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A method for in vitro construction of human brain microangiogenesis simulating blood-brain barrier

A technology of blood-brain barrier and construction method, which is applied in the direction of artificial cell constructs, biochemical equipment and methods, microorganisms, etc., can solve the problem that it is difficult to faithfully obtain cells in the body with 2D models, and achieve the goal of reducing the amount of samples and reducing the cost of experiments Effect

Active Publication Date: 2022-04-05
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although animal models have been widely used in the research of various diseases, some scholars have realized in recent years that there are still huge differences between animal models and humans. Even animals that are very similar to humans cannot be directly used to predict human diseases.
Recently, the field of cell biology has begun to recognize the dissimilarity between the environment in which cells grow on these flat surfaces and the complex environment in which 3D cells grow in vivo, so it is difficult for 2D models to faithfully capture the physiological behavior of cells in vivo

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Preparation and cultivation of 3D cell culture chip

[0074] (1) Cell solution preparation: Primary GFP-HBVEC cells (purchased from Science cell, USA) and primary mCherry-HA cells (purchased from Science cell, USA) were respectively used in endothelial cell growth medium EGM-2 (purchased from Lonza) and astrocyte culture medium AM (purchased from Science cell, USA) were revived and cultivated until the cell confluency reached 80-90%, and the GFP-HBVEC cell suspension and mCherry-HA cell suspension were collected respectively 1 mL each, corresponding to the obtained endothelial cell suspension and astrocyte suspension, for later use; wherein, the resuscitated culture conditions are: culture temperature 37°C, culture humidity 95%, CO 2 Concentration 5%; The cell concentration in the GFP-HBVEC cell suspension and mCherry-HA cell suspension is 1×10 6 cell / mL.

[0075] (2) Preparation of fibrinogen stock solution: After preheating DPBS (purchased from Gibco, USA)...

Embodiment 2

[0080] Morphological characterization of embodiment 2 3D cell culture model

[0081] During the continuous culture in step (6) of the above-mentioned embodiment 1, the 3D cell culture chip was tomographically scanned using a laser confocal microscope, and a 3D fluorescent image was collected for cell morphology identification (GFP green fluorescence was excited at 488nm, excited at 509nm Emission light imaging; mCherry red fluorescence is excited at 587nm and emitted at 610nm for imaging), and the 3D fluorescence image acquisition results are as follows Figure 4 to Figure 8 as shown, Figure 4 to Figure 6 3D fluorescence images collected when the cells were grown in the microfluidic chip for 1 day, 2 days and 3 days, respectively, Figure 7 to Figure 8 3D fluorescent images collected for cells grown in a microfluidic chip for 4 days, Figure 7 The results of cells growing in the microfluidic chip for 4 days showing the formation of vascular tubule-like structures, Figure ...

Embodiment 3

[0086] Example 3 Verification of functional fluidity of 3D cell culture model

[0087] After the step (6) of the above-mentioned Example 1, microparticles were loaded into the 3D cell culture chip formed with the microvascular network structure of the brain, and the trajectory of the loaded microparticles in the microvascular network was observed by tomographic scanning. The microvascular network moves and migrates through the blood vessels with the fluid flow, and the monitoring results of some of its running tracks are as follows: Figure 9 as shown ( Figure 9 The granular substances shown in C are partially loaded particles, which are evenly distributed in the formed vascular network), indicating that the brain microvascular network generated by endothelial cells and astrocytes shows good structural integrity and Vascular permeability.

[0088] In summary, the present invention uses the mixed cells of primary human brain microvascular endothelial cells and primary human ...

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Abstract

The invention discloses an in vitro construction method for simulating the blood-brain barrier of human brain microvascular formation, which comprises the following steps: preparing human brain microvascular endothelial cell suspension and human brain astrocyte suspension, and preparing fibrinogen mother liquor and thrombin mother liquor ; Mix endothelial cell suspension, astrocyte suspension, DMEM medium, fibrinogen mother solution and thrombin mother solution to prepare a mixed cell gel solution; inject the mixed cell gel solution into the microfluidic chip After incubating at a constant temperature until gelation, adding endothelial cell growth medium to the microfluidic chip to construct a 3D cell culture chip; continuously culturing the 3D cell culture chip so that the endothelial cells and astrocytes The plasmocytes grow to form the microvascular network structure of the brain, which corresponds to the generation of a simulated blood-brain barrier. The technical solution provided by the invention has successfully established an in vitro model of the blood-brain barrier, which more clearly and accurately reflects the characteristics of the blood-brain barrier.

Description

technical field [0001] The invention relates to the technical field of tissue engineering, in particular to an in vitro construction method of human brain microangiogenesis simulating blood-brain barrier. Background technique [0002] The blood-brain barrier refers to the barrier between the plasma and brain cells formed by the brain capillary wall and glial cells, and the barrier between the plasma and cerebrospinal fluid formed by the choroid plexus. These barriers can prevent harmful substances from entering the brain tissue from the blood. It plays an important biological role in maintaining the normal physiological state of the central nervous system. Therefore, the study of the formation of the blood-brain barrier is of great significance for the study of related diseases. [0003] Most of the current two-dimensional (2D) blood brain barrier (BBB) ​​models are composed of monocultured brain microvascular endothelial cells, or developed from glial cells or astrocytes i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/0697C12N2502/086C12N2502/28C12N2513/00C12N2533/56
Inventor 吴扬宫智勇李岩
Owner WUHAN CHOPPER BIOLOGY
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