3.2 Separate
the Deep Ectoderm
and Assemble 3D
Aggregates
Brief exposure to calcium-magnesium free DFA media loosens cell–
cell adhesions between the deep and superficial cell layers and
allows removal of the superficial sheet of the epithelial layer from
the multilayered deep cells of the animal cap (Fig. 1b) (see Note 2).
1. Transfer the isolated ectoderm explant into a dish filled with
calcium-magnesium free DFA. Keep explants away from the
air–water interface or air bubbles to prevent tissue damage.
2. Position the ectoderm tissue to face the pigmented animal side
up using hair tools.
3. Monitor tissue dissociation under the stereoscope. Within
5–10 min you should observe signs that the superficial layer is
delaminating from the deep cells as light colored, lesspigmented deep cells move out from the edge of the darker
pigmented cells of the superficial layer (see Note 3).
4. Carefully lift the pigmented superficial layer to detach from the
edge of deep cell layers using a hair knife. With practice, the
entire sheet of superficial layer can be peeled off in rapid fashion. Discard the superficial layers to prevent further dissociation and reduce chances of contamination with target deep
cells.
5. Collect the separated deep cells using 200P pipette and minimize the amount of calcium-magnesium free DFA media transferred with cells to a non-adherent PCR tube filled with 200 μl
of DFA with antibiotic (see Note 4).
6. Stand the PCR tube upright in a rack to induce deep ectoderm
cell aggregation (0 h post aggregation, hpa).
7. Deep ectoderm cells form a compact aggregate within 2 hpa
and become a spherical aggregate by 5 hpa.
8. To continue the culture of aggregates en masse, transfer the
aggregate after 5 hpa to a dish filled with DFA with antibiotic.
Position aggregate far enough from each other and avoid agitation of the culture dish to prevent conglomeration of individual aggregates.
9. Deep ectoderm aggregates that undergo successful epithelialization and sequential maturation to epidermis can be easily
recognized and scored using a simple stereoscope. By 24 hpa
the aggregate surface is populated by highly motile multiciliated cells which drive aggregate rotation or movement.
3.3 Imaging the 3D
Embryonic Aggregates
Progression of the MET can be visualized using end-point analysis
of fixed and immuno-stained aggregates (see steps 1–10) or can be
visualized dynamically from live aggregates expressing epithelialspecific proteins tagged with fluorescent proteins (see steps 11–16).
1. Fill the glass vial with a fixative (see Note 5).
280
Hye Young Kim and Lance A. Davidson
the Deep Ectoderm
and Assemble 3D
Aggregates
Brief exposure to calcium-magnesium free DFA media loosens cell–
cell adhesions between the deep and superficial cell layers and
allows removal of the superficial sheet of the epithelial layer from
the multilayered deep cells of the animal cap (Fig. 1b) (see Note 2).
1. Transfer the isolated ectoderm explant into a dish filled with
calcium-magnesium free DFA. Keep explants away from the
air–water interface or air bubbles to prevent tissue damage.
2. Position the ectoderm tissue to face the pigmented animal side
up using hair tools.
3. Monitor tissue dissociation under the stereoscope. Within
5–10 min you should observe signs that the superficial layer is
delaminating from the deep cells as light colored, lesspigmented deep cells move out from the edge of the darker
pigmented cells of the superficial layer (see Note 3).
4. Carefully lift the pigmented superficial layer to detach from the
edge of deep cell layers using a hair knife. With practice, the
entire sheet of superficial layer can be peeled off in rapid fashion. Discard the superficial layers to prevent further dissociation and reduce chances of contamination with target deep
cells.
5. Collect the separated deep cells using 200P pipette and minimize the amount of calcium-magnesium free DFA media transferred with cells to a non-adherent PCR tube filled with 200 μl
of DFA with antibiotic (see Note 4).
6. Stand the PCR tube upright in a rack to induce deep ectoderm
cell aggregation (0 h post aggregation, hpa).
7. Deep ectoderm cells form a compact aggregate within 2 hpa
and become a spherical aggregate by 5 hpa.
8. To continue the culture of aggregates en masse, transfer the
aggregate after 5 hpa to a dish filled with DFA with antibiotic.
Position aggregate far enough from each other and avoid agitation of the culture dish to prevent conglomeration of individual aggregates.
9. Deep ectoderm aggregates that undergo successful epithelialization and sequential maturation to epidermis can be easily
recognized and scored using a simple stereoscope. By 24 hpa
the aggregate surface is populated by highly motile multiciliated cells which drive aggregate rotation or movement.
3.3 Imaging the 3D
Embryonic Aggregates
Progression of the MET can be visualized using end-point analysis
of fixed and immuno-stained aggregates (see steps 1–10) or can be
visualized dynamically from live aggregates expressing epithelialspecific proteins tagged with fluorescent proteins (see steps 11–16).
1. Fill the glass vial with a fixative (see Note 5).
280
Hye Young Kim and Lance A. Davidson