3D Human Blood Brain Barrier.

3D BBB-Model” specifically regulates molecular and cellular flux between the blood and the nervous tissue. With a “three-dimensional” model of the “human blood-brain barrier” to analyse the transport of “nanoparticles” and “astrocyte” endothelial” interactions.

Product Details
3D Human Blood Brain Barrier
CATALOG NUMBER: 3DB33002

APPLICATIONS: In vivo Like Assays
TYPE: 3D Cell/Tissue Models
STORAGE: Keep frozen in liquid nitrogen until plating
SHIPPING: Frozen (Dry Ice/Liquid Nitrogen)
SPECIES REACTIVITY: Human

Catalog #: 3DB33002
Storage: -80°C
Format: 6, 12 or 24 well

Storing of BBB model thawing-sol and incubation medium.
BBB is stored at -80  and can be used within one month. Media is stored at below -20.

Product Sizes     List Price                A Special Price          Catalog #
6 well                    $2,035.5                   $1,770.00                RGBBB-RB006
12 well                  $2,875.00                 $2,500.00                RGBBB-RB0012
24 well                  $4053.75                  $3,525.00                RGBBB-RB0024

General Information,
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. We develop and characterize a highly reproducible Human in vitro model of the BBB using co-cultures of primary Human brain endothelial cells (HBEC), Human brain pericytes, and Human brain astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Many drugs developed to treat Central Nervous System (CNS) disorders are unable to reach the brain parenchyma in therapeutically relevant concentrations. The BBB protects brain nervous tissue from the fluctuation of plasma composition, from pathogenic agents, and maintains homeostasis of the brain parenchyma by restricting non-specific flux of ions, peptides, proteins and even cells into and out the brain.

Model Components:

  • Culture Medium,
  • Frozen Model,
  • Suture Removal Kits.

Astrocytes and brain pericytes help to develop and maintain specific BBB characteristics in brain capillary endothelial cells. Co-culture of the three cell types in our 3D Human BBB model led to the enhancement of barrier properties; an increase in expressions of tight junction proteins of occludin, claudin-5 and ZO-1 and continuous localizations of ZO-1 and claudin-5.

Our model mimic transport properties of the BBB due to the formation of tight junctions, higher expression of specific carriers, or great cell viability. We developed a 3D in vitro model of the BBB by culturing brain endothelial cells with pericytes and astrocytes layered in an insert. This model improves endothelial cell polarization and enhance the formation of tight junctions, provide better endothelial cell-to-cell contact that is important for barrier development, and prevent the dilution of secreted neurotrophic factors, and these conditions collectively led to the development of an in vitro model that can truly mimic the BBB.

Advantages:

     1. Cells used in the 3D model are all human cells; results obtained are                more relevant to human situations rather than those data from animal                  models, i.e. CAM et al
     2, The whole process can be monitored (from cell inoculation to the end              of experiment), therefore, more crucial information can be acquired at                multiple time points from a single experiment.
    3, No need to perform post-experimental staining for endothelial markers,            this is particularly important, if those markers are changed in                                experimental conditions involved in the studies.

The 3D Human BBB Model contains all the materials necessary to perform multiple angiogenesis assays in 6, 12, or 24 well formats.

The 3D Human BBB model can be use, but not limited to;

  • Drug BBB permeability assay
  • Research on BBB physiology
  • Cell-cell interactions
  • Transport pathway modulations
  • Research on BBB     toxicology
  • Brain endothelial toxicity assays                   Microvessels
  • Research on BBB pathology.
  • Disease modeling
  • Transport and permeability studies from ions to macromolecules: effect of physiological or pathogenetic factors
  • Paracellular barrier and cell polarity studies: TJ protein expression, distribution, polarized distribution of transport proteins, receptors, enzymes etc
  • Studies on endo- and transcytosis, receptor-ligand interactions
  • Drug transport, drug effect on permeability, localization of receptors, polarity of drug responses
  • Co-culture studies: cell-cell and cell-matrix interactions
  • Microbial pathogenesis: virus, bacteria, parasite attachment, invasion and penetration
  • Compounds screening neuroimmune targets.
    Protocol;
    We deliver the 3D Human BBB model in frozen packaged with dry ice. The 3D Human BBB model can be frozen as a whole and stored at -80?. 4 days prior to your experiment, you just thaw your 3D Human BBB model stored at –80?.
    ” Volume of media to use when feeding models after thawing:
    Plates and Inserts  Blood-side Volume (?L)  Brain-side Volume (?L)
    6 well plate                     2000                                             1500
    12 Well Plate                     500                                              1500

        24 well plate                      200                                               500

3). Protocol (procedure of activating BBB model)

thawing*1?8 ?medium 1*9?10 ?incubation*11? medium 2*12?14 ?incubation*15 ?experiment*16 Medium 3
medium 1 Cat. #RGBBB-RB006 ; Blood Brain Barrier Growth Media 100mL
medium 2 Cat. #RGBBB-RB0012; Endo-Neuro-Pharmaceuticals Media 100mL
Medium 3 Cat. #RGBBB-RB0024; Blood Brain Barrier Transportation Media 100mL

Thawing the 3D Model:

 

 

  1. Warm medium 1 (Blood Brain Barrier Growth Media) to 37 ?, prior to De-freezing and warm up BBB model. (Move frozen medium 1 to 37 ? water-bath.)
  2. Once at 37 ? move medium 1 to clean bench.
  3. Move frozen BBB model to clean-bench. Take off seals. (Do not take a minute.)
  4. Wipe up water drops (humidity) on BBB model with clean papers.
    [5. On thawing (day 0)]

 6.

 Do not touch membrane of insert with pipette,
 and do nnsert, during procedures
of #1 to #9.

7) Under a biosafety cabinet Add 1,000 ?L medium 1 to Brain-side (to all 6,12-wells), through an opening between Inserts.
8) Add 630 ?L thawing-sol (medium 1) to Blood-side (inside of Insert) (to all 6,12 or wells).
9) Place model in a 37° C incubator with 5% CO2. Note: DO NOT MOVE THE INSERTS UNTIL THE MODEL IS FULLY THAWED
10)  Once thaw removes old medium on both sides by gently pipetting.

11)  Please see chart above title: Volume of media to use when feeding models after thawing for correct working volume. If using a 24 well model add 200 microliters of 37° C Blood Brain Barrier Growth Media to the Blood side 
12) Add 500 microliters of 37° C Blood Brain Barrier Growth Media the Brain side.
13) Incubate for 2-3 hours in a 5% CO2 incubator.
14) After 2-3 hours, remove the media and repeat step 3 and 4 using 37° C Endo-Neuro-Pharmaceuticals Media.
15) Incubate overnight in a 37° C incubator with 5% CO2 until the BBB kit is activated on day 4,5,6, and 7. Use Medium 3 (Cat. #RBGBBB001); Blood Brain Barrier Transportation Media when testing the penetration of a molecule through the brain endothelial and pericytes layer of BBB.
16) You can perform your experiment in days, 4, 5, 6, and 7 after thawing

17) See cells with inverted microscope.
Endothelial cells on Polycarbonate membrane of BBB model cannot be seen by microscope, therefore.
microscopic examination for astrocytes on bottom side of lower compartment must be done to check cell- proliferation.

BBB model endothelial cells (high magnification) astrocytes (high magnification)

18) See astrocytes through polycarbonate membranes with inverted icroscope.
BBB model

TEER (trans endothelial electrical resistance) in BBB model.
TEER in BBB model reaches more than 150 ?×cm2, and maintains a plateau up to 7 days. (Thawing-sol/ medium 1, and medium 2 do not contain cAMP and its analogs.) *BBB model can be used from Day 4 to Day 6 after thawing.
3D Human Blood Brain Barrier Permeability assay

When testing the penetration of a molecule through the brain endothelial and pericytes layer of BBB representing the BBB, in a blood-to-brain direction, the molecule is applied to the upper (luminal, blood-side) compartment of the insert. Transport is measured after a given time (?T) by detecting the amount of compound from the lower (basal, brain-side) compartment.

Test compound
      

             

 

?t
Test compounds (not provided)                 Collect sampleMeasure Conc.
?Stop watch (not provided)                      Calculate Papp?
Orbital Shaker (100 rpm) in Incubator 37? (not provided)

Summary:
Check TEER of activated BBB Make sure   TEER >150? X CM²
– Prepare Assay buffer, test compounds, wash plate and assay plate Permeability Assay 

– Measure concentration of test compound in lower compartment – Calculate permeability coefficient.

  • TEER measurement?
    6 well TEER (? x cm²) = (Total R – Blank R) x 4.298
    12 well TEER (? x cm²) = (Total R – Blank R) x 0.33.
    24 well TEER (? x cm²) = (Total R – Blank R) x 0.189Blank insert should be soaked with medium (DMEM) prior to its use for accurate reading Only use inserts with TEER value of 150 ? x cm2 or more for assay.
    Assay?
    *   Make sure final concentration of DMSO is equal to or less than 0.2% (v/v) when test compound is dissolved in DMSO. Concentration of test compound used should not be at the concentration which cause any cytotoxic effects. For unknown compounds start with 1?M and adjust concentration as required.
    *  Use orbital shaker (100 rpm) during incubation period for obtaining accurate result.

Check TEER of activated 3D BBB Mode
Measure TEER of activated BBB. Make sure TEER > 150 ?x cm2 before assay. Please refer to the protocol for TEER measurement of BBB.

Prepare Assay Buffer (DPBS-H), test compounds, wash & assay plate

  1. Preparation of Assay Buffer (DPBS-H); Mix as follows.
               10 x Dulbecco’s PBS (Ca+/Mg+)      10 mL
                1M HEPES (pH 7.0 – 7.6)                 1 mL
                D-glucose                                        0.45 g 
                distilled water                                   89 mL                                                             total                                     100 mL
  2. Prepare test compounds in Assay Buffer to appropriate concentration, then keep them at 37 °C.

 

 

3. Add 900 ?L of Assay Buffer into 12 wells of wash and assay plate, then keep them at 37 °C.
Permeability Assay
1.  Remove all 12 inserts from BBB into wells of the Wash Plate containing                assay buffer with clean tweezer.

 

  1. One insert at a time:
  • Pick it up with tweezer and remove culture media from luminal side, then return the insert into Wash Plate.
  • Add 200 ?L of Assay Buffer containing test compound which is kept at 37 °C.
  • Note: Do not wash inside of the insert with Assay Buffer.
  • Quickly transfer the insert from Wash Plate into Assay Plate.
  • As you transfer the first insert into the Assay Plate start stopwatch.
  • Place on a shaker inside an incubator when all 12 inserts are transferred to Assay Plate.
  • Incubate at 37 °C, 100 rpm, for <30 minutes.

Wash Plate with insert            Assay Plate with inserts         Stop Watch
containing Test Compound

3.Collect Assay Buffer from inserts and Assay Plate for measuring concentration of Test Compound (Apical and Basal concentration). Make sure you perform pipetting action x10 times to have no test compounds remaining at the bottom of the well.

Mix collected sample with Vortex. Measure concentration of Test Compound and determine permeability coefficient using Excel form provided.

  • We recommend using Millipore Plate (Millipore corporation #PIMW S24 50).
  • The volume of Assay Buffer and Test Compound Dissolved-Assay Buffer varies if another Assay Plate is used. Please refer below.
  • Assay Time:

The amount of Test Compound which penetrate through to the brain-side will be greater when assay time is increased. Although concentration measurement will be easier this way especially when detection-limit is low, the barrier-function (tight junction-function) of BBB Kit will deteriorate with time and hence increase paracellular transport of Test Compound. For this we recommend completing the assay within 30 minutes for accurate evaluation.

Blood-side Volume (?L) Brain-side Volume (?L)
 

 

Millipore Plates

100 600
200* 900*
300** 1,200**
 

 

Corning Plates

100 1,000
200 1,300
300 1,600
 

 

BD Plates

100 1,000
200 1,300
300 1,600

*Recommendation to use for permeability assay. **Volume used for BBB model activation.

Analysis:
1. Calculation of apparent permeability

 

 

 

 

 

 

 

 

 

 

 

We obtained a very good correlation between the BBB and in vivo permeabilities of drugs. You can evaluate the BBB-permeability by our BBB model, quantitatively. When you design molecular modifications of your compound or vectors carrying your compound into the brain, you can easily evaluate the BBB-permeability, quantitatively.

Papp?×10-6cm/s? Permeability ex)
 

 

 

Permeability’s

>20 very good Antipyrine
10?20 good
2?10 low Na-F
<2 very low ESA, sucrose

* Trans well® is trademark of Corning, Incorporated, Corning,
Plate and Wells information:
Membrane material    Polyethylene Terephthalate    Polyester
Pore Size (?m)                                  3.0                             0.4
Membrane Diameter (mm)               6.5                               12
Membrane Surface Area (cm2)       0.33                           
1.12

Apical Volume (?L) 200/300 500
Basolateral Volume (?L) 900/1200 1500
Height of insert (mm) 16
Pore Density (pores/cm2) 2 x 106 4 x 106
Membrane Thickness (?m) 9 10
Optical Property Translucent Clear
Cell visibility Poor good

 

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