Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Abstract

Source: Fattakhov, N., et al. A triple primary cell culture model of the human blood-brain barrier for studying ischemic stroke in vitro. J. Vis. Exp. (2022)

This video demonstrates the co-culturing of human brain pericyte, astrocyte, and endothelial cells. Each cell layer is formed separately and then cultured together, creating a three-layered system that mimics the cellular arrangement of the blood-brain barrier

Protocol

1. Triple cell culture BBB model setting

1. Seeding pericytes
   1. Cultivate HBVP (Human brain vascular pericytes) in T75 culture flasks with an activated surface for cell adhesion within a 5% CO₂ incubator at 37 °C until confluent. Once confluence is reached, aspirate the old pericyte medium and wash the cells with 5 mL of warm Dulbecco's phosphate-buffered saline (DPBS). Aspirate the DPBS and detach the cells from the flask using a combination of 4 mL of warm trypsin-EDTA solution and 1 mL of DPBS.
      ​NOTE: Avoid using passages later than P7.
   2. Incubate the flask for 5 min at 37 &....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 mm Transwell with 0.4 µm Pore Polyester Membrane Insert	Corning	3450
35 mm Glass Bottom Dishes	MatTek Life Sciences (FISHERSCI)	P35GC-1.5-14-C
Astrocyte Medium	Science Cell	1801
Attachment Factor	Cell Systems (Fisher Scientific)	4Z0-201
Complete Classic Medium With Serum and CultureBoost	4Z0-500	Cell Systems
Corning 50 mL PP Centrifuge Tubes (Conical Bottom with CentriStar Cap	VWR	430829
Corning 75cm² U-Shaped Canted Neck Not Treated Cell Culture Flask	Corning	431464U
Corning CellBIND 96-well Flat Clear Bottom Black Polystyrene Microplates	Corning	3340
Countes Cell Counting Chamber Slides	Thermo Fisher Scientific	C10228
Countess II FL Automated Cell Counter	Thermo Fisher Scientific	ZGEXSCCOUNTESS2FL
DMEM Medium (No glucose, No glutamine, No phenol red)	ThermoFisher	A14430-01	Glucose-free medium
DPBS (No Calcium, No Magnesium)	ThermoFisher	14190250
EBM Endothelial Cell Growth Basal Medium, Phenol Red Free, 500 mL	Lonza	CC-3129
CO2 incubator
Centrifuge