Method for constructing glioma in-vitro 3D culture and analysis system and application

A construction method and technology for glioma, which is applied in the field of constructing an in vitro 3D culture and analysis system for glioma, and can solve the problems of single index for quantifying migration ability and inability to simulate completely.

Active Publication Date: 2021-06-08
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, they are all experiments on a 2D plane, which cannot fully simulate the actual situation in the body, and the indicators for quantifying the migration ability are also relatively simple: the scratch test is the change of the scratch area or width over time within a certain period of time, and the Transwell experiment is counting for a certain period of time. The amount of cells entering the lower chamber of the Transwell

Method used

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  • Method for constructing glioma in-vitro 3D culture and analysis system and application
  • Method for constructing glioma in-vitro 3D culture and analysis system and application
  • Method for constructing glioma in-vitro 3D culture and analysis system and application

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1. DBTRG, U251 cell lines can produce spheres with necrotic core cell sphere concentration

[0036] 1. The glioblastoma cell lines DBTRG and U251 cryopreserved in liquid nitrogen were recovered and subcultured;

[0037] 2. Obtain the cell suspension of DBTRG and U251 when the cells are subcultured, and use RPMI and DMEM complete medium containing 0.24% methylcellulose to dilute the cell suspension to 2.5kcell / 20μl, 5kcell / 20μl, Concentration of 1wcell / 20μl, 2w cell / 20μl;

[0038] 3. Pipette 20ul of cell suspension into the bottom center of the 24-well plate, cover the 24-well plate and turn the 24-well plate upside down. Finally, add about 5ml of PBS buffer solution to the 24-well plate cover to prevent the cell culture medium from evaporating during the culture process;

[0039] 4. Put the inverted 24-well plate into the cell incubator for culture, and image the cell suspension every 12 hours within 72 hours to observe the sphere formation of tumor multicellu...

Embodiment 2

[0040] Example 2. Quantification of elastic modulus parameters of matrix gels without and with hyaluronic acid

[0041] (1) Prepare a mixed gel of 0.5mg / ml Collagen I+3mg / ml Matrigel:

[0042] 1. Take out Collagen I and Matrigel matrigel, which are packed in 2ml centrifuge tubes and stored in refrigerators at 4°C and -20°C respectively, and store them in crushed ice (the whole process is performed on ice);

[0043] 2. Use 10×PBS, 1N NaOH, dH2O to dilute 3mg / ml Collagen I to 1mg / ml for later use. The specific method is as follows:

[0044] vt = total volume of collagen gel required, required collagen volume (v1) = (final concentration of collagen) x (total volume (vt)) divided by initial collagen concentration.

[0045] Required volume of 10xPBS (v2) = total volume (vt) / 10;

[0046] Required 1N NaOH volume (v3) = (v1) × 0.025;

[0047] Required dH2O volume (v4) = (vt) – (v1+v2+v3).

[0048] 3. Use RPMI and DMEM simple medium to dilute Matrigel to two concentrations of 6mg / m...

experiment example 1

[0057] Experimental example 1. DBTRG, U251 cell spheres embedded in 0.5mg / ml Collagen I+3mg / ml Matrigel+HA for migration

[0058] 1. Take a 96-well plate, add 50 μl of mixed gel to each well as an in vitro migration medium, embed DBTRG and U251 tumor multicellular spheres in it, and place them in a cell culture incubator for 30 minutes until the gel solidifies.

[0059] 2. Place the 96-well plate in the high-content cell imaging system, and perform imaging every 30 minutes for 60 hours.

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Abstract

The invention relates to the technical field of three-dimensional culture and analysis of tumor cells, and particularly discloses a method for constructing a glioma in-vitro 3D culture and analysis system and an application. The method comprises the steps that matrigel is prepared based on intracerebral components and elastic modulus, glioma tumor multicellular spheres obtained after hanging drop culture are embedded into the matrigel, and high-content cell imaging is combined for detection and quantitative analysis, so that the glioma in-vitro 3D culture and analysis system is obtained. According to the method for constructing the culture and analysis system, the biochemical composition and elasticity modulus of the matrigel are closer to the actual situation of the brain, the cell migration process in the model theoretically better conforms to the actual migration characteristics of glioma cells in the brain compared with a common scratch experiment and a Transwell experiment for detecting the cell migration ability at present, more information dimensions can be provided in the process of detecting the cell migration ability, and model support can be better provided for quantifying the migration ability of glioma cells and deeply exploring a glioma migration related mechanism.

Description

technical field [0001] The invention belongs to tumor cell three-dimensional culture and analysis technology, in particular to a construction method and application for constructing a glioma in vitro 3D culture and analysis system. Background technique [0002] Glioblastoma cells can often invade several centimeters away from the primary tumor and even migrate into the contralateral hemisphere (Michael D et al 2015)(K.Kallenberg 2013). However, unlike other malignant solid tumors, glioblastoma does not rely on vascular cavity or lymphatic cavity for metastatic spread. Instead, it mainly spreads infiltratively along the vessel wall and brain parenchyma (Vishnu Anand Cuddapah et al 2014). The large vessels of the brain and the postcapillary venules are surrounded by two layers of basement membrane, and the capillaries are surrounded by a layer of composite basement membrane (Michael Sixt et al 2001) (Reese T.S, Karnovsky M.J 1967), the basement membrane is composed of 50-100n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02
CPCC12N5/0693G01N33/5029G01N33/5011C12N2513/00C12N2533/80C12N2533/54C12N2533/90C12N2503/02G01N2500/10
Inventor 董志强王硕文
Owner HUAZHONG AGRI UNIV
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