dispersed phase 2 will volatilize on its own.
  • the organoid high-throughput culture method provided by the present invention completes the preparation of organoids through a microfluidic system, and combines the microfluidic system with a 3D printing system to easily, quickly and accurately transfer the organoids through printing. Go to the bottom of the vascular bed culture chamber 202 of the vascular bed microfluidic chip 200 for culture. This process is well controllable, saves time and effort, has good stability, and the organoids are uniform in size, ensuring the consistency of the organoids and reducing the cost of the organoids. The differences between individual organs.
  • the organoids are printed and cultured in the vascular bed culture chamber 202 of the vascular bed microfluidic chip 200, which can simulate the microenvironment in the human body in a short time (such as 3-5 days). Building a complete vascular network inside the organoids shortens the growth and development time of the organoids and improves the efficiency and accuracy of drug screening.

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Abstract

一种类器官高通量培养方法，包括以下步骤；S2、将细胞包裹于分散相1内；S4、将包裹有细胞的分散相1和分散相2分别注射到微流控系统的类器官前体制备区内，分散相1被分散相2剪切成大小均一的类器官前体液滴，剪切成的类器官前体液滴可依次进入到微流控系统的类器官液滴熟化区并可在该类器官液滴熟化区内熟化成类器官球体；S6、通过微流控系统的输送泵将类器官液滴熟化区内的第一个类器官球体经3D打印系统的输送管道输送至3D打印系统的打印喷头处。

Description

一种类器官高通量培养方法 技术领域
本发明涉及医疗器械领域，具体的是涉及一种类器官高通量培养方法。
背景技术
原代类器官的培养有助于抗癌药物的研发与患癌病人精准用药筛选。
目前原代类器官例如肿瘤类器官的培养方式主要分为两个步骤：第一、肿瘤类器官的制备，肿瘤类器官的制备通常依赖实验员的技术，第二、通过实验员手动将制备的肿瘤类器官转移至孔板的孔位内进行培养，该过程费时费力，操作过程不可控，制备的类器官大小不均一，无法保证类器官的一致性，类器官个体间的差异较大，并且在孔板的孔位内进行培养，不能模拟人体内微环境，类器官生长与发育时间较长。
发明内容
为了克服现有技术的不足，本发明提供一种类器官高通量培养方法，可控性好，省时省力，保证了类器官的一致性，降低了类器官个体间的差异，可模拟人体内微环境，缩短了类器官的生长与发育时间。
本发明解决其技术问题所采用的技术方案是：
一种类器官高通量培养方法，包括以下步骤：S2、将细胞包裹于分散相1内；S4、将包裹有细胞的分散相1和分散相2分别注射到微流控系统的类器官前体制备区内，分散相1被分散相2剪切成大小均一的的类器官前体液滴，剪切成的类器官前体液滴可依次进入到微流控系统的类器官液滴熟化区并可在该类器官液滴熟化区内熟化成类器官球体；S6、通过微流控系统的输送泵将类器 官液滴熟化区内的第一个类器官球体经3D打印系统的输送管道输送至3D打印系统的打印喷头处；S8、通过3D打印系统的类器官识别检测单元检测打印喷头处是否有类器官球体，当类器官识别检测单元检测到打印喷头处有类器官球体时，类器官识别检测单元输出类器官信号到3D打印系统的控制单元；S10、通过控制单元控制3D打印系统的驱动单元动作，通过驱动单元驱动打印喷头向下移动至第一个血管床微流控芯片的血管床培养室的底部，停留1-2秒钟后，第一个类器官球体会吸附于第一个血管床微流控芯片的血管床培养室的底部，如此第一个类器官球体即打印至第一个血管床微流控芯片的血管床培养室内，完毕后，通过驱动单元驱动打印喷头回到初始位置；S12、按照步骤S6至步骤S10的方法依次将类器官液滴熟化区内的剩下的类器官球体打印至剩下的血管床微流控芯片的血管床培养室内；S14、对所有血管床微流控芯片的血管床培养室内的类器官球体进行培养。
作为优选的技术方案，所述类器官前体制备区为一Y型结构，所述Y型结构包括第一入口管道、第二入口管道和连接管道，所述第一入口管道的第一端和第二入口管道的第一端分别与第一注射器、第二注射器连接，所述第一注射器连接有第一注射泵，所述第二注射器连接有第二注射泵，第一入口管道的第二端和第二入口管道的第二端均与所述连接管道的第一端连接，所述类器官液滴熟化区为一出口管道，所述连接管道的第二端与所述出口管道连接；所述步骤S4包括以下步骤；S42、将分散相1、分散相2分别置于第一注射器内和第二注射器内；S44、通过第一注射泵将第一注射器内的分散相1注射到第一入口管道内，通过第二注射泵将第二注射器内的分散相2注射到第二入口管道内，在连接管道内，分散相1被分散相2剪切成大小均一的类器官前体液滴，剪切成的类器官前体液滴可依次进入到出口管道内并可在该出口管道内熟化成类器官球体。
作为优选的技术方案，所述类器官前体制备区为一T型结构，所述T型结构包括第一入口管道、第二入口管道和连接管道，所述第一入口管道的第一端和第二入口管道的第一端分别与第一注射器连接、第二注射器连接，所述第一 注射器连接有第一注射泵，所述第二注射器连接有第二注射泵，第一入口管道的第二端和第二入口管道的第二端分别与所述连接管道的第一端连接，所述类器官液滴熟化区为一出口管道，所述连接管道的第二端与所述出口管道连接；所述步骤S4包括以下步骤：S42、将分散相1、分散相2分别置于第一注射器内和第二注射器内；S44、通过第一注射泵将第一注射器内的分散相1注射到第一入口管道内，通过第二注射泵将第二注射器内的分散相2注射到第二入口管道内，在连接管道内，分散相1被分散相2剪切成大小均一的类器官前体液滴，剪切成的类器官前体液滴可依次进入到出口管道内并可在该出口管道内熟化成类器官球体。
作为优选的技术方案，所述类器官前体制备区为一十字型结构，所述十字型结构包括第一入口管道、两个呈相对设置的第二入口管道和连接管道，所述第一入口管道和连接管道呈相对设置，所述第一入口管道的第一端与第一注射器连接，第一入口管道的第二端与所述连接管道的第一端连接，所述第一注射器连接有第一注射泵，两个第二入口管道的第一端分别与两个第二注射器连接，两个第二入口管道的第二端分别与所述连接管道的第一端连接，所述两个第二注射器分别连接有两个第二注射泵，所述类器官液滴熟化区为一出口管道，所述连接管道的第二端与所述出口管道连接；所述步骤S4包括以下步骤：S42、将分散相1置于第一注射器内，将分散相2分别置于两个第二注射器内；S44、通过第一注射泵将第一注射器内的分散相1注射到第一入口管道内，通过两个第二注射泵分别将两个第二注射器内的分散相2注射到两个第二入口管道内，在连接管道内，分散相1被注射到两个第二入口管道内的分散相2剪切成大小均一的类器官前体液滴，剪切成的类器官前体液滴可依次进入到出口管道内并可在该出口管道内熟化成类器官球体。
作为优选的技术方案，所述步骤S2中，所述细胞为原代肿瘤组织细胞、干细胞或细胞系。
作为优选的技术方案，所述分散相1的材质包括基质胶、胶原蛋白或明胶。
作为优选的技术方案，所述分散相2的材质包括氟油或植物油。
作为优选的技术方案，所述步骤S4中，将类器官前体液滴熟化成类器官球体的方法包括温度交联法、酶促交联法、离子交联或光交联法。
本发明的有益效果是：本发明提供的类器官高通量培养方法，通过微流控系统完成类器官的制备，并通过微流控系统与3D打印系统结合，能够简便、快速、精确地将类器官通过打印的方式转移至血管床微流控芯片的血管床培养室的底部进行培养，该过程可控性好，省时省力，稳定性好，类器官大小均一，保证了类器官的一致性，降低了类器官个体间的差异，同时，将类器官打印至血管床微流控芯片的血管床培养室内进行培养，可以模拟人体内微环境，在短时间(如3-5天)内即可在类器官内部构建完整的血管网络，缩短了类器官的生长与发育时间，提升筛药效率与精确度。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是本发明一实施例提供的一种类器官高通量培养方法的流程框图；
图2是图1所示一种类器官高通量培养方法的流程示意图；
图3是原代肿瘤组织细胞从原代肿瘤组织中提取的示意图；
图4是第一注射器和第二注射器的结构示意图；
图5是微流控系统的第一种方案的类器官前体制备区和类器官液滴熟化区的平面示意图；
图6是微流控系统的第二种方案的类器官前体制备区和类器官液滴熟化区的平面示意图；
图7是微流控系统的第三种方案的类器官前体制备区和类器官液滴熟化区的平面示意图；
图8是微流控系统的第四种方案的类器官前体制备区和类器官液滴熟化区的平面示意图；
图9是类器官球体在血管床微流控芯片的血管床培养室内进行培养的效果示意图。
具体实施方式
以下将结合实施例和附图对本发明的构思、具体结构及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。另外，专利中涉及到的所有联接/连接关系，并非单指构件直接相接，而是指可根据具体实施情况，通过添加或减少联接辅件，来组成更优的联接结构。本发明创造中的各个技术特征，在不互相矛盾冲突的前提下可以交互组合。
请参照图1和图2，本发明一实施例提供的一种类器官高通量培养方法，包括以下步骤：
S2、将细胞包裹于分散相1内。
步骤S2中，细胞为从原代肿瘤组织中提取的原代肿瘤组织细胞，如图3所示。可以理解地，细胞还可以是例如干细胞、细胞系等，细胞的类型、数量可根据实际情况进行设置。
本实施例中，包裹于分散相1中的细胞密度为1×10 7个/毫升，本发明所兼容的细胞密度包括但不限于1×10 5个/毫升至1×10 9个/毫升，可依据培养需求及细胞活性灵活调整。
分散相1的材质包括基质胶。可以理解地，分散相1的材质还可以是包括例如胶原蛋白、明胶等天然材料，分散相1的材质还可以是包括合成的材料，可根据实际情况进行设置。
S4、将包裹有细胞的分散相1和分散相2分别注射到微流控系统的类器官前体制备区内，分散相1被分散相2剪切成大小均一的的类器官前体液滴100。类器官前体液滴100的外部包裹有部分分散相2。剪切成的类器官前体液滴100可依次进入到微流控系统的类器官液滴熟化区并可在该类器官液滴熟化区熟化成类器官球体102，类器官即完成制备。
步骤S4中，分散相2的材质包括氟油。氟油能够更好的产生液滴现象，剪切力较好，使得包裹有细胞的分散相1形成大小均一的类器官前体液滴100。可以理解地，分散相2还可以是包括例如植物油等与细胞相容性较好且与水相互不相溶的材料，可根据实际情况进行设置。
将类器官前体液滴100熟化成类器官球体100的方法包括温度交联法、酶促交联法、离子交联法、光交联法等等方法。
本实施例中，类器官前体制备区为一T型结构，结合图5所示。T型结构包括第一入口管道42、第二入口管道43和连接管道44。第一入口管道42的第一端和第二入口管道43的第一端分别与第一注射器32连接(见图4)、第二注射器34(见图4)连接，第一注射器32连接有第一注射泵，第二注射器34连接有第二注射泵，第一入口管道42的第二端和第二入口管道43的第二端分别与连接管道44的第一端连接。第一入口管道42的轴线和连接管道44的轴线位于同一条水平线上，第二入口管道43与第一入口管道42、连接管道44呈垂直设置。第二入口管道43位于第一入口管道42和连接管道44的上方。类器官液滴熟化区为一出口管道50，连接管道44的第二端与出口管道50连接。连接管道44和出口管道50连接为一体。
步骤S4具体包括以下步骤：
S42、将分散相1、分散相2分别置于第一注射器32内和第二注射器34内。
S44、通过第一注射泵将第一注射器32内的分散相1注射到第一入口管道42内，通过第二注射泵将第二注射器34内的分散相2注射到第二入口管道43内，在连接管道44内，分散相1被分散相2剪切成大小均一的类器官前体液滴100，剪切成的类器官前体液滴100可依次进入到出口管道50内并可在该出口管道50内熟化成类器官球体102，如图2和图5所示。
本实施例中，分散相1的流速通常设置为20微升/分钟，分散相2的流速通常设置为120微升/分钟。本发明可依据需求调节两分散相流速与流速比，进而调节所生成类器官前体液滴的形状(例如为圆形或不等长度椭圆形)、类器官前体液滴100的间距与类器官前体液滴100制备的整体速度。
在第一种替换方案中，如图6所示，类器官前体制备区也为一T型结构，与图5的类器官前体制备区不同的是，第一入口管道42的轴线和第二入口管道43的轴线位于同一条水平线上，连接管道44与第一入口管道42、第二入口管道43呈垂直设置，连接管道44位于第一入口管道42和第二入口管道43的下方。
在第二种替换方案中，如图7所示，类器官前体制备区为一Y型结构。Y型结构包括第一入口管道42、第二入口管道43和连接管道44。第一入口管道42的第一端和第二入口管道43的第一端分别与第一注射器32、第二注射器34连接，第一入口管道42的第二端和第二入口管道43的第二端均与连接管道44的第一端连接。第一入口管道42和第二入口管道43形成V状结构，连接管道44位于第一入口管道42和第二入口管道43的下方。类器官液滴熟化区为一出口管道50，连接管道44的第二端与出口管道50连接。连接管道44和出口管道50连接为一体。
步骤S4包括以下步骤：
S42、将分散相1、分散相2分别置于第一注射器32内和第二注射器34内；
S44、通过第一注射泵将第一注射器32内的分散相1注射到第一入口管道42内，通过第二注射泵将第二注射器34内的分散相2注射到第二入口管道43内，在连接管道44内，分散相1被分散相2剪切成大小均一的类器官前体液滴100，剪切成的类器官前体液滴100可依次进入到出口管道50内并可在该出口管道50内熟化成类器官球体102。
在第三种替换方案中，如图8所示，类器官前体制备区为一十字型结构。十字型结构包括第一入口管道42、两个呈相对设置的第二入口管道43和连接管道44。第一入口管道42和连接管道44呈相对设置，第一入口管道42的第一端与第一注射器32连接，第一入口管道42的第二端与连接管道44的第一端连接。第一注射器32连接有第一注射泵。两个第二入口管道43的第一端分别与两个第二注射器34连接，两个第二入口管道43的第二端分别与连接管道44的第一端连接。两个第二注射器34分别连接有两个第二注射泵。类器官液滴熟 化区为一出口管道50，连接管道44的第二端与出口管道50连接。连接管道44与出口管道50连接为一体。
步骤S4具体包括以下步骤：
S42、将分散相1置于第一注射器32内，将分散相2分别置于两个第二注射器34内；
S44、通过第一注射泵将第一注射器32内的分散相1注射到第一入口管道42内，通过两个第二注射泵分别将两个第二注射器34内的分散相2注射到两个第二入口管道43内，在连接管道44内，分散相1被注射到两个第二入口管道43内的分散相2剪切成大小均一的类器官前体液滴100，剪切成的类器官前体液滴100可依次进入到出口管道50内并可在该出口管道50内熟化成类器官球体102。
S6、通过微流控系统的输送泵将类器官液滴熟化区内的第一个类器官球体102经3D打印系统的输送管道52输送至3D打印系统的打印喷头62处。
步骤S6中，3D打印系统为3D生物打印机。
S8、通过3D打印系统的类器官识别检测单元64检测打印喷头62处是否有类器官球体102，当类器官识别检测单元64检测到打印喷头62处有类器官球体102时，类器官识别检测单元64输出类器官信号到3D打印系统的控制单元。
步骤S8中，类器官识别检测单元64与打印喷头62相对，如图2所示，通过类器官识别检测单元64检测类器官球体102的例如颜色、几何轮廓、荧光信号等等，从而可检测出打印喷头62处有类器官球体102。类器官识别检测单元64例如为一CCD相机。
S10、通过控制单元控制3D打印系统的驱动单元动作，通过驱动打印喷头62向下移动至第一个血管床微流控芯片200的血管床培养室202的底部，停留1-2秒钟后，优选为1秒钟，在第一个类器官球体102与血管床培养室202底部的黏附作用下，第一个类器官球体102会吸附于第一个血管床微流控芯片200的血管床培养室202的底部，如此即实现将第一个类器官球体102打印至第一个血管床微流控芯片200的血管床培养室202内，完毕后，通过驱动单元驱动 打印喷头62向上移动以回到初始位置，初始位置即打印喷头62与类器官识别检测单元64相对的位置。
本实施例中，打印喷头62的移动速度范围通常为XY轴0-100mm/s(毫米/秒)，Z轴速度为0-80mm/s。打印喷头62的移动速度与与加速度可依据打印速度要求与精度要求实时灵活调整。
步骤S10中，血管床培养室202具有开口，打印喷头62经该开口移动至血管床培养室202的底部。
S12、按照步骤S6至步骤S10的方法依次将类器官液滴熟化区内的剩下的类器官球体102打印至剩下的血管床微流控芯片200的血管床培养室202内，如此即完成类器官的高通量打印。
S14、对所有血管床微流控芯片200的血管床培养室202底部的类器官球体102进行培养，类器官球体102即稳定存在于血管床培养室202的底部进行培养，如图9所示。
步骤S14中，对类器官球体102进行培养时，由于分散相2的材质包括氟油，分散相2会自行挥发。
本发明提供的类器官高通量培养方法，通过微流控系统完成类器官的制备，并通过微流控系统与3D打印系统结合，能够简便、快速、精确地将类器官通过打印的方式转移至血管床微流控芯片200的血管床培养室202的底部进行培养，该过程可控性好，省时省力，稳定性好，类器官大小均一，保证了类器官的一致性，降低了类器官个体间的差异，同时，将类器官打印至血管床微流控芯片200的血管床培养室202内进行培养，可以模拟人体内微环境，在短时间(如3-5天)内即可在类器官内部构建完整的血管网络，缩短了类器官的生长与发育时间，提升筛药效率与精确度。
以上是对本发明的较佳实施进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明精神的前提下还可做出种种的等同变形或替换，这些等同的变形或替换均包含在本申请权利要求所限定的范围内。

Claims (8)

  1. 一种类器官高通量培养方法，其特征在于，包括以下步骤：
    S2、将细胞包裹于分散相1内；
    S4、将包裹有细胞的分散相1和分散相2分别注射到微流控系统的类器官前体制备区内，分散相1被分散相2剪切成大小均一的的类器官前体液滴，剪切成的类器官前体液滴可依次进入到微流控系统的类器官液滴熟化区并可在该类器官液滴熟化区内熟化成类器官球体；
    S6、通过微流控系统的输送泵将类器官液滴熟化区内的第一个类器官球体经3D打印系统的输送管道输送至3D打印系统的打印喷头处；
    S8、通过3D打印系统的类器官识别检测单元检测打印喷头处是否有类器官球体，当类器官识别检测单元检测到打印喷头处有类器官球体时，类器官识别检测单元输出类器官信号到3D打印系统的控制单元；
    S10、通过控制单元控制3D打印系统的驱动单元动作，通过驱动单元驱动打印喷头向下移动至第一个血管床微流控芯片的血管床培养室的底部，停留1-2秒钟后，第一个类器官球体会吸附于第一个血管床微流控芯片的血管床培养室的底部，如此第一个类器官球体即打印至第一个血管床微流控芯片的血管床培养室内，完毕后，通过驱动单元驱动打印喷头回到初始位置；
    S12、按照步骤S6至步骤S10的方法依次将类器官液滴熟化区内的剩下的类器官球体打印至剩下的血管床微流控芯片的血管床培养室内；
    S14、对所有血管床微流控芯片的血管床培养室内的类器官球体进行培养。
  2. 根据权利要求1所述的类器官高通量培养方法，其特征在于，所述类器官前体制备区为一Y型结构，所述Y型结构包括第一入口管道、第二入口管道和连接管道，所述第一入口管道的第一端和第二入口管道的第一端分别与第一注射器、第二注射器连接，所述第一注射器连接有第一注射泵，所述第二注射器连接有第二注射泵，第一入口管道的第二端和第二入口管道的第二端均与所述连接管道的第一端连接，所述类器官液滴熟化区为一出口管道，所述连接管道的第二端与所述出口管道连接；
    所述步骤S4包括以下步骤；
    S42、将分散相1、分散相2分别置于第一注射器内和第二注射器内；
    S44、通过第一注射泵将第一注射器内的分散相1注射到第一入口管道内，通过第二注射泵将第二注射器内的分散相2注射到第二入口管道内，在连接管道内，分散相1被分散相2剪切成大小均一的类器官前体液滴，剪切成的类器官前体液滴可依次进入到出口管道内并可在该出口管道内熟化成类器官球体。
  3. 根据权利要求1所述的类器官高通量培养方法，其特征在于，所述类器官前体制备区为一T型结构，所述T型结构包括第一入口管道、第二入口管道和连接管道，所述第一入口管道的第一端和第二入口管道的第一端分别与第一注射器连接、第二注射器连接，所述第一注射器连接有第一注射泵，所述第二注射器连接有第二注射泵，第一入口管道的第二端和第二入口管道的第二端分别与所述连接管道的第一端连接，所述类器官液滴熟化区为一出口管道，所述连接管道的第二端与所述出口管道连接；
    所述步骤S4包括以下步骤：
    S42、将分散相1、分散相2分别置于第一注射器内和第二注射器内；
    S44、通过第一注射泵将第一注射器内的分散相1注射到第一入口管道内，通过第二注射泵将第二注射器内的分散相2注射到第二入口管道内，在连接管道内，分散相1被分散相2剪切成大小均一的类器官前体液滴，剪切成的类器官前体液滴可依次进入到出口管道内并可在该出口管道内熟化成类器官球体。
  4. 根据权利要求1所述的类器官高通量培养方法，其特征在于，所述类器官前体制备区为一十字型结构，所述十字型结构包括第一入口管道、两个呈相对设置的第二入口管道和连接管道，所述第一入口管道和连接管道呈相对设置，所述第一入口管道的第一端与第一注射器连接，第一入口管道的第二端与所述连接管道的第一端连接，所述第一注射器连接有第一注射泵，两个第二入口管道的第一端分别与两个第二注射器连接，两个第二入口管道的第二端分别与所述连接管道的第一端连接，所述两个第二注射器分别连接有两个第二注射泵，所述类器官液滴熟化区为一出口管道，所述连接管道的第二端与所述出口管道 连接；
    所述步骤S4包括以下步骤：
    S42、将分散相1置于第一注射器内，将分散相2分别置于两个第二注射器内；
    S44、通过第一注射泵将第一注射器内的分散相1注射到第一入口管道内，通过两个第二注射泵分别将两个第二注射器内的分散相2注射到两个第二入口管道内，在连接管道内，分散相1被注射到两个第二入口管道内的分散相2剪切成大小均一的类器官前体液滴，剪切成的类器官前体液滴可依次进入到出口管道内并可在该出口管道内熟化成类器官球体。
  5. 根据权利要求1所述的类器官高通量培养方法，其特征在于，所述步骤S2中，所述细胞为原代肿瘤组织细胞、干细胞或细胞系。
  6. 根据权利要求1所述的类器官高通量培养方法，其特征在于，所述分散相1的材质包括基质胶、胶原蛋白或明胶。
  7. 根据权利要求1所述的类器官高通量培养方法，其特征在于，所述分散相2的材质包括氟油或植物油。
  8. 根据权利要求1所述的类器官高通量培养方法，其特征在于，所述步骤S4中，将类器官前体液滴熟化成类器官球体的方法包括温度交联法、酶促交联法、离子交联或光交联法。
PCT/CN2022/111624 2022-07-21 2022-08-11 一种类器官高通量培养方法 WO2024016404A1 (zh)

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