A tumor cell three-dimensional culture pool and a cell observation method

Abstract

The application belongs to the technical field of medical basic research, and particularly relates to a tumor cell three-dimensional culture pool and a cell observation method. The tumor cell three-dimensional culture pool comprises a culture dish, the bottom of the culture dish is a conductive transparent glass plate, the conductive transparent glass plate is embedded with a PEEK microfluidic chip, the culture dish is paved with a bottom glue, a dispensing warehouse is arranged on the bottom glue through etching of a PET material support, the sidewall of the dispensing warehouse is a polyurethane TPU film, pool gaps are arranged between the dispensing warehouse and the sidewall of the culture dish and between adjacent two dispensing warehouses, the pool gaps are filled with tumor cell culture solution, and the cell observation method is provided. Through the arrangement of the PET material support and the dispensing warehouse, the tumor cells can be prevented from being pulled by semi-fluid flow, and the tumor cells can be conveniently gathered into balls.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical basic research, and particularly relates to a tumor cell three-dimensional culture pool, a cell observation method and a culture matrix glue. BACKGROUND

[0002] Tumor cells growing in vivo are in a complex three-dimensional (3D) environment. The shape and chemical composition of the extracellular matrix (ECM) surrounding the cells can determine many physiological responses. Traditional cell culture techniques and experimental protocols are carried out on two-dimensional (2D) surfaces usually made of glass or polystyrene. However, since traditional two-dimensional (2D) cell lines cannot well simulate the original tumor growth environment, 3D tumor organoids are more similar to the biological characteristics of the original tumor, and are more likely to accurately predict the patient's response to drugs.

[0003] Existing 3D tumor organoid models are mostly obtained by taking cancer cells from patients, simulating the in vivo microenvironment in the laboratory through 3D biological scaffolds and other materials, and culturing micro-organoids with three-dimensional structure in vitro to form a "double" of the tumor, that is, a tumor organoid. The organoid is highly similar to the patient's own tumor cells in both genetic background and histological characteristics. As a "drug double" of the patient, the tumor organoid has the characteristics of high throughput and easy operation, and can obtain the sensitivity detection results of chemotherapy drugs, targeted drugs, monoclonal antibody drugs, and combined drugs in a short time (an average of two weeks), so as to obtain more valuable treatment time for patients with no available drugs and multiple available drugs, and provide more accurate data support for the development of drug regimens.

[0004] The microenvironment of the organoid is composed of cell sources or substances added to the system exogenously, and these factors interact to form a dynamic environment with structure and function, which guides the self-renewal, differentiation and formation of the organoid in time and space.

[0005] At present, the scaffold-free system is still the preferred culture method for organoid culture, but the size of the 3D structure formed on the scaffold-free system is often irregular. Without a scaffold, some types of cells will not aggregate, and it is not convenient to observe cancer cells in real time. Therefore, a tumor cell three-dimensional culture pool and a cell observation method are needed to solve the problem. SUMMARY

[0006] The purpose of the present application is to provide a tumor cell three-dimensional culture pool, a cell observation method and a culture matrix glue to solve the above problems.

[0007] To achieve the above purpose, the present application provides the following solutions:

[0008] A tumor cell three-dimensional culture pool comprises:

[0009] A culture dish, the bottom of which is a conductive transparent glass plate, and a PEEK microfluidic chip is embedded in the conductive transparent glass plate; a bottom glue is laid in the culture dish;

[0010] A dispensing compartment is arranged on the bottom glue by etching a PET material support, the sidewall of the dispensing compartment is a polyurethane TPU film, and a gap is arranged between the dispensing compartment and the sidewall of the culture dish and between adjacent two dispensing compartments;

[0011] The gap is filled with a tumor cell culture solution;

[0012] A matrix glue containing a tumor is planted in the dispensing compartment.

[0013] Preferably, the culture dish is a regular hexagonal structure.

[0014] Preferably, the dispensing compartment is a triangular structure.

[0015] Preferably, six dispensing compartments are circumferentially and equally spaced in the culture dish.

[0016] Preferably, the tumor cell culture solution is DMEM / RPMI with L-glutamine 4.5g / L glucose+F12 basic culture medium+FBS fetal bovine serum.

[0017] Preferably, the matrix glue includes 68% glue component+32% tumor primary culture medium.

[0018] Preferably, the glue component is 58% Matrigel hydrogel+10% polyglutamine peptide.

[0019] Preferably, the glue component is simultaneously added with growth factors, hormones and antibiotics.

[0020] A tumor cell three-dimensional culture pool cell observation method based on the above-mentioned tumor cell three-dimensional culture pool, comprising the following steps:

[0021] Step one, before culture, tumor cells are added with protein fluorescent labels and grouped according to requirements;

[0022] Step two, the tumor cell three-dimensional culture pool in which tumor cells are cultured is placed in a culture box with a timing fluorescent detection system for culture;

[0023] Step three, connect electrodes and computer transmission system;

[0024] Step four, set up computer software to receive signals at a timing.

[0025] Preferably, the timing fluorescence detection system comprises an excitation light source and a fluorescence signal receiver arranged in sequence along the light path, the excitation light source is installed at the bottom of the inside of the incubator, the fluorescence signal receiver is installed at the top of the inside of the incubator, and the tumor cell three-dimensional culture pool is located between the excitation light source and the fluorescence signal receiver.

[0026] Compared with the prior art, the present application has the following advantages and technical effects:

[0027] In use, first, the conductive transparent glass plate is arranged at the bottom of the culture dish, the PEEK microfluidic chip is embedded in the conductive transparent glass plate, then the bottom glue is arranged on the conductive transparent glass plate, the dispensing bin is installed on the bottom glue through the etched PET material support and the position of the dispensing bin is fixed, the pool gap is arranged between the outer wall of the dispensing bin and the inner wall of the culture dish and between adjacent dispensing bins, the matrix glue is filled in the pool gap, the dispensing bin is supported through the PET material support, the semi-fluid containing tumor cells is shaped through the dispensing bin, compared with the traditional method of directly arranging the semi-fluid containing tumor cells on the bottom glue, since the semi-fluid containing tumor cells diffuses under the action of gravity, the tumor cells are pulled into a plane due to fluid tension, which is not conducive to the process of gathering tumor cells into a ball, the setting of the PET material support and the dispensing bin can avoid the pulling of tumor cells by the flow of semi-fluid, facilitate the gathering of tumor cells into a ball, and the matrix glue filled in the pool gap can also better simulate the human body environment for culture, and the setting of multiple dispensing bins can realize the culture of multiple groups of cells, which facilitates the control test or parallel test according to experimental requirements. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings:

[0029] Figure 1 It is a structural schematic diagram of the present application;

[0030] Figure 2 It is a structural sectional view of the present application;

[0031] Figure 3 It is a schematic diagram of the detection and analysis principle of the present application;

[0032] Figure 4 It is a 5-day culture effect diagram of the scaffold-free 3D organoid model of the present application;

[0033] Figure 5 It is a 10-day culture effect diagram of the scaffold-free 3D organoid model of the present application;

[0034] Figure 6 Culture effect diagram of the 3D organoid model 5 days with the scaffold of the present application;

[0035] Figure 7 Culture effect diagram of the 3D organoid model 10 days with the scaffold of the present application;

[0036] Figure 8 4-fold white light diagram of the 3D organoid model 10 days without the scaffold of the present application;

[0037] Figure 9 10-fold white light diagram of the 3D organoid model 10 days without the scaffold of the present application;

[0038] Figure 10 4-fold fluorescence diagram of the 3D organoid model 10 days without the scaffold of the present application;

[0039] Figure 11 10-fold fluorescence diagram of the 3D organoid model 10 days without the scaffold of the present application;

[0040] Figure 12 4-fold white light diagram of the 3D organoid model 10 days with the scaffold of the present application;

[0041] Figure 13 10-fold fluorescence diagram of the 3D organoid model 10 days with the scaffold of the present application;

[0042] Figure 14 Tumor cell growth curve diagram detected by real-time fluorescence signal of the present application;

[0043] 1, conductive transparent glass plate; 2, culture dish 3, etched PET material scaffold; 4, pool gap; 5, dispensing bin. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0045] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0046] Embodiment 1:

[0047] Reference Figures 1 to 14 The present embodiment discloses a tumor cell three-dimensional culture pool, comprising:

[0048] The Petri dish 2 is provided with a conductive transparent glass plate 1 at the bottom, and the conductive transparent glass plate 1 is embedded with a PEEK microfluidic chip; and the Petri dish 2 is provided with a bottom glue.

[0049] The dispensing cabin 5 is arranged on the bottom glue through the etched PET material support 3, and the side wall of the dispensing cabin 5 is made of polyurethane TPU film. The pool gap 4 is arranged between the dispensing cabin 5 and the side wall of the Petri dish 2 and between adjacent two dispensing cabins 5.

[0050] The pool gap 4 is filled with tumor cell culture solution.

[0051] The Matrigel containing tumor is planted in the dispensing cabin 5.

[0052] In use, first, the conductive transparent glass plate 1 is arranged at the bottom of the Petri dish 2, and the PEEK microfluidic chip is embedded in the conductive transparent glass plate 1. Then, the bottom glue is arranged on the conductive transparent glass plate 1, and the dispensing cabin 5 is installed on the bottom glue through the etched PET material support 3 and is fixed in position. The pool gap 4 is arranged between the outer wall of the dispensing cabin 5 and the inner wall of the Petri dish 2 and between adjacent two dispensing cabins 5. The pool gap 4 is filled with tumor cell culture solution. The dispensing cabin 5 is supported by the PET material support 3, and the semi-fluid containing tumor cells is shaped by the dispensing cabin 5. Compared with the conventional method of directly arranging the semi-fluid containing tumor cells on the bottom glue, the semi-fluid containing tumor cells is not pulled into a plane by the fluid tension under the action of gravity, which is not conducive to the process of gathering tumor cells into a ball. The arrangement of the PET material support 3 and the dispensing cabin 5 can avoid the pulling of tumor cells by the semi-fluid flow, facilitate the gathering of tumor cells into a ball, and better simulate the human body environment for culture by the tumor cell culture solution filled in the pool gap 4. Multiple dispensing cabins 5 can be arranged to realize the culture of multiple groups of cells and facilitate the control test or parallel test according to the experimental requirements.

[0053] The number of electrodes of the PEEK microfluidic chip matches the number of dispensing cabins 5. The electrodes of each PEEK microfluidic chip extend into the corresponding dispensing cabin 5 and act on the tumor cells in the dispensing cabin 5.

[0054] The bottom glue is 100% Matrigel hydrogel.

[0055] In a further optimization scheme, the Petri dish 2 has a regular hexagonal structure.

[0056] In a further optimization scheme, the dispensing cabin 5 has a triangular structure.

[0057] According to the principle of bionics, the Petri dish 2 has a regular hexagonal structure.

[0058] In a further optimization scheme, six dispensing cabins 5 are arranged in the Petri dish 2 at equal intervals in the circumferential direction.

[0059] According to the structure of the culture dish 2, six dispensing chambers 5 are arranged in a triangular structure, so that the six dispensing chambers 5 can be arranged at equal intervals in the circumferential direction in the culture dish 2.

[0060] The hexagonal structure better simulates the real tumor microenvironment in vivo. The etched PET material + polyurethane TPU film is used to construct a scaffold structure for permeating gas, nutrients and macromolecules, which is beneficial to the proliferation and differentiation of tumor cells and facilitates the formation of tumor cell spheres.

[0061] Further optimization scheme, tumor cell culture solution is DMEM40% / RPMI40% with L-glutamine 4.5g / L glucose+F12 basic medium 30%F+FBS fetal bovine serum 30%.

[0062] Further optimization scheme, Matrigel includes 68% base glue component + 32% tumor primary culture medium.

[0063] Further optimization scheme, the base glue component is 58% Matrigel hydrogel + 10% poly glue peptide.

[0064] Further optimization scheme, the base glue component is added with growth factors, hormones and antibiotics.

[0065] A tumor cell three-dimensional culture pool cell observation method based on the above-mentioned tumor cell three-dimensional culture pool, comprising the following steps:

[0066] Step one, before culture, add protein fluorescent label GFP / RFP to tumor cells according to requirements and group them;

[0067] Step two, place the tumor cell three-dimensional culture pool containing tumor cells in a culture box with a timing fluorescence detection system for culture;

[0068] Step three, connect the electrode and the computer transmission system;

[0069] Step four, set up the computer software and receive the signal at a regular time.

[0070] Further optimization scheme, the timing fluorescence detection system includes an excitation light source and a fluorescence signal receiver arranged in sequence along the light path. The excitation light source is installed at the bottom of the inside of the culture box, and the fluorescence signal receiver is installed at the top of the inside of the culture box. The tumor cell three-dimensional culture pool is located between the excitation light source and the fluorescence signal receiver.

[0071] Further, the culture box is a carbon dioxide culture box, the culture temperature is 37℃, and the culture box is filled with 5% CO2.

[0072] Through the setting of the PEEK microfluidic chip, the real-time detection function of the electrical signal of the tumor microenvironment can be realized, and through the design of the fluorescent electrical signal, the change of the total fluorescence value can be measured at a fixed time, so as to draw the growth curve graph in the whole tumor ball formation process.

[0073] Embodiment 2:

[0074] This embodiment is one of the application examples of embodiment 1.

[0075] Step one: construct tumor organoids by puncture biopsy and surgical resection of tumors, count 8000 cells / ml after tumor cell separation, wrap them in extracellular matrix (ECM), and need to add specific growth factors in the growth medium for culture.

[0076] Before culture, tumor cells can be added with protein fluorescent labels (GFP / RFP) according to specific needs and grouped.

[0077] After laying the bottom glue on the conductive transparent glass plate 1, the dispensing warehouse 5 is fixed by etching the PET material support 3, and the tumor cell culture solution composed of DMEM (40%) / RPMI (40%) with L-glutamine 4.5g / L glucose+F12 basic culture medium (30%F)+FBS fetal bovine serum (30%) is poured into the pool gap 4.

[0078] Step two, place the tumor cell three-dimensional culture pool in the incubator with a timing fluorescent detection system;

[0079] The bottom of the culture dish 2 is a conductive transparent glass plate 1, which is embedded with a PEEK microfluidic chip. The PEEK microfluidic chip includes a plurality of nanofiber microelectrodes. The electrodes of the PEEK microfluidic chip are correspondingly arranged in each dispensing warehouse 5, which is used for sensing and detecting the chemical signal molecules released by cells.

[0080] Step three, connect the electrodes and the computer transmission system;

[0081] Step four, set up computer software to receive signals at a fixed time.

[0082] The incubator is a carbon dioxide incubator. In the incubator, the light source, the tumor cell three-dimensional culture pool and the fluorescent signal receiver are excited in sequence along the light path. Since the incubator is in a low-light environment and is sealed, it is convenient to use fluorescent real-time monitoring of cell culture. Through live cell fluorescent imaging technology, the total fluorescence amount can be monitored in real time, and the real-time imaging of individual proteins with GFP / RFP labels on the cell membrane can be taken. It is of great significance for the study of cell signal transduction mechanism.

[0083] Take BRCA mutant breast cancer as an example, use CRISPR / Cas technology to construct Trip13 gene knockout ATCC verified BRCA mutant triple negative breast cancer primary tumor cell line (PJMMR1 / Trip13KO) and verify it at the protein (Western blot) level. PJMMR1 / Trip13WT without knocking out Trip13 protein and PJMMR1 / Trip13KO after knocking out, label Trip13 protein with GFP green fluorescent tag, respectively construct tumor spheroids with scaffolds, simulate 3D co-culture of extracellular matrix ECM and lymph node mononuclear cells (LMNCs), tumor macrophages (M1, M2) in vitro, verify the success rate of model construction by IHC, and observe the distribution changes of lymphocyte subgroups at different culture stages before and after Trip13 protein knockout, and regularly detect the content of related cytokines in the model. At the same time, detect the growth of tumor cells by fluorescence signal.

[0084] Group control, construct BRCA mutant triple negative breast cancer primary tumor cell line (PJMMR1 / Trip13KO), and PJMMR1 / Trip13WT tumor cell line without knocking out Trip13 protein. After incubation with fluorescent protein label, take 8000 cells / ml of each matrix gel, and point glue 100ul of pure matrix gel + tumor cell mixture 330ul / well into triangle point glue warehouse 5. Each group has three warehouses for repeated holes.

[0085] Connect the electrode and the computer transmission system and put them into the incubator. The incubator culture conditions are: temperature 37 degrees, 5% CO2.

[0086] Set the computer software to receive signals at regular intervals.

[0087] After completing the experiment, IHC staining is used to determine the effectiveness of the model.

[0088] In the description of the present application, it should be understood that the terms "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only used for the purpose of facilitating the description of the present application, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore it cannot be understood as a limitation on the present application.

[0089] The above-described embodiments are only preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A three-dimensional culture pool for tumor cells, characterized in that, include: The culture dish (2) has a bottom of a conductive transparent glass plate (1) with an embedded PEEK microfluidic chip; the culture dish (2) is lined with a base adhesive. The dispensing tank (5) is set on the base adhesive by etching a PET material support (3). The side wall of the dispensing tank (5) is a polyurethane TPU film. There are gaps (4) between the dispensing tank (5) and the side wall of the culture dish (2) and between two adjacent dispensing tanks (5). The pore (4) is filled with tumor cell culture medium; The matrix gel containing the tumor was implanted in the dispensing tank (5); The culture dish (2) has a regular hexagonal structure; The dispensing tank (5) has a triangular structure; The dispensing tank (5) is provided in six parts, which are arranged at equal intervals around the perimeter inside the culture dish (2); The tumor cell culture medium was DMEM (40%) / RPMI (40%) with L-glutamine 4.5 g / L glucose + F12 basal medium (30% F) + FBS fetal bovine serum (30%). The matrix gel comprises 68% matrix gel components + 32% primary tumor culture medium; The base gel composition is 58% Matrigel hydrogel + 10% polyacrylamide peptide; A method for observing tumor cells in a three-dimensional culture pool includes the following steps: Step 1: Before culturing, add fluorescent protein labels (GFP / RFP) to the tumor cells as needed and group them accordingly; Step 2: Place the three-dimensional culture pool containing tumor cells in an incubator equipped with a timed fluorescence detection system for further culture. Step 3: Connect the electrodes and the computer transmission system; Step 4: Configure the computer software to receive signals at regular intervals; The timed fluorescence detection system includes an excitation source and a fluorescence signal receiver arranged sequentially along the optical path. The excitation source is installed at the bottom inside the incubator, and the fluorescence signal receiver is installed at the top inside the incubator. The three-dimensional culture pool for tumor cells is located between the excitation source and the fluorescence signal receiver. By configuring the PEEK microfluidic chip, real-time detection of electrical signals in the tumor microenvironment can be achieved. By designing the fluorescent electrical signals and measuring the changes in total fluorescence value at regular intervals, a growth curve can be plotted throughout the tumor spheroidization process.

2. The three-dimensional culture pool for tumor cells according to claim 1, characterized in that: The base adhesive also contains growth factors, hormones, and antibiotics.

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