Protocols

Preparation of ES Cells
(for Blastocyst Injection on Friday)

Courtesy of Joanne Thorvaldsen from the laboratory of Dr. Marisa Bartolomei, Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine.

On Monday

  1. Seed ES cells onto one well of 24-well plate

    - Plate mouse embryonic feeder cells (MEFs) onto multiple wells of 24-well gelatinized plate.

    - After MEFs have attached to plate (2+ hrs), thaw ES cells and seed into one well of the prepared 24-well plate.

On Wednesday

  1. Split cells as necessary, onto wells of the prepared 24-well plate.

    - If well is confluent (50-60%), split to various densities into three wells of the 24-well plate e.g., approximately 1:2, 3:10 and 1:5 into each well. (See Note below.) The trypsinization method described below works well to ensure single-cell suspension.

    - If colonies are few or are not growing well, plate trypsinized cells onto one well. In addition, consider plating out an additional vial of ES cells onto another well, to ensure some population of ES cells will be ready for Friday.

On Thursday or Friday morning

  1. Confirm with Jean Richa that he is prepared for your ES cells. Laboratory Telephone: (215) 573-3023, Office Telephone: (215) 898-6064 (office)

On Friday
(The harvested cells need to be ready for injection by noon.)

Note: Two separate wells of ES cells which vary in confluence may be harvested simultaneously to provide Dr. Richa with the most optimum cell density required for the blastocyst injections.

  1. Feed cells around 8:00am.
  2. Seed ES cells onto one well of 24-well plate

    - Wash twice with PBS

    - Add trypsin (0.25% Trypsin, 1 mM EDTA; 0.2-0.3 ml/well of 24well dish) and incubate for 5 minutes at 37oC.

    - To disaggregate the cells, use a plugged Pasteur pipette and pipette cells up and down 5X. Then add 1 ml ES media. With a plugged Pasteur pipette inserted into a P-200 type narrow bore yellow tip, pipette entire cell suspension up and down (15-20X).

    Note: It is important to generate a single cell suspension for blastocyst injections. Avoid foaming of the medium!

  3. Adsorb MEFs on gelatinized 6-well plate and collect ES cells.

    - Transfer ES cell suspension to one well of gelatinized 6-well plate which contains 1-2 ml ES medium (final volume 2-3 ml) .

    - Leave plate in incubator for 1 hour. (The MEFs should sit on the plate after one hour while the stem cells are still in suspension.)

    - Carefully transfer supernatant to 15 ml conical tube and centrifuge for 5 minutes at 900 rpm (e.g. IEC HN-SII Centrifuge).

    - Discard medium and resuspend the cells in appr. 0.5 ml ES cell medium. To a separate tube, add 5 ml ES cell medium.

  4. Deliver 15 ml tube(s) containing ES cells and a tube with 5 ml ES cell medium to Jean Richa, Rm. 202A CRB. (Tubes may be delivered on ice in a Styrofoam box.)

Basics for Culturing ES Cells

  1. Colonies should be compact with a smooth outline (see ‘Manipulating the Mouse Embryo’, CSHL Press, Ed. Brigid Hogan et al.(1994)).
  2. Culture in the presence of LIF (1000U/ml, Gibco/BRL) on mitotically inactivated mouse embryonic feeder (MEF) cells that were plated onto gelatinized plates. Use ES medium as recommended for the specific ES cell-line in use.
  3. Feed cells daily (and 2-3 hr before cells are trypsinized).
  4. Do not grow cells for more than 3 days (2 days preferred) without splitting (1:3 to 1:6). Confluent/fast growing cells will need to be split one day after seeding/splitting.
  5. To prepare gelatinized plates (or wells), cover plate with 0.1% gelatin solution and leave plate in biosafety cabinet for 2 hrs. Remove gelatin and allow gelatin to dry for approximately 10 minutes. (Plates must be completely dry before use or storage at 4o C.)