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Preparation method of organoid spheres

A technology of organoids and spheroids, which is applied in the field of preparation of organoid spheroids, can solve the problems that organoids cannot be prepared in high throughput, affect the accuracy and repeatability of prediction results, and the shape and size of organoids are not uniform, reaching the modeling cycle Short, controllable shape and structure, low cost effect

Active Publication Date: 2019-07-12
TSINGHUA BERKELEY SHENZHEN INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing organoid spheroids, so as to solve the problem of inability to produce high-throughput organoids in the existing method, and the shape and size of the generated organoids are not uniform, which affects the prediction results. Questions of Accuracy and Repeatability

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  • Preparation method of organoid spheres

Examples

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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...

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Abstract

The invention provides a preparation method of organoid spheres. The preparation method comprises the steps that matrigel containing cells and fluorocarbon oil are introduced into a three-way device to obtain cell spheres, and after culture is performed, the organoid spheres are formed; according to the preparation method of the organoid spheres, the micro-fluid control method is adopted for generating mono-disperse biomaterial organs, high-throughput production is achieved, the sizes, shapes and structures of organoids are controllable and uniform, and compared with a 2D tumor sensitivity predicting model, the organoids are combined with tumor micro-environment factors, and the prediction result is more accurate; compared with a PDX mouse tumor sensitivity predicting model, the modeling period is shorter, and the cost is lower; compared with common gene sequencing, the clinic predicting rate is higher; the preparation method of organoid spheres has the important significance and valuefor clinic application, and the important basis is supplied to detection of related results.

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...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/06C12N2501/10
Inventor 马少华蒋盛威
Owner TSINGHUA BERKELEY SHENZHEN INST
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