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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







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

Documents

Blood-B rain Barrier ###,JU ä