Gregory C and Veeman M (2013), 3D-printed microwell arrays for Ciona microinje...

XB-ART-47714

PLoS One 2013 Jan 01;812:e82307. doi: 10.1371/journal.pone.0082307.

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3D-printed microwell arrays for Ciona microinjection and timelapse imaging.

Gregory C , Veeman M .

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Ascidians such as Ciona are close chordate relatives of the vertebrates with small, simple embryonic body plans and small, simple genomes. The tractable size of the embryo offers considerable advantages for in toto imaging and quantitative analysis of morphogenesis. For functional studies, Ciona eggs are considerably more challenging to microinject than the much larger eggs of other model organisms such as zebrafish and Xenopus. One of the key difficulties is in restraining the eggs so that the microinjection needle can be easily introduced and withdrawn. Here we develop and test a device to cast wells in agarose that are each sized to hold a single egg. This injection mold is fabricated by micro-resolution stereolithography with a grid of egg-sized posts that cast corresponding wells in agarose. This 3D printing technology allows the rapid and inexpensive testing of iteratively refined prototypes. In addition to their utility in microinjection, these grids of embryo-sized wells are also valuable for timelapse imaging of multiple embryos.

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Species referenced: Xenopus
Genes referenced: grap2 hopx

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References [+] :

Becker, Polymer microfabrication technologies for microfluidic systems. 2008, Pubmed

	Figure 2. Ciona injection mold, prototype 1.A) Top view of the design for the proposed device. B) Side view. C) Perspective view. D) Photograph of the device as built by micro-resolution stereolithography. E) Agarose dish with egg-sized microwells cast using the prototype mold device.
	Figure 3. Ciona injection mold, prototype 2.A) Top view of the design for the proposed device. B) Side view. C) Perspective view. D) Photograph of the device as built by micro-resolution stereolithography. E) Scanning electron micrograph of one subarray of posts on the 3D printed device. F) Confocal micrograph of micro-wells cast in agarose. The agarose is labelled with fluorescent beads. Orthogonal reconstructions through the confocal stack are as indicated. Scale bar=100µm.
	Figure 4. Eggs arrayed for microinjection.A) Uninjected eggs in the microwell array (prototype 2). B) Eggs injected with Alexa 568 conjugated dextran are evident by their red color (fluorescent overlay over bright field image).
	Figure 5. Embryos arrayed for timelapse imaging.Selected frames from a differential interference contrast time-lapse movie of fertilized eggs developing through to the mid-tailbud stage while arrayed in agarose microwells (prototype 1).
	Figure 1. Ciona egg diameter. A) Brightfield microscope image of fertilized Ciona intestinalis eggs. B) Histogram of egg diameter for 97 measured eggs.