Mar 24, 2020
Single Cell Seeding of BBB Stem Cell Model
- Ethan Lippmann1,
- Hannah Wilson2,
- Emma Neal1
- 1Department of Chemical Engineering, Vanderbilt University, Nashville, TN, USA;
- 2Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA
- Neurodegeneration Method Development CommunityTech. support email: ndcn-help@chanzuckerberg.com
Protocol Citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Single Cell Seeding of BBB Stem Cell Model. protocols.io https://dx.doi.org/10.17504/protocols.io.8j9hur6
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 22, 2019
Last Modified: March 24, 2020
Protocol Integer ID: 29025
Keywords: stem cell, single cell seeding, bbb stem cell model
Abstract
Standardized single cell seeding protocol for Blood-Brain Barrier (BBB) differentiation.
Attachments
Materials
MATERIALS
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
Countess™ II Automated Cell CounterThermo FisherCatalog #AMQAX1000
Y-27632 dihydrochloride (Rock Inhibitor)R&D SystemsCatalog #1254/10
Countess™ Cell Counting Chamber SlidesThermo Fisher ScientificCatalog #C10312
- Corning tissue culture plates
- 15 ml conical tubes
- Microfuge tubes
- E8 media prepared in-house
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
- Matrigel Plates should be ready to use at start of procedure.
- Procedure was optimized using IMR90-4 pluripotent stem cells. The procedure has successfully been extended to CC3, CD12, SM14, and DSP-mEGFP pluripotent stem cells.
Reagent Preparation
Reagent Preparation
- ROCK inhibitor:
Make 10 millimolar (mM) working stock solution by diluting 10 mg ROCK inhibitor into 3.12 mL ultrapure distilled water . Use at 1:1000 for 10 micromolar (µM) final concentration. Aliquots can be stored long term at -80 °C for up to 1 year and frozen/thawed as many times as necessary.
- E8 media
Seeding cells for BBB differentiation using single cell seeding (Day -1)
Seeding cells for BBB differentiation using single cell seeding (Day -1)
Manually transfer spent medium to a 15 ml conical. Save 1 mL of media for every well being passaged.
Wash each well once with 2 mL PBS .
Add 1 mL accutase (warmed to Room temperature ) to each well.
Incubate at 37 °C until the cells are beginning to detach (approx. 00:03:00 — 00:05:00 )
Using p1000, collect cells, and spray gently over surface 2 — 3x to dislodge any remaining cells.
Note
Pipetting more than this will reduce cell viability.
Collect cells in the 15 ml conical containing spent medium.
Spin down cells for 00:04:00 — 00:05:00 at 1000 rpm .
Aspirate media, resuspend cells in 1 mL E8 medium . Thoroughly triturate 2 — 3 times using p1000 to yield single cell suspension.
Take 10 µL of cells to count, drawing from the middle of the sample to prevent bias from settling cells. Transfer these cells to a clean microfuge tube.
Dilute the 10 µL of cells in 10 µL of 0.4 Mass Percent Trypan blue . Transfer 10 µL of this diluted suspension to a Countess cell counting chamber; allow to sit for 00:00:30 .
Note
Note that this step is performed outside the hood and is NONSTERILE.
Insert the slide into the Countess II automated cell counter. Note the calculated live cell density. You will use this density (not the total density) to calculate the number of cells needed for seeding.
Calculate appropriate volume of cells to add to each 6-well.
- Typical seeding number for IMR90-4 iPSCs is between 100,000 – 120,000 cells per well.
- Typical seeding number for CC3 and CD12 iPSCs is 150,000 cells per well
Resuspend cells in appropriate volume of E8 + 10 micromolar (µM) ROCK inhibitor (2 ml/well).
Note
If you have excess cells, discard the excess before resuspending.
Place plate in incubator, and shake plate quickly back and forth (not swirling) to distribute cells evenly.
Approximately 24 hours later (Day 0), initiate differentiation:
Protocol
NAME
Protocol for Differentiation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells
CREATED BY
Emma Neal
Note
Note: Cells are seeded for differentiation in E8 medium according to the standardized single cell seeding protocol
On day 0, aspirate E8 medium and add 2 mL of E6 per well.
Change medium every day using 2 mL of E6 per well.
At day 4 of E6 treatment, aspirate and add 2 mL of EC medium with bFGF (basic fibroblast growth factor) and 10 micromolar (µM) RA to each well.
Note
Medium is NOT changed during expansion phase.
BBB subculturing:
On day 6, subculture BBB onto plates and Transwell filters according to the following protocol:
Protocol
NAME
PROTOCOL FOR SUBCULTURE OF DIFFERENTIATED BLOOD-BRAIN BARRIER ENDOTHELIAL CELLS ONTO PLATES AND FILTERS
CREATED BY
Julia Roßmanith
.