Mar 24, 2020

Public workspaceProtocol for Subculture of Differentiated Blood-Brain Barrier Endothelial Cells onto Plates and Filters

DOI
dx.doi.org/10.17504/protocols.io.8g5hty6
  • 1Department of Chemical Engineering, Vanderbilt University, Nashville, TN, USA;
  • 2Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA
  • Neurodegeneration Method Development Community
    Tech. support email: ndcn-help@chanzuckerberg.com
Emma Neal
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DOI: dx.doi.org/10.17504/protocols.io.8g5hty6
Protocol Citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Protocol for Subculture of Differentiated Blood-Brain Barrier Endothelial Cells onto Plates and Filters. protocols.io https://dx.doi.org/10.17504/protocols.io.8g5hty6
Manuscript citation:
A Simplified, Fully Defined Differentiation Scheme for Producing Blood-Brain Barrier Endothelial Cells from Human iPSCs. Neal EH, Marinelli NA, Shi Y, McClatchey PM, Balotin KM, Gullett DR, Hagerla KA, Bowman AB, Ess KC, Wikswo JP, Lippmann S. Stem Cell Reports. 2019 Jun 11;12(6):1380-1388. doi: 10.1016/j.stemcr.2019.05.008
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group, and it is working. Though it is labeled as seeding for our BBB model, we follow this same procedure for seeding all of our differentiations with appropriate modifications to cell density.
Created: October 19, 2019
Last Modified: March 24, 2020
Protocol Integer ID: 28925
Keywords: human pluripotent stem cells, hPSCs, BBB, endothelial cells, blood-brain barrier, defined differentiation, human induced pluripotent stem cell, iPSC, in vitro model
Attachments
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Guidelines
Additional notes:

  • Cells should reach confluence 24 – 48 hours post-split.
  • If not treated with RA, cells should reach baseline TEER reading of 200 – 250 ohms (empty filter not subtracted) when confluent. If retinoic acid is used, initial TEER at 24 hours post-split can range anywhere from 200 – 2000 ohms depending on the cell fidelity. If using RA, TEER can reach 2000 – 4000 ohms, depending on the cell line used.
  • If using filters for permeability screens, 48 hours post-split is usually the optimal time (maximum TEER).


TROUBLESHOOTING (subculture onto plates and filters)

Problem: Large clumps of cells are accumulating on top of monolayer
Solutions:
  1. Increase trituration, particularly if you are not triturating vigorously. Cell clumps can accumulate on the top of the monolayer and decrease viability, while dispersed cells are more likely to remain in solution and can be removed the following day during the medium change.
  2. Try increasing length of enzyme treatment (i.e., if using 15 minutes accutase try 20 minutes). Singularized cells (as opposed to clumps of cells) are more likely to form an even monolayer.
  3. Try adjusting initial cell density at start of differentiation (day 0). Clumps can indicate that initial cell density was too high.

Problem: Cells have not filled in (resulting in low- to zero TEER if using filters)
Solutions:
  1. Treat cells more gently, too much trituration can hurt the cells (particularly if not using RA treatment, these cells tend to be more fragile).
  2. Decrease enzyme treatment length (i.e., if using 15 minutes, try 10 minutes). If using accutase, try versene instead.
  3. Try adding more cells to the filter. If using accutase, increase number of cells added to filter (i.e., if using 1 million cells try using 1.2 million cells).
  4. Try adjusting initial cell density at start of differentiation (day 0). Low attachment to plates/filters can indicate that initial cell density was too low.

Problem: Monolayer is intact but TEER is not spiking, even with RA addition
Solution:
Ensure that media is changed after 1 day post-split, and does not contain RA or bFGF. Addition of either RA or bFGF eliminates any potential spike in TEER.
Materials
MATERIALS
ReagentB-27 SupplementGibco - Thermo Fisher ScientificCatalog #17504044
ReagentGibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
ReagentRecombinant Human FGF-basic (154 a.a.)peprotechCatalog #100-18B
ReagentStemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
ReagentHuman Endothelial-SFMThermo FisherCatalog #11111044
ReagentRetinoic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #R2625-50MG
ReagentFibronectin bovine plasmaMerck MilliporeSigma (Sigma-Aldrich)Catalog #F1141-5MG
ReagentCollagen from human placentaMerck MilliporeSigma (Sigma-Aldrich)Catalog #C5533-5MG
Plasticware

FISHER
Corning Tissue Culture Plates (6- or 12-well, 3513 or 3516)
500 ml filter-top bottles (S2GPT05RE)
Corning Transwell Polyester Filters (07-200-161)


Equipment

EVOM2 located in tissue culture room
Chopstick electrodes or EndOhm chamber located in tissue culture room
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
REAGENT/MEDIUM PREPARATION:


B27 Supplement
Thaw 10 ml bottle and mix thoroughly. Aliquot into sterile microcentrifuge tubes at 280 μl/tube and store at Temperature-20 °C . Upon thawing, unused portions of an aliquot may be stored at Temperature4 °C for up to 1 week for further media preparation.

bFGF, 100 μg/ml (prepared according to E8 media protocol)
Thaw a 500 μl aliquot of bFGF and dilute 1:5000 in EC medium for a final concentration of Concentration20 ng/ml as described below. Divide the remaining bFGF in 100 μl aliquots and re-freeze at Temperature-80 °C . These remaining aliquots can be thawed and used for EC medium but cannot be refrozen a second time.

Retinoic acid (RA)
Dilute Amount50 mg RA in Amount16.6 mL DMSO to create a stock solution of Concentration10 millimolar (mM) and store 1 ml aliquots at Temperature-80 °C . To prepare working stocks, divide a 1 ml stock tube into 50 μl aliquots and store at Temperature-20 °C . Dilute working stocks 1:1000 in EC medium for a final concentration of Concentration10 micromolar (µM) .

EC medium w/ 200X B27 + 20 ng/ml bFGF
For 50 ml: add Amount250 µL of B27 and Amount10 µL bFGF to Amount50 mL of hESFM.
Good for up to two weeks at Temperature4 °C .

EC medium w/ 200X B27
For 50 ml: add Amount250 µL of B27 to Amount50 mL of hESFM.
Note
Note that after subculture, purified BMECs are changed to EC medium containing 200X B27, but without bFGF.


Collagen IV, 1 mg/ml
Dissolve Amount5 mg of collagen IV in Amount5 mL of sterile-filtered Concentration0.5 mg/ml acetic acid.

ECM solution for plate and filters
Mix 5 parts sterile ddH2O with 4 parts Concentration1 mg/ml collagen IV and 1 part fibronectin. Exact volume depends on the number of filters being coated (200 μl/filter) and number of plates being coated.

Please select between subculturing onto plates or filters.
Step case

Plates
15 steps

Subculturing onto Plates using Accutase.
Coat plates with ECM plate solution for at least Duration01:00:00 at Temperature37 °C . Volume depends on plate type (see Table):

Plate type for subculture phaseVolume of ECM solution for coatingWorking volume of EC media for cell culture
6-well800 μl2 ml
12-well250 μl1 ml
24-well200 μl500 μl
48-well100 μl400 μl
96-well50 μl200 μl

Note
If desired, plates may be coated DurationOvernight . 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.

Pipetting
Aspirate plates and allow to dry in sterile hood (place the plate in the back of the hood and leave the lid slightly ajar).
Note
Plates only need to dry for Duration00:05:00 (can be aspirated during accutase incubation). Do not over dry!

Retrieve cells from incubator and transfer equal volume of spent media to 15 ml conical corresponding to the number of wells being accutased.
Note
For example, if accutasing 4 wells, save 4 ml of spent media and discard the rest.

Pipetting
Wash each well once with Amount2 mL PBS.
Wash
Add Amount1 mL accutase (warmed to TemperatureRoom temperature ) to each well.
Pipetting
Incubate at Temperature37 °C , length of time depends on cell treatment:
Incubation
Step case

If cells have not been treated with RA
9 steps

If cells have not been treated with RA, incubate at Temperature37 °C for Duration00:20:00 , or until cells are dissociated from plate (whichever comes first).
Incubation
Using p1000, collect cells, and spray gently over surface 2–3x to dislodge any remaining cells. Triturate briefly to break up cell clumps.
Add cells to 15 ml conical containing spent media.
Pipetting
Spin down cells at Centrifigation1000 rpm, 00:04:00 .

Centrifigation
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.
Pipetting
Thoroughly triturate 3 – 4 times to yield single cell suspension.
Add appropriate volume of cells to each well.
Pipetting
Place plate in incubator, shaking plate back and forth to distribute cells evenly (do not swirl).
Incubation
24 hours later (i.e., day 7), aspirate spent media and add appropriate volume of EC medium (without bFGF or RA).
Pipetting