TITLE: Preparation of mouse embryonic fibroblast (MEF) feeder plates for hPSC cultures
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the preparation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture.

Protocol overview
A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional)
B. Starting with fresh irradiated or Mitomycin C inactivated MEFs


General notes
1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. Either fresh (start at step 4) or frozen stocks of irradiated or Mitomycin C inactivated MEFs can be used to prepare hPSC feeder cells. 
3. The indicated MEF density are recommended starting densities and might have to be adjusted for each hPSC line and hPSC medium formulation (KSR, serum-free versus serum-containing medium). 
4. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)
 



[BEFORE_START]
All cell culture plates which are used as feeders to maintain hPSCs are coated for at least 1 hour with autoclaved 0.2% gelatin solution at room temperature. Remove gelatin solution immediately before plating MEF cells. 

0.2% Gelatin Solution
 

 
 

[STEPS]
SECTION: A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional)
1. To recover frozen stocks of irradiated or Mitomycin C inactivated MEFs (up to passage P4), thaw MEF tubes in in a water bath at   37 °C by gently shaking.

SECTION: A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional)
2. Thawed cells are transferred into a 15 ml conical tube containing pre-warmed MEF medium and centrifuged at  250 x g, 5 min 

SECTION: A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional)
3. Re-suspend MEFs in fresh MEF medium
SECTION: B. Starting with fresh irradiated or Mitomycin C inactivated
4. Take two sets of 10 µl of inactivated MEFs suspension (either irradiated or Mitomycin C inactivated). Mix each set with 10 µl trypan blue dye, which comes with the Countess™ Cell Counting Chamber Slides. 

For a protocol on irradiation of MEFs or Mitomycin C inactivation of MEFs, refer to the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture." A link to this collection can be found in the title section of this protocol, located above.
SECTION: B. Starting with fresh irradiated or Mitomycin C inactivated
5. Count cells with Countess automated cell counter or hemocytometer, average the counts from the two sets.
SECTION: B. Starting with fresh irradiated or Mitomycin C inactivated
6. Dilute MEFs needed to 1.67x105cells/ml in MEF medium
SECTION: B. Starting with fresh irradiated or Mitomycin C inactivated
7. Add 2.5 ml diluted MEFs to each well of 6-well plates. This gives ~4x104 feeders/cm2.

The indicated MEF seeding density is a recommended starting density for growing hPSCs in serum-containing medium, and might have to be adjusted for each hPSC line and hPSC media formulation (KSR, serum-free versus serum-containing media). 
SECTION: B. Starting with fresh irradiated or Mitomycin C inactivated
8. Shake the plates to distribute cells evenly. Maintain plates in a humidified incubator (37°C; 5% CO2). Feeders shall be used within 2 weeks after plating.
SECTION: B. Starting with fresh irradiated or Mitomycin C inactivated
9. For the irradiated MEFs leftover, freeze at 10x106cells/cryovial for future use. Each vial usually can be thawed into three 6-well plates directly, if cells recover well.

For a protocol on freezing and thawing MEFs, refer to the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture." A link to this collection can be found in the title section of this protocol, located above.
SECTION: A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional)
2.1. MEF medium
 

SECTION: B. Starting with fresh irradiated or Mitomycin C inactivated
7.1. hPSCs Medium
     DMEM/F12      385 ml        Fetal Bovine   Serum (FBS)      75 ml        Knockout Serum Replacement      25 ml        L-Glutamine (100X)      5 ml        Penicillin & Streptomycin (100X)      5 ml        MEM Non-Essential Amino Acids (100X)      5 ml        2-Mercaptoethanol (10,000X)      50 ul        Heat Stable Recombinant Human FGF2 (25ug/ml)*      80 ul   
 

L-Glutamine (100X)
     L-Glutamine,   powder      14.6 g        MilliQ H2O      500 ml   
 
2-Mercaptoethanol (10,000X)
     2-Mercaptoethanol      0.78 ml        MilliQ H2O      9.22 ml   
 

Heat Stable Recombinant Human FGF2 (25µg/ml)
     Heat Stable Recombinant Human FGF2      500 ug        0.1% BSA      20 ml