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How to Use BBB Kit™ ( RBT-24H )

Thawing (De-freezing) and Activation of BBB Kit

RBE-24 Protocol
[ PDF-1.27MB ]

1. Schedule after thawing BBB Kit ™

  Amounts of solutions (to be added)
  Thawing-sol medium (1) medium (2*
Blood-side 200 μL 300 μL 300 μL
Brain-side 1000 μL 1200 μL 1200 μL

* Recipe: medium 10% PDS / DMEM F-12
  Dulbecco's Modified Eagle's Medium/ Nutrient Mixture F-12 Ham (DMEM F-12)  
Fetal Bovine Plasma Derived Serum (PDS) 10% (v/v)
Heparin 100 μg/mL
basic fibroblast growth factor (bFGF) 1.5 ng/mL
Insulin-Transferrin-sodium Selenite (ITS) insulin 5 μg/mL,
transferrin 5 μg/mL,
sodium selenite 5 ng/mL
Hydrocortisone 500 nM
Gentamicin 50 μg/mL

2. Storing of BBB Kit ™, thawing-sol and incubation medium
BBB Kit ™ is stored at -80 ℃, and can be used within one month.
Thawing-sol and incubation medium are stored at below -20℃.

3. Protocol (procedure of activating BBB Kit ™)
  [ On thawing (day 0) ]

1. Warm Medium to 37 ℃, prior to De-freezing and warm up BBB Kit ™.
(Move frozen thawing-sol to 37 ℃ water-bath.)
2. Move Medium to clean-bench.
3. Move a BB Kit ™ in frozen to clean-bench. Take off seals.
(Do not take a minute.)
4. Wipe up waterdrops (humidity) on BBB Kit with clean papers.
5. Add 1,000 μl Medium to Brain-side (to all 12-wells),
through an opening between Inserts.
Do not touch membrane of insert with pipette, and do not move insert, during procedures of
#1 to #5.
6. Add 200 μl Medium to Blood-side (inside of Insert) (to all 12-wells).
7. Wipe up humidity on surface and bottom of BB Kit ™.
8. Incubate BBB Kit for 2 to 3 hrs in CO2 incubator. During this incubation, Medium warm to 37 ℃
9. See cells with inverted microscope.

astrocytes enodthelium
Endothelial cells on Polyethylene terephthalate membrane of BBB Kit (RBT-24) cannot be seen by microscope, therefore microscopic examination for astrocytes on bottom side of lower compartment has to be done to check cell-proliferation.
10. Using a pincet to lift-up the insert, remove the present Medium of the brain-side. Return the insert to the plate, then remove the present Medium of the blood-side. Add 300 μL new medium to the blood-side very gently, and then 1200 μL new to the brain-side.
Notice that do not touch the cells or membrane of the insert.
Repeat this work to finish all 12 wells.
11. Incubate BBB Kit ™ with  Medium in CO2 incubator, overnight.

  [ On day 1 ( the next day after thawing of BBB Kit ™ ) ]

12. See astrocytes through polycarbonate membranes with inverted microscope.
13. Warm Medium to 37 ℃ in water-bath. (Move frozen Medium 37 ℃ water-bath. )
14. Remove Medium from Brain-side, and add 900μL ( (red arrow), then remove from Blood-side, and add 200μL (Do not touch cells, carelessly. Add Medium very gently.)
15. Incubate BBB Kit ™ with  Medium in CO2 incubator for 3 days. (from thawing day (Day 0) to Day 4)
16. On Day 4, BBB Kit ™ is activated functionally, and maintains BBB function until Day 7. Use activated-BBB Kit ™ on Day 4. (You can store activated- BBB Kit ™ in CO2 incubator at 37 ℃. We recommend you to use the BBB Kit ™ until Day 6.)

Schedule (example)

4. TEER (trans endothelial electrical resistance ) in BBB Kit ™ (RBT-24H)
TEER in BBB Kit ™ (RBT-24) reaches more than 150 Ω×cm2, and maintains a plateau up to 6 days.
(Medium does not contain cAMP and its analogs.)

*BBB Kit ™ can be used from Day 4 to Day 6 after thawing.

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