Oct 25, 2019
WTc11 iPSC Culture and Maintenance
- Connor Ludwig1,
- Ruilin Tian1,
- Martin Kampmann1
- 1University of California, San Francisco
- Neurodegeneration Method Development Community
- KampmannLab
Protocol Citation: Connor Ludwig, Ruilin Tian, Martin Kampmann 2019. WTc11 iPSC Culture and Maintenance. protocols.io https://dx.doi.org/10.17504/protocols.io.8rhhv36
Manuscript citation:
Tian et al (2019). CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. Neuron pii: S0896-6273(19)30640-3. [Epub ahead of print] PubMed PMID: 31422865.
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 and it's working
Created: October 25, 2019
Last Modified: October 25, 2019
Protocol Integer ID: 29193
Keywords: CRISPR interference, CRISPRi, functional genomics, neuron, stem cell, essential genes, CROP-seq, Perturb-Seq, single-cell RNA sequencing, high-content microscopy, maintenance of the wtc11 ipsc cell line, wtc11 ipsc culture, wtc11 ipsc cell line, maintenance this protocol, maintenance, protocol
Abstract
This protocol explains general culture and maintenance of the WTc11 iPSC cell line.
Attachments
Materials
MATERIALS
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
DPBS, no calcium, no magnesiumThermo FisherCatalog #14190136
Trypan Blue Solution, 0.4%Thermo FisherCatalog #15250061
StemFlex™ MediumThermo Fisher ScientificCatalog #A3349401
MatrigelCorningCatalog #356231
KnockOut™ DMEMThermo Fisher ScientificCatalog #10829018
Rock Inhibitor Y-27632 Dihydrochloride TocrisCatalog #1254
Troubleshooting
Safety warnings
Please refer to the Safety Data Sheets (SDS) for safety and health data.
Before start
Before use, warm complete medium required for that day at room temperature until it is no longer cool to the touch.
Alternatively, an aliquot for use that day may be pre-warmed in a 37 °C waterbath until no longer cool to the touch. Avoid extended dwell times at 37 °C .
Prepare complete StemFlex Medium
Thaw the frozen StemFlex Supplement 10X at Room temperature for ~02:00:00 or overnight at 2 °C to 8 °C .
Note
IMPORTANT! Do not thaw the frozen supplement at 37°C.
Mix the thawed supplement by gently inverting 3−5 times.
Aseptically transfer 50 mL of StemFlex Supplement 10X to the bottle of StemFlexTM Basal Medium (450 mL fill).
Gently invert the bottle several times to obtain 500 mL of homogenous complete medium.
Note
Following reconstitution, complete StemFlexTM Medium can be stored at 2°C to 8°C for up to 2 weeks or aliquoted and stored at -5 °C to -20 °C for up to 6 months. Alternatively, usage size aliquots of the supplement can be made and frozen at -5 °C to -20 °C for up to 6 months. Avoid multiple freeze-thaw cycles.
Feeding WTc11 iPSC
Feed the PSCs the day after seeding followed by every-other-day thereafter.
Note
If the cells are to be left without feeding for two days (for example, over a weekend), then double the feed volume (i.e., 4 mL added per well of 6-well plate).
Passaging WTc11 iPSC
iPSCs should be split when cells are ~ 80% confluent.
Thaw Matrigel on ice and dilute in pre-chilled Knockout DMEM for a final volume of 100 microgram per milliliter (μg/mL) .
Coat desired wells/plates with diluted Matrigel and incubate at 37 °C for 00:30:00 -01:00:00 using the following table for volumes to add per well:
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 40 μL | 200 μL | 0.5 mL | 1 mL | 5 mL | 10 mL |
Note
Matrigel may be re-used during this time only to coat additional plates. Original plates should
have PBS or media to prevent the matrix from drying out. Matrigel coated plates must be used within 14 days of coating.
Tilt cell-containing plate towards you and aspirate existing media.
Wash wells once with ample PBS (about 2x amount of media).
Add accutase to well(s) using the following table for volumes per well and incubate at 37 °C for 00:03:00 ; add another 01:00:00 -00:02:00 if cells have not mostly lifted/dissociated.
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 20 μL | 100 μL | 250 μL | 0.5 mL | 2 mL | 4 mL |
Add ample PBS to accutase-containing well(s) to dilute accutase using the following table for volumes per well:
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 200 μL | 1 mL | 2.5 mL | 5 mL | 10 mL | 20 mL |
Pipette up and down gently to mechanically release remaining cells, collect, and add to appropriately-sized conical tubes.
Spin cells at 200 x g for 00:05:00 at Room temperature .
Carefully aspirate supernatant from pelleted conicals.
Add appropriate volume of StemFlex + Rock inhibitor at 10 micromolar (µM) to conicals according to pellet size for counting.
Note
Rock inhibitor should only be used when cells are individualized or in small colonies (typically the first two days after passaging); the presence of Ri at higher densities results in cell stress/death, and in general, Rock inhibitor greatly reduces proliferation.
Note
For first time use of Rock inhibitor, it is suggested to aliquot at 10mM [1000x], diluting in DPBS, and use on cells at concentration 10 Mass Percent .
Triturate to resuspend cells in StemFlex + Rock inhibitor and remove 10μL and add this volume to a 1.5mL Eppendorf tube.
Note
Be careful to minimize contact of pipette with the side of the conical wall.
Count cells and calculate desired number of cells to seed, and dilute this volume with additional StemFlex + Rock inhibitor to plate using the following table for volume to add per well:
| Per: | 96-well | 24-well | 12-well | 6-well | 10-cm dish | 15-cm dish | |
| Volume to add: | 50-100 μL | 250-500 μL | 0.75-1 mL | 1.5-2 mL | 8-12 mL | 15-25 mL |
Note
For general passaging where exact cell number seeded is not important, adding resuspended cells at 1:100, 1:50, and 1:20 the final well volume typically provides near-confluency in 7 days, 5 days, and 3 days, respectively.
iPSCs can be frozen in StemFlex + 10% DMSO.