Culture Conditions

Cells were derived with feeders in low (2%) serum conditions to improve survival in early passages; however, cells should now be cultured in feeder-free, serum-free conditions (see below). Media, culture conditions, and procedures for thawing are provided below.

Serum free ES medium (SFES)- 500 mls

Component	Volume	Vendor	Catalog number
DMEM/F-12	242 mls	Thermo Fisher Scientific	11320082
Neurobasal medium	242 mls	Thermo Fisher Scientific	21103049
N-2 supplement	2.5 ml	Thermo Fisher Scientific	17502048
B27 supplement, minus vitamin A	5 ml	Thermo Fisher Scientific	12587010
PenStrep	5 ml	Thermo Fisher Scientific	15140122
7.5% BSA	3.33ml	Thermo Fisher Scientific	15260037

Complete “2i” medium- 50 mls

Use within 1 week, store at 4C, protect from light

Component	Volume	Vendor	Catalog number
SFES	49 mls*
GlutaMAX-1	500 ul	Thermo Fisher Scientific	35050061
Monothioglycerol (11.9 M)	0.63 ul	Sigma	M6145-25ML
Leukemia inhibitory factor (LIF) 10^7 units	5 ul	R&D Systems	8878-LF-500/CF
PD0325901	5 ul (stock solution)**	Stem Cell Technologies	72184
CHIR99021	5 ul (stock solution)***	Stem Cell Technologies	72054

*See recipe above

**Add 414 ul DMSO to 2mg of PD0325901 to make a 10mM stock solution. Avoid repeated freeze-thaws.

***Add 140ul DMSO to 2mg of CHIR99021 to make a 30mM stock solution. Avoid repeated freeze-thaws.

Additional reagents

Component	Vendor	Catalog number
0.1% Gelatin	Stem Cell Technologies	07903
PBS pH 7.2, no Ca or Mg	Thermo Fisher Scientific	20012050
TrypLE™ Express Enzyme (1X), phenol red	Thermo Fisher Scientific	12605010
ES-grade Fetal Bovine Serum (FBS)	Thermo Fisher Scientific	10439024
DMSO	Sigma	D2650

Cryopreservation Medium

50% culture medium, 40% FBS and 10% DMSO, prepare in quantities that can be used within a 2-week period

Thaw and culture of mES cells

1. Day 1. Gelatinize 1 35mm dish: coat the dish with 0.1% gelatin and incubate at room temperature for 15 minutes. Aspirate off the gelatin before use. Thaw one vial of mES cells rapidly in a 37C bead bath. Sterilize vial with 70% ethanol and then transfer cells to 6 mLs of pre-warmed (37C) media. Centrifuge the cells for 3 minutes at 1200 rpm. Resuspend the pellet in 6 mLs of media and create a single-cell suspension by gentle trituration with a serological pipet. Transfer the resuspended cells onto the gelatinized 35mm dish.
2. Day 2. Examine the cells under phase-contrast microscopy. Colonies should be visible and nicely distributed on the dish. Depending on the size and density of the colonies, either replace the culture medium to feed the growing cells or passage the cells ranging from 1:2 to 1:3 depending on colony size.
3. Passaging mES cells at a ratio range of 1:2 to 1:3: Remove and discard the ES cell culture medium. Add a quantity of TrypLE™ Express Enzyme (1X), phenol red to cover the cell layer, usually 1/3 the amount used to culture the cells. Return the cells to the incubator for 3-4 minutes. Observe cell dissociation using the microscope, with occasional swirling. As soon as colonies begin to dissociate, inactivate the TrypLE* by adding an equal amount of mESC culture medium. Collect the cells and then remove any remaining cells by rinsing the culture dish with additional mESC culture medium. Collect both the released cells and the rinse into one tube and create a single-cell suspension by gentle trituration with a serological pipet. Centrifuge for 3 minutes at 1200 rpm and resuspend the pellet, creating a single cell suspension by gentle trituration with a serological pipet in either cryopreservation medium if freezing (see step 4) or in culture medium and transfer to a new gelatinized dish.
4. Freeze: When desired, freeze at 70% confluency using the 50% culture medium, 40% FBS and 10% DMSO freeze media. Recommended freezing in 500uL with no more than 5-7x10^5 cells per vial.

*TyplE does not require inhibition with serum-containing media

The cell line was tested for bacterial and fungal growth and for the presence of mycoplasma using a PCR detection system. Cell culture contaminants were not detected. The sex and genotype of this cell line was determined by the Mouse Universal Genotyping Array (GigaMUGA) which consists of 140,000 SNP markers. These data are available on request. mES cells will spontaneously differentiate in the absence of LIF and supplemental growth factors. Karyotyping and germline testing were not performed.