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in vitro model ofBlood-Brain Barrier
BBB Kit™
www.pharmacocell.co.jp/en/
Reliable and Practical tool for CNS drug development BBB permeability Asssay BBB-targeted Drug discovery
The BBB is mainly composed of brain capillary endothelial cells (BECs). Recent studies have shown that the cross talk between BECs and neighboring cells like brain Pericytes and Astrocytes maintains BBB properties.BBB Kit™ is the worldʼs first in vitro BBB model not only using BECs but also brain Pericytes and Astrocytes.
The Blood-brain barrier (BBB) and BBB KitTM
BBB KitTM Ready-to-Use in vitro BBB model・ Containing Primary cultured BBB-related cells. Brain capillary Endothelial Cells (Passage 1) Brain Pericytes (Passage 3) Astrocytes (Passage 3)
・ All cells are pre-seeded and frozen at -80℃.
・ Delivered packed in dry ice.
・ Can be stored at -80℃ for a month after delivery.
-80℃
Time schedule of Experiments with BBB KitTM
1) Thaw BBB KitTM
2) Change MediumDay 0
3) Change MediumDay 1
Day 4to
Day 64) Check Integrity by TEER5) Carry out experiments
SUN MON TUE WED THU FRI SAT
Day0
Day1
Day4
Day6
TEER > 150 Ω x cm2
Simply thaw, incubate, do your experiments,in just a week!
TJs formation of Primary cultured BECs
Confluent monolayers of primary cultured brain capillary endothelial cells (BECs) in BBB KitTM give positive immuno stainings for von Willebrand factor (vWF, EC marker) and TJ proteins (claudin-5, occludin, ZO-1).
Day
0
100
200
300
2 3 4 765
Volt-Ohm meter
Ω
Trans Endothelial Electrical Resisitance (TEER) indicates the tightness of TJs between BECs.TEER can be measured with Ohm meter and EndOhm electrodes noninvasively.
Expressions of BBB-related transport-ers in BECs of BBB KitTM were detected by RT-PCR.
109841 2 3 65 7200 100
band size(bp)Lane Transporter Substrate
12345678910
ABCB1 (MDR1, P-gp)
ABCG2 (BCRP)
ABCC1 (MRP1)
ABCC2 (MRP2)
ABCC4 (MRP4)
ABCC5 (MRP5)
SLC2A1 (GLUT1)
SLC16A1 (MCT1)
SLC7A5 (LAT1)
MFSD2A docosahexaenoic acid (DHA)
cimetidine, imatinib, prazosin
glucose
digoxin, verapamil, quinidine
lactic acid, ketone bodies
vincristine, vinblastine
L-type amino acids
cisplatin, valsartan
acyclovir, methotrexate
methotrexate, rosuvastatin
90
125
116
104
110
102
116
123
126
103
Transporters expressing in BBB KitTM
0
R12
3 Pe
(% o
f A→
B)
50
100
200
150
250
**p < 0.01 vs. control (n=3), day 8
**
B
A
B
A
Functional analysis of P-gp using Rhodamine123, a P-gp substrate
BBB permeability assay with BBB KitTM
Compounds
~
Permeability assay for Small and large molecules Peptides and proteins Antibodies Nano particles
BBB KitTM - Product Information -
※ 12 wells are available as an in vitro BBB model
Product Name Product No. Cell type, origin Format
BBB KitTM
(Monkey)
BBB KitTM
(Rat)PCC-RBT-24H
PCC-MBT-24H
BECs : Wistar ratPericytes : Wistar rat
Astrocytes : Wistar rat
BECs : Macaca irusPericytes : Wistar rat
Astrocytes : Wistar rat
12 inserts (0.4 μm, PET) in
24-well plate ※
12 inserts (0.4 μm, PET) in
24-well plate ※
Customized BBB KitTM is also available. Please contact to “[email protected]”
Customization pointsCell arrangements (BECs-Astrocytes model, BECs-Pericytes model, BECs mono-model)Medium compositionCulture inserts (Membrane pore size, material) Multiwell plates (12-well plate, 6-well plate)
Nakagawa S et al. Neurochem. Int. 54: 253-263, 2009
●Development of BBB Kit™・ Nakagawa S et al. Cell. Mol. Neurobiol. 27: 687-694, 2007・ Nakagawa S et al. Neurochem. Int. 54: 253-263, 2009
●Review : in vitro BBB model・ Deli MA et al. Cell. Mol. Neurobiol. 25: 59-127, 2005・ Deli MA et al. Handbook of Neurochem. Mol. Neurobiol. 11: 29-56, 2007
●BBB research using BBB Kit™ and related in vitro BBB model・ Horai S et al. Cell. Mol. Neurobiol. 33: 291-307, 2013・ Takeshita T et al. Mol. Cell. Neurochem. 60: 1-9, 2014・ Muramatsu R et al. J. Biol. Chem. 290(18): 11515-11525, 2015・ Xu L et al. Int. J. Nanomedicine. 29(10): 6105-6118, 2015・ Miyazaki W et al. Neurotoxicology 52: 64-71, 2016・ Takagi T et al. J. Cereb. Blood Flow Metab. 37(1):123-139, 2017・ Hashimoto Y et al. J. Pharmacol. Exp. Ther. 363(2): 275-283, 2017
●Brain metastasis research using BBB Kit™ (Monkey type)・ Tominaga N et al. Nat. Commun. 6: 6716, 2015
●Permeability assay using BBB Kit™ (Rat type)・ Imamura S et al. Cell. Mol. Neurobiol. 31: 787-793, 2011・ Tabuchi M et al. Cell. Mol. Neurobiol. 32: 1139-1146, 2012・ Bohara M et al. J. Cereb. Blood Flow Metab. 34(4): 589-596, 2014・ Spencer B et al. J. Biol. Chem. 289(25): 17917-17931, 2014・ Pelisch N et al. PLoS One 10(4): e0124510, 2015・ Pervin M et al. Biochem. Biophys. Rep. 9: 180-186, 2017・ Kurokawa Y et al. Int. J. Nanomed. 12: 3967-3975, 2017・ Watanabe Y et al. Bioorg. Med. Chem. Lett. 27(15): 3495-3498, 2017・ Yoshimaru T et al. Sci. Rep. 7(1): 1821, 2017
●Permeability assay using BBB Kit™ (Monkey type)・ Salcedo Gómez PM et al. Antimicrob. Agents Chemother. 57(12): 6110-6121, 2013・ Amano M et al. Antimicrob. Agents Chemother. 59(5): 2625-2635, 2015
BBB KitTM - References -
www.pharmacocell.co.jp/en/
BBB KitTM-related Products Inquiry and Ordering from outside JapanPlease contact below,IWAKI & CO., LTD.Bio-Products Marketing, Research & Development Group30-17, Toshima 7-Chome, Kita-ku, Tokyo 114-0003 JapanTEL : +81-3-3913-4148 FAX : +81-3-3913-4149URL : https://www.iwaki-kk.co.jp/en.htmlE-mail : [email protected]
About usPharmaCo-Cell Company, Ltd. was established on Feb. 2005 as a bio venture spin off from Nagasaki University School of Medicine, Japan.
●R&D Partners ・ BBRC, Hungarian Academy of Sicences, Hungary ・ Nagasaki University, Japan ・ Fukuoka University, Japan
D-FLAG 305, 1-43 Dejima, Nagasaki 850-0862 JapanTEL: +81-95-895-7431 / FAX: +81-95-895-7432URL: www.pharmacocell.co.jp/en/E-mail: [email protected] Nagasaki, Japan
■Primary cells
Product Name Product No. Passage Format
Brain capillary Endothelial Cells(Wiatar Rat)
Brain capillary Endothelial Cells(Wiatar Rat)
Brain Pericytes(Wiatar Rat)
Brain Astrocytes(Wiatar Rat)
Brain capillary Endothelial Cells(Macaca irus monkey)
Brain capillary Endothelial Cells(Macaca irus monkey)
Brain Pericytes(Macaca irus monkey)
5 x 105 cells / vial
1 x 106 cells / vial
5 x 105 cells / vial
1 x 106 cells / vial
5 x 105 cells / vial
1 x 106 cells / vial
5 x 105 cells / vial
PCC-WRBEC-5105
PCC-WRBEC-1106
PCC-MIBEC-5105
PCC-MIBEC-1106
PCC-WRBPC-5105
PCC-WRBAC-5105
PCC-MIBPC-5105
1
1
1
1
2
2
2
■Cell Culture Reagents
Product Name Product No. Volume Notes
Brain capillary Endothelial CellMedium
Brain capillary Endothelial CellMedium
Brain capillary Endothelial CellCoating Solution
Type IV CollagenCoating Solution
PCC-BECM-100
PCC-BECM-500
PCC-BECCS-10
PCC-CL4CS-10
10 mL
10 mL
100 mL
500 mL
Fibronectin / Type IV Collagen