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25 (Chip Platform & Gel Specifications, Cell Seeding Densities and…
25
Chip Platform & Gel Specifications
Middle ECM:
neutralized rat collagen-I (5mg/mL)
1 M HEPES
37 g/L NaHCO3 at an 8:1:1 (v/v/v) ratio and dilute w PBS
Middle Channel Loading Volume: 3uL of collagen gel
Phaseguide Dimensions: 100um x 55um
Bottom Channel ECM: ES-qualified matrigel diluted on ice w LUHMES pre-differentiation medium to a final concentration of 5mg/mL
Channel Dimensions: 400um x 220 um (w x h)
bottom channel loading volume: 3uL of the cell-matrigel mixture
Chip Platform: 3-lane OrganoPlate (MIMETAS, #4004-400B)
coated w PBS-diluted collagen-IV (100ug/mL) and fibronectin (50ug/mL)
Cell Seeding Densities and Compartment Populations
Primary Human Brain Microvascular ECs (hBMECs) (Cell Systems, ACBRI 376)
Loading Volume: 3uL of cell solution added to the top channel inlet
Seeding density: resuspended in MV-2 medium at a density of 20,000 cells
Primary HA
5,000 cells/uL in the bottom channel
5,000 cells/uL embedded w/i bottom matrigel
LUHMES Neurons (ATCC, CRL-2927)
20,000 cells/uL w/i bottom matrigel
PBMCs
2,000,000 cells/mL for perfusion assays
culture media compositions
LUHMES Pre-differentiation medium
advanced DMEM/F12
0.5x GlutaMAX
1x N2 supplement
1x Antibiotic antimycotic
1mM db-cAMP
20ng/mL BDNF
20ng/mL GDNF
20ng/mL TGF-B3
0.2uM ascorbic acid
2ug/mL doxycycline
LUHMES Final Differentiation media
NeuroBasal (Thermo Fisher #21103049)
1x GlutaMAX
1x B27 supplement
1x antibiotic antimycotic
1mM db-cAMP
20ng/mL BDNF
20ng/mL GDNF
0.2 uM ascorbic acid
2ug/mL doxycycline
1/ muM DAPT
astrocyte growth medium (AGM) supplemented w 1x Antibiotic Antimycotic
PBMC Suspension Media
RPMI 1640 medium (Sigma-Aldrich, R8758)
2.5% (v/v) human serum
0.1% (v/v) 2-mercaptoethanol
50 IU/mL IL-2
1x antibiotic antimycotic
endothelial culture medium (MV-2)
MV-2 medium
initially 5% FBS to 2% FBS in the top channel after tube formation
1x Antibiotic Antimycotic