Limits...
Development-on-chip: in vitro neural tube patterning with a microfluidic device.

Demers CJ, Soundararajan P, Chennampally P, Cox GA, Briscoe J, Collins SD, Smith RL - Development (2016)

Bottom Line: Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity.In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development.Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo.

View Article: PubMed Central - PubMed

Affiliation: Microinstruments and Systems Laboratory, University of Maine, Orono, ME 04469, USA Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME 04469, USA The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus

Directed spatial patterning in the microfluidic device reveals a region of permissive differentiation. Representative images and average plots of spatial differentiation of HB9+ cells (GFP labeled) along SHH gradient (n=4). Vertical bars on the left diagrammatically indicate the concentration and spatial gradient of RA and/or SHH. Plots to the right indicate the average intensity distribution from at least four experiments as well as actual PM concentrations based on computer simulations (quantified as mean percent cells/bin ±s.d., n=3). (A) Control HB9+ MNs subjected to a uniform concentration of PM and RA. Red dashed lines indicate example bin width. (B-D) HB9+ MNs subjected to varying PM gradients. Inset in C illustrates higher magnification detail of the MN cluster (200× confocal image). (E) The addition of an opposing gradient of BMP4 (20 ng/ml) further narrows the MN domain. (F) High expression of the pluripotency marker OCT4 towards the dorsal end of the microdevice (outside of the permissive MN region) indicates the effect of early exposure to a cross-gradient of BMP4. (G) Live/dead staining with Hoechst 33342 and propidium iodide (PI) reveal that we have not simply created a zone of permissive cell growth. *P≤0.05, **P≤0.01, ***P≤0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920155&req=5

DEV126847F3: Directed spatial patterning in the microfluidic device reveals a region of permissive differentiation. Representative images and average plots of spatial differentiation of HB9+ cells (GFP labeled) along SHH gradient (n=4). Vertical bars on the left diagrammatically indicate the concentration and spatial gradient of RA and/or SHH. Plots to the right indicate the average intensity distribution from at least four experiments as well as actual PM concentrations based on computer simulations (quantified as mean percent cells/bin ±s.d., n=3). (A) Control HB9+ MNs subjected to a uniform concentration of PM and RA. Red dashed lines indicate example bin width. (B-D) HB9+ MNs subjected to varying PM gradients. Inset in C illustrates higher magnification detail of the MN cluster (200× confocal image). (E) The addition of an opposing gradient of BMP4 (20 ng/ml) further narrows the MN domain. (F) High expression of the pluripotency marker OCT4 towards the dorsal end of the microdevice (outside of the permissive MN region) indicates the effect of early exposure to a cross-gradient of BMP4. (G) Live/dead staining with Hoechst 33342 and propidium iodide (PI) reveal that we have not simply created a zone of permissive cell growth. *P≤0.05, **P≤0.01, ***P≤0.001.

Mentions: After 7 days, cells were imaged for GFP expression in order to identify spatial patterning within the cell chamber (Fig. 3). Except where noted, all images were taken under low magnification (50×) to capture the entire 1 mm×1 mm cell chamber and fluorescence intensity was quantified as a function of spatial distribution down the SHH/PM gradient. For analysis, the chamber was divided vertically (along the gradient) into ten 100-µm-wide bins, and the fluorescence intensity, which is proportional to the number of HB9+ cells, in each bin quantified. All experiments were repeated on at least four different devices, and the average cell counts for all experiments plotted as the percentage of total cells to the right of the figure as a function of distance (additional details can be found in Materials and Methods).


Development-on-chip: in vitro neural tube patterning with a microfluidic device.

Demers CJ, Soundararajan P, Chennampally P, Cox GA, Briscoe J, Collins SD, Smith RL - Development (2016)

Directed spatial patterning in the microfluidic device reveals a region of permissive differentiation. Representative images and average plots of spatial differentiation of HB9+ cells (GFP labeled) along SHH gradient (n=4). Vertical bars on the left diagrammatically indicate the concentration and spatial gradient of RA and/or SHH. Plots to the right indicate the average intensity distribution from at least four experiments as well as actual PM concentrations based on computer simulations (quantified as mean percent cells/bin ±s.d., n=3). (A) Control HB9+ MNs subjected to a uniform concentration of PM and RA. Red dashed lines indicate example bin width. (B-D) HB9+ MNs subjected to varying PM gradients. Inset in C illustrates higher magnification detail of the MN cluster (200× confocal image). (E) The addition of an opposing gradient of BMP4 (20 ng/ml) further narrows the MN domain. (F) High expression of the pluripotency marker OCT4 towards the dorsal end of the microdevice (outside of the permissive MN region) indicates the effect of early exposure to a cross-gradient of BMP4. (G) Live/dead staining with Hoechst 33342 and propidium iodide (PI) reveal that we have not simply created a zone of permissive cell growth. *P≤0.05, **P≤0.01, ***P≤0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920155&req=5

DEV126847F3: Directed spatial patterning in the microfluidic device reveals a region of permissive differentiation. Representative images and average plots of spatial differentiation of HB9+ cells (GFP labeled) along SHH gradient (n=4). Vertical bars on the left diagrammatically indicate the concentration and spatial gradient of RA and/or SHH. Plots to the right indicate the average intensity distribution from at least four experiments as well as actual PM concentrations based on computer simulations (quantified as mean percent cells/bin ±s.d., n=3). (A) Control HB9+ MNs subjected to a uniform concentration of PM and RA. Red dashed lines indicate example bin width. (B-D) HB9+ MNs subjected to varying PM gradients. Inset in C illustrates higher magnification detail of the MN cluster (200× confocal image). (E) The addition of an opposing gradient of BMP4 (20 ng/ml) further narrows the MN domain. (F) High expression of the pluripotency marker OCT4 towards the dorsal end of the microdevice (outside of the permissive MN region) indicates the effect of early exposure to a cross-gradient of BMP4. (G) Live/dead staining with Hoechst 33342 and propidium iodide (PI) reveal that we have not simply created a zone of permissive cell growth. *P≤0.05, **P≤0.01, ***P≤0.001.
Mentions: After 7 days, cells were imaged for GFP expression in order to identify spatial patterning within the cell chamber (Fig. 3). Except where noted, all images were taken under low magnification (50×) to capture the entire 1 mm×1 mm cell chamber and fluorescence intensity was quantified as a function of spatial distribution down the SHH/PM gradient. For analysis, the chamber was divided vertically (along the gradient) into ten 100-µm-wide bins, and the fluorescence intensity, which is proportional to the number of HB9+ cells, in each bin quantified. All experiments were repeated on at least four different devices, and the average cell counts for all experiments plotted as the percentage of total cells to the right of the figure as a function of distance (additional details can be found in Materials and Methods).

Bottom Line: Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity.In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development.Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo.

View Article: PubMed Central - PubMed

Affiliation: Microinstruments and Systems Laboratory, University of Maine, Orono, ME 04469, USA Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME 04469, USA The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, UK.

No MeSH data available.


Related in: MedlinePlus