The hiBBB90 Model
Setup Protocolver. 1.3(updated on Mar. 25, 2021)/10© The authors of Mol Pharmaceutics 2019 doi: 10.1021/acs.molpharmaceut.9b00519. (PMID:31573814)

Overview & Medium composition
Days

-2-1

Culturetemperature

01

233℃

37℃

33℃

Step1:

Step2:

Step3:

35/37 seeding

35/37 differentiation

18/35/37 co-culture

HASTR/ci35
HBPC/ci37
seedingDifferentiation medium
Culture temperature shift

HBMEC/ci18
seeding

5Assays

Cells
HBMEC/ci18

HBPC/ci37
HASTR/ci35

MediumPGM

AGM

PDM

Medium name
CellsBasal medium
Culture supplements
Pen/st (#0503)
1%
FBS (#0010)
2%
Pericyte growth factor (#1252) 1%Blasticidin S
4 μg/mL

ADM

PGM
PDM*1
HBPC/ci37
Pericyte medium(Sciencell, #1201-b)＋＋＋＋＋－＋－

EM-comp

EM-V/E free

BPM

Medium nameAGM
ADM*2Cells
HASTR/ci35
Basal medium
DMEM
Culture supplements
100 units/mL-100 µg/mL＋＋Pen/st
1%＋＋N2 (apo)10%＋－FBS＋－
Blasticidin S 4 μg/mL
1 mM－＋dBcAMP*1, Opitional. While we used FBS-containing medium in our initial*2, it may be better to further add Glutamax or L-Alanyl-L-work (Ito et al. 2019), you may change it to FBS-free medium.

Glutamine as we did in our initial work (Ito et al. 2019).

Medium name

BPM

Cells

HASTR/ci35, HBPC/ci37

Basal medium
Pen/stN2 (apo)L-glutamine

Culture supplements
100 units/mL-100 µg/mL
1%2 mM

Medium name
Cells

Neurobasal medium (LifeTechnologies #21103049)＋＋＋EM-comp
EM-V/E free
HBMEC/ci18
VascuLife BM (#LEB-LM0002)
Basal medium/ EBM-2 BM (#CC-3156)
Culture supplements
VascuLife VEGF Lfactors (#LEKLS1020) or EGM-2 SingleQuots＋＋Supplements (#CC-4176) *3＋＋hFGF-b
5 ng/mL
Ascorbic acid＋＋
50 μg/mLHC＋＋
1 μg/mLhIGF-1＋＋
15 ng/mLhEGF＋－
5 ng/mL
VEGF＋－
5 ng/mL
Heparin＋＋0.75 unit/mL
L-glutamine＋＋
10 mM
FBS＋＋2%100 units/mL-100 µg/mL
Pen/st＋＋
Blasticidin S 4 μg/mL＋－EM-comp, Endothelial complete medium;
EM-V/E free, VEGF- and hEGF-free EM-comp
FBS, fetal bovine serum
HC, hydrocortisone
hFGF-b, human fibroblast growth factor-basichIGF-1, human insulin-like growth factor-1
hEGF, human epidermal growth factor
VEGF, vascular endothelial growth factor
Pen/st, penicillin streptomycin
AGM, astrocyte growth medium
ADM, astrocyte differentiation medium
DMEM, Dulbecco’s modified Eagle medium
dBcAMP, dibutyryl cyclic adenosine
monophosphate
PGM, pericyte growth medium
PDM, pericyte differentiation medium
BPM, brain parenchyma medium*3, other types of endothelial medium

may be used.

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Summary of each preparation step (for 12-well format)
Day

Step1-1: Extracellular matrix (ECM) coating-2

Add ECM sol
Remove ECM
Wash with PBS(-)
Incubation
Dried by air
Dried by airfor 30 min
for 30 min
37℃, 1 h

ECM coated33℃

Step1: 35/37 seeding

Step1-2: HBPC/ci37 (pericyte) seeding
Seed HBPC/ci37 cells

Replace insert

Pericyte medium (PM)Stand for30 min

33℃ culture for 1 day

100 mm dishStep1-3: HASTR/ci35 (astrocyte) seeding
Seed HASTR/ci35 cells
Astrocyte growth medium(AGM)-1

Step2-1:HBPC/ci37 (pericyte) differentiation
Seed HBPC/ci37 cells

37℃

Step2: 35/37 differentiation

Medium change
Pericyte growth medium(PGM)

HBPC/ci37 maturation

HASTR/ci35 cells
37℃ culture
for 1 day

PBS(-) wash
Medium change

Astrocyte differentiation medium(ADM)

HASTR/ci35 maturation

Step3-1:HBMEC/ci18 (endothelial cells) seeding

Step3: 18/35/37 co-culture

33℃
1Pericyte differentiation medium(PDM)

Step2-2: HASTR/ci35 (astrocyte) differentiation

Astrocyte growth medium(AGM)

037℃ culture
for 1 day

PBS(-) wash

Collect BMECs

HBMEC/ci18 cells

Vas-V/E freemedium

Step3-3:Combine insert and receiver plate
33℃ culture
for 1 dayStep3-2:Medium change to BPM
Brain parenchyma medium(BPM)

EPA model
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Step1-1: Extracellular matrix coating
Step1-1: Extracellular matrix (ECM) coating
Add ECM sol
Remove ECM
Wash with PBS(-)
Incubation
Dried by air
Dried by airfor 30 min
for 30 min
37℃, 1 h

ECM coated Set the inserts on the culture plates※1, then add 250 μL and 750
μL ECM solution※2 to the apical and the basolateral side,
respectively. Stand at 37℃ for one hr.
 Remove the ECM solution and dry the inserts and the plates byair for 30 min.
 Wash the inserts and the plates with PBS (-).
 Dry the inserts and the culture plates well for 30 min※3.
※1
In our study, 12-well culture plate and insert (translucentpolyethylene terephthalate, 0.4 μm high-density pores, BD Falcon)
are used. However, other culture materials can be used.
For 24-well, adjust the volume proportionally.
※2
ECM solution is a mixture of type-IV collagen and fibronectin (finalconcentration: 0.1 mg/mL of each).
Prepare at the time of use.
※3Make it sure that the inserts are completely dried out.

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Step1-2: HBPC/ci37 (pericyte) seeding
Step1-2: HBPC/ci37 (pericyte) seeding
Seed HBPC/ci37 cells

Replace insert

Stand for30 minPericyte medium (PM)33℃ culture for 1 day

100 mm dish

 Place the insert in a container (which is sufficiently larger than theinsert: e.g. 100-mm dish) in an upside-down manner.
 Prepare 300 μL HBPC/ci37 cell suspension at 3.0×104 cells/cm2using the pericyte medium and place them gently on the insert※4.
 Stand it in the clean bench until the cells adhere (approx. 30 min)※5.
 Turn over the insert upside down and carefully set it in the culture
plate, where the pericyte medium are filled in advance※6.
 Culture at 33℃ in 5% CO2/95% air.
※4
Seed gently, or the cell suspension may fall into the bottom. For a 24well format, the volume should be adjusted according to themanufacturer’s instruction.
※5
No problem if a small portion of cell suspension drops to the bottom
during the incubation, unless most cells remain attached.
※6Confirm that a large number of the cells cannot be found in the bottomof the plate with a microscope.
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Step1-3: HASTR/ci35 (astrocyte) seeding
Step1-3: HASTR/ci35 (astrocyte) seeding
Seed HASTR/ci35 cells

33℃ culture for 1 day

Astrocyte growth medium(AGM) Prepare 750 μL HASTR/ci35 cell suspension at 2.5×104 cells/cm2
using the astrocyte growth medium and place it on the culture
plate※7.
 Culture at 33℃ in 5% CO2/95% air.
※7Important! To prevent the cells from clustering in the center of theplate, shake gently the plate and make them uniformly attached.
For a 24-well format, the volume should be adjusted according to
the manufacturer’s instruction.

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Step2-1: HBPC/ci37 (pericyte) differentiation
Step2-1:HBPC/ci37 (pericyte) differentiation
HBPC/ci37 cells
PBS(-) wash

37℃ culture
for 1 dayMedium change
Pericyte growth medium(PGM)

Pericyte differentiation medium(PDM)

HBPC/ci37 maturation

 Confirm that a large number of HBPC/ci37 cells are not found inthe culture plate with a microscope.
 Gently wash twice with PBS (-).
 Change the medium from PGM (for cell proliferation) to PDM (forcell differentiation)※8.
 Culture at 37℃ in 5% CO2/95% air※9.

※8Please note that in our published paper, we use PGM only. While
we assume PDM would be better, you can follow the publishedprotocol. In such case, please skip the medium change and just dotemperature change alone.

※9Make sure that the temperature is set at 37℃.

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Step2-2: HASTR/ci35 (astrocyte) differentiation
Step2-2: HASTR/ci35 (astrocyte) differentiation
HASTR/ci35 cells
PBS(-) wash

37℃ culture
for 1 dayMedium change
Astrocyte growth medium(AGM)

Astrocyte differentiation medium(ADM)

HASTR/ci35 maturation

 Gently wash twice with PBS (-)※10.
 Change the medium from AGM (for cell proliferation) to ADM (forcell differentiation).

 Culture at 37℃ in 5% CO2/95% air※11.
 Confirm that the HASTR/ci35 cells show differentiated morphology
at several hours after the medium change※12.

※10
Important! Wash out FBS completely, or the differentiation wouldbe insufficient.
※11Make sure that the temperature is set at 37℃.
※12
Please refer to Kitamura et al. J Pharmacol Sci. 2018;137(4):350358.

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Step3-1: HBMEC/ci18 (endothelial cells) seeding
Step3-1:HBMEC/ci18 (endothelial cells) seeding
HBMEC/ci18 cells

Collect BMECs

Vas-V/E freemedium

 Prepare 750 μL HBMEC/ci18 cell suspension at 1.3×105 cells/cm2
using the VEGF/EGF-free endothelial medium, and gently place iton the insert※13.

※13
For a 24-well format, the volume should be adjusted according tothe manufacturer’s instruction.

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Step3-2: Medium change to BPM
Step3-2:Medium change to BPM

Brain parenchyma medium(BPM) Change the medium from ADM to BPM (2250 μL) ※14.

※14
For a 24-well format, the volume should be adjusted according to
the manufacturer’s instruction.

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Step3-3: Tri-coculture
Step3-3:Combine insert and receiver plate
HBMEC/ci18 cells

33℃ culture
for 1 dayAssaysBrain parenchyma medium(BPM)

EPA model

 Carefully transfer the inserts to the culture plate and start coculturing.
 Culture at 33℃ in 5% CO2/95% air※14.

※14Make sure that the temperature is set at 33℃.

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The hiBBB90 model setup protocol ver. 1.3 Mar. 25, 2021.
This is the first version and will be updated without any prior notices.
Any questions and comments would be greatly appreciated.
Contact Information:
Tomomi Furihata, Ph.D.
Department of Clinical Pharmacy and Experimental Therapeutics
School of Pharmacy
Tokyo University of Pharmacy and Life Sciences
1432-1 Horinouchi, Hachioji, Tokyo, JAPAN 192-0355
Tel/Fax: +81-42-676-8969/5809
tomomif@toyaku.ac.jp
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