A kind of preparation method of organoid spheroid

An organoid and spheroid technology, applied in the field of organoid spheroid preparation, can solve problems such as the uneven shape and size of organoids, affecting the accuracy and repeatability of prediction results, and the inability of high-throughput preparation of organoids to achieve the modeling cycle The effect of short, low cost, high throughput production

CN110004111BActive Publication Date: 2021-10-08TSINGHUA BERKELEY SHENZHEN INST
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2021-10-08

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Abstract

The invention provides a method for preparing organoid spheroids. The preparation method comprises: passing cell-containing Matrigel and fluorine oil into a three-way device respectively to obtain cell spheroids, and forming organoid spheroids after culturing; the present invention provides The preparation method of organoid spheroids uses microfluidic method to generate monodisperse biomaterial organoids, which realizes high-throughput production. The size, shape and structure of organoids are controllable and uniform, compared with 2D tumor sensitivity Predictive model, the organoids prepared by the present invention combine tumor microenvironmental factors to predict more accurate results; compared with the PDX mouse tumor sensitivity prediction model, the present invention has a shorter modeling period and lower cost; compared with ordinary Gene sequencing, the clinical prediction rate of the present invention is higher; the preparation method of organoids provided by the present invention is of great significance and value for clinical application, and provides an important basis for the detection of related results.
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Description

technical field

[0001] The invention belongs to the field of medical devices and relates to a method for preparing organoid spheroids. Background technique

[0002] Organoids are multicellular clusters constructed by three-dimensional (3D) culture in vitro, which have the ability of self-renewal and self-organization, and maintain the characteristics of the physiological structure and function of their source tissues. Tumor organoids are organoids derived from tumor tissues, which are new and powerful tools for studying tumor biology. Organoid technology is the fastest-growing type of in vitro culture technology in the past decade, and was once hailed as one of the most important scientific advances in 2013. In 2014, Lancaster and Knoblich formally made a systematic scientific definition of organoids, laying a theoretical foundation for the future development of organoid technology.

[0003] The current organoid culture technology is to encapsulate pluripotent embryonic st...

Examples

Embodiment 1

[0049] This embodiment provides a method for preparing organoid spheroids, comprising the following steps:

[0050] Use the primary cell extraction method to extract primary cells, and count the cells; in 4°C environment, 2.0×10 6 Mix one primary cell with 100 μL matrigel, inject it into a 1mL syringe, inject fluorine oil into another syringe, place the fluorine oil and matrigel in two syringe pumps and put them in a 4°C refrigerator; It is a 800μm polytetrafluoroethylene (PTFE) tube, and the two tubes are merged into a polydimethylsiloxane (PMDS) three-way device to obtain cell spheroids, and then the three-way device is connected to another PTFE tube with a diameter of 800 μm, and the entire platform Place in a refrigerator at 4°C; the flow rate of the injection pump containing Matrigel is 20 μL / min, and the flow rate of the fluorine-containing oil injection pump is 50 μL / min, and then the cell spheroids are placed at 37°C with a concentration of 5% CO 2 The solidification ...

Embodiment 2

[0053] This embodiment provides a method for preparing organoid spheroids, comprising the following steps:

[0054] Use the primary cell extraction method to extract primary cells, and count the cells; in an environment of 4°C, 5.0×10 6 Mix one primary cell with 200μL Matrigel, inject it into a 1mL syringe, inject fluorine oil into the other syringe, place the fluorine oil and Matrigel in two syringe pumps and put them in a 4°C refrigerator; It is a 800μm polytetrafluoroethylene (PTFE) tube, and the two tubes are merged into a polydimethylsiloxane (PMDS) three-way device to obtain cell spheroids, and then the three-way device is connected to another PTFE tube with a diameter of 800 μm, and the entire platform Place in a refrigerator at 4°C; the flow rate of the injection pump containing Matrigel is 25 μL / min, and the flow rate of the fluorine-containing oil injection pump is 55 μL / min, and then the cell spheroids are placed at 37°C with a concentration of 5% CO 2 The solidifi...

Embodiment 3

[0056] This embodiment provides a method for preparing organoid spheroids, comprising the following steps:

[0057] Use the primary cell extraction method to extract primary cells and count the cells; in an environment of 4°C, 1.0×10 6 1 primary cell and 80 μL of Matrigel were mixed thoroughly, injected into a 1mL syringe, and the other syringe was injected with fluorine oil. The fluorine oil and Matrigel were respectively placed in two syringe pumps and placed in a 4°C refrigerator; the syringes were respectively connected with diameters It is a 600μm polytetrafluoroethylene (PTFE) tube. The two tubes are merged into a polydimethylsiloxane (PMDS) three-way device to obtain cell spheroids, and then the three-way device is connected to another PTFE tube with a diameter of 600 μm. The entire platform Place in a refrigerator at 4°C; the flow rate of the injection pump containing Matrigel is 15 μL / min, and the flow rate of the fluorine-containing oil injection pump is 45 μL / min, a...