Jun 07, 2020
Protocol to culture mESCs (LSCB, UPLUT)
- Stefano D Vianello1,
- Mehmet Girgin2,
- Giuliana Rossi3,
- Matthias Lutolf4
- 1Marine Research Station, Institute of Cellular and Organismic Biology (ICOB), Avademia Sinica;
- 2Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV);
- 3Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland;
- 4Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences (SV) and School of Engineering (STI), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland and Institute of Chemical Sciences and Engineering, School of Basic Science (SB), EPFL, Lausanne, Switzerland
Protocol Citation: Stefano D Vianello, Mehmet Girgin, Giuliana Rossi, Matthias Lutolf 2020. Protocol to culture mESCs (LSCB, UPLUT). protocols.io https://dx.doi.org/10.17504/protocols.io.7xbhpin
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 03, 2019
Last Modified: June 07, 2020
Protocol Integer ID: 28355
Keywords: cell culture, mouse embryonic stem cells, mESCs
Abstract
Standard protocol used for culturing mouse embryonic stem cells (mESCs) in the Lutolf Lab, EPFL.
Guidelines
This protocol assumes standard cell culture aseptic technique.
Materials
MATERIALS
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
PBS, pH 7.4Thermo FisherCatalog #10010023
The following recipes were used to prepare culture and splitting media:
10% Serum Medium (home made fresh):
to make 500mL
- 434mL DMEM, high glucose, with GlutaMAX [CAT# 61965059, Gibco™/Life Technologies]
- 50mL ES-grade Foetal Bovine Serum [CAT#16141-079, Gibco™/Life Technologies]
- 5mL Penicillin-Streptomycin 10000U/mL [CAT#15140122, Gibco™/Life Technologies]
- 5mL Non Essential Amino Acids 100X [CAT#11140035, Gibco™/Life Technologies]
- 5mL Sodium Pyruvate 100mM [CAT#11360039, Gibco™/Life Technologies]
- 1mL beta-mercaptoethanol 50mM [CAT#31350010, Gibco™/Life Technologies]
for final concentrations of:
- Penicillin: 100U/mL
- Streptomycin: 100ug/mL
- Non Essential Amino Acids: 0.1mM
- Sodium Pyruvate: 1mM
- GlutamaMAX (= L-Alanyl-Glutamine, included in DMEM): 3.97mM
- beta-mercaptoethanol: 0.1mM
to make 10% Serum 2i/LIF Medium (made fresh), add:
- 3uM CHIR99021 (1:1000 from our 3mM stock,) [CAT#361559, Merck/Millipore ]
- 1uM PD0325901 (1:500 from our stock) [CAT#S1036, Selleck Chemicals]
- 100u/mL LIF (1:1000 from our stock), [sourced in house]
Before start
- Bring Accutase and small molecule inhibitor solutions to Room Temperature
- Bring medium and PBS to 37°C (preferably) or Room Temperature.
Cell detachment
Cell detachment
Cell detachment:
Using a vacuum pump+glass pipette, aspirate all culture medium out of the well (but do not dry out the cells completely!)
10s
Add 2mL PBS-/- (against side of the well) ti wash medium traces, and then aspirate it out again
10s
Add 500uL Accutase, and let it digest ~00:03:00 min, Room temperature , or until most cells are detached
Note
You want to let Accutase digest until almost all cells have detached from the bottom of the well, even if they are still forming clumps in suspension. By the time you start collecting them up, they will have further dissociated into single cells.
Softly tapping with your hand on the side of the plate will aid detachment of loosely-adherent cells.
Note
While the digestion is taking place, and if not done previously, you can start preparing the solutions and tubes required later in the protocol.
- ~6mL 10% Serum Medium to wash cells from the dish and, later, resuspend the pellet
- 1 empty 15mL Falcon tube where you will collect the cells
- 2mL "10%Serum 2i/LIF": 10% Serum Medium + 3uM CHIR99021 (1:1000 from our 3mM stock), 1uM PD0325901 (1:500 from our stock), 100u/mL LIF (1:1000 from our stock),
3m
With a P1000, collect all cells while breaking down clumps. To do so, pipette repeatedly up and down (without introducing bubbles) and use the ejected liquid to wash the surface of the well. The goal is to collect as many cells as possible in suspension. At the end of the process, transfer to a 15mL Falcon tube.
1m
Further collect leftover cells by washing the well one last time with with 4.5mL 10%Serum Medium; then adding such suspension to the tube with the accutase/cell suspension. The total volume in the tube should now be ~5mL.
30s
Centrifuge200 x g (1000rpm on our centrifuge), 00:04:00 min , 4 °C
Note
While cells are centrifuging, add the 2mL of 10%Serum/2iLIF medium you prepared before to a new well of the 6well plate. Transfer this plate to the incubator to equilibrate it and have it ready to accept the new cells later on.
4m
Counting and plating
Counting and plating
10m 30s
10m 30s
Cell counting:
Check that a nice pellet has formed at the bottom of the tube, and use a vacuum pump+glass pasteur pipette to remove all medium.
30s
Resuspend the pellet in 1mL 10%Serum Medium and pipette up and down for ~20 times. Change to a P200 and pipette up and down again for ~20 times to break down cells even finer.
1m 30s
Using a P20 or a P10 pipette, pipette up and down the cell suspension solution for ~5 times, and load 10uL of the suspension into a chamber of the haemocytometer. Count cells and calculate their concentration in the original solution.
Note
To count cells in the haemocytometer:
- tally the cells in each of the 4 big (4x4) squares at the four corners of the central cross
- divide the resulting total by 4, to find the average number of cells per square
- multiply *10'000
- this is your # cells/mL
- if you then need e.g. 70'000 cells, divide 70k by the number you just found, to know how many uL of cell suspension to use
7m
Plate 70k cells in the new well of 2iLIF +10%Serum you had previously put to equilibrate in the incubator
1m
Slide the plate back and forth (cross pattern) across the surface of the laminar flow cabinet to distribute them evenly, and then leave the cells undisturbed in the incubator to allow them to attach
30s
Cells can be split every 2 or 3 days (choose one) without medium change in between. Daily checks are still recommended to monitor overcrowding and other possible issues.