Methods for the derivation of mES cell lines

The classical and standard method for deriving mES cell lines is to culture intact day 3.5 p.c blastocysts on feeder layers for 2-5 d (during which time the blastocysts hatch and attach to the substratum), and then to collect, dissociate, and subculture the cells of the explanted ICM (Protocol 7, Method A). With blastocysts from strain 129 or C57BL/6 mice, lines should be obtained efficiently by this technique without recourse to other methods. Otherwise, improved efficiencies may be achieved by culturing the isolated epiblast after microsurgi-cal removal of the primitive endoderm and trophectoderm. This procedure may be carried out on either implanting day 4.5 p.c. blastocysts (Method B), or on delayed implanting blastocysts (Method C). As outlined in Table 1, each method offers practical advantages depending on the mouse strain of choice; but it should be emphasized that the microsurgical techniques require considerable effort to master, and a substantial investment in equipment.

4.1 Derivation of mES cells from intact blastocysts (Method A)

Blastocysts are collected from day 3.5 p.c. pregnant mice by Protocol 6, explanted intact into culture, and mES cells derived by Protocol 7, which is based on refer ences (4), (5), (24) and (25). Stages in the derivation are shown in Figure 1, and a schedule for manipulations and culture is given in Figure 2.

4.2 Derivation of mES cells from isolated epiblast

By day 4.5 p.c., the ICM of the mouse embryo develops into the epiblast and the primitive endoderm: the epiblast is invested on the blastocoelic surface by the layer of primitive endoderm, and is overlaid externally by trophectoderm. The aim of the microsurgical technique is to separate the epiblast cleanly from endoderm and trophectoderm, prior to culture. The procedure is the same for both day 4.5 p.c. and delayed blastocysts, but the subsequent schedule for epiblast culture differs slightly (Figure 2). Delayed blastocysts are produced by ovariectomy of mice on day 2.5 p.c. followed by the administration of a progestagen, and are recovered 5-7 d later. Protocols for ovariectomy may be found elsewhere (24). An alternative and effective procedure that avoids the need for this major

Figure 1 Stages in the derivation of mES cells from an Intact, explanted blastocyst.

(A) Appearance of the blastocyst after 4d of culture on feeder cells: the ICM has formed a central, rounded mass of proliferating, undifferentiated cells surrounded by outgrowths of trophoblast giant cells. (B) One mES-cell colony 5d after the dissociation of the cultured ICM: this colony shows the classic multilayering and clearly demarcated boundary with the feeder cells. (C) Several mES-cell colonies 3d after the dissociation of the colony shown in

(B). (D) The resultant culture of mES cells at passage 1: it is apparent that the colonies are composed of small cells with a high nuclear-to-cytoplasmic ratio. Scale bars: 100mm.

Figure 1 Stages in the derivation of mES cells from an Intact, explanted blastocyst.

(A) Appearance of the blastocyst after 4d of culture on feeder cells: the ICM has formed a central, rounded mass of proliferating, undifferentiated cells surrounded by outgrowths of trophoblast giant cells. (B) One mES-cell colony 5d after the dissociation of the cultured ICM: this colony shows the classic multilayering and clearly demarcated boundary with the feeder cells. (C) Several mES-cell colonies 3d after the dissociation of the colony shown in

(B). (D) The resultant culture of mES cells at passage 1: it is apparent that the colonies are composed of small cells with a high nuclear-to-cytoplasmic ratio. Scale bars: 100mm.

Method A Method B Method C

Day 3.5 p.c. blastocyst Epiblast isolated from day 4.5 Epiblast isolated from delayed blastocyst

Plate onto feeder wells

4-5 days

Disaggregation of ICM

1 7 days

Subculture of mES cell-like colonies into feeder wells

3-4 days p.c. blastocyst

Plate onto feeder wells

Disaggregation of epiblast colony

7 days

Subculture of mES cell-like colonies into feeder wells

Passage into 35 mm feeder dish (P1)

3 days

Passage into 25 cm2 feeder flask (P2)

Passage and expand (P3)

Freeze down

3 days

3 days

3-4 days

Passage into 35 mm feeder dish (P1)

3 days

Passage into 25 cm2 feeder flask (P2)

Passage and expand (P3)

Freeze down

3 days

3 days

Plate onto feeder wells 6 days

Disaggregation of epiblast colony

4 days

Passage into 35 mm feeder dish (P1)

3 days

Passage into 25 cm2 feeder flask (P2)

Passage and expand (P3)

Freeze down

3 days

3 days

Figure 2 Schedules of manipulations and culture for the derivation of mES cell lines using the methods described in the text.

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