Cultivation of Undifferentiated ES Cells on Feeder Layer

3.1.1. Feeder Layer Culture (see Note 4)

1. Remove embryos from a mouse pregnant for 15 to 17 d (e.g., NMRI or CD-1 outbred strains), rinse in PBS, and remove placenta and fetal membranes, head, liver, and heart. Rinse the carcasses in trypsin solution.

2. Mince the tissue in 5 mL of fresh trypsin solution and transfer to an Erlenmeyer flask containing a stir bar.

3. Stir on a magnetic stirrer for 25 to 45 min (use a longer incubation time if the embryos are older), filter the suspension through a sieve or a screen, add 10 mL of culture medium I, and spin down (5 min at 1000g).

4. Resuspend the pellet in approx 3 mL of culture medium I and plate on 100-mm tissue culture plates (~2 x 106 cells per 100-mm dish) containing 10 mL of cultivation medium I, and incubate at 37°C and 5% CO2 for 24 h.

5. Change the medium to remove debris, erythrocytes and unattached cellular aggregates, and cultivate for an additional 1 to 2 d.

6. Passage the primary culture of mouse embryonic fibroblasts: split 1/2 to 1/3 on 100-mm tissue culture plates and grow in cultivation medium I for 1 to 3 d. The cells in passages 2 to 4 are most suitable as feeder layer for undifferentiated ES cells.

7. Incubate feeder layer cells with MC buffer for 2 to 3 h, aspirate the MC solution, wash 3X with PBS, trypsinize the feeder cells, and replate to new gelatin (0.1%)-treated microwell plates or Petri dishes. Feeder layer cells prepared one day before ES cell subculture are optimal!

3.1.2. Culture of Undifferentiated ES Cells (see Notes 5 and 6)

It is important to passage ES cells every 24 or 48 h. Do not cultivate longer than 48 h without passaging or the cells may differentiate and be unsuitable for differentiation studies. Selected batches of FCS have to be used for ES cell culture (see Note 6).

1. Change the medium 1 to 2 h before passaging.

2. Aspirate the medium, add 2 mL of trypsin-EDTA, and incubate at room temperature for 30 to 60 s.

3. Carefully remove the trypsin-EDTA mixture and add 2 mL of fresh cultivation medium II.

4. Resuspend the cell population with a 2-mL glass pipet into a single cell suspension and split 1/3 to 1/10 to freshly prepared (60 mm) feeder layer plates.

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