TITLE: Protocol for Subculture of Differentiated Blood-Brain Barrier Endothelial Cells onto Plates and Filters
AUTHORS: Ethan Lippmann, Hannah Wilson, Emma Neal

[STEPS]
?. [Subculturing (Day 6)]
Please select between subculturing onto plates or filters.
?. [Subculturing (Day 6)]
Coat plates with ECM plate solution for at least  at . Volume depends on plate type (see Table):  ABC1Plate type for subculture phaseVolume of ECM solution for coatingWorking volume of EC media for cell culture26-well800 μl2 ml312-well250 μl1 ml424-well200 μl500 μl548-well100 μl400 μl696-well50 μl200 μl
37 °C
ABC1Plate type for subculture phaseVolume of ECM solution for coatingWorking volume of EC media for cell culture26-well800 μl2 ml312-well250 μl1 ml424-well200 μl500 μl548-well100 μl400 μl696-well50 μl200 μl
If desired, plates may be coated  . If coating overnight, add necessary volume of ECM and an equal volume of ddH2O to each well to prevent excessive evaporation. If using glass plates, overnight incubation is needed to achieve adequate protein adsorption.
?. [Subculturing (Day 6)]
Aspirate plates and allow to dry in sterile hood (place the plate in the back of the hood and leave the lid slightly ajar).
Plates only need to dry for   (can be aspirated during accutase incubation). Do not over dry!
?. [Subculturing (Day 6)]
Retrieve cells from incubator and transfer equal volume of spent media to 15 ml conical corresponding to the number of wells being accutased.
For example, if accutasing 4 wells, save 4 ml of spent media and discard the rest.
?. [Subculturing (Day 6)]
Wash each well once with  PBS.
2 ml
?. [Subculturing (Day 6)]
Add  accutase (warmed to ) to each well.
1 ml
37 Room temperature
?. [Subculturing (Day 6)]
Incubate at , length of time depends on cell treatment:
37 °C
?. [Subculturing (Day 6)]
Using p1000, collect cells, and spray gently over surface 2–3x to dislodge any remaining cells. Triturate briefly to break up cell clumps.
?. [Subculturing (Day 6)]
Add cells to 15 ml conical containing spent media.
?. [Subculturing (Day 6)]
Spin down cells at .
Centrifuge: 1000 33, 4 min
?. [Subculturing (Day 6)]
Aspirate media, and resuspend cells in appropriate volume of EC media (with or without  RA). For 6- and 12-wells, cells are seeded based on a split ratio:1 well of a 6-well plate is split to 1 well of a 6-well plate [1:1] 1 well of a 6-well plate is split to 3 wells of a 12-well plate [1:3]For smaller plates (24-, 48-, or 96-wells), seed 1 million cells/cm2.Multiply split ratio by the working volume found in the table to arrive at total volume of EC media (with or without  RA) in which to resuspend cells.
?. [Subculturing (Day 6)]
Thoroughly triturate 3 – 4 times to yield single cell suspension.
?. [Subculturing (Day 6)]
Add appropriate volume of cells to each well.
?. [Subculturing (Day 6)]
Place plate in incubator, shaking plate back and forth to distribute cells evenly (do not swirl).
?. [Subculturing (Day 6)]
24 hours later (i.e., day 7), aspirate spent media and add appropriate volume of EC medium (without bFGF and RA).
?. [Subculturing (Day 6)]
Coat Transwell filters with  of ECM filter solution.
Note: owing to the complexity of filter washing and separate addition of media to the top and bottom chambers of filters, make sure you calculate the necessary volumes of EC media before starting the passaging step.
200 µl
?. [Subculturing (Day 6)]
Aspirate filters and allow to dry in sterile hood, similar to the description for plates. Filters should dry for at least  but no longer than . If cells are still dissociating after , move to step 10.1 before performing steps 5 – 9.
?. [Subculturing (Day 6)]
Retrieve cells from incubator and transfer equal volume of spent media to 15 ml conical corresponding to the number of wells being accutased.
For example, if accutasing 4 wells, save   of spent media and discard the rest.
?. [Subculturing (Day 6)]
Wash each well once with  PBS.
2 ml
?. [Subculturing (Day 6)]
Incubate at . The length of time depends on cell treatment:
37 °C
?. [Subculturing (Day 6)]
If cells have been treated with RA, incubate at  for  – , or until cells are dissociated from plate.
37 °C
?. [Subculturing (Day 6)]
Following accutase incubation, use p1000 to collect cells and spray gently over surface 2–3x to dislodge any remaining cells. Triturate briefly to break up cell clumps.
?. [Subculturing (Day 6)]
Add accutased cells to the conical containing spent medium.
?. [Subculturing (Day 6)]
Aspirate media, resuspend cells in appropriate volume of EC media (with or without  RA). Cells are seeded based on a 1:3 ratio (i.e. 1 well of a six well plate seeds three Transwell filters). For 3 filters, resuspend cells in  EC medium, for 6 use , etc.
1.5 ml
3 ml
?. [Subculturing (Day 6)]
Place plate in incubator, shaking plate back and forth to distribute cells evenly (do not swirl).
?. [Subculturing (Day 6)]
24 hours later (i.e., day 7), aspirate and change to EC medium lacking bFGF or RA. Gently add EC medium lacking bFGF or RA,  in top chamber and  in bottom chamber. DO NOT include bFGF or RA in the medium – these factors prevent the monolayer from tightening.
0.5 ml
1.5 ml
?. [Subculturing (Day 6)]
If cells have been treated with RA, incubate at  for  to  typically suffices, though up to  may be necessary for some differentiations.
37 °C
?. [Subculturing (Day 6)]
If cells have not been treated with RA, incubate at  for , or until cells are dissociated from plate (whichever comes first).
37 °C
?. [Subculturing (Day 6)]
Using p1000, collect cells, and spray gently over surface 2–3x to dislodge any remaining cells. Triturate briefly to break up cell clumps.
?. [Subculturing (Day 6)]
Add cells to 15 ml conical containing spent media.
?. [Subculturing (Day 6)]
Spin down cells at .
Centrifuge: 1000 33, 4 min
?. [Subculturing (Day 6)]
Aspirate media, and resuspend cells in appropriate volume of EC media. For 6- and 12-wells, cells are seeded based on a split ratio:1 well of a 6-well plate is split to 1 well of a 6-well plate [1:1] 1 well of a 6-well plate is split to 3 wells of a 12-well plate [1:3]For smaller plates (24-, 48-, or 96-wells), seed 1 million cells/cm2.Multiply split ratio by the working volume found in the table to arrive at total volume of EC media in which to resuspend cells.
?. [Subculturing (Day 6)]
Thoroughly triturate 3 – 4 times to yield single cell suspension.
?. [Subculturing (Day 6)]
Add appropriate volume of cells to each well.
?. [Subculturing (Day 6)]
Place plate in incubator, shaking plate back and forth to distribute cells evenly (do not swirl).
?. [Subculturing (Day 6)]
24 hours later (i.e., day 7), aspirate spent media and add appropriate volume of EC medium (without bFGF or RA).
?. [Subculturing (Day 6)]
Incubate at least  in  incubator, up to overnight.
37 °C
?. [Subculturing (Day 6)]
Add  accutase (warmed to ) to each well.
1 ml
37 Room temperature
?. [Subculturing (Day 6)]
If cells have not been treated with RA, incubate at  for , or until cells are dissociated from plate (whichever comes first). Do not go over .
37 °C
?. [Subculturing (Day 6)]
During accutase incubation, prepare filters:Add  hESFM to the top of each filter for approximately  to wet filters. After , aspirate hESFM from the top of each filter. Add  EC medium (with or without  RA) to the basolateral chamber of each filter.
0.5 ml
1.5 ml
?. [Subculturing (Day 6)]
Add  hESFM to the top of each filter for approximately  to wet filters.
0.5 ml
?. [Subculturing (Day 6)]
After , aspirate hESFM from the top of each filter.
?. [Subculturing (Day 6)]
Spin down for  at .
Centrifuge: 1000 33
?. [Subculturing (Day 6)]
Aspirate EC media from both chambers (do not touch monolayer with pipet tip!).
?. [Subculturing (Day 6)]
Gently add EC medium lacking bFGF or RA,  in top chamber and  in bottom chamber. DO NOT include bFGF or RA in the medium – these factors prevent the monolayer from tightening.
0.5 ml
1.5 ml
?. [Subculturing (Day 6)]
During accutase incubation, prepare filters:Add  hESFM to the top of each filter for approximately  to wet filters. After , aspirate hESFM from the top of each filterAdd  EC medium (with or without  RA) to the basolateral chamber of each filter.
0.5 ml
1.5 ml
?. [Subculturing (Day 6)]
Add  hESFM to the top of each filter for approximately  to wet filters.
0.5 ml
?. [Subculturing (Day 6)]
After , aspirate hESFM from the top of each filter.
?. [Subculturing (Day 6)]
Following accutase incubation, use p1000 to collect cells and spray gently over surface 2–3x to dislodge any remaining cells. Triturate briefly to break up cell clumps.
?. [Subculturing (Day 6)]
Add accutased cells to the conical containing spent medium.
?. [Subculturing (Day 6)]
Spin down for  at .
Centrifuge: 1000 33
?. [Subculturing (Day 6)]
Aspirate media, resuspend cells in appropriate volume of EC media (with or without  RA). Cells are seeded based on a 1:3 ratio (i.e. 1 well of a six well plate seeds three Transwell filters). For 3 filters, resuspend cells in  EC medium, for 6 use , etc.
1.5 ml
3 ml
?. [Subculturing (Day 6)]
Place plate in incubator, shaking plate back and forth to distribute cells evenly (do not swirl).
?. [Subculturing (Day 6)]
24 hours later (i.e., day 7), aspirate and change to EC medium lacking bFGF and RA. Gently add EC medium lacking bFGF and RA,  in top chamber and  in bottom chamber. DO NOT include bFGF and RA in the medium – these factors prevent the monolayer from tightening.
0.5 ml
1.5 ml
?. [Subculturing (Day 6)]
Aspirate EC media from both chambers (do not touch monolayer with pipet tip!).
?. [Subculturing (Day 6)]
Gently add EC medium lacking bFGF and RA,  in top chamber and  in bottom chamber. DO NOT include bFGF and RA in the medium – these factors prevent the monolayer from tightening.
0.5 ml
1.5 ml
?. [Subculturing (Day 6)]
Add  EC medium (with or without  RA) to the basolateral chamber of each filter.
1.5 ml
?. [Subculturing (Day 6)]
Add  EC medium (with or without  RA) to the basolateral chamber of each filter.
1.5 ml