TROPHOBLAST STEM CELL PROTOCOLS
The protocols and reagents described here are for the maintenance and derivation of trophoblast stem (TS) cell lines. Slight variations from the procedures or the suppliers of reagents are probably acceptable. If you have any questions or concerns contact Tilo Kunath: tilo.kunath@ed.ac.uk. If you have made improvements or simplifications to the protocols please let us know about these too.
reference:
Tanaka, S. Kunath, T, Hadjantonakis, A-K, Nagy, A and Rossant, J.
Promotion of Trophoblast Stem Cell Proliferation by FGF4.
Science (1998) 282, 2072-2075
The following reagents should be prepared before attempting to culture or derive TS cell lines:

1000x FGF4
Human recombinant FGF4 R & D systems cat #235-F4-025, 25 ug
Other sources of FGF4 would likely work as well. We found that aFGF (Sigma #F5542) and bFGF (Sigma #F0291) also maintained TS cells in culture at 25 ng/ml. However, derivation of TS cell lines with these othere FGFs have not been attempted.

Resuspend lyophilized FGF4 in its vial with 1.0 ml of PBS/0.1% w/v fraction V BSA*.
Mix well with P200 and make 10 aliquots of 100ul into 1.5 ml microfuge tubes and freeze at -70oC.
Thaw each aliquot as needed and store at 4oC; do not re-freeze
   *10 ml PBS/0.1% (w/v) BSA is prepared by dissolving BSA (Sigma cat# A3311) in PBS without Ca2+/Mg2+; filter through 0.45u syringe filter, and make 1 ml aliquots in microfuge tubes; store at -70oC and thaw one tube when a vial of FGF4 needs to be reconstituted.

1000x Heparin
heparin (Sigma cat # H3149 10,000 units)
Resuspend heparin in PBS to a final concentration of 1.0 mg/ml (1000X) and store at -70oC. Thaw aliquots as needed and store at 4oC.
Heparin can also be prepared as a 10,000X (10 mg/ml) stock in PBS without CA2+/Mg2+ and stored at -70oC. This can be used multiple times to make batches of 1000X heparin.

TS medium
TS medium is prepared by adding the following reagents to 500 ml RPMI 1640 + antibiotics (pen/strep @ 50 ug/ml each final conc)
130 ml fetal bovine serum (final conc 20%) (Life Technologies)
6.5 ml 100mM sodium pyruvate (final conc 1 mM) (Life Technologies)
6.5 ml 10 mM B-mercaptoethanol (final conc 100uM) (Sigma)
6.5 ml 200 mM L-glutamine (final conc 2 mM) (Life Technologies)

Feeder-Conditioned medium
Feeder-conditioned medium (CM) is used to culture TS cells in the absence of EMFIs. Prepare mitomycin-treated primary embryonic fibroblasts (EMFIs) in 100 mm dishes (2 x 10 6 cells; 2 x 105 cells/ml and culture in approx. 10 ml TS medium. Culture for at least 72 hours and then collect the medium. Spin to remove floating cells and debris, filter (0.45um), and sotre at -20oC in 10 ml aliquots. Thaw each aliquot as needed and store it at 4oC; do not re-freeze. Use the EMFIs to prepare two more batches of feeder-CM, then discard the cells. EMFIs are only used up to 10 days after the mitomycin treatment.

Culturing TS cells
TS cells may be grown up on either EMFIs (A) or regular tissue culture plastic (B). Gelatin-coating is not required.

Culturing TS cells on EMFIs
Plate mitomycin-treated EMFIs at half the density (1x 105 cells/ml) used to culture embryonic stem (ES) cells and for the preparation of feeder-CM. TS cells may be plated on the EMFIs after they have adhered or co-plated with the EMFIs.
Prepare fresh TS + FGF4 medium (10 ml TS medium, 10 ul 1000x FGF4 stock, 10 ul 1000x heparin stock)
Culture TS cells in the above medium in a standard tissue culture incubator (37oC, 5% CO2 incubator. The medium is normally changed every two days, and the cells are passaged (1:10 to 1:20) every 4th day, or when the culture has reached approximately 80% confluency. Passaging TS cells at higher densities (eg, 1:3 or 1:5) may lead to precocious differentiation. The cells are trypsinized to very small clumps with some single cells. A complete single-cell suspension is not required and it may even be detrimental to the culture. Trypsinizing for 3-4 minutes at 37oC with some pipetting up and down is usually sufficient.

Culturing TS cell on tissue culture plastic
TS cells grow well on standard tissue culture dishes if the medium is supplemented with feeder-CM. The dishes do NOT need to be gelatin-coated as mentioned in Tanaka et al.
Prepare fresh TS + FGF4 medium (7 ml feeder-CM medium, 3 ml TS medium,10 ul 1000x FGF4 stock, 10 ul 1000x heparin stock)
Culture the TS cells in the above medium; feed and passage the cells as above.

Freezing TS cells
Prepare 2x freezing medium (50% FBS, 30% TS medium, 20% DMSO), and cool to 4oC.
Lift and pellet TS cells. Resuspend in TS medium and add an equal volume of 2x freezing medium. Freeze the cells slowly at -70oC overnight and then transfer to liquid nitrogen after 24 to 48 hours. A confluent 100 mm dish has enough cells for about 5 vials.

Thawing TS cells
Hand-thaw the vial and put the contents over 1.0 ml TS medium, and pellet. Resuspend the cells in 5 ml TS + F4H medium. Plate over EMFIs in 60 mm tissue culture dish, and change medium after 8 hours to get rid on non-adherent cells. Feed two days later.

Removing EMFIs from TS cell cultures
When switching from EMFI cells to EMFI-CM it may be desirable to get rid of the EMFI cells immediately. The different adherence rates of EMFI cells (fast) and TS cells (slow) can be used to obtain a pure TS cell population.

Passage cells to a new plate and incubate the culture for 1.5 hr at 37oC in 5% CO2. Remove the supernatant and plate onto another dish. This population of cells should consist almost entirely of TS cells. Since some TS cells do adhere along with the EMFIs, the desired passage density may be increased a bit (e.g. 1 in 14 instead of 1 in 16).

Derivation of TS cell lines from Blastocysts