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Method for establishing human blood-brain barrier model in vitro through 3D co-culture of four cells

A cell model and blood-brain barrier technology, applied in the field of cell models, can solve the problems of malnutrition, difficult treatment and intervention, growth restriction, etc., and achieve blood-brain barrier function breakthrough, easy treatment and intervention, and cell contact uniform effect

Pending Publication Date: 2022-04-05
广州市第一人民医院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages: Only 3 types of cells can be co-cultured at most, the cell growth environment is a 2D plane, and the growth of co-culture is limited
Disadvantages: The cell growth environment is a 2D plane, and the growth is limited. During in vitro co-culture, the cells are easily squeezed to death or starved to death due to insufficient growth space and excessive growth.
Disadvantages: Cells grow freely in a liquid environment without a tissue-like structure, and it is difficult to deal with and intervene in the later research on a certain single cell, and it is also impossible to truly simulate the establishment of the blood-brain barrier in the human body environment

Method used

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  • Method for establishing human blood-brain barrier model in vitro through 3D co-culture of four cells
  • Method for establishing human blood-brain barrier model in vitro through 3D co-culture of four cells
  • Method for establishing human blood-brain barrier model in vitro through 3D co-culture of four cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cell Culture and Treatment

[0032] Human primary brain microvascular endothelial cells (hpBEC) were purchased from Angio-Proteomie, USA. Human primary cerebral perivascular cells (hpPs) and primary human cerebral astrocytes (hpA) were purchased from ScienCell, USA.

[0033]Human neuroblastoma cell line SH-SY5Y (American Type Culture Collection) was grown in modified Eagle medium + F12 cell medium (1:1) (PAA Laboratories, Pasching, Austria), supplemented with 15% fetal Bovine serum, 1% penicillin / streptomycin. HpBECs were grown in endothelial basal medium (EGM-2) (Lonza, UK) supplemented with growth factors and 0.4% fetal calf serum. HpPs were grown in Pericyte Growth Supplemented Medium (ScienCell, USA), whereas hpA were grown in Astrocyte Growth Supplemented Medium (ScienCell, USA). HpBECs, hpPs and hpA were used for experiments between passage 2 to passage 4. Co-cultivation was performed by the RAFT 3D cell culture system (Lonza, London, UK) and cells w...

Embodiment 2

[0036] Example 2 Using 4 different cell types to co-culture with or without 6-OHDA and (or) glucose to establish NVU, PD, DM, PD-DM and down-regulated BBB cell models of the PDGFRβ gene

[0037] The present invention first established in vitro the 3D cell culture system (UK, Lonza) of the neurovascular unit (NVU) model composed of hpBEC, hpP, hpA and dopaminergic (DAergic) neuron cells (SH-5Y) (co-cultivation 6 sky). The model of the present invention can well simulate the arrangement of blood-brain barrier cells in vivo according to the order of hpBEC, hpP, hpAs and SH-SY5Y cells from inside to outside ( figure 1 ).

[0038] SH-SY5Y cells were divided into 1.0×10 5 Cells / ml were seeded in 24-well plates for 24 hours. SH-SY5Y cells received different concentrations of 6-OHDA (0, 50, 100, 150μmol / L) at different time points (0,8,24,48h). The concentration of 6-OHDA was selected as 50 μmol / L, and co-cultured for 24 hours (the survival rate of SH-SY5Y cells detected by trypan...

Embodiment 3

[0041] Example 3 Fluorescein Sodium (Na-FLU) Permeability Measurement

[0042] After the in vitro BBB cell model is completed, wash the inside and outside of the model 2 to 3 times with serum-free EBM-2 medium, and then add 200 μL of Na-FLU at a concentration of 100 mg / L to the inside of each well. Add 1.2 mL of EBM-2 medium externally. After 2 h and 24 h, remove 100 μL of medium from outside the pool. Use a microplate reader to measure the amount of Na-FLU in the BBB model under the condition that the excitation wavelength is 460nm and the emission wavelength is 515nm ( figure 2 ).

[0043] The beneficial effects of this embodiment are: the blood-brain barrier in vivo can be well simulated, and the arrangement order of the cells is consistent with the arrangement order of the blood-brain barrier in vivo, so as to better study the damage of Parkinson's and diabetes on the blood-brain barrier and how to protect it in the future Human blood-brain barrier research. The healt...

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Abstract

The invention relates to the field of cell models, in particular to a method for establishing a human blood-brain barrier model in vitro through 3D co-culture of four cells. The healthy blood-brain barrier cell model is obtained by co-culturing four cells; the four types of cells comprise a human primary microvascular endothelial cell hpBEC, a human primary pericyte hpPs (with or without a down-regulated PDGFR beta gene), a human primary astrocyte hpA and a human neuroblastoma cell line SH-SY5Y; the co-culture is carried out through an RAFT (Reversible Addition-Fragmentation Transfer) 3D cell culture system. The cell model provided by the invention can truly simulate the growth of cells in tissues rather than only in liquid, and can better simulate the growth environment of the cells in a human body and the arrangement sequence of the blood-brain barrier cells in the human body, so that the cell model has a great breakthrough compared with a traditional cell model when being used for researching the blood-brain barrier function and the damage of the blood-brain barrier function.

Description

technical field [0001] The invention relates to the field of cell models, in particular to a method for establishing a human blood-brain barrier model in vitro by 3D co-culture of four kinds of cells. Background technique [0002] Transwell model: The most traditional model used to study the blood-brain barrier, in which endothelial cells are grown on a filter cartridge with small holes in order to study the permeability between the two compartments. Under co-culture conditions, pericytes normally growing on the other side of the filter membrane may allow some cell-cell interactions through the pores, and astrocytes grow at the bottom of the lower chamber, thereby possibly influencing other cells through secreted factors. Disadvantages: Only 3 types of cells can be co-cultured at most, and the cell growth environment is a 2D plane, so the growth of co-culture is limited. Very easy to cause cell contamination and death. In fact, the human blood-brain barrier is composed of ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09C12N5/0793C12N5/079
Inventor 王婷
Owner 广州市第一人民医院
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