WO2020215487A1 - Method for preparing organoid spheroid - Google Patents

Abstract

Provided is a method for preparing an organoid spheroid. The preparation method comprises: matrigel containing cells, and a fluorocarbon oil being respectively introduced into a three-way device so as to obtain a cell spheroid; and forming an organoid spheroid by means of cultivation. According to the provided method for preparing an organoid spheroid, a mono-dispersed biological material organoid is produced by means of a microfluidic method, thereby realizing high-throughput production, and the size, shape and structure of the organoid are controllable and uniform. In comparison with a 2D tumor sensitivity prediction model, the prepared organoid is combined with a tumor microenvironment factor such that a prediction result is more accurate; in comparison with a PDX mouse tumor sensitivity prediction model, the present invention has a shorter modeling period and lower cost; and in comparison with normal gene sequencing, the present invention has a higher clinical prediction rate. The provided method for preparing an organoid has a significant meaning and value with regard to clinical application, and provides an important basis for the detection of related results.

Description

Method for preparing organoid sphere Technical field

The application belongs to the field of medical devices and relates to a method for preparing organoid spheres.

Background technique

Organoids are multi-cell clusters constructed by three-dimensional (3D) culture in vitro, which have the ability to self-renew and self-organize, and maintain the physiological structure and function of their source tissues. Tumor organoids are organoids derived from tumor tissues and are a new and powerful tool for studying tumor biology. Organoid technology is the fastest growing type of in vitro culture technology in the past ten years, and was once hailed as one of the most important scientific developments 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.

The current organoid culture technology is to encapsulate pluripotent embryonic stem cells or induced pluripotent stem cells in an extracellular matrix matrigel, and then culture them in a medium containing specific cytokines, such as epidermal growth factor, R-spondin protein and noggin. Long-term maintenance of the growth of small intestine organoids, and colon-derived organoids need to add nicotinamide, p38 inhibitor SB202190, prostaglandin E2 and Alk inhibitor A83-01. After a period of time, stem cells will form a collection of organ-specific cell types and have certain specific functions of the organ, such as secretion and contraction. Organoids that have been successfully cultured include brain, lung, liver, kidney, prostate, pancreas, colorectal, etc.

Cancer has always been the most important issue affecting human health in the world. Although in recent years, cancer research and treatment have achieved many breakthrough results, including the discovery of immune checkpoints and the birth of CAR-T immunotherapy. However, as more and more cancer drugs and treatment methods are available, more and more drugs are available for clinical patients. Individualized treatment based on patients is increasingly being valued by doctors and patients. The traditional tumor screening model is mainly a 2D tumor cell line model. Although this model is simple and fast for modeling, it does not respond well to tumor growth under physiological conditions due to the lack of tumor microenvironment leading to changes in tumor gene expression, so it is based on 3D The cultured tumor organoids can provide a reliable model for tumor research and treatment. At present, most tumor organoid models remain in the experimental stage, and it is difficult to carry out high-throughput drug screening. At the same time, organoids of different sizes affect the accuracy of experimental results to a certain extent.

Microfluidic technology is a technology that manipulates fluids in micron-scale space. It has attracted the attention of researchers due to its advantages of high throughput, high sensitivity, and low consumption. It is a field involving chemistry, fluid physics, microelectronics, and new technologies. The emerging interdisciplinary of materials, biology and biomedical engineering. Microfluidic technology mainly applies fluid laminar flow and droplet phenomena. Laminar flow corresponds to turbulent flow, which refers to the laminar flow of fluid, the streamline of which is parallel to the pipe wall. When the viscous force is far greater than the inertial force, or the Reynolds number is less than 3000, laminar flow will appear. When fluids of several phases and different colors enter the same microchannel from different inlets, even if they dissolve each other, a multi-phase parallel flow with distinct layers will be formed. Using this geometric regularity of laminar flow, an orderly arrangement of materials, chemical environment and cells in the microchannel can be realized. Microfluidics can prepare highly monodisperse droplet emulsions with very high throughput. The common microchannel structures are T-type and ψ-type. In some cases, the aqueous liquid containing different high molecular polymers will also form immiscible droplets in the microfluidic channel.

CN109136185A discloses a preparation method and application of a kind of organ organs of the brain, and specifically provides a method and culture conditions for stem cells to differentiate and develop in vitro to form a cortical organoid with a three-dimensional structure and a vascular structure. Moreover, with continuous cultivation, a 3D system with neuronal functions and mature neural circuit connections can be established, making it closer to the developmental process of the cerebral cortex from embryonic neurogenesis to postnatal synaptic development and functional establishment. The products cultivated with this culture system can be used in disease research and drug screening.

CN108486035A discloses a droplet culture method of three-dimensional organoids, which relates to a droplet culture method of three-dimensional organoids and its application in biomedicine. Combining the pipette tip with conventional cell culture methods, using the pipette tip to form droplets in a spherical conformation as a culture carrier, culture and prepare millimeter-level three-dimensional organoid tissues. By screening and analyzing factors such as the cutting position of the pipette tip, the volume of the injected liquid, and the contact angle between the droplet and the cross-section, the ideal organoid droplet culture conditions were obtained. However, this method cannot guarantee uniformity and the handling is poor.

Therefore, how to develop an organoid with uniform shape and size, achieve high-throughput production, and make the prediction accuracy of anticancer drugs in clinical application is of great significance for the application of organoids.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of this application is to provide a method for preparing organoid spheres to solve the problem of the existing methods for preparing organoids that cannot be prepared with high throughput, and the resulting organoids are uneven in shape and size, which affects the prediction results. Issues of accuracy and repeatability.

To achieve the purpose of this application, this application adopts the following technical solutions:

This application provides a method for preparing a kind of organ spheroids. The preparation method includes: passing matrigel and fluorine oil containing cells into a three-way device to obtain cell spheroids, which are cultured to form organoid spheroids.

The preparation method of organoid spheres provided in the present application uses a microfluidic method to generate monodisperse biomaterial organoids, which realizes high-throughput production. The size, shape and structure of the organoids are controllable and uniform, compared to 2D Tumor sensitivity prediction model. The organoids prepared in this application combine tumor microenvironmental factors to predict the results more accurately; compared with the PDX mouse tumor sensitivity prediction model, the modeling period of this application is shorter and the cost is lower; Compared with ordinary gene sequencing, the clinical prediction rate of this application is higher.

At present, there are many anti-tumor drugs, and at the same time, the heterogeneity of tumors and the differences in the sensitivity of patients to drugs make it impossible for patients to receive optimal individualized treatment. Existing anti-cancer drug sensitivity test models include 2D tumor cell models and PDX models. The 2D cell tumor cell model lacks the tumor tissue microenvironment, so the prediction accuracy of anti-cancer drugs is low, while the PDX mouse model is over Long, the cost is too high, the success rate is too low, and there are species differences that cause inaccurate predictions.

In addition, the traditional tumor organoid production process cannot achieve high-throughput applications and cannot be applied to a large number of drug screening. At the same time, the generated organoids have different shapes and sizes, which affects the accuracy and repeatability of the results to a certain extent.

Preferably, the cell-containing matrigel is prepared by mixing cells and matrigel.

Preferably, the number of the cells is 1.0×10 ⁵ to 1.0×10 ⁷ ; for example, it can be 1.0×10 ⁵ , 1.0×10 ⁶ , 2.0×10 ⁶ , 3.0×10 ⁶ , 4.0 ×10 ⁶ pieces, 5.0×10 ⁶ pieces, 1.0×10 ⁷ pieces, etc., preferably 2.0×10 ⁶ pieces.

In this application, the number of cells cannot be too high or too low. If the number of cells is too low, the cells can hardly grow; if the number of cells is too high, the formed sphere will be explosive.

Preferably, the cell is a primary cell.

The primary cells described in this application are obtained through extraction. Primary cell extraction method: The cancer tissue or animal tissue obtained by surgery or puncture is cut into small pieces of about 1 mm under the condition of 4 degrees, and digested with type I collagenase in a 37 degree cell incubator for 1 hour, using a 100 μm filter Filter the cells with a mesh, lyse the red blood cells with the red blood cell lysate, centrifuge and resuspend the cells in culture medium, and count the cells for later use.

Preferably, the volume of the matrigel is 10-1000 μL, for example, 10 μL, 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 550 μL, 600 μL, 650 μL, 700 μL, 750 μL, 800 μL , 850 μL, 900 μL, 950 μL, 1000 μL, etc., preferably 100 μL.

Preferably, the mixing temperature is 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc., preferably 4°C.

In this application, fluorine oil can better produce droplets, and can produce good liquid-liquid interfacial tension and shear force for the cell-containing Matrigel, so that the cell-containing Matrigel fluid forms a highly uniform intermittent flow. The fluorine oil and the cell-containing matrigel will form an emulsion. Through this microfluidic method, the organoids are sheared and wrapped into spheres from the bottom up, so that the organoid spheres can finally be formed.

Preferably, the method of access is injection.

The method of access described in this application is not limited to injection, and any method that can add two liquids to the tee is applicable to this application.

Preferably, the injection method is as follows: placing the cell-containing Matrigel and fluorine oil in two injection devices respectively, and then injecting them into the three-way device through a pipe.

In this application, the injection device is generally connected to two of the three-way devices through a pipe, and the other pipe of the three-way device is used to transfer the formed organoids.

Preferably, the flow rate of the cell-containing matrigel is 1-100 μL/min, for example, 1 μL/min, 5 μL/min, 10 μL/min, 15 μL/min, 16 μL/min, 17 μL/min, 18 μL/min, 19μL/min, 20μL/min, 21μL/min, 22μL/min, 23μL/min, 24μL/min, 25μL/min, 30μL/min, 40μL/min, 50μL/min, 60μL/min, 70μL/min, 80μL/ min, 90 μL/min, 100 μL/min, etc., preferably 20 μL/min.

Preferably, the flow rate of the fluorine oil is 1-500 μL/min; for example, it can be 1 μL/min, 5 μL/min, 10 μL/min, 15 μL/min, 20 μL/min, 25 μL/min, 30 μL/min, 40 μL/min. , 45μL/min, 46μL/min, 47μL/min, 48μL/min, 49μL/min, 50μL/min, 51μL/min, 52μL/min, 53μL/min, 54μL/min, 55μL/min, 75μL/min, 100μL /min, 200 μL/min, 300 μL/min, 400 μL/min, 500 μL/min, etc., preferably 50 μL/min.

Preferably, the temperature of the injection device is 2-8°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc., preferably 4°C.

Preferably, the diameter of the pipe is 200-1000 μm, for example, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm or 1000 μm, preferably 800 μm.

Preferably, the method of culturing is culturing in a culture medium after coagulation.

Preferably, the solidification temperature is 35-40°C, for example, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C, etc., preferably 37°C.

In this application, the heating process causes the organoid to solidify. If the heating process is not passed, the organoid cannot form a regular spherical shape, which affects its uniform, stable and controllable effect.

Preferably, the solidification is performed in an atmosphere containing CO ₂ at a concentration of 5%, and then organoids are obtained.

This application is the concentration of 5% CO ₂ refers to the ordinary air, into an amount such that the mixed gas CO CO ₂ accounted for 5% by volume after _2.

Preferably, the culture medium is DMEM/F12 medium.

Preferably, the modified DMEM/F12 medium contains growth factors.

Preferably, the preparation method includes the following steps: the preparation method includes the following steps: mixing 1.0×10 ⁵ to 1.0×10 ⁷ cells with 10 to 1000 μL Matrigel at 2 to 8° C. to obtain a cell-containing Matrigel , And then put the cell-containing Matrigel and fluorine oil in two injection devices at a temperature of 2～8℃, and inject them into the three-way device through a pipe with a diameter of 200～1000μm to obtain cell spheroids. The flow rate of Matrigel is 1-100μL/min, and the flow rate of fluorine oil is 1-500μL/min. Then, the cell spheres formed are solidified in an atmosphere with a temperature of 35-40°C and a concentration of 5% CO ₂ . The modified DMEM/F12 medium was added to culture the organoid spheroids.

Compared with the prior art, this application has the following beneficial effects:

The preparation method of organoid spheres provided in the present application uses a microfluidic method to generate monodisperse biomaterial organoids, which realizes high-throughput production. The size, shape and structure of the organoids are controllable and uniform, compared to 2D Tumor sensitivity prediction model. The organoids prepared in this application combine tumor microenvironmental factors to predict the results more accurately; compared with the PDX mouse tumor sensitivity prediction model, the modeling period of this application is shorter and the cost is lower; Compared with ordinary gene sequencing, the clinical prediction rate of this application is higher; in summary, the preparation method of organoids provided in this application has important significance and value for clinical application, and provides an important basis for the detection of related results.

Description of the drawings

Figure 1 is a schematic diagram of an organoid preparation process provided by the present application.

Figure 2 is a partial enlarged view of the confluence of the water phase and the fluorine oil in this application.

Figure 3 is the organoid sphere prepared in Example 1 of the present application.

Detailed ways

The technical solution of the present application will be further explained through specific implementations below. It should be understood by those skilled in the art that the described embodiments are only to help understand the application and should not be regarded as specific limitations to the application.

A schematic diagram of one of the preparation processes provided in this application is shown in Figure 1. It can be seen from Figure 1 that the matrigel containing cells in the water phase enters the tee from one of the syringes, and the fluorine oil enters the tee from the other syringe. Then converge in the three links to form organoids. Among them, Fig. 2 is a partial enlarged view of the two-phase confluence of the three links in Fig. 1, and the formation process of organoids of uniform shape and size can be seen from Fig. 2.

The brand of Matrigel used in this application: R&D systems, item number: 3533-010-02.

The composition of the modified (Advanced) DMEM/F12 medium described in this application: 20% fetal bovine serum, 100ng/mL Noggin, 50ng/mL FGF4, 50ng/mL FGF-basic, 200ng/mL R-spondin 1.

Example 1

This embodiment provides a method for preparing an organoid sphere, which includes the following steps:

Use the primary cell extraction method to extract the primary cells and count the cells; mix 2.0×10 ⁶ primary cells with 100 μL Matrigel in a 4 ℃ environment, and inject them into a 1 mL syringe. Inject fluorine oil into the other syringe. Fluorine oil and matrigel were placed in two syringe pumps and placed in a refrigerator at 4°C; the syringes were connected to a polytetrafluoroethylene (PTFE) tube with a diameter of 800 μm, and the two tubes were joined to polydimethylsiloxane (PMDS) Cell spheres are obtained from the three-way device, and then another PTFE tube with a diameter of 800μm is connected to the three-way device, and the entire platform is placed in a 4℃ refrigerator; the flow rate of the Matrigel-containing injection pump is 20μL/min, and the flow rate of the fluorine-containing oil injection pump is 50μL/min. Then, the cell spheroids were solidified in an atmosphere containing 5% CO ₂ at 37° C., and finally the modified DMEM/F12 medium was added to culture the organoid spheroids. The obtained organoid sphere microscope observation chart is shown in FIG. 3.

It can be seen from Fig. 3 that the obtained organoid spheres are uniform in shape and size.

Example 2

This embodiment provides a method for preparing an organoid sphere, which includes the following steps:

Use the primary cell extraction method to extract the primary cells, and count the cells; mix 5.0×10 ⁶ primary cells with 200μL Matrigel at 4℃ and inject them into a 1mL syringe. Inject fluorine oil into the other syringe. Fluorine oil and matrigel were placed in two syringe pumps and placed in a refrigerator at 4°C; the syringes were connected to a polytetrafluoroethylene (PTFE) tube with a diameter of 800 μm, and the two tubes were joined to polydimethylsiloxane (PMDS) Cell spheres are obtained from the three-way device, and then the three-way device is connected to another PTFE tube with a diameter of 800μm. The entire platform is placed in a 4℃ refrigerator; the flow rate of the Matrigel-containing injection pump is 25μL/min, and the flow rate of the fluorine-containing oil injection pump is 55μL/min. Then, the cell spheroids were solidified in an atmosphere containing 5% CO ₂ at 37° C., and finally the modified DMEM/F12 medium was added to culture the organoid spheroids.

Example 3

This embodiment provides a method for preparing an organoid sphere, which includes the following steps:

Use the primary cell extraction method to extract the primary cells, and count the cells; mix 1.0×10 ⁶ primary cells with 80 μL Matrigel in a 4 ℃ environment, and inject them into a 1 mL syringe. Inject fluorine oil into the other syringe. Fluorine oil and matrigel were placed in two syringe pumps and placed in a refrigerator at 4°C; the syringes were connected to a 600μm polytetrafluoroethylene (PTFE) tube, and the two tubes were joined to polydimethylsiloxane (PMDS) Cell spheres are obtained from the three-way device, and then another 600μm diameter PTFE tube is connected to the three-way device, and the entire platform is placed in a 4℃ refrigerator; the flow rate of the Matrigel-containing injection pump is 15μL/min, and the flow rate of the fluorine-containing oil injection pump is 45μL/min. Then, the cell spheroids were solidified in an atmosphere containing 5% CO ₂ at 37° C., and finally the modified DMEM/F12 medium was added to culture the organoid spheroids.

Example 4

The difference between this embodiment and Embodiment 1 is that the number of primary cells in this embodiment is 3.0×10 ⁷ , and the rest are the same as Embodiment 1.

Due to the excessive number of cells, the viscosity of the matrigel increases, and the shearing force generated by the fluorine oil is not enough to cut the matrigel at a uniform speed, resulting in the formation of organoid spheres of different sizes. At the same time, the formed spheres are not easily cross-linked and easily damaged.

Example 5

The difference between this embodiment and Embodiment 1 is that the number of primary cells in this embodiment is 0.1×10 ⁵ , and the rest are the same as in Embodiment 1.

Because the number of cells is too low, the interaction between cells is reduced, cell growth is slow, and the purpose of rapid drug screening cannot be achieved.

Comparative example 1

The difference between this comparative example and Example 1 is that instead of using a three-way device, the fluorine oil and the matrigel containing primary cells are directly mixed.

Since there is no three-way device, the fluorine oil and the Matrigel containing primary cells are incompatible, and the fluorine oil will be separated from the water phase on the top.

The applicant declares that this application uses the above-mentioned embodiments to illustrate the preparation method of the organoid spheres of this application, but this application is not limited to the above-mentioned process steps, which does not mean that this application must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvements to this application, the equivalent replacement of raw materials selected in this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

Claims (15)

1. A preparation method of organoid spheroids includes: passing matrigel and fluorine oil containing cells into a three-way device to obtain cell spheroids, which are cultured to form organoid spheroids.
2. The preparation method according to claim 1, wherein the cell-containing matrigel is prepared by mixing cells and matrigel.
3. The preparation method according to claim 1 or 2, wherein the number of the cells is 1.0×10 ⁵ to 1.0×10 ⁷ ; preferably 2.0×10 ⁶ cells.
4. The preparation method according to claim 1, wherein the cell is a primary cell.
5. The preparation method according to any one of claims 1 to 4, wherein the volume of the matrigel is 10-1000 μL; preferably 100 μL.
6. The preparation method according to any one of claims 1-5, wherein the temperature of the mixing is 2-8°C; preferably 4°C.
7. The preparation method according to any one of claims 1 to 6, wherein the method of access is injection.
8. The preparation method according to any one of claims 1-7, wherein the method of injection is: placing the matrigel containing cells and the fluorine oil in two injection devices respectively, and then injecting them into the three through the pipes. Pass in the device.
9. The preparation method according to any one of claims 1-8, wherein the flow rate of the cell-containing matrigel is 1-100 μL/min; preferably 20 μL/min.
10. The preparation method according to any one of claims 1-9, wherein the flow rate of the fluorine oil is 1-500 μL/min; preferably 50 μL/min.
11. The preparation method according to any one of claims 1-10, wherein the temperature of the injection device is 2-8°C; preferably 4°C.
12. The preparation method according to any one of claims 1-11, wherein the diameter of the pipe is 200-1000 m, preferably 800 m.
13. The preparation method according to any one of claims 1-12, wherein the method of culturing is culturing in a culture medium after coagulation.
14. The preparation method according to claim 13, wherein the solidification temperature is 35-40°C; preferably 37°C;

    The solidification is carried out in an atmosphere containing CO ₂ at a concentration of 5%;

    The culture medium is a modified DMEM/F12 medium; and

    The modified DMEM/F12 medium contains growth factors.
15. The preparation method according to any one of claims 1-14, wherein the preparation method comprises the following steps: 1.0×10 ⁵ ～1.0×10 ⁷ cells and 10～1000 μL Matrigel are heated at 2～8°C The matrigel containing cells is obtained by mixing, and then the matrigel containing cells and the fluorine oil are respectively placed in two injection devices at a temperature of 2 to 8°C, and injected into the three-way device through a pipe with a diameter of 200 to 1000 μm. Cell spheroids, in which the flow rate of the matrigel containing cells is 1-100μL/min, and the flow rate of the fluorine oil is 1-500μL/min; then the cell spheres are generated at a temperature of 35-40°C and contain a concentration of 5% CO Solidify under the atmosphere of ₂ and add modified DMEM/F12 medium to culture to obtain organoid spheroids.

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