Methods

Explantation of blastocysts into culture:

1 Aspirate MEF medium from feeder layers in 4-well plates, and replace with 0.6 ml/ well of LIF-supplemented mES-cell medium.

2 Transfer blastocysts singly onto feeder layers in wells, using a pulled Pasteur pipette under mouth control; and incubate for 4-5 d.a

3 Change medium after 3-4 d, or when it starts to become acidic. Dissociation of cultured ICMs:

After 4-5 d of culture the ICM is ready to be picked and dissociated (Figure 1A). The cultured ICMs will vary in size and appearance, with some having grown or differentiated more than others; and it is impossible to predict precisely from their morphological appearance at this stage which are more likely to give rise to mES cell lines. However, those cultured ICMs that have a dense core of material surrounded by a 'rind' of endoderm-like cells, or which have elongated into an egg cylinder-like structure, are not likely to produce undifferentiated mES cell lines.

4 Dispense (~30 ml) microdrops of trypsin solution in rows in a 60 mm Petri dish. Carefully pipette mineral oil down the side of the dish to just cover the drops.

5 Aspirate medium from the blastocyst cultures, and replace with 0.5 ml PBS.

6 Under a dissecting microscope, use the blunt pipette to dislodge the ICM from the underlying trophoblast. Transfer the ICM into a microdrop of trypsin solution, using a pulled pipette under mouth control.

7 Leave the ICM in trypsin solution for 5 min at r.t.; introduce a small amount of mES-cell medium to neutralize the trypsin; and using a pipette which has been pulled and broken at a diameter about half that of the ICM, draw the ICM up and down several times to dissociate it into a mixture of small clumps and single cells.

should be heat-sealed to give a blunt, closed tip

• Aspirator tube assembly (Protocol 6)

• Trypsin solution (Protocol 3)

8 Transfer the disaggregated ICM into a fresh feeder well, and resume incubation.

Identification and harvesting of mES-cell colonies

9 After 2-3 d of incubation of the disaggregated ICM, discrete colonies of cells may be observed under an inverted microscope with either x 10 or x 20 objectives.b

10 By 4-7 d of culture, definitive mES-cell colonies should be discernible in a proportion of dissociated ICM cultures (Figure 1B). These colonies, which are multilayered and have a sharp, clear boundary, are composed of characteristic, small cells with a high nuclear-to-cytoplasmic ratio, and which are difficult to distinguish individually by light microscopy. Definitive mES-cell colonies may be picked and dissociated individually following the method used for ICMs (above).c 4 e

11 Transfer each disaggregated colony into a fresh feeder well containing LIF-supple-mented mES-cell medium for expansion.

Expansion of mES cells:

After 2 d of culture small, secondary colonies of mES cells will be visible in the majority of wells or dishes. Discard cultures consisting only of differentiated cells. By 3-4 d of culture, the undifferentiated colonies will have reached a size suitable for expansion (Figure IC), which should carried out even if few colonies are present.f

12 Aspirate medium, wash cells with 1 ml PBS, add 0.2 ml trypsin solution, and incubate for 5 min. Add 0.5 ml mES-cell medium to neutralize the trypsin, and using a Pasteur pipette draw the cell suspension up and down several times to break up the colonies into single cells.g

13 When a single-cell suspension is achieved, transfer this onto a feeder layer in a 35 mm dish containing LIF-supplemented mES-cell medium. Contents of wells originating from the same blastocyst may be combined at this stage, and the mES cell culture is designated as 'passage 1' (Figure ID).

14 After a further 3-4 d of culture, trypsinize the dish and transfer the contents into a 25 cm2 feeder flask (passage 2). Cells are now cultured as described in Protocol 3, and aliquots should be frozen down as soon as sufficient cells are available (usually by passage 3).

aThe blastocysts hatch from the zona pellucida and attach to the dish during overnight culture. Thereafter, trophoblast cells grow outwards over the surface of the dish whilst the cells of the ICM form a clump in the middle of the outgrowth.

b At this stage most colonies will consist of differentiated cells, and may include trophoblast, endoderm and epithelial-like cells. Illustrations of these are given in (4), (5), and (24). Before becoming giant cells, trophoblast colonies in particular may be mistaken for mES-cell colonies; and so it is important to monitor periodically the development of putative mES-cell colonies.

c If the dissociated ICM cells have been blown hard via the mouthpiece into the well, mES-cell colonies become located near the edge of the well where they are difficult to see; and so it is advisable to search the periphery particularly carefully.

Protocol 7 continued d These primary mES-cell colonies dissociate more readily than cultured ICMs, and form a single cell suspension with a minimum of pipetting.

e Alternatively, one may trypsinize the entire contents of a well in situ and transfer the whole onto another feeder well (5, 26) or a larger feeder layer (if there are many mES-cell colonies). fWhen allowed to become too large, these secondary mES-cell colonies fail to dissociate into single cells on trypsinization.

gCare should be taken to minimize formation of air bubbles, which make it difficult to monitor the progress of dissociation under a dissecting microscope.

surgical intervention has been described (17). Briefly, this uses tamoxifen as a pharmacological block to oestrogen action, thus obviating the need for ovariectomy; and progesterone activity is maintained as in the ovariectomy method by the coinjection of Depo-Provera.

Microsurgical dissection is performed with glass needles and requires a micromanipulator assembly, consisting of a pair of micromanipulators attached to a base-plate and a fixed stage microscope with image-erected optics (Figure 3). The reader is referred to primary sources for comprehensive descriptions of the preparation of apparatus, and for rudiments in micromanipulation (23, 24).

4.2.1 Culture of epiblast from day 4.5 p.c. blastocysts (Method B)

Epiblasts isolated from day 4.5 p.c. blastocysts are cultured (on feeder layers, with LIF-supplemented mES-cell medium) for 3 d, by which time a single colony of undifferentiated cells should be present in each well (Figure 6A). If contaminating primitive endoderm was transferred with the epiblast there may be several additional, small colonies of differentiated cells (Figure 6B). After 3 d, the epiblast colonies are picked and dissociated as described in Protocol 7 for cultured ICMs. However, detaching the epiblast colony from the dish is not as straightforward as with ICM clumps, but can usually be achieved by cutting through the feeder layer around the colony with the blunt-tipped pipette, and then pushing the colony with attached feeder cells away from the surface of the dish. The epiblast colonies should dissociate in trypsin solution more easily than ICMs to give mostly single cells, which are transferred into a fresh feeder well and cultured for 7 d in LIF-supplemented mES-cell medium. The medium is changed after 4 d.

Colonies of cells usually can be found after ~3d. As occurs following ICM dissociations, many of these colonies are of differentiated cells, often endo-derm-like. In contrast to ICM dissociations, epiblast dissociations give rise to fewer colonies in total, and trophectoderm and giant cells are rarely encountered. This facilitates the identification of individual mES-cell colonies, which appear from ~4 d of culture and may vary in number from one to 30 per well. After 7 d, mES-cell colonies are harvested and dissociated as in Protocol 7, and seeded into fresh feeder wells. Numerous mES-cell colonies should be apparent 2 d after this second dissociation. After 3-4 d (Figure 6D), the well is trypsinized

(B)

Figure 3 Micromanipulator assembly for microsurgical dissection of epiblasts. (A) Here, Leitz micromanipulators are attached to a baseplate, and a microscope with erect optics is set between them. (B) Close-up to show a Puliv chamber on the microscope stage, with glass needles mounted in each micromanipulator.

Figure 3 Micromanipulator assembly for microsurgical dissection of epiblasts. (A) Here, Leitz micromanipulators are attached to a baseplate, and a microscope with erect optics is set between them. (B) Close-up to show a Puliv chamber on the microscope stage, with glass needles mounted in each micromanipulator.

Protocol 8

Solutions required for epiblast isolation

Ca2+ — and Mg*+ — free Tyrode's saline: For 1L, combine the following reagents:

NaCl 8.00 g KH2PO4 0.025 g

KCl 0.3 g NaHCO3 1.0 g

NaH2PO4.5H2O 0.093 g Glucose 2.0 g

(or NaH2PO4.2H2O 0.05 g)

Make up in AnalaR® water, filter sterilize,

aliquot (e.g. 5 ml), and store at 4 °C.

Hepes-buffered KSOM, or KSOM-Hepes (27): For 1 L, combine the following reagents:

NaCl 5.5518 g

CaC12.2H2O 0.2514 g

KCl 0.1868 g

EDTA 0.0038 g

KH2PO4 0.0476 g

Hepes 5.206 g

MgSO4.7H2O 0.0493 g

BSA 4.0g

Pyruvate (sodium salt) 0.022 g

Penicillin 0.060 g

Glucose 1.002 g

Streptomycin 0.050 g

NaHCO3 0.4201 g

Phenol Red 0.01 g

Lactate (Na salt) syrup, 60% (w/w) 0.8623 ml

Glutamine (200 mM) 5.0 ml

Make up in AnalaR® water, pH to 7.2, filter sterilize, aliquot (e.g. 10 ml), and store at 4 °C.

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