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Biochem Biophys Res Commun
2021 Sep 10;569:29-34. doi: 10.1016/j.bbrc.2021.06.049.
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Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.
Nam SW
,
Chae JP
,
Kwon YH
,
Son MY
,
Bae JS
,
Park MJ
.
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Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of eggcleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.
Fig. 1. (a)–(d) Xenopus chip fabrication and (e)–(h) egg-loading process. (a) 3D design structure created using the Autodesk software. (b) Digital light processing (DLP) method to print the 3D mold structure. (c) Polydimethylsiloxane (PDMS) chamber created by soft lithography. (d) Xenopus chip created by the assembly of the upper PDMS chamber and lower slide glass. Snapshots of the Supporting movie S1 are summarized in (e)–(h). (e) Xenopus eggs are stored in the pipette tip. (f) Xenopus eggs are loaded into the chip by manual pipetting. (g) As one trapping site was filled with an egg, the other eggs moved to the next sites via the bypass channel. (h) All trapping sites are filled with eggs. The scale bar is 10 mm.
Fig. 2. In vitro fertilization of Xenopus eggs in the chip. (a) A schematic of the sperm-circulating system via a peristaltic pump connected between the Xenopus chip and the sperm-mixed solution. (b) A picture of the in vitro fertilization system. (c)–(g) Development of fertilized eggs. (c) Cleavage of the egg at stage 2. (d) 4-cell conditions at stage 3. (e) Blastula at stage 7. (f) Early stage of tadpole development at stage 25. (g) Tadpole incubated in the Xenopus chip. All scale bars are 1 mm.
Fig. 3. Heartbeat of a trapped tadpole that has been in vitro fertilized inside the chip. Snapshot of the Supporting movie S2. The scale bar is 1 mm
Fig. 4. (a)–(d) Trapping process. (e)–(h) Escaping process. Three tadpoles were in vitro fertilized inside the chip. Via a peristaltic pump, the flow directions inside the chip were controlled, in either a forward or reverse direction. The arrows in (a)–(d) represent the flow direction. Snapshots of the Supporting movie S3 are summarized in (a)–(d). (a) Forward directional flow leads the tadpoles to become trapped in the trapping sites. (b) Reverse directional flow leads the tadpoles to escape from the trapping site. (c) The next forward directional flow guides the tadpoles to the trapping sites, where they become trapped. (d) All tadpoles are trapped. To spare the life of the in vitro fertilized tadpoles, a tadpole escaping process was developed. Snapshots of the Supporting movie S4 are summarized in (e)–(h). (e) The inlet port is separated from the tube, and a strong reverse flow is applied. (f) With a water droplet, the 1st tadpole escaped through the inlet port. (g) The 2nd tadpole escaped. (h) All tadpoles escaped. All scale bars are 10 mm.