Here we describe a reliable, controllable and scalable 3D cell
aggregate consisting of mesenchymal cells isolated from the deep
ectoderm of Xenopus laevis embryos that provides ready access to
the processes driving MET [33]. The ectoderm of early Xenopus
embryos is composed of one or more rows of deep mesenchymal
cells that actively express and assemble the extracellular matrix
protein fibronectin [34] and are covered by a superficial epithelial
sheet with stereotypical tight junctions [35] and distinctive keratin
intermediate filaments [36–38]. Upon isolation, deep mesenchymal cells adhere to one another to form a dense aggregate. After
only 5 h surface cells on the mesenchymal aggregate progressively
transition to adopt an epithelial phenotype which is marked by
elevated epithelial gene expression along with newly established
apical basal polarity and the formation of tight junctions. The
intrinsic nature of 3D culture, self-assembly, rapid development,
scalability in size and number, and accessibility for both live imaging and mechanical testing all contribute to the utility of this 3D
tissue model of MET.
2 Materials
2.1 Embryos
Xenopus laevis embryos are obtained by general procedure
[39]. Embryos may be reliably cultured at a variety of temperatures
from 14 to 26
C and staged by external criteria [40].
2.2 Reagents
1. 1x MBS (Modified Barth’s Saline): 88 mM NaCl, 1 mM KCl,
2.4 mM NaHCO 3 , 0.82 mM MgSO 4 , 0.33 mM (CaNO 3 )
2 ,
0.41 mM CaCl 2 , 10 mM HEPES in distilled water. Adjust pH
to 7.4 with NaOH. Use 1/3Â MBS for embryo culture.
2. Danilchik’s for Amy (DFA): 53 mM NaCl, 5 mM Na 2 CO 3 ,
4.5 mM potassium gluconate, 32 mM sodium gluconate,
1 mM CaCl 2 , 1 mM MgSO 4 in distilled water. Adjust pH to
8.3 with granular Bicine. Filter with 0.22 μm filter. Just prior to
use, add 1% of antibiotic/antimycotic (A5955, Sigma-Aldrich)
to prevent bacterial growth. DFA is formulated to match the
ionic composition of interstitial media of gastrula stage
embryos [41]. Use 1Â DFA for aggregate culture.
3. Calcium-magnesium free DFA: DFA made without CaCl 2 and
MgSO 4 .
4. 3% Ficoll (Sigma-Aldrich) in 1Â MBS. Culture media used
during microinjection.
5. Phosphate-buffered saline (PBS).
6. Triton X -100.
Xenopus Deep Cell Aggregates: A 3D Model for MET
277
aggregate consisting of mesenchymal cells isolated from the deep
ectoderm of Xenopus laevis embryos that provides ready access to
the processes driving MET [33]. The ectoderm of early Xenopus
embryos is composed of one or more rows of deep mesenchymal
cells that actively express and assemble the extracellular matrix
protein fibronectin [34] and are covered by a superficial epithelial
sheet with stereotypical tight junctions [35] and distinctive keratin
intermediate filaments [36–38]. Upon isolation, deep mesenchymal cells adhere to one another to form a dense aggregate. After
only 5 h surface cells on the mesenchymal aggregate progressively
transition to adopt an epithelial phenotype which is marked by
elevated epithelial gene expression along with newly established
apical basal polarity and the formation of tight junctions. The
intrinsic nature of 3D culture, self-assembly, rapid development,
scalability in size and number, and accessibility for both live imaging and mechanical testing all contribute to the utility of this 3D
tissue model of MET.
2 Materials
2.1 Embryos
Xenopus laevis embryos are obtained by general procedure
[39]. Embryos may be reliably cultured at a variety of temperatures
from 14 to 26
C and staged by external criteria [40].
2.2 Reagents
1. 1x MBS (Modified Barth’s Saline): 88 mM NaCl, 1 mM KCl,
2.4 mM NaHCO 3 , 0.82 mM MgSO 4 , 0.33 mM (CaNO 3 )
2 ,
0.41 mM CaCl 2 , 10 mM HEPES in distilled water. Adjust pH
to 7.4 with NaOH. Use 1/3Â MBS for embryo culture.
2. Danilchik’s for Amy (DFA): 53 mM NaCl, 5 mM Na 2 CO 3 ,
4.5 mM potassium gluconate, 32 mM sodium gluconate,
1 mM CaCl 2 , 1 mM MgSO 4 in distilled water. Adjust pH to
8.3 with granular Bicine. Filter with 0.22 μm filter. Just prior to
use, add 1% of antibiotic/antimycotic (A5955, Sigma-Aldrich)
to prevent bacterial growth. DFA is formulated to match the
ionic composition of interstitial media of gastrula stage
embryos [41]. Use 1Â DFA for aggregate culture.
3. Calcium-magnesium free DFA: DFA made without CaCl 2 and
MgSO 4 .
4. 3% Ficoll (Sigma-Aldrich) in 1Â MBS. Culture media used
during microinjection.
5. Phosphate-buffered saline (PBS).
6. Triton X -100.
Xenopus Deep Cell Aggregates: A 3D Model for MET
277